CHAPTER 4 EFFECT OF SWIFT HEAVY ION IRRADIATION ON HYDROXYAPATITE

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1 67 CHAPTER 4 EFFECT OF SWIFT HEAVY ION IRRADIATION ON HYDROXYAPATITE 4.1 INTRODUCTION Hydroxyapatite (HAp) is the main mineral component of human bone and teeth. The synthetically prepared HAp has biocompatibility and bioactivity, hence used for biomedical implants (Vallet-Regi and Gonzalez- Calbet 2004). Bulk form of stoichiometric HAp while implanting into the animal body has low bioactivity and poor mechanical properties. Hence, there is a need to improve the bioactivity of the ceramic implants by modification of the surface. The modifications of surface energy, roughness, topography and surface chemistry determine the bonding between the implant and the host tissue (Vallet-Regi and Gonzalez-Calbet 2004, Lahann et al 2003). Many methods have been developed to modify the surface of the materials viz. chemical etching, laser, plasma, electron beam, ion beam implantation and irradiation (Elayaraja et al 2010, Balanzat et al 1995). Ion beam irradiation is a useful tool for tailoring the surface characteristics such as physical, chemical and mechanical properties connected to the biocompatibility and functionality of the materials (Romanov and Tsaiova 2002). In addition, ion beam irradiation was found to improve the adhesion and densification of HAp thin films (Lopatin et al 1998). The osseointergration property of HAp was significantly improved by ion beam irradiation (Li et al 2005). Nowadays surface modification by ion

2 68 beam irradiation has been identified as a probable technique to improve bioactivity of HAp (Girija et al 2008, Prakash et al 2008). In the nuclear industry, the storage of nuclear waste over geological periods is one of the main problems. The synthesis of new material with better sensitivity of radiation storage material was investigated by Soulet et al (2001). The apatites (Fluoroapatite and HAp) could be used as matrices for storing nuclear waste elements like Sr, I, Cs, Np, Pu, etc (Veilleux et al 2001). The effect of swift heavy ion (SHI) irradiation on fluoroapatite pellets was carried out by Miro et al (2005) to explore its suitability for nuclear waste treatment. Suljovrujic et al (2003) examined the influence of gamma irradiation on the structure and properties of the HAp/Poly-L-lactide composites. Balanzat et al (1995) studied the physico-chemical modifications in polymers by performing swift heavy ion irradiation. The advantages of ion beam irradiation on bioceramic materials were selective surface modification without affecting the bulk properties. The oxygen incorporated HAp may produce oxy-hap, which have very good bioactivity and osteointegration (Gross et al 1998). Silicon is an essential trace element in the normal metabolism of human being and it was required in the formation of bones, cartilages, connective tissues and several important metabolic processes (Carlisle et al 1986). Silver has shown excellent antimicrobial property (Kim et al 1998). In this chapter, the surface modification of the samples was carried out by swift heavy oxygen, silicon and silver ions irradiation. The irradiated samples were characterized by glancing angle X-ray diffraction (GXRD), photoluminescence (PL) and scanning electron microscopy (SEM). In vitro bioactivity and antimicrobial activity was also evaluated on pure and irradiated samples.

3 EXPERIMENTAL METHODS The helical ribbon (HAp) and 0.05 M strontium substituted HAp (Sr-HAp) obtained from gel method described in chapter 2 and 3 were subjected to the ion beam irradiation. The samples were washed with distilled water and then they were dried and powdered. The powder form of the samples was pressed into pellets of about 8 mm diameter and 1 mm thickness. The pellets were subjected to swift heavy ion irradiation of oxygen, silicon and silver delivered from the 15UD pelletron accelerator at the Inter-University Accelerator Centre, New Delhi (India). The irradiation was carried out at the Material Science beam line with background pressure of 3x10-7 Torr. The ion beam was scanned over the sample surface of cm 2 by magnetic scanner for uniform irradiation. (a) Oxygen ion irradiation HAp was irradiated with 100 MeV O 7+ ions with various fluences of and ions/cm 2 with the beam current of 4 particles nano-ampere (pna) during the irradiation. (b) Silver ion irradiation HAp was irradiated with 100 MeV Ag 7+ ions with various fluences of , and ions/cm 2 and the beam current was maintained at 3 pna during the irradiation. (c) Silicon ion irradiation HAp and Sr-HAp were irradiated with 60 MeV Si 5+ ions with various fluences of 1x10 11, 1x10 12 and 1x10 13 ions/cm 2 and the beam current was maintained at 4 pna during the irradiation.

4 70 The in vitro drug loaded experiment was performed using 25 mg of amoxicillin (AMX) drug dissolved in 0.5 ml of deionized water. With and without silicon and silver ion irradiated samples were immersed into drug solution and it was kept at 37 C with shaking speed of 100 rpm using incubator-cum-orbital shaker for 24 h. The antibacterial activity of the drug loaded samples was investigated by standard disc diffusion method (Raahave 1974). Muller-Hinton agar (MHA, Merck) was used as a base medium and nutrient broth was used for the preparation of inoculum. A sterile cotton swab was dipped into the standardized bacterial culture of S. aureus containing the cell of CFU/ml. These cells were inoculated on the entire surface of the MHA plate and allowed for 10 min at the room temperature. The drug loaded pristine, irradiated samples were represented as (D) and samples without drug were represented as (C) respectively. The plates were incubated at 37 C for 24 h. After incubation, inhibition zone diameters were measured. Phase analyses of the pristine and irradiated samples were performed by glancing angle X-ray diffraction (GXRD) studies using Bruker AXS Diffract Plus-D8 Advance diffractometer with Cu K radiation = Å) over a 2 scan range of 20º-50 with increment steps of A glancing angle was fixed at 2. Photoluminescence (PL) spectroscopy was carried out using a He-Cd laser source (KIMMON) with an excitation wavelength of 325 nm at the room temperature. Photoluminescence spectra were recorded in the range of 300 to 900 nm using Mechelle900 spectrograph. The PL setup had a cooled CCD array-based detection system. The laser light was an incident on the sample at 45 and the luminescence light was collected using a collector assembly and transmitted to the spectrograph through an optical fiber for detection and analysis. The surface morphology of the pristine, irradiated and SBF treated samples were examined under scanning electron microscope (SEM) using LEO 440 STEROSCAN (LEICA). In vitro bioactivity test was done as described in the previous chapter.

5 RESULTS AND DISCUSSION SHI Energy losses and Penetration depth (a) Oxygen ion irradiation Based on the SRIM-2008 (Stopping and Ranging of Ions in Matter) simulation program (Zeigler et al 1985), the calculated electronic energy loss (de/dx) e and nuclear energy loss (de/dx) n for various energy of O 7+ ion on HAp are as shown in Figure 4.1 and the values are listed in Table 4.1. Energy loss (ev/å) 250 S e S n O ion Energy (MeV) Figure 4.1 Electronic and Nuclear energy loss in HAp for various energies of O 7+ ions From the Figure 4.1 it is clearly evident that the electronic energy loss (S e ) increases as the ion energy increases. It reaches a maximum at 6.5 MeV and then it decreases gradually. The maximum electronic energy loss is 243 ev/å. At 100 MeV, the calculated electronic energy loss is ev/å. Hence, surface modification of HAp occurs due to the electronic energy loss.

6 72 (b) Silver ion irradiation The calculated (de/dx) e and (de/dx) n as a function of Ag 7+ ion energy interacting with HAp samples are shown in Figure 4.2. The (de/dx) e increases as the ion energy increases. On the other hand nuclear energy loss (de/dx) n shows low value for all the energies and it is almost negligible. This implies that the whole energy gets transmitted into the sample through electronic excitations. Hence, the surface modification of HAp is due to the electronic energy loss during the ion beam irradiation Ag ion S e S n Energy loss (ev/å) Energy (MeV) Figure 4.2 Electronic and Nuclear energy loss in HAp for various energies of Ag 7+ ions (c) Silicon ion irradiation From the SRIM-2008 program, the electronic energy loss (de/dx) e and nuclear energy loss (de/dx) n are calculated for various energies of Si 5+ ion on HAp as shown in Figure 4.3. The electronic energy loss (S e ) increases as

7 73 the ion energy increases and reaches a maximum at 20 MeV then it gradually decreases. The maximum energy loss is ev/å. At an energy of 60 MeV, the calculated electronic energy loss is approximately ev/å. On the otherhand nuclear energy loss (S n ) shows low value for all energy and it is found to be almost negligible. 500 Si ion S e Energy loss (ev/å) S n Energy (MeV) Figure 4.3 Electronic and Nuclear energy loss in HAp for various energies of Si 5+ ions The estimated range (using SRIM-2008) of oxygen, silver and silicon ions in HAp was found to be 70.33, and m respectively (Figure 4.4). These energetic ions would pass through some depth and get embedded into the samples. Thickness of the sample was 1 mm which is larger than the ions penetration depth.

8 74 80 O 7+ ion Si 5+ ion Ag 7+ ion depth (µm) Energy (MeV) Figure 4.4 Penetration depth of oxygen, silicon and silver ions as a function of energy in HAp Table 4.1 Energy loss and range of oxygen, silver and silicon ions in HAp Parameters O 7+ Ag 7+ Si 5+ Electronic energy loss, S e (ev/å) Nuclear energy loss, S n (ev/å) Range ( m) Glancing angle XRD Analysis (a) Oxygen ion irradiation The GXRD spectra of pristine and O 7+ ion irradiated HAp samples were shown in Figure 4.5. The XRD peaks of pristine and irradiated samples showed pure phase of HAp (JCPDS, ). All the planes were indexed using XRDA 3.1 software. HAp was identified by the isolated (002) plane at = 25.9º and the broad peak around 32º was composed of overlapped (211) and (112) planes. The (002) plane showed maximum intensity, compared to

9 75 the characteristic (211) plane of HAp. This is because the crystals were highly oriented along the c-axis (Zollfrank et al 2005) (Figure 4.5a). The irradiated sample at a fluence of 1x10 12 ions/cm 2 exhibited high intensity compared to pristine which indicated the increase in crystallinity (Figure 4.5b). The intensity of (002) peak decreased in the case of 1x10 13 ions/cm 2 irradiated samples (Figure 4.5c). The 1x10 13 ions/cm 2 fluence was 10 times higher than the fluence of 1x10 12 ions/cm 2, a very high heat regime was formed during irradiation, which could lead to partial amorphization. (002) c Intensity (a.u) (002) (002) (211) (112) (211) (112) (211) (112) (212) (310) (310) (212) (310) b a theta (deg) Figure 4.5 GXRD spectra of (a) HAp and O 7+ ions irradiated at fluences (b) (c) ions/cm 2 (b) Silver ion irradiation Figure 4.6 shows the GXRD spectra of the pristine and Ag 7+ ion irradiated samples. The peaks observed at 25.9, 31.7 and 32.1 correspond to (002), (211) and (112) planes of the HAp for all the samples (Figure 4.6a). There was no additional peak observed on the samples due to Ag 7+ ion irradiation, which revealed the absence of any phase change. Intensity of the peaks was increased when irradiated with fluences of 1x10 11 and 1x10 12

10 76 ions/cm 2 (Figure 4.6b and 4.6c). In addition, (211) and (112) planes were well resolved than pristine and (002) plane had high intensity. This might be due to the Ag 7+ ion transferring the required energy to (211) and (112) plane of HAp lattice. The peak intensity was decreased when increasing the ion fluence to 1x10 13 ions/cm 2 (Figure 4.6d). The occurrence of partial amorphization at 1x10 13 ions/cm 2 fluence was due to the creation of defect inside the lattice (Hemon et al 1997). The O 7+ (100 MeV) ion irradiated samples with fluence of ions/cm 2 on HAp showed more crystallinity than Ag 7+ ion (100 MeV) irradiated samples. This is because O 7+ ion having low atomic number, creates less surface modification than the higher atomic number Ag 7+ ion. (002) (111) (211) (112) d Intensity (a.u) (111) (111) (002) (002) (102) (102) (211) (112) (211) (112) (202) (202) (212) (310) (221) (212) (310) (221) c b (002) (211) (112) a theta (deg) Figure 4.6 GXRD spectra of (a) HAp and Ag 7+ ions irradiated at fluences (b) (c) (d) ions/cm 2

11 77 (c) Silicon ion irradiation The GXRD spectra of pristine and Si 5+ ion irradiated samples were as shown in Figure 4.7. Pristine sample has been already discussed in section 4.3.2(a). The fluence of 1x10 11 ions/cm 2 irradiated sample showed high intensity compared to pristine (Figure 4.7b). The (002) plane of HAp showed high intensity at the fluence of 1x10 12 ions/cm 2 (Figure 4.7c). The observations are similar as observed for the case of O 7+ and Ag 7+ ions. This was due to the recrystallization of the HAp during the ion beam irradiation to produce the local heat on the sample. In addition, (211) and (112) planes were well resolved than the pristine that similar to the observations in Ag 7+ ion irradiated samples. When the ion fluence is increased to 1x10 13 ions/cm 2, the intensity of all the peaks was gradually decreased (Figure 4.7d). (002) (211) (112) d Intensity (a.u) (111) (111) (002) (002) (102) (210) (102) (210) (211) (112) (211) (112) (202) (202) (212) (310) (221) (212) (310) (221) c b (002) (211) (112) a theta (deg) Figure 4.7 GXRD spectra of (a) HAp and Si 5+ ions irradiated at fluences (b) 1x10 11 (c) 1x10 12 (d) 1x10 13 ions/cm 2 The fluence of 1x10 13 ions/cm 2 of Si 5+ ions led to partial amorphization as observed for Ag 7+ ions irradiated samples. The O 7+ ion

12 78 irradiated HAp has shown better crystallinity than Si 5+ ion irradiated one. This was due to the low atomic number of O 7+ ion, which may cause less surface modification than higher atomic number of Si 5+ ion (Miro et al 2005). The GXRD patterns of pristine and Si 5+ ion irradiated Sr-HAp samples are as shown in Figure 4.8. The intensity of Sr-HAp planes gradually decreased on increasing ion fluences (Figure 4.8b and 4.8c). At the fluence of 1x10 13 ions/cm 2, the sample was converted to fully amorphous nature (Figure 4.8d). This was due to the creation of more defects in Sr-HAp lattice during the Si 5+ irradiation. These results suggested that the metal ion substituted HAp (Sr-HAp) showed low stability than pure HAp at 1x10 13 ions/cm 2 irradiation. d (002) (102) (211) (300) c Intensity (a.u) (111) (002) (102) (211) (300) b (111) (002) (102) (211) (300) (221) (310) (222) (213) a theta (deg) Figure 4.8 GXRD spectra of (a) Sr-HAp and Si 5+ ions irradiated at fluences (b) 1x10 11 (c) 1x10 12 (d) 1x10 13 ions/cm 2

13 79 The crystallite size (X s ) and crystallinity (X c ) of the above described samples were calculated using Scherrer s equation and empirical formula which were explained in chapter 3. All the planes of pristine and different ion irradiated samples of GXRD patterns were analysed by XRDA 3.1 software. The crystallinity of the irradiated samples increased from 50 to 70 % when compared to pristine (HAp) (Table 4.2), whereas Sr-HAp irradiated samples decreased to around 20 % of crystallinity at ions/cm 2. At higher fluences ( ions/cm 2 ), the samples became amorphous. Table 4.2 Crystallinity of various ion irradiated samples Samples Pristine ions/cm ions/cm ions/cm 2 Crystallinity along (002) plane, X c (%) Si 5+ O 7+ Ag 7+ HAp Sr-HAp amorphous amorphous amorphous From the Table 4.3, the crystallite size of HAp was found to decrease for O 7+ and Ag 7+ ion irradiation, while Si 5+ ion irradiation enhanced the crystallite size both in HAp and Sr-HAp. This could be attributed to the electronic energy loss which creates more defects on the surface of the samples.

14 80 Table 4.3 Average crystallite size of various ion irradiated samples Samples Pristine ions/cm ions/cm ions/cm 2 Average crystallite size along (002) plane, X s (nm) (±1) Si 5+ O 7+ Ag 7+ HAp Sr-HAp amorphous amorphous amorphous Photoluminescence Studies Ionoluminescence was observed in all samples during irradiation. Presence of phosphorous and inclusion of silica in HAp are likely to be the origin of ionoluminescence. To investigate this property, PL spectra of the samples were recorded. Figure 4.9 shows the PL spectra of HAp before and after O 7+ ion irradiation. The excitation energy is greater than that of the emission. The band gap was determined to be 3.52 ev which was consistent with the reports available in the literature (Rosenman et al 2007). PL intensity (a.u) pristine ions/cm ions/cm 2 PL intensity (au) Wavelength (nm) Wavelength (nm) Figure 4.9 PL spectra of the HAp before and after O 7+ ions irradiation

15 81 The luminescence intensity considerably increased in the case of irradiated sample due to the combined effect of surface state emission as well as emission from the large defects created by the O 7+ ion during SHI irradiation (Chowdhury et al 2005). With an increase in ion dose, there was no significant shift in the emission wavelength. Such surface state emission has been reported earlier in the case of ZnS nano crystallites (Nanda and Sarma 2001). The peak positions and corresponding energies from PL spectra of pristine and irradiated samples with different fluences were listed in Table 4.4. Table 4.4 Photoluminescence emission of HAp and O 7+ ions irradiated samples Excitation Peak positions Samples wavelength (nm) 1 (nm) E (ev) 2 (nm) E (ev) 3 (nm) E (ev) 4 (nm) E (ev) HAp ions/cm ions/cm SEM Studies (a) Oxygen ion irradiation The SEM micrograph of the pristine sample showed smooth surface (Figure 4.10a). O 7+ ion irradiated sample with the fluence of 1x10 12 ions/cm 2 showed several pores with the diameter of about 4.5 µm (Figure 4.10b). At higher fluence of 1x10 13 ions/cm 2, the pores of around 7 µm as well as cluster of particles were clearly seen on the surface (Figure 4.10c). The pores were attributed to the HAp atoms pile up on its surface produced by heating due to the electronic energy loss (Singh et al 2000).

16 82 Figure 4.10 SEM micrograph of (a) HAp and O 7+ ions irradiated at fluences (b) (c) ions/cm 2

17 83 (b) Silver ion irradiation The surface of the Ag 7+ ion irradiated with fluence of 1x10 11 ions/cm 2 sample contained platy crystals of length 2 m and diameter 1 m (Figure 4.11a). When the ion fluence was increased to 1x10 12 ions/cm 2, the dimension of platelets was also increased around 5 m length and 3.5 m diameter on the surface (Figure 4.11b). This was due to the recrystallization of HAp by local heating during irradiation. Further, it was confirmed by the GXRD results (Figure 4.6c). The samples irradiated with 1x10 13 ions/cm 2 showed interconnected pores on the surface (Figure 4.11c). Irradiation with Ag 7+ ions have shown increased surface modification than O 7+ irradiated samples. This was due to the electronic energy loss variation between O 7+ and Ag 7+ ion (Table 4.1). (c) Silicon ion irradiation At 1x10 11 ions/cm 2 fluence, the hexagonal platy (1-5 m) and small needle shaped crystals of size 400 nm were observed on the surface (Figure 4.12a). At 1x10 12 ion/cm 2, irregular platy crystals were observed (Figure 4.12b). Large clusters were observed on the surface of 1x10 13 ions/cm 2 irradiated sample (Figure 4.12c). Si 5+ irradiated samples had significant surface modification compared to O 7+ irradiated samples. This could be due to 60 MeV Si 5+ ions, producing maximum electronic energy loss on HAp surface.

18 84 Figure 4.11 SEM micrograph of Ag 7+ ions irradiated HAp at fluences (a) (b) (c) ions/cm 2

19 85. Figure 4.12 SEM micrograph of Si 5+ ions irradiated HAp at fluences (a) (b) (c) ions/cm 2

20 86 The SEM images of Sr-HAp irradiated with 60 MeV Si 5+ ions are shown in Figure The smooth surface was observed in pristine sample (Figure 4.13a). The surface became rough and porous on 1x10 11 ions/cm 2 irradiation. In addition, the platy crystals of size 2 m were found on the surface (Figure 4.13b). At 1x10 12 ions/cm 2, the whole surface contained platy crystals of 5 m size (Figure 4.13c). Porous granular structure of size 500 nm was observed at 1x10 13 ions/cm 2 irradiation (Figure 4.13d). The formation of pores is due to the large amount of electronic energy loss in the sample by Si 5+ ions irradiation. The local heating produced by ion beam irradiation, converted the surface into amorphous one. Si 5+ ion irradiated Sr-HAp sample displayed high surface modification than HAp. Figure 4.13 SEM micrograph of (a) Sr-HAp and Si 5+ ions irradiated at fluences (b) 1x10 11 (c) 1x10 12 (d) 1x10 13 ions/cm 2

21 Figure 4.13 (continued) 87

22 In vitro Bioactivity Test (a) Oxygen ion irradiation Figure 4.14 shows SEM micrographs of the samples after immersion in SBF, the surface of both pristine and O 7+ ion irradiated samples showed apatite deposition. The pristine sample surface contained deposits of 3 µm globules (Figure 4.14a). The surface of the 1x10 12 ions/cm 2 fluence samples showed deposits of globules of size 8 µm (Figure 4.14b) and also showed better surface coverage of apatite deposition. The 1x10 13 ions/cm 2 fluence samples showed spheres of apatite deposits of size 4.5 µm (Figure 4.14c). Figure 4.14 SEM micrograph of SBF soaked (a) HAp and O 7+ ions irradiated at fluences (b) (c) ions/cm 2

23 89 (b) Silver ion irradiation In vitro bioactivity tests of the Ag 7+ ion irradiated samples revealed the formation of apatite layer on the surface (Figure 4.15). After immersion, the layer of spherulites (500 nm) and 250 nm were formed at 1x10 11 and 1x10 12 ions/cm 2 irradiation pellet surface respectively (Figure 4.15a and 4.15b). Whereas, fluence of 1x10 13 ions/cm 2 irradiated samples showed elongated crystals (700 nm) (Figure 4.15c). The deposition of apatite was found to be less due to the surface amorphization. Figure 4.15 SEM micrograph of SBF soaked Ag 7+ ions irradiated HAp at fluences (a) (b) (c) ions/cm 2 (c) Silicon ion irradiation The SEM micrograph of Si 5+ ion irradiated HAp were as shown in Figure The layer of apatite consisted of spherulites (450 nm) on the 1x10 11 ions/cm 2 irradiated surface (Figure 4.16a). The surface covered with the globules of size 500 nm (Figure 4.16b), and further increase of fluence

24 ions/cm 2 resulted in the formation of ball-like apatite (Figure 4.16c). In addition, the large number of pores also observed on the bioactivity tested surface. The apatite deposition increased with increase in ion fluence upto ions/cm 2. Figure 4.16 SEM micrograph of SBF soaked Si 5+ ions irradiated HAp at fluences (a) 1x10 11 (b) 1x10 12 and (c) 1x10 13 ions/cm 2 The SEM micrograph of Si 5+ ion irradiated Sr-HAp were as shown in Figure Sr-HAp showed more bioactivity than pure form of HAp (Figure 4.17a) which consisted of spherical apatites of size 1 m. Pores (200 nm) were observed at lower fluence ( ions/cm 2 ) (Figure 4.17b). At ions/cm 2, the large number of spherulites (600 nm) was formed on the surface and its size also increased (Figure 4.17c). At the higher fluence of ions/cm 2, the sample showed lower bioactivity when compared with pristine (Figure 4.17c). In addition, the Sr-HAp had lower bioactivity than Si 5+ ion irradiated HAp at higher fluence ( ions/cm 2 ).

25 91 Figure 4.17 SEM micrograph of SBF soaked (a) Sr-HAp and Si 5+ ions irradiated at fluences (b) 1x10 11 (c) 1x10 12 and (d) 1x10 13 ions/cm 2 It is established by Kim et al (2004) that the synthetic HAp when immersed in SBF, led to the deposition of apatite on its surface. HAp has the ability to promote bone-like apatite formation directly on their surface (Itoh et al 2006). The formation of HAp is governed by factors such as density, porosity, surface area, surface composition and its structure (Kim et al 2004). SHI irradiation, due to large electronic energy loss can produce significant changes on the surface. The densification of the HAp surface on irradiation was due to electronic energy loss. Based on SRIM-2008 calculations, the electronic energy loss (S e ) and nuclear energy loss (S n ) values are listed for oxygen, silver and silicon ion (Table 4.1). Since S e is large for three different ions, this energy is transferred to the surface layers which promote the densification (Lopatin et al 1998).

26 92 The mechanism for the formation of apatite is schematically shown in Figure The HAp surface reveal negative surface charges due to the presence of hydroxyl (OH - ) and phosphate (PO 3-4 ) groups on its surface (Figure 4.18a). These negative ions interact with the positive calcium ions in the SBF to form the Ca-rich ACP (amorphous calcium phosphate), which gain positive surface charge (Figure 4.18b). The Ca-rich ACP interact with the negative phosphate ions in the SBF to form the Ca-poor ACP (Figure 4.18c), which stabilizes by being crystallized into bonelike apatite in the SBF (Figure 4.18d). Nanoscale variations in the surface potential can affect the formation of apatite layer from SBF (Vandiver et al 2005). Figure 4.18 Schematic presentations of the process of bonelike apatite formation on HAp (a) HAp surface in SBF under normal conditions (b) calcium ions settle on HAp surface from the fluid to form Ca-rich ACP (c) Ca-rich ACP surface attracts phosphate groups from SBF to form Ca-poor ACP and (d) apatite layer on the HAp surface

27 93 The oxygen, silver and silicon ion irradiated HAp pellets showed more bioactivity at the fluence of 1x10 12, ions/cm 2. In addition, Sr-HAp had shown enhanced bioactivity at 1x10 12 ions/cm 2 of Si 5+ ion. The enhancement of bioactivity on irradiation could be qualitatively explained from the energy loss mechanism. The difference in the energy loss between S e and S n clearly indicated that the changes occurring on the surface on irradiation, were due to electronic energy losses without any change in phase of HAp. Also, the ionization plot (Figure 4.19) gave some important information about the energy loss mechanism. From the plot it was clear that the energy loss was only due to the incident ions. The energy loss due to the recoiling ions was negligible. The target (HAp) atoms do not recoil due to the stable HAp lattice. Irradiated samples in general showed enhanced bioactivity compared to pristine sample. On irradiation the surface became more negative which provided a large number of negative groups (PO 3-4, OH - ) for the Ca 2+ ions in SBF to form Ca-rich apatite on the irradiated HAp surface. This layer further leads to the formation of Ca-deficient apatite and finally an apatite layer develops on ageing. The incident ions might interact with the electrons of the target atoms (HAp), particularly the electron-deficient calcium atom and may make them electron rich leading to a highly negative HAp surface. Moreover, the creation of pores due to irradiation exposed more number of HAp atoms which increased the negativity of the surface. HAp and Sr-HAp samples showed enhanced bioactivity at the above described fluences. The lesser fluences of oxygen, silver and silicon ion irradiated samples was too less to create the required negative surface to enhance the bioactivity. In the case of Ag 7+ ion irradiated on HAp with ions/cm 2 fluence had low apatite formation compared to other fluences. The reason may be heavy dosage of ions led to the low crystallinity and saturation of surface negativity of the HAp.

28 94 Figure 4.19 Energy loss due to ionization on HAp with O 7+ irradiation ions Antibacterial Activity (a) Silver and Silicon ion irradiation Antibacterial activity of the samples determined by standard disc diffusion method against S. aureus as shown in Figure 4.20 and Figure 4.21, the samples without drug (C) did not show any zone of inhibition. In contrast, the drug loaded HAp, Ag 7+ and Si 5+ ion irradiated samples (D) showed antibacterial activity with a relatively large zone of growth inhibition (Figure 4.20a to 4.20d, Figure 4.21a and 4.21c). While increasing the fluence of Ag 7+ and Si 5+ ion irradiation increased the zone of inhibition (Table 4.5) against S. aureus due to the partial amorphization.

29 95 a b C D C D c d C D C D Figure 4.20 Disc diffusion test results of (a) HAp and Ag 7+ ions irradiated at fluences (b) (c) (d) ions/cm 2 Figure 4.21 Disc diffusion test results of Si 5+ fluences (a) (b) (c) ions/cm 2 ions irradiated HAp at

30 96 Antibacterial activity of with (D) and without (C) drug loaded Si 5+ ion irradiated Sr-HAp were as shown in Figure The Si 5+ ion irradiated samples (C) were did not show any antibacterial activity. Whereas, AMX drug loaded irradiated samples were showed various diameter of inhibition zone (Table 4.5). The drug loaded Sr-HAp has lower activity than drug loaded Si 5+ ion irradiated samples (Figure 4.22a). The zone of growth inhibition for irradiated samples was increased with increase in ion fluence (Figure 4.22b to 4.22d). The disc diffusion test suggested that the bactericidal effect of Si 5+ ion irradiated samples was higher than Ag 7+ ion irradiated samples. Figure 4.22 Disc diffusion test results of (a) Sr-HAp and Si 5+ ions irradiated at fluences (b) (c) (d) ions/cm 2

31 97 Table 4.5 Antibacterial activity of drug loaded samples against S. aureus Samples HAp Ag ions/cm 2 Ag ions/cm 2 Ag ions/cm 2 Diameter of zone of inhibition (± 0.5mm) 12 h 24 h Si ions/cm 2 Si ions/cm 2 Si ions/cm 2 Sr-HAp Si ions/cm 2 Si ions/cm 2 Si ions/cm CONCLUSIONS The effect of SHI oxygen, silver and silicon ion irradiation was studied for gel grown HAp. The O 7+ (100 MeV) ion irradiated HAp with fluence of 1x10 13 ions/cm 2 showed more crystallinity than Ag 7+ (100 MeV) and Si (60 MeV) ion irradiated samples. The Sr-HAp showed low stability than HAp at 1x10 13 ions/cm 2 irradiation. The SHI irradiation improved the crystallinity along with the production of pores. The PL intensity significantly increased with O 7+ ion irradiation, which could be attributed to the defects caused by the ion beam during irradiation. The photoluminescence exhibited by HAp could make it suitable for biosensor applications. Si 5+ and Ag 7+ irradiated HAp had significant surface modifications compared to O 7+ irradiated HAp. Si 5+ ion irradiated Sr-HAp sample showed high surface modification than HAp. Moreover irradiated samples exhibited better

32 98 bioactivity when they were soaked in SBF, compared to pristine samples. The more bactericidal effect was observed for drug loaded irradiated samples with increasing ion fluence and Si 5+ ion irradiated samples showed higher bactericidal activity against S. aureus. This result indicated that the samples modified with irradiation could be used as promising bactericidal candidates for implant applications.