CHAPTER 3 FIBROUS GROWTH OF STRONTIUM SUBSTITUTED HYDROXYAPATITE

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1 51 CHAPTER 3 FIBROUS GROWTH OF STRONTIUM SUBSTITUTED HYDROXYAPATITE 3.1 INTRODUCTION Calcium phosphate has many different phases, such as CaHPO 4.2H 2 O (DCPD), Ca 3 (PO 4 ) 2 (TCP) and Ca 10 (PO 4 ) 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). Ca 2+ ions can be replaced by various divalent cations including Sr 2+, Ba 2+, Cd 2+, Mg 2+ 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).

2 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 ( 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

3 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 (CaCl 2.2H 2 O, Merck) and disodium hydrogen phosphate (Na 2 HPO 4, 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 (Na 2 SiO 3.9H 2 O, Qualigens) of specific gravity 1.03 g/cc and Na 2 HPO 4 (0.6 M) was adjusted to the ph 7.4 using glacial acetic acid. After gelation, about 1 M CaCl 2.2H 2 O mixed with strontium chloride (SrCl 2, 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 ( = 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

4 54 (PERKIN ELMER spectrum RXI using KBr pellet technique) within the scanning range 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 HNO 3 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).

5 55 Figure 3.1 Liesegang patterns with various Sr concentrations (a) Sr0, (b) Sr01, (c) Sr05 and (d) Sr 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).

6 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 Length (µm) Width (µm) Aspect code (±0.5) (±0.1) ratio Sr ±3 Sr ±19 Sr ±7 Sr ± 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 ). Sr05 and Sr1 showed the broad and

7 57 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 Sr 2+ (1.13 Å) has higher ionic radius than that of Ca 2+ 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. (211) d Intensity (a.u.) (200) (200) (111) (111) (111) (002) (002) (002) (102) (102) (102) (211) (202) (202) (301) (211) (112) (202) (301) (221) (221) (221) (213) c (213) (213) b (200) (111) (002) (102) (210) (211) (112) (202) (212) (310) (221) (203) (222) (312) a (213) Figure t w o t h e t a ( d e g ) XRD patterns of (a) Sr0, (b) Sr01, (c) Sr05 and (d) Sr1 The crystallite size was calculated using Scherrer s equation, that is, X s = 0.9 cos, where X s is the average crystallite size in nm, is the

8 58 full width of the peak at half of its maximum intensity (radian), is the wavelength of X-rays ( 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 (X c ) of the samples was determined by an empirical relation between X c and 002 (i.e., X c = K A ), where X c is the crystallinity degree, 002 is full width of the peak at half intensity of (002) plane in degree and K A 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 Sample code Sr0 Crystallite Size, X s (nm) (± 1) 9 Crystallinity, X c (%) 64 Lattice parameters a = b (Å) c (Å) (±0.02) (±0.02) Sr Sr Sr 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

9 59 cm -1 may correspond to HPO 2-4 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 CO 2-3 absorption peak at 1394 cm -1 except for the sample Sr0, probably contaminated by the CO 2 absorption from the air. The peak at 863 cm characteristic for HPO 4 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). d 3730 Transmittance (%) c b 3730 a Wavenumber (cm -1 ) Figure 3.4 FT-IR spectra of (a) Sr0, (b) Sr01, (c) Sr05 and (d) Sr1

10 60 Table 3.3 FT-IR Assignments of functional groups of HAp Vibrational frequency (cm -1 ) Assignments O-P-O bending O-P-O bending 2- O-H stretching of HPO 4 P-O Asymmetric stretching P-O Asymmetric stretching O-H In-plane bending HPO 2-4 groups O-H Stretching O-H Stretching 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 Wilson et al (2005) reported that the Ca-deficient apatite with Ca/P molar ratios from was due to the incorporation of HPO 2-4 and CO 2-3 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).

11 61 Table 3.4 Elemental analysis of the control and Sr-HAp Sample Ca (ppm) P (ppm) Sr (ppm) Si (ppm) Ca/P Sr/Ca (Ca+Sr)/P Sr Sr Sr Sr Strontium concentration (ppm) Sr01 Sr05 Samples Sr1 Figure 3.5 Strontium concentration of the samples in ppm BET Analysis The N 2 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 m 2 /g, pore volume of 0.41 cm 3 /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).

12 62 Table 3.5 Pore volume, pore size and surface area of the samples Sample Pore volume Pore size Surface area code (cm 3 /g) (nm) (m 2 /g) Sr0 0.41± ± ±0.26 Sr ± ± ±0.14 Sr ± ± ±0.32 Sr1 0.27± ± ± Sr0 250 Sr01 Quantity Adsorbed (cm 3 /g) Quantity Adsorbed (cm 3 /g) Relative pressure (P/Po) Relative pressure (P/Po) Sr Sr Quantity Adsorbed (cm 3 /g) Quantity Adsorbed (cm 3 /g) Relative pressure (P/Po) Relative pressure (P/Po) Figure 3.6 Nitrogen adsoption-desorption isotherm of control and Sr-HAp

13 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 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

14 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 m 2 /g) compared with other samples. The Sr0 sample with the surface area of about m 2 /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. Amoxicillin release (%) Sr0 Sr01 Sr05 Sr Time (hrs) Figure 3.8 Cumulative drug release of AMX from the samples

15 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 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

16 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 strain Diameter of zone of inhibition (± 0.5 mm) Sr0D Sr01D Sr05D Sr1D B. subtilis S. aureus 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.