Materials Science and Engineering C 36 (2014) 194–205

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Effect of Sr on the bioactivity and corrosion resistance of nanoporous niobium oxide coating for orthopaedic applications S. Anne Pauline, N. Rajendran ⁎ Department of Chemistry, Anna University, Chennai 600025, India

a r t i c l e

i n f o

Article history: Received 14 August 2013 Received in revised form 15 November 2013 Accepted 6 December 2013 Available online 15 December 2013 Keywords: Implants Strontium Niobium oxide Nanostructures Polarization Electrochemical impedance spectroscopy

a b s t r a c t In this study, strontium incorporated Nb2O5 was synthesized in two different proportions by sol–gel methodology and was deposited on 316L SS by spin coating method. The synthesis conditions were optimized to obtain a nanoporous morphology. The prepared Sr-incorporated Nb2O5 coatings were uniform, smooth and well adherent on to the substrate 316L SS. The coatings were characterized by attenuated total reflectance-infrared spectroscopy (ATR-IR), X-ray diffraction analysis (XRD), scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), atomic force microscopy (AFM) and transmission electron microscopy (TEM) and the formation of Sr-incorporated Nb2O5 coatings with nanoporous morphology was confirmed. Static water contact angle measurements showed an enhancement in the wettability of the obtained coatings. In vitro bioactivity test of the coated substrates showed that 0.05 M Sr-incorporated Nb2O5 coating had better bioactivity compared to 0.1 M Sr-incorporated coating. Solution analysis studies confirmed the controlled release of Sr ions from the coating, which aid and enhance hydroxyapatite (HAp) growth. Electrochemical studies confirmed that the coatings provided excellent corrosion protection to the base material as increased charge transfer resistance and decreased double layer capacitance was observed for the coated substrates. © 2013 Elsevier B.V. All rights reserved.

1. Introduction An orthopaedic implant is a biomaterial used to replace missing or deceased biological structures. An implant material should have superior corrosion resistance, wear resistance, good biocompatibility, high hardness and non-toxic. Materials such as austenitic stainless steels, Co–Cr alloys, Ni–Cr alloys and Ti alloys fulfil these requirements [1,2]. After implantation into the human body, an implant interacts with the host tissue through its surface. It has been reported that the chemical, biomechanical and topographical features of the implant surface control the initial stage of implant integration with the surrounding tissue [3,4]. When a bioactive material is implanted in the human body, a thin bone like apatite layer will deposit on the surface. Owing to the chemical similarities, natural bone may not recognize the apatite layer as foreign, and bonds directly with the implant [5]. The topography of an implant material plays a critical role in deciding this bond formation known as early peri-implant fixation. The combined process of osteoconduction and bone formation on the implant surface is known as contact osteogenesis [6]. An implant with nanoporous topography can positively assist the process by increasing the available surface area for recruitment of osteogenic cells involved in this process. Although, metals and alloys satisfy most of the requirements of an implant material, they fail to create a functional interfacial bond between the metallic surface and the surrounding tissue. In addition, ⁎ Corresponding author. Tel.: +91 44 2235 8659; fax: +91 44 2220 0660. E-mail address: [email protected] (N. Rajendran). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.12.016

release of metallic ions by corrosion leads to aseptic loosening after long-term implantation [7]. These are the two major reasons for a metallic implant failure and hence re-surgery becomes unavoidable. The implantable materials used currently, last for a maximum of 10– 12 years. By depositing a nanoporous ceramic oxide coating on the implant surface, the tissue response to the implant surface can be improved and hence the life of the implant material can be increased. Many researchers have worked on ceramic oxides such as titania (TiO2), silica (SiO2), niobia (Nb2O5), zirconia (ZrO2), mixed oxides etc. They have found that the oxides when applied as a coating over the metallic implant surface, exhibited positive response to HAp growth in vitro, osteoblast adhesion and proliferation in vivo and they also enhanced wear and corrosion resistance of the implant material [8–10]. These reports suggest that, ceramic oxide coated metallic prosthesis combines the mechanical properties of the bulk metal and increased bioactivity and corrosion resistance of the oxide coating. However, there is a constant need for functional implant materials with better osseointegrative properties. Functional coatings can be achieved by incorporating bone stimulating ions such as Zn, Mg, Si, Sr, etc., into their chemical composition [11]. Sr, a trace element in the human body, plays a key role in the stimulation of bone formation and reduction of bone resorption [12]. Clinically, Sr in the form of strontium ranelate when taken orally favours bone healing after surgery. A low dose of Sr can have a beneficial effect on bone healing whereas increased Sr levels can lead to the development of osteomalacia [13]. Further, reports suggest that Sr is also considered to be effective in enhancing bioactivity and have potential effect in the

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treatment of osteoporosis [14]. E. Landi et al. [15] reported that Srsubstituted HAp had better performance in vitro and higher resorption in vivo. Saeed Hesaraki et al. [16] reported that the incorporation of Sr into calcium silicophosphate glass stimulated the proliferation of osteoblastic cells and enhanced their alkaline phosphatase activity. Based on these reports, the present study aims to investigate in detail the bone formation effect of Sr ions by incorporating it into Nb2O5. Nb, a refractory metal has high corrosion resistance, is hypo allergic and is the safest metal tolerated by the human body [17,18]. The biocompatibility and corrosion resistance of Nb2O5 coating on various metallic materials have been reported [19,20]. The Sr-incorporated Nb2O5 was synthesized by sol–gel methodology. This technique is superior to other coating techniques as it can be operated at low temperatures, can coat complex shapes, thin films can be deposited and also homogeneous products at atomic to molecular level can be achieved without the inclusion of impurities [21,22]. Further reports suggest that, protective and bioactive functional coatings can be obtained by addition of particles in the sol [23]. Hence, the aim of the present study was to synthesise Srincorporated Nb2O5 in two different compositions by sol–gel methodology and to deposit them on 316L SS by spin coating technique. The phase composition, microstructure, bioactivity and electrochemical behaviour of the Sr-incorporated coatings on 316L SS were studied and analyzed based on the concentration of Sr ions. Tape adhesion test and Vickers micro hardness test were carried out to assess the adhesion and hardness of the coatings. In vitro bioactivity test was carried out in SBF to evaluate the coatings ability to favour HAp growth. Finally, electrochemical studies like potentiodynamic polarization and electrochemical impedance spectroscopy were carried out on the coated substrates to evaluate the coating ability to resist corrosion.

heating rate of 10 °C min−1. The Brunauer–Emmett–Teller (BET) specific surface area analysis by N2 adsorption–desorption isotherms at liquid nitrogen temperatures were carried out using a Quantachrome quadrawin version 5.02 instrument to determine the specific surface area of Sr-incorporated Nb2O5. The ATR-IR spectral analysis was carried out to determine the functional groups present on the coated substrates using Perkin Elmer FT-IR Spectrometer Spectrum Two with UATR Two accessory and KBR window in the 4000–400 cm−1 range. X-ray diffraction analysis was carried out to analyze the phase composition using Pan Analytical X-pert Pro Diffractometer using Cu Kα radiation (λ = 0.15406 nm), with 40 kV and 30 mA, at a scan rate of (2θ) 0.02° over the range of 10–80°. Surface morphology was investigated using SEM with a Hitachi Model-S 3400. Silicon cantilevers with force constants 0.02–0.77 N/m and tip height 10–15 nm was used. The 3-D topography was examined by AFM using Agilent Technologies Pico LE SPM in contact mode. The microstructure of the coating was examined by TEM with JEOL 2000FX. Hydrophilicity of the substrates were measured using contact angle meter, OCA 15EC, Data Physics Instruments, Germany. Drops of ultrapure water were delivered onto the substrate surface with a set drop volume of 10 μL at a dosing rate of 1 μL s−1. An average of 10 readings was taken for each sample and triplicate measurements were made for each type of coating. Coating thickness was measured using Elcomaster thickness meter. Adhesion of the coating to the substrate was measured by Tape Adhesion test according to ASTM D 3359. For each individual substrate, 25 grids were generated. Adhesive tape was placed on the grids, using a soft eraser; the tape was then removed with a firm and steady pulling action. The equation given below was used for evaluating the percentage of adhesion remaining:

2. Experimental section

AR % ¼ ðn=25Þ  100

The Sr-incorporated Nb2O5 sol was synthesized by sol–gel methodology as follows: Stoichiometric amount of acetylacetone (ACA) was mixed with polyethylene glycol 400 (PEG) and iso propanol (i-PrOH) and the solution was stirred vigorously at 80 °C for 15 min. Later, niobium ethoxide (NbE)–i-PrOH solution was added and vigorous stirring was continued for another 30 min. Strontium in the form of strontium ethylhexanoate (SrEH) was added to the reaction mixture and stirring was continued. After 1 h, water was added and the mixture was allowed to stir vigorously for another 3 h. Finally, the resultant clear sol was aged for 8 h to facilitate gelation. The molar ratio of each chemical in the sol was 1:0.2:30:0.05/0.1:0.02 (NbE:ACA:i-PrOH:SrEH:H2O). All the chemicals used in the synthesis were of analytical grade. Niobium oxide sol was synthesized as a control, by the same procedure without the addition of SrEH. Prior to the deposition process, the surface of 316L stainless steel (316L SS) substrates of size 30 × 15 × 3 mm were abraded with silicon carbide papers (upto 400 grit), degreased with acetone, cleaned with double distilled water and dried in air. The substrates were then treated with a mixture of 15% HNO3 and 5% HF for 2 min to remove the surface oxides, washed thoroughly with double distilled water and ultrasonically degreased with acetone. The composition of 316L SS is given in Table 1. The aged sol was deposited on 316L SS substrates by spin coating at a rotation speed of 2000 rpm for 2 min. The coated substrates were then dried in air at 60 °C and then sintered at 500 °C for 1 h at a slow heating rate of 2 °C min−1. The thermal effects associated with the heat treatment were analyzed using Netzsch STA 409 instrument in nitrogen atmosphere at a

where, AR represents adhesion remaining and n is the average number of squares of undetached coating [24]. The bond strength of the coatings was evaluated according to ASTM C 633 standard. The test was carried out using a material tester, Instron 1196, USA. An average of five readings was recorded as the bonding strength of the coating. Coating hardness was measured with a Vickers Micro hardness Tester with a loading force of 50 g and duration of 5 s. In vitro bioactivity test was carried out by soaking the coated substrates in simulated body fluid (SBF) to evaluate the coating ability to favour apatite deposition. SBF was prepared according to the procedure proposed by Kokubo et al. [25]. Sodium azide was added to SBF to avoid bacterial growth. Throughout the experimental time, the temperature of the samples immersed in SBF was maintained at 37 °C, similar to human body temperature. The solution after bioactivity test was analyzed by ICP-OES analysis after 7 and 14 days to measure the amount of Sr released from the coatings as well as to measure the amount of Ca and P ions taken up by the coating from the solution. During the experimental time, temperature was maintained at 37 °C to mimic the body temperature. Electrochemical tests were carried out to evaluate the corrosion resistance of the coatings. Potentiodynamic polarization curves were recorded in SBF using Electrochemical Workstation, Version 2.0.0.1, CH Instruments, Inc. USA. A conventional three electrode cell of volume 250 mL, fitted with a Pt sheet counter electrode, a saturated calomel reference electrode and a Luggin capillary bridge was used. The polarization curves were recorded at a sweep rate of 1 mV s− 1. The workstation was controlled by a personal computer with software (Electrochemical Software, chi760d beta, version 1.0.0.1) for conducting the experiments. Electrochemical impedance measurements were carried out using a small AC signal of ± 10 mV in the frequency range 100 kHz–0.01 Hz. Prior to testing, the substrates with an exposed surface area of 1 cm2 were immersed in SBF for 60 min and the open circuit potential was measured during the period. Triplicate measurements

Table 1 Composition of 316L stainless steel. Element

Cr

Ni

Mo

Mn

N

C

Fe

Wt %

17.20

12.60

2.40

1.95

0.02

0.03

Balance

ð1Þ

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Fig. 1. TGA and DSC curves of (a) 0.05 M and (b) 0.1 M Sr-incorporated Nb2O5.

were made to check the reproducibility of the results. The impedance spectra were analyzed using equivalent circuits. The contact angle and bond strength of the coatings are reported as means ± standard deviation. Comparison of the experimental data was made using one-way analysis of variance (ANOVA) and Turkey post hoc (multiple comparisons) test using the SSP statistical analysis package (version 2.80). A p value of p b 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Material characterization The TGA plot of 0.05 and 0.1 M Sr-incorporated Nb2O5 is given in Fig. 1. The initial weight loss region was observed at around 323 °C for 0.05 M Sr-incorporated Nb2O5 whereas the same was observed at around 330 °C for 0.1 M Sr-incorporated Nb2O5. This region signifies the removal of solvent and other organic components present in the

sol. A minimal weight loss of 3 and 4% was observed in this step for 0.05 and 0.1 M Sr-incorporated Nb2O5 respectively. The second stage weight loss was observed at around 636 °C for 0.05 Sr-incorporated Nb2O5. However, in the case of 0.1 M Sr-incorporated Nb2O5, the second stage weight loss was found to be around 663 °C. The shift in this weight loss region may be due to increase in the amount of incorporated strontium. The minimal weight loss observed during the second stage is indicative of the phase transformation from amorphous to crystalline oxide and of crystal growth. Based on the TGA analysis, sintering of the coating was carried out at 500 °C, as this temperature would yield a coating free of all organic components and further crystallization of the coatings can happen [26]. Further, it is well known that, when 316L SS is heat treated above 500 °C, it will undergo intergranular corrosion due to chromium depletion [27]. The sintering process ensures removal of organic components as it may have a negative influence on biocompatibility [28]. The N2 gas adsorption and desorption isotherms of 0.05 and 0.1 M Srincorporated Nb2O5 are given in Fig. 2. The average pore size of 0.05 M

Fig. 2. N2-gas adsorption–desorption isotherm of (a) 0.05 M and (b) 0.1 M Sr-incorporated Nb2O5.

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Fig. 3. ATR-IR spectra of Sr-incorporated Nb2O5 coated 316L SS (i) before and (ii) after in vitro bioactivity test.

Sr-incorporated Nb2O5 is 17 Å whereas that of 0.1 M Sr-incorporated Nb2O5 is 15 Å. The total pore volume was 8.49 × 10−2 cm3g–1 for 0.05 M Sr-incorporated Nb2O5 and that of 0.1 M Sr-incorporated Nb2O5 was found to be 1.74 × 10−2 cm3g–1. The hysteresis loops similar to typical H1-type isotherms are observed for the mesoporous samples which have a surface area of 96 and 44 m2g–1 for 0.05 and 0.1 M Srincorporated Nb2O5 respectively. The appreciable surface area of 0.05 M Sr-incorporated Nb2O5 indicates that the material itself is porous in nature due to ageing process. It is reported that, surface area and pore size increase with the ageing time employed prior to film deposition in the sol–gel process [29]. 3.2. Surface characterization The ATR-IR spectrum of Sr-incorporated Nb2O5 coated 316L SS substrates are given in Fig. 3(a). The coatings exhibit a strong broad band in the range of 800–400 cm−1, which could be attributed to the stretching vibration of Nb–O band [30]. After in vitro bioactivity test, the recorded ATR-IR spectrum is given in Fig. 3(b). As evident from the figure, 0.05 M Sr-incorporated Nb2O5 coated substrates exhibited a broad band in the region of 750–400 cm−1. This band arises due to the presence of PO3− 4

group. The band at 1041 cm− 1 is attributed to the presence of P\O bending vibration. Another broad band in the region of 1700– 1400 cm−1 is also seen. This band arises as a result of overlapping of bands at around 1468 cm−1 due to C\O stretching vibration with the band at 1625 cm− 1 attributable to the presence of adsorbed water. The presence of broad band at 3700–3000 cm− 1, is due to the O\H stretching vibration of OH group in the apatite. The presence of these bands indicates the deposition of HAp on 0.05 M Sr-incorporated Nb2O5 coated 316L SS after in vitro bioactivity test. In contrast to this observation, 0.1 M Sr-incorporated Nb2O5 coated substrates showed no characteristic band of apatite, indicating lack of HAp growth. The XRD spectra of the coatings are given in Fig. 4 (a). There are no visible peaks corresponding to the coating material and the obtained peaks are those of the substrate 316L SS. To confirm the phase composition and crystallinity of the coating material, Sr-incorporated Nb2O5 powders sintered at 500 °C were subjected to XRD analysis. The representative XRD plot of Sr-incorporated Nb2O5 powder was given in the inset of Fig. 4(a). Interestingly, the obtained spectra for both the Sr compositions revealed the semi crystalline nature of the coating material. This might be due to the incorporation of Sr as it is reported that incorporation of foreign atoms pushes the crystallization temperature

Fig. 4. XRD pattern of Sr-incorporated Nb2O5 coated 316L SS (i) before and (ii) after in vitro bioactivity test.

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towards higher value [31]. The peaks disclose the presence of Nb2O5 in monoclinic system in accordance with JCPDS No. 43-1042. The peaks at 2θ values of 28.4, 32.8, 49.8, 50.6, 53.4, 64.2 and 72.3° correspond to the planes (100), (102), (113), (113), (006), (022) and (024) respectively. Still, no crystalline strontium compound peak was observed in the XRD spectra. The reason for the absence of visible peaks on the coated

substrates may be due to the negligible thickness of the coating compared to that of the substrate 316L SS. The XRD spectra of 0.05 M Sr-incorporated Nb2O5 coating [Fig. 4(b)] after in vitro bioactivity test showed main diffraction peaks related to (102), (220), (302) and (143) orientations of primitive crystal structure [JCPDS no. 74-0565]. These peaks represent crystalline HAp

Fig. 5. SEM and EDX profiles of (a, b) uncoated, (c, d) Nb2O5 coated, (e, f) 0.05 M Sr-incorporated Nb2O5 coated and (g, h) 0.1 M Sr-incorporated Nb2O5 coated 316L SS before in vitro bioactivity test.

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[Ca10(PO4)6(OH)2]. On the contrary, 0.1 M Sr-incorporated Nb2O5 coated steel did not exhibit any characteristic peak of HAp after bioactivity test. The SEM images and EDX profiles of uncoated and Sr-incorporated Nb2O5 coated substrates before and after in vitro bioactivity test are given in Figs. 5 & 6 respectively. As can be seen from Fig. 5, asperity and grid lines arising from mechanical polishing are seen on the surface

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of uncoated 316L SS. The uncoated substrate is clearly not bioactive as it does not show the presence of HAp particles after bioactivity test. Nanoporous Nb2O5 coating was prepared as a control in order to verify whether Nb2O5 coating without Sr incorporation could enhance the formation of apatite. A detailed discussion about the bioactivity and corrosion resistance of nanoporous Nb2O5 coating on 316L SS was reported in our previous work [32]. For comparison, the SEM image of Nb2O5 coated

Fig. 6. SEM and EDX profiles of (a, b) uncoated, (c, d) Nb2O5 coated, (e, f) 0.05 M Sr-incorporated Nb2O5 coated and (g, h) 0.1 M Sr-incorporated Nb2O5 coated 316L SS after in vitro bioactivity test.

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Fig. 7. 3-D AFM image of (a) 0.05 M Sr-incorporated Nb2O5 coated and (b) 0.1 M Sr-incorporated Nb2O5 coated 316L SS.

316L SS was given and its bioactivity was discussed. The SEM image reveals that the surface was smooth and has a uniform structure with mesopores and nanofissures. The EDX analysis indicates the presence of Nb and O peaks along with the substrate peaks confirming the presence of Nb2O5 on 316L SS surface. Considering 0.05 M Sr-incorporated Nb2O5 coating, the SEM image clearly shows that the coating is continuous, smooth, uniform, and has mesoporous morphology without any cracks or fissures. The pores are partially interconnected forming a network like structure. The coating has retained the appearance of the substrate through calcination. The mesopores are observed to be only on the top surface of the coating and the substrates were well covered by the coating material as an inner compact layer is observed from SEM images. It has been reported that, implant materials possessing a bilayer passive film with an inner barrier and outer porous layer display better osseointegration [33]. The EDX analysis shows the presence of Sr element peak along with the peaks for Nb, O and the base material. This confirms the successful incorporation of Sr into the Nb2O5 coating. After bioactivity test, the surface of the Nb2O5 coating was covered with newly nucleated mineral layer of HAp. The chemical nature of the formed HAp as analyzed by EDX analysis, confirms the presence of HAp due to the presence of characteristic peaks of Ca and P along with the coating element peak. SEM observation of the 0.05 M Srincorporated Nb2O5 coated substrate after bioactivity test reveals the presence of dense deposition of uniform ball like particles of HAp. Exorbitant growth of HAp was observed, where the HAp particles

have clustered and have also formed scale like precipitates covering the entire mesoporous surface. This newly formed layer of HAp was confirmed by EDX analysis, showing the characteristic peaks of Ca and P along with the coating element peak. The intensity of Nb peak in the EDX profile was very less compared to Ca and P elemental peak. This further confirms the enhanced growth of HAp on Sr-incorporated Nb2O5 coated substrate as compared to Nb2O5 coated one. The mesoporous topography of the coating facilitated the nucleation and accelerated growth of HAp on 0.05 M Sr-incorporated Nb2O5 coated 316L SS [34,35]. Comparing the SEM images and EDX profiles of Nb2O5 and 0.05 M Srincorporated Nb2O5 coated 316L SS, it is obvious that Sr incorporation into the Nb2O5 coating has accelerated the growth of HAp as both the coatings exhibited nanoporous surface. The surface of 0.1 M Srincorporated Nb2O5 coating showed a fine mesoporous topography with nanoscale fissures. However, after bioactivity test, the surface appears smooth but deposition of HAp is not seen. This is further evident from EDX profile showing the absence of Ca and P element peaks. Fig. 7(a) & (b) gives the 3-D AFM images of 0.05 and 0.1 M Srincorporated Nb2O5 coated 316L SS. The topographic images reveal the presence of uniform and smooth surface with no lump formation. The root mean square roughness (Rrms) values of 0.05 and 0.1 M Srincorporated Nb2O5 coatings were found to be 13.97 and 11.83 nm respectively. The average roughness (Ra) values of 0.05 and 0.1 M Srincorporated Nb2O5 coatings are 13.28 and 11.34 nm respectively. The roughness values match well with the higher porosity observed in the SEM images. The small differences between Rrms and Ra values may be

Fig. 8. Bright field TEM image of (a) 0.05 M and (b) 0.1 M Sr-incorporated Nb2O5 coated 316L SS.

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380 and 260 HV of 0.1 M Sr-incorporated Nb2O5 coated and uncoated 316L SS respectively. The higher hardness value of the coating indicates that it can offer excellent corrosion resistance to 316L SS [40]. 3.4. Wettability and solution analysis studies

Fig. 9. Vickers Micro hardness of uncoated, 0.05 M and 0.1 M Sr-incorporated Nb2O5 coated 316L SS.

due to the strong topographical variations with higher peaks on the surface [36]. The roughness of the prepared coatings is within the limit of 0.08–1 μm, over which cell proliferation might be difficult in vivo [37]. Fig. 8 shows the Bright-field TEM images of 0.05 and 0.1 M Srincorporated Nb2O5 coatings. It can be seen that the particles have agglomerated and have uniform foam like porous structure. The particles exhibit uniform pore size distribution and the channels are disordered in the nanoscale. 3.3. Mechanical characterization The thickness of Sr-incorporated Nb2O5 coatings on 316L SS was found to be in the range of 2–3 μm. The adhesion strength of the coatings on 316L SS was evaluated by Tape Adhesion test. The coated substrate showed no failed region and AR was deliberated to be 95%, which indicated that the coating has excellent adhesion strength of 5B, according to ASTM standard 3359 D. The coating–substrate bonding strength plays a vital role in deciding the performance and reliability of the coated substrate as an orthopaedic implant [38]. Bond strength of 48.5 ± 4.2 and 42.6 ± 3.7 MPa was observed for 0.05 M and 0.1 M Sr-incorporated Nb2O5 coatings respectively. The decreased bond strength of 0.1 M Sr-incorporated Nb2O5 coating on 316L SS may be due to the presence of nanoscaled blisters [as observed from SEM images], as it is reported that, microcracks can induce macroscopic failure when a load of appropriate value is applied [39]. The Vickers micro hardness values of uncoated and Sr-incorporated Nb2O5 coated 316L SS is given in Fig. 9. The hardness value of 0.05 M Srincorporated Nb2O5 coated substrate increased to 390 HV compared to

The wettability of the coated substrates was evaluated after sintering process. Fig. 10 shows the images of water droplets on uncoated and Srincorporated Nb2O5 coated 316L SS. The coatings exhibited contact angles ranging from 77.2 ± 2.3° for 0.05 M Sr-incorporated Nb2O5 coating to 75.8 ± 3.5° for 0.1 M Sr-incorporated Nb2O5 coating indicating that both the coatings are hydrophilic compared to uncoated substrates with a contact angle value of 80.7 ± 1.2°. A decrease in the contact angle value was observed with increasing Sr concentration in the coating. The low contact angle value is a positive indicator of accelerated HAp growth in vitro and good cell attachment, spreading and proliferation in vivo [41]. The release of Sr ions from the Sr-incorporated coatings was measured by ICP-OES. The results revealed that, Sr ion release decreased with the increase in time. As evident from Fig. 11(a), on the 7th day, Sr release was high and it has decreased considerably on the 14th day. This was due to the nucleation and growth of HAp. However, the dissolution process was still active and goes along with the precipitation process. The precipitation of HAp on 0.05 M Sr-incorporated Nb2O5 coated 316L SS was confirmed by the simultaneous decrease in the concentrations of Ca and P ions from the solution, as noticeable from Fig. 11(b & c). Conversely, a very high increase in the ionic concentration of Sr and only a slight decrease in the ionic concentration of Ca and P was observed for 0.1 M Sr-incorporated Nb2O5 coated 316L SS. The results indicate that, bioactivity is also dependent on the concentration of Sr ions released from the coated substrates. The Sr-incorporated Nb2O5 coatings on 316L SS release Sr ions through exchange with H3O+ ions in SBF and they form Nb-OH groups on their surfaces. The release of Sr ions is expected to accelerate HAp nucleation by increasing the ionic activity product (IAP) of apatite in the fluid [42]. The Nb-OH groups can further react with SBF and form calcium niobate similar to calcium titanate, an intermediate for HAp nucleation [43]. Once nucleation of apatite has been induced, HAp can grow spontaneously by taking up calcium and phosphate ions from SBF as the body fluid is highly supersaturated with respect to apatite [44]. The nanoporous morphology of the coating is expected to increase the available surface area for ion exchange process between the coating and SBF. In our study, 0.05 M Sr-incorporated Nb2O5 coating is found to accelerate HAp growth, whereas no trace of HAp was found on 0.1 M Srincorporated Nb2O5 coated steel. However, it was further reported that a low dose of Sr release accelerates bone formation, whereas, high doses may pose deleterious effects on bone mineralization [45]. Further, it has been reported that, initial burst release of Sr will lead to reduced cell proliferation in vivo, as cells avoid contact with a surface having highest doses of early released Sr [46]. The high increase in the

Fig. 10. Contact angle images of (a) Uncoated, (b) 0.05 M Sr-incorporated Nb2O5 coated and (c) 0.1 M Sr-incorporated Nb2O5 coated 316L SS.

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Fig. 11. ICP-OES analysis of concentrations of (a) Sr, (b) Ca and (c) P ions in SBF after in vitro bioactivity test.

ionic concentration of Sr in SBF after 7 days indicates that there has been initial burst release of Sr. Further, only a slight decrease in the ionic concentration of Ca was observed during the same time period. This indicates reduced Ca absorption by the coating. Hence, the inability of 0.1 M Sr-incorporated Nb2O5 coated 316L SS to support HAp growth may be due high dose of Sr release and that too specifically, initial burst release of Sr and reduced Ca absorption by the coating. 3.5. Electrochemical characterization Typical polarization curves of uncoated and Sr-incorporated Nb2O5 coated substrates in SBF are presented in Fig. 12 and the polarization parameter values are listed in Table 2. It has been reported that, EOCP at the coating–electrolyte interface varies with time and is dependent on various factors, namely, temperature, pH, surface state of the metal, etc. [47]. As a result, by comparing the values of EOCP, it is possible to evaluate the corrosion resistance behaviour of the coatings. The EOCP values revealed that the coated samples are nobler than the uncoated one. For the coated substrates, the active region of the anodic branch of the polarization curves showed significant shift towards the lower current

region. The Ecorr of the coated substrates is high indicating that the coating acts as an insulative barrier and reduces the effective electroactive area responsible for the corrosion process, thereby reducing corrosion. The breakdown potential, Eb of the coated substrates is more electropositive as compared to the uncoated substrates. However, of the coatings studied, 0.05 M Sr-incorporated Nb2O5 coating provided much better resistance to corrosion, as indicated by the extended passivation region and significantly higher Eb value of +458 mV. The shift in Eb can be attributed to the incorporation of Sr into Nb2O5 coating, as the Eb obtained for amorphous Nb2O5 coating obtained by magnetron sputtering on 316L SS in 8.9 g/l NaCl (ionic concentration similar to body fluid) at pH 7.4 is less than +300 mV [48]. An increase in resistance and a decrease in capacitance values are observed for the coated substrates which indicates the corrosion resistance offered by the coating to the underlying material [49]. The results validate that, of the coatings studied, comparatively, a greater increase in Rp and a decrease in Icorr are observed for 0.05 M Sr-incorporated Nb2O5 coating, confirming that this coating offers better corrosion resistance to the underlying material. The electrochemical impedance responses of uncoated and Srincorporated Nb2O5 coated 316L SS on immediate immersion and after in vitro bioactivity test are given in Fig. 13, in the form of BodeImpedance and Bode-Phase angle plots. From the Bode-Impedance plots, it can be observed that the coated substrates exhibited a tenfold increase in impedance module compared to the uncoated substrate. As noticeable from Fig. 13(b), the phase angle of the coated substrates exhibited a significant shift to −80° in the mid frequency region and remained constant in the low frequency region. Thus, the coated substrates exhibited a highly capacitive behaviour illustrative of passive materials. Of the coatings studied, 0.05 M Sr-incorporated Nb2O5 coating exhibited superior insulating and protective property. This may be due to the incorporation of Sr into the coating. However, the

Table 2 Potentiodynamic polarization parameters of uncoated and Sr-incorporated Nb2O5 coated 316L SS in SBF.

Fig. 12. Polarization plots of uncoated, 0.05 M and 0.1 M Sr-incorporated Nb2O5 coated 316L SS in SBF.

316L SS

Eocp mV vs SCE

Eb mV vs SCE

Icorr μA cm−2

Rp kΩ cm2

Uncoated 0.05 M Sr-Nb2O5 0.1 M Sr-Nb2O5

−262 −190 −208

+275 +458 +386

79.60 51.44 69.17

72.96 110.42 76.28

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203

Fig. 13. Bode-Impedance and Bode-Phase angle plots of uncoated, 0.05 M and 0.1 M Sr-incorporated Nb2O5 coated 316L SS in SBF before and after in vitro bioactivity test.

concentration of Sr alters the corrosion resistance offered by these coatings as evident from the decreased insulation provided by 0.1 M Srincorporated Nb2O5 coating. This might be due to addition of Sr beyond the optimal concentration, as it was reported that when a metal is incorporated into a system beyond the optimal concentration, it may lead to high coefficient of expansion of its oxide which aids the probability of cracking [50]. Further, in the case of 0.1 M Sr-incorporated Nb2O5 coating, the electrolyte can easily reach the alloy surface through conductive pathways in the pores. Whereas, in the case of 0.05 M Sr-incorporated Nb2O5 coating, as the pores are smaller in size as evident from SEM images, the formation of conductive pathways is comparatively difficult and hence the electrolyte would need a longer period of time to reach the alloy surface. The Bode-Impedance and Bode-Phase angle plot of the coatings after in vitro bioactivity test are given in Fig. 13(c & d). An increase in impedance value at low frequency is observed for 0.05 M Sr-incorporated Nb2O5 coating. Further, a new time constant is also observed for the same in Bode-Phase angle plot, in the low frequency region with a maximum phase angle value of − 63°. The appearance of a new time constant indicates the initiation of a new layer i.e., HAp over the nanoporous 0.05 M Sr-incorporated Nb2O5 coating [51]. The impedance spectra were analyzed with equivalent circuits and an excellent agreement was observed between the experimental and

fitted results. In the present study, the constant phase element (Q) is used, as it allows the simulation of phenomena that deviates from ideal capacitive behaviour. Exponent n of the CPE varies between 0 b n b 1, due to fractal geometry i.e., surface inhomogeneity [52]. The equivalent circuits used for fitting the experimental data are given in Fig. 14. The obtained impedance parameters for uncoated and Srincorporated Nb2O5 coated 316L SS before and after in vitro bioactivity test are given in Tables 3 & 4. The simple Randles circuit, Rs[RbQb], represents the impedance response of uncoated 316L SS with thin native oxide film, where Rs, Rb and Qb correspond to solution resistance, charge transfer resistance and double layer capacitance. The low Rct value of the uncoated substrate indicated poor protective nature of the native oxide layer. The Sr-incorporated Nb2O5 coated substrates can be fitted with a two time constant, equivalent to the inner barrier and outer porous layer. The higher Rb and lower Qb values of 0.05 M Sr-incorporated Nb2O5 coated substrate confirm that the coating is insulative and prevents the ingress of corrosive ions through the coating. The 0.05 M Sr-incorporated Nb2O5 coated 316L SS after in vitro bioactivity test can be fitted with a three-time constant resulting from the formation of a new apatite layer over the barrier layer. Whereas, the 0.1 M Sr-incorporated Nb2O5 coated 316L SS can only be fitted with a two time constant. The equivalent circuit used to fit the uncoated and 0.1 M Sr-incorporated Nb2O5 coated substrates after immersion in SBF

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Fig. 14. Equivalent circuits used to fit (a) uncoated, (b) coated and (c) coated substrates after HAp growth.

Table 3 Electrochemical impedance parameters of uncoated and Sr-incorporated Nb2O5 coated 316L SS in SBF. 316L SS

Rs Ω cm2

Rc kΩ cm2

Qc μF cm−2

nc

Rb kΩ cm2

Qb μF cm−2

nb

Uncoated 0.05 M Sr-Nb2O5 0.1 Sr-Nb2O5

84.73 33.21 57.97

– 362 135

– 3.46 11.38

– 0.84 0.44

918 3.01 × 109 8.46 × 107

17.45 1.01 1.15

0.81 0.88 0.85

is Rs(RcQc)(RbQb) and the equivalent circuit used to fit 0.05 M Srincorporated Nb2O5 coated 316L SS is Rs(RaQa)(RcQc)(RbQb) respectively. Here Rc and Ra represent the charge transfer resistance of the coated samples and the newly formed apatite layer. Qc and Qa represent the double layer capacitance of the coating and the apatite layer. The 0.05 M Sr-incorporated Nb2O5 coated 316L SS has very high resistance value after immediate immersion in SBF and it has not been altered much after in vitro bioactivity test. This is due to the growth of HAp which imparts certain degree of corrosion resistance to the coated substrate by preventing the dissolution of metal ions [53]. The results confirm that 0.05 M Sr-incorporated Nb2O5 coating exhibited unaltered barrier effect even after immersion in SBF. 4. Conclusions Sr-incorporated Nb2O5 sols were prepared by sol–gel process and the coatings were deposited on 316L SS by spin coating technique.

Calcination of the coated substrates leads to the formation of nanoporous morphology as confirmed by SEM and AFM analysis. The 0.05 M Sr-incorporated Nb2O5 coating supported the nucleation and accelerated the growth of apatite layer over its surface during in vitro bioactivity test in SBF. The bioactivity depends on the nanoporous morphology of the coating and the Sr release rate. Of the coatings studied, 0.05 M Sr-incorporated Nb2O5 coating exhibited enhanced bioactivity due to controlled and continuous release of strontium. Mechanical studies confirmed that the coatings are well adherent and have high hardness value. The shift in the Eb value to + 458 mV for 0.05 M Srincorporated Nb2O5 coating defined that the coating acts as a barrier layer and prevents the underlying material from corrosion. The excellent corrosion protection offered by the coating to 316L SS is further demonstrated by the high Rb and low Qb obtained from EIS studies. Hence, it can be concluded that, incorporating 0.05 M Sr into Nb2O5 coating with nanoporous morphology is an appropriate method of surface modification of 316L SS to improve its bone bonding ability and corrosion resistance.

Acknowledgement One of the authors S. Anne Pauline is thankful to Council of Scientific and Industrial Research (CSIR), India for financial assistance under Senior Research Fellowship (SRF) scheme. Instrumentation facilities provided under DST-FIST and UGC-DRS to Department of Chemistry, Anna University, Chennai, India are great fully acknowledged.

Table 4 Electrochemical impedance parameters of uncoated and Sr-incorporated Nb2O5 coated 316L SS after in vitro bioactivity test in SBF. 316L SS

Rs Ω cm2

Ra kΩ cm2

Qa μF cm−2

na

Rc kΩ cm2

Qc μF cm−2

nc

Rb kΩ cm2

Qb μF cm−2

nb

Uncoated 0.05 M Sr-Nb2O5 0.1 M Sr-Nb2O5

7.94 12.87 12.42

1281 22,670 –

37.83 3.04 –

0.71 0.70 –

44.60 7.31 × 109 1040 × 106

9.83 1.87 27.59

0.89 0.98 0.92

77.00 115.20 90.17

37.20 1.48 15.66

0.69 0.48 0.88

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