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Fabrication of minerals substituted porous hydroxyapaptite/ poly(3,4-ethylenedioxy pyrrole-co-3,4-ethylenedioxythiophene) bilayer coatings on surgical grade stainless steel and its antibacterial and biological activities for orthopedic applications Ramya Subramani, Elangomannan Shinyjoy, Kavitha Louis, soundarapandian kannan, and Dhanaraj Gopi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b01795 • Publication Date (Web): 29 Apr 2016 Downloaded from http://pubs.acs.org on May 4, 2016

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ACS Applied Materials & Interfaces

Fabrication of minerals substituted porous hydroxyapaptite/poly(3,4-ethylenedioxy pyrrole-co3,4-ethylenedioxythiophene) bilayer coatings on surgical grade stainless steel and its antibacterial and biological activities for orthopedic applications Ramya Subramani†, Shinyjoy Elangomannan†, Kavitha Louis⊗, Soundarapandian Kannan §, Dhanaraj Gopi †,‡,* †

Department of Chemistry, Periyar University, Salem 636 011, Tamilnadu, India Department of Physics, School of Basic and Applied Sciences, Central University of Tamilnadu, Thiruvarur 610 101, Tamilnadu, India § Proteomics and Molecular Cell Physiology Laboratory, Department of Zoology, Periyar University, Salem 636 011,Tamilnadu, India ‡ Centre for Nanoscience and Nanotechnology, Periyar University, Salem 636 011, Taminadu, India * Corresponding author. Tel.: +91 427 2345766; fax: +91 427 2345124. E-mail address: [email protected] (D. Gopi). ⊗

ABSTRACT Current strategies of bilayer technology have been designed mainly at the enhancement of bioactivity, mechanical property and corrosion resistance. In the present investigation, the electropolymerisation of poly (3,4-ethylenedioxypyrrole-co-3,4-ethylenedioxythiophene) (P(EDOPco-EDOT)) with various feed ratio of EDOP/EDOT on surgical grade stainless steel (316L SS) and the successive electrodeposition of strontium (Sr2+), magnesium (Mg2+) and cerium (Ce3+) (with 0.05, 0.075 and 0.1M Ce3+) substituted porous hydroxyapatite (M-HA) are successfully combined to produce the bioactive and corrosion resistance P(EDOP-co-EDOT)/M-HA bilayer coatings for orthopaedic applications. The existence of as-developed coatings was confirmed by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), proton nuclear magnetic resonance spectroscopy (1H-NMR), high resolution scanning electron microscopy (HRSEM), energy dispersive X-ray analysis (EDAX) and atomic force microscopy (AFM). Also, the mechanical and thermal behaviour of the bilayer coatings were analyzed. The corrosion resistance of the asdeveloped coatings and also the influence of copolymer (EDOP:EDOT) feed ratio were studied in Ringer’s solution by electrochemical techniques. The as-obtained results are in accord with those 1 ACS Paragon Plus Environment


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obtained from the chemical analysis using inductively coupled plasma atomic emission spectrometry (ICP-AES). In addition, the antibacterial activity, in vitro bioactivity, cell viability and cell adhesion tests were performed to substantiate the biocompatibility of P(EDOP-co-EDOT)/M-HA bilayer coatings. On account of these investigations, it is proved that the as-developed bilayer coatings exhibit superior bioactivity and improved corrosion resistance over 316L SS, which is potential for orthopedic applications. Keywords: Poly (3,4-ethylenedioxypyrrole), Poly (3,4-ethylenedioxythiophene), Substituted hydroxyapatite, Bilayer, Electrodeposition, Bioactivity 1. INTRODUCTION Currently, metallic biomaterials are widely being used in several medical devices to aid with bone replacement or repair.1 The most acceptable metallic biomaterials such as titanium and its alloys, stainless steels and cobalt-chromium based alloys are widely used as surgical implants owing to their favourable mechanical strength and toughness.2 As the reduction of costs in health care services is of vast significance, stainless steel (316L SS) is immensely employed for manufacturing dental, orthopaedic and other implantable biomedical devices.3 Though 316L SS possesses several advantages such as ease of fabrication, good mechanical strength, reasonable corrosion resistance and low cost, it is easily prone to localized corrosion when used in certain environment such as in human body.4-6 Moreover, the leach out of metal ions such as chromium, nickel and iron in the physiological environment from the 316L SS implants results in a decreased bioactivity and biocompatibility.7-8 Hence, it is very important to enhance the bioactivity of such 316L SS biometallic implants in physiological body fluid. Calcium phosphate bioceramics and mainly, hydroxyapatite (HA, Ca10(PO4)6(OH)2) are the extensively studied materials for coating metallic orthopaedic and dental implants since 1980s, owing to their chemical similarities with the mineral component of bone, osteogenic properties and 2 ACS Paragon Plus Environment

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their ability to form direct bonding with host bone tissues.9,10 Though HA is bioactive, biocompatible and osteoconductive it has a poor solubility in human body fluids and hence it cannot be replaced by newly formed bone tissues.11-17 Unfortunately, HA possesses poor osteogenic capacity to bone and inferior mechanical properties. 18-24 The major hard tissues in human body such as teeth and bone are composed of HA mineral phase containing cationic (ie., Sr2+, Mg2+,Zn2+, Ba2+, Al2+, Na+, K+) and/or anionic (ie., F-, Cl-, SiO42and CO32-) ions in low traces. In actual fact, the above ions are naturally present in human bone tissues and, thus, play an imperative role in osteogenesis.20-22 Mainly, Sr2+ and Mg2+ play a significant role in improving the bioactivity of HA bioceramics by inducing an enhanced in vitro osteoblast cell proliferation, attachment and growth. 11 In detail, strontium (Sr2+) ions possess dual effects in promoting bone cell growth and inhibiting the osteoclast activity, bone resorption and also in reducing the risk of fractures in osteoporotic patients as well as in suppressing osteoporosis. 25-27 In particular, the high concentration of Sr2+ induces defective bone mineralization and also changes the mineral profile in rats. In contrast, the low concentration of Sr2+, stimulates bone formation and enhances the replication of preosteoblastic cells. Hence, the concentration of Sr2+ ions is an imperative factor for bone formation.28,29 In fact, the partial substitution of Ca2+ by Sr2+ can enhance the biological properties of HA based bioceramic materials.30 Mg2+ is an essential trace element for all living organisms such as enamel, dentin and bone despite its low concentration (0.44, 0.123, 0.72 wt.% of Mg2+ in human body respectively).31 Mg2+ depletion adversely affects all phases of bone metabolism causing cessation of bone growth, decrease of osteoclastic and osteoblastic activities and also its deficiency results in bone loss, skeletal fragility and generation of osteopenia.32 Substituting HA with Mg2+ has been the subject of extensive research because of its potential for developing artificial bone and other biomedical 3 ACS Paragon Plus Environment


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applications. 30-34 Among the various trace elements which have been used as antibacterial agents in different clinical fields, cerium (Ce3+) exhibits the antibacterial activity at very low concentrations without toxicity to human cells. 35-40 Several biological studies such as in vitro studies demonstrated that the Ce3+ ions in the HA coated biometallic implants play a main role in preventing or minimizing initial bacterial adhesion.41 As a result Sr2+, Mg2+ and Ce3+, as essential foreign ions can play a key role in improving the bioactivity and antibacterial activity of HA for orthopedic applications. In the present research work, we have proposed the fabrication of Sr2+, Mg2+ and Ce3+ substituted HA (M-HA) coating on 316L SS by electrodeposition. Electrodeposition is attracting increasing attention as a superior coating technique since it provides better control of coating morphology (i.e.,) in achieving porosity of the coatings, uniformity in deposition, well adherent coating, etc.36,42-47 Recently, Gopi et al., reported that the electrodeposition of minerals substituted HA on polymer coated 316L SS material at high current density of 9 mA/cm2 resulted with an uniform porous morphology.48 One of the limitations of porous M-HA coatings on pristine 316L SS implant material is the leach out of toxic metal ions through pores into the tissues. Furthermore, it is reported that the adhesion between the coating of porous HA and 316L SS is very poor. One of the important strategies used to generate more protective and adherent interfaces includes the use of conducting polymers. Conducting polymers are widely used for a variety of potential applications in several fields, including anticorrosion coatings.48,49 The electropolymerisation of conducting polymer on 316L SS substrate in presence of salicylate, forms a first layer for the coating of additional porous layer containing M-HA. While the porous M-HA coating surface can improve the implant fixation by tissue growth, the first layer of conducting polymer with an underneath salicylate passive film can act as an effective barrier for corrosion of 316L SS substrate in physiological fluid.

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Poly(3,4-ethylenedioxypyrrole) (PEDOP) has been extensively utilized as a biocompatible and anticorrosion coating for metallic implants due to its superior properties such as excellent conductivity, good environmental stability, easy polymerizability and biocompatibility with mammalian cells.50 In spite of these advantages, PEDOP has some inherent limitations such as poor mechanical stability, water permeability, lower bone-bonding ability and poor long term stability that limits its applications as a protective coating material for orthopaedic implant strategies.51-54 It is reported that the copolymerization enhances various properties of PEDOP films and it offers higher corrosion protection due to compact film formation.55 Recently, many researchers are focused on the electrochemical copolymerisation of thiophene and its derivative to improve their antistatic properties.56 The unique properties of Poly(3,4-ethylenedioxythiophene) (PEDOT), such as excellent environmental stability, high conductivity, excellent biocompatibility make it a desirable material for the application in orthopaedic implantable devices. 57-60 In the present work porous M-HA coating is electrodeposited on P(EDOP-co-EDOT) copolymer coated 316L SS. The influence of P(EDOP-co-EDOT) feed ratio and M-HA concentration on the structure and surface morphology of the P(EDOP-co-EDOT)/M-HA bilayer coatings on 316L SS was analysed. The corrosion protection performance of the P(EDOP-coEDOT)/M-HA bilayer coatings on 316L SS in Ringer’s solution was investigated using electrochemical techniques. Consequently, antibacterial activity and in vitro cell viability tests were carried out to confirm the biological performance of P(EDOP-co-EDOT)/M-HA bilayer coated 316L SS for orthopaedic applications. It can be noted that, to the best of authors knowledge, the electrodeposition of P(EDOP-co-EDOT)/M-HA bilayer coatings on 316L SS has not been reported so far. Finally, all the results showed that the P(EDOP-co-EDOT)/M-HA bilayer coated 316L SS can be potential for orthopaedic applications with enhanced corrosion protection performance and improved mechanical and biological properties. 5 ACS Paragon Plus Environment


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2. MATERIALS AND METHODS 2.1. Materials. The chemicals used for P(EDOP-co-EDOT) electropolymerization were 3,4ethylenedioxypyrrole (EDOP), 3,4-ethylenedioxythiophene (EDOT) and sodium salicylate procured from Sigma-Aldrich, USA. The purification of monomers like EDOP and EDOT were by performed by distillation process under reduced pressure and then stored at 4 ºC in a refrigerator. All the reagents used in the experiments were of analytical grade and no further purification is needed before using them. For M-HA electrolyte, calcium nitrate tetrahydrate (Ca(NO3)2.4H2O, 99.9 % purity, Sigma– Aldrich, USA), strontium nitrate hexahydrate (Sr(NO3)2.6H2O, 99.8 % purity, Sigma–Aldrich, USA), magnesium nitrate hexahydrate (Mg(NO3)2.6H2O, 99.8 % purity, Sigma–Aldrich, USA), cerium nitrate hexahydrate (Ce(NO3)3.6H2O, 99.9% purity, Sigma–Aldrich, USA), diammonium hydrogen phosphate ((NH4)2HPO4, 99.9% purity, Sigma–Aldrich, USA), ethanol (99.99% purity, Alfa Aesar, USA), ammonium hydroxide (NH4OH, Sigma Aldrich, UK) and hydrochloric acid (HCL, Sigma Aldrich, UK) were used. For the development of various coating and for the electrochemical characterization, deionized water and ethanol were used as the solvents. Also, the sterile distilled water was used for the assessment of antibacterial activity of the coatings.61 Deionized water was used in the preparation of all the aqueous electrolytes employed in the electrosynthesis of the P(EDOP-co-EDOT)/M-HA coatings and the whole experiments were performed at room temperature. 2.2. Preparation of 316L SS specimens. In present study, 316L SS which was purchased from Steel Authority of India Ltd.,(SAIL), India, was used as a substrate the composition of elements was (wt.%): C - 0.0222, Si - 0.551, Mn - 1.67, P - 0.023, S - 0.0045, Cr - 17.05, Ni - 11.65, Mo - 2.53, Co - 0.136, Cu - 0.231, Ti - 0.0052, V - 0.0783, N - 0.0659 and rest Fe, respectively.48 The substrates, 316L SS were cut into 10 × 10 × 3 mm3 dimensions and were then embedded in epoxy 6 ACS Paragon Plus Environment

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resin by leaving area of 1 cm2 for its contact to the electrolyte.7 Prior to each experiment, the working electrodes (316L SS) surface was ground mechanically using silicon carbide (SiC) sheets of different grades from 400 to 1200 grits. Then, the 316L SS substrates were thoroughly cleaned in 1:1 ethanol/acetone mixture in an ultrasonic bath to remove impurities, then rinsed with distilled water and then air dried for improved adhesion of coating onto the substrate. 2.3. Preparation of electrolyte. The electrolyte for the electropolymerization of P(EDOP-coEDOT) on 316L SS was prepared by mixing analytical grade 0.5 M sodium salicylate in a solution containing three different co-monomer feed ratio (EDOP:EDOT (90:10, 70:30, 50:50)) in an airtight container. The preparation of electrolyte for M-HA was done by dissolving analytical reagents like (0.25-Z) M Ca(NO3)2.4H2O, 0.2 M Sr(NO3)2.6H2O, 0.05 M Mg(NO3)2.6H2O, various concentrations (0.05, 0.075 and 0.1 M (Z)) of Ce(NO3)3.6H2O and 0.3 M (NH4)2HPO4. In brief, for the preparation of M-HA (with 0.05, 0.075 and 0.1 M of Ce3+) electrolyte, the pH solution was constantly monitored and attuned to 4.5 by adding either a diluted solution of 0.1 M HCl (or) a solution of NH4OH, as reported in detail elsewhere.41 The (NH4)2HPO4 solution was added dropwise into the mixed solution (Ca(NO3)2.4H2O, Sr(NO3)2.6H2O, Mg(NO3)2.6H2O and Ce(NO3)3.6H2O) to generate a colloidal solution and then subjected to magnetic stirring for 3 h at room temperature. The above solution was prepared to produce the proper molar ratio ((Ca+Sr+Mg+Ce)/P) of 1.67 which was used as the electrolyte for M-HA coating. During the M-HA deposition process, the electrolyte was de-aerated for 20 min with nitrogen (N2) in order to diminish the sum of dissolved carbon dioxide (CO2) and thus to reduce the formation of calcium carbonate (CaCO3) deposits. The reaction for the formation of M-HA (with 0.05, 0.075 and 0.1 M of Ce3+) is expressed as follows; (10-X-Y-Z) Ca(NO3)2.4H2O + X Sr(NO3)2.6H2O + Y Mg(NO3)2.6H2O+ Z Ce(NO3)3.6H2O + 6 (NH4)2HPO4 + 8 NH4OH → Ca10-X-Y-ZSrXMgYCeZ (PO4)6(OH)2-Y(O)Y +6 H2O + 20 NH4NO3 (where X=0.25 M, Y= 0.05 M, Z= 0.05, 0.075 and 0.1 M) 7 ACS Paragon Plus Environment


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2.4. Electrochemical deposition 2.4.1. Electropolymerization of P(EDOP-co-EDOT)-1 to P(EDOP-co-EDOT)-3 on 316L SS. The P(EDOP-co-EDOT)-1 (EDOP:EDOT (90:10)), P(EDOP-co-EDOT)-2 (EDOP:EDOT (70:30)) and P(EDOP-co-EDOT)-3 (EDOP:EDOT (50:50)) coating was developed by electropolymerization on 316L SS substrate from aqueous 0.5 M sodium salicylate solution containing different monomer feed ratio of EDOP and EDOT, respectively by cyclic voltammetry method with the potential ranging from -0.8 V and 2.0 V vs. SCE for about 20 cycles with a fixed scan rate of 100 mV/s.48 The electropolymerization was performed in a regular three electrode cell configuration using an electrochemical workstation (CHI 760C (CH Instruments, USA)) in which the 316L SS substrate, saturated calomel electrode (SCE) and platinum was used as the working electrode, reference electrode and counter electrode, respectively. All the measured potential values in the text are reported with respect to the SCE. After electropolymerization of the P(EDOP-co-EDOT), the coated electrodes were removed from the polymerization medium and washed with deionized water for several times to eliminate adsorbed electrolytes, monomers/oligomer molecules before being dried in air. 2.4.2.

Electrodeposition

of

M-HA

on

P(EDOP-co-EDOT)-3

coated

316L

SS.

The electrodeposition of porous M-HA (with 0.05, 0.075 and 0.1 M Ce3+) on P(EDOP-co-EDOT)-3 coated 316L SS was performed galvanostatically by electrochemical workstation (CHI 760C, CH Instruments, USA) at constant current density of 9 mA cm-2 for 30 min which was obtained as the optimum current density in our previous paper to produce uniform porous coating.48 Three series of M-HA electrolytes were utilized for the development of M-HA coatings (with 0.05, 0.075 and 0.1 M Ce3+), which are, Series 1 (M-HA-1 with 0.05 M Ce3+): 0.2 M Ca(NO3)2.4H2O, 0.2 M Sr(NO3)2.6H2O, 0.05 M Mg(NO3)2.6H2O, 0.05 M Ce(NO3)3.6H2O and 0.3 M (NH4)2HPO4 8 ACS Paragon Plus Environment

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Series 2 (M-HA-2 with 0.075 M Ce3+): 0.175 M Ca(NO3)2.4H2O, 0.2 M Sr(NO3)2.6H2O, 0.05M Mg(NO3)2.6H2O, 0.075 M Ce(NO3)3.6H2O and 0.3 M (NH4)2HPO4 Series 3 (M-HA-3 with 0.1 M Ce3+): 0.15 M Ca(NO3)2.4H2O, 0.2 M Sr(NO3)2.6H2O, 0.05 M Mg(NO3)2.6H2O, 0.1 M Ce(NO3)3.6H2O and 0.3 M (NH4)2HPO4 The deposition of P(EDOP-co-EDOT)/M-HA-1 to P(EDOP-co-EDOT)/M-HA-3 on 316L SS was carried out from the above series of solution under magnetic stirring at room temperature. Then the as-coated 316L SS substrates were softly rinsed with deionized water and then dried for 24 h at room temperature. 2.5. Characterization of the P(EDOP-co-EDOT)/M-HA bilayer coatings. Fourier transform-infrared spectroscopy (FT-IR, Impact 400 D Nicholet Spectrometer) was employed to categorize and confirm the functional groups of the as-formed P(EDOP-co-EDOT), M-HA and P(EDOP-co-EDOT)/M-HA bilayer coatings on 316L SS samples in the frequency range from 4000 cm-1 to 400 cm-1 with 32 scans and spectral resolution of 4 cm-1 in the transmittance mode. The coating samples scraped from the surface of 316L SS were mixed with KBr, pressed into discs and then analyzed. The structural elucidation of the as-synthesized P(EDOP-co-EDOT) copolymer coatings with three different co-monomer feed ratio (EDOP :EDOT (90:10, 70:30, 50:50)) were further supported by proton nuclear magnetic resonance spectroscopy (1H-NMR: Bruker DRX -400 MHz) at 25 ͦC, and the chemical shifts are reported in parts per million (ppm) using the dimethyl sulphoxide as solvent in a 5 mm diameter NMR tube and TMS as an internal reference. The phase identifications of the as-developed coatings were performed using X-ray diffraction (XRD, Seifert, X-ray diffractometer Siemens D500 Spectrometer) in the scattering angle range between 20° ≤ 2θ ≤ 60° with CuKα radiation generated at 40 kV and 30 mA with a step size of 0.02o at a scanning rate of 1o min-1. The XRD measurements were examined using powders scraped from 9 ACS Paragon Plus Environment


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the surfaces of the samples with standard data compiled by the International Centre for Diffraction Data (ICDD).9 The surface morphology of the as-deposited coatings were studied using a high resolution scanning electron microscopy (HRSEM, JEOL JSM-6400, Japan) with secondary electron detectors after gold sputtering at an accelerating voltage of 15.0 kV. The surface elemental composition of the P(EDOP-co-EDOT)/M-HA bilayer coatings was investigated by using an energy dispersive X-ray spectroscopy (EDAX). For the estimation of coating thickness, cross sectional SEM morphology of the P(EDOP-co-EDOT)/M-HA bilayer coatings on 316L SS was examined. The surface topography of the P(EDOP-co-EDOT) and P(EDOP-co-EDOT)/M-HA bilayer coated samples were assessed by AFM and the morphologies were obtained using a multimode scanning probe microscope (NTMDT, NTEGRA prima, Russia) operating in the semi contact mode with a spring constant of 1.6 N/m at a resonance frequency of 26 kHz. All the AFM images were documented under air atmosphere at room temperature. The root-mean-square (rms) roughness was determined from statistical application of the Nanoscope software, which evaluates the average considering all the values recorded in the topographic image with the exception of the maximum and the minimum. 2.6. Mechanical properties of the coatings 2.6.1. Adhesion tests. The adhesion strength can presents significant information about the mechanical properties of the coating and the underlying implant and also is one of the most vital properties for in vivo implantation.34 The adhesion measurements were performed on P(EDOP-coEDOT), M-HA and P(EDOP-co-EDOT)/M-HA(at optimum concentration of Ce3+) bilayer coated substrates, respectively by pull-out tests with at least ten measurements for each experiment according to the American Society for Testing Materials (ASTM) international standard F 1044-05. All the as-coated 316L SS cylindrical samples (25 mm in diameter) were bonded to the uncoated 10 ACS Paragon Plus Environment

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316L SS cylinder surface using quick set epoxy adhesive, the samples were cured in an oven at 100 ᵒ

C during 50 min and then the fixtures were subjected to pull-out tests using a Universal Instron

Mechanical Testing system (Model 5569, Instron Co. USA) at a crosshead speed of 1 mm min−1. 2.6.2. Vickers micro-hardness tests. The mechanical performance (Vickers micro-hardness (HV) tests) of the P(EDOP-co-EDOT), M-HA and P(EDOP-co-EDOT)/M-HA (at optimum concentration of Ce3+) bilayer coated 316L SS samples were carried out with a Vickers pyramid indenter, using an Akashi AAV-500 series hardness tester (Kanagawa, Japan).62

The HV measurements were

performed on the surfaces at a load 490.3 mN for a dwell time of 20 s and the HV value was calculated for each sample which was subjected to the average of ten measurements. 2.7. Investigation of electrochemical properties of the coatings. The corrosion protection performance of uncoated, P(EDOP-co-EDOT), M-HA and P(EDOP-co-EDOT)/M-HA (at optimum concentration of Ce3+) bilayer coated samples were examined by using potentiodynamic polarisation and electrochemical impedance spectroscopy at 28 ± 1 oC in Ringer’s solution (NaCl 8.6 g l-1, CaCl2.2H2O 0.66 g l-1 and KCl 0.6 g l-1) 48. Electrochemical corrosion experiments were done in the typical three-electrode system (CHI 760C, USA) using the electrochemical workstation (CHI 760C (CH Instruments, USA)) with 316L SS specimens of dimension 1 cm2 as the working electrode, SCE as the reference and platinum electrode as the counter electrodes and all the measured corrosion potential is related to the SCE. Before each electrochemical corrosion experiment, the working electrode samples were allowed to become stable until it reached a steady potential, which is denoted as the open circuit potential (OCP). The potentiodynamic polarisation studies were performed out at a potential range from -1000 to 1000 mV vs. SCE for uncoated, PEDOP, P(EDOP-co-EDOT)-3 and P(EDOP-co-EDOT)/M-HA-3 (at optimum concentration of Ce3+) bilayer coated samples. The electrochemical impedance studies (EIS) were recorded over the frequency range of 10-2 to 105 Hz at an OCP condition, with applied ac 11 ACS Paragon Plus Environment


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perturbation amplitude of about 5 mV. The analysis of the EIS spectra was evaluated by fitting the electrochemical experimental results to equivalent circuits. To ensure the reproducibility of the results, all the electrochemical experiments were executed in triplicate and the measurements were recorded using internally available software. 2.8. ICP-AES. The leach out characteristics of the uncoated, P(EDOP-co-EDOT), M-HA and P(EDOP-co-EDOT)/M-HA (at 0.1M of Ce3+) bilayer coated samples was evaluated in an impressed potential of 455 mV (vs. SCE) (just above the breakdown potential (Eb) of working electrode) in Ringer’s solution for 1 h. After experiments, the concentration of the leached out metal ions in the Ringer’s solution was investigated using ICP-AES (Thermo Jarrel Ash-Atom Scan (USA)). 48 2.9. TGA analysis. The thermal behaviour of the P(EDOP-co-EDOT), M-HA and P(EDOP-coEDOT)/M-HA(at optimum concentration of Ce3+) bilayer coated samples were investigated by TGA using Perkin Elmer, Diamond TG/DTA apparatus with heating rate of temperature ranging from 28 °C to 700 °C at 20 °C min-1 in air under a nitrogen atmosphere. 2.10. Biological characterization 2.10.1 Bacteria preparation and characterization. Freeze-dried E.coli (ATCC 25922) and S. aurus (ATCC 43300) were obtained from the American Type Culture Collection (Rockefeller, MD). Pure cultures of two strains were cultured aerobically at 37 C on sheep blood agar (SBA) plates for about 24 h. A single colony of the pure cultures was collected and incubated in 6 ml of sterile Trypticase Soy Broth (TSB) with agitation at 220 rpm for 12 h at 37 C. The inocula of the above strains were prepared by adjusting the concentration of bacterial broth culture to 1x106 colony forming units (CFUs) mL-1 with Dulbecco's modified Eagle's medium (DMEM) in TSB, supplemented with 10% fetal bovine serum (FBS) for in vitro experiments.


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2.10.2. Antibacterial activity. The antibacterial activity of all the coated samples are tested for the microorganisms that are responsible for implant associated infections.63 Majority of the infections in the hip and knee joints are caused by the S. aureus and E. coli, most commonly S. Aureus about 23% and coagulase-negative Staphylococci about 25%.64 The antibacterial activity of the P(EDOP-coEDOT)/M-HA (with 0.05, 0.075 and 0.1 M of Ce3+) bilayer coated samples was qualitatively studied by the disc diffusion test. By adopting the standard protocols the assessment of antibacterial activity was carried out. The agar disc diffusion study was carried out in the Muller-Hinton agar and the antibacterial activity test was done by pouring agar for 4 mm thick layers to the plates and then the nutrient medium were evenly inoculated with E.coli (Gram-negative bacteria) and S. aureus (Grampositive bacteria) and then incubated for 24 h in tryptic soy broth at 37 °C. Discs of 6 mm diameter were prepared from Whatman filter paper and were immersed into different volumes of (25, 50, 75, 100, 125 µl) P(EDOP-co-EDOT)/M-HA (with 0.05, 0.075 and 0.1 M of Ce3+) bilayer coated samples. The discs were placed at equal distance on the petri plates and then incubated at 37 °C for 24 h after which the region around the discs was observed for the inhibition of bacterial growth against two different bacterial strains.41 2.10.3. In vitro antibacterial assay. The in vitro antibacterial activity of the as-developed P(EDOP-co-EDOT)-3 and P(EDOP-coEDOT)/M-HA (with 0.05, 0.075 and 0.1 M Ce3+) bilayer coated 316L SS samples at 6, 12 and 24h was quantitatively analyzed by spread plate method. A total of 125 µL of the bacterial suspension (1x106 CFUs/mL) was added to each well in 24-well plate containing samples, and incubated at 37 C for 6, 12 and 24 h. To remove loosely adherent bacteria from the tested sample surface and for the evaluation of the number of viable adherent bacteria, the samples were mildly washed with PBS three times and the adhered bacteria on each sample were detached into 1 ml of PBS by ultrasonic shaking at 50 Hz (100 W) for 3 min. Then, the number of viable bacteria adhered on the samples 13 ACS Paragon Plus Environment


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were counted from the resulting bacterial suspension. The bacterial solutions were serially diluted ten-fold, plated in triplicate onto SBA and incubated at 37 C for 6, 12 and 24h. The number of CFUs on the SBA plates for various hours was counted in accordance with the ASTM E 2149 protocol. The antibacterial rates (Ra) for the adhered bacteria on the tested samples were calculated according to the formula. 2.10.4. Immersion of bilayer coated 316L SS in simulated body fluid (SBF). In vitro tests are performed to assess the bioactivity of the P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS in physiological medium. The preparation of SBF solution was done in accordance with Kokubo’s recipe.65 The P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS samples are immersed into airtight beaker containing 50 mL of SBF solution for time periods ranging from 1 to 14 days. After incubation at regular time period (i.e., after 1, 7 and 14 days), the samples were separated from SBF solution, then gently rinsed with de-ionized water and then dried at room temperature. The formation of bone-like apatite on the surfaces of the P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS can be obtained by HRSEM. 2.10.5. In vitro cytotoxicity study. MG63 osteoblast, a human osteosarcoma cell line (HOS MG63, ATCC CRL-1427 TM) purchased from National Centre for Cell Science (NCCS), Pune, India, is used in the investigation of in vitro cytocompatibility of the P(EDOP-co-EDOT)/M-HA (at 0.1 M Ce3+) bilayer coated 316L SS samples. All the cells were grown in culture media (Dulbecco's modified Eagle medium (DMEM, Gibco), containing minimal essential medium which is further supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), and 10 % FBS, respectively. The cells were then cultured in a 5 % CO2 incubator at 37 °C according to the protocol from ATCC. Then the culture medium was removed, and rinsed with PBS solution to eliminate all traces of serum. Then about 2 ml of Trypsin–EDTA solution was added in order to detach cells from flask and to that 6 ml of medium is added to aspirate cells by gentle pipetting. The cells were then counted 14 ACS Paragon Plus Environment

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with a hemocytometer, and 200 µl of suspension was seeded in each well with the cell density of 1 X 105 cells/mL, and then cultured in a 5 % CO2 incubator at 37 °C for 48 h. Then, HOS MG63 cells were incubated with P(EDOP-co-EDOT)/M-HA (at 0.1 M Ce3+) bilayer coated 316L SS sample. After 1, 4 and 7 days of incubation, the media was separated and the wells were washed three times with phosphate buffered saline (PBS, OXOID Limited, England)) prior to any toxicity assays.46 An MTT assay was carried out for the incubation time period of 1, 4 and 7 days. The viability of cells were evaluated using a modified MTT (3-(4,5-dimetyl-2-tiazolyl)-2,5diphenyl-2H tetrazolium bromide) assay. After the incubation period, MTT solution in 1 ml serum free medium was added to it and incubated at 37 °C for 4 h in a humidified 5% CO2 atmosphere. The above solution was then removed, dimethyl sulfoxide was added, and before measuring absorbance value the well plate was shaken for 15 min. The absorbance was recorded by using an ELISA microplate reader at 570 nm wavelength, and the cell viability (%) of the P(EDOP-co-EDOT)/M-HA (at 0.1 M Ce3+) bilayer coated 316L SS sample was calculated with respect to control wells using: % Cell viability = [A] Test / [A] Control × 100. 2.10.6. Cell adhesion test. Human osteoblast (HOS MG63) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin-streptomycin cocktail. Prior to cell seeding, the P(EDOP-co-EDOT)/M-HA (at

0.1 M Ce3+) bilayer coated

samples were kept for sterilization in autoclave (121 ºC, 0.1 MPa pressure for 15 min) and subsequently, the cells with the density of 5X 105 mL-1 were seeded on the bilayer coated samples. The samples with cells were then incubated in CO2 incubator under standard culture conditions. After the stipulated time period of incubation (36-48 h), the samples were washed twice with phosphate buffer saline (pH 7.4). The culture medium was removed at every 2 days interval and then fresh culture medium was added into each well. For the evaluation of cell morphology, the HOS MG63 cells seeded samples were fixed with 2% glutaraldehyde at room temperature for 1 h, and 15 ACS Paragon Plus Environment


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then dehydrated with a series of ethanol/water solution for 10 min. Then, 0.5 mL of hexamethyldisilazane (HMDS, Hi Media) was added to each well, to preserve the original morphology of the cells. The samples were coated with gold (for conduction) for observing the morphology of the cells on the samples by HRSEM. 3. RESULTS AND DISCUSSION 3.1. Electropolymerisation of P(EDOP-co-EDOT) copolymer coatings. The electropolymerization of EDOP and EDOT in 0.5 M sodium salicylate solution was carried out on

cycling

the

potential

between

-0.5

and

+2.0

V

at

a

fixed

scan

rate

of

100 mV s-1 and for 20 cycles. In order to identify the changes in cyclic voltammetric behavior of 316L SS in the copolymerization of EDOP and EDOT with optimum concentration of monomer feed ratio (50:50), the voltammogram was obtained over 20 cycles and is shown in Figure 1. During the cycle, the oxidation peak observed around 1.3 V vs. SCE is ascribed to the electrochemical polymerisation of comonomers as well as the oxidation of electrolyte (ie., salicylate) forming a passive film beneath the P(EDOP-co-EDOT) copolymer coating. Separate voltammetric peaks cannot be detected owing to overlapping of anodic peaks of comonomers (EDOP and EDOT) and salicylate.34,48 Application of anodic potential could improve the oxidation of monomers (EDOP and EDOT) to form the active diradical cations, which is unstable and thus polymerizes with other active group to form dimer radical cations, followed by additional reaction with another group to form trimer radical cations and so on. This oxidation of monomers takes place adjacent to the 316L SS electrode surface and the growth of P(EDOP-co-EDOT) copolymer is intensified during reverse sweep. Gopi et.al., found that the quality of the passivating layer o n the 316L SS is very effective on adhesion behavior and physicochemical properties of the electrochemical polymerized coating. The electrochemical polymerization mechanism proposed in recent literature is normally used to 16 ACS Paragon Plus Environment

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elucidate the polymerization of conducting monomer. Based on the obtained voltammetric results, the P(EDOP-co-EDOT) copolymer formation mechanism on the 316L SS surface could be clarified in two stages: (i) decrease of metal ion dissolution by forming a metal-salicylate layer and (ii) radical cation formation followed by the electropolymerization on the working electrode surface.66 The oxidation of sodium salicylate which forms a passive film is advantageous for the electropolymerisation of monomers (EDOP and EDOT) and the formation of P(EDOP-co-EDOT) copolymer layer on the 316L SS surface.

34

With increasing number of potential cycles,

34

the

intensity of the oxidation peak of monomers (EDOP and EDOT) gradually decreases while a slight increase in current density was observed at the end of each cycle which is attributed to the process of monomer oxidation that occurs in a step by step manner indicating that a homogeneous

34,67,68

and

strong adherent P(EDOP-co-EDOT) copolymer film is grown over 316L SS surface with increasing thickness.48 3.2.

Characterisations

3.2.1. FT-IR analysis. The FT-IR spectra for the P(EDOP-co-EDOT) copolymers (with EDOP: EDOT (90:10, 70:30, 50:50)) and P(EDOP-co-EDOT)/M-HA (with 0.05, 0.075 and 0.1 M Ce3+) bilayer coated 316L SS, respectively are shown in Figure

2 (a-f). It is observed that the

characteristic peaks of both EDOP and EDOT appeared in the spectra (Figure 2 (a-c)) and are intensified by the concentration of monomers. As seen from the spectrum (Figure 2 (a)) of P(EDOPco-EDOT), the characteristic peaks found at 642 and 1142 cm−1 are ascribed to the stretching of N−H and C−N groups in the pyrrole ring, respectively.66 Apart from the EDOP characteristic peaks, the EDOT peaks are clearly found at 842, 1194, 1387 and 1576 cm−1, which originate from the stretching modes of C-S-C, C-O-C groups, and vibration modes of the C=C and C-C bonds in thiophene ring, respectively. In addition, a characterized strong peak at 1619 cm-1 is responsible for the formation of metal-sodium salicylate complex.

48

All the P(EDOP-co-EDOT) copolymer



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coatings spectra (Figure 2 (a-c)) strongly demonstrate the characteristic peaks of both EDOP and EDOT units. It is importance noting that the intensity of the corresponding EDOT peaks was increased with increasing feed ratio of EDOT in the copolymer, which is in well agreement with the fact that P(EDOP-co-EDOT) copolymer structure depends only on the monomer feed ratio in the sodium salicylate solution. The appearance of these FT-IR characteristic peaks confirmed the formation of a pure P(EDOP-co-EDOT) copolymer coatings on 316L SS substrates. Besides the all above peaks belonging to P(EDOP-co-EDOT), the FT-IR spectra of P(EDOP-co-EDOT)/M-HA (with 0.05, 0.075 and 0.1 M Ce3+) bilayer coated 316L SS, exhibited peaks at 947 cm-1 (ν1) and 472 cm-1 (ν2) as well as at 1018 & 1086 cm-1 (ν3) and 561 & 590 cm-1 (ν4), respectively which are due to the phosphate groups of M-HA unit. The stretching and bending mode of OH- groups can be found at 3580 and 631 cm-1, respectively. In addition, the characteristic peaks observed at 3446 cm-1 and 1628 cm-1 reveal the stretching and bending modes of water molecule, indicating the presence of M-HA in the bilayer matrix.48 All the above features imply that the P(EDOP-co-EDOT)/M-HA bilayer coatings contain both the P(EDOP-co-EDOT) and M-HA units. These results strongly demonstrate the formation of P(EDOP-co-EDOT)/M-HA bilayer coatings on 316L SS surface. 3.2.2. XRD analysis XRD patterns were used to confirm the phase of the P(EDOP-co-EDOT) copolymers (with EDOP: EDOT (90:10, 70:30, 50:50)) and P(EDOP-co-EDOT)/M-HA(with 0.05, 0.075 and 0.1 M Ce3+) bilayer coated 316L SS, as depicted in Figure 3(a-f). The broad diffraction peaks in the region of 2θ° at 23.6° and 25.2° in the XRD pattern of P(EDOP-co-EDOT) are attributed to the EDOP and EDOT units, respectively which indicates the amorphous structure of the electrodeposited P(EDOP-co-EDOT) coating and are in well agreement with the previous report.68 As seen from the Figure 3 (a-c), all the P(EDOP-co-EDOT) copolymer coatings spectra strongly 18 ACS Paragon Plus Environment

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demonstrate the peaks for both EDOP and EDOT units. Further, the intensity of the corresponding EDOT diffraction peak is increased with increasing ratio of EDOT in P(EDOP-co-EDOT)-1 to P(EDOP-co-EDOT)-3, which shows that the P(EDOP-co-EDOT) structure depends only on the EDOT monomer feed ratio in the copolymer electrolytic solution. These XRD results strongly indicates the formation of a pure P(EDOP-co-EDOT) coating on 316L SS substrate.67 In the XRD patterns for P(EDOP-co-EDOT)/M-HA(with 0.05, 0.075 and 0.1 M Ce3+) bilayer coated 316L SS (Figure 3 (d-f)), a broad peak of P(EDOP-co-EDOT) existing along with the peaks for M-HA reveals that the bilayer coatings have a more ordered arrangement than the pure P(EDOPco-EDOT) copolymer due to the presence of M-HA. These XRD patterns indicate that the M-HA coating strongly influenced the crystalline behaviour of the P (EDOP-co-EDOT)/M-HA bilayer coatings. The peaks (Figure 3(f)) of M-HA correspond to the 2θ values of 25.8°, 30.4°, 31.9°, 32.5° and 35.9 and no other secondary characteristic peaks were found. The XRD peaks identified for MHA match well with the standard XRD data for HA and also well consistent with International Centre for Diffraction Data (ICDD card No. 09-0432).41 Whereas the XRD diffraction peak positions deviated slightly towards the lower angles from the standard XRD patterns for HA, which may be due to crystal lattice distortion that has occurred as a result of substitution of minerals (Sr, Mg and Ce) in the HA lattice. This could be recognized due to the presence of M-HA in the bilayer coatings. Thus the presence of diffraction peaks corresponding to P(EDOP-co-EDOT) and M-HA in the XRD pattern of P(EDOP-co-EDOT)/M-HA bilayer coatings (Figure 3(d-f)) suggests that the P(EDOP-co-EDOT) have been coated additionally with M-HA coating to form the bilayer. 3.2.3. 1H-NMR spectral analysis. In order to identify the structure and formation of P(EDOP-coEDOT) copolymer with three different ratios of monomers (EDOP: EDOT (90:10, 70:30, 50:50), 1

H-NMR spectra were performed at room temperature in DMSO-d6 solvent and the results are 19 ACS Paragon Plus Environment


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showed in Figure 4(a-c). These spectra are characterized by four main signals which exactly correspond to the four types of protons on the P(EDOP-co-EDOT) copolymer chain. The two strongest signals for protons of DMSO and water in DMSO were observed at δ 2.7 and 3.3 ppm, respectively. The signal observed in the comparatively shielded region around 5.2-5.7 ppm can be ascribed to the protons of N-H group, which means that the pyrrole group is present in the P(EDOPco-EDOT) copolymer chain. The copolymer exhibited the signals from 6.3 to 6.7 ppm which are assigned to the thiophene 67 protons in EDOT unit. While the signals observed at 4.1-4.3 are ascribed to the ethylenic protons of copolymers, which confirm the presence of EDOT monomer units in copolymer P(EDOP-co-EDOT) chain. The intensity of signals corresponding to the thiophene proton increased with increasing EDOT feed ratio from P(EDOP-co-EDOT)-1 to P(EDOP-co-EDOT)-3, which indicates that the number of EDOT unit is increased in P(EDOP-co-EDOT) copolymer chain. The protons of EDOP are overlapped with the protons of EDOT. Thus, the participation of EDOP and EDOT comonomeric units in the P(EDOP-co-EDOT) copolymers is confirmed. 66 The XRD and 1H-NMR results substantiated a suitable structure (Figure

3 & 4) for

copolymer P(EDOP-co-EDOT) with monomer of EDOP and EDOT rings in adjacent positions which is also similar to the findings of other researchers. 3.2.4. SEM and EDAX analysis. The morphology and structure of conducting polymers and calcium phosphate strongly influence their properties. Representative SEM micrographs and EDAX images of the P(EDOP-co-EDOT) copolymers (with EDOP: EDOT (90:10, 70:30, 50:50)) and P(EDOP-co-EDOT)/M-HA(with 0.05, 0.075 and 0.1 M Ce3+) bilayer coated 316L SS, respectively are displayed in Figure 5(a-h). The micrographs of P(EDOP-co-EDOT) copolymer coating showed a slight difference with varying monomers feed ratio in P(EDOP-co-EDOT) chain. The morphology of P(EDOP-co-EDOT)-1 coating on 316L SS (Figure

5(a)) exhibited a typical granular-like

particles with irregular pores in between them. 20 ACS Paragon Plus Environment

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In the case of P(EDOP-co-EDOT)-2 coating, aggregated and irregularly arranged particles were observed as shown in Figure 5(b). With increasing EDOT and decreasing EDOP ratio, the nucleation and accumulation of granules were exactly seen in the copolymer coatings. When the feed ratio of EDOT is increased from 10 to 50, it is clearly seen that the P(EDOP-co-EDOT)-3 coated 316L SS surface (Figure 5(c)) revealed an uniform and more compact coatings with wellordered three dimensional granular structure formation without any pores in nature and these results are in well agreement with the AFM results. Thus the copolymer coating on the substrate can act as an adherent barrier coating and reduce the rate of corrosion.67 Figure 5(d) shows the EDAX spectrum of the P(EDOP-co-EDOT)-3 copolymer sample which indicates the presence of C, N, O and S there by confirming the existence of P(EDOP-co-EDOT)-3 copolymer coating on the 316L SS sample. Figure 5(e-g) shows the surface morphology of the electrodeposited M-HA with 0.05, 0.075 and 0.1 M Ce3+ on P(EDOP-co-EDOT)-3 copolymer coated 316 L SS at 9 mA cm-2 for the duration of 30 min, respectively. While comparing the morphology of the M-HA coatings at three different concentrations of Ce3+ (0.05, 0.075 and 0.1 M), the coating obtained at 0.1 M Ce3+ (Figure 5g) consisting of uniform distribution of porous network structure is considered as optimum. The similar porous morphology has been obtained for all the three M-HA samples on P(EDOP-co-EDOT)-3 copolymer coated 316L SS (Figure 5(e-g)). The results (Figure 5(e-g)) revealed that the influence of Ce3+ concentration on the morphology of the M-HA coating is less appreciable. The porous nature of the M-HA coating is a result of H2 bubbles evolution at the working electrode (316L SS) surface. The interconnected pores can be beneficial for circulation of the physiological fluid throughout the coating when it is used as a bone implant for biomedical application.48 The interconnected porosity can allow the proliferation and attachment of various cell types accountable for bone tissue formation. 21 ACS Paragon Plus Environment


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The EDAX spectrum (Figure 5h) of the P(EDOP-co-EDOT)/M-HA bilayer coatings on 316L SS reveals the presence of Ca, Sr, Mg, Ce, O and P there by confirming the existence of P(EDOP-co-EDOT)/M-HA bilayer coatings on 316L SS surface. The HRSEM image shown in Figure 5(i) presents the cross section of P(EDOP-coEDOT)/M-HA (with 0.1M Ce3+) bilayer coatings on 316L SS. The formation of bilayer with distinct porous morphology of M-HA over the P(EDOP-co-EDOT) coated 316L SS sample is noticed from the cross-sectional image and the thickness of the porous M-HA coating was about ~140 µm on the P(EDOP-co-EDOT) thickness (~14 µm) coated 316L SS specimen (Figure 5i). However the top MHA coating demonstrated a porous network and a continuous interface without any pores or cracks along its length is observed between the P(EDOP-co-EDOT) layer and substrate. Hence P(EDOPco-EDOT)/M-HA (with 0.1M Ce3+) bilayer coated 316L SS surface may be more corrosion protective because there may not be any leach out of metal ions from 316L SS in physiological body fluids. 3.2.5. Surface roughness by AFM. The surface topography of P(EDOP-co-EDOT)-3 and P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coatings on 316L SS samples was studied using AFM and the Figure

S1 (a&b) shows the observed topographic images. The AFM

topographical image for P(EDOP-co-EDOT)-3 coating with optimum feed ratios of monomers (50:50) on 316L SS sample (Figure S1(a)) clearly showed that the coating surface is more compact, dense, continuous and uniform in nature (Figure S1 (a)). The P(EDOP-co-EDOT)/M-HA(with 0.1 M Ce3+) bilayer coated 316L SS topography is presented in Figure S1(b). The topographical image of the bilayer coatings Figure S1(b), revealed the uniform distribution of porous network structure on the compact polymer layer. The average root mean squared roughness value for the P(EDOP-coEDOT)-3 compact layer measured for 1.6 x 1.6 µm2 was of 350±28.6 nm whereas for the P(EDOPco-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coatings measuring the value for 5 x 5 µm2 was found to 22 ACS Paragon Plus Environment

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be 1.6±0.18 µm, respectively. Comparatively, an increased average roughness value was observed for the bilayer coatings surface. 3.3. Mechanical characterisation of the P(EDOP-co-EDOT)/M-HA bilayer coatings 3.3.1. Adhesion strength.

The adhesion strength of the as-developed coatings over 316L SS

specimens was examined using the ASTM F 1044-05 adhesion test method and the values of the adhesion strength are shown in Figure S2(a) and Table.1. Here, the adhesion strength (Figure S2(a) and Table.1) of the M-HA (at 0.1M Ce3+), P(EDOP-co-EDOT)-3 and P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coatings on 316L SS specimen, respectively was evaluated. The adhesive strength of the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated 316L SS (14.5 ± 1.1 MPa) was found to be greater than that of the P(EDOP-co-EDOT)-3 and M-HA (at 0.1M Ce3) coatings, which were exhibited as 13.8± 1.2 and 10.9± 0.7 MPa, respectively. Hence, the results clearly reveal that the P(EDOP-co-EDOT)/M-HA bilayer coatings possessed good adhesion to the 316L SS surface. This adhesion strength of the as-developed P(EDOP-co-EDOT)/M-HA bilayer coatings will make it appropriate for orthopaedic applications. 3.3.2. Vickers micro-hardness. The micro-hardness (Hv) of the coating is an important requirement for the biomedical implants for providing the information about the load bearing tendency when it is implanted into human body under stress. In our present study, the hardness values of the uncoated 316L SS and M-HA (at 0.1M Ce3+), P(EDOP-co-EDOT)-3 and P(EDOP-coEDOT)/M-HA (with 0.1 M Ce3+) bilayer coated 316L SS substrates, respectively were evaluated using the Vickers micro hardness test and the results are shown in Figure S2 (b) and Table.1. The M-HA (at 0.1M Ce3+) and P(EDOP-co-EDOT)-3 coated substrates exhibited higher hardness values of 341.6± 10.2 and 110.8 ± 13.2 Hv, respectively. The Hv value for the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated 316L SS sample was observed to be 391.8 ± 12.3 Hv which is still higher than that of the M-HA (at 0.1M Ce3+) and P(EDOP-co-EDOT)-3 coated specimens. This is 23 ACS Paragon Plus Environment


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owing to the indivisible and intact bonding strength between the M-HA and P(EDOP-co-EDOT) layers of the as-developed bilayer. 3.4.

Electrochemical characterisation

3.4.1. Potentiodynamic polarisation. Corrosion resistance is an important requirement for the biomedical applications of 316L SS, and, hence, the corrosion protection performance of uncoated and bilayer coated 316L SS is investigated in Ringer’s solution for long-term biomedical applications. Figure 6 shows the potentiodynamic cyclic polarisation curves obtained for the uncoated 316L SS, PEDOP, P(EDOP-co-EDOT) copolymer and P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated 316L SS samples, respectively in Ringer’s solution in a potential range of -1000 mV to +1000 mV vs. SCE at an OCP condition. The values of the corresponding corrosion parameters such as corrosion potential (Ecorr), breakdown potential (Eb) and repassivation potential (Epp) derived from these curves are presented in the Table 2. The Ecorr, Eb and Epp values obtained from the potentiodynamic polarization curves for the uncoated 316L SS specimen were -870, +449 and -90 mV (vs. SCE), respectively. For the PEDOP coated speciment, the Ecorr, Eb and Epp values were observed to be as -510 mV, 534 mV and 138 mV vs. SCE, respectively. An analysis of polarization curves showed a positive shift in the Ecorr, Eb and Epp values of the electrochemically synthesised P(EDOP-co-EDOT) copolymers (with three different feed ratios of monomers) coatings on 316L SS, which indicates the corrosion protection efficiency of the copolymers. In particular, the polarization values of P(EDOP-co-EDOT) coated 316L SS increased with increasing EDOT units into EDOP. The Ecorr, Eb and Epp values of the copolymers were found to be increased in the following order P(EDOP-co-EDOT)-1 < P(EDOP-co-EDOT)-2 < P(EDOP-co-EDOT)-3 This implied that the P(EDOP-co-EDOT)-3 copolymer coating has greater protection efficiency for 316L SS against corrosion in Ringer’s solution when compared to the P(EDOP-co24 ACS Paragon Plus Environment

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EDOT)-2 and P(EDOP-co-EDOT)-1 copolymer coatings due to its more compact morphology with uniform arrangement of granules and this is in well agreement with HRSEM and AFM results. The polarisation curve of the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated 316L SS specimen showed the Ecorr value of -361 mV (vs. SCE) while the Eb and Epp values were found to be 962 mV and 291 mV vs. SCE, respectively. These Ecorr, Eb and Epp values of the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated specimen showed a maximum nobler shift when compared with that of the uncoated, PEDOP, and P(EDOP-co-EDOT) (with EDOP: EDOT (90:10, 70:30, 50:50)) 316L SS specimens (Table 2). The maximum positive shift in the values of Ecorr, Eb and Epp in the noble direction demonstrate that the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated 316L SS specimen exhibited the maximum corrosion protection performance in Ringer’s solution. The significant higher values of Ecorr, Eb and Epp for the P(EDOPco-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated 316L SS when compared with the other coated specimens permit a significant improvement in the corrosion protection performance by hindering the access of the electrolyte to the substrate. The higher protection performance of the P(EDOP-coEDOT)/M-HA (with 0.1 M Ce3+) bilayer coated substrate is attributed to the uniform and compact surface coverage of the primary layer P(EDOP-co-EDOT)-3. Thus, the copolymer coated 316L SS can serve as a protective barrier layer between the top porous layer of M-HA (with 0.1 M of Ce3+) and the 316L SS substrate which is in correlation with the cross-sectional HRSEM and AFM analysis. 3.4.2. Electrochemical impedance spectroscopy. The EIS measurements were also performed in Ringer’s solution, in order to get better understanding in corrosion performance aspects of all the uncoated and as-coated 316L SS specimens. Figure 7(a&b) shows the fitted equivalent circuits obtained for P(EDOP-co-EDOT)-3 and P(EDOP-co-EDOT)/M-HA (with

0.1 M Ce3+) bilayer

coated 316L SS samples in Ringer’s solution. The EIS spectrum obtained for P(EDOP-co-EDOT)-3 25 ACS Paragon Plus Environment


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coated 316L SS was fitted using the equivalent circuit represented as Rs(R1Cdl) (R2Cdl1) in Figure 7(a) in which the two combinations of resistor and capacitor are in series with the solution resistance. In Figure 7(a), Rs represents the solution resistance, whereas R1 and Cdl represent the charge transfer resistance and double layer capacitance of the uncoated 316L SS specimen and R2 and Cdl1 represent the resistance and double layer capacitance of the P(EDOP-co-EDOT)-3 layer, respectively. The impedance spectrum for P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated specimens was fitted using equivalent circuit (Figure 7(b)) consisting of three combinations of resistor and capacitor in series with solution resistance and represented as Rs(R1Cdl) (R2Cdl1) (R3Cdl2), where, R3 and Cdl2 represent the resistance and double layer capacitance for the porous MHA (with 0.1 M Ce3+) layer on the P(EDOP-co-EDOT)-3 coated 316L SS specimen, respectively. It strongly demonstrated the two time constant, which corresponds to the top porous M-HA layer and the underneath compact P(EDOP-co-EDOT)-3 copolymer layer on 316L SS. This suggests an effective barrier property for this P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coatings, which is indicating its physical ability to inhibit the leach out of metallic ions from the substrate. Figure 8(a–c) shows Bode and phase plots which represent the uncoated, PEDOP, P(EDOPco-EDOT) (with three different feed ratios of monomers) and P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated specimens in Ringer’s solution at an OCP condition. As discussed earlier, the EIS parameters such as polarisation resistance (Rp) and the total impedance (|Z|) were calculated after fitting the spectra to the equivalent circuits (Figure 8a and b). The corresponding EIS parameters such as polarisation resistance (Rp) and the total impedance (|Z|) are shown in Figure 8 and their values are presented in the Table 2. The values of Rp and |Z| for the uncoated 316L SS specimen were obtained from Figure 8 as 41 and 58 Ω cm2, respectively. The Rp and |Z| values obtained for the PEDOP coated 316L SS were found to be 2494 and 2612 Ω cm2, respectively which are greater when compared with that of uncoated 316L SS sample. It can be clearly observed from 26 ACS Paragon Plus Environment

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the Figure 8 that the EIS plots showed positive shift in Rp and |Z| values of the P(EDOP-co-EDOT) copolymers (with three different feed ratios of monomers) coatings on 316L SS, which strongly demonstrates the effective corrosion protection performance of the copolymers and also the values increased with increasing EDOT units into EDOP chain. The Rp and |Z| values were found to be increased in the following order, P(EDOP-co-EDOT)-1 < P(EDOP-co-EDOT)-2 < P(EDOP-co-EDOT)-3 For the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated 316L SS sample the Rp and |Z| values are obtained as 3372 and 3491 Ω cm2, respectively which are greater than that of the P(EDOP-co-EDOT)-3 coated 316L SS samples. Two capacitive semicircles were obtained for the P(EDOP-co-EDOT)/M-HA(with 0.1 M Ce3+) bilayer coated 316L SS sample. Out of these two, the first semicircle at higher frequencies is attributed to porous M-HA (with 0.1 M Ce3+) coating and the second semicircle at low frequencies corresponds to the compact copolymer coating of P(EDOP-coEDOT)-3. Furthermore, the comparison of the EIS values of all the coated 316L SS specimens revealed that the highest value of Rp and |Z| obtained for the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coatings are owing to the greater effective barrier of P(EDOP-co-EDOT)-3 coating and the top layer of porous M-HA (with 0.1 M Ce3+). These results show that P(EDOP-co-EDOT)-3 coating acts as an effective physical barrier between the 316L SS and top porous M-HA coating (with 0.1 M Ce3+) and thus enhance its corrosion resistance in the physiological environment. As a result of these electrochemical investigations, it can be concluded that the P(EDOP-co-EDOT)/MHA (with 0.1 M Ce3+) bilayer coatings is superior in corrosion protection for 316L SS bioimplants. 3.5. ICP-AES analysis. Figure 9 shows the level of metallic ion release from uncoated 316L SS, P(EDOP-co-EDOT)-3 and P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated 316L SS samples, respectively in Ringer’s solution after ageing of 1 h at an impressed potential of 455 mV (vs. SCE).34 The ICP-AES investigation made for the uncoated 316L SS sample which was subjected 27 ACS Paragon Plus Environment


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to the impressed potential of 455 mV in the Ringer’s solution, shown a major amount of leached metallic ions such as Cr, Fe, Ni and Mo. 34 This is ascribed to the absence of barrier film on the 316L SS surface to avoid the attack of chloride ions present in the Ringer’s solution. The P(EDOP-coEDOT)-3 coated 316L SS sample in the Ringer’s solution shown that the leach out of metallic ions was noticeably reduced compared to that of the uncoated 316L SS

34

which is due to the as formed

passive film by the P(EDOP-co-EDOT)-3 in its underneath that protects the 316L SS. Thus, the P(EDOP-co-EDOT)-3 layer plays a double role of forming the passive film in its underneath and to protect the breakdown of passive film from the harsh environment which in turn offers superior corrosion protection performance of 316L SS implant with longer life time in human body.34 Moreover, in the case of P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated 316L SS sample, the leach out tendency of metallic ions are significantly reduced than uncoated and P(EDOP-co-EDOT)-3 coated 316L SS (Figure 9) samples at the impressed potential of 455 mV (vs. SCE). Thus, the coating of P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer on the 316L SS sample can certainly reduce the rate of leach out of metallic ions and their adverse effects in the Ringer’s solution.34 So, this P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated 316L SS sample with excellent anti-corrosion resistance diminishes the leach out of metal ions when implanted into the real biological system with longer life time. 34 3.6. Thermal analysis. The P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coatings was scratched from the 316L SS surface and its thermal stability analysis was studied using thermo gravimetric method as shown in Figure S3. The thermal stability of the as-coated sample is very important for their potential application. Also, TGA is significant dynamic method for detecting the degradation behaviour of P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coatings. The TGA analysis was carried out under a nitrogen steam at a heating rate of 20 oC min-1 in the temperature range from 28 °C to 850 °C. In the TGA plot of the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+), 28 ACS Paragon Plus Environment

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the initial weight loss starting at 100 °C is attributed to the trapped water bonded to the copolymer chain through a hydrogen-bonding interaction. In a P(EDOP-co-EDOT) copolymer units, each monomer is generally independently degraded in response to heating. The TGA plot of the copolymer would comprise a plateau starting at a temperature of 350 °C, and a mass loss of 41% is obtained at 750 °C according to the thermal properties of monomers such as EDOP and EDOT which may occur from the fact that the EDOP and EDOT rings in the P(EDOP-co-EDOT)-3 copolymer are carbonized which converts the polymeric chains to char residuals and the total weight loss of the bilayer coatings is found to be 20.24%. The weight loss can be associated with the structurally incorporated polymers and evaporation of adsorbed water and solvent molecules. There is no major weight loss observed above 600 °C, demonstrating that the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coatings generate a stable phase after heat-treatment at temperatures above 600 °C. These results indicated the good thermal stability of P(EDOP-co-EDOT)-3 copolymer coating on 316L SS. 3.7. Biological characterizations 3.7.1 Antibacterial activity by disc diffusion method. In recent year, research has been focused on the coating of bioceramic based material that provide antibacterial activity to prevent surgical site infections associated with the implants.69 In vitro antibacterial activity of the P(EDOP-co-EDOT)/MHA (with 0.05, 0.075 and 0.1 M Ce3+) bilayer coated 316L SS was qualitatively evaluated against the bacterial strains such as E. coli and S. aureus, respectively, which are often accountable for post surgical illness in the orthopedic surgery. All the coated samples exhibited antibacterial activity and the zone of inhibition around the P(EDOP-co-EDOT)/M-HA (with 0.05, 0.075 and 0.1 M Ce3+) bilayer samples at five different concentrations such as 25, 50, 75, 100 and 125 µL against E. coli and S. aureus are depicted in Figure 10. These data revealed that as the Ce3+ concentration in the coating material increases, the antibacterial activity of the coated sample increases. When compared 29 ACS Paragon Plus Environment


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to the P(EDOP-co-EDOT)/M-HA (with 0.05M Ce3+) and P(EDOP-co-EDOT)/M-HA (with 0.075 Ce3+) coated samples, the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) coated sample demonstrated an improved anti-bacterial activity which is apparent from the zone of inhibition (Figure 10). The inhibition zones for the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) sample against E. coli and S. aureus were found to be 11.5 & 10.3 mm for 25µL, 12.4 & 11.8 mm for 50 µL, 13 .9& 12.7 mm for 75 µL, 14.7 & 13.4 mm for 100 µL and 15.7 & 14.3 mm for 125 µL volumes, respectively. At 125 µg /ml, the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) coated sample recorded the highest level of antimicrobial activity against E. coli by exhibiting the highest inhibition zone diameter. The activity of P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coatings against E. coli strain was slightly higher than that of activity against S. aureus due to the differences observed in the cell wall structure. It is obvious that the antibacterial property of the bilayer coated samples was dependent on the molecular structure of the material, the type of bacterial strains used and the solvents involved in the process. Thus, the substitution of minerals like Sr2+ and Ce3+ plays a significant role in improving the antibacterial activity.48 3.7.2. In vitro antibacterial property by spread plate method. The antibacterial rate (Ra) of P(EDOP-co-EDOT)-3 and P(EDOP-co-EDOT)/M-HA (with 0.05, 0.075 and 0.1 M Ce3+) bilayer coated 316L SS samples at 6, 12 and 24h, respectively was quantitatively assessed by the spread plate method (Figure 11). From the figure, the Ra values of P(EDOP-co-EDOT)-3 against S. aureus and E. coli were found to be 45.6%, 51.9% and 59% at 6, 12 and 24 h for S. aureus and 53.2%, 62.7% and 69.3% at 6, 12 and 24 h for E. coli, respectively, representing that P(EDOP-co-EDOT)-3 can inhibit bacteria adhesion and proliferation. When compared to the P(EDOP-co-EDOT)-3, P(EDOP-co-EDOT)/M-HA (with 0.05M Ce3+) and P(EDOP-co-EDOT)/M-HA (with 0.075 Ce3+) coated samples, the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated sample exhibited an improved bacterial inhibition which is clear 30 ACS Paragon Plus Environment

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from the Ra values (Figure 11). The Ra values for the P(EDOP-co-EDOT)/M-HA (with 0.1 M of Ce3+) sample against S. aureus and E. coli were found to be increasing as 80.7%, 90.2% and 96.1% at 6, 12 and 24 h for S. aureus and 83.5%, 91.7% and 99.9% at 6, 12 and 24 h for E. coli, respectively, indicating that P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) has the strongest antibacterial activity. 3.7.3. In vitro bioactivity assessment of the coating in SBF. Generally, when bioceramic coated implant materials were immersed in SBF solution, a thick apatite layer is observed over the surface. This is because, when a bioceramic material is immersed in SBF solution, the apatite layer on the surface undergoes a series of chemical reactions like precipitation, nucleation and formation of calcium phosphate. The structure of HA consists of Ca2+, PO43-, and OH- groups that are tightly packed to each other. Among these, the OH− and PO43− groups are accountable for negative group of HA surface whereas, Ca2+, Sr2+ and Ce3+ ions form the positive group. The apatite formation procedure mainly depends on the negative group on the surface of the coating material. During incubation process, the positively charged ions (Ca2+, Sr2+ and Ce3+) from the SBF are attracted by the negatively charged (OH− and PO43−) ions present on the surface of the coated material which tends to the development of apatite layer. Figure 12(a-c) shows the morphology of bone-like apatite layer on the surfaces of the P(EDOP-co-EDOT)/M-HA bilayer coated 316L SS at 1, 7 and 14 days of immersion in SBF solution. Figure 12(a) depicts the morphology of the bilayer coated sample after immersion in SBF solution for 1 day. Not much appreciable changes were observed and in fact the morphology remained the same. When the immersion period was extended to 7 days (Figure 12(b)), globule like apatite was observed on the surface and it is well evident that the bilayer coated samples accelerate the process of mineralization in the SBF solution. Further these globule like apatite material got interlinked with each other after 14 days (Figure 12(c)) of immersion which clearly reveals the 31 ACS Paragon Plus Environment


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enhanced growth of apatite layer over the surface of the P(EDOP-co-EDOT)/M-HA bilayer coated 316L SS. Further, to substantiate the growth of apatite, EDAX analysis was performed and the spectrum obtained for the apatite growth at 14 days of immersion is presented in Figure 12(d). The peaks for Ca, P and O confirm the growth of calcium phosphate on the bilayer coated surface. From EDAX analysis it is revealed that the steady development of apatite layer has occurred on the surfaces of the bilayer coated 316L SS specimen when immersed in SBF for various days. The cross sectional HRSEM image of the P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS after immersion in SBF solution for 14 days is shown in Figure 12(e). From the figure, the formation of apatite layer with compact morphology over the P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS sample can be clearly seen. In particular, the top apatite layer exhibited a compact layer whereas the interface between the apatite layer and porous bilayer coating is found to be continuous without any pore which is observed along its length. Hence, the bone-like apatite material could eventually enhance the osteointegration and osteoconduction properties. These properties are believed to provide firm fixation between the P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS implants and human body bone. Thus, from this result it can be clear that the bioactive bone-like apatite is formed on the bilayer coated 316L SS implant during the immersion in SBF. Therefore, the as developed P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS implant is biocompatible and hence can be used for various biomedical applications. 3.7.4. MTT assay test. The biocompatibility of P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated 316L SS sample was evaluated in vitro by measuring the absorbance value from MTT test after 1, 4 and 7 days of culture and observing the behavior of osteoblasts in close contact with these sample. The % cell viability was calculated for the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated 316L SS with respect to control and is shown as bar diagram in Figure S4. The viability of cells improved on increasing the days of culture from 1 to 7 days. Among the coatings, 32 ACS Paragon Plus Environment

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the 125 µg/mL of the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated 316L SS at 7 days of incubation revealed improved cell viability when compared with that for 1 and 4 days of incubation. The improved viability of cells over the surface of the coated samples is mainly due to the existence of minerals like Sr2+, Mg2+ and Ce3+. The optical microscopic images supported for the viability of cells at 1, 4 and 7 days of incubation over the bilayer coated 316L SS as shown in Figure 12. From Figure 13(a-d), it can be noted that, the number of viable cells was found to be high in the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coatings and also the cell viability comparable to the control group was observed. The P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coatings at 7 days of culture (Figure 13d) illustrated the existence of more number of viable cells which evidences that the biocompatibility of the bilayer has not been affected by the P(EDOP-co-EDOT) copolymer present in the P(EDOP-co-EDOT)/M-HA(with 0.1 M Ce3+) bilayer coatings. Thus, the bilayer coatings on 316L SS extensively increased the viability of cells (98.1 %) and is shown to be non-cytotoxic and has good cytocompatibility which is favourable for the orthopaedic applications. 3.7.5. Cell adhesion. Figure S5 shows the typical HRSEM micrographs of HOS MG63 cells after 1,4 and 7 days of culture on the P(EDOP-co-EDOT)/M-HA(with 0.1 M Ce3+) bilayer coatings. A slight change was observed in the cell morphology on different days of culture on the bilayer coatings (Figure S5(a-d)). The cells had initially started growing on the bilayer coated 316L SS which can be clearly seen in Figure S5(a). After culturing for 4 days, the cells were found to be more flattened and spread across the surface of the coating (Figure S5(b)). The cells were spread over the surface with lamellapodia on day 7 of culture (Figure S5(c)) and the cells exhibited their phenotypic morphology. Among different days of culture, it is possible to observe that, cells tended to spread more on the P(EDOP-co-EDOT)/M-HA (with 0.1 M Ce3+) bilayer coated surface on day 7 than on the other days of incubation. As shown in Figure S5d, the cells were seen packed and attached 33 ACS Paragon Plus Environment


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tightly on the coating surface with their filopodium and lamellipodium, suggesting excellent cell growth on the bilayer coatings. It may also be observed that all cells were broaden and grew favourably across the coating surface. Thus, it is confirmed that the P(EDOP-co-EDOT)/M-HA(with 0.1 M Ce3+) bilayer coatings showed better biocompatibility without any toxicity due to the mineral ions substituted in HAP coating and also due to the polymer layer over the metallic substrate. 4. CONCLUSION Fabrication of P(EDOP-co-EDOT)/M-HA-3 bilayer coatings on 316L SS was successfully demonstrated by electrodeposition for improved corrosion protection performance and bioactivity. The functional group, phase and surface analysis using FT-IR, XRD and 1H-NMR, spectra confirmed the formation of bilayer coatings. The morphology, topography and thermal results revealed the compact nature, increased roughness and high thermal resistance, respectively of the asdeveloped coatings. The electrochemical analysis confirmed the improved corrosion protection performance of P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS. The results of antibacterial activity, in vitro bioactivity, cell viability and cell adhesion studies revealed that the bilayer coatings facilitate very good antibacterial property, excellent bone-like apatite formation, excellent promotion of the cell viability and cell adhesion by spreading of MG-63 cells due to the more compact and porous morphology of the P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS. Based on the above findings, it is evident that the P(EDOP-co-EDOT)/M-HA-3 bilayer coatings has better anticorrosion, antibacterial and superior bioactivity properties and hence the P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS is considered as the prospective candidate for orthopedic applications.

ASSOCIATED CONTENT Supporting Information


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AFM three dimensional images, Bar diagrams of Adhesion strength and Vickers micro-hardness, TGA curve, Bar diagram of % viability of HOS MG63 cells and SEM images of cell adhesion. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Tel.: +91 427 2345766. Fax: +91 427 2345124. Email: [email protected]. Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGEMENTS D. Gopi acknowledges the major financial support from the Defence Research and Development Organisation, New Delhi, India, (DRDO, No. ERIP/ER/1103949/M/01/1513), Department

of

Science

and

Technology,

New

Delhi,

India

DST/TSG/NTS/2011/73), DST-EMEQ, Ref. No.:SB/EMEQ-185/2013)).

(DST-TSD,

Ref.

No.:

Also, D. Gopi and L.

Kavitha acknowledge the UGC (Ref. No. F. 30-1/2013 (SA-II)/RA-2012-14-NEW-SC-TAM-3240 and Ref. No. F. 30-1/2013(SA-II)/RA-2012-14-NEW-GE-TAM-3228) for the Research Awards. 5. REFERENCES (1)

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Cyclic voltammogram for P(EDOP-co-EDOT)-3 coating obtained on 316L SS for cycles 1-20 at a scan rate of 100 mV s-1 in EDOP:EDOT (50:50) containing 0.5 M sodium salicyclate.

Figure 2.

The FT-IR spectra of copolymer coatings (a) P(EDOP-co-EDOT)-1 (b) P(EDOP-coEDOT)-2 and (c) P(EDOP-co-EDOT)-3 and bilayer coatings (d) P(EDOP-coEDOT)/M-HA-1 (e) P(EDOP-co-EDOT)/M-HA-2 and (f) P(EDOP-co-EDOT)/MHA-3 obtained on 316L SS. 44 ACS Paragon Plus Environment

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Figure 3.

The XRD spectra of copolymer coatings (a) P(EDOP-co-EDOT)-1 (b) P(EDOP-coEDOT)-2 and (c) P(EDOP-co-EDOT)-3 and bilayer coatings (d) P(EDOP-coEDOT)/M-HA-1 (e) P(EDOP-co-EDOT)/M-HA-2 and (f) P(EDOP-co-EDOT)/MHA-3 obtained on 316L SS.

Figure 4.

The 1H-NMR spectra of copolymer coatings (a) P(EDOP-co-EDOT)-1 (b) P(EDOPco-EDOT)-2 and (c) P(EDOP-co-EDOT)-3 obtained on 316L SS.

Figure 5.

HRSEM images of copolymer coatings (a) P(EDOP-co-EDOT)-1 (b) P(EDOP-coEDOT)-2 and (c) P(EDOP-co-EDOT)-3 on 316L SS, (d) EDAX spectrum of P(EDOP-co-EDOT)-3 coated 316L SS and HRSEM images of bilayer coatings (e) P(EDOP-co-EDOT)/M-HA-1 (f) P(EDOP-co-EDOT)/M-HA-2 (g) P(EDOP-coEDOT)/M-HA-3 and (h) EDAX spectrum and (i) HRSEM cross sectional micrograph of P(EDOP-co-EDOT)/M-HA-3 bilayer coatings on 316L SS.

Figure 6.

Cyclic polarisation curves for uncoated, PEDOP, P(EDOP-co-EDOT)-3 and P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS in Ringer’s solution.

Figure 7.

Equivalent circuits for (a) P(EDOP-co-EDOT)-3

and (b) P(EDOP-co-EDOT)/

M-HA-3 bilayer coated 316L SS in Ringer’s solution. Figure 8.

(a) Nyquist, (b) Bode and (c) Phase plots for uncoated, PEDOP, P(EDOP-co-EDOT)3 and P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS in Ringer’s solution.

Figure 9.

ICP-AES analysis of uncoated, P(EDOP-co-EDOT)-3 and P(EDOP-co-EDOT)/M-HA3 bilayer coated 316L SS in Ringer’s solution.

Figure 10.

Qualitative antibacterial activity of P(EDOP-co-EDOT)/M-HA-1,

P(EDOP-co-

EDOT)/M-HA-2 and P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS against E. coli and S. aureus bacteria.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 11.

Page 46 of 61

Quantitative antibacterial activity of P(EDOP-co-EDOT)/M-HA-1,

P(EDOP-co-

EDOT)/M-HA-2 and P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS against E. coli and S. aureus bacteria for 6, 12 and 24h.. Figure 12.

HRSEM images of apatite growth on P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS at (a) day 1, (b) day 7 and (c) day 14 and (d) EDAX spectrum of 14 day immersion

of

P(EDOP-co-EDOT)/M-HA-3

bilayer coated 316L SS and (e)

HRSEM cross sectional micrograph of 14 days immersion of

P(EDOP-co-

EDOT)/M-HA-3 bilayer coated 316L SS in SBF solution. Figure 13.

Optical images showing the viability of HOS MG63 cells on P(EDOP-co-EDOT)/MHA-3 bilayer coatings for (a) 1, (b) 4 and (c) 7 days of incubation.

Supplementary Information Figure Captions: Figure S1.

AFM three dimensional images of (a) P(EDOP-co-EDOT)-3 coated and (b) P(EDOPco-EDOT)/M-HA-3 bilayer coated on 316L SS.

Figure S2.

(a) Adhesion strength and (b) Vickers micro-hardness of M-HA (at 0.1M of Ce3+), copolymer P(EDOP-co-EDOT)-3 and P(EDOP-co-EDOT)/M-HA-3 bilayer coated 316L SS.

Figure S3.

TGA curve obtained for copolymer P(EDOP-co-EDOT)-3 and P(EDOP-coEDOT)/M-HA-3 bilayer coated 316L SS.

Figure S4.

Bar diagram showing the % viability of HOS MG63 cells on P(EDOP-co-EDOT)/MHA-3 bilayer coatings for 1, 4 and 7 days of incubation.

Figure S5.

Typical morphology of cells attached on (a) control, P(EDOP-co-EDOT)/M-HA-3 bilayer coatings on 316L SS at day (b) 1, (c) 4 and (d) 7. Yellow circles represent the area of cell adhesion on bilayer coating.


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Table 1 Adhesion strength and hardness values of the M-HA, P(EDOP-co-EDOT)-3 and P(EDOP-coEDOT)/M-HA (with 0.1 M Ce3+) bilayer coating on 316L SS.

Sample condition

Adhesion strength (MPa)

Vickers micro-hardness (Hv)

M-HA (at 0.1M of Ce3+)

10.9± 0.7

341.6± 10.2

P(EDOP-co-EDOT)-3

13.8± 1.2

110.8 ± 13.2

P(EDOP-co-EDOT)/M-HA (with 0.1 M of Ce3+)

14.5 ± 1.1

391.8 ± 12.3

Table 2 Electrochemical parameters of the uncoated 316L SS, PEDOP coated, P(EDOP-co-EDOT)-1to 3 coated and P(EDOP-co-EDOT)/M-HA-3 coated 316L SS in Ringer’s solution.

Sample condition Uncoated 316L SS PEDOP P(EDOP-co-EDOT)-1 P(EDOP-co-EDOT)-2 P(EDOP-co-EDOT)-3 P(EDOP-co-EDOT)/M-HA-3

Polarisation parameters Ecorr Eb (mV vs.SCE)

-870 -510 -457 -442 -427 -361

Impedance parameters Epp

Rp

(mV vs. SCE)

(mV vs. SCE)

(Ω cm )

(Ω cm2)

+449 +534 +708 +816 +910 +962

-90 +138 +169 +194 +213 +291

41 2494 2730 2890 2970 3372

58 2612 2852 2905 3062 3491


|Z| 2


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Figures:

Fig. 1


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Fig. 2 49 ACS Paragon Plus Environment


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Fig. 3


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Fig. 4. 51 ACS Paragon Plus Environment

ACS Applied(e) Materials & Interfaces

(a) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(f)

(b)

(c)

(g)

(h)

(d)

(i)

Fig.5. 52 ACS Paragon Plus Environment

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Fig. 6.



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(a)

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(b)

Fig. 7.


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Fig. 8.

Fig. 8. 55 ACS Paragon Plus Environment


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Fig. 9.


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Fig. 10.



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Fig. 11.


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(b)

(a) (a)

(b)

(c)

(d)

(e)

Fig. 12.



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Fig. 13.


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173x120mm (96 x 96 DPI)

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