51

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).

52

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

53

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

54

(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).

55

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).

56

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

57

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

58

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

59

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

60

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).

61

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).

62

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

63

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

64

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

65

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

66

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.