Volume 23 Preprint 38


IN VITRO EVALUATION and ELECTROCHEMICAL ANALYSIS of HYDROXYAPATITE/TANTALUM NANOLYER COATINGS on Ti-6Al-4V IMPLANTS for ORTHOPEDIC APPLICATIONS

Mahboobeh Mahmoodi, Peyman Mahmoudi Hashemi, Rana Imani and Javed Iqbal

Keywords: Ti-6Al-4V, Corrosion resistance, Tantalum, Hydroxyapatite, Electron beam physical vapor deposition, Electrophoretic deposition

Abstract:
Many different kinds of surface modification methods are applying to Ti-6Al-4V implant surfaces to improve surface specifications and corrosion resistance. In the present study, Ti-6Al-4V was coated first with tantalum (Ta) and then hydroxyapatite (HA) particles. A 200 nm thick Ta layer coated on Ti-6AL-4V surface (Ti-6Al-4V/Ta) was deposited using an Electron Beam-Physical Vapor Deposition (EB-PVD) to increase the corrosion resistance and mechanical properties. The second layer, HA particles were coated on the Ti-6Al-4V/Ta surface (Ti-6Al-4V/Ta-HA) by electrophoretic deposition (EPD) to improve the osteoconduction and biocompatibility of Ti-6Al-4V/Ta. After coating, X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) were conducted to determine phase compositions, surface morphology, and elemental analysis of the samples. The surface hardness and roughness of Ti-6Al-4V/Ta in comparison to Ti-6AL-4V increased from 341 HV to 375 HV and from 0.056 µm to 0.108 µm, respectively. Moreover, the corrosion test in Hank's solution revealed that the corrosion current density of Ti-6Al-4V/Ta decreased from 1.9 µA/cm2 to 0.7 µA/cm2 for Ti-6Al-4V sample. Following the corrosion test, the release rate of V, Al and Ti ions was examined. The results showed that the concentration of the released elements was halved after coating. The correction test indicated that Ti-6Al-4V/Ta-HA has desirable corrosion resistance and excellent surface properties in comparison to Ti-6Al-4V/Ta. Additionally, proliferation and cytotoxicity of MG-63 osteoblast-like cells on the surface of samples in DMEM media was carried out using MTT assay. The in vitro studies determined that MG-63 cells were attached and proliferated in greater numbers on Ti-6Al-4V/Ta-HA and Ti-6Al-4V/Ta surface compared to Ti-6Al-4V sample. In conclusion, the findings demonstrate that Ti-6Al-4V/Ta-HA implants could be suggested for orthopedic applications.

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ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 IN VITRO EVALUATION and ELECTROCHEMICAL ANALYSIS of HYDROXYAPATITE/TANTALUM NANOLYER COATINGS on Ti-6Al-4V IMPLANTS for ORTHOPEDIC APPLICATIONS Mahboobeh Mahmoodi1*, Peyman Mahmoudi Hashemi1, Rana Imani2, Javed Iqbal3 1 Department of Biomedical Engineering, Yazd Branch, Islamic Azad University, Yazd, Iran 2 Department of Biomedical Engineering, Amirkabir University of Technology, Tehran, Iran 3 Department of Plant Sciences, Quaid-i-Azam University Islamabad, 45320, Pakistan Mahboobeh Mahmoodi: 0000-0002-9763-6587 Javed egbal : 0000-0002-9032-2622 Peyman Mahmoudi Hashemi: 0000-0003-0133-9789 Rana Imani: 0000-0002-5337-2903 * Corresponding author: m.mahmoodi@iauyazd.ac.ir ABSTRACT Many different kinds of surface modification methods are applying to Ti-6Al-4V implant surfaces to improve surface specifications and corrosion resistance. In the present study, Ti-6Al-4V was coated first with tantalum (Ta) and then hydroxyapatite (HA) particles. A 200 nm thick Ta layer coated on Ti6AL-4V surface (Ti-6Al-4V/Ta) was deposited using an Electron Beam-Physical Vapor Deposition (EB-PVD) to increase the corrosion resistance and mechanical properties. The second layer, HA particles were coated on the Ti-6Al-4V/Ta surface (Ti-6Al-4V/Ta-HA) by electrophoretic deposition (EPD) to improve the osteoconduction and biocompatibility of Ti-6Al-4V/Ta. After coating, X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS) were conducted to determine phase compositions, surface morphology, and elemental analysis of the samples. The surface hardness and roughness of Ti-6Al-4V/Ta in comparison to Ti-6AL-4V increased from 341 HV to 375 HV and from 0.056 µm to 0.108 µm, respectively. Moreover, the corrosion test in Hank's solution revealed that the corrosion current density of Ti-6Al-4V/Ta decreased from 1.9 µA/cm2 to 0.7 µA/cm2 for Ti-6Al-4V sample. Following the corrosion test, the release rate of V, Al and Ti ions was examined. The results showed that the concentration of the released elements 1 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 was halved after coating. The correction test indicated that Ti-6Al-4V/Ta-HA has desirable corrosion resistance and excellent surface properties in comparison to Ti-6Al-4V/Ta. Additionally, proliferation and cytotoxicity of MG-63 osteoblast-like cells on the surface of samples in DMEM media was carried out using MTT assay. The in vitro studies determined that MG-63 cells were attached and proliferated in greater numbers on Ti-6Al-4V/Ta-HA and Ti-6Al-4V/Ta surface compared to Ti-6Al-4V sample. In conclusion, the findings demonstrate that Ti-6Al-4V/Ta-HA implants could be suggested for orthopedic applications. Keywords: Ti-6Al-4V, Corrosion resistance, Tantalum, Hydroxyapatite, Electron beam physical vapor deposition, Electrophoretic deposition. Introduction The increasing demand for implants necessitates rapid developments in the field of biomaterials, research on biomaterials has expanded in recent years [1-7]. Biomaterials must be biocompatible, nontoxic, non-carcinogenic and don’t exhibit undesirable chemical reactions with body fluids [8-11]. Stainless steels, cobalt-chrome alloys and titanium (Ti) alloys are the used common metal implants [12, 13]. Ti alloy has attracted special attention in biomedical applications due to their fascinating properties including physical, mechanical, possess long fatigue life, corrosion resistance and appropriate density [14-17]. Furthermore, the lower elastic modulus of Ti alloys (55-110 GPa) compared to 316L stainless steel and cobalt-chrome alloys have significantly attracted the attention of researchers in different applications [18]. Despite the favorable properties of Ti alloys, especially Ti-6Al-4V, their long use may release aluminum and vanadium and finally lead to Alzheimer's and neuropathy diseases [18, 19]. Tantalum (Ta) as a biocompatible metal has attracted the attention of scientific community due to their remarkable properties such as high radiopacity [20-22], high fracture toughness, high workability [23] and high corrosion resistance [24, 25]. Tantalum is one of the most chemically inert and biologically biocompatible material with excellent corrosion resistance properties. Ta also possess wide range uses in sensors [26], electrical resistors, capacitors [27], stents [28], artificial heart valves [29] and medical imaging [30]. In spite of its great clinical results, application of Ta has numerous limitations; high density, high relative elastic modulus, and low friction characteristics. Therefore, different methods, e.g. sputtering [31], electron beam [32, 33], and ion implantation [34] are recommended to create layers of Ta and its oxides on silicon, titanium and titanium alloys substrates. 2 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 They also inhibit the release of toxic alloying elements such as vanadium and nickel from Ti alloys in physiological environment. Different research studies have confirmed that Ta biologically binds to bones by forming a layer of apatite in simulated body fluid (SBF) [8, 23]. According to research study, Ta has been proven to have great biocompatibility when implanted in mouse skin tissue [35] and determined excellent corrosion resistance in highly acidic environments [36]. Recent studies have also reported the cell adhesion and cell proliferation on the Ta surface reflecting biocompatible nature of Ta [37]. Different methods have been performed to develop thin Ta layers on various substrates, such as stainless steels and Ti [38]. According to the best of knowledge, no study has assessed the formation of Ta nanolayers on Ti-6Al-4V alloy using Electron Beam-Physical Vapor Deposition (EB-PVD) method. Host tissue response to Ti and Ta alloy is not always desirable as they get encapsulated by fibrous tissue after implantation, which separated them from surrounding tissue [39]. For fabrication of new long lasting orthopedic and dental implants, there is an urgent need to combine materials with potential mechanical properties of metals and significant bioactive and biological properties of bioceramics. Bioceramics coated metals stimulate the growth of bone cells to increase osseointegration of orthopedic and dental implants [40, 41]. There are different methods to increase the bioactivity and biological properties of implants. Among theme, one of the main methods to increase bioactivity of implants is surface modification with bioceramic coatings [42]. Hydroxyapatite (HA) is a bioactive calcium phosphate ceramic with chemical and crystallographic similarity to that of natural apatite in bone[43]. HA has been currently used in hard tissue engineering for bone regeneration, and as bioactive coating on Ti alloy surface for orthopedic applications to improve integration between the orthopedic implant and bone tissue [44]. HA particles increase bone formation and help in the absorption of protein on implant surface. However, delamination, poor mechanical strength, low fracture toughness and cracking of HA and weak bonding strength between substrate/coating interface limit its use in applications subjected to mechanical stress [45]. The Ta layer is a remarkable option as it is an intermediate layer between the Ti-6AL-4V surface and HA coating due to high mechanical integrity of Ta with HA. Marti et al. [41] has investigated the growth of HA coatings on Ta and observed that the HA coating improve the bioactivity of Ta. Different methods have been reported for coatings such as sol–gel, plasma spray and electrophoretic deposition (EPD) [46]. Among theme, EPD is the most common method due to simplicity, low cost, and its flexible nature can be used to coat bioceramic on the material surface. Bioceramic coatings lead 3 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 to bioactivity and osteoconductivity, because they provide more active sites for the ions absorption such as calcium, and phosphate [47, 48]. Although different materials have been utilized for coating of Ti-6Al-4V surface however, no Ta/HA coatings on Ti-Al6-4V substrate has not been reported to improve the corrosion resistance, and biocompatibility. The aim of this current study was to enhance biocompatibility and corrosion resistance of Ti-6Al-4V through metal/ceramic coatings for orthopedic and dental implants. For this purpose, Ta and HA layers were coated on Ti-6Al-4V substrate using EB-PVD method and the EPD process, respectively. The corrosion resistance, ion release, biological properties in comparison with single-layer Ta and double-layer Ta-HA coatings on Ti-6V-4V surface were evaluated using electrochemical test, inductively coupled plasma optical emission spectroscopy, and MTT test. Sample Preparation Circular substrates of Ti-6Al-4V (ASTM F136) with a diameter of 18 mm and a thickness of 2 mm were prepared. Afterward, 99.95% pure bulk Ta (ASTM F560) with dimensions of 3 mm×10 mm×20 mm was prepared and deposited on Ti-6Al-4V substrate. Before deposition, the samples were polished using an emery paper (from 400 to 1000 grit) thoroughly cleaned using an ultrasonic bath and placed in the presence of acetone for 10 minutes. Further, the samples were washed with deionised water twice and were completely dried. Surface Coating 2.2.1 EB-PVD process In this study, Electron Beam-Physical Vapor Deposition (EB-PVD) method was selected due to high melting and evaporation points of Ta. An electron gun (EDS160) with 3000 W was used to evaporate Ta. At first, chamber was evacuated using a diffusion pump with a pressure of 2 × 10-2 mbar. Once the system was ready, the beam was aimed at the bulk Ta and system power was increased until Ta reached a temperature of 3017°C. After evaporation of Ta, temperature of the chamber was decreased to 400°C. Measurements with a beam electron device revealed the deposition rate of Ta particles reported to be 2 A°/sec. After 20 minutes, the Ta layer were coated on Ti-6Al-4V surface was ~200 nm thick. 4 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 EPD Process After EB-PVD process, the HA particles were deposited on Ti-6Al-4V/Ta surface through electrophoretic deposition (EPD) method. Sodium dodecyl sulfate (SDS) as an anionic surfactant and HA powder with a particle size of 10 μm were obtained from Sigma-Aldrich. In order to prepare homogeneous HA suspension as electrolyte for EPD, 0.5 g SDS and 1 g HA particles were dispersed in 100 mL deionised water and ultrasonically treated for 2 h. Ti-6Al-4V/Ta and stainless steel sheet were used as a working electrode and a counter electrode in EPD cell, respectively. The distance between the electrodes was set to be 15 mm. The EPD process was conducted under 20V direct-current power at deposition time of 3 min in a glass beaker containing HA suspension. After EPD, the coated Ti-6AL4V/Ta (Ti-6AL-4V/Ta-HA) samples were taken out from the suspension and were dried at room temperature. A schematic diagram of the EB-PVD and EPD setup is illustrated in Figure 1. Figure 1. Schematic illustration of EB-PVD and EPD system. A beaker cell containing hydroxyapatite (HA) and SDS, Ti-6Al-4V/Ta as a working electrode and stainless steel sheet as a counter electrode is used in EPD system. Surface Characterization The morphology, microstructure and elemental compositions of the uncoated samples (Ti-6Al-4V), Tacoated Ti alloy (Ti-6Al-4V/Ta) and HA-coated Ti alloy (Ti-6Al-4V/Ta-HA) were studied using scanning electron microscope (SEM) coupled with energy dispersive X-ray spectroscopy (EDS) (Vega II XMU, Tescan, Czech Republic) at 20 kV. The Fourier transform infrared (FTIR) (BRUKER5 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 TENSOR-27) analysis was performed in the range of 400–4000 cm−1 to confirm the HA particle on the Ti-6Al-4V/Ta surface. The X-ray diffraction (XRD) pattern test (Philips, X' Pert Pro) was performed to detect the crystallinity, quality of coatings and phase formation in the samples. The phases were assessed using X'Pert High score Plus software (PANalytical, The Netherlands) and a PDF-2 file [49]. The crystallite size was calculated by Scherrer equation (Eq. 1) [45]. 𝐷 = 𝑘𝜆/𝛽𝑐𝑜𝑠𝜃 (1) In the equation (1), K indicates shape factor (∼0.9), λ is for the wavelength of X-ray (∼15 nm), β is the full width at half maximum (FWHM) of XRD peak, and θ indicates angle of diffraction. Further, the surface roughness of Ti-6Al-4V and Ti-6Al-4V/Ta samples were measured using mobile micro roughness instrument (MARSURF M300C-Mahr-Germany) in accordance with DIN EN ISO 4287(1998) at room temperature with 40% humidity. A micro-hardness tester (FM700 M, FUTURE-TECH CORP., Japan) was used to evaluate the surface hardness of Ti-6Al-4V and Ti-6Al-4V/Ta samples. The samples were placed under the Vickers indenter (a square pyramid with opposite faces at an angle of 136 degrees) and a load of 50 gf with 2 mN maximum load was applied. The unloading rate was kept at 50 µN s–1. After a dwell time of five seconds, surface micro-hardness was measured at three different points of each sample surface and a mean value was calculated. Electrochemical Tests In order to evaluate the corrosion behavior (as an index of biocompatibility) of the Ti-6Al-4V and Ti6Al-4V/Ta samples, the potentiodynamic polarization test was conducted using an Autolab PGSTAT12 (Metrohm Autolab B.V., Netherlands) with frequency response analysis (FRA) module. The samples were degreased with acetone, rinsed with double-distilled water and immersed in Hank's solution (H9269-Sigma). Platinum as the counter electrode, saturated calomel as reference electrode, samples with surface area 25 mm2 as a working electrode and Hank's solution as physiological solution or electrolyte were used in this system [50]. The Hank’s solution was degassed using argon and the experiment was performed at pH:7.4 and 37°C. Finally, the corrosion current density (icorr) and potential value for each sample were calculated using Tafel extrapolation method. Further, anodic ( 𝛽𝑎 ) and 6 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 cathodic ( 𝛽𝑐 ) Tafel slopes were obtained from the potentiodynamic polarization curve at a scan rate of 1 mVs-1 and polarization resistance (Rp) calculated using equation (2) [51]. 𝑅𝑃= 𝛽𝑎 𝛽𝑐 2.303(𝛽𝑎 + 𝛽𝑐 )𝑖𝑐𝑜𝑟𝑟 (2) Ion Release Test Following chemical polarization in Hank's solution, Ti-6Al-4V and Ti-6Al-4V/Ta samples were removed from the solution and remaining solution was quantitatively analyzed using inductively coupled plasma optical emission spectroscopy (ICP-OES, Varian Vista-Pro). The measured concentrations of the released elements were compared to determine the efficacy of Ta coating in preventing the release of toxic elements such as V and Al. In vitro Cell Culture The in vitro cytotoxicity behavior of samples was evaluated and compared for a minimum incubation period of 3 days using human MG-63 osteoblast-like cells (osteosarcoma cell line, purchased from Iranian National Cell Bank of Pasteur Institute). All samples were sterilized by autoclaving at 121°C for 20 min prior to the cell culture experiment. For this purpose, cells were seeded on the surface of samples and a negative control (i.e. MG-63 cells only in the cell culture medium) then placed in a 24well plates. The initial cell density was 3× 104 cells well-1. One milliliter of Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Karslruhe, Germany) provided with 10% fetal bovine serum (FBS) and antibiotics (penicillin/strepto- mycin/mL; Invitrogen) was added to each well. Further, the cells cultures plates were maintained at 37°C in a humidified 5% CO2 incubator for up to 3 days. The culture media were changed every other day [52, 53]. Cell Morphology Cell morphology was assessed by SEM after 3 days of incubation. The cultured samples for SEM observation were rinsed with 0.1 M phosphate-buffered saline (PBS) and fixed with 2% paraformaldehyde, 2% glutaraldehyde in 0.1 M cacodylate buffer overnight at 4°C. Following this, postfixation was done for each sample with 2% osmium tetroxide (OsO4) for 2 h at room temperature. Fixed samples were then dehydrated in an ethanol series (60%, 70%, 80%, 90% and 100%) three times 7 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 followed by a hexamethyldisilane (HMDS) drying procedure. Further, dried samples were mounted on aluminum stubs, coated in gold and observed by SEM [52]. Cell Proliferation The proliferation of MG-63 cells attached to Ti-6Al-4V, Ti-6Al-4V/Ta and Ti-6Al-4V/Ta-HA surface was evaluated by MTT assay (Invitrogen) after 3 days of incubation. A 5 mg/mL MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium) solution was prepared by dissolving MTT in phosphate buffer saline (PBS). MTT solution was diluted (100 µL into 900 µL) in a DMEM culture medium and added to each sample to form formazan through the action of mitochondrial dehydrogenases. After 4 h incubation at 37°C, DMEM medium was removed and 200 µL of dimethyl sulfoxide (DMSO, Invitrogen) was added to dissolve the formazan crystals. Further, 100 µL of the resulting supernatant was transferred into 96-well plate and three data points were obtained from each sample. The optical density of the solution in each well was measured at a wavelength of 570 nm using a microplate reader (BioTek, Elx808, USA) [54-56]. Statistical Analysis Statistical analysis was performed using ANOVA test followed by GraphPad Prism 8 software. Error bars represent the mean ± standard deviation of measurements for 3 replicates (*p<0.05, **p<0.01, ***p<0.001and ****p<0.0001). Results and Discussion Surface Characterization Developing new biomaterials for biomedical applications is one of the main concerns of researchers around the globe. Orthopedics is a branch of medicine that has a high demand for new materials to treat and replace injured parts of the body [1-4]. The current study provides deep insights and sought to improve the surface and corrosion properties of Ti-6Al-4V by coating with thin layers of Ta and HA via EB-PVD and EPD processes. The biocompatible coatings with high hardness and high corrosion can be deposited by PVD method. The surface microstructure and morphology of Ti-6Al-4V, Ti-6Al-4V/Ta and Ti-6Al-4V/Ta-HA samples were compared based on the obtained SEM images (Fig 2). Assessments revealed Ta particles have coated the Ti-6Al-4V surface as a fully integrated phase. On the other hand, comparison of the 8 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 images taken before and after Ta coating process (Fig. 2(a) and (b)) revealed fewer surface cracks subsequent to coating i.e. a number of cracks had been coated with Ta particles. Different coefficients of thermal expansion of Ti-6Al-4V and Ta may result in exfoliation and formation of microcracks on the surface. However, such thermal stress at the interface of the substrate and the Ta coating was prevented by pre-warming the substrate in the chamber at 400°C. In addition, at the end of the EB-PVD, samples were gradually cooled to room temperature within the chamber in order to avoid possible thermal stress [54]. The SEM micrograph in Figure 2 (c) shows that HA particles were successfully coated on the surface of Ti-6Al-4V/Ta. The coating displayed a continuous porous film with dense topography on the substrate. As mentioned in previous research[57] , that during EPD process with increasing the voltage of deposition results in an increase in the porosity of coating. The results suggest, that 20V for 3 min are optimal voltage applied and time of deposition for electrophoretic deposition of HA particles on Ti-6Al-4V/Ta surface, respectively. Further, using SDS solution improved solubilization and separation of the HA particles and prevent agglomeration of particles. The SDS solution is a surfactant with the amphiphilic properties that can interact with HA particles through its hydrophobic chain and negative charge[58]. The adsorption of SDS onto the HA particles results in an improved solubilization of HA in deionized water and provides some negative charges to the HA surface. These negative charges are very important for EPD process, as HA particles in electrolyte solution led towards working electrode (Ti-6Al-4V/Ta) with a positive charge. 9 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 Figure 2. SEM micrographs (a) Ti-6Al-4V, (b) Ti-6Al-4V/Ta and (c) Ti-6Al-4V/Ta-HA samples. The results of EDS analysis are shown in table 1. The elements present on the surface of Ti-6Al-4V were Ti, Al, and V elements with amount of 86.33±0.05, 7.70±0.1, and 5.96±0.04 wt%, respectively. The concentration of Ta, Ti, Al and V elements in Ti-6Al-4V/Ta was 23.95±0.1, 66.81±0.13, 5.14±0.25, and 4.10±0.09 wt%, respectively. In fact, increased Ta concentration on Ti-6Al-4V/Ta surface was associated with decreased concentrations of other elements. The increase of the Ta weight percentage on Ti alloy surface proves the successful Ta coating on the Ti-6Al-4V surface. Moreover, as EDS analysis results from different points of the coated sample surface showed close weight percentages of the elements, such compound uniformity throughout the surface could have eliminated the probability of galvanic corrosion in the samples. Table 1. Weight percent (Wt%) of chemical elements in the surface samples. Sample Ti Al V Ta Ca P C O Ti 86.33±0.05 7.70±0.1 5.96±0.04 - - - - - Ti-6Al-4V 56.71±0.13 5.14±0.25 4.10±0.09 23.95±0.1 - - - 10.1±1.2 Ti-6Al4V/Ta 10.6±0.065 - - 1.3±0.01 20.8±0.05 13.1±0.04 1.8±0.02 52.4±2.3 The elemental analysis from different parts of the Ti-6Al-4V/Ta-HA surface by EDS showed the presence of HA coating on samples. In the Ti-6Al-4V/Ta-HA surface, weight percentage of Ta, Ca, and P were 1.3±0.01, 20.8±0.05, and 13.1±0.04 wt%, respectively. The EDS spectrum indicated the 10 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 presence of intense peaks for Ca, and P and small peaks corresponding to Ta present. So, the intense peaks for Ca and P suggested the presence of strong HA coating on the surface of Ti-6Al-4V/Ta. Also, Ca/P ratio was 1.59 which is close to stoichiometric bone apatite (1.67) [45, 59]. The FTIR spectrums of HA coating is presented in Figure 3. The presence of PO43− and OH− functional groups in FTIR spectra indicates HA coating. The bands at 1042 cm−1 and 610-450cm−1 ranges are corresponding to PO43− and P-O. The absorption peaks at 3570 cm−1 and 1630cm−1 are related to the vibration mode of OH−. The presence of carbonate ion (CO32−) at 2331 cm−1 is a normal phenomenon in the apatite formation. This process happens through the dissolution and interaction of SDS with HA. According to studies [45, 60], carbonated HA is more bioactive than pure HA and it has a higher solubility. Figure 3. The FTIR spectra of Ti-6Al-4V/Ta-HA samples. Figure 4 shows the XRD patterns of Ti-6Al-4V and Ti-6Al-4V/Ta samples in the range of 2θ =10°-80°. The sharp peaks were observed at 2θ =35.526°, 40.93°, 57.5°, 64.1194°, and 70.97° attributed to Ti- 𝛼 phase, represent the degrees of crystallinity. Peaks related to Ti-6Al-4V/Ta sample appeared around the 11 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 angles 2θ = 35.526°, 38.93°, 40.40°, 40.93°, 53.244°, 55.77°, 64.1194° and 70.97°. The detected peaks of the Ta in XRD patterns match with Ta2O5 phase (00-001-1182, database of X'Pert High score Plus software) and detected peaks were observed at 2θ = 38.93°, 55.77°, 64.18, 73.16° and 78.38 [61]. According to Scherrer equation, the crystallite size of Ti was calculated ~32.9 nm and that smaller crystallite size results in a more stable phase. Figure 4. X-ray diffraction pattern of samples (a) Ti-6Al-4V and (b) Ti-6Al-4V/Ta. Surface morphology and roughness are two important factors for cells growth and adhesion between an implant and bone. [62, 63]. Figure 5 shows the graphs and images of surface hardness and roughness of Ti-6Al-4V and Ti-6Al-4V/Ta samples that were assessed via Vickers microindenter and mobile micro 12 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 roughness instrument, respectively. After coating of surface with Ta, surface roughness of Ti-6Al-4V changed from 0.056 µm to 0.108µm (Fig. 5(b)). The increased roughness can facilitate cell anchorage and enhance cell adhesion. The surface hardness value for Ti-6Al-4V/Ta was 375 HV, which is greater than Ti-6Al-4V (341HV) (Fig. 5(c)). Therefore, the results showed micro-hardness of Ti-6Al-4V samples was improved through surface coating with Ta. The presence of Ta2O5 phases on the surface may be reason for the observed increased hardness of Ti-6Al-4V/Ta [61]. Figure 5. (a) Surface roughness images of Ti-6Al-4V and Ti-6Al-4V/Ta samples, (b) Surface roughness of Ti-6Al-4V and Ti-6Al-4V/Ta samples. Statistical significance was shown as ****p<0.0001, (c) Surface hardness of samples. Statistical significance was shown as *p<0.05, and (d) Optical microscopy images of Vickers indentation. In vitro Corrosion Studies The corrosion characteristics of an alloy are influenced by the passive layer formed on its surface and the presence of alloying elements. The vanadium in the passive layer of Ti-6Al-4V contains empty spaces which lead to the corrosion of the implant and decrease the corrosion resistance of Ti-6Al-4V. There is a large number of studies to improve the corrosion resistance of Ti implants [18, 64, 65]. In 13 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 this study, Ta coating as a new method was used for the enhancement of corrosion resistance of Ti-6Al4V. The potentiodynamic polarization curves of Ti-6Al-4V and Ti-6Al-4V/Ta in Hank’s solution at 37 °C are shown in Figure 6. The corrosion potential of Ti-6Al-4V/Ta (Ecorr =-0.21 V) was higher than Ti6Al-4V (Ecorr = -0.304V). The positive potential of Ti-4V-6Al/Ta might be justified by eliminating or covering of the pits and cracks on alloy surface by Ta. The corrosion parameters of samples by Tafel extrapolation method are summarized in table 2. The reduced corrosion rate of the Ti-6Al-4V/Ta can be explained with increased level of Ta on the surface of Ti-6Al-4V. The corrosion current density (Icorr) reduced from 2 µA/cm2 to 0.7 µA/cm2 after Ta coating, thus, this process could double the corrosion resistance of the Ti alloy. The low corrosion current density of Ti-6Al-4V/Ta showed the presence of a corrosion-resistant tantalum-pentoxide (Ta2O5) layer which is more stable than the TiO2 layer existing on Ti-6Al-4V surface [66]. Figure 6. Tafel potentiodynamic polarization curves of Ti-6Al-4V and Ti-6Al-4V/Ta samples in Hank’s solution. Tantalum coating also decreased the current of the passivation region from 500 µA/cm 2 in Ti-6Al-4V to 250 µA/cm2 in Ti-6Al-4V/Ta. The polarization resistance of Ti-6Al-4V/Ta (Rp=39.88×103 Ω/cm2) was higher than the samples without coating, because of forming Ta2O5 on the substrate surface. The cathodic Tafel slope (𝛽𝑐 ) of Ti-6Al-4V/Ta and Ti-6Al-4V samples were calculated 118 and 65 mV/decade, respectively. It shows the corrosion resistance of Ti alloy with Ta coating is higher than samples without coating. Also, The larger anodic Tafel slope (𝛽𝑎 ) value (141.3) in comparison with the 𝛽c value for Ti-6Al-4V/Ta samples proved the existence of the inert film (Ta2O5), which formed a passive area on the Ti-6Al-4V surface with a low corrosion current density. 14 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 Table 2. The average corrosion parameters of the samples in Hanks’ solution. Sample Icorr (µA/cm2) Ecorr (V vs. SCE) 𝜷𝒂 (mV/decade) -𝜷𝒄 (mV/decade) Rp (Ω/cm2) Ti-6Al-4V 2 -0.304 120.6 65 9.16×103 Ti-6Al-4V/Ta 0.7 -0.21 141.3 118 39.88×103 Once the corrosion test was performed, inductively coupled plasma (ICP) of Hank’s solution that had been in contact with the samples was examined separately in order to better understand the corrosion behavior of the samples in physiological environment. The results demonstrated different concentrations of released ions from Ti-6Al-4V and Ti-6Al-4V/Ta (Fig. 7), i.e. Ta-coating could halve the release rate of toxic elements such as vanadium, during corrosion test by forming stable Ta2O5 layer as an inhibitor. The corrosion behavior of materials depends on their electrical, chemical and mechanical properties. Due to stable Ta2O5 on Ti-6Al-4V surface, tantalum has the stable chemical properties, which resistant to attack by all acids except HF. However, TiO2 forms on Ti-6Al-4V without coating which dissolves in the HCl solution especially with increasing electrochemical potential [56]. So, Ta coated Ti-6Al-4V samples had a higher chemical stability and biocompatibility compared to Ti-6Al4V. Consequently, Ta-coating improves the corrosion resistance and mechanical properties of Ti-6Al4V, therefore it can be used as a coating on dental and orthopedic implant surface. 15 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 Figure 7. ICP analysis of elements in Ti-6Al-4V and Ti-6Al-4V/Ta samples after immersion in Hanks’ solution. Statistical significance was shown as ****p<0.0001. In vitro Biocompatibility The MTT assay was used to determine the MG-63 cell proliferation on the surface of Ti-6Al-4V/Ta/HA, Ti-6Al-4V/Ta and Ti-6Al-4V samples. The cell proliferation of samples in comparison to control sample is shown in Figure 8. The cell proliferation on HA coated-Ti-6Al-4V/Ta samples was similar to the control sample (100%). The cell culture on the surface of Ti-6Al-4V/Ta-HA showed the highest rate of proliferation compared to Ti-6Al-4V (86%) and Ti-6Al-4V/Ta (87%). This indicates that compared with Ti-6Al-4Vsamples, the presence of HA could promote the proliferation of MG63 cells. There was no significant difference observed in cell proliferation behavior on the HA and Ta coatings after 3 days of incubation. The MTT results showed excellent biocompatibility for samples with Ta and HA coatings. The in vitro cell culture analysis showed that Ta and HA coatings are bioactive and can play an effective role to increase the performance of medical implants. The bone formation on the surface of implants with HA coatings is higher than Ti alloy [65]. HA coatings promotes and accelerate osseointegration of orthopedic and dental implants into the body, due to its similarity to natural bone, high biocompatibility, osteoconductivity, chemical stability [60]. 16 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 Figure 8. MTT assay for MG-63 cells on samples after 3 days of incubation. Statistical significances were shown as *p<0.05 and ns (nonsignificant). SEM micrographs reveal cell attachment and cell growth on the surface of samples after 3 days incubation (Fig.9). The present in vitro MG-63 cell–material interaction results clearly showed that Ti6Al-4V/Ta and Ti-6Al-4V/Ta-HA samples have excellent biocompatibility. MG-63 cells on the three samples with cellular micro-extensions were attached. However, fewer cells were observed on Ti-6Al4V surface. The surface of Ti-6Al-4V/Ta-HA and Ti-6Al-4V/Ta were covered with cells that they were attached to the surface with the numerous filopodia extensions. Also, the presence of a relatively high extracellular matrix (ECM) mineralization on Ti-6Al-4V/Ta-HA samples also showed that HA and Ta coating enhanced in vitro biocompatibility of Ti-6Al-4V [34, 67, 68]. 17 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 Figure 9. SEM images with different magnifications illustrating MG-63 cell morphology after 3 days of culture on Ti-6Al-4V, Ti-6Al-4V/Ta, and Ti-6Al-4V/Ta-HA surface. Conclusion In this study, Ti-6Al-4V was coated with Ta and HA using the EB-PVD and EPD methods. The corrosion resistance, surface properties and biocompatibility of Ti-6Al-4V with HA and Ta coatings were investigated using different characterization techniques. The passive behaviors were observed for Ta-coated Ti-6Al-4V and the corrosion resistance of Ti-6Al-4V was improved after coating with Ta, because the more stable Ta2O5 passive films strengthen the TiO2 passive films. Ta coating increased the corrosion potential and decreased the corrosion current density and release of alloy elements such as vanadium. The concentration of elements released from the Ta coated Ti-6Al-4V was half of that from Ti-6Al-4V samples. Also, the surface roughness and hardness of Ti-6Al-4V increased after coating with Ta. SEM/EDS analysis of HA -coated Ti-6Al-4V showed that the Ca/P ratio which was close 18 © 2020 University of Manchester and the authors. This is a preprint of a paper that has been submitted for publication in the Journal of Corrosion Science and Engineering. It will be reviewed and, subject to the reviewers' comments, be published online at http://www.jcse.org in due course. Until it has been fully published it should not normally be referenced in published work. ISSN 1466-8858 Volume 23, Preprint 38 first submitted 01 June 2020 stoichiometric of bone apatite. EDS point analysis confirmed that bond coat of Ta was covered with the hydroxyapatite top coat layers. In vitro cell culture showed that MG-63 cells were proliferated on Ta and HA coated-Ti-6Al-4V surface for a period of 3 days. HA coatings showed a more favorable cellular response in terms of cell proliferation compared to Ti-6Al-4V. Based on these results, it was concluded that both HA and Ta coatings enhanced the biocompatibility and surface properties of the Ti-6Al-4V. Therefore, Ti-6Al-4V/Ta-HA can be suggested for use in dental and orthopedic implants. Acknowledgments The authors would like to thank the facilities provided at Department of Materials, Islamic Azad University, Yazd Branch. References [1] C. Arnould, J. Denayer, M. Planckaert, J. Delhalle, Z. 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