chapter 3 fibrous growth of strontium ...shodhganga.inflibnet.ac.in/bitstream/10603/11503/8/08...51...
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CHAPTER 3
FIBROUS GROWTH OF STRONTIUM SUBSTITUTED
HYDROXYAPATITE
3.1 INTRODUCTION
Calcium phosphate has many different phases, such as
CaHPO4.2H2O (DCPD), Ca3(PO4)2 (TCP) and Ca10(PO4)6(OH)2 (HAp)
(Vallet-Regi and Gonzalez-Calbet 2004). Among these, particular attention
has been drawn towards HAp, since it is the main mineral constituent of
natural bone and teeth. It is widely used in various biomedical applications
and many undesirable cases of pathological mineralization of the articular
cartilage, cardiac valves and kidney stones (Sivakumar et al 1998, Anee et al
2004, Dieppe and Calvert 1983). Previous reports have stated that the fibrous
HAp reinforced composites could be a promising material for hard tissue
replacement implants (Suchanek and Yoshimura 1998, Cui et al 2008, Lin et
al 2007). However, the bioactive process in HAp implants has drawbacks
when compared with other materials such as bioactive glasses and glass
ceramics because of their solubility (Ducheyne et al 1993). The possibility to
perform ionic substitution in CaPs will induce the complex structures at the
unit cell level and alter its bioactivity (Porter et al 2003). Ca2+ ions can be
replaced by various divalent cations including Sr2+, Ba2+, Cd2+, Mg2+ etc.
These substitutions alter its thermal stability, solubility and surface reactivity.
Strontium plays a significant role in the biomineralization of bone (Saint-Jean
et al 2005, Guo et al 2005).
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In addition, strontium is used for the treatment of osteoporosis
(Meunier et al 2004). It was found to induce osteoblast activity by
stimulating bone formation and inhibiting bone resorption both in vitro and
in vivo (Pors Nielsen 2004). Strontium has various effects on bone
metabolism depending on its dosage used. Low strontium concentration
(2-10 µg/ml) stimulate bone formation, whereas, high concentration (20-100
µg/ml) of strontium induces mineralization defect (Verberckmoes et al 2004).
The in vitro crystallization of CaPs has been carried out using gel
under physiological conditions by Ashok et al (2003). The influence of
various ions on the crystallization of DCPD and HAp has been reported
(Kanchana and Sekar 2010, Parekh et al 2008). Crystal structure of Sr-HAp
is reported by Kikuchi et al (1994). A combination of strontium and fluoride
elements seems to be the proper treatment of osteoporosis (Rotika et al 1999).
The capability of Sr-HAp to improve osteointegration is also reported by
Ni et al (2006). Recently, Xue et al (2006) have demonstrated the enhanced
adhesion and differentiation of osteoprecursors cells in contact with Sr -HAp.
Strontium ranelate (Protelos®) is the drug that can induce bone cell replication
and inhibit the osteoclasts activity (Marie 2006). In addition, strontium
containing toothpaste was developed to enhance the remineralization of the
dental enamel (Surdacka et al 2007).
Semisynthetic, orally absorbed broad spectrum antibiotic drug,
amoxicillin (AMX) has been extensively used against bacterial infections.
Slow and continuous release of an antibiotic during the bone implantation is
essential to prevent infections. The drug release kinetics of HAp, other
calcium phosphates, porous HAp blocks and HAp coating on metals has been
reported in the literature (Joosten et al 2005, Kim et al 2005, Radin et al
1997). In these cases, drug release is too rapid and a sustained release in a
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controlled manner is very difficult to attain. Alkhraisat et al (2010) have
investigated the loading and release of the doxycycline hyclate from strontium
substituted -TCP which provide a way to switch from the rapid and complete
release to slower and prolonged drug delivery. Recently, mesoporous
strontium HAp nanorods synthesized by hydrothermal method, were shown to
have controlled release property (Zhang et al 2010). In this chapter, we have
investigated the effect of strontium on the mineralization of HAp at
physiological temperature along with its drug release properties.
3.2 EXPERIMENTAL METHODS
The analytical grade calcium chloride (CaCl2.2H2O, Merck) and
disodium hydrogen phosphate (Na2HPO4, Merck) were used as reagents. The
single diffusion silica gel method was employed to crystallize the HAp, as
described in chapter 2. The mixture of the aqueous solution of sodium
metasilicate (Na2SiO3.9H2O, Qualigens) of specific gravity 1.03 g/cc and
Na2HPO4 (0.6 M) was adjusted to the pH 7.4 using glacial acetic acid. After
gelation, about 1 M CaCl2.2H2O mixed with strontium chloride (SrCl2, 0, 10,
50 and 100 mM) was used as a supernatant solution and were labeled as Sr0,
Sr01, Sr05 and Sr1, respectively. The crystallization was carried out at 27 °C
(±0.1 °C) in an incubator. The samples were harvested and thoroughly
washed with distilled water, dried and kept in a dessicator. The bactericidal
experiments were carried out with gram positive bacteria Bacillus subtilis and
Staphylococcus aureus in nutrient media.
The phase analysis of the powders was done by XRD (Model PW
1729, Philips, Holland) using 35 mA/40 kV current, with monochromatic
CuK (target) radiation ( = 1.5405 Å) with increment step size of 0.04°, scan
rate of 0.02° and a scan range from 2 = 20 to 50°. The identification of
functional groups in the HAp powder was analyzed by FTIR analysis
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(PERKIN ELMER spectrum RXI using KBr pellet technique) within the
scanning range 4000 - 450 cm 1. The elemental analyses of the samples were
done using ICP-AES (Inductively coupled plasma-atomic emission
spectrometer, 5300DU, PERKIN-ELMER) by dissolving 0.1 gm of the
sample in 0.5 ml of HNO3 and make upto 50 ml by adding Milli-Q water.
The surface morphology of the samples was investigated by scanning electron
microscopy (SEM) (Model JSM-5800, JEOL, scanning electron microscope,
Japan). The samples were sputter coated with gold before examination. The
specific surface area of samples was determined by the Brunauer-Emmett-
Teller (BET) method using an ASAP 2020 V3.00 H model (Micromeritics)
surface area analyzer. The samples were outgassed under vaccum for 12 h at
200 °C before the analysis. In vitro bioactivity, drug release and
antimicrobial test were done as described in the previous chapter.
3.3 RESULTS AND DISCUSSION
After the addition of supernatant solution in the control and
strontium doped setups, a dense white precipitate of thickness 0.2 cm were
observed at the gel solution interface. For control test tubes, helical ribbon
was observed just below the interface precipitate and continued to develop
over a period of time (Figure 3.1a). The formation of helical ribbon was
found to be inhibited in strontium doped setups. For Sr01 and Sr05, periodic
well defined discs of precipitate along with small platy crystals were found
inside the gel (Figure 3.1b and 3.1c). For higher concentration (Sr1), thick
continuous precipitation followed by periodic precipitation was observed just
below the gel solution interface, without any platy crystals (Figure 3.1d).
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Figure 3.1 Liesegang patterns with various Sr concentrations (a) Sr0,
(b) Sr01, (c) Sr05 and (d) Sr1
3.3.1 SEM Studies
The HAp platy crystals of approximately 2.8 µm length and 0.2 µm
width was arranged radially from a central point for control sample
(Figure 3.2a). Presence of strontium changed the morphology of HAp from
plates to fibers. With low strontium concentration (Sr01), HAp fibers of
length 9 µm and width 1 µm are formed (Figure 3.2b). Further increase of
strontium (Sr05), produced dense fibers of 5 µm length and 500 nm width
(Figure 3.2c). Bunched fibers were observed in Sr1 (Figure 3.2d). The aspect
ratio and length of the fibers decreased significantly with increasing strontium
content (Table 3.1).
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Figure 3.2 SEM micrographs of (a) Sr0, (b) Sr01, (c) Sr05 and (d) Sr1
Table 3.1 Particle size of the samples by SEM
Sample
code
Length (µm)
(±0.5)
Width (µm)
(±0.1)
Aspect
ratio
Sr0
Sr01
Sr05
Sr1
2.8
9.0
5.0
2.3
0.2
1.0
0.5
0.4
6.20±3
27.46±19
16.14±7
5.75±1
3.3.2 XRD Analysis
The XRD patterns of the samples crystallized at 27 °C are
presented in Figure 3.3a to 3.3d which is in good agreement with the standard
data for HAp (JCPDS No. 09-0432). Sr05 and Sr1 showed the broad and
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2 0 3 0 4 0 5 0
(213
)
(221
)
(112
)
(200
)
t w o t h e t a ( d e g )
Inte
nsi
ty (
a.u
.)
(312
)
(222
)
(203
)
(310
)(2
12)
(210
)
(111
)
(202
)
(211
)
(102
)
(002
)
(202
)
(221
)
(200
)
(111
) (112
) b
a
c
(102
)
(002
)
(200
)
(211
)
(213
)(2
13)
(221
)
(111
)
(301
)
(202
)(211
)
(102
)
(002
)
(111
)
(213
)
(221
)
(301
)(202
)
(211
)
(102
)
d
(002
)
intense peak centered at 31.6°, due to the contributions of the (211), (112) and
(300) lattice planes. The increase in the intensity of the (002) plane with the
increase of strontium concentration, indicated the preferred orientation growth
of the crystals along the c-axis. The peak positions shifted slightly from the
standard XRD patterns for HAp, indicating the incorporation of strontium. The
lattice parameters determined by XRDA 3.1 software (Desgreniers and Lagarec
1994) were as given in Table 3.2. The lattice parameters varied with the
increase in the strontium content, which may be due to the replacement of
calcium by strontium in the apatite structure, inducing an increase in the lattice
constants, as Sr2+ (1.13 Å) has higher ionic radius than that of Ca2+ ions
(0.99 Å) (O’Deonnell et al 2008). Pan et al (2009) reported that the
crystallinity increased with an increase of strontium due to the formation of
strontium substituted apatite.
Figure 3.3 XRD patterns of (a) Sr0, (b) Sr01, (c) Sr05 and (d) Sr1
The crystallite size was calculated using Scherrer’s equation, that
is, Xs = 0.9 cos , where Xs is the average crystallite size in nm, is the
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full width of the peak at half of its maximum intensity (radian), is the
wavelength of X-rays (1.5406 Å), and is the Bragg’s diffraction angle (Klug
and Alexander 1974). The size of apatite crystals was found to be in the range
of 8-26 nm which is similar to the apatite crystals found in bone (Table 3.2).
The crystallinity (Xc) of the samples was determined by an empirical relation
between Xc and 002 (i.e., 002 × 3 Xc = KA), where Xc is the crystallinity
degree, 002 is full width of the peak at half intensity of (002) plane in degree
and KA is a constant (0.24) (Landi et al 2000). The crystallinity of the
samples was found to increase with strontium doping (Table 3.2). Strontium
is more electropositive (less electronegative) than calcium and as a result, the
bonding between strontium and oxidic site is more ionic. Hence introduction
of strontium in HAp increased the crystallite size and crystallinity.
Table 3.2 Crystallite size, Crystallinity and Lattice parameter of HAp
Samplecode
Crystallite Size,Xs (nm) (± 1)
Crystallinity,Xc (%)
Lattice parameters
a = b (Å)(±0.02)
c (Å)(±0.02)
Sr0
Sr01
Sr05
Sr1
9
16
20
27
64
79
82
87
9.30
9.48
9.36
9.27
7.08
6.84
6.86
6.87
3.3.3 FT-IR Analysis
FT-IR spectrum of the sample (Figure 3.4a to 3.4d, Table 3.3)
showed the small peak above 3500 cm-1 corresponds to the stretching
vibration of OH- group in apatite. A broad peak in the region 3445 cm-1,
which is assigned to the stretching and the band at 1645 cm-1 is ascribed to the
bending mode of adsorbed water on the sample. The peaks at 2927 and 2842
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cm-1 may correspond to HPO42- groups. The absorption peak at 1111 and
1034 cm-1 might be due to the stretching vibrations of phosphate group and
peaks at 596 and 563 cm-1 were due to bending vibrations of phosphate group.
There was no noticeable CO32- absorption peak at 1394 cm-1 except for the
sample Sr0, probably contaminated by the CO2 absorption from the air. The
peak at 863 cm-1 characteristic for HPO42- was observed at Sr0. A sharp
bending mode doublet around 600 cm-1 indicated that Sr-HAp samples were
highly crystallized (Canham et al 1996). Further, XRD analysis revealed the
increase in crystallinity on strontium incorporation (Table 3.2).
4000 3500 3000 2500 2000 1500 1000 500
3730
Wavenumber (cm-1)
Tra
nsm
itta
nce
(%
)
3445
1645 1
394
1111
1034
863
563
596
3730
2842
2842
2927
a
b
3445
1642
1110
1039
56060
1
3730
2842
2927
3445
d
c
1642
1107 10
39
561601
3730
2927
3445
1641
1111
1039
56160
1
Figure 3.4 FT-IR spectra of (a) Sr0, (b) Sr01, (c) Sr05 and (d) Sr1
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Table 3.3 FT-IR Assignments of functional groups of HAp
Vibrational frequency (cm-1
) Assignments
563
596
863
1034
1111
1645
2927
3445
3730
O-P-O bending
O-P-O bending
O-H stretching of HPO42-
P-O Asymmetric stretching
P-O Asymmetric stretching
O-H In-plane bending
HPO42- groups
O-H Stretching
O-H Stretching
3.3.4 Elemental Analysis
The ICP-AES results of Sr0, Sr01, Sr05 and Sr1 are presented in
Table 3.4. From elemental analysis, Ca/P ratio in Sr0 was found to be 1.34.
Wilson et al (2005) reported that the Ca-deficient apatite with Ca/P molar
ratios from 1.33-1.66 was due to the incorporation of HPO42- and CO3
2- in to
the apatite. The calcium content decreased gradually due to its incorporation
by strontium. Hence in response to the decrease in calcium content, strontium
content gradually increased and the proportional decrease in the strontium
content points to the isomorphic substitution. The Ca/P molar ratio of the
doped samples was similar to that of the biological apatite (1.50 to 1.85)
(Elliott 1994). Further, increase in the incorporation of strontium into HAp
crystals was seen, as the ion concentration increased in the growth medium
(Figure 3.5).
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Table 3.4 Elemental analysis of the control and Sr-HAp
SampleCa
(ppm)
P
(ppm)
Sr
(ppm)
Si
(ppm)Ca/P Sr/Ca (Ca+Sr)/P
Sr0
Sr01
Sr05
Sr1
479.6
756
723.5
654
357.4
472.75
470.8
438
0.00
13.24
70.31
86.00
3.38
10.55
14.86
11.91
1.34
1.59
1.53
1.49
-
0.01
0.09
0.13
1.34
1.62
1.68
1.68
0
20
40
60
80
100
Str
onti
um c
once
ntra
tion
(pp
m)
SamplesSr1Sr05Sr01
Figure 3.5 Strontium concentration of the samples in ppm
3.3.5 BET Analysis
The N2 adsorption/desorption isotherms of Sr0 and strontium doped
samples were as shown in Figure 3.6. The samples exhibited similar IV
isotherms and the typical H1-hysterisis loops, indicating the mesoporous
nature. The Sr0 sample had a specific surface area of 15.41 m2/g, pore
volume of 0.41 cm3/g and average pore size of 20 nm. The specific surface
area increased with the increasing concentrations of strontium ions, except for
Sr01. The difference in specific surface area was not significant with
increasing concentration of strontium (Table 3.5).
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Table 3.5 Pore volume, pore size and surface area of the samples
Sample
code
Pore volume
(cm3/g)
Pore size
(nm)
Surface area
(m2/g)
Sr0
Sr01
Sr05
Sr1
0.41±0.02
0.37±0.01
0.26±0.01
0.27±0.01
20.46±1.02
21.39±1.06
20.60±1.03
20.15±1.00
15.41±0.26
14.51±0.14
15.96±0.32
20.78±0.37
0.0 0.2 0.4 0.6 0.8 1.0
0
50
100
150
200
250
Qua
ntit
y A
dsor
bed
(cm
3 /g)
Relative pressure (P/Po)
Sr0
0.0 0.2 0.4 0.6 0.8 1.0
0
50
100
150
200
250
Qua
ntit
y A
dsor
bed
(cm
3 /g)
Relative pressure (P/Po)
Sr01
0.0 0.2 0.4 0.6 0.8 1.0
0
20
40
60
80
100
120
140
160
180
Qu
anti
ty A
dsor
bed
(cm
3 /g
)
Relative pressure (P/Po)
Sr05
0.0 0.2 0.4 0.6 0.8 1.0
0
20
40
60
80
100
120
140
160
180
Qua
ntit
y A
dso
rbed
(cm
3 /g)
Relative pressure (P/Po)
Sr1
Figure 3.6 Nitrogen adsoption-desorption isotherm of control and
Sr-HAp
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3.3.6 In vitro Bioactivity Test
The in vitro bioactivity test was performed by immersing the
samples into the SBF and maintained at 37 °C. The Sr0 sample before
immersion in SBF showed smooth surface (Figure 3.7a). In control (Sr0),
globules of size 3 m was randomly deposited on the surface after immersion
in SBF (Figure 3.7b), whereas in the strontium doped samples, porous layer,
consisting of sphere-like clusters were observed. A layer with irregular pores
of size varying from 4-5 m and the size of the spheroids was 500-700 nm
were observed on the Sr01 (Figure 3.7c). The surface of Sr05 and Sr1,
induced the deposition of homogeneous apatite layer (Figure 3.7d and 3.7e).
Based on these results, Sr-HAp is considered to have an enhanced bioactivity
compared to the native samples.
Figure 3.7 SEM micrograph of the samples in SBF (a) Sr0, (b) Sr01,
(c) Sr05 and (d) Sr1
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3.3.7 Drug Release Studies
The cumulative in vitro drug release profiles for the various
samples as a function of release time in PBS are as shown in Figure 3.8. The
initial rapid release of about 35.4, 37, 31 and 30 % respectively were observed
for Sr0, Sr01, Sr05 and Sr1 samples for a time period of 12 h. This rapid release
may be due to physical adsorption of drug molecules onto HAp surface. The
initial rapid release followed by a gradual slow release was observed for all
samples. It revealed that 100 % AMX was released in 72 h from the Sr01,
whereas, 84 and 73 % was released from Sr05 and Sr1 samples for the same
period. The Sr01 sample showed the fastest AMX release due to the lowest
surface area (14.51 m2/g) compared with other samples. The Sr0 sample with
the surface area of about 15.41 m2/g showed the faster release and reached 100 %
after 85 h. The Sr05 and Sr1 reached 100 % drug release after 104 and 118 h,
respectively.
0 20 40 60 80 100 120
0
20
40
60
80
100 Sr0 Sr01 Sr05 Sr1
Am
oxic
illi
n re
leas
e (%
)
Time (hrs)
Figure 3.8 Cumulative drug release of AMX from the samples
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Low concentration of strontium (Sr01) may increase the solubility
of HAp crystals which leads to the rapid release. In contrast to Sr01, Sr05
and Sr1 exhibit slow release. As the concentration of incorporated Sr
increases in the samples, it reduces the solubility of the samples, thereby
exhibiting slow rate of drug release (Dedhiya et al 1972). The burst release in
the initial phase and maintenance of an appropriate concentration would be
favourable to prevent the disease after surgery.
3.3.8 Antibacterial Activity
The antibacterial activity of AMX drug incorporated Sr0, Sr01,
Sr05 and Sr1 samples (Sr0D, Sr01, Sr05 and Sr1) were determined by disk
diffusion method using B. subtilis and S. aureus bacterial strains
(Figure 3.9 and 3.10). No bacterial resistance observed on non-drug loaded
samples. The inhibition zone of drug incorporated HAp samples on
B. subtiles and S. aureus were in the range of 13 to 22 mm and the results are
summarized in Table 3.6. The highest resistance was observed on Sr01D,
while Sr05D and Sr1D showed lesser sensitivity against both bacteria upto 24
h. The reason may be due the low solubility of the samples (Sr05D and Sr1D)
(Lin et al 2008). When compared with both bacterial strains, S. aureus was
less susceptible for all samples than B. subtilis (Stanic et al 2010).
Figure 3.9 Inhibition zone of control and Sr-HAp samples against
B. subtilis
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Figure 3.10 Inhibition zone of control and Sr-HAp samples against
S. aureus
Table 3.6 Antibacterial activity of drug incorporated samples against
B. subtilis and S. aureus
Bacterial strainDiameter of zone of inhibition (± 0.5 mm)
Sr0D Sr01D Sr05D Sr1D
B. subtilis 21 22 20 19
S. aureus 16 17 15 13
3.4 CONCLUSIONS
Strontium substituted HAp with fibrous morphology were
crystallized by a single diffusion silica gel method at 27 °C and pH 7.4. The
incorporation of the strontium led to the formation of fibrous HAp. The
incorporation of strontium increased the crystallite size and crystallinity of
HAp. The strontium in HAp accelerated the formation of biological apatite
and enhanced the in vitro bioactivity of HAp. The presence of strontium
(86 ppm) increased the surface area leading to the prolonged releases of drug
compared to the control HAp. Sr-HAp could be used as a drug carrier which
simultaneously improves osteointegration and prevents infection. The
bactericidal activity results show that all the drug incorporated samples are
strongly active against B. subtilis and S. aureus bacterial strains. The fibrous
HAp may be used as a reinforcement material to improve the mechanical
properties of HAp based biomaterial composites.