Combined effect of surface polarization and ZnO addition on antibacterial and cellular response of Hydroxyapatite-ZnO composites

Journal Pre-proof Combined effect of surface polarization and ZnO addition on antibacterial and cellular response of Hydroxyapatite-ZnO composites Angaraj Singh, Kuppili Reshma, Ashutosh Kumar Dubey PII:

S0928-4931(19)32285-4

DOI:

https://doi.org/10.1016/j.msec.2019.110363

Reference:

MSC 110363

To appear in:

Materials Science & Engineering C

Received Date: 20 June 2019 Revised Date:

22 September 2019

Accepted Date: 22 October 2019

Please cite this article as: A. Singh, K. Reshma, A.K. Dubey, Combined effect of surface polarization and ZnO addition on antibacterial and cellular response of Hydroxyapatite-ZnO composites, Materials Science & Engineering C (2019), doi: https://doi.org/10.1016/j.msec.2019.110363. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Combined effect of surface polarization and ZnO addition on antibacterial and cellular response of Hydroxyapatite-ZnO composites Angaraj Singh, Kuppili Reshma and Ashutosh Kumar Dubey* Department of Ceramic Engineering, Indian Institute of Technology (BHU) Varanasi - 221005, INDIA

Abstract Bacterial infection is among the serious concerns in orthopaedic during/ after surgery. Here, we demonstrate a novel technique to induce the antibacterial response in biomaterial substrates via surface polarization. In the present work, hydroxyapatite, HA-xZnO (x= 3.0, 4.5 and 7.5 wt. %) composites were processed by solid state sintering route at 1250 ºC for 2 h. After phase evolution analyses, the detailed dielectric and electrical measurements were performed over a wide range of temperature (30-500 C) and frequency (1 Hz to 1 MHz). The impedance spectroscopic analyses suggest the activation energies for grains and grain boundaries for HA and HA-3 wt.% ZnO are (1.36, 1.44 eV), and (1.18, 1.98 eV), respectively. The sintered samples were polarized under polarizing temperature and voltage of 500 C and 20 kV, respectively. The viability of Escherichia Coli (E. Coli) and Staphylococcus Aureus (S. Aureus) bacteria is observed to reduce significantly for polarized HA-x ZnO (x = 4.5 and 7.5 wt. %) composites as compared to their respective counterparts. On the other hand, polarization supports the proliferation of SaOS2 cells. Overall, the combination of surface polarization and optimal ZnO addition in HA has been demonstrated to significantly improve the antibacterial as well as osteoblast-like SaOS2 cellular response. Keywords: Hydroxyapatite, zinc oxide, polarization, antibacterial, cell viability 'Declarations of interest: none' *

Corresponding Author: [email protected] ; Phone: +91 8726823415 1

1. Introduction In recent years, bacterial infection in orthopaedic implants has been recognized as one of the serious concerns [1]. In the past few decades, a wide variety of prosthetic implants have been developed to repair, augment or replace injured/defective bone tissue [2]. Among various existing biomaterials, hydroxyapatite (HA) has striking similarity with natural bone in terms of its mineral composition [3]. HA exchanges its ions with host bone which accelerates the bone tissue formation at the interface [4]. Despite of having excellent biocompatibility, HA supports bacterial adhesion on its surface [5]. Therefore, HA is being used along with antibiotics to prevent infections at implantation sites [6]. However, it has been recognized that the bacteria develop resistance with time against certain antibiotics [7]. Sometimes, antibiotics may have limited ability to kill the bacteria at the implant site and also, the antibiotic coatings may not be effective for longer durations [8]. Number of antibacterial inorganic metals and metal oxides such as, Ag, Ti, Au, CuO, ZnO, MgO etc., are being incorporated with HA to overcome these limitations [9,10]. The antibacterial ratio of Escherichia Coli (E. Coli) and Staphylococcus Aureus (S. Aureus) bacteria is found to be 99.8 and 99.6 % on Ti implant, coated with polydopamine/graphene oxide/ silver nanoparticles (PDA/GO/ AgNps- Ti)[11]. In another study, it has been reported that the combined effect of visible light irradiation at 660 nm for 15 min and electrophoretic deposited Stable Chitosan@MoS2 coating on Ti implant increases the antibacterial efficiency of E. Coli and S. Aureus bacteria by 91.58 and 92.52 % , respectively [12]. However, the atomic layer coating of nano-structure ZnO on Ti implant and further modification with chitosan and carbon nanotubes (CNTs) increases the antibacterial ratios of E. Coli and S. Aureus bacteria by 73 % and 98 %, respectively [13]. Among various metal oxides, ZnO has superior antibacterial spectrum, stability and durability [14,15]. Moreover, Zn is the

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second most abundant trace elements in the human body, majority of which is present in muscles and bones [16]. The addition of ZnO as secondary phase increases the antibacterial effect for both, gram positive and gram negative bacteria [17]. However, the addition of such antibacterial agents above a minimum threshold raises the concern of potential toxic effect to the cells [18]. It has been reported that the addition of ZnO more than 10 wt. % in HA matrix becomes toxic to cells [19]. However, ZnO content up of to 7.5 wt. % in HA-x ZnO (x = 3, 4.5, and 7.5 wt. %) composites have been reported to support cellular proliferation [19]. The application of external electrical and magnetic stimuli has been suggested as potential alternatives to induce the antibacterial response [20,21]. The external stimuli enhance ROS generation which subsequently, improves the antibacterial response [22]. It has been reported that direct contact of bacterial cells with electric field produces toxic elements like, H2O2 and Cl2, which can also damage the mammalian cells [23]. Similarly, the exposure of high intensity magnetic field changes the hormonal concentration and gene expression [24]. Electrical properties of bacterial cells play an important role during interaction of bacterial cells with the implant surface [25]. It has been reported that both, gram positive and gram negative bacterial cells possess negative charge [26]. Therefore, the surface polarization of substrate has been anticipated to reveal antibacterial response [27]. As for as the influence of surface charge towards cellular functionality is concerned, number of studies demonstrated that the negatively charged surfaces accelerate cell growth and proliferation of Osteoblast-like cells as compared to positively charged or uncharged surfaces [28,29]. These studies indicate that the effect of surface polarization on the functionality of mammalian cell and bacteria are diametrically opposite. In view of above, the present work investigated the effect of ZnO (up to 7.5 wt. %) addition in HA on dielectric and electrical properties. The existing theoretical models were use to compare the

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measured values of the dielectric constant with calculated values. Further, the combined effect of surface polarization and optimal ZnO (up to 7.5 wt. %) addition on antibacterial (both, S. Aureus and E. Coli bacteria) and cellular (SaOS2 cell line) response have been examined. 2. Experimental 2.1. Materials and Methods 2.1.1. Synthesis of pure hydroxyapatite In this work, pure HA has been prepared using a suspension precipitation route [30]. Calcium oxide powder (Merck) and orthophosphoric acid (Himedia) were used as precursors. H3PO4 solution was added drop wise in aqueous CaO solution at 80

C, while the solution was

magnetically stirred at 200 rpm. During the entire procedure, the pH (8-10) of the solution was maintained by adding few drops of ammonia solution. The solution was kept overnight to form precipitate, which was then filtered and dried at 100

C for 24 h. The dried cake was crushed

using agate mortar and pestle and the powder was calcined at 800

C for 2 h.

2.1.2. Synthesis of HA-ZnO composites For the composite preparation, varying amounts of ZnO (3.0 - 7.5 wt %) have been blended with HA. Initially, the above-mentioned compositions were mixed using mortar and pestle for 30 min. It was then followed by ball milling using acetone and zirconia balls as supporting media (ball to powder ratio kept at 1:4) for 16 h at 600 rpm. Further, the ball milled slurry was dried in air oven at 100

C for 1 day and crushed again to get fine powders. The calcined composite powders

were used to prepare pellets of 10 mm diameter and 1 mm thickness, which were sintered at a temperature of 1250

C for 2 h and at a heating rate of 5

C/min. The sintered and polished

pellets were used for further characterization.

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2.2. Phase evolution X-ray diffraction analysis (XRD, Rigaku Miniflex II Desktop X-ray Diffractometer) was performed using Cu-Kα radiation at the diffraction angle ranging from 30-60º. Fourier transform infrared spectroscopy (FTIR) was performed using FTIR Spectrometer (Bruker Model Tensor 27, Germany) in the wavelength range of 4000-400 cm-1. 2.3. Microstructural analyses Scanning electron microscopy (SEM) study was performed to observe the microstructure of all sintered surfaces. For microstructural analyses, the samples were mirror polished and thermally etched at a temperature of 1050 C for 10 min and Au sputtered for 120 sec. 2.4. Dielectric measurements The dielectric and electrical measurements of pure HA as well as composites were performed using Alpha-A High-Performance Frequency Analyzer, in the temperature and frequency range of 30- 500 ºC and 0.1 Hz-1 MHz, respectively. For dielectric measurements, 10 mm diameter and 1.5 mm thickness pellets were prepared. The samples were mirror polished and electroded using Ag paste, which was then cured at 700

C for 5 min. The AC conductivity of HA and HA-

ZnO composites were calculated with the help of recorded data in dielectric measurement as [31], σ

d = ωC tan δ (1) A

Where, ω is angular frequency (ω = 2πf), tan δ is dielectric loss, C, d and A are capacitance, thickness and area of the samples, respectively.

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2.5. Polarization The samples were corona polarized at the temperature and voltage of 500 ºC and 20 kV, respectively, for 30 min. The samples were then cooled under constant exposure of field (20 kV). 2.6. Antibacterial Assessment 2.6.1. MTT Assay The quantitative assessment of antibacterial response on HA and HA-x ZnO (x = 3, 4.5, 7.5 wt. %) composites in triplicates was performed using MTT (3(4, 5- dimethylthiazol-2-Yl)-2, 5diphenyl tetrazolium bromide) assay. The E. Coli (MTCC#1673) and S. Aureus (MTCC# 435) bacteria were procured in the freeze-dried condition from microbial type culture collection (MTCC), Chandigarh, India. The bacterial cells were cultured in their respective growth media (Nutrient Agar for S. Aureus and Luria broth for E. coli) before seeding on the samples and incubated for 12 h at 37

. The autoclaved samples were washed with 70 % ethanol and rinsed

with phosphate buffer saline (1x PBS), before seeding. The samples were seeded with 200 µl bacterial solution (with 0.1 optical density) per well in 24 tissue culture well plate and incubated for 8 h at 37

. After incubation, samples were washed with 1x PBS and reconstitute MTT

(MTT: PBS in the ratio of 1:10) was added to the cultured samples and further incubated for 2 h. MTT reacts with the viable cells and forms purple coloured formazan crystal [32]. These crystals were dissolved with dimethyl sulfoxide (DMSO) and the absorbance was determined using ELISA micro plate reader (iMark Bio-red) at 595 nm. The absorbance of formazan crystal is directly proportional to the number of viable cells [33]. The viability of bacterial cells was calculated as, [33] % Viability =

Mean absorbance of sample (2) mean absorbance of uncharged HA

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Furthermore, the antibacterial ratio of E. Coli and S. Aureus bacteria was calculated as [34], Antibacterial ratio (%) =

#$%&'() *#$+',-&. #$%&'()

× 100

(3)

The statistical analyses of the absorbance values were performed using SPSS software with oneway analysis of variance (ANOVA) method. Tukey test at significant value, p < 0.05 were performed for comparison of means. 2.6.2. Live/ dead assay Live/ dead assay was performed to understand the simultaneous effect of polarization and ZnO addition on the bacterial stain. Syto 9 and propidium iodide (PI) dyes were used for staining the live and dead cells, respectively. The unpolarized and polarized samples were seeded with 200 µl bacterial solution (0.1 optical density) per well in 24 well tissue culture plate and incubated for 8 h. After stipulated incubation period, the samples were washed twice with 1x PBS and dried. A mixture of both dyes (Syto 9: PI in the ratio of 1:1) were added for 15 minutes in dark. The samples were then observed under fluorescence microscopy (Nikon Eclipse LV 100 ND). 2.6.3. Disc Diffusion Method The antibacterial activity of unpolarized and polarized HA-x ZnO (x = 3, 4.5, and 7.5 wt. %) composites were also investigated using disc diffusion method. This assay was performed according to Kirby-Bauer Disk Diffusion method using agar plate. Both, E. Coli and S. Aureus bacteria were cultured in agar plates. Following this, unpolarized and polarized samples were placed over the cultured agar plate and the inverted plate was incubated for 12 h at 37 ºC. The colony formation on the unpolarized and polarized surface of the samples was observed. The scrub from the area under the samples were transferred with inoculation loop to a new agar plate and further incubated for 12 h. The bacterial colonies were observed after incubation.

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2.6.4. Nitro blue tetrazolium (NBT) assay The NBT assay was performed to quantify the polarization induced superoxide anions (O2-) production [35]. The samples were cultured with E. Coli and S. Aureus bacteria and after required incubation period, 300 µl of NBT solution was added in the samples and incubated further for 1 h. The O2- ions dilute NBT and produce a blue color precipitate (diformazan) which was dissolved in DMSO solution [36]. The absorbance of these dissolved diformazan was taken at 595 nm, which is directly proportional to the produced O2- [36]. 2.7. Atomic absorption spectroscopy (AAS) test The release of Zn2+ ions in culture media were examined using AAS test. The samples were dipped in nutrient broth (culture media) and incubated for 12 h. After incubation, the samples were removed and the culture solution was filtered using 0.22 micron syringe filter. The filtered solution was used for AAS test. The AAS test was performed using AA7000 Shimadzu atomic absorbance spectrometer. 2.8. Cell culture study Human Osteoblast-like SaOS2 cells have been used to assess in-vitro viability of unpolarized and polarized HA and HA-x ZnO (x = 3, 4.5, and 7.5 wt. %) composites. The SaOS2 cell line was procured from National Centre for Cell Sciences (NCCS) Pune, India. The growth media for the cells was McCoy's 5A medium supplemented with 15 % fetal bovine serum (FBS) and 1 % antibiotic. The samples were seeded with the 104 cells/well in 24 well plates and incubated for 3, 5 and 7 days in CO2 incubator. After the respective incubation periods, MTT assay was performed. The samples were washed twice with 1xPBS after incubation and incubated further with reconstitute MTT (MTT: media in a ratio of 1:10) for 6 h to form the purple coloured formazan crystals. The formazan crystals were dissolved in DMSO and the optical density of

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viable cells was measured using ELISA micro plate reader (iMark Bio-red). The viability of SaOS2 cell was calculated as [33], % Viability =

Mean absorbance of sample × 100 (4) mean absorbance of control

To evaluate the effect of polarization on adherence of SaOS2 cells, morphological observation were performed. After the incubation for desired period, the cells were fixed in formaldehyde (3.7%) for 30 min. Triton X-100 was added for permeabilization of cells. DAPI was used to stain the nucleus of adhered cells which were observed under fluorescent microscope (Nikon Eclipse LV 100 ND). 3. Results and Discussion 3.1. Phase evolution Fig. 1 represents the x-ray diffraction patterns for HA and HA-x ZnO (x = 4.5 and 7.5 wt. %) composites. The major diffraction peaks of HA is observed at, 2θ = 31.86º, 32.28º, 32.98º and 34.12º. These values correspond to the single phase hexagonal HA structure (JCPDS # 09-0432). On the other hand, the diffraction peaks corresponding to ZnO appears at 2θ =31.83 , 36.36 , and 56.62 , which is similar to the XRD pattern of wurtzite hexagonal ZnO (JCPDS # 79-2205). In the composite samples, the diffraction peaks, corresponding to only HA and ZnO phases were observed which represents the thermochemical stability of HA-ZnO composite under the optimal processing parameters. Fig. 2. illustrates the FTIR spectra of sintered HA-x ZnO (x = 0, 3, 4.5 and 7.5 wt. %) composites. The characteristic bands, corresponding to PO43- in HA appears at 1089, 1023, 962, 604, and 560 cm-1[37]. The adsorption bands, corresponding to CO32- appears at 1415 and 1450 cm-1[38]. The band at 874 cm-1corresponds to HPO42- [39]. The stretching and bending modes of vibration correspond to hydroxyl group (OH-) observed at 3572 and 630 cm-1, respectively [40]. 9

3.2. Microstructural analysis Fig. 3 represents the SEM images of fractured HA and HA-x ZnO (x = 3, 4.5, 7.5 wt. %) composite surfaces. The brittle mode of fracture has been observed in HA and composite samples. The average grain size of HA and HA-x ZnO (x = 3, 4.5, and 7.5 wt. %) composites were calculated to be 0.89, 0.96, 0.99 and 1.28 µm, respectively. The energy-dispersive X-ray (EDX) analyses confirm the presence of ZnO in the composite samples. The fractured surfaces reveal good densification of the sample at the optimal processing parameter. 3.3. Dielectric behaviour The dielectric response of HA and HA-x ZnO (x = 3, 4.5, 7.5 wt. %) composites in the temperature range of 35 to 500 ºC at a few frequencies are represented in Fig. 4. The dielectric constant of HA [Fig. 4(a)] is observed to be poorly dependent on temperature up to 200 ºC. However, it increases with temperature in the high temperature region (>200 ºC). The dielectric loss curve shows almost similar behavior. In case of HA-3 ZnO composite, diffused maxima at about 100 ºC and another in the higher temperature (350- 400

C) region are observed [Fig.

4(b)]. For HA-x ZnO (x = 4.5 and 7.5 wt. %) composites, almost temperature independent dielectric constant [Fig. 4(c and d)] has been observed up to ~ 250 C, which is followed by peak with further increase with temperature. The peak at about 400

C, represents the dynamic

stabilization of hexagonal phase due to reorientation of OH- ions [41].The dielectric loss of HA-x ZnO (x = 4.5 and 7.5 wt. %) composites shows the similar behavior to that of dielectric constant. At the lower temperature (< 100

C) the dielectric constant of HA depends upon the processing

based structural defects such as, OH- and O2- ions, and vacancies [42]. However, at higher temperature (> 300

C), migration of OH- ions are responsible for conduction [43]. The phase

transformation from monoclinic to hexagonal phase in HA occurs at 210

C, because of the

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reorientation of OH- ions. Above 300

C, formation of thermal defects in HA [Eq. (5)] are

responsible for the observed dielectric response [44].

Ca2 (HPO5 )(PO5 )6 (OH) = 3Ca8 (PO5 )9

+H9 O (5)

Oxygen in form of O2- and O- ions are adsorbed on the surface of ZnO at lower temperature. [45] However, with increase in the temperature, the rate of release of adsorbed oxygen increases. [46] Consequently, a large number of oxygen vacancies are formed. These oxygen vacancies are accumulated at grain boundary which increases the polarization at higher temperature [46]. The dielectric constant and loss values at room temperature for HA, HA-3 ZnO, HA- 4.5 ZnO and HA-7.5 ZnO composites are (17.9, 0.1137), (16.3, 0.531), (4.6, 0.0377) and (17, 0.105) respectively, measured at a frequency of 10 kHz. The dielectric constant of ZnO has been reported to be 8.5 at room temperature and frequency of 10 kHz. The dielectric constant of natural human bone has been suggested to be around 10 [47]. The experimental values of dielectric constant have been compared with the existing theoretical models. The dielectric constant values for HA-3 ZnO, HA-4.5 ZnO and HA- 7.5 ZnO composites were calculated using Wiener parallel and series bounds [Eqs.(6) and (7)] as well as using logarithmic mixture rule [Eq. (8)] as [48],

ε

<=>
1 ε

<=>
= VCD εCD + VEF# εEF# (6)

=

VCD VEF# + (7) εCD εEF#

log εIJ,-JKLM. = VCD log εCD + VEF# log εEF# (8)

Where, ε and V is the dielectric constant and volume fraction, respectively. 11

It is clearly observed from Fig. 5 that the experimental values of dielectric constant for HAxZnO composites are lower than those, calculated using the Eqs. (6), (7) and (8) at room temperature and frequency of 10 kHz. The connectivity between HA and secondary phase (ZnO) can be one of the reasons for such deviation in dielectric constant values. The small amount of ZnO in HA results in 0-3 connectivity between ZnO and HA phases in HA-ZnO composite. The Landaure’s expression for 0-3 connectivity for binary system is given as [49],

F = VP

εP − ε ε9 −ε + V9 (9) εP + 2ε ε9 +2ε

Where, V1 and V2 are the volume fractions of constituent phases, ε1, ε2 and εc are the dielectric constant of ZnO, HA and composite, respectively. It is suggested that F should be zero for perfect 0-3 connectivity. The values of F for HA-3 ZnO, HA-4.5 ZnO and HA-7.5 ZnO composites are calculated [Eq. 9] to be 0.027, 0.49, and 0.078, respectively. The effective dielectric constant of polyphase materials can also be calculated by Maxwell- Garnet equations [50].

εCD VCD [ εBSS =

9 8 9

+

εT(U 8εVW εT(U

VCD [ 8 + 8ε

VW

] + εEF# VEF# ] + VEF#

(10)

The effective dielectric constant for HA- 3 ZnO, HA- 4.5 ZnO and HA- 7.5 ZnO composites were calculated using the above expression at the frequency of 10 kHz and room temperature are 17.1, 17.6 and 19.9, respectively. The measured value of dielectric constant for HA-x ZnO (x =3, 4.5 and 7.5 wt. %), composites are deviated about 4.6 %, 72 % and 1.45 %, respectively, with those of the calculated [Eq. 10] values.

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3.4. AC conductivity behavior Fig. 6 represents the variation of ac conductivity of HA and HA- x ZnO (x = 4.5 and 7.5 wt. %) composites, with inverse of temperature at frequencies of 10 kHz, 100 kHz, and 1 MHz. A diffused maxima in the temperature range of 100- 250

C is observed for HA. However, for

HA-3 ZnO and HA-7.5 ZnO composites, the maxima is observed in the temperature range of 60200

C and 60-120

about 400

C, respectively. The sharp peaks [Fig. 6 (b, c), @ 10 kHz], observed at

C due to dielectric relaxation in the samples [51]. The diffused maxima shift towards

higher temperature with increase in the frequency. The ac conductivity values for HA, HA-3 ZnO, HA-4.5 ZnO and HA-7.5 ZnO composites at room temperature and frequency of 10 kHz are 1.29 × 10-8, 5.65 × 10-9, 6.82 × 10-8 and 4.29×10-9 (ohm cm)-1, respectively. For natural bone, ac conductivity has been reported to be in the range of 10-10 to 10-9(ohm cm)-1[52]. It has been suggested that the conduction of HA at lower temperature (< 100

C), is due to the migration of

protons (H +) in adsorbed water [53]. However, at elevated temperature (> 300

C), dehydration

of OH- ions are responsible for conduction [54]. The increase in conductivity at higher temperature is associated with the hopping of H+ at O2- sites [Eq. (11)] [55]. In HA, two proton conduction mechanisms have been suggested. Firstly, protons conduct via OH- sites along c-axis (Eq. 12). Another mechanism involves conduction of protons via PO43- tetrahedra [56].The second mechanism is feasible due to comparatively shorter distance (0.307 nm) between the PO43- tetrahedra and OH- ions (Eq. 12) with that of adjacent OH- ion (0.344 nm) sites [57]. CaPX (PO5 )Y (OH)9 = CaPX (PO5 )Y OZ + H9 O (11) OH * + OH * → O9* + HOH (12) * 9* 2OH * + [PO8* + OH → O9* + [PO5 ]8* + HOH +Z 5 ] → O9 + [HPO5 ]

Where,

#C* (13)

represents the vacancy. 13

3.5. Impedance analysis Fig. 7 represents the complex plane impedance response for HA and HA-3 ZnO composite. The impedance analyses were performed to understand the behavior of grain and grain boundary in the entire spectrum as a function of resistance and capacitance [58]. It is clearly observed from Fig. 7 (a, b) that the centers of semicircular arcs appear below the x-axis which shows the multiple relaxation processes i.e., non-Debye type relaxation in the samples [59].The intercepts of semicircles on the real axis give information about the grain and grain boundary resistances [60]. In case of non-Debye type relaxation behavior, the constant phase element (CPE) explains the variation from Debye type relaxation process [61]. The value of CPE were calculated using the expression, C = (RP*F C_ )P/F, where, R and C_ are the resistance and capacitance, respectively, n > 0 for non-ideal case [62]. The resistance and CPE are represented as parallel combination and each combination has a time constant to describe the relaxation behavior. The maxima in each semicircular arc are used to obtain the relaxation frequency of the circuit. The relaxation frequencies of grains and grain boundaries for HA have been calculated to be (8, 0.3 kHz), (1.9, 0.3 kHz), (22, 0.4 kHz), (52, 0.5 kHz) and (140, 0.4 kHz) at temperatures of 400,425, 450,475 and 500

C, respectively. Similarly, for HA-3 ZnO composite, the relaxation

frequencies of grains and grain boundaries have been calculated to be (41, 0.32 kHz), (18, 0.3 kHz), (20, 0.18 kHz), (8.1, 0.23 kHz) and (60, 0.84 kHz), respectively at similar temperature. Fig.7 (c, d) represents the plot of grain and grain boundary resistances (log Rg and log Rgb) with inverse of temperature for HA and HA-3 ZnO composite, respectively. The activation energies of both samples were calculated with the help of linear fitting of [log Rg /log Rgb Vs 1000/T] plot. For HA and HA-3 ZnO composite, the activation energies of grains and grain boundaries are (1.36, 1.44 eV), and (1.18, 1.98eV), respectively. Yamashita et al. [63] reported the

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activation energies of HA to be 1.86 eV [64]. The activation energy for H+ and O22- conduction has been reported to be 0.5 and 1.5 eV, respectively [64]. 3.6. Antibacterial Response of HA-x ZnO (x= 3, 4.5, 7.5 wt. %) Composites 3.6.1. MTT assay The viability of E. Coli and S. Aureus bacteria on unpolarized and polarized HA and HA-xZnO (x = 4.5 and 7.5 wt. %) composites are shown in Figs. (8) and (9), respectively. It is observed that the viability of E. Coli and S. Aureus bacteria decreases significantly with addition of ZnO in HA-xZnO (x= 3, 4.5, 7.5 wt. %) composites. As for as the combined effect of ZnO addition and polarization is concerned, more pronounced reduction in viability of both, E. Coli and S. Aureus has been observed on negatively and positively charged HA- 7.5 ZnO composites, respectively. These results suggest that the antibacterial response of the charged surface depends upon the charge polarity and bacteria. The population of E. Coli and S. Aureus bacteria are reduced by (9.62, 14, and 37.5%) and (8.69, 14.19, and 24.69 %) with the addition of ZnO in HA-x ZnO (x = 3, 4.5 and 7.5 wt. %) composite, respectively as compared to HA. After polarizing the samples, viability of E. Coli and S. Aureus bacteria decreased by 23, 10, and 15.4, 19 % respectively, on negatively and positively charged HA as compared to uncharged HA. The viability of E. Coli bacteria on negatively and positively charged HA-x ZnO (x = 3, 4.5 and 7.5 wt. %) composites are reduced by (31.5, 43.7, 53%) and (20, 23, 41.3 %) respectively, as compared to uncharged HA. However, for S. Aureus bacteria, negatively and positively charged surfaces of similar composition demonstrate (26, 29.6, 39.7 %), and (36, 41, 52%) reduction in viability, respectively with respect to uncharged HA. Overall, it has been observed that the combined action of ZnO addition and polarization significantly reduces the viability of E. Coli and S. Aureus bacteria.

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Fig. 10 (a and b) represents the antibacterial ratios of E. Coli and S. Aureus bacteria on unpolarized and polarized HA-x ZnO composites (x = 3, 4.5, 7.5 wt. %), respectively. The statistical analyses revealed that the antibacterial ratios of E. Coli and S. Aureus bacteria HA-x ZnO composites (x= 3, 4.5, 7.5 wt. %), increases with incorporation of ZnO in HA matrix. In addition, all polarized samples demonstrate significant enhancement in antibacterial ratios of both the bacteria as compared to unpolarized HA [represented as (*) in Fig.10]. However, with respect to polarized HA, the polarized composite as well as unpolarized HA-7.5 ZnO composite demonstrated significant enhancement in antibacterial ratio of E. Coli and S. Aureus bacteria [represented as (**) and (***) in Fig. 10, respectively]. Overall, it has been observed that the addition of ZnO in HA matrix as well as polarization significantly increases the antibacterial ratios of both the bacteria. 3.6.2. Live-dead assay The fluorescent microscopy images for both, E. Coli and S. Aureus bacteria, adhered on uncharged, positively and negatively charged surfaces of HA, HA-x ZnO (x = 3, 4.5 and 7.5 wt. %) composites are shown in Figs. 11 and 12, respectively. The population of live bacteria decreases with increasing the ZnO content in composite for both, E. Coli and S. Aureus bacteria. The density of E. Coli bacteria on negatively charged surface is observed to be lower than that of positively and uncharged surfaces. However, for both the bacteria, positively charged surface reflects more dead bacteria as compared to uncharged and negatively charged surfaces (Figs.11 and 12). It is observed that positively charged HA-7.5 ZnO composite have more dead bacterial cells for both, E. Coli and S. Aureus bacteria as compared to other samples. Overall, the combined action of ZnO addition in HA-x ZnO (x= 3, 4.5, 7.5 wt. %) composites and polarization increases the number of dead bacterial cells for both, E. Coli and S. Aureus bacteria.

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It has been reported that the chemical composition and structure of outer cell wall of both, gram negative and gram-positive bacteria are different [65]. The cell wall of gram-negative bacteria has an outer layer of lipopolysaccharides [66]. However, a thick peptidoglycan layer is present in gram positive bacteria. Gram negative bacteria have more negative charge as compared to gram positive bacteria due to the presence of lipopolysaccharides layer [67]. The zeta potential for E. Coli and S. Aureus bacteria has been reported to be -49 and -31.7 mV, respectively [68]. The adhesion of E. Coli bacteria on negatively charged surfaces reduces due to electrostatic repulsion [69]. In another study, it has been reported that ZnO dissolved in culture media and produces reactive oxygen species (ROS) such as OH-, H2O2, O2- and Zn2+ ions [70]. ROS reacts with lipid layer of the cell wall and destroys the cell structure which leads to the death of bacterial cells [71]. Zn2+ions diffuse into the cell wall and damage the outer peptidoglycan layer [72]. It has been suggested that the polarization increases the hydrophilicity of surfaces, irrespective of charge polarity [73]. Such hydrophilic surfaces reduce the adhesion of bacterial cells [74]. Tan et al.[75] reported that positively charged surface promotes the ROS generation through the electrolysis reactions (Eq. 14-17) as. 2H9 O → O9 + 4H a + 2e* (14) 2H9 O → OH * + H a (15) * O9 + H9 O + 2e* → HO* 9 + OH (16)

* * HO* 9 + H9 O + 2e → H9 O9 + OH (17)

It has been demonstrated that the generation of ROS increases the permeability of cell which can penetrate the cell wall and disrupts the bacterial cell membranes [76]. Fig.13 schematically

17

represents the mechanism for antibacterial response on polarized HA and HA-x ZnO (x = 4.5 and 7.5 wt. %) composites, as discussed above. 3.6.3. Disc diffusion method Figs. 14 and 15 represent the result of Kirby-Bauer test for antibacterial activity of both, E. Coli and S. Aureus bacteria on unpolarized and polarized HA and HA- x ZnO (x= 0, 3, 4.5, and 7.5 wt. %) composites, respectively. It is clearly observed that the colony formation on agar plates for both bacteria decreases with increasing the ZnO content in HA matrix. In addition, the polarization of samples further reduces the bacterial growth for both the bacteria. The zone of inhibition observed under the samples were not significantly differentiable [Figs 14 (e-h) and 15 (e-h)] and therefore, the swab under the samples was further used for striking using sterilized inoculation loop in a new agar plate to visualize the bacterial growth [Figs. 14 (i-l) and 15 (i-l)]. It is clearly observed that the bacterial growth on agar plates decreases with increase the ZnO content in HA matrix for both E. Coli and S. Aureus bacteria, which decreases further after polarization treatment. It is evident that the swab under negatively charged samples has lower growth as compared to positively charged and uncharged samples for E. Coli bacteria. However, for S. Aureus bacteria, positively charged surface demonstrates lower bacterial growth as compared to uncharged and negatively charged samples. Overall, this result demonstrates that the growth of both the bacteria decreases remarkably with ZnO addition in HA and subsequent, polarization. 3.6.4. NBT assay Figs. 16 (a) and (b) represent the production of O2- ions in E. Coli and S. Aureus bacteria, cultured on unpolarized and polarized surfaces of HA and HA-x ZnO (x = 3, 4.5 and 7.5 wt. %) composites, respectively, after specific incubation period. The statistical analyses reveal that the

18

production of O2- ions on positively charged surface is significantly higher than negatively charged and uncharged surfaces. It has been reported that polarized piezoelectric potassium sodium niobate (KNN) surface produces micro electric field on its surfaces which generates ROS and kill the bacteria [79]. Overall, the NBT assay suggests that positively charged surface produces more O2- ions for both, E. Coli and S. Aureus bacteria as compared to negatively charged and uncharged surfaces. 3.7. Atomic absorption spectroscopy (AAS) test Fig. 17 represents the concentration of Zn2+ ion, released from HA-x ZnO (x = 3, 4.5, and 7.5 wt. %) composite, after incubation of 12 h in culture media. It is clearly observed the Zn2+ concentration increases with increasing the ZnO content in the composite. The statistical analyses reveal the significant enhancement in the concentration of Zn2+ ions for HA- (4.5-7.5) wt. % ZnO as compared to HA-3 wt. % ZnO composite [represented as (*) in Fig.17]. It is observed that HA-7.5 wt. % ZnO composite released more Zn2+ ions as compared to both HA - x ZnO (x = 3, 4.5 wt.%) composites after incubation of 12 h at 37 ºC. 3.8. Cellular response The viability of osteoblast-like SaOS2 cells on unpolarized and polarized HA and HA-x ZnO (x = 3, 4.5 and 7.5 wt. %) composites are demonstrated in Fig.18. The statistical analyses reveal that the viability of SaOS2 cells significantly increases with addition of ZnO in HA-x ZnO (x = 3, 4.5, and 7.5 wt. %) composites. The viability of SaOS2 cells significantly increases on all unpolarized and polarized samples after 3, 5 and 7 days of incubation as compared to uncharged HA [represented as (*) in Fig 18]. As far as the influence of polarization is concerned, negatively charged surface demonstrate significant enhancement in viability of SaOS2 cells with respect to uncharged and positively charged surfaces for the same sample after 3, 5 and 7 days of

19

incubation[represented as (**) and (***) in Fig 18]. It has been observed that the viability of SaOS2 cells on HA-x ZnO (x = 3, 4.5 and 7.5 wt. %) composites are increased by (32, 25, 12 %), (23, 61, 41 %), and (11, 19, 28 %) with respect to HA after incubation of 3,5 and 7 days, respectively. In contrast, the viability of SaOS2 cells on negatively and positively charged HA increased by (31, 18, 75 %), (9, 7, 46 %) after incubation of 3, 5 and 7 days, respectively. As for as the combined effect of ZnO addition and polarization is concerned, the negatively charged HA-x ZnO (x = 3, 4.5 and 7.5 wt. %) composite shows (47.2, 68, 77 %), (44.7,74,70%) and (70.3, 64, 62.5 %) enhancement in viability of SaOS2 cells as compared to uncharged HA after 3, 5, and 7 days of incubation, respectively. However, positively charged surfaces of similar composition demonstrate (41.4, 32, 34 %), (33, 67, 65 %) and (41, 28, 51 %) enhancement in viability of SaOS2 cells after similar incubation conditions. Overall, the negatively charged HAx ZnO composite demonstrate the pronounced enhancement in the viability of SaOS2 cells as compared to uncharged and positively charged surfaces. Fig. 19 represents the fluorescent microscopy images of stained nucleus of adhered SaOS2 cells on unpolarized and polarized HA and HA - x ZnO (x = 3, 4.5, and 7.5 wt. %) composites, after incubation of 3 days. The cell density of SaOS2 cells increases with increasing the ZnO content in HA-x ZnO (x = 3, 4.5, and 7.5 wt. %) composites. In addition, the negatively charged surface evident more cell density as compared to positively charged and uncharged surfaces of each composition. Overall, the combined effect of ZnO addition and polarization can be suggested to increase the proliferation of SaOS2 cells. It has been reported that the proliferation of MC3T3-E1 osteoblast-like cells enhances on negatively charged HA as compared to uncharged HA after 7 days of incubation [77,78]. Bodhak et al. [79] demonstrated that the proliferation of human osteoblast cells (hFOB) on negatively

20

charged HA is almost doubled as compared to positively charged HA after incubation of 11 days. The adhesion of cell with substrate depends upon the interaction between media proteins and nature of the substrate surface [80]. Selective adsorption of cations such as, Ca+, Na+, Mg+ and K+ on negatively charged surfaces attract with proteins (Fig. 20) and promotes the formation of bone like apatite layer which increase the cell proliferation [81,82]. In contrast, positively charged surfaces, interact with anionic groups such as HPO42-and HCO32- which act as antiadhesive molecules [83]. These anions do not promote the formation of apatite layer [84]. 4. Conclusion The present study reveals that the combined effect of polarization as well as optimal ZnO (up 7.5 wt. % ZnO) addition in HA can induce the antibacterial response without affecting the cellular response. In the present work, HA and HA-x ZnO (x = 3, 4.5 and 7.5 wt. %) composites were successfully synthesized by co-precipitation and solid state mixing route, respectively. Phase analyses reveal the presence of single phase HA and ZnO in the composite. The ac conductivity and dielectric constant of developed composites are measured to be almost similar to those of the natural bone. The addition of ZnO in HA matrix significantly reduces the viability of E. Coli and S. Aureus bacteria. The viability of E. Coli bacteria on negatively charged HA-7.5 wt. % ZnO composite reduces by approximately 53 %. However, positively charged HA-7.5 wt. % ZnO composite shows 52 % reduction in viability of S. Aureus bacteria. The viability of human osteoblast-like SaOS2 cells on negatively charged HA and HA-7.5 ZnO composites increases by approximately 31.4 and 70.5 %, respectively, with respect to uncharged HA, after incubation of 7 days. 5. Acknowledgements The financial support from SERB, DST, Govt. of India is gratefully acknowledged.

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[78] D. Kumar, J.P. Gittings, I.G. Turner, C.R. Bowen, A. Bastida-Hidalgo, S.H. Cartmell, Polarization of hydroxyapatite: Influence on osteoblast cell proliferation, Acta Biomater, 6 (2010) 1549–1554. 10.1016/j.actbio.2009.11.008 [79] S. Bodhak, S. Bose, A. Bandyopadhyay, Role of surface charge and wettability on early stage mineralization and bone cell–materials interactions of polarized hydroxyapatite. Acta Biomater, 5 (2009) 2178–2188. 10.1016/j.actbio.2009.02.023 [80] F. Grinnell, and M.K/ Feld, Fibronectin adsorption on hydrophilic and hydrophobic surfaces detected by antibody binding and analyzed during cell adhesion in serum-containing medium, J. Biol. Chem. 257 (1981), 4888-1982. [81] E.G. Hayman, M.D. Pierschbacher, S. Suzuki, and E. Ruoslahti, Vitronectin a major cell attachment-promoting protein in fetal bovine serum. Exp. Cell Res. 160 (1985) 245-258. https://doi.org/10.1016/0014-4827(85)90173-9 [82] W. Chen, Yu. Zunxiong, J. Pang, Yu. Peng, T. Guoxin, and C. Ning, Fabrication of Biocompatible Potassium Sodium Niobate Piezoelectric Ceramic as an Electroactive Implant, Materials, 10, (2017) 345. https://doi:10.3390/ma10040345 [83] C.J. Wilson, R.E. Clegg, D.I. Leavesley, M.J. Pearcy, Mediation of biomaterial-cell interactions by adsorbed proteins: A review. Tissue Eng. 11 (2005) 1–18. DOI: 10.1089/ten.2005.11.1 [84] C. Yoshida-Noro, N. Suzuki, M. Takeichi, Molecular nature of the calcium-dependent cellcell adhesion system in mouse teratocarcinoma and embryonic cells studied with a monoclonal antibody. Dev. Biol. 101 (1984) 19–27.

32

*#

#

** #

*

Intensity (au)

HA (09-0432) ZnO (79-2205)

*

*

*

*

*

*

* *

#

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*

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(a)

30

35

40 45 50 2θ θ (degree))

55

60

Fig.1. X ray diffraction patterns for (a) HA, (b) HA-3 wt. % ZnO, (c) HA-4.5 wt. % ZnO and (d) HA-7.5 wt. % ZnO composite samples.

Transmittance

* - OH (d) *

(c)

*

(b)

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3000

2000

°

• ° • •

4000

°

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1000 500

Wave Number (cm-1) Fig.2. Fourier transform infra-red (FTIR) spectra for (a) HA, (b) HA-3 wt. % ZnO, (c) HA4.5 wt. % ZnO and (d) HA-7.5 wt. % ZnO composite samples.

1

Fig.3. SEM images of fractured (a) HA, (b) HA-3 wt. % ZnO, (c) HA-4.5 wt. % ZnO and (d) HA-7.5 wt. % ZnO composite surfaces.

2

150 (a) εr

100 50

1 MHz 100 kHz 10 kHz

60 εr

(b)

1 MHz 100 kHz 10 kHz

40 20 1.0

1

d

d

0.5

0 0.0

0

14 (c) 12 εr 10 8 6

100 200 300 400 500 Temperature (°C)

1 MHz 100 kHz 10 kHz

0

1.0

d 0.5

d 0.5

0.0

0.0 100 200 300 400 500 Temperature (°C)

200 300 400 Temperature (°C)

400 (d) 300 εr 200 100

1.0

0

100

0

500

1 MHz 100 kHz 10 kHz

100

200 300 400 Temperature (°C)

500

Fig. 4. Variation of dielectric constant and loss with temperature for (a) HA, (b) HA-3 wt. % ZnO, (c) HA- 4.5 wt. % ZnO and (d) HA- 7.5 wt. % ZnO at few selected frequencies.

3

26

22 (a)

Parallel

Series

Series

24

ε r @ 100 kHz

ε r @ 10 kHz

Present study

22 20 18 0

1

2 3 4 5 6 7 ZnO wt. % in HA

22

log mixture rule

20

log mixture rule

16

Parallel

(b)

present study

18 16 14

8

0

1

2 3 4 5 6 7 ZnO wt. % in HA

8

Parallel

(c)

Series

ε r @ 1 MHz

20

log mixture rule Present study

18 16 14

0

1

2 3 4 5 6 7 ZnO wt. % in HA

8

Fig. 5. Comparison of experimental values for HA-xZnO (x= 3,4.5 and 7.5 wt.%) with those of the values, obtained using theoretical models (Eqs. 6-8) at frequencies of (a) 10 kHz, (b) 100 kHz and (c) 1 MHz.

4

-6.0

-7.0

-7.0

-7.5

-7.5

-8.0

-8.0 -8.5 1.0

logσ σ ac(ohm cm)-1

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-6.5

-6.5

-6

(b)

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1.5

2.0 2.5 1000/T (K)-1

(c)

3.0

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1 MHz 100 kHz 10 kHz

-7 -8 -9

-10 -11 1.0

1.5

2.0

2.5 1000/T(K)-1

3.0

-8.5 1.0

3.5

logσ σ ac(ohm cm)-1

logσac(ohm cm)-1

-5.5

-5.5

logσ σ ac(ohm cm)-1

1 MHz 100 kHz 10 kHz

-5.0 (a)

3.5

1.5

2.0

2.5

1000/T(K)-1

(d)

3.0

3.5

1 MHz 100 kHz 10 kHz

-6.5 -7.0 -7.5 -8.0 -8.5 -9.0 1.0

1.5

2.0 2.5 3.0 1000/T (K)-1

3.5

Fig.6. Variation of ac conductivity with inverse of temperature for compositions (a) HA, (b) HA-3 wt. % ZnO, (c) HA-4.5 wt. % ZnO and (d) HA-7.5 wt. % ZnO.

5

20

400 °C 425 °C 450 °C 475 °C 500 °C

(b)

Z"(10 6 Ω )

15 10 5 0

(c)

7

8 Rg

R gb

Egb= 1.44 eV

6 5 4

Eg = 1.36 eV

3 2

1.30

1.35 1.40 1.45 1000/T (K)-1

1.50

log Rg, log Rgb(Ω Ω)

log R , log R (Ω Ω) g gb

8

0

5

(d)

7

10 15 Z'(106 Ω)

Rg Rgb

20

Egb= 1.98 eV

6 5 Eg= 1.18 eV

4 3

1.30

1.35 1.40 1.45 1000/T (K)-1

1.50

Fig.7. Complex plane impedance plots for compositions (a) HA and (b) HA-3 wt. % ZnO and (c) and (d) represent the variations of resistances of grain and grain boundaries with the inverse of temperature.

6

Fig.8. The viability of E. Coli bacteria on unpolarized and polarized HA- xZnO (x = 3, 4.5, 7.5 wt. %) composites. Asterisk (*) mark represents the significant difference among all samples w.r.t uncharged HA at p < 0.05. Asterisk marks (**) and (***) represents the significant difference among all samples w.r.t negatively charged and positively charged HA, respectively, at p < 0.05.

Fig.9. The viability of S. Aureus bacteria on unpolarized and polarized HA- xZnO (x = 3, 4.5, 7.5 wt. %) composites. Asterisk (*) mark represents the significant difference among all samples w.r.t HA at p< 0.05. Asterisk marks (**) and (***) represents the significant 7

difference among all samples w.r.t negatively charged and positively charged HA, respectively, at p < 0.05.

Fig.10. The antibacterial ratio for (a) E. Coli bacteria and (b) S. Aureus bacteria on unpolarized and polarized HA-xZnO (x = 3, 4.5, 7.5 wt. %) composites. Asterisk (*) mark represents the significant difference among all the samples w.r.t HA at p< 0.05. Asterisk marks (**) and (***) represent the significant difference among all the samples w.r.t negatively charged and positively charged HA, respectively, at p < 0.05.

8

Fig.11. Fluorescent microscopy images of live and dead E.Coli bacteria on uncharged, positively and negatively charged HA and HA-xZnO (x = 3, 4.5, 7.5 wt. %) composites. Live bacteria (green) are stained with syto 9 dye and dead bacteria (red) are stained with propidium iodide. Scale bar corresponds to 100 µm.

9

Fig.12. Fluorescent microscopy images of live and dead S. Aureus bacteria on uncharged,

positively and negatively charged HA and HA-xZnO (x = 3, 4.5, 7.5 wt. %) composites. Live bacteria (green) are stained with syto 9 dye and dead bacteria (red) are stained with propidium iodide. Scale bar corresponds to 100 µm.

10

Fig.13. Schematic diagram, illustrating the proposed mechanism for antibacterial response of gram positive and gram negative bacteria on polarized HA-x ZnO (x = 3, 4.5, 7.5 wt. %) composites.

11

Fig.14. The disc diffusion test (Kirby Bauer test) performed for unpolarized (U), positively (P) / negatively polarized (N) HA-x ZnO (x = 3, 4.5, 7.5 wt. %) composites on agar culture plate, using E. Coli bacteria, (a-d) represent the cultured agar plates after incubation of 24 h, (e-h) area under the samples and (i-l) represent the colony formation, after transferring swab from the area under the samples on agar plates.

Fig.15. The disc diffusion test (Kirby Bauer test) performed for unpolarized (U) positively (P) / negatively polarized (N) HA-x ZnO (x = 3, 4.5, 7.5 wt. %) composites on agar culture plate, using S. Aureus bacteria, (a-d) represents the cultured agar plates after incubation of 24 h, (e-h) area under the samples and (i-l) represent the colony formation, after transferring swab from the area under the samples on agar plates.

12

Fig.16. The measurement of superoxide production in (a) E. Coli and (b) S. Aureus bacteria on unpolarized and polarized HA-x ZnO composites. Asterisk (*) mark represents the significant difference among all samples w.r.t uncharged HA at p< 0.05. Asterisk marks (**) and (***) represents the significant difference among all samples w.r.t negatively charged and positively charged HA, respectively, at p < 0.05.

Fig.17. The measurement of Zn2+ ion released from HA-x ZnO (x = 3, 4.5, and 7.5 wt. %) composites in nutrient broth after incubation of 12 h. Asterisk marks (*) the significant difference among all samples w.r.t HA -3 ZnO, at p < 0.05. 13

Fig.18. The percentage cell viability of SaOS2 cells on unpolarized and polarized HA- xZnO

(x = 3, 4.5 and 7.5 wt. %) composites after (a) 3, (b) 5 and (c) 7 days of incubation, respectively. Asterisk (*) mark represents the significant difference among all the samples w.r.t uncharged HA at p < 0.05. Asterisk (**) mark represents the significant difference between polarized surfaces w.r.t unpolarized surfaces for the same sample at p < 0.05 and Asterisk (***) mark represents the significant difference between negatively charged surfaces w.r.t positively charged surfaces for the same sample at p < 0.05.

14

Fig.19. Fluorescent microscopy images of SaOS2 cells (stained nucleus), adhered on uncharged, positively and negatively charged HA and HA-xZnO (x = 3, 4.5 and 7.5 wt. %) composites. Scale bar corresponds to 100 µm.

15

Fig.20. Schematic diagram illustrating the proliferation of SaOS2 cells on HA-xZnO (x = 3, 4.5 and 7.5 wt. %) composite (a) on negatively charged surface, cations interact with proteins and promote cell proliferation, (b) on positively charged surface, adhesion of anionic groups reduces cell proliferation, and (c) on unpolarized surfaces, cations and anions floats and normal cell adhesion take place. [(Idea adopted from the ref. (77)]

16

Highlights 1. Combined effect of optimal ZnO (< 7.5 wt. %) addition and polarization treatment enhances the antibacterial as well as cellular response. 2. The reduction in viability of bacteria is charge specific. 3. The negatively charged surfaces enhance the proliferation of Saos2 cells.

Dr. Ashutosh Kumar Dubey Assistant Professor and Ramanujan Fellow Department of Ceramic Engineering Indian Institute of Technology (BHU) Varanasi-221005 (U.P.), India June 24, 2019 Conflict of Interest

This is to confirm that there is no conflict of interest to declare. 'Declarations of interest: none'

Also, this is to confirm that the submitted paper is original and has not been or is not being submitted to the peer review process in any other journal.

Ashutosh Kumar Dubey, PhD (Corresponding Author)

Department of Ceramic Engineering, Indian Institute of Technology (BHU), Varanasi-221005 Email: [email protected], Mobile: +91-8726823415