Electrical Properties of Newly Calcified Tissues on the Surface of Silver Ion Administrated Hydroxyapatite Scaffolds

The application of electric field to graft materials has significant contribution in bone healing mechanism. Hence, the aim of this study is to develop conductive hydroxyapatite (HAp) scaffolds by introducing different concentrations of silver ion into its structure and demonstrate its impact on in vitro bioactivity and electrical properties. Hydroxyapatite was synthesized by wet chemical method and calcium ions from HAp structure have been partially replaced by silver ions. The HAp and Ag-HAp nanocomposites were characterized by Fourier-transform infrared, Raman spectroscopy, XRD and EDAX for functional group and phase formation analysis as well as to confirm existence of silver ions in HAp structure respectively. Bioactivity of these scaffolds was assessed by using simulated body fluid. The surface morphology, structural analysis and electrical properties of scaffolds before and after formation of newly calcified tissues on its surface were examined via scanning electron microscopy (SEM), XRD, FTIR, dielectric and impedance spectroscopy techniques. Overall, our finding suggests that the administration of silver ions in HAp scaffold boosts bioactivity and has strong correlation with electrical properties.

cal field [8]- [15]. However, among various trace metal ions, introduction of Ag + ion in HAp can have an advantage of excellent biocompatibility, satisfactory stability, antibacterial nature and noncytotoxicity and this can help to prevent post transplantation infection risk of implant material [16] [17]. The inclusion of silver ions in HAp structure promotes bioactivity and such coating on metal offers corrosion resistance [18] [19] [20]. Therefore, silver particles are widely used in various medical applications like bone prostheses, artificial teeth, and bone coating [21] [22]. Furthermore, the cation exchange rate of HAp is found to be very high with silver ions.
Recent advances in orthopedic therapy have shown that application of electrical field to bone stimulates bone growth, promotes osseointegration, and boosts bone density [23] [24] [25] [26]. Further, few studies demonstrated that accumulation of electrical charges on HAp graft plays a significant role in osteoconduction mechanism and in reconstruction of bone occupancy at damaged part of bone after implantation [27] [28] [29]. Also, Maharbiz et al. have observed that impedance measurements can be used to monitor early stage bone healing process and recovery of bone fracture [30]. Similarly, Tian et al. detected the variations in HAp electrical impedance value due to structural changes during bone regeneration process and illustrated that impedance spectroscopy can hold considerable potential for quantitative bone healing analysis [31]. The dielectric properties of HAp, Sr-HAp and Mn-HAp incubated in simulated body fluid as a function of incubation period have been studied by our group [9] [32] [33]. These studies have shown that electrical properties, specifically dielectric properties of biomaterials are of great interest to understand bone healing mechanism.
Hence, by considering the coalition between the electrical properties and bone healing process, efforts have been made to investigate the in vitro electrical property of newly calcified tissue on silver ion incorporated HAp scaffold. Emphasis has also been given to correlate the dielectric, photoluminescence properties of Ag-HAp with the bioactivity.

Hydroxyapatite Modification
Nano crystalline hydroxyapatite (HAp) bioceramic was synthesized by wet-chemical precipitation method [32]. Further, HAp structure was modified by partial replacement of calcium ions by silver ions (Ag + ) via ion exchange process, carried out at room temperature. Synthesized Ca-HAp and Ag-HAp nano-bioceramic materials were characterized by XRD, FTIR, SEM/EDAX, Raman spectroscopy and BET techniques. The Ag-HAp samples with variable silver concentrations (0.001 M, 0.005 M, and 0.025 M) were uniaxially pressed at 5-ton pressure to form compact disc shaped scaffolds of 13 mm diameter and 2 mm thickness.
The prepared scaffolds were then heat treated at 500˚C for 2 h and used as scaffolds to study further in vitro bioactivity and electrical properties.

Preparation of Simulated Body Fluid (SBF) and In Vitro Bioactivity
In vitro bioactivity study of HAp and Ag-HAp scaffolds was carried out using pseudo body fluid (SBF) in static mode condition.

Dielectric and Electrical Characterization
The dielectric measurements such as dielectric constant, dissipation factor, and impedance were carried out using Quad Tech make LCR 7600 meter at room temperature in frequency range of 10 Hz to 1 MHz for already prepared scaffolds.

Characterization
The structural analysis was carried out using Rigaku to make X-ray diffractometer with CuKα radiation (K = 1.543Å). FTIR and Raman spectroscopic techniques were used for identification and conformation of the functional groups.
Fourier transform infrared (FTIR) spectra of scaffolds were recorded in 400 to 4000 cm −1 range with a resolution of 4 cm −1 by using Shimadzu make spectrophotometer. Raman spectra of scaffolds were analyzed in the spectral range of 100 to 1100 cm −1 with the spectral resolution of 4 cm −1 by using Raman microscope (Horiba 800, France). Surface morphology and elemental analysis of Ag-HAp scaffolds before and after SBF treatment were examined by scanning electron microscope (SEM) (with LiecaStereoscan 440 model SEM) coupled with Energy dispersive X-ray analyzer (EDAX). Further, surface area and porosity of these scaffolds were decided by Bruaauer-Emmett-Teller (BET) method (Supplementary information). The PL spectra for Ag-HAp scaffolds were conducted, before and after SBF incubation, at room temperature using Horiba FL3-22-1186C-2609 (λ exc = 415 nm).

XRD Analysis
The XRD profiles of Ag-HAp samples as a function concentration is presented in Figure 1 with XRD pattern of the parent Ca-HAp (JCPDS 09-0432) wherein major characteristic apatite peaks are found to be present. This not only indicates the development of hexagonal hydroxyapatite phase but also confirms that ionic substitution does not change the apatite structure. No evidence is found for any other phases. It is also observed from Figure 1(a) that due to the incorporation of silver ions in hydroxyapatite matrix, intensities of almost all peaks decrease compared to Ca-HAp. In particular the intensity of peak assigned to (211) plane decreases with increase in silver concentration. The XRD spectra of Ag-HAp samples after incubation in SBF are presented in Figure 1

FTIR Analysis
The FTIR spectra of ion exchanged hydroxyapatite nano-bioceramics, in the range of 500 -1300 cm −1 , are presented in Figure 2 PO − ) functional group [35]. The bands appearing at 967 and 941 cm −1 are attributed to ν 1 symmetric stretching mode of ( 3 4 PO − ) [36]. The absorption band near 632 cm −1 corresponds to hydroxyl liberation mode. The absorption bands due to ν 4 fundamental bending mode of ( 3 4 PO − ) group are present near 543 cm −1 and 603 cm −1 [35] [36]. It is found that the absorption increases with increase in ion concentration.
Hence, it can be concluded that higher the ion concentration, higher is the absorption.
Typical FTIR spectra of incubated HAp samples, showing change in absorption peak intensities of various groups, are presented in Figure 2 durations, and the appearance of absorption peak near 1120 cm −  rate. The surfaces of Ag-HAp scaffolds are found to be fully covered with newly formed calcified tissues wherein size of new formed grains scaffold surface observed to be increasing with silver ion contents. This indicates dominance of higher growth rates and coalescence phenomenon with silver ion concentration. It is also observed that for fixed silver ion loading and variable incubation periods, agglomeration phenomenon plays important role for higher incubation period (Figure 3(c)). Further, the EDAX data for incubated Ag-HAp samples reveals that Ca/P ratio reaches to a maximum value of 1.43 after 24 days. These results show that Ag-HAp nano-ceramic scaffolds have ability to act as templates for apatite layer formation on their surfaces and can be considered as superior bioactive nano-ceramics than Ca-HAp.

SEM Analysis
Since the process of apatite layer formation takes place at the bioactive HAp scaffold surface/SBF solution interface, it depends on majorly on surface ions. Due to presence of hydroxyl (OH − ) and phosphate (  Figure 4(a) presents the change in dielectric constant for Ag-HAp scaffolds with change in frequency of an applied ac field as a function of concentration and the plots are compared with that for Ca-HAp. All the scaffolds show similar behavior of decrease in dielectric constant with increase in frequency. The dielectric constant of Ca-HAp drastically changes after addition of silver ions into HAp structure. The dielectric constants for Ag-HAp scaffolds are found to be smaller than that for Ca-HAp as depicted in Figure 4(a). It is observed that as the silver ion content increases, dielectric constant decreases. The dielectric constant for higher silver ion concentration (0.025 M) is observed to the smallest (approximately near to 8) in comparison with other silver concentrations. The study clearly reveals the dependence of dielectric constant on ionic concentration.

Dielectric Constant
The effect of incubation on dielectric constant of Ag-HAp as a function of frequency of applied ac field is depicted in Figure 4 It is well known that the dielectric properties of hydroxyapatite are mainly due to the motions of OH − ions with applied ac field and inclusion of silver ions may be responsible for change of electrical dipoles of OH − ions leading to the decrease in dielectric permittivity of Ag-HAp.
The dielectric properties of Ag-HAp, before and after incubation, display high dielectric permittivity at low frequencies which falls off with increase in frequency reaching a constant value for all samples. High values of dielectric constants at low frequencies are obvious because the hydroxyapatite belongs to bio-ceramic category and the behavior of dielectric permittivity is related to free dipoles oscillating in an applied alternating field. At very low alternating field frequencies, electric dipoles specifically due to hydroxyl ions follow the field al-terations and contribute to high value of dielectric constant at lower frequency. With increase in frequency of ac field, the dipoles start lagging behind the field reversal leading to slight decrease in dielectric constant followed by abrupt drop in dielectric constant. At still higher frequencies, the ions cannot follow the alternating field. As a result, polarization decreases and capacitor offers low reactance to the sinusoidal signal minimizing the conduction losses in the resistor and hence, dielectric constant decreases. The high dielectric constant for lower incubation period at lower frequencies may be due to the fact that the free charges buildup at interfaces within bulk of the scaffold (interfacial Maxwell-Wagner polarization) indicating apatite layer formation.

Dielectric Loss
Typical variation in dielectric loss with frequency for Ag-HAp scaffolds, prior to incubation, is presented in Figure 5 The dielectric loss for incubated HAp scaffolds, for fixed & variable incubation periods, as a function of frequency of applied ac field is presented in Figure  5(b) and Figure 5 The Debye relaxation theory states that the loss peak appears when the alternating field is in phase with dielectrics since the condition of τω = 1 (where ω = 2πf & τ is relaxation time) is satisfied.

Cole-Cole Plot
Cole-Cole plots for Ag-HAp scaffolds are presented in Figures 6(a)-(c). The horizontal axis is the resistance (Z') i.e. the real part of the impedance and vertical axis is the reactance (Z") i.e. imaginary part of the impedance. The presence of two semicircles for all scaffolds reveals the poly-dispersed nature of the material and represents two RC elements. The low frequency arcs are due to grain boundaries and higher frequency arcs indicate grain effect. The intercepts of low frequency arc & high frequency arc on the real axis designate grain boundary resistance (R gb ) and bulk resistance (R g ) respectively. The values of R g , τ and R gb are listed in Table 1. Similarly, grain capacitance (C g ), grain boundary capacitance (C gb ) values are calculated from following equation: × π Journal of Biomaterials and Nanobiotechnology  where, R g is grain resistance determined from the intercept of high frequency arc on the real Z' axis of impedance spectra and f c is the relaxation frequency corresponding to the maximum value of Z". The C g and C gb values calculated for Ca-HAp and Ag-HAp scaffolds, before and after incubation in SBF, are shown in Table 2.
High frequency arcs for all scaffolds before incubation as a function of concentration are depicted in Figure 6(a). The heights of high frequency arcs of impedance spectra do not show any appreciable change for variations in ion concentration excluding scaffold with 0.005 M silver ion content. This scaffold exhibits lowest amplitude with the lowest grain resistance effect.
Cole-Cole plots for SBF incubated Ag-HAp scaffolds as a function of incubation period and silver ion concentration are depicted in Figure 6(b) and Figure  6 The change in amplitude & position of low frequency arcs in impedance spectra for incubated scaffolds may be due to the development of calcified tissues on the surface of scaffolds. These changes in scaffolds, after SBF treatment, affect the gain boundary resistance. These findings suggest that the impedance spectroscopy can be useful technique to determine in-vitro bioactivity of scaffolds.

Photoluminescence Analysis
The photoluminescence spectra of SBF treated Ag-HAp scaffold (0.005 M) as a function of immersion period is shown in Figure 7. From the figure, it can be concluded that all Ag-HAp scaffolds follow similar trend. With an excitation wavelength of 415 nm, the emission spectrum of Ag-HAp, prior to incubation, exhibits a very intense, sharp and narrow blue emission band localized at ~470 nm. It can also be seen clearly that the relative photoluminescence intensity of this peak changes with change in SBF incubation period. Higher the period of incubation, higher is the intensity. It may be due to increase in thickness of additional biologically active layer formed on the surface upon incubation.

Conclusions
Ag-HAp scaffolds can be a potential bioactive biomaterial. The in-vitro bioactiv-