hydroxyapatite composite film on zirconium

Surface & Coatings Technology 238 (2014) 58–67

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Fabrication, characterization and in-vitro evaluation of nanostructured zirconia/ hydroxyapatite composite film on zirconium Sandhyarani M. a, Rameshbabu N. a,⁎, Venkateswarlu K. a,b, Rama Krishna L. c a b c

Department of Metallurgical and Materials Engineering, National Institute of Technology, Tiruchirappalli 620015, Tamilnadu, India Department of Physics, National Institute of Technology, Tiruchirappalli 620015, Tamilnadu, India International Advanced Research Centre for Powder Metallurgy and New Materials, Balapur P.O., Hyderabad, 500005, India

a r t i c l e

i n f o

Article history: Received 15 July 2013 Accepted in revised form 19 October 2013 Available online 1 November 2013 Keywords: Zirconia/hydroxyapatite film Plasma electrolytic oxidation Electrophoretic deposition Corrosion resistance Bioactivity Cell adhesion

a b s t r a c t The present study aims to fabricate ZrO2/hydroxyapatite [HA] composite film on Zr by plasma electrolytic oxidation coupled with electrophoretic deposition process in a single step. Further, ZrO2/HA film formation mechanism was studied as a function of treatment time in the range of 2 to 6 min in an aqueous electrolyte system consisting of dissolved tri sodium orthophosphate and suspended HA nanoparticles. The phase composition, surface morphology and elemental composition of the formed films were examined by X-ray diffraction [XRD], Raman spectroscopy and scanning electron microscopy equipped with energy dispersive X-ray spectroscopy. Surface roughness, wettability, corrosion resistance, bioactivity and osteoblast cell adhesion characteristics of the ZrO2/HA film were also investigated. Uniform and dense ZrO2/HA films with thickness varying from 42 to 75 μm were formed at 2 to 6 min of treatment time. XRD results revealed that the films were comprised of nanocrystalline cubic zirconia and monoclinic zirconia. During the film growth process, HA particles were dragged into the discharge channels and subsequently entrapped into the oxide film by electrophoretic deposition. Additionally, calcium that originated from partial melting of HA enters the sites of Zr thereby stabilizing cubic ZrO2 phase. Raman spectrum confirmed the presence of HA phase along with ZrO2 phases. ZrO2/HA film exhibited better wettability and high surface energy compared to untreated Zr. Potentiodynamic polarization test revealed that the ZrO2/HA film formed at 6 min treatment time showed superior pitting corrosion resistance compared to untreated Zr in simulated body fluid [SBF] environment. Bone-like apatite layer was formed on entire surface of ZrO2/HA film after soaking in SBF for 8 days, indicating its significantly enhanced in-vitro bioactivity. Cell adhesion test results showed that the human osteosarcoma cells could attach, adhere and propagate well on the surface of ZrO2/HA film, suggesting the potential application of ZrO2/HA coated Zr as orthopedic implant material. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Zirconium [Zr] and its alloys have been the material of choice for orthopedic and dental implants, hard tissue replacements due to its excellent technological properties including chemical and dimensional stability, high flexural strength, fracture toughness, lower cytotoxicity, good corrosion resistance and biocompatibility [1–4]. Zr is expected to be a surgical implant material due to its low elastic modulus [92 GPa] which is viewed as a biomechanical advantage as it can result in minimal stress shielding of the host bone compared to titanium and its alloys [100–110 GPa], stainless steel [189–205 GPa] and CoCr alloys [230 GPa] [5]. The good corrosion resistance of Zr has been mainly attributed to its native oxide film. Nevertheless, this oxide film shows morphological fixation with the surrounding tissues without producing any chemical or biological bonding when implanted, because of its poor bioactivity and due to the encapsulation phenomena by the fibrous tissues [6]. Additionally, this native oxide film can be lost soon when Zr is used in long ⁎ Corresponding author. Tel.: +91 431 2503464; fax: +91 431 2500133. E-mail addresses: [email protected], [email protected] (R. N.). 0257-8972/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.surfcoat.2013.10.039

term load bearing applications. The improvement of the bioactivity and corrosion resistance of Zr implants has been the key research focus in the recent years. Plasma electrolytic oxidation [PEO], also called microarc oxidation [MAO] or spark plasma anodization, is gaining attention as a novel and unique technique for development of firmly adherent and relatively thick oxide films on light metals such as Zr, Al, Mg and Ti [6–10]. Further, uniform coatings can be obtained on the relatively complex geometries in the PEO technique, as it is not a line of sight process [11] and it has been proven as an effective, eco-friendly substitute to conventional techniques primarily in terms of oxide films being formed in environmental friendly electrolytes [12]. PEO fabricated zirconia [ZrO2] films have demonstrated promising in-vitro corrosion resistance, acting as a chemical barrier against the release of metal ions from the implants [13,14]. However, being bioinert, the chemical bond with the living bone in the body is not very strong with ZrO2. Some researchers have focused on improving bioactivity of PEO treated Zr by secondary treatments. For instance, Yan et al. [15] reported that chemical treatments in which immersion in acid or alkaline solutions after PEO treatment can enhance the apatite formation ability of Zr in SBF medium. Zhang

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et al. [16] investigated the PEO treated Zr when photo excited by UV light of energy greater than its band gap resulted in abundant basic Zr–OH groups on the surface and exhibited an enhanced apatite formation during immersion in SBF solution. Further, calcium ions are successfully incorporated into the ZrO2 film during MAO process using an electrolyte solution containing calcium precursor, which resulted in a significant enhancement in the apatite forming ability of Zr in SBF medium [5]. Yet another approach to improve the bioactivity and corrosion resistance of Zr is by forming ZrO2/HA composite film, as hydroxyapatite [HA, Ca10(PO4)6(OH)2] can spontaneously bond to and integrate with bone in living body. ZrO2/HA composite coating has been proved to be an excellent coating option on nitinol and stainless steel substrates to simultaneously impart diverse properties such as corrosion resistance and bioactivity [17,18]. However, available literature on the fabrication of ZrO2/HA coatings on Zr is limited. Plasma spraying is one of the most widely adopted processes. ZrO2/HA composite coating on Zr by plasma spraying revealed that the addition of HA to ZrO2 showed undoubtedly superior biological properties to that of ZrO2 coating, while maintaining its biocompatibility [19]. However, plasma sprayed coatings lack reliability as a result of residual stress and further, it is not possible to prepare uniform coatings on Zr implants with complex geometries. In recent years, electrophoretic deposition [EPD] is gaining more interest to produce ZrO2/HA composite coatings. EPD is a simple, inexpensive and highly versatile coating technique for the development of ceramic coatings, in which coatings are formed on the electrode by migration of charged ceramic particles under the influence of an electric field [20]. Additionally, in the context of orthopedic and dental implant coatings, EPD has been proposed especially for the fabrication of bioactive glass coatings or composite coatings combining bioactive glass with other ceramics from organic or aqueous suspensions [21]. However, EPD requires a post-heat-treatment process to improve the adhesion of the coatings to the substrate. Thus, in order to combine the potential advantages of PEO and EPD processes, in this study, a novel and rather simple approach based on coupling the PEO and EPD processes [hereafter named as “PEOEPD”] has been employed to fabricate bioactive ZrO2/HA composite film on Zr substrate in a single step. For this purpose, charged HA nanoparticles were added to base electrolyte to develop ZrO2/HA film on Zr substrate. In order to study the ZrO2/HA film formation during the process and also to simultaneously understand the nature of phases being formed and associated phase transformations, PEOEPD was performed on Zr substrate as a function of treatment time in the duration of 2, 4 and 6 min. Based on the analysis of experimentally obtained data pertaining to the surface morphology, phases evolution and surface roughness as a function of PEOEPD treatment time, the ZrO2/HA composite film formation mechanism has been explained. Further, the efficacy of ZrO2/HA composite film on in-vitro corrosion resistance, bioactivity and biocompatibility of PEOEPD treated Zr substrate has been investigated. 2. Materials and methods 2.1. Materials Commercially available Zr [purity N 99.5 wt.%] coupons with the dimensions of 20 mm × 15 mm × 1.5 mm were used as substrate material in the present study. Prior to PEOEPD treatment, the specimens were ground with SiC abrasive paper [with five grades: 80, 240, 400, 800 and 1200 grit] and then cleaned ultrasonically with acetone and deionized water bath to avoid surface contamination. The development of ZrO2/HA composite film involves three steps, namely synthesis of HA nanoparticles, preparation of electrolyte and finally PEOEPD treatment. In the present study, HA nanoparticles were prepared by microwave synthesis method. Analytical grade calcium hydroxide [Ca(OH)2, E. Merck, Germany] and di-ammonium hydrogen phosphate [DAP, (NH4)2HPO4, E. Merck, Germany] were used for the synthesis of HA nanoparticles.

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The experimental procedure for microwave synthesis of nanosized HA [15–20 nm width and 60–80 nm length] particles was reported in one of the author's previous study [22]. 2.2. Preparation of PEOEPD electrolyte HA aqueous suspension was prepared by adding 5 g of HA powder, 10 ml of ethylene glycol [C2H6O2, Merck, India] and 5 ml of triethanolamine [C6H15NO3, Merck, India] simultaneously to 1 l of distilled water taken in a conical flask. Ethylene glycol was used to charge HA nanoparticles negatively, while triethanolamine was used to stabilize the suspension of nanoparticles. The aqueous HA suspension was then sonicated using an ultrasonic vibrator [Sonics, 750 W, 20 kHz, USA] for 45 min to obtain a stable dispersion. Later, 5 g of tri-sodium ortho phosphate [TSOP; Na3PO4·12H2O, Merck India Pvt. Ltd.] was added and then dissolved in the prepared aqueous HA suspension under uniform magnetic stirring conditions. This TSOP dissolved aqueous solution with negatively charged HA particle suspension serves as electrolyte for PEOEPD process. 2.3. Development of ZrO2/HA films by PEOEPD treatment For PEOEPD treatment, the electrolyte prepared in the previous step was taken into a stainless steel bowl which serves as cathode and Zr coupons immersed in electrolyte serve as anode. The PEOEPD treatment was performed at constant current density of 150 mA/cm2, frequency of 50 Hz and duty ratio of 95% for 6 min of treatment time using a pulsed DC power supply unit [Milman Thin Films Pvt. Ltd., Pune, India] of 900 V/15 A capacity. More technical details about the experimental set up employed in the present study were reported in our previous work [13]. During the PEOEPD process, at higher voltages, the negatively charged HA particles that are dispersed in the electrolyte are attracted and migrate toward the anode (Zr substrate) due to the electric field between anode and cathode, and eventually gets deposited into the oxide film grown via PEO process. Thus, formation of oxide layer via PEO and deposition of charged HA particles into oxide film via EPD will take place simultaneously, thereby forming ZrO2/HA composite layer. In order to study the ZrO2/HA composite film formation mechanism, PEOEPD process was interrupted at 2 and 4 min of treatment time. After the PEOEPD treatment, the samples were taken out from the electrolyte, washed with distilled water, air dried at room temperature and stored in vacuum desiccator for further characterization. The identification codes for the PEOEPD treated Zr samples at 2, 4 and 6 min treatment time were hereafter represented as HA2, HA4 and HA6, respectively, while the substrate was referred as “S”. 2.4. Surface characterization of ZrO2/HA composite films The phase composition of HA2, HA4 and HA6 films were studied by a Rigaku Ultima III X-ray diffractometer [40 kV, 30 mA] with a Cu Kα [λ = 1.5406 Å] radiation over a 2θ range from 20° to 70° with a scan speed of 1° min−1 and a step size of 0.05°. The corresponding phases were identified by matching relevant data from the JCPDS [Joint Committee on Powder Diffraction Standards] cards. To provide further information on phase composition, Raman spectral analysis was performed on the surfaces of HA2, HA4 and HA6 films by WiTec Raman spectroscopy [Alpha300, Germany] using Nd:YAG laser light of 532 nm wavelength. The surface morphology, film thickness, elemental composition and mapping of HA2, HA4 and HA6 films were observed using scanning electron microscope [SEM, Hitachi, S3000 N] equipped with energy-dispersive X-ray spectroscopy [EDS]. Before SEM analysis, PEOEPD treated samples were sputtered with a thin gold layer to make the coating surfaces conductive. The average surface roughness [Ra] of untreated Zr and HA2, HA4 and HA6 samples was measured using surface profilometer [Surtronic 25, Taylor-Hobson, Precision, UK] with accuracy of 0.01 μm. Contact angle of untreated Zr and HA2,

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HA4 and HA6 samples was observed by sessile drop method with Easy DROP instrument [DSA100, KRUSS, Germany] using distilled water as the contacting solvent. The drop image was stored by a video camera and an image analysis system was employed to calculate the contact angle [θ] based on the average of left and right angles of each drop. Measurements were obtained at ten different locations on the sample surface and the average values were reported [Table 3]. 2.5. In-vitro tests 2.5.1. Electrochemical corrosion study The corrosion resistance properties of the untreated Zr and HA6 film were assessed by potentiodynamic polarization [PDP] test using a computer controlled potentiostat/galvanostat/frequency response analyzer [Gill AC, ACM Instruments, Cumbria, UK] unit. The three electrode cell, with sample as working electrode, saturated calomel as reference electrode and platinum foil as counter electrode, was used in the present study. All experiments were carried out under simulated body fluid [SBF] conditions [7.4 pH and 37 °C]. The SBF test medium was prepared following the procedure suggested by Kokubo and Takadamo [23]. The exposed area of the working electrode with SBF solution was 0.5 cm2. Prior to these tests, all the test samples were exposed to the test solution for 4 h to attain a stable open circuit electrode potential [OCP]. Subsequently, the current density was measured over a potential range of ±200 mV [Tafel region] with reference to OCP by employing a scan rate of 0.166 mV/s. The corrosion current density and corrosion potential were obtained using Tafel extrapolation method. The polarization resistance [Rp] of all the test samples is calculated using Stern–Geary equation [8]: Rp ¼

βa  βc 2:303 jcorr ðβa þ βc Þ

ð1Þ

where βa and βc are slopes of anodic and cathodic Tafel plots, respectively, and jcorr is the corrosion current density. From the obtained Rp values, the protection efficiency of HA6 film was calculated according to the formula [24]: Protection efficiency ð% Þ ¼

Rp ðPEOEPD treated ZrÞ−Rp ðuntreated ZrÞ

 ð100Þ:

Rp ðPEOEPD treated ZrÞ

ð2Þ

Further, the passivation behavior of all the samples in the SBF medium was studied over a potential range of −500 mV to 3000 mV versus OCP at a scan rate of 100 mV/min by performing PDP test.

using well-characterized human osteoblast cell line [human osteosarcoma (HOS) cells, National Centre for Cell Science, Pune, India]. HOS cells seeded on conditioned material [glass cover slip] were used as a test control. These cells were subcultured and seeded at a density of 1 × 104 cells/cm2 on both the samples in minimum essential medium supplemented with 10% fetal bovine serum under standard cell culture conditions [sterile chamber maintained at 37 °C and a humidified environment: 5% CO2/95% air]. Both untreated Zr and HA6 sample were autoclaved for sterilization purposes at 121 °C for 20 min. After 48 h, samples were rinsed three times with phosphate buffered saline to remove non-adherent cells, while adherent cells were fixed with 2.5% glutaraldehyde. Samples were then dehydrated through graded concentration of alcohol followed by critical point drying. Dried samples were sputter-coated with gold, and were subjected to SEM examination to evaluate adhesion of HOS cells. 3. Results and discussion 3.1. Phase structure of ZrO2/HA films The XRD patterns of HA2, HA4 and HA6 samples are shown in Fig. 1. All the films composed of cubic zirconia [c-ZrO2, JCPDS card no. 491642] as a major phase and monoclinic zirconia [m-ZrO2, JCPDS card no. 37-1484] as a minor phase. Further, as the PEOEPD treatment time increases from 2 to 6 min, the m-ZrO2 peaks gradually decrease and c-ZrO2 peaks gradually increase and at 6 min treatment time, m-ZrO2 is almost converted to c-ZrO2. The decreasing tendency of m-ZrO2 in the films with treatment time is expected to be related to two factors. It is reported that, during the PEO process, the instantaneous localized temperature in the arc discharge region reaches around 2500 K [25]. Further, the higher voltages [535 to 542 V at 2 to 6 min of treatment time in the present study] can yield higher temperatures, enhancing the transformation of m-ZrO2 to c-ZrO2, because the later is a high temperature phase. On the other hand, during PEOEPD film formation process, Ca that originated from partial melting of HA in discharges can occupy Zr sites in ZrO2 and stabilizes c-ZrO2 phase. In addition to oxide peaks, the XRD patterns also showed diffraction peaks that correspond to Zr substrate [JCPDS card no. 05-0665]. It can be seen from Fig. 1(a, b, c), that the intensity of Zr peaks gradually decreases with increase in treatment time which could be attributed to the increase in film thickness at higher treatment time. The size of all films  crystallite  was calculated using Scherer's formula for 111 and (111) reflections of m-zirconia and c-zirconia, respectively, and the obtained values are

2.5.2. In-vitro bioactivity study The apatite forming ability of the substrate and the HA6 sample was evaluated by immersing the samples in SBF with ion concentrations reported elsewhere [23]. The SBF solution was buffered to pH of 7.4 with tris [hydroxymethyl] amino methane and concentrated HCl at 36.5 °C. Each sample was vertically immersed in a plastic vial containing 62 ml of SBF solution and was kept under static conditions inside a biological thermostat at 36.5 °C to a certain period. The ratio of the sample's surface area [in mm2] to SBF solution volume was set equal to 10. The SBF solution was renewed every 24 h and has been kept colorless and stable without any deposit during immersion period to maintain the ionic concentration. After immersing for 4, 8 and 12 days, the samples were taken out from the SBF, gently washed with distilled water, dried in air and stored in vacuum desiccator and analyzed by scanning electron microscopy equipped with energy dispersive X-ray spectroscopy. 2.5.3. Cell adhesion test To study the biocompatibility of ZrO2/HA composite film, an in-vitro cell adhesion test was performed on HA6 sample and untreated Zr,

Fig. 1. XRD patterns of the PEOEPD fabricated (a) HA2, (b) HA4 and (c) HA6 films.

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reported in Table 1. The films consist of c-ZrO2 phase with crystallite size ranging from 38 to 41 nm and m-ZrO2 phase with crystallite size ranging from17 to 22 nm. From Fig. 1, it can be noted that the peaks corresponding to HA or decomposition products of HA are not observed in any of the samples. The absence of diffraction peaks corresponding to HA phase in XRD patterns suggests that either low concentration of HA particles is incorporated in the oxide film, or the HA particles entrapped in discharge channels during PEOEPD process might be melted and re-solidified as amorphous phase. To validate the presence of HA, Raman spectroscopy data was recorded for HA2, HA4 and HA6 samples (Table 2). X-ray diffraction can only detect crystalline phases within a large area [in accordance to the classical Bragg–Brentano geometry], and does not provide sufficient information about amorphous phases [26]. On the other hand, Raman spectroscopy provides valuable information about the structural changes that take place locally on the various calcium phosphate phases. Thus, the detailed structural information obtained using the Raman spectroscopy technique could be essentially important. The Raman spectrum of HA2, HA4 and HA6 films [Fig. 2] shows that all bands correspond to those of cubic zirconia and monoclinic zirconia are present as in clear support of XRD results presented. In HA2 sample, besides the bands corresponding to the ZrO2 phase, there is a band at 959 cm−1 [assigned from P\O vibration], which is associated with HA, even though in small quantity. However, the formed HA is amorphous in nature, which is reflected by the large broad band around 959 cm−1. As the treatment time increases, the relative intensity of the HA band [~960 cm−1] increases proving that the films are composed of HA phase along with ZrO2 phases. In Fig. 2(c), the bands at vibration modes υ1 [961 cm−1], υ2 [444 cm−1], υ3 [1044, 1061, and 1135 cm−1] and υ4 [581, and 611 cm−1] are associated with the internal modes of phosphate ions surrounded by Ca2+ and OH− ions in HA [27]. 3.2. Surface morphology and elemental composition of ZrO2/HA films The surface morphology, film thickness and elemental composition of the HA2, HA4 and HA6 films were studied by scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy facility and the results are presented in Fig. 3. The films show variation in morphological features with treatment time and film thickness increases with increase in treatment time. It can be seen from Fig. 3(a), HA2 film surface is covered with fine discharge channels appearing as circular spots distributed uniformly over entire surface. The low content of Ca and high content of Zr and O from elemental composition spectra [Fig. 3(a′)] reveals that oxidation rather than EPD is predominant in the initial stages of PEOEPD process. The concentration of phosphorous content is due to the incorporation of electrolyte borne element into the oxide film during the PEO process. As treatment time increases from 2 to 4 min, the number of discharge channels decreases and the discharge channel diameter concurrently increases as shown in Fig. 3(b). Additionally, along with oxide discharge channels, electrophoretic deposition of HA is taking place by forming deposits on to the surface. Table 1 Sample codes, process parameters and phase details obtained from XRD and Raman spectra of ZrO2/HA composite film formed for different treatment time. PEOEPD treatment time (min)

Sample code

Final voltage (volts)

Thickness (μm)

XRD Phase

Crystallite size (nm)

2

HA2

535

42 ± 2

M, C, Z

4

HA4

539

58 ± 3

M, C, Z

6

HA6

542

75 ± 5

M, C, Z

M(−111)–17.6 C(111)–38.6 M(−111)–19.8 C(111)–40 M(−111)–22.5 C(111)–41.5

Raman phases

M, T, C, HA M, T, C, HA M, T, C, HA

Note: M refers to monoclinic zirconia; T refers to tetragonal zirconia; C refers to cubic zirconia; Z refers to Zr substrate; and HA refers to hydroxyapatite.

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Table 2 Major Raman band assignments of PEOEPD fabricated HA2, HA4 and HA6 films. Raman bands

Ag Raman active mode B1g Raman active mode Bg Raman active mode PO3− 4 υ2 (bend) (asymmetric stretch) Ag Raman active mode PO3− 4 υ4 (bend) (asymmetric stretch) Ag Raman active mode PO3− 4 PO3− 4

υ1 (symmetric stretch) υ3 (stretch)

Phase

Wave number (cm−1)

Reference

HA2

HA4

HA6

m-ZrO2 t-ZrO2 m-ZrO2 HA

178 255 381 –

173 255 – –

173 255 – 444

[4,14] [4] [4,14,26] [27]

m-ZrO2, t-ZrO2 HA

471

471

–

[4,14]

–

611

581, 611

[27]

m-ZrO2, c-ZrO2 HA HA

633

–

–

[4,14,26]

959 –

961 –

961 1044, 1061, 1135

[22,27] [27]

The increase in Ca concentration in HA4 film confirms that HA is getting incorporated into the oxide film [Fig. 3(b′)]. It can be observed from Fig. 3(a′, b′, c′), the decrease in Zr concentration and concurrent increase in Ca and P concentrations with increase in treatment time from 2 to 6 min. The changes in concentration of Ca and P can be attributed to the changes in processing voltages with increase in treatment time [Table 1]. Thus at higher voltages, the negatively charged HA particles are attracted towards the anode [Zr substrate] and gets deposited into the oxide film formed by PEO. It can be noticed from SEM surface micrographs that the oxide phase formation is predominant at lower anodic potentials through PEO and HA deposition is predominant at higher potentials through EPD. The thickness of HA2, HA4 and HA6 films were measured using SEM cross-sectional micrographs shown in Fig. 3(a′), (b′) and (c′) and the values obtained are reported in Table 1. At 2 min of treatment time, a relatively dense and uniform film with 42 μm thickness is obtained. Further, the thickness increased with increase in PEOEPD treatment time and the final thickness attained at the end of 4 and 6 min of treatment time are 58 and 75 μm, respectively. The elemental mapping of Zr, O, P and Ca elements for HA6 film is shown in Fig. 4 for instance. From Fig. 4, it can be observed that the Ca and P are uniformly present over the surface of the film. 3.3. Surface roughness and wettability of ZrO2/HA films The surface roughness [Ra] of untreated Zr and HA2, HA4 and HA6 films is presented in Table 3. The Ra of PEOEPD treated Zr sample(s) is significantly higher than that of untreated Zr. Further, as the PEOEPD treatment time increases from 2 to 6 min, the Ra value increases from 1.24 ± 0.04 μm to 2.28 ± 0.05 μm. The film formed at 6 min treatment time exhibits significantly increased surface roughness due to the thicker ZrO2/HA composite layer formation in comparison to HA2 and HA4 films. The higher roughness of HA6 film at 6 min of treatment time could be as a result of rapid deposition of HA particles due to EPD [Fig. 3(c)]. It was reported in literature that implant surfaces having Ra value in the range of 0.3 to 2 μm are expected to improve bone responses favorably [28]. Contact angle measurement of untreated Zr and HA2, HA4 and HA6 films was shown in Fig. 5 and the corresponding values were presented in Table 3. From Fig. 5, it was observed that all PEOEPD treated Zr films show good wettability compared to untreated Zr. The water contact angles of untreated and PEOEPD films are 55° [SD ± 4.4, N = 10] for untreated Zr, 35.8° [SD ± 2.2, N = 10] for HA2, 35.5° [SD ± 1.1, N = 10] for HA4 and 28.8° [SD ± 3.1, N = 10] for HA6 films, respectively [Fig. 5]. The surface energy [Es] has been calculated from these contact angles using the equation [29] ES ¼ Evl cos θ

ð3Þ

Fig. 2. Raman spectra of the PEOEPD fabricated (a) HA2, (b) HA4 and (c) HA6 films.

62 S. M. et al. / Surface & Coatings Technology 238 (2014) 58–67

S. M. et al. / Surface & Coatings Technology 238 (2014) 58–67

63

Fig. 3. Surface micrographs, EDS spectra and cross-sectional SEM images of the PEOEPD fabricated (a) HA2, (b) HA4 and (c) HA6 films.

where Evl is the surface energy between water and air under ambient condition, [i.e., 72.8 mJ/m2 at 20 °C] for pure water and θ is the static contact angle. The surface energy values of untreated Zr, HA2, HA4

and HA6 films were reported in Table 3. HA6 film has been found to have higher surface energy [63.7 ± 1.9 mJ/m2 at 20 °C] and untreated Zr has found to have lower surface energy [41.6 ± 4.6 mJ/m2 at

Fig. 4. The EDS elemental mapping images of the PEOEPD treated Zr at 6 min treatment time.

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Table 3 Surface roughnesses, contact angle measurements and surface free energy values of untreated Zr and HA2, HA4 and HA6 films. Sample code

Surface roughness (Ra) (μm)

Contact angle (°)

Surface energy (mJ/m2)

S HA2 HA4 HA6

0.6 1.24 1.78 2.28

55.0 35.8 35.5 28.8

41.6 59.0 59.3 63.7

± ± ± ±

0.06 0.04 0.06 0.05

± ± ± ±

4.4 2.2 1.1 3.1

± ± ± ±

4.6 1.6 0.8 1.9

20 °C], and among PEOEPD treated samples, surface energy has been found to increase with increasing treatment time. The low contact angle and high surface energy for PEOEPD treated Zr are due to the ZrO2/HA composite film formed on its surface. It has been reported in literature, surface free energy plays an important role that guides the first events occurring at the biomaterial/biological interface [such as interaction and adsorption of water and proteins with biomaterial implants] and in general, cellular adhesion strength on metallic implants is proportional to their surface free energy [30,31]. Thus in the present study, the HA/ZrO2 composite film on Zr formed by PEOEPD with high surface energy is expected to influence the initial cell attachment and spreading of human osteoblastic cells at the surface. 3.4. ZrO2/HA film formation mechanism Based on the experimental data obtained through XRD, Raman, SEM and EDS analysis, the mechanism of the ZrO2/HA composite film formation has been postulated. ZrO2 has three known polymorphs under atmospheric pressure, namely monoclinic [temperature below 1000 °C], tetragonal [temperature between 1000 °C and 1500 °C], and cubic [temperature above 1500 °C], in which m-ZrO2 is thermodynamically the most stable phase at ambient temperature and t-ZrO2 and c-ZrO2 phases are stable at high-temperatures [32]. In addition to temperature, ZrO2 phase transformations are influenced by the presence of dopants [Ca, Mg, Y] in its structure. During the initial stage of PEOEPD process, as the voltage increases, the OH− ions in the electrolyte tend to move towards Zr substrate and take part in the formation of oxide layer. When the thickness of the oxide layer is growing with treatment time, higher voltages need to be applied to reach the dielectric breakdown of the grown oxide layer [33]. Thus, due to the occurrence of higher voltages in our experiments [N 500 V in 60 s], charged HA particles [Ca10(PO4)6O− 2 ] in the electrolyte solution will migrate towards the Zr anode surface through electrophoretic forces. Because of the molten oxide in high temperature plasma, it is possible for the negatively charged Ca10(PO4)6O− 2 particles present near the Zr surface to be incorporated into the newly forming oxide layers during the process. Thus, ZrO2/HA composite film is being formed. However, HA phase was not observed in the film by XRD [Fig. 1]. It was reported in literature that when the particles size is very small, these particles would undergo

plasma decomposition and thereby get incorporated not in the form of particles, but simply as constituent elements in the oxide film [34]. However, HA peak could be observed by the Raman spectrum. The appearance of HA peak in Raman spectrum indicates that the HA particles are not completely decomposed. The complete or partial melting of particles depends on its size, i.e., the bigger the particles the melting will be incomplete. In the present case, it may be partial melting by which calcium entered into ZrO2 films and the un-melted portion of HA got entrapped into the film. During the PEO process, the temperature of the molten oxide due to plasma discharge reaches as high as 2500 K [25] which can melt HA particles [whose melting point is 1550 °C] reaching near Zr anode surface. Consequently, the release of Ca2+ ions from decomposed HA phase whose ionic radius [0.99 Å] is close enough to the ionic radius of Zr4+ [0.79 Å] therefore enters into the lattice sites of Zr4+ during ZrO2 oxide formation. However, due to the difference in ionic charge, presence of Ca2+ ions leaves crystal defects around the ZrO2 matrix, which can stabilize the otherwise high temperature c-ZrO2 phase at room temperature [19]. Thus XRD results show the presence of c-ZrO2 phase with minor amount of m-ZrO2 phase at 2 min treatment time. The presence of c-ZrO2 and HA phases from Raman results confirms that both oxide growth and electrophoretic deposition takes place at 2 min treatment time. This oxide/HA composite film formation on Zr through PEO coupled with EPD is slightly different from the oxide/HA formation mechanism observed with Ti [33], where oxide formation was dominant during the initial stages of process without significant EPD of HA particles. This difference in composite film formation mechanism on Zr is mainly attributed to the higher voltages [500 V at 1 min and 535 V at 2 min of treatment time] that are reached at the initial stages of process in the present work [Table 1]. Further, as the treatment time increases from 2 to 6 min, the increase in voltage from 535 to 542 V is not significant and during the longer treatment time, the EPD process continues more actively than the former stage, although PEO process also contributes to the increased film thickness. Thus, at longer treatment times, more number of charged HA [Ca10(PO4)6O2¯] particles from the electrolyte dragged towards the Zr anode surface by electrophoretic forces. Raman spectra revealed the increase in intensity of major P\O vibration band at 961 cm−1 with increase in treatment time from 2 to 6 min. This can be attributed to the incorporation of bigger HA particles with only partial melting on its surface, whereas the smaller HA particles can completely melt and incorporated into the oxide phase in the initial stages of process. In addition, as the treatment time increases, EPD increases and the decrease in the density of plasma discharges can also be noticed. Further, the SEM micrographs of HA4 and HA6 samples show the coarse structure with the deposition of more number of HA particles in the composite film formed on its surface. Thus, from Raman spectra and SEM analysis it was understood that oxide formation along with electrophoretic deposition took place at the initial stages of PEOEPD process and electrophoretic deposition became dominant at higher treatment time.

Fig. 5. Water droplet images on surfaces of (a) untreated Zr, and PEOEPD fabricated (b) HA2, (c) HA4 and (d) HA6 films.

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Fig. 6. (a) Potentiodynamic polarization curves and (b) Tafel plots of untreated Zr and PEOEPD fabricated HA6 sample in 7.4 pH SBF environment.

3.5. Electrochemical tests The passivation behavior of untreated and HA6 sample was studied by conducting PDP test in 7.4 pH SBF medium at 37 °C. The PDP curves of Zr and HA6 samples are displayed in Fig. 6 and the data obtained from the curves are reported in Table 4. As shown in Fig. 6, the untreated Zr shows an active state in the beginning and then pitting corrosion happens immediately. The pitting potential [Epitt] of untreated Zr is 341.5 mV and when the polarization potential is over 757.23 mV, passivation at the surface takes place. On the other hand, HA6 sample shows excellent pitting corrosion resistance over a potential range of −500 mV to 3000 mV. Additionally, the HA6 sample shows higher corrosion resistance than untreated Zr, which means that the ZrO2/HA film formed on Zr is less susceptible to corrosion in the SBF environment. This improvement in the corrosion resistance of the HA6 sample strongly depends on the surface morphology and phases formed in composite film. Denser and thicker film with fine porosity is more useful for improving the corrosion resistance by inhibiting the attack from Cl− ions in the 7.4 pH SBF solution [13]. According to the surface and cross-sectional SEM micrographs [Fig. 3(c) and (c″)] of HA6 sample, a 75 ± 5 μm thick ZrO2/HA composite film with no obvious deep pores is formed on its surface. As a consequence, the film overlying on HA6 shows highest protection efficiency against the corrosive ions present in physiological environment. It is well known that the plasma electrolytic oxidation involves more complex plasma enhanced physicochemical processes, in which micro-arc discharges can generate high localized temperature in the discharge channels, forming molten oxides and subsequently quenched by the electrolyte bath to form relatively dense and adherent oxide coatings on substrates. In the present PEOEPD process, the HA particles added to the electrolyte are attracted towards the substrate (anode) and entrapped into the oxide coatings formed by PEO on its surface. In view of this, the composite film formed by PEOEPD is expected to be compact. The Tafel plots obtained over a potential range of ± 200 mV with reference to the stable OCP are shown in Fig. 6(b) and the obtained kinetic parameter values are reported in Table 4. After PEOEPD treatment, the corrosion potential [Ecorr] of the Zr samples increases and their jcorr

decreases significantly than that of the untreated Zr. From the reported values in Table 4, it can be seen that jcorr of untreated Zr and HA6 sample is 0.28 μA/cm2 and 0.02 μA/cm2, respectively, depicting that the jcorr of HA6 sample is one order of magnitude lower than that of untreated Zr. The protection efficiency of HA6 sample calculated by Eq. (2) is found to be 91%. Thus by considering the Ecorr, jcorr, Rp and corrosion rate of untreated and PEOEPD treated Zr, the PEOEPD treated Zr shows highest corrosion resistance over a potential range of −500 mV to 3000 mV with reference to the stable OCP in 7.4 pH SBF environment. 3.6. Bioactivity of ZrO2/HA film Bioactivity of ZrO2/HA composite film on Zr was studied by immersing HA6 sample in SBF medium for 4, 8 and 12 days and the samples were characterized by SEM equipped with EDS to examine the growth of apatite on its surface. For comparison, untreated Zr was also tested for its bioactivity under similar experimental conditions. The surface morphology and composition of HA6 film immersed in SBF for 4, 8 and 12 days and untreated Zr for 12 days are displayed in Fig. 7. Fig. 7(a) shows the morphology of HA6 film immersed in SBF for 4 days. It can be noticed that the rough morphology of HA6 film (Fig. 3(c)) is changed and the entire surface is covered by a precipitate layer after immersion in SBF for 4 days. After 8 days of immersion in SBF, thick and dense precipitate layer is formed and island like deposits started nucleating on HA6 film surface as shown in Fig. 7(b), but none can be observed on the untreated Zr surface within 8 days [corresponding image is not provided]. After 12 days of immersion in SBF, the entire surface of HA6 sample is fully covered by apatite spherulites and their size becomes larger [Fig. 7(c)]. On the other hand, few spherical like particles are observed on untreated Zr in 12 days of immersion in SBF exhibiting its poor capability of apatite mineralization (Fig. 7(d)). From the aspects of composition, the Ca and P content increases and Zr content decreases for HA6 film [Fig. 7(a′, b′, c′)] after SBF immersion, compared to the data in Fig. 3(c′). From Fig. 7(a′, b′, c′), it can be observed that the Ca and P counts increase with immersion time, and the appearance of strong signals for Ca and P [with Ca/P ratio of 1.68

Table 4 The OCP and kinetic parameter values obtained from Tafel plots of untreated Zr and HA6 sample in 7.4 pH SBF medium. Sample Code

βa (mV/dec)

|βc| (mV/dec)

Ecorr (mV)

jcorr (μA/cm2)

Corrosion rate (mm/year)

Rp (kΩ·cm2)

Epitt (mV)

PE (%)

S HA6

455 613

278 258

−491.6 −177.6

0.28 0.02

9.7 5.0E−03

271 3179

341.5 –

– 91

66

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Fig. 7. Surface SEM micrographs and respective EDS spectra of the PEOEPD fabricated HA6 sample after immersion in 7.4 pH SBF solution for 4 days (a, a′), 8 days (b, b′) and 12 days (c, c′) and untreated Zr for 12 days (d, d′).

which is nearly equal to Ca/P ratio of stochiometric apatite 1.67] indicates that significantly thicker apatite layer is formed uniformly on HA6 film after 12 days of immersion in SBF. On the other hand, EDS of untreated Zr [Fig. 7(d′)] shows very less counts of Ca and P on its surface even after 12 days of immersion in SBF. The EDS result corroborates with the SEM micrograph shown in Fig. 7(d) indicating its poor bioactivity. From this bioactivity test, it can be concluded that ZrO2/HA composite film on Zr is able to induce apatite formation in SBF solution within short period of time.

3.7. Cell adhesion characteristics of ZrO2/HA film The HOS cell activity on the untreated Zr and HA6 sample after 48 h of culture [Fig. 8] shows that both untreated Zr and HA/ZrO2 film surfaces were able to support the attachment of HOS cells. However, the HOS cell growth on HA6 sample [Fig. 8(b)] was significantly higher than that of untreated Zr [Fig. 8(a)], indicating that ZrO2/HA film is capable of promoting cell adhesion and proliferation better than untreated Zr. The SEM micrographs showed that HOS cells were able to attach

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Fig. 8. Surface SEM micrographs showing human osteosarcoma cells' attachment and spreading on (a) untreated Zr, and PEOEPD fabricated (b) HA6 sample in 48 h.

and grow on HA6 film surface and maintained their osteoblastic morphology [Fig. 8(b)]. It is well known that directed cell adhesion and spreading ability of cells [osteoblast] are important for bone implants. Surface morphology, wettability, roughness, chemical composition and phase composition strongly affect the cellular responses in contact with the implants. Many studies have proved that surface energy and roughness are proportional to cellular adhesion strength [3,31]. Thus, in our present study, the HA/ZrO2 film with good wettability, high surface energy coupled with optimum surface roughness offered more sites for cell attachment and is responsible for increasing the adhesion of HOS cells and their subsequent growth over the exposed surface. It is thus further supports the hypothesis that surface modification of Zr by ZrO2/HA film augments a cell's response in addition to apatite forming ability and excellent pitting corrosion resistance. 4. Conclusions PEO coupled with EPD process was successfully employed to fabricate ZrO2/HA composite film on Zr in a single step. The formation of ZrO2/HA composite layer was strongly influenced by the PEOEPD treatment time. Uniform, dense and thick [42 to 75 μm] ZrO2/HA composite films were formed at 2 to 6 min of treatment time. XRD results illustrate that the films formed by PEOEPD mainly composed of c-ZrO2 with small amount of m-ZrO2. Raman spectra revealed the presence of HA phase in the films with intense P\O vibration band at 961 cm−1 in addition to cubic and monoclinic ZrO2 phases. The Ca2+ ions originated from partial melting of HA in plasma discharges occupy the sites of Zr, thereby stabilizing c-ZrO2 phase at room temperature. The formed ZrO2/HA composite film shows good wettability, high surface energy and roughness compared to Zr substrate. ZrO2/HA film formed at 6 min of treatment time illustrates excellent pitting corrosion resistance in SBF environment compared to untreated Zr. The ZrO2/HA film shows good bioactivity by the formation of bone like apatite layer within 12 days of immersion in 7.4 pH SBF medium. Further, ZrO2/HA film supports the attachment and growth of HOS cells over entire surface within 48 h. The ZrO2/HA composite film formed on Zr by PEOEPD process at 6 min treatment time with higher corrosion resistance, excellent bioactivity and good biocompatibility can serve as a potential candidate material for orthopedic and dental implants. Acknowledgments The authors would like to acknowledge the facilities procured through the grants received from the Department of Science and Technology, New Delhi (SR/S3/ME/0024/2011, dated 3rd July 2012) and

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