Synthesis, characterization and cytocompatibility assessment of hydroxyapatite-polypyrrole composite coating synthesized through pulsed reverse electrochemical deposition

Accepted Manuscript Synthesis, characterization and cytocompatibility assessment of hydroxyapatite-polypyrrole composite coating synthesized through pulsed reverse electrochemical deposition

Rajib Chakraborty, Venkata S. Seesala, Jhimli Sarkar Manna, Partha Saha, Santanu Dhara PII: DOI: Reference:

S0928-4931(18)30481-8 doi:10.1016/j.msec.2018.10.001 MSC 8927

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

13 February 2018 13 September 2018 1 October 2018

Please cite this article as: Rajib Chakraborty, Venkata S. Seesala, Jhimli Sarkar Manna, Partha Saha, Santanu Dhara , Synthesis, characterization and cytocompatibility assessment of hydroxyapatite-polypyrrole composite coating synthesized through pulsed reverse electrochemical deposition. Msc (2018), doi:10.1016/j.msec.2018.10.001

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ACCEPTED MANUSCRIPT Synthesis, characterization and cytocompatibility assessment of hydroxyapatite-polypyrrole composite coating synthesized through pulsed reverse electrochemical deposition Rajib Chakrabortya*, Venkata S. Seesalab, Jhimli Sarkar Mannac , Partha Sahaa , Santanu Dharab

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a Department of Mechanical Engineering, Indian Institute of Technology Kharagpur, Kharagpur 721302, India b School of Medical Science and Technology, Indian Institute of Technology Kharagpur, Kharagpur 721302, India c Department of Metallurgical and Materials Engineering, Indian Institute of Technology Kharagpur, Kharagpur 721302, India

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Abstract:

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Composite coating of hydroxyapatite-polypyrrole is synthesized with the help of pulsed reverse electrochemical deposition method from aqueous bath through in-situ formation and co-deposition of both phases simultaneously over metallic stainless steel surface. The inter phase bonding along with surface energy variation and morphology is tuned with the help of deposition current density, deposition time and reverse duty cycle. Hydroxyapatite (HA) lattice exhibits unidirectional growth along the highest atomic plane of <111> parallel to the coating surface. Different kind of deposited hydroxyapatite structures, namely lamellar and spherical particle scaffold, are observed at moderate and high current densities respectively together with the incorporation of polypyrrole (PPy) phase in between. Pyrrole ring stretching and bond strengthening represent the bonding with hydroxyapatite lattice, which in turn helps to increase the overall corrosion resistance of composite coating by ten fold as compared to bare PPY coating. The coating deposited with moderate current density (10 mA/cm2) seems to be the optimum one regarding the faster-interconnected growth of MG63 cells over the coating surface along with highest corrosion resistance and anodic passivation capability. Presence of sub-micron level ceramic hydroxyapatite scaffold along with polymer filler material makes this composite biocompatible coating as a potential candidate to use over the load bearing metallic implant surfaces due to its sufficient elasticity along with superior toughness.

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Keyword: Polypyrrole; hydroxyapatite; composite coating; pulsed reverse electrochemicaldeposition; biocompatible coating; load bearing implant 1. Introduction:

On account of high mechanical strength, corrosion resistance and excellent process ability, metallic implants are the most preferred materials for orthopedic and dental implantation applications. Although the significant drawbacks with the metallic implants on the long-term basis are corrosion and subsequent metal particles removal from outer interface surfaces under contact with corrosive body fluid. The local pitting is further expedited with the heterogeneous structure of most metal alloys. This local corrosion leads to the release of metal ions or particles in surrounding live Page 1 of 24

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tissues/organs which subsequently lead to inflations/damage to live biological systems in the human body [1-4]. On the other hand, the surface energy of implant material interaction surfaces with live tissues plays an important role on account of cytocompatibility and uniform interconnected growth of living cells. All the metal implant surfaces lack the presence of uniform surface energy throughout interaction surfaces due to the presence of grains on account of the formation of different crystallographic planes during solidification. Most of the implant failures at the initial stage are found on account of the absence of improper bonding with surrounding tissues vis-à-vis non-uniform growth and spreading of tissues over implants [5-8]. Therefore to improve the biocompatibility, corrosion resistance, longevity and desired tissue growth, a bio-ceramic coating similar to that of the natural bone structure is essential over metallic implant surfaces. Researchers have been able to find out hydroxyapatite coating (compound present in human bone along with other calcium phosphates phases) over metallic implant surfaces through various ways like plasma arc melting with preplaced powder, electrophoretic deposition of HA powder dispersed under non-aqueous medium, laser cladding etc. [9-13]. Out of these techniques, electrochemical depositions seem to be the most outstanding candidate in view of in-situ formation and simultaneous deposition of hydroxyapatite and its group components in nano-crystallite form along with better control on surface morphology vis-à-vis uniform surface energy for faster cell growth and better bonding of surrounding tissues with implant surfaces [14-15].

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In recent days, various conductive polymers gained significant interest amongst research community for various applications. Polypyrrole (PPY) is one of the most promising candidates in this field due to its various advantages such as relatively easy synthesis for mass production, excellent physical stability and corrosion protection performance [16-18]. Besides that PPY possesses many other qualities and stimulus-responsive properties, which make it one of the interesting ‘smart’ biomaterial [19-20]. Most importantly, its good in-vitro and in-vivo biocompatibility were proved along with superior corrosion resistance under contact with human body fluid [21-24]. Furthermore, PPY can be easily synthesized through various methods, namely, electro-deposition and wet chemical method in aqueous medium from its monomer pyrrole with the variation of surface area, porosity and functionalization with different bio-molecules [25-28]. However, as a polymeric material PPY has some inherent limitation on account of the poor mechanical strength and osteoconduction ability for application as an individual biocompatible layer over the metallic implant surface under direct interaction with live tissues/cells. On the other hand, the ceramic hydroxyapatite material, which is having superior biocompatibility in terms of osteoconduction ability over the metallic implant surfaces, has its limitation on account of brittleness and generation of internal stresses during formation which in turn deteriorate its corrosion performance. Based on the above, the idea of this work was to synthesize a composite ceramicpolymer coating consisting of hydroxyapatite–polypyrrole over metallic surfaces in terms of superior biocompatibility, corrosion resistance along with mechanical properties between of polymer and ceramic material. Page 2 of 24

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Various research works were reported till date about the formation of hydroxyapatite-polypyrrole composite coatings through the electro-deposition process in layer by layer structure along with the presence of only hydroxyapatite layer on the outer surface [29-31]. There are no previous reports available on an in-situ formation and subsequent deposition of hydroxyapatite and polypyrrole together on to implant metal surfaces, which in turn is presumed to increase the overall mechanical strength of the coating layer by incorporating soft polymer phase in between the brittle hydroxyapatite ceramic structure. Since the top layer would consist of both the polypyrrole along with hydroxyapatite phase under contact with body fluid/tissues, this can exhibit enhanced osteoconduction along with corrosion protection performance simultaneously. Since polypyrrole is anodic deposition and hydroxyapatite is obtained through cathodic deposition, these stand as major constraints to co-deposit both the materials on the same surface, which is overcome with the idea of pulsed reverse electrochemical-deposition in this study.

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The objective of this study is to in-situ synthesis and subsequent co-deposition of hydroxyapatite – polypyrrole composite bio-compatible coating over the SS316 substrate under various deposition conditions with varying nucleation rate in order to compare the surfaces regarding their physical properties along with osteoconduction, cytocompatibility and corrosion protection performance behaviour under contact with body fluid.

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2. Materials and methods: 2.1. Materials: Commercial grade stainless steel (SS316) sheet of 180 µm thickness was selected as substrates for electro-deposition. The substrate surface was prepared for electro-deposition by polishing with SiC emery papers (600 and 800 grit) followed by ultrasonic cleansing in distilled water and acetone for several times. Epoxy resin was applied to cover the required surface so that it left an exact exposed area of 6 cm2 on one side of the SS316 plate for deposition.

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2.2. Deposition of coatings: The deposition was carried out with the help of a conventional two-electrode system. SS316 substrate was used as the working electrode, and another SS316 substrate plate with an exposed area of 10 cm2 was used as the counter electrode. The electrolyte solution was prepared by mixing 0.15 M of CaCl2, 2H2O (Merck, >98% purity) along with 0.1 M of NH4H2PO4 (Merck, >98% purity) salt in distilled water. 0.5 M potassium nitrate along with 0.1 M pyrrole monomer was added into the solution and stirred well for 15 min just before electro deposition. A typical pulsed reverse technique was followed to carry out electro polymerization followed by in-situ formation and co-deposition of hydroxyapatite and calcium phosphate phases during each integrate pulse of around 5 sec. Altogether 300 integrated pulses were applied for each coating surface. The coating was carried out at three different cathode current density namely 5 mA/cm2, 10 mA/cm2 and 20 mA/cm2 to identify and establish the relationship between the effect of nucleation on in-situ formations and their physical performance characteristics. Page 3 of 24

ACCEPTED MANUSCRIPT The electro-deposition was carried out with the help of Biologic SP150 electro-deposition set up in the pulsed DC mode in constant cathode current density. All the depositions were performed at room temperature which was around 25o C. 2.3.

Surface Characterization of coatings:

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The phase presence and crystallinity of the coatings were analysed with the help of X-ray diffraction (Pan Analytical Empyrean diffractometer) technique, using CuKα radiation (wavelength= 0.1546 nm), at a step angle of 0.02 degree, scanning rate 0.05 degree per second and two theta range between 10 to 60 deg. The average crystallite size was determined with the help of the Scherrer equation. The microstructure, composition and surface morphology of the coatings at different deposition current densities were characterized with the help of SEM (EVO 18 , Zeiss) combined with EDX (EDAX, USA). Further, in order to identify the bonding strength of phosphate and hydroxyl group in the coating composite material, which was major constituent of the complex chemical structure of hydroxyapatite, Fourier transform infrared spectroscopy (FTIR) was performed in a Nicolet 6700 spectrometer using KBr pallet technique in the range of 4000 to 400 cm-1, with a resolution of 4 cm-1. All measurements were performed at room temperature which was around 25 o C. 2.4. In vitro cytocompatibility assessment: 2.4.1. Cell culture: For cell adhesion and proliferation studies on various coated samples, MG-63 human osteosarcoma cell line received from National Center for Cell Science (NCSS, Pune, India) was used. MG-63 cells are relatively immature osteosarcoma cell line exhibiting osteoblast like phenotypic expression and therefore are considered suitable for the study of the effect on implant material surface [35]. The cells were cultured in the Dulbecco’s Modified Eagle medium (DMEM) (Himedia, Mumbai, India), supplemented with 10% fetal bovine serum (FBS) containing L- glutamine and Na- pyruvate and 1% penicillin –streptomycin in tissue culture flasks at 37oC with 5% CO2 environment. The culture medium was interchanged every alternate day. Sub culturing was performed on every 4 th day by detaching the cell from the flasks using trypsin- EDTA (ethylene diamine tetra acetic acid) solution. Prior to each experiment, the samples were sterilized for 30 min under UV radiation and were immersed under 70% ethanol for 2 h. The cell viability was tested with trypan blue staining. All the cell culture experiments were carried out for cell viability study.

2.4.2. Cell viability assay (MTT assay): For cell proliferation studies, MG-63 cells were applied at a cell density of around 10 4 cells/well on the coatings placed in the wells and also on the bare SS316 steel surface in a 48 well tissue culture plate (TCP). The proliferation of MG-63 on the coating surfaces was measured after 1, 3 and 5 days with the help of MTT assay. The culture media was changed at every day. The coated samples with attached cells were incubated in a mixture of 360 µL of PBS (phosphate buffer solution) and 40 µL Page 4 of 24

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MTT solution (5 mg/mL in PBS) for 4 h at 37oC in 5% CO2 atmosphere. The intense purple coloured formazan derivatives formed for living cells were dissolved in 300 µL dimethyl sulfoxide for 15 min, and the absorbance was measured with a micro plate reader at a wave length of 590 nm. For MTT assay, the absorbance values were compared with a standard curve to estimate the number of active cells and the same is represented with mean and variance. 2.4.3. Cell adhesion assessment: The morphology of the adherent cells proliferating on the various coatings are the indication of cell attachment and the same was examined under the secondary electron image mode of scanning electron microscope. To prepare the samples for SEM analysis, the samples were soaked in 2.5 % glutaraldehyde in PBS solution for around 1 hour for cell fixing and further dehydrated in aqueous ethanol solution at room temperature. 2.4.4. Cytotoxicity test and live/dead straining: Attachment of seeded cells on various coating samples was verified using fluorescence microscopy after staining through FITC loaded nano-gel incubation for 4 hours in media and subsequently washed with PBS solution and DAPI using manufacturer’s protocol (Invitrogen, USA). Fluorescence images of stained constructs were taken using a fluorescence microscope (Zeiss Axio observer Z1, Carl Zeiss, Germany). 2.5. Electrochemical behaviour assessment of coatings:

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The electrochemical behaviour under immersion in simulated body fluid was evaluated by electrochemical impedance spectroscopy (EIS) with the help of Biologic SP 150 device set up. A three electrode system, consisting of an Ag/AgCl electrode as the reference electrode, platinized platinum as the counter electrode and coated sample as the working electrode was used. EIS measurement was performed at OCP condition with the surface area of around 1 cm 2 for the working electrode at a frequency range of 106 to 10-2 Hz with 10 mV amplitude. The impedance spectra were analyzed with the help of biologic EC Lab software, and the results were reported in the form of standard Nyquist and Bode plots. An equivalent circuit was chosen for this analysis based on the review of similar research work in the recent past on this type of biocompatible coating [29].

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Corrosion behaviour of deposited coatings in simulated body fluid solution was evaluated based on potentiodynamic polarization measurements, by using a potentiostat/galvanostat (Bio Logic SP150) with a standard three electrode cell. All potentials were measured to the saturated Ag/AgCl electrode (RE), in which SS316 strip was used as the counter electrode. The potentiodynamic polarization curve was obtained by scanning from -1.0 V to +1.0 V at a scanning rate of 1 mV/s. The polarization resistance was derived from the software directly by fitting the Tafel curve method. Stabilization time of around 30 min in SBF was allowed for each sample. The surface topography and composition of the corrosion samples were also assessed with the help of SEM micrograph and EDX spectroscopy respectively. The simulated body fluid (SBF) prepared using the following compositions, NaCl- 7.996 g/l, KCl-0.224 g/L, CaCl2, 2H2O-0.278 g/L, MgCl2, 6H2O- 0.305 g/L, NaHCO3–0.350 g/L,

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2.6. Mechanical properties evaluation: The mechanical properties of the coatings were assessed with the micro-indentation technique (Anton Paar MCT). The tests were performed with the help of a Berkovich indenter (tip radius 100 µm) to determine the hardness and elastic modulus of the individual coatings with different proportions of phase elements. Three indentation tests were performed on the randomly selected sites of each sample. The loading penetration depth curve was plotted under a controlled maximum load of 100 mN along with loading, holding and unloading time of 30 s, 10 s and 30 s respectively. The hardness and elastic modulus of the coatings were derived from the recorded curves by applying the Oliver-Pharr method. 3. Results and discussions: 3.1. Phase analysis: The diffraction pattern [fig 1] of all composite coatings deposited with varying current densities were compared and are in good agreement with the standard data of hydroxyapatite (ICDD: 00-009-0432) and calcium hydrogen phosphate (ICDD: 04-013-3344) phase. The presence of an initial hump in the background is evident in all the coating surfaces, and it is on account of amorphous PPY phase in the composite coatings.

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At low current density, a broadened peak of <111> crystal plane of hydroxyapatite is present along with the sharp low-intensity peak of <002> and <222> planes of calcium hydrogen phosphate phase. Along with the increase in current density to 20 mA/cm 2, peak related to <222> plane of calcium hydrogen phosphate gets stronger in intensity along with the presence of additional planes of hydroxyapatite namely <002>, <301> and <101> parallel to the coating top surface. However, at 10 mA/cm2 current density, only single broadened peak corresponding to <111> crystal plane of hydroxyapatite phase is evident in the coating. Thus it may be presumed that the coating deposited with 10 mA/cm2 current density comes with one single crystal plane parallel to the top surface which will be in interaction with surrounding live cells/tissues and thus, it may be beneficial in terms of faster and uniform growth of tissues on account of uniform surface energy all across the coating top surface. Also <111> crystallographic plane is presumed to have the capability of superior corrosion resistance and mechanical stability as compared to all other crystallographic planes on account of higher atomic density, and thus, it would subsequently help to improve the overall corrosion protection performance of the coating surface under contact with body fluid [41-42]. The comparison of crystallographic data indicates that nucleation rate achieved at 10 mA/cm2 current density is optimum under pulse reverse technique to synthesize high corrosion resistance single crystal plane (111) of hydroxyapatite phase parallel to the coating surface. However, deposition of composite hydroxyapatite coatings by other researchers lead to the formation of multiple crystal planes parallel to the coating surface as compared to as stated composite coating with polypyrrole [29]. Further, the broadened peak is evident from the formation of nano crystalline structure of around 4-10 nm size which is beneficial for live cell interaction and attachment.

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Coating surface morphology was observed and compared with the help of secondary electron images of SEM [fig 3]. In 5 mA/cm2 coating surface, the spherical form of polypyrrole deposition of around 660-900 nm diameter was observed in a textured manner along with calcium phosphate and hydroxyapatite matrix in between of polypyrrole layer. Due to the low nucleation rate, less amount of hydroxyapatite phase was formed, and only a few sites were available along with exposure of the hydroxyapatite phase on the top surface of the coating. On magnified image (fig. 2b), the presence of hydroxyapatite phase is evident in the form of small scaffolds of 100-200 nm diameter particles. The individual scaffolds were not well connected in between, and their presences were random in nature. At moderate current density coating of 10 mA/cm2, well interconnected scaffolds of hydroxyapatite phase were observed all along the coating surfaces. The scaffolds were mostly made of a lamellar structure with strong bonding between each other like sharing of arms. Also due to porous scaffold structure of hydroxyapatite phase, it provided void to deposition of PPY in the next cycle and thus strengthened the overall structure by making a composite of ceramic and polymeric material. On the other hand, the completely different set of hydroxyapatite and calcium phosphate scaffolds structure made of the spherical particle of around 100 nm diameter was observed in the high current density coating of 20 mA/cm2. The change in physical shape of in-situ formed and deposited hydroxyapatite Page 7 of 24

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phase was on account of the presence of calcium phosphate phase. It was evident that 10 mA/cm 2 coating, due to lack of presence of any significant amount of calcium phosphate phases, were formed with lamellar kind of structure whereas the presence of other calcium phosphate phase made the spherical shape of structure. The porosity in 20 mA/cm 2 coating scaffold was found to be more as compared to 10 mA/cm2 coating due to the spherical shape of particles in the scaffold. This, in turn, reduced the overall bonding strength of the entire scaffold. On comparison of surface morphology, it appeared that 10 mA/cm2 coating creates a uniformly distributed and well-connected strong scaffold of hydroxyapatite phase on the top surface of the coating along with strong bonding with PPY and hydroxyapatite phase in the coating base. Thus, it may help in a strong bonding of surrounding tissues with the exposed coating surface over metallic implant along with strong bonding of the coating layer with metallic implant surface. In other research work carried out on the similar field, mostly hydroxyapatite phase are observed on the outer surface as the separate layer of hydroxyapatite and polypyrrole were deposited without having any chemical bonding between two phases [29-31].

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3.3. FTIR: Functional group analysis of bare PPY and its co-deposition with HA was carried out with the help of FTIR analysis, and the same is compared in fig 3. In bare PPY coating, characteristics peak at 3621 cm 1 and 1650 cm-1 indicate the N-H stretching and N-H in-plane bending vibrations. The peaks at 1449 cm-1 and 1556 cm-1 are attributed to the C=C and C-N stretching vibration of pyrrole ring. The peaks at 1314 cm-1 and 1182 cm-1 correspond to the C-N bending vibration and C-H in-plane bending vibration respectively. The appearance of a peak at 912 cm-1 signifies the presence of C-H out-ofplane bending vibration. The five-member heterocyclic ring presence can be identified with a sharp peak at 795 cm-1[36].

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The alpha-carbonyl group region (1700 cm-1) of pyrrole is broadened and gradually decreased in intensity with increasing current density along with finally settling into small low energy peak shifting in 20 mA/cm2 coating, implies that increased over-oxidation with increasing current density produces new oxygen functionality on the alpha carbon of polypyrrole during hydroxyapatite co-deposition [fig 4b]. At the moderate current density of 10 mA/cm2, polymerization is sufficient without overoxidation as no such intense peak has been found. The band located at 1531 to 1561 cm -1 is attributed to the CC/C=C stretching vibration of the PPy chains, which is first shifted to low frequency for 5 mA/cm2 coating from bare PPy signifying that conjugation length of polymer chain is increased whereas, at moderate and high current density composites, the same is decreased along with shifting of the peak at the higher frequency. This data suggest that over-oxidation may lead to oxygen defect and shorter polymerization length in high current density as shorter polymer chains are more prone Page 9 of 24

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to carbonyl defects. At low current density composite along with higher conjugation in the polymer chain, higher defect state in alpha position has also been found. The broadening of the carbonyl region in these composites implies that during co-deposition and hydroxyapatite formation the nascent polymer chains are bonded through this alpha position. As the current density increases, the increase in polymerization rate of polypyrrole makes the shorter nascent polymer chains to be available for bonding with hydroxyapatite and thus reduces the carbonyl peak along with shifting the conjugation peak towards high frequency. In the 10 mA/cm2 composite coating, the polymerization rate and hydroxyapatite conjugation with the alpha carbon finds an equilibrium, which can provide a balanced matrix towards mechanical property enhancement. The band at 1470 cm-1 is a predominantly C=C and C–N stretch mode corresponding to various interring C-C, C-N, C-N-C and in-plane deformation and C=N-C-C stretching. Broadening of the peak in all the composites other than 20 mA/cm2 coating, Indicates non-planarity of polymer chains, i.e., change in polymer symmetry; which activates some additional mode in the spectrum in these composites. But at the highest current density, the alternative symmetry restored with intense inplane deformation peak signifies the presence of homo symmetry which may occur due to faster deposition. At higher current density, the polypyrrole formation is unidirectional and especially restricted by the growth of crystalline hydroxyapatite. Band around 890 cm -1 to 960 cm-1 are of transoligomer which is very complex and coming from mainly C=C stretch, C–C in-ring stretch, C–N stretch, C–C inter-ring stretch, C=C–C, C–N–C ring deformation, C–H and N–H in-plane bending along with C– C inter-ring bending [fig 4d]. Interestingly at highest current density, non-planar or trans-oligomer has been observed whereas cisoligomerisation is most evident in 10 mA/cm2 coating, which may help to improve the overall mechanical property of this polymer-ceramic composite. Peaks around 1200-1300 cm-1 are assigned to various in- ring and inter ring bending/stretching of polymer chains, which are broadened due to the addition of various in plane mode arising from intense interaction with hydroxyapatite with that of the polymeric chain. Thus, it indicates the strong amalgamation with hydroxyapatite phase during deposition. In the 10 mA/cm2 coating, the highest amount of broadening is evident vis-à-vis the highest amount of in-plane interaction in these composite [fig 4c]. The bands in the region of 875880 cm-1 indicate the presence of HPO4, which can only be found in 5 mA/cm2 composite coating and thus signifies incomplete hydroxyapatite formation at low current density. Another very important observation which marks the presence of CO3-2 at low current density composite is band vibration at 2300 cm-1 range [fig 4a]. On the other hand, the H-O-H vibration [3548 cm-1] is most prominent in 20 mA/cm2 coating, which also helps the formation of alpha carbonyl of nascent polymer chain during polymerization as evident from the intense peak around. The 10 mA/cm2 composite coating neither has the water presence, nor the unreacted carbonyl peaks corroborate the overall mechanical property enhancement along with uniform surface energy. This may help in uniform tissue growth along with strong bonding with both the metallic implant surface as well as live tissues.

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Conclusively the formation of the symmetric polymer chain, with less water and no unreacted ions, signifying complete hydroxyapatite formation, augmented with the signature of peak broadening as evidence of composite formation makes the 10 mA/cm2 composite coating a better scaffold towards strong mechanical supports along with osteogenic differentiation and tissue regeneration. Stretching of polypyrrole ring are observed in this study due to the bonding of in-situ formed hydroxyapatite phases with the polymer ring structure along with the growth of hydroxyapatite phase, in particular, <111> plane, which is not experienced by the other researchers in the absence of co-deposition through pulsed reverse technique [29-31].

Fig 4: Detailed comparison of individual FTIR peak relates the formation of N-H, O-H, PO4, and Pyrrole ring with different bond energy under various deposition conditions 3.4. Electrochemical behavior assessment: 3.4.1. Open circuit potential behavior: PPY, as a standalone coating over the metallic surface, shows cathodic passivation under contact with simulated body fluid. The initial sign of corrosion is evident from the coating surfaces followed by stabilization at higher open circuit voltage. All three PPY composite coatings with hydroxyapatite phase show anodic passivation under contact with body fluid [fig 5]. It is presumed that anodic passivation is more preferred over other coating material regarding slower corrosion/reaction rate. Page 11 of 24

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The shifting of open circuit potential from cathodic to anodic region is a clear indication of bonding of hydroxyapatite phase with PPY co-deposition in the composite coating at all three current densities. On comparison of composite coating deposited at different current densities with varying nucleation rates, it is observed the coating with less amount of hydroxyapatite phase shows the lower level of passivation as compared to the other two coatings. The unique phenomenon of shifting the OCP towards cathodic region for initial contact period along with the stable recovery of the same towards anodic region was observed only in 10 mA/cm2 coating. The formation of initial hump can be presumed on account of the dissolution of single PPY phase from the coating surface along with left out of only composite phase on the top surface, which makes the coating more passive under contact with simulated body fluid.

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Fig 7: Comparison of electrochemical corrosion resistance and stability of various composite coatings with the help of (a) Nyquist (b) Bode phase and (c) Bode impedance plots

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The Nyquist plots of all the coatings, as shown in fig 7, compare their electrochemical behaviour under contact with SBF. All the composite coatings show higher electrochemical corrosion resistance under contact with SBF as compared to bare PPY coating over metallic surfaces. The enhanced corrosion resistance is on account of strong bonding of hydroxyapatite phase with PPY during in-situ formation and co-deposition. On comparison of coatings with varying deposition rates, it is observed that 10 mA/cm2 coating, which is having lamellar scaffold formation of hydroxyapatite as evident through SEM, exhibits 10-fold high corrosion resistance throughout the entire lower frequency range [fig 7c]. The higher corrosion resistance vis-à-vis strong bonding of the hydroxyapatite phase with PPY in the composite, transforms the coating into more passive and robust under contact with body fluid. Bode plots signify the presence of capacitance peaks in all three composite coatings including the bare PPY [fig 7c]. The degree of capacitance varies along with deposition current density. All composite coatings exhibit twin peak, one at the lower frequency and other at the higher frequency. The intensity of capacitance peak at higher frequency range is getting sharper with increasing Page 13 of 24

ACCEPTED MANUSCRIPT amount of hydroxyapatite phase in the composite coating. The lower frequency capacitance peak has no direct relation either with the amount of hydroxyapatite phase in the coating or PPY. Rather this capacitance peak gets broadened and deeper at a specific deposition current density of 10 mA/cm 2. Thus, it can be presumed that 10 mA/cm2 is the optimum deposition current density for this composite coating on account of better amalgamation bonding between two phases and accordingly to obtain the highest corrosion resistance coating. However, the electrochemical impedance of as prepared composite coatings is few thousand times higher than that of reported literature data [40].

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The potentiodynamic polarization curves for all the composite coatings in SBF are shown in fig 8, and their results are compared with bare PPY coating surface in terms of Ecorr and Icorr values which in turn signify the relative corrosion rate under applied potential range. It is observed that bare PPY coating exhibits lower or almost equal corrosion rate under SBF as compared to the composite coating deposited at lower and higher current density respectively. Also, a good amount of anodic passivation phenomena is observed for bare PPY coating. Thus, it can be presumed that bare PPY coating as an individual shows a good amount of resistance under contact with SBF, which is reported in the various literature. It can be concluded from the results that the amount of hydroxyapatite presence in the composite has no direct relationship with the corrosion resistance of the overall coating. Rather, the bonding of the hydroxyapatite phase with PPY structure plays an important role to decide the corrosion protection performance of the overall coating. Thus, it can be evident that only the coating deposited at the moderate current density (10 mA/cm 2) shows improved corrosion resistance behaviour as compared to stand alone bare PPY coatings. The results for 10 mA/cm2 coating are also in well agreement with the SEM and EIS results. PPY HA+PPY 5 mA/sq. cm HA+PPY 10 mA/sq. cm. HA+PPY 20 mA/sq.cm

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Fig 9: Comparison of surface topography post-potentiodynamic polarization test through SE_SEM images

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Cyto-compatibility assessment with MG-63 cell line:

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In this study, MG-63 cells were used for examining early reaction in the response of bone cells to different composite coating surfaces along with bare PPY in terms of toxicity, attachment and growth of live tissues/cells after implementation in the human body [39].

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Fig 10: MTT assay trend of different coatings after 1, 3 and 5 days of incubation with MG63 cell line

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At the initial period of incubation (1 day), no major differences in the number of cells attached amongst the different composite coating surfaces, including control and bare PPY surfaces, were observed [fig 10]. After 3 days of incubation, all the coating surfaces exhibited superior growth in the number of cells as compared to the bare SS316 surface used as the control surface. Amongst the four coated samples, a slight difference, in terms of presence of larger number of cells on the coating surfaces were observed in case of 10 mA/cm2 and 20 mA/cm2 coating surfaces as compared to 5 mA/cm2 and bare PPY coatings. This phenomenon of slightly faster cell growth on the third day can be explained with the help of the presence of higher amount of hydroxyapatite in the coating surfaces for 10 mA/cm2 and 20 mA/cm2 coating surfaces. After 5 days of incubation, a significant stable growth of cell was observed only in 10 mA/cm2 coating surfaces. Whereas, a steep decrement in the number of live cells were observed in bare PPY and 20 mA/cm 2 composite coatings. A decrement in the total number of cells can only happen either when cells do not sustain on the coating surface or when the continuous cell spreading and growth are restricted with the amalgamation of a large number of live cells into a hump kind of pattern. The formation of cell humps on the surface indicates the combined presence of superior biocompatible zone along with less biocompatible zone on to the coating surfaces, and thus it restricts the uniform growth of cells all along the contact surface. 3.5.2. Cell morphology on coating surfaces:

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Fig 11: Comparison of spread cell surface morphology on different coating surfaces Cell spreading, bonding interaction and growth morphology over the coating surfaces are compared and studied with the help of secondary electron images of scanning electron microscope, and the Page 16 of 24

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same is presented in fig 11. Discrete/localized growth and spreading of cells are evident in both bare PPY and 5 mA/cm2 composite coating surfaces. Apparently well spreading of cells all across the coating surfaces along with strong attachment with adjacent cells as well as with coating materials are evident in 10 mA/cm2 coating surface. In 20 mA/cm2 coating surface, the presence of a large number of cells is observed along with the formation of several humps like structures made by cells due to over confluence of cells at that specific areas. The cell hump structure is indicated with the yellow arrow in fig 11d. Based on these results, it can be presumed that 10 mA/cm 2 composite coating was formed with optimum surface energy along with the uniform distribution of the uniform PPy all across the coating surface, which is beneficial for cell attachment in terms of uniform and faster cell growth over the coating surfaces.

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Fig 12: Comparison of spreading of live-dead cell on different coating surfaces with fluorescence microscopy Fluorescence microscopy images help to track the spreading and condition of live cells over the coating surfaces, and the same is compared in fig 12. Sustenance and growth of live cells in isolation along with very few interconnections are identified for bare PPY and 5 mA/cm 2 coating surfaces. This is presumed that on account of non-uniform surface energy vis-à-vis cytocompatibility of coating surfaces for these two coatings, which have a high amount PPY deposition on their surfaces under contact with the cell membrane. Stretched morphology of the cell cytoskeleton arrangement which is indicated with green colour and nucleus which is indicated with blue colour, along with cell migration and interconnected growth, is well evident in all areas on the contact surface of 10 mA/cm 2 Page 17 of 24

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composite coating. The strong bonding between in-situ formed hydroxyapatite and PPY phase is thus responsible for maintaining the uniform and optimum surface energy which in turns help in uniform cell spreading and faster growth of live cells over the coating surfaces. A large concentration of cells is observed with the presence of a large amount of green cytoskeleton in 20 mA/cm2 coating surfaces which is further analyzed with the help of fig 13 comparing the live nucleus distribution pattern. It is observed that monolayer unidirectional growth and spreading of cells occurred over the 10 mA/cm2 coating surfaces which are evident with uniformly distributed round shape nucleus all across the 10 mA/cm2 coating surface. On the other hand, several distorted and amalgamated nucleus structure were identified in 20 mA/cm2 coating, which signifies the presence of clusters of live and dead cells together on account of over confluence in few areas over the coating surfaces. These clusters of live and dead cells were also identified as the presence of several humps in SE-SEM image of 20 mA/cm2 coating surface (fig 11d). This composite coating exhibits the similar level of cell viability along with strong bonding of live cells with the coating surfaces as compared to the results produces by the other researchers for hydroxyapatite-polypyrrole layer by layer sandwich coating over stainless steel surfaces [29-30]. (b) HA+PPY_20mA/cm2

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Fig 13: Comparison of cell nucleus spreading between 10 mA/cm 2 and 20 mA/cm2 coating surfaces

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Fig 14 compares the typical load-indentation depth curves for different composite coating surfaces on three randomly selected sites. The calculated elastic modulus and hardness values of composite coatings are compared with the help of fig 15. It is observed that elastic modulus and surface hardness follow the same trend with deposition current density. The maximum elastic modulus and surface hardness are observed in 10 mA/cm2 coating, wherein strong bonding of in-situ formed hydroxyapatite and PPY are evident from the FTIR spectrum and surface morphology study. The lamellar scaffold formation of hydroxyapatite phase seems to be responsible for providing additional strength to the coating surface and thus help to enhance its surface hardness and elastic modulus. In both low and high current densities, a low value of hardness and elastic modulus are obtained. It indicates the absence of strong bonding between hydroxyapatite and PPY at both the case of slower nucleation rate and higher nucleation rate on account of the absence of bonding reaction force and Page 18 of 24

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short bonding reaction time, respectively. Thus, it is presumed that 10 mA/cm 2 current density provides optimum bonding force and bonding reaction time, which in turn help to improve the overall composite coating strength in terms of better amalgamation during in-situ formation and codeposition over the metallic implant surface. Less variation of load indentation curve of 10 mA/cm 2 coating also signifies the uniform mechanical properties vis-à-vis uniform composite phase present all across the coating surfaces as compared to other two composite coating surfaces deposited at lower and higher current densities. The elastic modulus of 10 mA/cm 2 coating comes to nearly 90 GPa which is in between metallic surface (~200 GPa) and bone (6-30 GPa) modulus and thus acts as a good gradient coating for use in between metallic implant and bone. Also due to the presence of polymer structure (PPY) in composite, this coating exhibits superior elasticity and spring back properties, which in turn help to take care of biocompatible coating over the surfaces of high loadbearing implants. The polymer-ceramic composite coating exhibits superior improvement in modulus of elasticity along with hardness as compared to both the individual material coating as reported by other researchers [30]. 20 mA/cm2

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Fig 15: Elastic modulus and surface hardness trend of various coating surfaces with respect to deposition current density 4. Conclusions:

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In this present study, polypyrrole-hydroxyapatite composite coatings were successfully prepared with the help of pulsed reverse electro-deposition technique through the in-situ formation and codeposition of both the phases simultaneously along with process optimization concerning reverse duty cycle, deposition time and current density. Effects of nucleation and reaction rate were successfully correlated with the bonding structure along with overall physical and biocompatible characteristics of the composite coatings. The unidirectional growth of hydroxyapatite crystal along with high atomic density <111> plane parallel to the contact surface took place in all the composite coatings. Based on this entire study, the following conclusions were made: Nucleation rate and reaction time greatly influence the bonding strength and uniform distribution of the hydroxyapatite phase with PPY. It is observed that coating deposited at 10 mA/cm2 current density lead to the formation of an optimum coating through a balancing between both the formations of in-situ hydroxyapatite along with subsequent bonding with pyrrole ring structure. b. Hydroxyapatite phase is present in the form of scaffold structure made out of lamellar or spherical shape of sub-micron size particle along with internal voids filled up with polypyrrole and thus help to increase the stability of the overall coating. c. 10 mA/cm2 coating exhibits faster interconnected growth and strong bonding of MG-63 cell over the coating surfaces as compared to all other composite coatings. d. Shifting of open circuit potential towards anodic passivation region from the cathodic region under contact with SBF took place in all the composite coatings compared to bare PPY coating. e. The surface hardness and elastic modulus of 10 mA/cm2 coating, which are highest amongst all other composite coatings, fall in between the metallic implant and human bone.

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ACCEPTED MANUSCRIPT Thus, these composite coatings can act as an interconnected biocompatible layer between human bone and metallic implant surfaces with gradient variation of elastic modulus. Presence of PPY in the composite coating matrix provides sufficient elasticity along with required toughness. Composite coatings with such properties could be an ideal sustainable biocompatible coating over the load bearing implant surfaces. References:

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1. S. Nagarajan, N. Rajendran, Surface characterization and electrochemical behavior of porous titanium dioxide coated 316L stainless steel for orthopedic applications, Appl. Surf. Sci. 255 (2011) 3927–3932. 2. D. Gopi, S. Ramya, D. Rajeswari, L. Kavitha, L. Kavitha, Corrosion protection performance of porous strontium hydroxyapatite coating on polypyrrole coated 316L stainless steel, Colloids Surf. B: Biointerfaces 107 (2013) 130–136. 3. K. Grandfield, I. Zhitomirsky, Electrophoretic deposition of composite hydroxyapatite–silica– chitosan coatings, Mater. Charact. 59 (2008) 61–67. 4. P.N. Minoofar, B.S. Dunn, J.I. Zink, Multiply doped nanostructured silicate sol–gel thin films: spatial segregation of dopants, energy transfer, and distance measurements, Chem. Soc. 127 (2005) 2656–2665. 5. M. Gonzalez, S. Saidman, Electrodeposition of polypyrrole on 316L stainless steel for corrosion prevention, Corros. Sci. 53 (2011) 276–282. 6. A. Parsapour, S.N. Khorasani, M.H. Fathi, Effect of surface treatment and metallic coating on corrosion behavior and biocompatibility of surgical 316L stainless steel implant, Sci. Technol. 28 (2012) 125–131. 7. Y.-Ch. Tang, S. Katsuma, S. Fujimoto, S. Hiromoto; Electrochemical study of Type 304 and 316L stainless steels in simulated body fluids and cell cultures; Acta Biomater. 2 (2006) 709 8. Y. Okazaki, E. Gotoh, T. Manabe, K. Kobayashi; Comparison of metal concentrations in rat tibia tissues with various metallic implants; Biomaterials 25 (2004) 5913. 9. C. Monika, E. Klas, P. Jinshan, K. Andrzej, Silane–parylene coating for improving corrosion resistance of stainless steel 316L implant material, Corros. Sci. 53 (2011) 296–301 10. M. Haidopoulos, S. Turgeon, G. Laroche, D. Mantovani, Surface modifications of 316 stainless steel for the improvement of its interface properties with RFGD-deposited fluorocarbon coating, Surf. Coat. Technol. 197 (2005) 278–287 11. M. Maryam, M.M. Diego, Biodegradable metals for cardiovascular stent application: interests and new opportunities, Int. J. Mol. Sci. 12 (2011) 4250–4270. 12. D.G. Wang, C.Z. Chen, J. Ma, G. Zhang, Colloids Surf. B: Biointerfaces 66 (2008) 155. 13. D. Gopi, et al., A comparative study on the direct and pulsed current electrodeposition of hydroxyapatite coatings on surgical grade stainless steel, Surf. Coat. Technol. 206 (2012) 2859– 2869

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14. Rajib Chakraborty, Srijan Sengupta, Partha Saha, Karabi Das, Siddhartha Das, Synthesis of calcium hydrogen phosphate and hydroxyapatite coating on SS316 substrate through pulsed electrodeposition, Mater. Sci. Eng. C 69 (2016) 875–883. 15. Rajib Chakraborty, V.S. Seesala, Srijan Sengupta, Santanu Dhara, Partha Saha, Karabi Das, Siddhartha Das; Comparison of Osteoconduction, cytocompatibility and corrosion protection performance of hydroxyapatite-calcium hydrogen phosphate composite coating synthesized insitu through pulsed electro-deposition with varying amount of phase and crystallinity; Surfaces and Interfaces 10 (2018) 1–10 16. Cui X, Hetke JE, Wiler JA, Anderson DJ, Martin DC. Electrochemical deposition and characterization of conducting polymer polypyrole/PSS on multichannel neural probes. Sens Actuator A-Phys 2001;93:8–18. 17. Lee JY, Lee JW, Schmidt CE. Neuroactive conducting scaffolds: nerve growth factor conjugation on active ester-functionalized polypyrrole. J R Soc Interface 2009; 6:801–10. 18. Ateh DD, Vadgama P, Navsaria HA. Culture of human keratinocytes on polypyrrole-based conducting polymers. Tissue Eng 2006;12: 645–55. 19. Garner B, Georgevich A, Hodgson AJ, Liu L, Wallace GG. Polypyrrole–heparin composites as stimulus-responsive substrates for endothelial cell growth. Inc. J Biomed Mater Res 1999;44:121– 9. 20. Garner B, Hodgson AJ, Wallace GG, Underwood PA. Human endothelial cell attachment to and growth on polypyrrole–heparin is fibronectin dependent. J Mater Sci Mater Med 1999;10: 19–27 21. Kim DH, Richardson-Burns SM, Hendricks JL, Sequera C, Martin DC. Effect of immobilized nerve growth factor on conductive polymers: electrical properties and cellular response. Adv Funct Mater 2007;17:79–86. 22. Li Y, Neoh KG, Kang ET. Plasma protein adsorption and thrombus formation on surface functionalized polypyrrole with and without electrical stimulation. J Colloid Interf Sci 2004;275:488–95. 23. George PM, LaVan DA, Burdick JA, Chen CY, Liang E, Langer R. Electrically controlled drug delivery from biotin-doped conductive polypyrrole. Adv Mater 2006;18:577–81. 24. Meng S, Rouabhia M, Shi G, Zhang Z. Heparin dopant increases the electrical stability, cell adhesion, and growth of conducting polypyrrole/poly(L,Llactide) composites. J Biomed Mater Res 2008;87A:332–44. 25. Akkouch A, Shi G, Zhang Z, Rouabhia M. Bioactivating electrically conducting polypyrrole with fibronectin and bovine serum albumin. J Biomed Mater Res 2010;92A:221–31. 26. Ferraz N, Strømme M, Fellstrom B, Pradhan S, Nyholm L, Mihranyan A. In vitro and in vivo toxicity of rinsed and aged nanocellulose–polypyrrole composites. J Biomed Mater Res A 2012;100a:2128–38. 27. Zhang X, Manohar SK. Bulk synthesis of polypyrrole nanofibers by a seeding approach. J Am Chem Soc 2004;126:12714–5.

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28. Chronakis IS, Grapenson S, Jakob A. Conductive polypyrrole nanofibers via electrospinning: electrical and morphological properties. Polymer 2006;47:1597–603. 29. K. Prem Ananth , A. Joseph Nathanael b, Sujin P. Jose, Tae Hwan Oh , D. Mangalaraj; A novel silica nanotube reinforced ionic incorporated hydroxyapatite composite coating on polypyrrole coated 316L SS for implant application; Materials Science and Engineering C 59 (2016) 1110–1124 30. D. Gopi, S. Ramya, D. Rajeswari, L. Kavitha ; Corrosion protection performance of porous strontium hydroxyapatite coating on polypyrrole coated 316L stainless steel; Colloids and Surfaces B: Biointerfaces 107 (2013) 130– 136 31. R. Ma , K.N. Sask , C. Shi , J.L. Brash, I. Zhitomirsky; Electrodeposition of polypyrrole-heparin and polypyrrole-hydroxyapatite films ; Materials Letters 65 (2011) 681–684 32. D Gopi, A karthika; development of lotus like hydroxyapatite coating on HELCDEB treated titanium by pulsed electrodeposition; material letters 105 ( 2013) 216-219 33. A. Oyane, H.M. Kim, T. Furuya, T. Kokubo, T.Miyazaki, T. Nakamura, Preparation and assessment of revised simulated body fluids, J. Biomed. Mater. Res. A 56 (2003) 188–195 34. L. Muller, F.A.Muller, Preparation of SBF with different HCO−3 content and its influence on the composition of biomimetic apatites, Acta Biomater. 2 (2006) 181–189 35. L. Feng, K.Z. Li, Z.G. Zhao, H.J. Li, L.L. Zhang, J.H. Lu, Q. Song, Mater. Des. 92 (2016) 120–128 36. W.X. Zhang, X.G. Wen, S. Yang, Synthesis and characterization of uniform arrays of copper sulfide nano rods coated with nano layers of polypyrrole, Langmuir 19 (2003) 4420–4426. 37. R. Drevet, O. Aaboubi, H. Benhayoune, In vitro corrosion behavior of electrodeposited calcium phosphate coatings on Ti6Al4V substrates, J Solid State Electrochem 16 (2012) 3069 38. X. Zhang, Q. Li, L. Li, P. Zhang, Z. Wang, F. Chen, Fabrication of hydroxyapatite/stearic acid composite coating and corrosion behavior of coated magnesium alloy, Materials Letters 88 (2012) 76 39. S.H. Yim, Effect of titanium surface roughness on cell adhesion of human osteoblast like cells (MG63), J. Korean Acad. Prosthodont. 42 (2004) 261–266 40. Shaoyun Chen, Hui Liu, Yuan Wang, Shaoqin Xu, Wenfeng Liu, Dan He, Xueqing Liu, Jiyan Liu, Chenglong Hu; Electrochemical Capacitance of Spherical Nanoparticles Formed by Electrodeposition of Intrinsic Polypyrrole onto Au Electrode; Electrochimica Acta 232 (2017) 72– 79 41. Heywood, B. R.; Sparks, N. H.; Shellis, R. P.; Weiner, S.; Mann, S. Ultrastructure, Morphology and Crystal Growth of Biogenic and Synthetic Apatites Connect. Tissue Res. 1990, 25, 103– 119 42. Nimshi L. Fernando, Nilwala Kottegoda, Sumedha Jayanetti, Veranja Karunaratne, Dilushan R.Jayasundara; Stability of nano-hydroxyapatite thin coatings at liquid/solid interface; surface and coating technology 349 (2018), 24-31

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ACCEPTED MANUSCRIPT Highlights: a. In-situ formation along with subsequent co-deposition of both the ceramic and polymer phases was achieved through pulse reverse electrochemical deposition. b. Strong amalgamation of calcium phosphate ceramic phase with pyrrole ring are evident through a shifting of FTIR peak compared to bare polypyrrole coating c. 10 mA/cm2 coating, which comes with uniform lamellar scaffold of sub-micron size

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