3.1. X-ray Diffraction (XRD) Analysis
Figures S1 and S2 show the XRD patterns of pure and ZnHAP, pure and MgHAP samples respectively (Refer supplementary). Figure 1 illustrates the XRD patterns of the pure, 5% Zn, and 5% Mg-doped HAP. From the XRD pattern, the calculated lattice constants (a and c), unit cell volume (V) are in Table 2 and the crystallite size (D), microstrain (ε), dislocation density (ρ), the fraction of crystallinity (Xc) and specific surface area (S) are in Table 3 [13, 14]. All the XRD results are in good accord with the joint committee for powder diffraction standard (JCPDS) card # 09-432. The (0 0 2) peak position in pure HAP slightly shifts due to the Zn or Mg dopant. Lattice parameters a and b decrease while the c almost remains the same due to the substitution ions, hence the (210) and (300) peak positions shift to higher angles [15]. In HAP structure, Zn2+or Mg2+ can occupy any one of the following Ca (I), Ca (II), P and OH positions. The radius of Ca2+, P5−, Zn2+ and Mg2+ ions are 0.099, 0.031, 0.074, and 0.065 nm respectively. The Ca (I) and P5− atoms form an octahedral structure with their O2− atoms. But Ca (II) atoms form a tetrahedral structure with O2− atoms. To form a tetrahedral structure less energy is required than to form an octahedral structure. However, the difference in ionic radius and charge of Zn2+and Mg2+ ions are more likely to replace Ca2+ ions. The Zn2+ and Mg2+ ions prefer Ca (II) ion to replace, due to lesser energy required in the formation of tetrahedral structure [16]. The ionic radius of Ca2+ is greater than Zn2+ and Mg2+. Therefore, the crystallite size decreases with an increase in dopant concentration. This result is consistent with the previous reports [8, 17, 18]. The mismatch of the ionic radius of Zn2+ and Mg2+ with the Ca2+creates a defect in the crystal array. Hence from the calculated values, it is found that, when the dopant concentration increased, the surface area, dislocation density and strain of the prepared samples are increasing whereas the crystallite size decreases. If the substitution ion replaced the OH ion in HAP, the crystallite size might get increased; the decrease in crystallite size, confirms that the dopant ion replaced Ca2+. These results are in good accord with Miculescu et al.[19].
Table 2
Calculated lattice constants a, c and unit cell volume of the samples.
Sample
|
Parameter a (Å)
|
Parameter c (Å)
|
Unit cell volume (Å)3
|
Pure HAP
|
9.541
|
6.872
|
541.816
|
1%ZnHAP
|
9.51
|
6.828
|
534.907
|
3%ZnHAP
|
9.504
|
6.817
|
533.257
|
5%ZnHAP
|
9.501
|
6.812
|
532.529
|
1%MgHAP
|
9.616
|
6.85
|
547.544
|
3%MgHAP
|
9.52
|
6.828
|
535.917
|
5%MgHAP
|
9.474
|
6.813
|
529.585
|
Table 3
The reported values of crystallite size (D), dislocation density (ρ), strain (ε),
Sample
|
Crystallite Size (nm)
|
Dislocation Density × 1015 (lines/m2)
|
Strain×10− 4 (line− 2.m− 4)
|
Surface Area × 1011 m2/g
|
Fraction of Crystallinity (Xc)
|
Pure HAP
|
65.39
|
0.259
|
1.096
|
2.903
|
7.702
|
1%ZnHP
|
62.85
|
0.276
|
1.548
|
3.021
|
6.851
|
3%ZnHP
|
56.87
|
0.309
|
2.257
|
3.338
|
3.813
|
5%ZnHP
|
53.83
|
0.345
|
2.603
|
3.527
|
2.418
|
1%MgHP
|
61.4
|
0.265
|
2.113
|
3.092
|
5.435
|
3%MgHP
|
55.86
|
0.321
|
2.703
|
3.399
|
4.299
|
5%MgHP
|
52.35
|
0.382
|
2.854
|
3.626
|
3.677
|
surface area (S) and the fraction of crystallinity (Xc) for pure, Zn and Mg-doped HAP.
3.2. FT-IR Analysis
The supplementary Figs. S3 and S4 illustrate the Fourier Transform infrared (FTIR) spectra for the prepared samples. Figure 2 illustrates the FTIR spectra of pure, 5% Zn and 5% Mg doped HAP. Table 4 depicts the FT-IR vibrational assignment of pure, Zn and Mg doped HAP. The presence of OH− vibrations and ν1 to ν4 vibrations of PO43− in all the FT-IR spectra confirm the hydroxyapatite structure. The FT-IR spectra of Zn/Mg doped samples are similar to the pure HAP, but the intensity of the peak at 563 cm− 1 slightly decreases with an increase in dopant concentration. The FT-IR bands at 1590 and 1418 cm− 1 represent the carbonate in the samples. The presence of CO32− in the samples is due to CO2 in the air mixed with water during the preparation of HAP [20]. The bone structure consists of carbonate, so the presence of CO32− increases the bioactivity of the HAP. The intensity of the carbonate peak decreases with the increase of concentration of Zn in HAP whereas the intensity of the carbonate peak increases with the increase of concentration of Mg in HAP. The peaks related to the PO43− and OH− become broad while raising the concentration of both Zn2+ and Mg2+ and it indicates the decrease in crystallinity of the HAP as obtained in XRD. Table 4 represents the PO43−, OH− and CO32− vibrational assignment. There is no vibrational indication for Zn and Mg in FT-IR spectra.
Table 4
FT-IR vibrational bands assigned to pure, Zn and Mg doped HAP.
Pure HAP
|
Zinc doped HAP
|
Magnesium doped HAP
|
Assignment cm− 1
|
Reference
|
1%
|
3%
|
5%
|
1%
|
3%
|
5%
|
3574
|
3561
|
3566
|
3565
|
3571
|
3573
|
3573
|
OH− Stretching
|
15
|
1590
|
1587
|
1588
|
1588
|
1578
|
1579
|
1579
|
Carbonate ions (CO32−)
|
20
|
1418
|
1416
|
1422
|
1422
|
1416
|
1410
|
1424
|
Carbonate ions (CO32−)
|
2
|
1090
|
1089
|
1091
|
1086
|
1088
|
1087
|
1090
|
υ3 Asymmetric stretching of P-O in PO43−
|
8
|
1040
|
1042
|
1043
|
1041
|
1047
|
1047
|
1047
|
υ3 Asymmetric stretching of P-O in PO43−
|
15
|
961
|
955
|
958
|
959
|
978
|
965
|
964
|
υ1 Symmetric stretching mode of P-O in PO43−
|
17
|
604
|
602
|
605
|
605
|
605
|
605
|
605
|
υ4 asymmetric bending mode of O-P-O in PO43−
|
8
|
563
|
559
|
563
|
563
|
565
|
567
|
567
|
υ4 asymmetric bending mode of O-P-O in PO43−
|
15
|
472
|
470
|
471
|
467
|
461
|
471
|
471
|
υ2 Symmetric bending mode of O-P-O in PO43−
|
1
|
3.3. Micro Raman Analysis
Figures S5 and S6 represent the Raman spectra of pure, ZnHAP and MgHAP samples respectively. Figure 3 shows the Raman spectra of pure, 5% ZnHAP and 5% MgHAP. Table 5 depicts the Raman vibrational assignment of pure, Zn and Mg doped HAP. Phosphate posses four vibrational modes υ1, υ2, υ3, and υ4. The vibrational modes of PO43− present in all the Raman spectra confirm the hydroxyapatite structure in the samples. Table 5 depicts the Raman vibrational assignment of pure, Zn and Mg doped HAP. The FWHM of υ1 peak (962 cm− 1) increases with the dopant concentration. The υ2 peak position in doped HAP samples shift from 459 cm− 1 to 485 cm− 1 and it becomes strong. The υ3 (1035 cm− 1) and υ4 (583 cm− 1) peaks shift to higher wavenumber. The shift of υ2, υ3, and υ4 peak from lower to higher wavenumber is due to the following reasons; (i) the ionic radius of Ca2+ is more than dopant ions (ii) metal-oxygen distance of Zn and Mg, is smaller than Ca-O distance (iii) dopant ions decrease the cell volume of HAP. The metal-oxygen distance of Ca, Zn, and Mg are 2.668, 1.875, and 1.743 Å respectively. The continuous increase in FWHM reveals an increase in crystal disorder provoked by the Zn and Mg dopants in HAP, so the intensity of the vibrations decreases. Furthermore, the peak at 1119 cm− 1 and 1464 cm− 1 indicate CO32− present in HAP. The υ1 (1119 cm− 1) and υ3 (1119 cm− 1) peaks are symmetric and asymmetric stretching vibrational modes of CO3 respectively [22]. The insert figure f in Fig. 3 shows the υ3 asymmetric stretching vibration of CO3. The peaks appearing at 519 (vw) and 440 (w) cm− 1 are the representatives of dopants Mg and Zn respectively [23, 24]. The peaks of Zn and Mg are illustrated as insert figures d and e in Fig. 3 respectively. These results are confirmed by Popa and his groups [4]. Raman spectra results support the FT-IR results.
Table 5
Raman vibrational mode assignment of pure, Zn and Mg doped HAP.
Pure HAP
|
Zinc doped HAP
|
Magnesium doped HAP
|
Assignment
|
References
|
1%
|
3%
|
5%
|
1%
|
3%
|
5%
|
429
|
430
|
432
|
443
|
431
|
435
|
440
|
υ2 doubly degenerate bending mode of PO43−
( O-P-O bond)
|
4, 21
|
459
|
486
|
485
|
485
|
480
|
484
|
485
|
υ4triply degenerate bending mode of PO43− ( O-P-O bond)
|
4
|
583
|
584
|
585
|
589
|
589
|
588
|
594
|
υ4 triply degenerate bending mode of PO43− ( O-P-O bond)
|
4, 21
|
962
|
964
|
962
|
965
|
962
|
966
|
966
|
υ1 nondegenerate symmetric stretching mode of PO43−
( P-O bond)
|
4, 21
|
1035
|
1040
|
1036
|
1036
|
1032
|
1035
|
1053
|
υ3 triply degenerate asymmetric stretching mode of PO43− ( P-O bond) and υ1 symmetric stretching mode of CO32−
|
4, 21, 22
|
1119
|
1123
|
1119
|
1123
|
1123
|
1129
|
1113
|
υ1 symmetric stretching mode of CO32−
|
22
|
1466
|
1466
|
1470
|
1468
|
1470
|
1467
|
1466
|
υ3 asymmetric stretching mode of CO32−
|
22
|
3.4. X-ray Photoelectron Spectroscopy (XPS)
XPS analysis is used to determine the oxidation state and the structural information of ZnHAP and MgHAP. The survey scan spectra of XPS in Fig. 4 indicates the presence of Ca, P, O, and C in all the samples and their corresponding binding energy is 347.76, 133.76, 531.37 and 285.26 eV [25, 26]. Fig. S7 shows the Ca, P, O, and C individual XPS spectra. In this spectra P, C and O have a sharp single peak, but due to spin-orbit splitting Ca has Ca2p3/2 and Ca2p1/2 two sharp peaks. The peak at 531 eV stands for O present in phosphate and the peak at 285 eV is for carbon (NIST database). The C1s (285.31 eV) peak is for adsorbed carbon. In the synthesis process, a very small amount of dopant was used, so the peaks corresponding to Zn and Mg are very weak. The XPS peak for the dopants Zn and Mg are shown as inset figures b and c in Fig. 4. Table 6 represents the BE (binding energy) of the elements in the samples. Phosphorus possesses 2p3/2 and 2p1/2 states due to spin-orbit splitting in ZnHAP. The binding energy of Zn2p1/2, Zn2p3/2, 3p, and 3d in ZnHAP XPS spectra is 1045, 1022, 45.18 and 10.35 eV respectively. Among the two Zn peaks, the 2p3/2 peak is tapered and is more intensive than 2p1/2 and the area of the Zn2p3/2 peak is more than that of Zn2p1/2 due to spin-orbit (j-j) coupling. Zn2p3/2 shows degeneracy in four states, whereas Zn2p1/2 shows only in two states. The Zn2p3/2 peak is associated with a satellite peak. The satellite peak of Zn2p1/2 is located at higher binding energy approximately 23 eV than the main Zn2p3/2 peak. The binding energy of Zn2p3/2 is 1022 eV. It is nearly three times greater than the binding energy of Ca2+. Hence, more energy is required to substitute Ca2+ by Zn2+ during the synthesis of ZnHAP, the required energy may be obtained either through pressure or temperature, or both. The binding energy of Mg2s and Mg2p are 88.02 and 49.4 eV respectively. There is no satellite peak for Mg. Zn and Mg binding energy results are very close to the result obtained by Negrila et al. and Predoi et al. [27, 28]. There is no remarkable change observed in the binding energy of Ca, P, and O due to the dopants. The incorporation of dopant with HAP decreases the intensity of the Ca2p3/2 peak. It implies that dopant ions decrease the Ca2+ ions present on the surface. The addition of dopant ions decreases crystallinity. These results support the XRD result. No other foreign element is found in the XPS spectra. The XPS result confirmed that the dopant elements occupied the Ca2+ position in the HAP structure. The XPS analysis successfully detected the presence of Zn and Mg in doped HAP, but XRD and FT-IR did not detect these elements. XPS results support the micro Raman analysis.
Table 6
The binding energy of the Ca, P, O, C, Zn, and Mg.
Element Name
|
1s
|
2s
|
2p
|
2p3/2
|
2p1/2
|
3s
|
3p
|
3d
|
References
|
Ca
|
|
|
|
347.76
|
351.2
|
48.9
|
25.09
|
|
25, 26
|
P
|
|
|
|
133.76
|
|
|
|
|
25, 26
|
O
|
531.4
|
|
|
|
|
|
|
|
25, 26
|
C
|
285.3
|
|
|
|
|
|
|
|
25, 26
|
Zn
|
|
|
|
1022
|
1045.02
|
|
45.18
|
10.35
|
27
|
Mg
|
|
88.02
|
49.4
|
|
|
|
|
|
28
|
3.5. FE-SEM Analysis
Figures S8 and S9 show the FE-SEM images of pure and Zn doped HAP and Mg doped HAP samples respectively. Figure 5 shows the FE-SEM images of pure, 5% Zn and 5% Mg doped HAP. Pure and doped HAP showed cylindrical and hexagonal morphology. In Zn2+ doped HAP, one-dimensional growth has been promoted. The one-dimensional growth leads to the higher thermodynamic stability of the HAP. Because of decreasing crystallite size, grain size decreases. Micropores and porosity increase with the increase in Mg concentration in HAP. But the addition of zinc in HAP decreases porosity. The osteoconductivity attains an adequate level when the micropores allow the flow of extracellular fluid through the inner structure of the biomedical device. The change in grain size and a slight variation in morphology are due to the dopant ion replacing Ca2+ in HAP. The grain size of pure HAP, 5% Zn doped HAP, and 5% Mg doped HAP are 275, 510 and 251 nm respectively. The 5% Zn added HAP shows highly defined rod shape morphology. Figure 6 (a, b, c,) shows the EDX spectra of pure HAP, ZnHAP and MgHAP samples respectively. The sharp Ca, P, and O peaks in all the spectra indicate the presence of HAP. EDX results confirm the presence of Zn, Mg in doped HAP. The FE-SEM results are in good accord with Kanasan et al. and Iqbal et al. findings [29, 30].
3.6. TEM Analysis
Figure 7 shows the TEM images of pure, 5% Zn and 5% Mg doped HAP separately. The TEM images show the non-uniform and hexagonal morphology of HAP. In Mg doped samples, the spherical morphology is more dominant than the hexagonal morphology (indicated in 7f ). So, the dopant changes the morphology of HAP. The TEM image of 5% ZnHAP shows rod shape morphology and this result matches with the FE-SEM result. The agglomeration of particles is to decreases energy due to interfacial bonding formed by dopants [15]. Therefore, particles are composed of smaller crystallites. Figure 7 (g, h, i) represents the selected area electron diffraction (SAED) pattern of pure HAP, 5% ZnHAP, and 5% MgHAP respectively. The spots and the ring patterns conform to the polycrystalline nature of hydroxyapatite. The morphology observed in TEM is in resemblance with the FE-SEM morphology.
3.7. Electrical Analysis
3.7.1. Dielectric constant
The variation of electrical capacitance and the dielectric constant, due to applied ac field is analysed. The dielectric constant (ε') is determined using the following formula [31].
![](data:image/png;base64,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)
1
Here C, Ɛ˳, A and t represent the capacitance, permittivity of the vacuum, area and the average thickness of the disc respectively. Figs. S10 and S11 show the variation of dielectric constant with frequency and dopant concentration. The frequency dependents dielectric constant of the pure, 5% Zn, and 5% Mg doped HAP are given in Fig. 8. The estimated dielectric constant (ε') values for different frequencies are given in Table 7. The dielectric constant decrease when the frequency increases, but it increases with the increase in the concentration of dopant. In the ac field, an induced electric dipole causes polarization. The change in dielectric constant is associated with four types of polarization they are electronic polarization, ionic polarization, orientation polarization and interfacial polarization. At low frequency, the storage of charges at the grain and in grain boundary causes large interfacial polarization so, the dielectric constant is high. At higher frequencies, dipoles cannot harmonize with the applied ac field so the decrease in interfacial and orientation polarization causes a reduction in the dielectric constant. At higher frequencies, the dielectric constant is only due to electronic and ionic polarization. In HAP electrical conduction is due to the movement of a proton between O2− or an OH− interacting with the PO4 group along the c-axis. And also at elevated temperatures, OH− ion in HAP is responsible for electric conduction. At room temperature, electric conduction is due to the movement of proton in adsorbed or condensed water in HAP [31]. The incorporation of Zn/Mg into the Ca site in the HAP structure changes the dipole moment of the OH− ions. The addition of dopant in HAP causes shrinkage in structure, crystallographic defects and mobility of charge carriers hence the dielectric constant increases. The XRD result clearly shows that, as the dopant concentration in HAP increases, the crystallite size decreases but defects increase. When the dielectric constant is changed the spreading of electromagnetic fields in bone fractures is also changed. The increase in dielectric constant supports bone growth and promotes fracture healing. The Zn doped HAP possesses a more dielectric constant than the Mg doped HAP.
Table 7
Dielectric constants of pure and metal ions doped HAP for different frequencies
Sample/ Frequency
|
30kHz
|
40kHz
|
50kHz
|
60kHz
|
Pure HAP
|
1.0
|
0.89
|
0.8
|
0.78
|
1% ZnHAP
|
11.66
|
10.46
|
9.62
|
8.92
|
3% ZnHAP
|
12.25
|
11.06
|
10.14
|
9.52
|
5% ZnHAP
|
13.23
|
11.95
|
11.03
|
10.26
|
1% MgHAP
|
8.31
|
7.54
|
7.19
|
6.46
|
3% MgHAP
|
9.42
|
8.48
|
7.86
|
7.30
|
5% MgHAP
|
10.76
|
9.71
|
8.97
|
8.36
|
3.7.2. Dielectric loss
The dielectric material absorbs energy from the ac field, based on its capacity it holds one part of the energy and the remaining energy is lost in the form of heat at a particular frequency. This dissipated energy is considered as dielectric loss. The best dielectric material possesses the least amount of dielectric loss [32]. Figs. S12 and S13 represent the variations in dielectric loss with frequency for pure, Zn doped and Mg doped HAP samples respectively. Figure 9 represents the variation of dielectric loss with the frequency of the sample of pure and 5% ZnHAP, and 5% MgHAP. The factors affecting the dielectric loss are crystal structure, applied AC frequency, temperature, stricture defects, imperfections in the crystal lattice, micro cracks, dislocations and etc. Therefore the dielectric loss can be minimized by using proper material. The dielectric loss goes on decreasing with the increase of frequency and the increase in the concentration of dopant material. At low frequency, the movement of charge carriers dominating the polarization, to rotate the dipoles more energy is required hence dielectric loss increases. In all the cases, at lower frequencies, the dielectric loss increases drastically and then decreases gradually with an increase in frequency. The deviation in dielectric loss depends on the concentration of dopants and their nature. Among Zn and Mg, at a particular frequency dielectric loss is more for ZnHAP, less for MgHAP. The high dielectric loss at low frequencies is due to the oscillations of the dipole. At a higher frequency, the ionic polarization ceases. Hence, the energy is not spent in rotating the dipole of the ions present in HAP. Therefore, dielectric loss lowers.
3.7.3. AC Conductivity
Figures S14 and S15 show the variation of ac conductivity due to the applied frequency and dopant concentration of the prepared samples. The frequency dependent of ac conductivity of pure, 5% Zn, and 5% Mg doped HAP are given in Fig. 10. Table 8 shows the ac conductivity of pure, Zn and Mg doped HAP at different frequencies. The ac conductivity of the materials is determined by the following relation [32].
σac = 2πfε′ε˳tanδ (2)
Where f is the applied frequency of ac signal, tanδ, ε′ and ε˳ are the dielectric loss, dielectric constant and permittivity of vacuum respectively. Alternating current conductivity increase with increasing frequency, due to proton jumping of O2− or due to the interaction of OH− ion bonded with PO4 group [33]. The AC conductivity of the sample decreases with the increase in dopant material concentration. The increase in dopant concentration causes an increase in dielectric constant and a decrease in conductivity. The results of the dielectric constant give well supports for the conductivity result. The calculated ac conductivity values are in order of 10− 6 Sm− 1.
Table 8
Ac conductivity of the samples at different frequencies is given in10− 6 S/m.
S. No.
|
Sample
|
Frequency
|
30 kHz
|
40 kHz
|
50 kHz
|
60 kHz
|
1
|
Pure HAP
|
6.32
|
6.68
|
6.99
|
7.29
|
2
|
1% ZnHAP
|
5.26
|
5.53
|
5.63
|
5.76
|
3
|
3% ZnHAP
|
4.45
|
4.59
|
4.73
|
4.82
|
4
|
5% ZnHAP
|
3.81
|
3.95
|
4.05
|
4.14
|
5
|
1% MgHAP
|
4.36
|
4.62
|
4.68
|
4.80
|
6
|
3% MgHAP
|
3.76
|
3.93
|
4.04
|
4.12
|
7
|
5% MgHAP
|
3.25
|
3.42
|
3.49
|
3.56
|
3.8. Micro-hardness
The Vickers hardness tester is used to find the micro-hardness. All the prepared samples are made into discs separately, each of diameter 13 mm and average thickness of 2 mm. The estimated micro-hardness values, for loads 25, 50 and 100 grams are given in Table 9. The Vickers hardness (HV) can be determined by using the following relation [34].
![](data:image/png;base64,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)
3
Where P is the applied load in kg and d is the diagonal length in µm. The hardness of all the samples is increasing with the increase in load as well as the increase in dopants concentration. The addition of dopants decreases the crystallite size and increases the surface area, the driving force for densification, leading to higher mechanical properties. As the dopants are smaller in size as compared to Ca, the replacement of Ca by dopants would reduce the bond length and increase the bond strength with O. The increase in bond strength is the cause of the increase in hardness. So the samples become more compact and harder in structure [35]. The hardness of 5% Zn2+ doped HAP and 5% Mg2+ doped HAP are 1059 MPa and 1081MPa respectively for a 100 gram load. At higher loads, the plastic flow of the material is less, hence the resistance offered by the material is more. Therefore, the magnitude of microhardness increases with an increase in load. The higher hardness enhances cell attachment and proliferation.
Table 9
Micro-hardness for a different load of pure, Zn and Mg doped HAP.
Sample
|
Micro-hardness (Hv)
|
Load 25 g.
|
Load 50 g.
|
Load 100 g.
|
Pure HAP
|
20.1
|
38.8
|
71.1
|
1%ZnHAP
|
56.6
|
67.6
|
85.3
|
3%ZnHAP
|
59.2
|
70.6
|
95
|
5%ZnHAP
|
67.1
|
78
|
108
|
1%MgHAP
|
59.6
|
64.8
|
79.4
|
3%MgHAP
|
64.2
|
68.6
|
85.7
|
5%MgHAP
|
71.7
|
86.9
|
110.3
|
3.9. Vibrating Sample Magnetometer (VSM)
The magnetic behavior of the prepared samples is analyzed by using a vibrating sample magnetometer with the magnetic field of – 3 to + 3 T. Figs. S16, S17, S18 and S19 represent the as-prepared and annealed sample's magnetic behavior, in the external magnetic field. Figures 11 and 12 represents the pure, 5%Zn and 5%Mg doped HAP as prepared and annealed samples respectively. The slope of the magnetic moment and field curve gives the susceptibility of the material. It is evident from the M-H curve all the prepared samples possess negative susceptibility. The pure, doped HAP, of as-prepared, annealed samples are diamagnetic [36]. The susceptibility of the material changes with dopant concentration. Hence, the addition of Zn2+/ Mg2+ with pure HAP increases the mechanical properties without any change in the magnetic property.
3.10. Antibacterial Activity
The antibacterial activity of the samples is analyzed against the Gram-positive Staphylococcus aureus and Streptococcus pneumoniae and Gram-negative Escherichia coli and Shigelladysenteriae bacteria by using the disc diffusion method. Erythromycin is used as positive control and pure HAP is used as a negative control. The zone of inhibition for each bacteria is measured and recorded in Table 10. The antibacterial activity of the samples is increased due to an increase in the concentration of dopants. And also antibacterial activity depends on the nature of the dopants [37]. The 5% ZnHAP and 5% MgHAP samples show the highest zone of inhibition against S. aureus and S. dysenteriae respectively. Antibacterial activity is related to the dielectric constant and is based on charge density. The increase in dielectric constant with an increase in the concentration of dopant causes increases in charge density hence antibacterial activity increases. The positive ion interacts with negative bacteria by giving a bacterial zone. The cation of the doped element exhibits antibacterial activity and leads the biomaterial towards medical applications [38]. Figs. S20 and S21 represent the antibacterial activity zone of inhibition of ZnHAP and MgHAP.
Table 10
Zone of inhibition of antibacterial activity.
Sample name
|
Bacteria name
|
Zone of inhibition (mm)
|
|
1%
|
3%
|
5%
|
HAP
|
Erythromycin
|
|
ZnHAP
|
S. dysentery
|
13.5
|
14.5
|
15.5
|
12
|
12.5
|
E.coli
|
16.5
|
16.5
|
17
|
15
|
17
|
S.aureus
|
14
|
16
|
18.5
|
13
|
14
|
S.pneumonia
|
16
|
15.5
|
18
|
15
|
14
|
|
MgHAP
|
|
S.dysentery
|
16
|
18
|
20
|
13.5
|
14
|
E.coli
|
16
|
17
|
18.5
|
14.5
|
13
|
S.aureus
|
17
|
18
|
19
|
14
|
15
|
S.pneumonia
|
16
|
18
|
18.5
|
15
|
14
|
3.11. Antifungal activity
The antifungal activity of the samples is investigated against the human pathogenic fungal strains Aspergillusniger and Candida albicans by well diffusion method. Here Fluconazole is used as positive control and pure HAP is used as a negative control. The obtained antifungal results are recorded in Table 11. Furthermore, the antifungal potential of nanoparticles depends on the size and shape of the particles. The antifungal activity of Aspergillusniger showed a better result than Candida albicans in both ZnHAP and MgHAP. The zone of inhibition increases with the increase in the concentration of dopants. Among Zn and Mg dopants, 5%Mg doped HAP shows the highest zone of inhibition against Aspergillusniger. The actual mechanism of the antifungal activity is, ZnHAP and MgHAP penetrate the fungal cell and hyphae, affecting the integrity of the plasma membrane. It leads to cell damage due to blocking nutrient uptake, promotion of chromosomal aberrations, mitochondrial oxidative stress and adenosine triphosphate (ATP) production is reduced. Besides, it is interrupted by fungal intracellular communication and it reduces the lifespan of fungi. Zn and Mg-doped hydroxyapatite nanoparticles are found to damage cell membranes leading to leakage of cellular components and finally cell death [39]. Figs. S22 and S23 represent the antifungal activity zone of inhibition for ZnHAP and MgHAP.
Table 11
Zone of inhibition of the Zn2+ and Mg2+ doped HAP.
Sample name
|
Fungus name
|
Zone of inhibition (mm)
|
HAP
|
1%
|
3%
|
5%
|
Fluconazole(PC)
|
ZnHAP
|
Candida albicans
|
12
|
13
|
16
|
18
|
11
|
Aspergillusniger
|
15
|
16
|
18
|
19
|
13
|
MgHAP
|
Candida albicans
|
12
|
13
|
13
|
14
|
11
|
Aspergillusniger
|
15
|
17
|
19
|
21
|
13
|
3.12. Antibiofilm activity
The Staphylococcus aureus (Gram-Positive) and Escherichiacoli (Gram-Negative) bacterial strains are used to analyse the antibiofilm activity of the prepared samples. Pure HAP is used as a control. The antibiofilm activity of ZnHAP and MgHAP are as shown in Figs. 13 and 14 respectively. The pure HAP treated biofilm showed dense and strong adhesive biofilm formation on the glass substrates. The density of bacterial growth decreased with an increase in dopant concentration. Notably, ZnHAP showed a better result than MgHAP [39]. The antibiofilm activity can be explained by stating that the ZnHAP samples enabled in migration of radical anions into the cell membrane and reacted with the thiol group of peptides/amino acid leading to loss of cell division, mesosoma function, intracellular cell signaling, intracellular electrolytes potential, protein and DNA synthesis.