Influence of Mg Incorporation on Mechanical, Dielectric and Biological Properties of Hydroxyapatite Ceramic


 This research focused on the mechanical electrical and biological properties of magnesium doped (Mgx, x = 0.5 - 2.5 mol%) hydroxyapatite(Hap) using chemical method. It was observed that addition of magnesium produced the secondary phase (whitlockite) depended on the concentration of magnesium using the XRD, FTIR and Raman techniques. These two phases forming BCP(Biphasic) are beneficial from the implant point of view. The dielectric properties were measured as a function of frequency for different concentration of Mg. For 1.0 -2.0mol% doped Mg samples showed dielectric constant value within the range required for implant material. The bioactivity and However, beyond 2mol% of Mg third phase of magnesium oxide was also observed which enhanced the micro-hardness and bioactivity of specimen.


Introduction
Calcium phosphate based bioceramics are suitable as synthetic bone substitutes due to their bioactive and biocompatible that occur in different phases [1]. Hydroxyapatite(Hap) and beta tricalcium phosphate(β-Tcp) are the naturally occurring calcium phosphate bioceramics in bones and teeth of mammals. These two phases makes an exceptional combination for bone implantation with bone-bonding ability of Hap and good bioresorbability of β-Tcp in physiological environment [2,3] . An optimal ratio of these two can enhance the regeneration of bone in the body environment [4,5,6]. It is noteworthy that research is not only concentrated on the biological properties of these ceramics but also electrical and mechanical properties are of great interest. It has been reported that electrical stimulation enhance the growth rate thus reduce the healing time [7,8]. The electrical response of Hap is analogous to bone [9], thus it is important to study the behavior under the influence of electric field. The crystalline structure(hexagonal) of Hap is quite flexible to accommodate the impurities that in turn can enhance the biological, mechanical and dielectric properties of Hap. According to the previous studies, dielectric properties help in bone regeneration. Little work has been previously done to study dielectric properties for doped calcium phosphate. Magnesium is a vital element in bone formation and its deficiency causes brittleness and bone loss [10,11]. Thus Mg can be incorporated into Hap structure to augment its properties.
In this study, Mg doped hydroxyapatite was synthesized using wet chemical method. Thermal, Structural, Chemical, mechanical, dielectric and biological properties of undoped and Mg doped samples will be discussed.

Materials and Method
All the samples were synthesized by chemical precipitation method [12,13]. Calcium nitrate, diammonium hydrogen phosphate and Magnesium nitrate were used as a calcium(Ca 2+ ), phosphate and magnesium ions respectively, all of analytical grade. Ammonia solution was used to control the pH. The reaction will take place according to the equaton(1). The schematic for the synthesis is illustrated in Figure 1.   To determine the thermal characteristics of the samples TGA-DSC (SDT Q600) was used with a heating rate of 10°C/min. The chemical bond characteristics of samples were determined by Midac M2000, recorded in the range of 450 to 4000cm -1 . To investigate the effects of incorporation of Mg 2+ in the structure of the samples Raman spectroscopy(Advantage 532 Raman Spectrometer) was performed. The structure of undoped and Mg-doped samples were characterized using XRD( Bruker D8 Advance X-ray diffractometer) with step size of 0.02 in the 2θ range from 10 o -80 o . The experimental XRD patterns were identified by comparing them with the standard JCPDS(Joint committee in Powder Diffraction and Standards). Scanning electron microscope(JSM 6480 VL, Joel) was used to study the surface morphology of the samples. The hardness of the samples were determined using Vickers hardness(Suntech Clark Model Cv-700at) with a load of 9.8N applied for 10s on the surface of each compressed polished disc. Dielectric properties were studied by measuring the capacitance and resistance in parallel plate configuration with Precision Impedance Analyzer(Wayne Kerr 6500B) .The bioactivity of the samples was examined by immersing them in Simulated Body Fluid(SBF) [ 14] solution for two weeks incubated at 37 o C under static condition. The antioxidant activity of the samples were studied by measuring the absorbance at 517 nm using UV-VIS spectrophotometer (JEOL JSM-6480 LV).

Thermal Analysis
Thermal analysis predicts the phase purity and behavior of the material at high temperature. The DSC curves for the samples are illustrated in Figure 2

IR Spectra
Several vibrational bands were observed in IR spectra of pure and Mg doped samples as shown in Figure 3. For undoped sample, Mg0.0, the band at 1023(v1) cm -1 and 969(v2) cm -1 correspond to PO4 stretching band while PO4 bending bands relate to 605(v4) and 562(v4) cm -1 that are characteristics of hydroxyapatite [18]. For Mg(1.0 -2.5mol%) doped samples the IR spectra showed a new band at 1118cm -1 which correspond to characteristic β-TCP along with hydroxyapatite characteristic bands [19], listed in Table 2. It was observed that with Mg doping the broadness of vibrational bands were reduced which indicates alignment of atoms.

Raman Spectra
The Raman spectra of undoped and Mg doped samples is shown in Figure 4. In all samples hydroxyapatite was prominent by a very robust band at 961cm -1 and within 400cm -1 (v2) range of Hap that ascends from the symmetric stretching mode(v1) of PO4 group. Other Raman-active band for PO4 could be seen at 1036cm -1 (v3) [ 22,23]. With the addition of 0.5mol% of Mg the distinct peak at 961cm -1 splits into double peaks, 948cm -1 and 964cm -1 , which are attributed to PO4 stretching mode of β-TCP [24,25]. Another difference observed was the disappearance of 1036cm -1 peak(stretching mode P-O) with addition of Mg up to 1.0mol%. With further addition of Mg(1.5 -2.5 mol%) the double peaks at 948cm -1 and 946 cm -1 combined into a single intense peak. The reappearance of peak corresponding to phosphate vibration at 1051cm -1 was observed.

XRD Analysis
X-ray diffraction data was used to determine the lattice parameters, phase analyses and crystallite size of the samples. The phase percentage of undoped and Mg-doped samples is listed in Table 3. With the addition of Mg the secondary phase whitlockite /appeared whose percentage is Mg concentration dependent. (1) Where d is interplaner distance and hkl are Miller indices. The crystallite size(D) and crystallinity percent was calculated using the following relations [ 30] = 0.9 ℎ cos ( ℎ ) The lattice parameters(a, c) decreased with the increase in concentration of Mg as listed in Table 4 and Table 5, thus suggested the substitution of Mg ion for Ca ion Since Mg ion radius is smaller than Ca ion radius; substitution of Mg ion for Ca ion produced the decrease in the lattice parameters [31,32]. Substitution of Mg ion for Ca ion produces destabilization/decomposition of the structure. The crystallites became smaller, more irregular and form agglomerates when Mg was substituted [33].
The crystallite size was found to increase for Mg 2.5mol% due to increase of the lattice disorder as a result of the presence of Mg ions and formation of MgO due to surplus Mg. The value of lattice parameters reduced with the addition of Mg 2+ for Mg 2+ 0.5 -1.5 mol% that confirmed the incorporation of Mg into the lattice. For Mg 2+ 2.0 -2.5mol% the value of 'c' slightly increased identifying the appearance of third phase that caused the lattice disorder and strain. However, the volume decreased due to smaller bond length of Mg 2+ with O 2as compared to Ca 2+ . The crystallite size and crystallinity of the samples showed the decreasing trend with the addition of Mg 2+ (0.5 -2.0mol%) which agreed well with the previous Raman analysis. But for Mg2.5 sample the crystallite size and crystallinity increased this mighty be due to the formation of MgO that reduced the availability of Mg 2+ for replacement of Ca 2+ in Hap. The XRD peak intensity at 17.08 o for Mg2.5 decreased due to non-availability of Mg 2+ hence Mg-β-Tcp formation also reduced.

SEM
SEM micrographs as shown in Figure 6 illustrates the morphology of sintered undoped and Mg-doped samples.

Figure 6 SEM micrograph of Mg doped samples
It can be seen that undoped Hap(Mg0.0) is in form of small-size crystal, by increasing the Mg 2+ concentration, the densification of particles increases. This densification reduces the elongation and widening of particles so the grain size reduces gradually. After the addition of Mg, these particles have plate like or flakes like morphology. The results of researches indicate that by increasing Mg concentration in Hap, secondary phase (whitlockite) formed so the morphology also varies.

Mechanical Analysis
The microhardness of the samples was measured using Vickers hardness method. The Vickers hardness was measured using the following formula [34] = 1.854 2 (5) where F is the load applied and d is mean diameter of the indent. Average value was taken from five indents on each sample. It was observed that the hardness of the doped samples was higher than the undoped samples as shown in Figure 7, having the highest indentation hardness value of 1.01GPa as listed in Table 7. The values are comparable to the human bone ~0.3-0.9 GPa [35].  The optimal ratio was observed for the Mg content at 2.0mol%, after which the trend showed the decline. The increase in hardness for Mg2.0 sample is due to the appearance of the third phase MgO that enhances the hardness as reported by Satoshi [36]

Dielectric Properties
The dielectric properties of the implant are of interest in order to augment the bone growth.
The dielectric properties of the samples were studied as a function of frequency. The capacitance values were used to calculate the dielectric constant(ε). The dielectric constant, alternating current conductivity (σac) and dielectric loss(tan(δ)) were determined using the following formula [37] = (6) = (7) Where Cp is the capacitance, o the free space dielectric constant (8.854 × 10 -12 F/m), A is the area, t is the thickness and Z is the impedance of the dielectric samples. The dielectric constant determines the efficiency of the material to store energy(electrical). It is a frequency dependent variable and decreases as the frequency increases.
The dielectric properties of the samples were studied by measuring resistance and capacitance using Impedance Analyzer at room temperature. Figure 8a showed the dielectric constant as a function of frequency from 1KHz to 1MHz. The values of dielectric constant varied as the concentration of dopant varies. It has been observed that the value of ε increases as the conc. of Mgx increase from 0 ≤ x ≤ 1.5 mol% after which it showed the decreasing trend. The dielectric constant decreased as the frequency increased for all the samples. Since the applied electric field produced polarization in the material, as the frequency increased the dielectric constant decreased which is attributed to the failure of the dipoles to follow the alternating electric field frequency and lag behind the applied frequency [38]. The alternating current conductivity increased with the increase in frequency as shown in Figure 8(b), which is attributed either due to H + ion bouncing between O 2or OH -1 ions with PO4 group. Tangent loss, absorption of electrical energy by the material, also showed( Figure 8c ) the same trend as of dielectric constant and conductivity. The high value of tangent loss for Mg1.0 and Mg1.5 at low frequency are attributed due to defects/impurities present in the samples. All the curves exhibited bulge around 13kHz that is attributed to the resonance between the ions frequency and the frequency of applied field. Thus these results suggest that Hap samples can also be used as dielectric material in biosensors and as a dielectric material in electronic devices.

Bioactivity
Simulated body fluid(SBF) solution, prepared by Kokubo method [39] was used to assess the bioactivity of the samples. The samples were immersed in SBF solution for two weeks at 37 o C, the pH and the concentration of Ca, P and Mg ions in the solution were recorded periodically as shown in Figure 9. Initially there was rise in pH due to release of cations(Ca 2+ ) from the samples this was justified as there was increase in Ca concentration as shown in Figure 9(d) and 9(a) respectively. After four days the pH and Ca concentration started to decrease as the leakage of Ca ions reduced which is attributed to the formation of apatite layer.

Figure 9 Ca Concentration(a), P concentration(b) Mg concentration(c) and Variation of pH(d) in SBF Sol. For 14 days
The formation of apatite layer in physiological environment depends on the resorbability, which increases the pH. From Figure 9(d) it can be observed that the pH of Mg0.0(undoped) was lower as compared to doped samples the same trend can be seen for Ca and P content. The maximum bioresorbalility was observed for Mg1.5, which is a mixture of Hap and whitlockite having greater percentage of whitlockite phase as compared to the other sample. The FTIR spectra of the samples after soaking in SBF-solution for 14 days is shown in Figure  10. The same trend can be seen from the figure as was the case of when the samples were not immersed( Figure 3). However some band become clear and prominent like the bands at 500 -560cm -1 which are due to bending vibration mode(crystalline) of P-O bonds in PO4 3group [40,41] and the band around 470 cm -1 correspond to double degenerated bending mode(v2). These bands identify the Hap. It can be observed that the widening of the main broad band centered at 1028cm -1 did not occurred for the immersed samples in FTIR transmittance spectrum as compared to the un-immersed. In case of un-immersed samples, the widening occurred due to Mg 2+ that increased with increasing concentration of Mg 2+ . The band in the around 1121cm -1 related to β-Tcp vibrational mode. Thus, the FTIR analysis showed that the apatite layer formed on the samples were of biological Hap due to appearance of carbonate group.

Antioxidant Activity
Free radicals produce during oxidation chain reaction that can damage cells. Cell metabolism, ecological pollutants and infective toxins through these oxidative stress exhibit toxic effects [42]. The scavenging of free radicals by antioxidant control the deterioration of the cells. the prevention of many chronic diseases, such as cancer, diabetes and cardiovascular disease, has been suggested to be associated with the antioxidant activity DPPH(2,2-diphenyl-1picrylhydrazyl) radical scavenging assays were used to determine the radical scavenging effect of the samples. The absorbance was measured at 517 nm and the percentage scavenging activity of radicals was calculated using the formula % Scavenging activity =  Scavenging of free radicals increased with doping as compared to the undoped sample. The antioxidant property of Hap could be beneficial for orthopedic and dental implant.
Pristine and Mg doped hydroxyapatite were synthesized using chemical method. Incorporation of Magnesium subvert the Hap crystal structure with the formation of whitlockite which was confirmed by XRD, FTIR and Raman spectroscopy. These results illustrated the phase formation, Mg ions substitution in hydroxyapatite and transformation of Hap into other phases with the increase in Mg content. The effects of Mg were linked to the variation of lattice parameters and crystallinity of Hap. Beginning from pristine Hap with regular incorporation of Mg, admixture of the two phase was obtained. However, as the amount of Mg exceeded 2mol% new phase (MgO) appeared due to excess of magnesium. These phases favored the hardness and bioactivity, and for Mg 1.5mol% gave the better result as compared to others. It is possible to obtain the particular ratio of Hap and whitlockite by controlling the Mg concentration during synthesis. The dielectric properties were also affected by the addition of Mg. For 1.0 -2.0mol% of Mg the dielectric constant value is within the range required for implant material (18 -68[43]) at lower frequency range. The loss factor of all the samples were at 13kHz. The AC conductivity increased at higher frequency obeying the Jonsher's power law. Scavenging of free radicals increased with doping as compared to the undoped sample. These outcomes could be useful for implant material under the influence of electric field, as well as for biosensor.