Antibacterial effects, biocompatibility and electrochemical behavior of zinc incorporated niobium oxide coating on 316L SS for biomedical applications

Applied Surface Science 427 (2018) 1166–1181

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Antibacterial effects, biocompatibility and electrochemical behavior of zinc incorporated niobium oxide coating on 316L SS for biomedical applications K. Pradeep PremKumar a , N. Duraipandy b , Manikantan Syamala Kiran b , N. Rajendran a,∗ a b

Department of Chemistry, College of Engineering Guindy, Anna University, Chennai, 600025, India Biological Materials Laboratory, CSIR-Central Leather Research Institute, Adyar, Chennai, 600025, India

a r t i c l e

i n f o

Article history: Received 15 May 2017 Received in revised form 28 August 2017 Accepted 30 August 2017 Available online 6 September 2017 Keywords: Zn Nb2 O5 316L SS Bacterial inhibition Biocompatibility Corrosion resistance

a b s t r a c t In the present study, Nb2 O5 (NZ0) composite coatings with various concentrations of zinc (NZ2, NZ4 & NZ6) are produced on 316L SS by sol-gel method with the aim of improving its antibacterial activity, bone formability and corrosion resistance properties. This work studied the surface characterization of NZ0, NZ2, NZ4 & NZ6 coated 316L SS by ATR-FTIR, XRD, HR-SEM with EDAX. The synthesized coatings were different in the morphological aspects, NZ0 shows mesoporous morphology whereas irregular cluster like morphology was observed for the zinc incorporated coatings. The chemical composition of the NZ0 and NZ4 composite coatings were studied by XPS and the results revealed that the zinc exist as ZnO and Nb as Nb2 O5 in the coatings. The increase in the concentration of zinc in Nb2 O5 increases the hydrophilic nature identified by water contact angle studies. The potentiodynamic polarization studies in simulated body fluid reveals the increase in polarization resistance with decrease in current density (icorr ) and electrochemical impedance spectroscopic studies with increase in charge transfer resistance (Rct ) and double layer capacitance (Qdl ) were observed for NZ4 coated 316L SS. The inhibition of Staphylococcus aureus and Escherichia coli bacteria were identified for NZ4 coated 316L SS by bacterial viability studies. The NZ4 coated 316L SS showed better Osseo-integration by spreading the MG 63 osteoblast cells. The study results imply that zinc incorporated Nb2 O5 (NZ4) composite coating exhibits antibacterial activity and also enhance the corrosion resistance and biocompatibility of the 316L SS. © 2017 Published by Elsevier B.V.

1. Introduction 316L austenitic stainless steels have a wide range of preferred properties like high mechanical strength under elevated temperatures, acceptable corrosion resistance, excellent ductility, good processability and low price. In addition, 316L SS also has good biocompatibility and is therefore an ideal material for orthopedic applications [1–3]. However, when it is used as long-term implant material in human body, release of metal (Ni, Fe and Cr) ions ensues. The released metal ions in turn trigger the coagulation of blood and chronic inflammation leading to restenosis in blood vessels and fibrinogenesis at surrounding tissues [4]. Hence, coating on 316L SS with a suitable material becomes inevitable to avoid the straight contact of metal with blood plasma and to achieve

∗ Corresponding author at: Department of Chemistry, Anna University, Chennai, Tamilnadu, 600025, India. E-mail addresses: [email protected], [email protected] (R. N.). http://dx.doi.org/10.1016/j.apsusc.2017.08.221 0169-4332/© 2017 Published by Elsevier B.V.

improved corrosion resistance and biocompatibility. Coating plays a very important role for ion interchange process and its surface topography develops a competition between bone regeneration and fibrous tissue formation at the Bone–Implant interface [5]. Bone-Implant interface induces the cell attachment by developing stable three dimensional interlocking systems at surrounding tissues [6], further bone-implant interface helps implant fixation to the bone and prevents from the loosening of bone [7]. Previous studies suggest that nano structured surface topographies can effectively enhance the protein adsorption and cell response to the implant [8]. Bioceramic coatings possessed bone-binding ability with the bone tissue in the body. Bioceramics, hard and crystalline (as compared to natural bone) have received considerable attention because of their important role in hard tissue repair and tissue engineering [9]. Application of bioceramic coating is an effective method to increase the corrosion resistance as well as biocompatibility of the material [10]. One of the biggest complications that rose after implant fixation through surgical implant procedures is the implant – associ-

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ated infections [11]. Once infection starts, the hydrated matrix of implant interface supports bacteria to aggregate and develops bio-film over the surface. It is a tough job to eliminate infection completely using antibiotics from outside [12]. Infection on implant surface leads to failure of implant which results in revision surgery and high medical costs [13]. In complicate matters, implant therapies could not be implemented to the patients due to many factors like aging, osteopenia and diabetes [14]. Therefore, it is important to develop implant material that possesses both Osseo-integration inducing ability and antibacterial activity to prevent the implant from the initial bacterial adhesions. Various bioactive ceramic oxide coatings like TiO2 , ZrO2 , SiO2 , Nb2 O5 and the combination of mixed oxide coatings like CaO2 SiO2 , ZrO2 -TiO2 and Nb2 O5 -Sr have been developed to improve the biocompatibility and corrosion resistance of the implant materials [15–18]. Even though several types of coatings have been developed and reported, there is an increasing need for bone implant formulations that have anti-bacterial activity along with bioactivity and biocompatibility [19]. It is well known that some metal ions like silver, zinc and copper etc., have antibacterial activity to the microorganisms. Silver ions have high antibacterial activity compared to other metal ions due to their wide antibacterial spectrum but the application of silver ions is hindered due to high cost and discoloration [20]. Zinc ions have some antibacterial effect and they are heat-resistant, stable towards colour change and inexpensive [21]. Zinc is also an essential biological element involved in many cellular signaling neuro behavior, metabolic functions, and important to immune functions and normal growth [22]. Zinc incorporation into biomaterials can enhance bone formation and mineralization. The effect of Zinc incorporated TiO2 coatings have been studied for their antibacterial activity and biocompatibility [23]. But there was no report on the effect of zinc incorporated Nb2 O5 for antibacterial activity and biocompatibility. Niobium oxide is non-toxic, soft, semi-permicious refractory and hypo allergic material highly tolerated by human body [24]. Niobium oxide ceramics are potential coating materials extensively used in biomedical applications due to their high corrosion resistance; wear resistance, thermodynamic stability and strongly influencing the endothelial cell response [25]. Hence, application of zinc incorporated Nb2 O5 coating on 316L SS can fulfill the desired objectives of implant materials. There are many coating techniques such as plasma spray, chemical vapor deposition, electrophoretic deposition, thermal spray and magnetron sputtering for the deposition of bioactive coatings. All these techniques have their own limitations and they require complex procedures for developing nano structured surface and adhesion strength to the substrate. Sol-gel is a highly versatile technique used for the synthesis of powders, fibers, films, aerogels and xerogels etc., with high purity and homogeneity [26]. Mixed oxide films derived from sol-gel technique also attracted the attention of many researchers to work in this field due to their simplicity with ease of control on the film composition, low cost and advantages for fabrication on large area deposition of films [27]. Sol-gel based coatings on metal surface improve the adhesion of coating by forming chemical bonding. Additives like zinc in the precursor solutions helps in tailoring the functional surface morphology. Hence in the present investigation, nano structured niobium oxide coating without and with zinc at different concentrations were developed by sol-gel methodology and fabricated on 316L SS by dip coating method. The effect of zinc incorporation in increasing anti-bacterial activity, corrosion resistance and biocompatibility of Nb2 O5 coating over 316L SS was studied. The nanostructured surface and its chemical states were evaluated by various surface characterization techniques. The mechanical strength of the coatings on the substrate was measured and identified from Vicker’s microhardness test. Corrosion resistance of the coated substrates

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Table 1 Composition of 316L stainless steel. Elements

Cr

Ni

Mo

Mn

Si

C

N

Fe

Wt (%)

17.20

12.60

2.40

1.95

1

0.03

0.02

Balance

was investigated by potentiodynamic polarization and electrochemical impedance spectroscopic studies. Anti-bacterial activity and viability of MG 63 cell cultures by MTT Assay for the uncoated and coated 316L SS substrates were examined. 2. Materials and methods 2.1. Fabrication of Nb2 O5 and zinc incorporated Nb2 O5 coatings Nb2 O5 coating [28] and zinc incorporated Nb2 O5 composite coatings [29] were synthesized from sol-gel method using the precursors, niobium ethoxide (Alfa aesar > 99.999%) and zinc nitrate hexa-hydrate (Sigma Aldrich > 99.99%). Initially, ethanol (MP Chemicals) (as solvent), acetyl acetone (Merck) (as complexing agent) and ethylene poly glycol (SRL chemicals) (as templating agent for porous morphology) were mixed in the ratio 20:1:1 in four vials separately. The solutions were allowed to stirring continuously for 20 min at 80 ◦ C in order to get homogenous mixture. A stabilized concentration of niobium ethoxide was added equally to the prepared solutions and then zinc nitrate hexa hydrate was added with varying concentration to each solution. The final stoichiometric molar ratios of niobium and zinc were 0.5:0, 0.5:0.25, 0.5:0.5 and 0.5:0.75 respectively in the solutions. The prepared coating solutions were named as NZ0, NZ2, NZ4 and NZ6 respectively. Further 0.2 ml of dilute HCl was added to the solutions in order to initiate the hydrolysis process. The solutions were allowed for refluxion at a temperature about 80 ◦ C for 24 h and kept stand for 8 h ageing in order to aid gelation. The procedures for the sol-gel coatings have been developed in the laboratory of the investigators [30,31]. 2.2. Preparation of substrates Austenitic 316L stainless steel material with specification ASTM A240 was procured from the Salem Stainless Steel Suppliers (P) Ltd, India. The detailed elemental composition of austenitic 316L stainless steel in percentage is given in Table 1. Prior to coating, the substrates were subjected to the mechanical grinding with silica carbide sheets from 100 to 600 grades in order to remove the organic debris accumulated on the surface and to bring uniformity. The substrates were then thoroughly washed with water and ultra sonicated in acetone for 5 min and used. The synthesized sol-gels were coated on prepared 316L SS substrates by dip coating method with a withdrawal speed of 10 mm/min. The coated specimens were then air dried and subjected to sintering in a heating box furnace, having inert atmosphere and temperature at 500 ◦ C for 1 h with constant raise of 3 ◦ C/min. 2.3. In vitro bioactivity test For conducting the In vitro immersion studies, simulated body fluid (SBF) solution was prepared as per the literature described by Kokuboet al. [32]. The concentration of the inorganic ions and the pH of the SBF solution were maintained as similar to the human blood plasma and its detailed composition is given in Table 2. Intensive care was taken while preparing the SBF solution in order to avoid the turbidity and deposition of the ions at the bottom of the beaker. The bioactivity for the uncoated, niobium oxide and zinc

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Table 2 Composition of SBF. Chemicals

Composition, (g/L)

NaCl NaHCO3 KCl K2 HPO4 ·3H2 O MgCl2 ·6H2 O 1 M HCl CaCl2 Na2 SO4 ((HOCH2 )3 CNH2 ) pH = 7.4

8.035 0.355 0.225 0.231 0.311 39 ml 0.292 0.072 6.118 adjusted with appropriate amount of 1 M HCl

incorporated Nb2 O5 bio ceramic coated 316L SS were evaluated by immersing the specimens in SBF solution for 7 days. 2.4. Physical characterization 2.4.1. Thermal studies (TGA & DSC) In order to find the thermal variations and the crystallization process of the prepared sol-gel, the gel was dried inside an oven for 24 h at 80 ◦ C. The powder thus obtained was subjected to Thermo Gravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) studies in nitrogen atmosphere between room temperature to 800 ◦ C with a heating rate of 10 ◦ C/min using SDT Q600 V8.0 Build 95 TGA instrument and diamond DSC (Perkin Elmer Model) respectively with a continuous nitrogen flow of 30 ml/min purging of the heating chamber to avoid the sample oxidation. 2.4.2. Zeta potential The particle size of the prepared niobium oxide and zinc incorporated Nb2 O5 sols were characterized by dynamic light scattering (DLS) technique using Malvern Zetasizer 3000 series at room temperature and 633 nm wavelengths fixed at 90 p as scattering angle. The stability of the prepared sol and their surface charge were also measured by the same Zetasizer instrument. 2.4.3. ATR-FTIR studies The presence of functional groups for uncoated, Nb2 O5 and zinc incorporated Nb2 O5 coated 316L SS surfaces were identified with ATR-FTIR spectrometric analysis in the wavenumber 4000–400 cm−1 region using the instrument UATR with two Accessory and KBr window Perkin Elmer ATR-FTIR spectrometer. 2.4.4. XRD analysis The crystalline state and phase composition of the coated substrates and the calcined powder were studied by scanning with monochromatic Cu K␣ radiation in XPERT PAN PRO X-ray diffractometer system having the radiation wavelengths (␭) 1.5418 A´˚ at 30 kV and 30 mA ratings over a Bragg angle (2␪) range of 5◦ –80◦ . 2.4.5. HR-SEM/EDAX studies The surface morphology and elemental composition of the coated specimens were observed with high resolution scanning electron microscopy (HRSEM) Model FEI Quanta FEG 200 attached with energy dispersive x-ray analysis (EDAX) Hitachi Model S3400 Japan. 2.4.6. Vickers hardness test Micro hardness measurements were made on the 316L SS and coated specimens (NZ0, NZ2, NZ4 and NZ6) using micro hardness tester Buehler make with a Vickers diamond indenter (25gf load). Three readings were taken for each deposit and the values were then averaged.

2.4.7. Wettability studies The wettability of the specimens was studied by performing the water contact angle measurements by placing a water drop of volume 8 ␮l with contact angle analyzer Phoenix 300 Plus model. 2.4.8. XPS analysis XPS data were obtained in a SPECS surface nano analysis XPS system with 150 W non-monochromatic Al K␣ radiation of 1486.6 eV. Core level spectra of Nb, O, Zn and C were obtained at pass energy of 25 eV. 2.5. Electrochemical characterization Electrochemical studies were carried out for uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS substrates by using Potentiostat/Galvanostat (model PGSTAT 302 N, Metrhom Autolab B.V., the Netherlands) with the help of GPES version 4.9.005 and FRA software. The conventional three electrode glass cell was used to conduct the electrochemical studies. Simulated body fluid was used as electrolyte to carry out the electrochemical studies at room temperature where the sample was kept as working electrode (1 cm2 ) area; platinum foil and Saturated Calomel Electrode (SCE) were used as counter and reference electrodes respectively. The working and reference electrodes were placed very near (about 1 cm) to each other. After the sample preparation, the samples were subjected to Open Circuit Potential (OCP) studies for 45 min to obtain steady state potential. Frequency Response Analyzer (FRA) was used for the electrochemical impedance spectroscopic measurements (EIS) from the frequency range 0.01 Hz to 100 kHz with an applied sinusoidal potential of ±10 mV on OCP. The plots obtained from the acquired data after each experiment were displayed as Bode plots, consisting of plots with log f vs log |Z| and log f vs – phase angle (␪), where log f is frequency and |Z| is real impedance. The plots thus acquired were curve fitted by ZSimpWin 3.21 version software (Princeton Applied Research, USA) in order to get the suitable equivalent circuit parameters. Based on the ␹2 values (10−3 ), the quality of the fit parameters were checked. Potentiodynamic polarization studies were carried out on the same specimens from the potential of −0.6 V (cathodic) to 1 V (anodic) with a scan rate of 1 mV/s. The obtained results were tafel plotted (potential vs log (i)), from which corrosion current (icorr ) and corrosion potential (Ecorr ) were obtained by the extrapolation of anodic and cathodic curves. From the icorr and Ecorr values the corrosion rate and the polarization resistance were calculated using the Stern Geary Eq. (1) [33]. icorr =

␤a × ␤c



2.3 × Rp ␤a + ␤c



(1)

Where RP is the polarization resistance, icorr is corrosion current ␤a is anodic slope and ␤c is cathodic slopes. 2.6. Antibacterial studies Antibacterial activity of the uncoated, zinc free Nb2 O5 (NZ0) and zinc incorporated Nb2 O5 (NZ4) coated 316L SS specimens were studied against gram positive Staphylococcus aureus and gram negative Escherichia coli bacteria. The survival of bacterial cells was examined on specimens by bacterial viability test. Luria Broth Agar media was prepared for 200 ml with composition of 5 gms LB broth from HiMedia chemicals, Bangalore and 1.6 g agar plus in 193 ml distilled water and 0.2 ml of S. aureus and E. coli (1 × 10−6 cfu/ml) mixed in the medium separately. The uncoated, NZ0 and NZ4 coated 316L SS specimens of two sets were placed in sterile plates; subsequently 8 ml LB agar containing S. aureus and E. coli separately were poured on to each sample plate immersed and overlaid. After

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air drying for 30 min, the sample plates were incubated at 37 ◦ C for 16 h and visible bacteria colonies on LB agar plates were measured. The bacterial growth was calculated by colony forming (cfu) units. The bacterial adherence number on the surface was identified from the ratio of total bacterial growth on the area of surface to the measured area of the surface. 2.7. Biocompatibility 2.7.1. Cell culture studies Human Osteosarcoma (MG63) cells were purchased from the National Centre for Cell Science (NCCS), Pune, India. The cells were maintained in specified Minimal Essential Medium (MEM) (Sigma Aldrich, India), supplemented with recommended 10% Fetal Bovine Serum (FBS) (Sigma Aldrich, India) mixed for their normal cell growth without using antibiotic. The cells were incubated with 5% CO2 in a CO2 incubator for 24 h. The cells were detached from the culture flask with the help of 0.05% trypsin-ethylene-diaminetetraacetic acid (EDTA) (Sigma Aldrich, India) after reaching the confluency. 2.7.2. Cell morphology and cell proliferation studies For cell morphology studies using SEM, the cells were seeded onto 12 Kcells/well culture plates which contained pre-sterilized uncoated, NZ0 and NZ4 coated 316L SS specimens and incubated for 24 h at 37 ◦ C in a 5% CO2 and 95% O2 humidified incubator. After incubation, the uncoated, NZ0 and NZ4 coated 316L SS specimens were washed with phosphate-buffered saline (PBS) and cells were fixed with 2.5% of glutaraldehyde solution for 1 h. The specimens were further dehydrated in ethanol solution in series (25, 50, 75 and 100%) dried and observed under SEM. 2.7.3. MTT assay In order to investigate the biocompatibility of the uncoated, NZ0 and NZ4 coated 316L SS specimens, MTT assay was performed. Briefly 12 K cells/well MG63 cells were seeded onto cell culture plates which contained pre sterilized uncoated, NZ0 and NZ4 coated 316L SS and allowed to grow for 24 h at 37 ◦ C in a 5% CO2 and 95% O2 humidified incubator. For comparison, same number of cells (12 K cell/well) was grown separately along with loaded samples and optical density was measured. After 24 h time period of incubation, the seeded cells were examined under phase contrast microscope and the cell morphology was observed using Leica systems. Subsequently, the medium was removed and the cells were treated with 0.5 mg/ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide salt) and 1X PBS (150 ␮l/well) was added further and incubated for 4 h in a CO2 incubator. After incubation, the solution was removed and solubilized with purple colored formazan crystal in 200 ␮l DMSO and the absorbance was measured at 570 nm using BioRad ELISA plate reader. 3. Results and discussions 3.1. Thermal studies Thermo gravimetric studies for the dried gel powders of Nb2 O5 (NZ0) and zinc incorporated Nb2 O5 (NZ4) were carried out in order to establish the sintering temperature for the coatings. The systematic thermal changes and transformation to various phases were observed during the rise in temperature and the plots are given in supplementary data Fig. S1. The temperature applied from room temperature to 850 ◦ C using 10 ◦ C/min rises in temperature for the sample gel powders, and the significant weight losses were observed. The first major weight loss of 70.75% occurred at 85 ◦ C for NZ0 and 18% at 120 ◦ C for NZ4 gel powders, could be due to the evaporation of solvent (ethanol). This decrease in percentage

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of weight loss for solvent in NZ4 powder may due to the association of solvent to the other remaining organic residues. The second major weight losses were occurred at 330 ◦ C and 350 ◦ C for NZ0 and NZ4 gel powders and the percentage of weight losses were observed to be 27.61 and 65% respectively due to the decomposition of organic residues in gel powders. This also indicates the initiation of crystalline phase from the amorphous. The NZ0 and NZ4 TGA plots suggest that the significant changes in mass were observed by the incorporation of zinc in Nb2 O5 . Further there was no significant weight loss was observed but gradual decrease of 2–4% of weight loss was noticed in both gel powders due to the presence of various crystalline phases in ceramic structures [34]. The increase in temperature in ceramics improves the crystalline nature. Similar thermal changes were observed from the DSC plots by change in heat flow along with increase in temperature up to 800 ◦ C for NZ0 and NZ4 gel powders and the plots obtained are given in supplementary data in Fig. S2. The decomposition of all the organic residues and solvent solution was noticed at 360 ◦ C for NZ0 and NZ4 gel powders. The crystalline phase attained at 360 ◦ C and melting point occurred at 500 ◦ C for both the gel powders. The temperature higher than 600 ◦ C, which results in chromium depletion from the surface of 316L SS [35]. The sintering temperature was established at 500 ◦ C from the results of TGA and DSC plots in order to remove the complete organic residues and to prevent the intergranular corrosion caused due to the depletion of ions from the metal [36]. 3.2. Particle size analysis The particle size distribution and the zeta potential for NZ0 and NZ4 sol-gels were investigated and the resulted plots are given in Fig. S3 in electronic supplementary data. The information concerning the completion of hydrolysis, condensation process and stability of the sol-gel prepared were predicted from the distribution of particles in aqueous suspensions at different pH and scattering of light from different sized particles [37,38]. The DLS measurements were carried and found to be high at 50 mV for pH 3, and a scattered light intensity weighted average particle sizes were 80 nm and 83 nm for NZ0 and NZ4 sol-gels respectively [39,40]. The particle size analysis clearly states that the synthesized mixed oxide sol-gels existed as individual particles against to their natural tendency to form agglomeration and coagulation and exhibited higher stability. 3.3. Surface characterization studies 3.3.1. ATR-FTIR analysis The ATR-FTIR spectra obtained for NZ0, NZ2, NZ4 and NZ6 coated 316L SS before and after 7 days immersion in SBF solution for In vitro bioactivity are given in Fig. 1(a & b). The strong overlapped broad band exhibited for all the coatings in the wavelength range 1000–450 cm−1 representing the fundamental stretching vibrational peaks for Nb O, Nb O and bridging Nb O Nb groups [41]. From the plots it is observed that there is no change in the stretching vibrations of Nb-O by the addition of zinc but the intensity of the bands is affected by increase in the concentration of zinc which is incorporated in Nb2 O5 . The small band appearing around 1680 cm−1 resembles the bending vibration of O H group. The uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS specimens were subjected to In vitro bioactivity studies for 7 days in SBF solution. ATR-FTIR was carried out to find the deposition of hydroxyapatite on the coated surfaces and resulted plots are given in Fig. 1(b). The spectra for NZ0, NZ2, NZ4 and NZ6 coated 316L SS after immersion in SBF were exhibited additional peaks along with stretching vibrational peaks of Nb-O group. The presence of short sharp bands in the

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Fig. 1. ATR-FTIR spectra for uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS (a) before and (b) after In vitro bioactivity studies in SBF solution.

range at 660 and 1030 cm−1 resembles the PO4 3− group. The band at 1280 cm−1 is attributed to the presence of stretching vibration of CO3 2− group. The stretching sharp O H band and broad O H band observed in the range at 1640 cm−1 and 3700–2990 cm−1 respectively, are formed due to the water adsorbed from the atmosphere. Whereas for uncoated 316L SS does not exhibit the distinctive peaks for phosphate groups and it further reveals that 316L SS had less bioactivity. The formation of phosphate and carbonate bands on zinc incorporated Nb2 O5 (NZ2, NZ4 & NZ6) coated substrates after seven days immersion in SBF solution confirms the bioactivity by deposition of HAp [28]. 3.3.2. XRD analysis The uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS specimens and gel powders for NZ0 and NZ4 sol-gels after sintering at 500 ◦ C were subjected to X-ray diffraction analysis and the resulted spectra are given in Fig. 2(a & b) respectively. All coatings exhibited strong diffraction peaks for substrate 316L SS due to the formation of thin layer of coating on its surface and coating peaks are very minute and are given in Fig. 2(a). In order to identify the crystalline structure and phase composition of the coating materials, the gel powders were sintered at 500 ◦ C and subjected to XRD analysis and their spectra are given in Fig. 2(b). The NZ0 exhibited strong diffraction peaks at 2␪ values 22.71◦ , 28.43◦ , 36.74◦ , 46.21◦ , 50.89◦ , 55.13◦ , 56.26◦ and 59.01◦ representing (001), (100), (101), (002), (110), (102), (111) and (200) planes respectively. These are the characteristic peaks of Nb2 O5 present in monoclinic crystalline structure as identified from the JCPDS No 28-0317 [42]. The XRD spectra for NZ4 shows the peaks at 2␪ values 30.92◦ , 36.12◦ , 56.61◦ , 62.10◦ , 66.62◦ and 72.35◦ with corresponding planes (100), (101), (110), (103), (200) and (004) respectively, representing the characteristic peaks for ZnO present in hexagonal crystalline phase along with the characteristic monoclinic crystalline peaks for Nb2 O5 identified from JCPDS No 36-1451 [43]. The above preferred crystallographic orientations from the XRD analysis found for the Nb2 O5 and Zn incorporated Nb2 O5 coatings on 316L SS [44,45]. The incorporation of zinc in Nb2 O5 does not change the crystalline nature and phase composition of Nb2 O5 . The monoclinic and hexagonal crystalline structures mostly facilitate the deposition of HAp on its surface by continuous immersion in SBF solution.

3.3.3. HR-SEM/EDAX The morphological features of sintered specimens and their elemental composition were investigated by HR-SEM with EDAX spectroscopy and the micrographic images of uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS are given in Fig. 3(a–e). The surface of uncoated 316L SS is rough and appeared with grid lines raised due to mechanical polishing. All the micrographic images showed different morphology. Microcracks were observed in the Nb2 O5 (NZ0) coating on 316L SS due to the difference between thermal coefficient of expansion of metal (316L SS) and the coating material. However, microcracks were not observed in Zn incorporated Nb2 O5 (NZ2, NZ4 & NZ6) coatings on 316L SS. In the clinical environment there could be considerable probability of ionic migration through the micro cracks in Nb2 O5 coating, which would eventually result in corrosion of 316L SS substrate. The probability of this eventuality will be at its minimum in the case of Zn incorporated Nb2 O5 coated 316L SS as there is hardly any micro cracks on its surface and hence ensures clinical safety. Incorporation of zinc in Nb2 O5 coating on 316L SS brings morphological changes with change in concentration of zinc [46,47]. The NZ2 coated 316L SS is showed the short elongated irregular cluster like nanostructures formed uniformly with mesoporous morphology. The NZ4 coated 316L SS is appeared to be hard, elongated fused crystalline clusters with porous morphology covering a dense layer on the surface. Precipitates are observed on the surface of NZ6 coated 316L SS, which further states that high concentration of zinc in Nb2 O5 forms agglomeration. The SEM micrographs revealed that the concentration of zinc in Nb2 O5 coating strongly influenced the surface morphology by improving the surface roughness. The increase in surface roughness further helps in improving the bone binding ability to the implant material [48]. The corresponding EDAX profile for uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS specimens were given in Fig. 3(1–5) respectively. The presence of Nb and O peaks in NZ0 and Zn, Nb, O peaks in NZ2, NZ4 and NZ6 coatings on 316L SS revealed the formation of Nb2 O5 coating and successful incorporation of zinc in Nb2 O5 coating on 316L SS. The thickness of the Nb2 O5 (NZ0) and Zn incorporated Nb2 O5 (NZ2, NZ4 & NZ6) coatings on 316L SS were measured after coating and sintered at 500 ◦ C. The thickness of the NZ0 coating on 316L SS was around 460 nm, but whereas the incorporation of Zinc into Nb2 O5 brought the variation

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Fig. 2. XRD spectra for (a) uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS and (b) Nb2 O5 and zinc incorporated Nb2 O5 sol-gel powders.

in the coating thickness slightly. The thickness of the NZ2, NZ4 and NZ6 coatings on 316L SS were around 550 nm. All the specimens were subjected to In vitro bioactivity studies for seven days immersion in SBF solution and the deposition of hydroxyapatite on the surfaces of specimens were identified by HRSEM with EDAX analysis to confirm the growth of hydroxyapatite and the SEM micrographs of uncoated, NZ0 and NZ4 coated 316L SS are given in Fig. 4(a–c). The surface morphology of uncoated 316L SS shows the absence of hydroxyapatite growth due to its bio inert nature, and hence the grid lines raised due to mechanical polishing were clearly visible on the surface however, the traces of hydroxyapatite were observed on 316L SS. The micrographic images of NZ0 and NZ4 coatings on 316L SS showed the deposition of spherical clusters like micro structures forming the mineral hydroxyapatite layer. Comparatively, zinc incorporated Nb2 O5 coated 316L SS shows more bioactivity by favoring more deposition of hydroxyapatite. The presence of phosphate and calcium peaks along with the coating peaks identified by EDAX spectra is given in Fig. 4(d–f) confirmed the HAp deposition which was high for the NZ4 coated 316L SS. The presence of porous nanostructures in NZ0 and NZ4 coated 316L SS specimens facilitated the growth of HAp layer on their surfaces [49]. 3.3.4. XPS studies XPS spectra for zinc free (NZ0) Nb2 O5 and zinc incorporated (NZ4) Nb2 O5 coated 316L SS are given in Fig. 5(a) and (b) respectively. The XPS survey spectra of NZ0 coated 316L SS showed the presence of Nb, O, C and Fe peaks. The peak position, binding energy values, percentage of area under the peak and their related phases are given in Table 3. The changes in binding energy values and percentage of different O, Nb and C were observed from de-convoluted core spectra are also given in Fig. 5(a). The binding energy values were corrected with respect to carbon binding energy value of 284.6 eV which was used as reference. The XPS core spectra of niobium showed two 3d peaks, Nb 3d5/2 and Nb 3d3/2 were found at 206.5 and 209.3 eV respectively, revealing that the peaks correspond to Nb2 O5 [50]. The XPS core spectra of oxygen showed two

Table 3 Chemical states and values of Nb2 O5 and zinc incorporated Nb2 O5 coated 316L SS. Sample Core Elements Peak number Center eV Area (%) Spin state

Nb 3d NZ0

O 1s C 1s Nb 3d

NZ4

O 1s Zn 2p C 1s

1 2 3 4 1 2 1 2 1 2 3 4 1 2 3 1 2 1 2 3

206.42 206.99 209.28 210.00 529.71 531.54 284.51 285.60 206.46 207.18 209.13 209.64 529.63 531.00 532.20 1021.10 1044.51 284.43 285.79 287.74

38.78 20.99 34.56 05.68 65.80 34.20 73.28 26.72 52.81 06.54 27.80 12.85 59.85 17.42 22.72 57.29 42.71 44.79 37.92 17.28

Nb2 O5 3d5/2 Nb2 O5 ·2OH 3d5/2 Nb2 O5 3d3/2 Nb2 O5 ·2OH 3d3/2 O2 − (Nb2 O5 ) OH− C 1s CO Nb2 O5 3d5/2 Nb2 O5 ·2OH 3d5/2 Nb2 O5 3d3/2 Nb2 O5 ·2OH 3d3/2 O2 − OH− H2 O ZnO 2p3/2 ZnO 2p1/2 C 1s CO COH

de-convoluted peaks, the first sharp peak at 529.7 eV representing to the oxygen, present in metal oxide i.e., Nb2 O5 and the short broad peak at 532 eV representing the presence of hydroxyl group that resulted due to the chemisorption of water or hydroxide from atmosphere or by sample handling. Only a short peak for Fe2 O3 was identified at 712.3 eV from XPS survey spectra due to the complete coverage of coating on 316L SS surface. It is clear that the Nb2 O5 coating was successfully fabricated on 316L SS. Fig. 5(b) illustrates the XPS complete survey spectra of NZ4 coated 316L SS. In this spectrum the presence of Zn peak was identified along with Nb, O and C peaks. The spectrum was corrected with binding energy of carbon taken as reference to determine the binding energies of each peak and the area under the peak was used to determine the percentage. The core spectra for oxygen 1s is given in Fig. 5(b), which exhibited three de-convoluted peaks at 529.6,

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Fig. 3. HRSEM images with corresponding EDAX spectra respectively for uncoated (a & 1), NZ0 (b & 2), NZ2 (c & 3), NZ4 (d & 4) and NZ6 (e & 5) coated 316L SS.

531.0 and 532.2 eV corresponding to metal oxide (O2− ), hydroxide group (OH− ) and water molecule (H2 O) adsorbed from atmosphere, respectively. The de-convoluted core spectra for zinc was exhibiting two 2p peaks at 1021.1 eV and 1044.5 eV found to be 2p3/2 and 2p1/2 spin states confirms that zinc exists as ZnO [51]. The binding energy peaks of niobium core spectra in Fig. 5(b) shows the presence of two sharp 3d peaks, the first peak at 206.5 eV found to be 3d5/2 and second peak at 209.2 eV found to be 3d3/2 corresponding to the presence of Nb2 O5 . Further the incorporation of zinc is not affected the oxidation state of Nb in Nb2 O5 coating on 316L SS.

3.3.5. Mechanical behavior The hardness of the coatings NZ0, NZ2, NZ4 and NZ6 on 316L SS was measured by Vickers micro hardness test. The hardness value of 316L SS substrate was calculated to be 269 HV whereas the coated substrates showed improved hardness values of 341, 362, 363 and 294 HV for NZ0, NZ2, NZ4 and NZ6 coated 316L SS respectively. All the coatings added extra strength to the 316L SS by forming dense compact layer. The NZ2 and NZ4 coated 316L SS showed comparatively more hardness and were greater than the strength of human bone [52]. This improved hardness could be due to the incorpora-

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Fig. 4. HRSEM images with EDAX spectra after in vitro bioactivity for uncoated (a & d), NZ0 (b & e) and NZ4 (c & f) coated 316L SS.

Fig. 5. (a) XPS plots for Nb2 O5 full survey spectra and core spectra for Oxygen, Niobium and Carbon. (b) XPS plots for zinc incorporated Nb2 O5 full survey spectra and core spectra for Oxygen, Zinc, Niobium and Carbon.

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Fig. 5. (Continued)

tion of low concentration of zinc in Nb2 O5 on 316L SS mold the surface into a uniform hard composite layer that further improved the corrosion resistance.

3.3.6. Wettability studies Wettability studies play an important role in understanding the surface phenomena of the substrates. The wettability of substrates illustrated by water contact angle measurements was carried out for uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS after heating and the obtained ␪ values and their corresponding images are

given in Fig. 6. The polished smooth surface of 316L SS exhibited a contact angle value 92◦ revealing the hydrophobic nature. The contact angle value reduced to 56◦ for NZ0 coated 316L SS substrate due to increase in the roughness and porous morphology of the coating that facilitated absorption of water. The coatings NZ2, NZ4 and NZ6 on 316L SS showed hydrophilic nature and following a decreasing trend in contact angle measurements viz., 36◦ , 31◦ and 26◦ respectively, indicating the increase in the concentration of zinc in Nb2 O5 coating increased the surface roughness and facilitates the absorption of water on its surface. It has been reported

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Fig. 6. Contact angle images for uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS.

that the hydrophilic surface facilitates the cell spreading and rapid hydration of cells [53]. 3.4. Electrochemical characterization 3.4.1. Potentiodynamic polarization studies The potentiodynamic polarization curves for uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS, with immediate and after In vitro bioactivity in SBF solution respectively are given in Fig. 7(a & b). The electrochemical polarization parameters like corrosion potential (Ecorr ), corrosion current density (icorr ), break down potential (Ebreak ) and polarization resistance (Rp ) for uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS were obtained from potentiodynamic curves using Tafel extrapolation and are summarized in Table 4. It is observed that the positive shift in Ecorr for the coated substrates with respect to the uncoated 316L SS revealed that the coatings acted as a barrier layer to the corrosive ions from reaching the metal surface [54]. The incorporation of zinc in Nb2 O5 formed composite coating of mixed oxides (Nb2 O5 and ZnO) on 316L SS further enhanced the Ecorr towards nobler direction than the porous Nb2 O5 (NZ0) coating on 316L SS. Break down potential of the coatings was observed by increasing anodic potential up to 1 V. The shift of break down potential from 0.162 V for the uncoated 316L SS to 0.633 V for NZ0, 0.561 V for NZ2 and 0.701 V for NZ4 coated 316L SS revealed the increasing compactness of the coating by inclusion of zinc in Nb2 O5 but higher concentration of zinc led to precipitation resulting in decreased compactness and hence showing break down at less potential (0.336 V) for NZ6 coated 316L SS. The icorr value of a material is proportional to the corrosion rate, the higher icorr value results more corrosion [55]. The uncoated 316L SS showed higher icorr value and the presence of resistive metal oxides (Nb2 O5 and ZnO) in the coatings reduced the icorr of 316L SS. The greater compactness and adequate thickness of the coatings of NZ2 and NZ4 declines the current density highly on 316L SS due to their more adhesion strength. The polarization resistance (Rp ) values further supported the fact that zinc incorporated Nb2 O5 coated 316L SS results in improved corrosion resistance and decreased corrosion rate of 316L SS. After In vitro bioactivity studies for uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS specimens in SBF solution for seven days, the specimens were subjected to potentiodynamic polarization studies to evaluate the changes that took place during immersion. The polarization curves in Fig. 7(b) shows the shift in Ecorr values of the NZ0, NZ2, NZ4 and NZ6 coated 316L SS specimens towards positive direction after In vitro bioactivity. The shift in Ecorr could be

Fig. 7. Potentiodynamic polarization curves for (a) uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS and (b) uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS after In vitro bioactivity studies.

due to the growth of hydroxyapatite on the coated surfaces which further enhanced the corrosion resistance. The extended anodic regions of the NZ2 and NZ4 coated specimens showed the break down potential shift towards more positive potential than before In vitro bioactivity confirming the formation of new HAp layer over the surface [56]. For the NZ0 and NZ6 coated 316L SS specimens, the aggression of corrosive ions from electrolyte took place and attacked the metal surface which resulted in Ecorr values being shifted towards less positive direction. The decrease in icorr values for NZ2 and NZ4 coated 316L SS specimens when compared to uncoated, NZ0 and NZ2 coated specimens could be due to its compact crystalline nature enhanced the growth of HAp that further improved the corrosion resistance of 316L SS. The incorporation of zinc into the Nb2 O5 enhanced the bioactivity of 316L SS by facilitating the HAp growth in SBF. 3.4.2. Electrochemical impedance spectroscopy studies EIS is a powerful technique used to get useful information of both surface resistance and capacitance behavior of specimens in SBF solution. Electrochemical impedance spectroscopy (EIS) studies were carried out for the uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS before and after In vitro bioactivity to investigate the surface electrode/electrolyte interface and the results obtained are shown as Bode plots in Fig. 8. It was observed that from Fig. 8(a) the impedance of 316L SS coated with NZ2 and NZ4 composites showed

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Table 4 Polarization parameters for uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS before and after In vitro bioactivity studies in SBF solution. Specimens

Ecorr (V vs SCE)

icorr × 10 −6 (A/cm2 )

Ebreak (V)

Rp (K cm2 )

Corrosion Rate (mm/year) × 10−2

316L SS NZ0 NZ2 NZ4 NZ6

−0.331 −0.274 −0.215 −0.172 −0.256

1.86 0.28 0.07 0.22 0.42

0.162 0.633 0.561 0.701 0.336

77.48 126.90 548.30 240.30 219.40

2.01 0.30 0.07 0.23 0.46

1.53 0.37 0.35 0.32 0.89

−0.044 0.324 0.358 0.905 –

30.18 82.72 93.73 133.20 54.50

1.64 0.41 0.38 0.34 0.95

After In vitro bioactivity studies for 7 days −0.321 316L SS −0.234 NZ0 NZ2 −0.179 −0.173 NZ4 NZ6 −0.192

ten folds improved magnitude than the uncoated 316L SS at the low frequency region (10−2 –100 Hz). The NZ0 and NZ6 coated 316L SS specimens also showed five folds improvement of magnitude than uncoated 316L SS. The compact barrier layer effectively protected the coating and improved the corrosion resistance by inhibiting the free access of ions from electrolyte [57]. In Fig. 8(b), the Bode-Phase plots represents the capacitance of NZ2 and NZ4 coated 316L SS which reached −80◦ phase angle which is near to the ideal capacitance i.e., −90◦ in the mid frequency region and extends up to low frequency region with good capacitance. The specimens NZ0 and NZ6 coated 316L SS showed higher capacitive behavior at mid frequency region compared to 316L SS. However it was declined to −50◦ and −30◦ respectively at low frequency region and this may be attributed to the interaction of ions from the electrolyte [58]. The low concentration of zinc in Nb2 O5 coatings on 316L SS formed a compact matrix which impeded the charge transfer process and resisted the penetration of corrosive ions through the coating from electrolyte thus confirming better corrosion resistance behavior. Fig. 8(c and d) represents the EIS plots for the specimens uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS after In vitro bioactivity studies for seven days in SBF solution. The Bode Modulus plots in Fig. 8(c) shows coated specimens with higher impedance even after seven days of immersion in SBF solution. The magnitude of impedance for the coated specimens showed higher corrosion resistance than for the uncoated 316L SS and appreciably constant in the low frequency region. This may be due to the deposition of hydroxyapatite on the NZ0, NZ2, NZ4 and NZ6 coated 316L SS surfaces providing similar trend in resistance. The Bode-Phase plots in Fig. 8(d) also confirmed the changes in the phase angle of coated specimens with the formation of new humps due to formation of new HAp layer. The higher corrosion resistance and the greater phase angle at low frequency region for the coated specimens indicated the stronger intact nature of the coatings with the surface of 316L SS [59], which further resisted the corrosive ions attack even after continuous immersion in SBF solution. The obtained Bode plots were analyzed by circuit fitting with Zsimpwin software and the circuit parameters are summarized in Table 5. The fitted circuits for uncoated, coated 316L SS specimens before and after In vitro bioactivity studies are given in Fig. 8(e). Uncoated 316L SS plots were curve fitted with simple Randles circuit Rs(Rb Qb ) given in Fig. 8(e (1)) where Rs is the solution resistance, Rb and Qb are the charge transfer resistance and double layer capacitance of the thin passive oxide film on the surface of 316L SS. The less charge transfer resistance of the specimen indicated the poor resistive nature of the passive oxide layer. The EIS plots of all the coated specimens were fitted with two time constant circuit Rs (Rc Qc ) (Rb Qb ) given in Fig. 8(e (2)), consisting two resistances (Rcoat and Rbarrier ) and two capacitances (Qcoat and Qbarrier ) connected in series with Rs . Rcoat is the resistance of outer coated layer and Rbarrier is the resistance of inner barrier layer whereas Qcoat is the capacitance of outer coated layer and Qbarrier is the capacitance

of inner barrier layer. The higher coating resistance (Rcoat ) of the coated specimens indicated greater ability to resist the corrosive ions thus protecting the substrate to a greater extent. NZ2 and NZ4 coated specimens showed the greater coating resistance (Rcoat ) values compared to remained coatings. Fig. 8(e (3)) shows three time constant circuit Rs (Ra Qa ) (Rc Qc ) (Rb Qb ) connected in series to Rs fitted for all coated 316L SS specimens after seven days In vitro bioactivity studies. The newly formed apatite layer on coated surfaces providing additional resistance Ra and capacitance Qa along with Rcoat , Qcoat and Rbarrier , Qbarrier . The charge transfer resistance and double layer capacitance values from Table 5 clearly support the fact that the 316L SS coated with NZ2 and NZ4 have high corrosion resistance and capacitance and help in developing the apatite layer. 3.4.3. Anti-bacterial studies After the completion of 24 h for bacteria growth in the specimens loaded culture plates, the area on the surface of specimens was used to calculate the bacterial colony forming units (CFU). The number of colonies present on the surface (the area of bacterial spread) was measured by counting them using colony counter. The uncoated 316L SS was used as control to calculate the percentage of inhibition of bacteria on Nb2 O5 (NZ0) and Zn incorporated Nb2 O5 (NZ2, NZ4 and NZ6) coated 316L SS. The percentage inhibition of S. aureus and E. coli on Nb2 O5 and Zn incorporated Nb2 O5 coated 316L SS were calculated using the followed Eq. (2) [60]. % inhibition of bacteria =

Contol CFU − Test CFU × 100 Control CFU

(2)

Antibacterial activity of the uncoated and zinc free (NZ0) Nb2 O5 and zinc incorporated (NZ4) Nb2 O5 coated 316L SS was investigated by bacterial viability test and the percentage of inhibition of S. aureus and E. coli bacterial growth on the surface of specimens was measured and its images are given in Fig. 9. Based on the results obtained from the bacterial colony counting experiments, it was observed from that 41% for S. aureus and 90% for E. coli bacteria were inhibited for the NZ4 coated 316L SS specimens whereas NZ0 and uncoated 316L SS showed considerably less inhibition efficiency. The increased bacterial inhibition efficiency for NZ4 coated 316L SS was due to the presence of ZnO in the coating, which reduced the bacterial adhesion [61] and inhibited the growth of bacteria by forming reactive oxygen species (ROS) [62]. The antibacterial mechanism of ZnO could most likely be the generation of ROS (such as H2 O2 , OH− , and O2− ) which are harmful to the bacteria. From the bacterial colony counting experiments it can be seen that the release of ROS from zinc incorporated Nb2 O5 coated 316L SS, which further enhanced the antibacterial efficiency. 3.4.4. Biocompatibility Fig. 10 shows the SEM images of MG 63 cells over uncoated, NZ0 and NZ4 coated 316L SS at different resolutions. The surface

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Fig. 8. EIS plots for uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS (a) Bode-Impedance, (b) Bode-Phase, (c) Bode-Impedance after In vitro bioactivity and (d) Bode-Phase after In vitro bioactivity studies in SBF solution. Equivalent circuits used to fit (a) uncoated 316L SS, (b) NZ0, NZ2, NZ4 and NZ6 coated 316L SS and (c) NZ0, NZ2, NZ4 and NZ6 coated 316L SS after In vitro bioactivity studies.

morphology, topography and roughness play important roles in cell attachment, proliferation and differentiation of MG 63 osteoblast cells. The MG 63 cells were cultured on uncoated, NZ0 and NZ4 coated 316L SS for 24 h and studied because they are very important in the initial cell response for implants to establish stable bioactive interface for further cell proliferation, differentiation and attachment [63]. From the SEM images in Fig. 10(a–c), it was observed that there was increase in number of osteoblast cells spread over

NZ0 and NZ4 coated 316L SS surface compared to uncoated 316L SS. The spreading of large number of cells on coated surfaces could be due to the chemical composition of coating and presence of functional groups, which facilitated the cell proliferation [64]. The images also showed the presence of healthy osteoblast cells over the sample surfaces with pseudopodia extensions, indicating the strong attachment of cells with active proliferation. Zn incorporated Nb2 O5 coated 316L SS provided more surface roughness and

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Table 5 EIS curve fitted parameters for uncoated, NZ0, NZ2, NZ4 and NZ6 coated 316L SS before and after In vitro bioactivity studies in SBF solution. Specimens/Circuit elements

2

Rs ( cm ) Rb (K cm2 ) Qb (F cm−2 ) Qn Rc /Rp (K cm2 ) Qc /Qp (F cm−2 ) nc Ra (K cm2 ) Qa (F cm−2 ) na

Before In vitro bioactivity

After In vitro bioactivity

316L SS

NZ0

NZ2

NZ4

NZ6

316L SS

NZ0

NZ2

NZ4

NZ6

62.24 15.85 1.108 × 10−4 0.79

87.19 26.13 1.63 × 10−4 0.83 190.1 9.38 × 10−5 0.80

73.8 17.50 3.15 × 10−3 0.80 239.1 1.90 × 10−3 0.80

89.40 19.28 5.01 × 10−5 0.82 290.4 1.62 × 10−5 0.83

81.81 24.97 6.82 × 10−8 0.90 101.1 7.41 × 10−5 0.97

84.80 6.09 7.91 × 10−5 0.69 11.02 1.19 × 10−4 0.67

78.72 22.45 2.08 × 10−5 0.81 220.6 1.32 × 10−3 0.83 21.20 3.21 × 10−4

80.78 27.12 1.52 × 10−8 0.92 303.1 3.91 × 10−4 0.81 23.48 9.87 × 10−5

85.74 29.18 4.50 × 10−6 0.78 405.3 5.53 × 10−5 0.82 47.38 7.36 × 10−4 0.77

72.78 31.67 1.38 × 10−8 0.77 127.6 3.16 × 10−4 0.87 89.45 8.93 × 10−5

Fig. 9. Bacterial viability studies for uncoated, Nb2 O5 and zinc incorporated Nb2 O5 coated 316L SS against to S. aureus and E. coli bacteria.

functional active groups for better cell attachment and proliferation and facilitated improved biocompatibility than the uncoated and Nb2 O5 coated 316L SS. The optical density of MTT assay performed for only cells, uncoated, NZ0 and NZ4 coated 316L SS after 24 h cell culture in MEM and the values were origin plotted and given in electronic supplementary data Figure S4. From the plot, it was clearly observed that 316L SS showed low optical density value compared to only cell revealing the decrease in the cell number due to toxicity exhibited by substrate [65,66], whereas NZ0 and NZ4 coated 316L SS showed increase in the number of cells providing high optical density values. From the results it can be identified that, large numbers of live cells were present in the coated substrates giving high optical density values and facilitating biocompatibility. Comparatively, zinc incorporated Nb2 O5 coated 316L SS exhibited higher biocompatibility than uncoated and Nb2 O5 coated 316L SS. 4. Discussion Microcracks were initiated on the coating, when the samples sintered above 500 ◦ C, due to difference in thermal coefficient of expansion of the base metal (316L SS) and the coatings. The micro cracks formed in Nb2 O5 coating due to variation in the thermal coef-

ficient of expansion, was overcome by the Zn incorporated Nb2 O5 composite coating on 316L SS. This type of coating systems is suitable for load bearing application for non-union defects. However, it will not be suitable for dental implants and can be used for hip and knee replacement implants with necessary modification. From the investigations, the enhancement of corrosion resistance, biocompatibility and mechanical properties for Zn incorporated Nb2 O5 coating on 316L SS were observed. In addition, the antibacterial activity of the Zn incorporated Nb2 O5 coatings on 316L SS was higher than that of the uncoated and Nb2 O5 coated 316L SS. Hence the Zn incorporated Nb2 O5 coated 316L SS sample could be recommended for further in vitro and in vitro studies to be considered for future investigations.

5. Conclusion The nanostructured porous Nb2 O5 coating and Zn incorporated Nb2 O5 composite coatings, prepared by sol-gel methodology were successfully fabricated on 316L SS. All the coatings achieved phase transformation and crystallinity around 350 ◦ C confirmed by thermal studies. X-ray diffraction studies showed that Nb2 O5 was in monoclinic and ZnO was in hexagonal crystalline structure. The phase transformation and crystalline nature of the Nb2 O5

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Fig. 10. MG 63 osteoblast cell spreading over (a, b) uncoated, (c, d) Nb2 O5 and (e, f) zinc incorporated Nb2 O5 coated 316L SS.

were not apparently altered by the incorporation of zinc. The surface morphology and elemental compositions of the NZ0, NZ2 and NZ4 coated 316L SS were nano porous and nano structured identified from SEM with EDAX. The NZ0, NZ2, NZ4 and NZ6 coated 316L SS showed appreciably high hardness and hydrophilic nature confirmed by the Vicker’s micro hardness test and contact angle measurements respectively. The microstructure, surface roughness, hardness and wettability of the coatings were greatly enhanced by incorporation of zinc in Nb2 O5 . The chemical states of Nb2 O5 remained constant even after incorporation of zinc which is in the form of zinc oxide (ZnO) confirmed by XPS. Electrochemical studies revealed enhanced corrosion resistance for the zinc incorporated Nb2 O5 coatings than Nb2 O5 and 316L SS. The stoichiometrically low and equal concentrations of zinc with Nb2 O5 (i.e., NZ2 & NZ4) developed compact matrix which prevented the aggression of corrosive ions and facilitated good resistance to 316L SS in SBF solution with immediate immersion and even after In vitro bioactivity studies. Zinc incorporated Nb2 O5 coated 316L SS was effective in inhibiting the S. aureus and E. coli bacterial adhesion identified by counting the CFU by bacterial viability test. The Zn incorporated Nb2 O5 coated 316L SS exhibited more biological activity in promoting cell adhesion, proliferation and differentiation of MG 63 osteoblast cells which was confirmed with MTT Assay and cell spreading over the surface matrix observed from the SEM.

Therefore, it can be concluded that the porous nanostructured Zn incorporated Nb2 O5 coated 316L SS possesses enhanced corrosion resistance, antibacterial activity and hence can promote Osseointegration and a promising material for biomedical applications. Acknowledgement One of the authors, K. Pradeep PremKumar gratefully acknowledges the department of Chemistry, Anna University, Chennai, India for instrumentation facilities provided under DST-FIST and UGCDRS. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.apsusc.2017.08. 221. References [1] K.P. Ananth, S. Shanmugam, S.P. Joseb, A.J. Nathanael, T.H. Oh, D. Mangalaraj, A.M. Ballamurugan, Structural and chemical analysis of silica-doped ␤-TCP ceramic coatings on surgical grade 316L SS for possible biomedical application, J. Asian Ceram. Soc. 3 (2015) 317–324.

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