Surface & Coatings Technology 324 (2017) 153–166 Contents lists available at ScienceDirect Surface & Coatings Technology j ourna l homepage: www.e lsev ie r .com/ locate /sur fcoat Afirst insight on the bio-functionalizationmechanisms of TiO2 nanotubes with calcium, phosphorous and zinc by reverse polarization anodization Sofia A. Alves a,b, André L. Rossi c, Ana R. Ribeiro b,d,e, Jacques Werckmann b,f, Jean-Pierre Celis g, Luís A. Rocha a,b,h,⁎, Tolou Shokuhfar i,j,⁎ a CMEMS – Center of MicroElectroMechanical Systems, Department of Mechanical Engineering, University of Minho, Azurém, 4800-058 Guimarães, Portugal b IBTN/BR – Brazilian Branch of the Institute of Biomaterials, Tribocorrosion and Nanomedicine, Faculty of Sciences, UNESP – Universidade Estadual Paulista, 17033-360 Bauru, SP, Brazil c Brazilian Center for Research in Physics, 22290-180 Rio de Janeiro, Brazil d Directory of Life Sciences Applied Metrology, National Institute of Metrology, Quality and Technology, 25250-020 Duque de Caxias, RJ, Brazil e Postgraduate Program in Translational Biomedicine, University of Grande Rio, 25070-000 Duque de Caxias, RJ, Brazil f Institute of Biomedical Sciences, UFRJ – Federal University of Rio de Janeiro, 21941-901 Rio de Janeiro, Brazil g Department of Materials Engineering, KU Leuven, 3001 Leuven, Belgium h Faculdade de Ciências, Departamento de Física, UNESP – Universidade Estadual Paulista, 17033-360 Bauru, SP, Brazil i Department of Bioengineering, University of Illinois at Chicago, 60607 Chicago, IL, USA j IBTN/US – American Branch of the Institute of Biomaterials, Tribocorrosion and Nanomedicine, University of Illinois at Chicago, 60612 Chicago, IL, USA ⁎ Corresponding author. E-mail addresses: lrocha@fc.unesp.br (L.A. Rocha), tolo http://dx.doi.org/10.1016/j.surfcoat.2017.05.073 0257-8972/© 2017 Elsevier B.V. All rights reserved. a b s t r a c t a r t i c l e i n f o Article history: Received 3 March 2017 Revised 18 May 2017 Accepted in revised form 25 May 2017 Available online 26 May 2017 The decoration of titanium (Ti) implant surfaceswith TiO2 nanotubes has emerged as a promising strategy to im- prove osseointegration and avoid infection. Nevertheless, it has been reported that nanotubular films are prone to peeling off from the Ti substrate due to the poor interfacial adhesion. The knowledge on the interfacial prop- erties of such interface, although notwell explored, is crucial for understanding themechanisms behind the poor adhesion problem of these films and to further achieve an easy and effective solution to solve it. This paper is focused on the bio-functionalization of TiO2 nanotubular films with zinc (Zn) as an antimicrobial and bone healing agent, togetherwith twomajor components of bonematrix, namely calcium (Ca) and phospho- rous (P). The main aim is, for the first time, the thorough characterization of the interface between TiO2 nano- tubes and the Ti substrate, along with the better understanding of the bio-functionalization mechanisms of TiO2 nanotubes and their influence on the interfacial features of the films. TiO2 nanotubes were successfully synthesized by two-step anodization and their bio-functionalizationwith Ca, P and Zn was achieved by reverse polarization anodization treatments. The in-depth characterization of the mor- phological and chemical features of TiO2 nanotubes was carried out along their length by scanning transmission electron microscopy (STEM) and energy dispersive X-ray spectroscopy (EDS), before and after bio- functionalization treatments. STEM images showed that the interface between conventional TiO2 nanotubes and Ti is non-continuous due to the existence of a hollow space. However, bio-functionalized TiO2 nanotubes ev- idenced an interface with different features, due to the formation of an interfacial oxide film as a consequence of anodization, with a thickness comprised between 230 and 250 nm. The results presented in this work may inspire the emergence of novel surface treatment strategies seeking the long-term performance of metallic-modified osseointegrated implants. © 2017 Elsevier B.V. All rights reserved. Keywords: TiO2 nanotubes Interface Reverse polarization Anodization Osseointegrated implants 1. Introduction Dental implants require the use of materials that are beyond fulfill- ing requirements such asmechanical, chemical and physical properties, must provide excellent biocompatibility and avoid foreign body re- sponses [1,2]. Along with the discovery of Ti implants, introduced by Brånemark in 1964, revealing the ability of titanium (Ti) to induce u@uic.edu (T. Shokuhfar). osseointegration, the exploration of this material for use in dentistry and orthopedic fields has undergone a global boom [1]. In fact, nowa- days, Ti-based materials represent the most widely used in dental and orthopedic fields, owing to their good mechanical properties, excellent biocompatibility and high corrosion resistance, resulting from the spon- taneous formation of a thin (of 3–10 nm in thickness) and stable titani- um dioxide (TiO2) film on its surface [1,3,4]. In spite of the high success rate that Ti-based dental implant thera- pies have reached to replace tooth loss due to traumaor periodontal dis- eases, a significant number of failures have still been reported to be http://crossmark.crossref.org/dialog/?doi=10.1016/j.surfcoat.2017.05.073&domain=pdf http://dx.doi.org/10.1016/j.surfcoat.2017.05.073 mailto:tolou@uic.edu http://dx.doi.org/10.1016/j.surfcoat.2017.05.073 http://www.sciencedirect.com/science/journal/02578972 www.elsevier.com/locate/surfcoat 154 S.A. Alves et al. / Surface & Coatings Technology 324 (2017) 153–166 comprised between 1 and 20% [3,5,6]. Dental implant failures are gener- ally ascribed both to biological (e.g. bacterial infection and inadequate implant-to-bone contact) and biomechanical factors (e.g. occlusal overloading leading to fracture and/or damage of dental implant mate- rial) [5,7]. Despite the good biocompatibility of Ti, insufficient osteogen- ic activity and the lack of antimicrobial properties are the main factors leading to delayed osseointegration and complicated bacterial infec- tions, which may conduce to implant failures, essentially in patients with complex pathologies [4,8–11]. Aiming to overcome the current bone-loss and infection related complications, several studies have been devoted to functionalization of Ti implant surfaces by modifying their features regarding morphology, topography and chemistry [12– 15]. Nanotechnology has emerged in the last years as an exciting and successful way to engineer Ti surfaces with nanoscale features for fast integration with bone that is also considered a nanostructured compos- ite matrix [16–19]. Studies have shown that the decoration of Ti-based materials with TiO2 nanotubes through electrochemical anodization, is a simple and effective way to promote cellular functions, which may be ascribed to their unique morphological, physical and chemical prop- erties [20–23]. The benefits of bio-functionalization of conventional TiO2 nanotubes have been demonstrated through in vitro and in vivo studies, as they have led to an enhancement on osteoblastic cell func- tions [24–28], ability to impair bacterial adhesion [29–32] or even both, simultaneously [33,34]. Apart the outstanding properties that bio-functionalized TiO2 nanotubes have revealed for osseointegrated implants applications [35,36], it has been reported that these films are prone to peeling off from the Ti substrate due to the poor interfacial ad- hesion between them [37]. This might have catastrophic consequences since, during and after implantation, osseointegrated implants are ex- posed to tribological and tribocorrosive conditions, which may induce to film degradation accompanied by the release of wear debris and cor- rosion products to implant surroundings, triggering harmful biological effects and ending up in implant failure [38–45]. A few studies have been reported seeking to understand the poor adhesion of TiO2 nanotubes to Ti substrate. In accordance with Miraghaei et al. [46] TiO2 nanotubes detach easily from the substrate due to the dissolution of a fluoride-rich layer existing between the tubes and Ti. Moreover, a hydrogen-assisted crack mechanism induced by the existence of Ti\\H and hydrogen blisters in the bottom layer of the nanotubes was proposed by Zhao et al. [47]. The beneficial effect of anodization of TiO2 nanotubes on their adhesion strength to Ti was reported by Yu et al. [37], whichwas ascribed to the formation of a com- pact layer near the nanotube bottom. However, the characteristics of Ti/ TiO2 film interface before and after anodization, were not reported. The knowledge of the characteristics of Ti/TiO2 nanotubes interface is still very limited in literature, which is an issue of crucial importance to well-understand the poor adhesion problem of these films and to further achieve an effective solution to solve it. A new methodology for TiO2 nanotubes bio-functionalization through reverse polariza- tion anodization was described by our group in previous works [48, 49]. After bio-functionalization treatments, biocompatible TiO2 nanotubes were synthesized displaying superior corrosion behavior than conventional nanotubes [48]. Additionally, the tribo-electro- chemical behavior of TiO2 nanotubes was significantly improved after bio-functionalization treatments, which was correlated with their improved adhesion strength to the Ti substrate, granted by the formation of a film at the interface region [49]. The focus of the present contribution is on the bio-functionalization of the TiO2 nano- tubes with Ca, P and Zn by reverse polarization anodization. The main aim relies, for the first time, on the in-depth morphological and chemical characterization of the TiO2 nanotubes along their length, with special focus at the interface region, before and after bio-functionalization treatments. A first insight on the bio- functionalization mechanisms of TiO2 nanotubes by reverse polari- zation and anodization processes is presented. 2. Materials and methods 2.1. Surface pre-treatment Commercially pure titanium (cp-Ti grade 2) (American Society for Testing of Materials – Grade 2) (MacMaster-carr, IL, USA) rods cut into discs of 15 mm diameter and 2 mm thickness were the substrates used in this study. A series of silicon carbide (SiC) sandpapers #240, #320, #400, #600 and #800 were used to ground cp-Ti surfaces follow- ed by their polishing with alumina suspension untill achieve a mirror finishing. After polishing, the Ti samples were ultrasonically cleaned in ethanol (10 min) and distilled (DI) water (5 min), followed by drying at room temperature. 2.2. Synthesis of TiO2 nanotubes by two-step anodization Titanium dioxide (TiO2) nanotubes were synthesized by two-step anodization of Ti in an optimized electrolyte constituted of ethylene gly- col (EG), 0.3 wt% ammonium fluoride (NH4F) (VETEC, Xerém, Rio de Janeiro, Brazil) and 3 vol% DI water. The electrolyte was continuously stirred (150 rpm) at room temperature (22 to 24 °C). The anodic treat- ments were conducted using a dc power supply (KEYSIGHT, N5751A) with a limiting current of 2.5 A. Firstly, Ti polished samples (anode, surface area: 4.5 cm2) and a graphite rod (cathode, surface area: 1.5 cm2) were almost completely immersed in the EG-based electrolyte, and only a small area was not in contact with the solution, i.e., the correspondent placewhere theme- tallic alligator clip was holding the electrodes. To control the reproduc- ibility of the anodic film features, one must be highlighted that the side of the Ti sample intended to be treated and considered for further char- acterization studies was always facing the graphite rod, and these two electrodes were placed parallel to each other separated at a fixed dis- tance of 2 cm. The electrochemical treatments were conducted at 60 V for 1 h. The resulting nanotubes grown from Ti through this first anodiza- tion step, were intentionally removed by ultrasonication in isopropanol for 15 min followed by cleaning in DI water for 5 min. Secondly, the resulting nanopatterned Ti surfaces were anodized at the previous con- ditions for 30 min. The second anodization step resulted in the growth of self-ordered TiO2 nanotube arrays, which were named as NT. Imme- diately after the second anodization step, NT samples were rinsed with DI water and dried at room temperature. 2.3. Bio-functionalization of TiO2 nanotubeswith calcium, phosphorous and zinc by reverse polarization and anodization The TiO2 nanotubular samples were bio-functionalized by reverse polarization and anodization processes, aiming the doping of nanotubes with calcium (Ca), phosphorous (P) and zinc (Zn) elements. Cathodic and anodic treatments were performed in an aqueous electrolyte con- stituted of calcium acetate (CaA) (Calcium acetate monohydrate, VETEC, Xerém, Rio de Janeiro, Brazil) and β-glycerolphosphate (β-GP) (β-glycerolphosphate disodium salt pentahydrate, Sigma-Aldrich, St. Louis, MO, USA) as the source of Ca and P, respectively, and this electro- lyte was named as Ca/P-based electrolyte (pH= 7.92± 0.06). The con- centrations of CaA (0.35 M) and β-GP (0.04 M) in the electrolyte were established in accordance with the experimental procedures followed in previous works [48,50–52], with Ca/P ratio of 19.75. Zinc acetate (Zinc acetate dihydrate, Sigma-Aldrich, St. Louis, MO, USA) at a concen- tration of 0.35 M was added to the previous Ca/P-based electrolyte aiming the additional incorporation of Zn in the nanotubular structure, and this solutionwas named as Ca/P/Zn-based electrolyte (pH=6.52± 0.01). The reverse polarization and anodization treatments were conduct- ed using a dc power supply (KEYSIGHT, N5751A) setwith a limiting cur- rent of 2.5 A. For reverse polarization step, the NT samples were set as 155S.A. Alves et al. / Surface & Coatings Technology 324 (2017) 153–166 the cathode (surface area: 4.5 cm2) and a graphite rod as the anode (surface area: 1.5 cm2), and these were immersed in the Ca/P-based electrolyte, at the previously described configuration in section 2.2. The power suply was set at 20 V for 30 s. After 1min, the electrodes po- larity was inverted, and the anodization step was carried out in the same electrolyte for 30 min at 100 V. These samples were named as NT-Ca/P. Additionally, NT samples were treated in the Ca/P/Zn-based electrolyte, at the previous reverse polarization and anodization condi- tions, and were named as NT-Ca/P/Zn. All the reverse polarization and anodization treatments were carried out under magnetic stirring at 200 rpm. 2.4. Characterization of TiO2 nanotubular films TiO2 nanotubular samples before and after bio-functionalization treatments were mounted on a stub with double sided conductive car- bon tape, and their morphology was analyzed by scanning electron mi- croscopy (SEM) using a FEI Helios NanoLab 650. This instrument was equipped with a detector for energy dispersive X-ray spectroscopy (EDS). EDS spectra and elemental maps were acquired to evaluate the chemical composition of the nanotubular samples and the distribution of the elements. For a better understanding of the bio-functionalization mechanisms of TiO2 nanotubes, the morphological and chemical features of the nanotubes were evaluated along their length. For this purpose, thin cross-sections of the nanotubular films (around 100 nm thick) were ob- tained in a dual beam instrument equippedwith focused ion beam(FIB) (TESCAN LYRA 3) operated with gallium (Ga) ion source. A thin gold (Au) layer was previously deposited to the film surface to improve the electrical conductivity. A platinum (Pt) layer of 1 μmwas locally depos- ited in situ using a gas injection system and 1 nA Ga+ ion current accel- erated at 30 keV. Initial etching was performed with 5 and 2 nA at 30 keV. The lamella was then transferred to a copper (Cu) transmission electron microscopy (TEM) grid using a nanomanipulator and Pt depo- sition. Thinning was performed in 3 steps to obtain a lamella of ~100 nm: 1) 1 nA/30 keV; 2) 0.1 nA/10 keV; 3) 10 pA/5 keV. A final step was accomplished with ~5 pA/3 keV to reduce the damaged layer produced during the thinning process. Fig. 1. SEM micrographs and EDS spectra showing the surface morphology and elemental FIB cross-sectionswere investigated by TEM and dark-field scanning transmission electron microscopy (STEM-DF) using a JEOL 2100F oper- ating at an accelerating voltage of 200 kV. EDS spectra and elemental maps were obtained in the same instrument with an EDS detector (Noran Seven), in STEM mode (STEM-EDS). Selected area electron dif- fraction patternswere obtained to investigate the crystallinity of the an- odic oxide films. 3. Results 3.1. Surface characterization Well-defined and well-organized TiO2 nanotube arrays were fabri- cated by two-step anodization in a fluoride (F−) containing electrolyte, whose surface morphology is depicted in Fig. 1a. These samples are mainly composed of Carbon (C), Ti, Oxygen (O) and Fluorine (F) as ob- served in the correspondent EDS spectrum in Fig. 1b. As demonstrated by XPS studies carried out in a previouswork [48], NT surfaces are com- posed of Ti and O mainly as TiO2. TiO2 nanotubular samples were submitted to cathodic and anodic treatments aiming their functionalization with bioactive elements, namely calcium (Ca), phosphorous (P) and zinc (Zn). For this purpose, NT samples were reverse polarized in a Ca/P-based electrolyte and, im- mediately after, anodized in the same solution. The surface morphology of NT-Ca/P samples is shown in Fig. 1c, and the correspondent EDS spec- trum shows the presence of Ca and P elements (Fig. 1d). To achieve the incorporation of Zn, togetherwith Ca and P, NT sampleswere submitted to reverse polarization and anodization processes in a Ca/P/Zn-based electrolyte, and the morphology of the fabricated nanotubes is shown in Fig. 1e. The presence of Zn is confirmed by the EDS spectrum shown in Fig. 1f. No significant differences are observed on the nano- tube surface morphology before and after bio-functionalization treatments. The EDS elemental maps in Fig. 2a show the homogeneous distribu- tion of Ti, O and F along the surface of NT samples. Similar results were obtained for the elemental distribution of these elements on NT-Ca/P and NT-Ca/P/Zn samples (results not shown). The elemental maps of Ca and P extracted from NT-Ca/P samples are shown in Fig. 2b, while composition of (a) and (b) NT; (c) and (d) NT-Ca/P; (e) and (f) NT-Ca/P/Zn samples. Fig. 2. (a) Elementalmaps representative of Ti K,O K and FK extracted fromNT samples. In (b) the elementalmaps of CaK andP Kobtained fromNT-Ca/P samples are depicted,while in (c) are presented the maps for Ca K, P K and Zn L elements acquired from NT-Ca/P/Zn samples. The elemental maps were obtained from the samples shown in Fig. 1. 156 S.A. Alves et al. / Surface & Coatings Technology 324 (2017) 153–166 in Fig. 2c the elemental distribution of Ca, P and Zn on NT-Ca/P/Zn sam- ples is depicted. 3.2. Cross-sectional characterization of nanotubular films Thin cross-sectional slices of the nanotubular films with approxi- mately 100 nm thickness were obtained by FIB, then imaged by TEM and STEMand analyzed by EDS. The general overview of TiO2 nanotubes produced by two-step anodization is depicted in Fig. 3a. From this TEM image the thickness of the filmwasmeasured as 6.1± 0.1 μm. In Fig. 3b a higher magnification STEM-DF image representative of the cross-sec- tional view of TiO2 nanotubes in the top region of the film is shown. The region from which the magnified image was taken is indicated by the inset square in Fig. 3a named as A1. From this image the wall and the hollow part of the nanotubes can be observed and, in general, the tubes arewell aligned and present a uniformmorphology. Furthermore, the insertion in this image shows the electron diffraction pattern com- posed of diffuse rings indicating an amorphous nature of TiO2 nano- tubes for this region of the film. To study the elemental distribution along the nanotubular films thickness, STEM-EDS elemental maps of Ti, O and F were acquired in the upper part and at the interface region. As observed from elemental maps depicted in Fig. 3c – A1, representa- tive of the upper part of TiO2 nanotubular films, it is observed that Ti, O and F are uniformly distributed along the nanotube thickness. Similar features are observed at the interface region shown in Fig. 3c – A2, where the distribution of these elements is also uniform. The interface between TiO2 nanotubes and Ti can be easily identified in the maps by the lower O and F colour intensity in the area related to Ti substrate and, on the contrary, by the higher intensity of the colour for Ti in this region. The morphological and chemical features of TiO2 nanotubes were also investigated after bio-functionalization treatments with Ca and P. The TEM image representative of the FIB cross- section of NT-Ca/P film is shown in Fig. 4a. The nanotube length, i.e. the thickness of the film, was measured as 4.8 ± 0.1 μm. The upper part of the film was imaged at highermagnification as shown in Fig. 4b, inwhichwell-aligned single nanotubes are clearly observed. The STEM-EDS spectrum obtained from the region indicated by the inset red square A in Fig. 4b, is shown in Fig. 4c. From this spectrum it is observed that C, Ti, O, F and Cawere detected in the superficial region of the film (until approximately 1 μm depth). Additional chemical elements such as Cu, Ga, Silicon (Si), Au and Pt were also detected in the uppermost regions of NT-Ca/P (Fig. 4c) film. The presence of Cu is related to the Cu grid used for TEM and STEManal- yses, while the Ga is related to the Ga primary ion beamused by FIB sys- tem. Additionally, the detection of a small signal of Si is probably related with the internal fluorescence peak from Si dead layer of Si\\Li detector [53]. Finally, the presence of Au and Pt are related to the Au surface coat- ing performed before sample preparation by FIB, and to the Pt protec- tion against Ga ions during polishing. The inset spectrum in Fig. 4c, with energy values comprised between 2 and 5 keV, intends to show in more detail the peak of Ca. In this spectrum, the escape peak for Ti K (Ti K - Si K = 2.77 keV) is also observed. After bio-functionalization processes in the Ca/P/Zn-based electrolyte, nanotubular films with a length of 4.6 ± 0.1 μm were produced, as shown in Fig. 5a. The STEM- DF image of the uppermost region of NT-Ca/P/Zn film is shown in Fig. 5b, fromwhich well-ordered and single nanotubes are observed. Bright dots are Pt and Au particles that penetrated the TiO2 nanotubes during samples preparation for FIB sectioning. The STEM-EDS spectrum ac- quired from the area highlighted by the inset red square in Fig. 5b is depicted in Fig. 5c. From this spectrum, elements such as C, Ti, O and F were identified, including Ca and Zn as shown by the more detailed spectrum added in the figure, with energy values comprised between 3 and 10 keV. Once again, the presence of elements such as Cu, Ga, Si, Au and Pt were detected, whose source was previously explained in the above description of Fig. 4c. Additionally, Chromium (Cr), Iron (Fe) and Cobalt (Co) were also present in the upper part of these films, which are most likely related to contamination from the metallic alligator clip used as the electrical conductive holder of Ti samples Fig. 3. TEM and STEM-DF images of the FIB cross-section of TiO2 nanotubular film synthesized by two-step anodization: (a) general overview of the film; (b) upper region of the film. The inset in (b) shows the electron diffraction pattern obtained for TiO2 film. In (c) are shown the STEM-EDS elementalmaps of Ti K, O K and F K obtained from twodifferent regions in the TiO2 nanotubular film shown in (a): A1 – the upper region of the film; A2 – the region at the Ti/film interface. 157S.A. Alves et al. / Surface & Coatings Technology 324 (2017) 153–166 during anodization processes. It is noteworthy to highlight that addi- tional contributions were found for both films at the elemental energy of P, which are related to Pt and Au. Therefore, it is not possible to accu- rately identify this element in both spectra (Fig. 4c and Fig. 5c). Howev- er, from the EDS surface spectra obtained from NT-Ca/P (Fig. 1d) and NT-Ca/P/Zn (Fig. 1f) samples (prepared without Au and Pt), it is expect- ed that P compounds are present in this region of the films. For a better understanding on the elemental distribution along the length of the nanotubes in the top region of thefilms, STEM-EDS spectra were acquired fromdifferent regions along the STEM images depicted in Fig. 4b and Fig. 5b (Supplementary material). From these results, the el- ements were found uniformly distributed along the nanotube length, and no gradient on their atomic concentrationwas observed. Similar re- sultswere found for STEM-EDS analysis carried out in the central part of the bio-functionalized nanotubular films (Supplementary material). Aiming to study the interfacial features of TiO2 nanotubular films, before and after bio-functionalization processes, STEM-DF images were taken at the interface regions. The lower and higher magnification STEM-DF images at the interface of TiO2 nanotubular films are shown in Fig. 6. The region of Ti substrate is easily identified as the brighter area contrastingwith the darker region related to TiO2 nanotubes, as indicat- ed in the image. A non-continuous interface is observed between TiO2 nanotubes and the Ti substrate, as shown by the darker region appearing between TiO2 nanotubes and Ti. This interface is character- ized by a hollow space as indicated by the inset white arrow in Fig. 6b, and is found along the extension of the film with a thickness at a nano- scale range (35 ± 4.3 nm). After bio-functionalization treatments, remarkable changes were observed at the interface, as the hollow space became partially filled, with some defects still present. The interface regions of NT-Ca/P and NT-Ca/P/Zn films are shown in Fig. 7a and b, respectively, with both films presenting an interface with similar morphological features. From higher magnification STEM-DF images shown in Fig. 7c and d, it is observed a porous interface characterized by the presence of an oxide film between, below and above the pores. A second interface was found in these films, as observed from the lines appearing along the films length, which are indicated by the inset white arrows in the figures. This second interface delimitates the thickness of newly-formed oxide films, whose values are comprised between 230 and 250 nm for both nanotubular films. NT-Ca/P andNT-Ca/P/Zn films are overall amor- phous, as confirmed from electron diffraction patterns obtained from the lower, middle and upper regions of the films, characterized by broad and diffuse rings in every case (results not shown). For a better knowledge of the differences observed at the interface region before and after bio-functionalization treatments, the current vs. time curves were recorded during all the anodization processes (Fig. 8). The current vs. time evolution recorded during the second step of anodization for TiO2 nanotube formation is shown in Fig. 8a. This is a typical curve showing the threemain stages of current achieved during anodization of Ti for nanotube formation [54]. Firstly, there is a decrease in the current values from 60mA (13.3mA/cm2) until approx- imately 15mA(3.3mA/cm2). Afterwards, a slight increase in the current is observed until 20 mA (4.4 mA/cm2) followed by a period of stabiliza- tion until the end of the anodization process. The current evolution achieved during bio-functionalization of NT samples in the Ca/P-based electrolyte is observed in Fig. 8b. In this curve it is shown the initial pe- riod of reverse polarization applied for 30 s, in which the current values were kept approximately at 750mA (166.7mA/cm2). In the inset graph Fig. 4.TEMand STEM-DF images of the FIB cross-section ofNT-Ca/Pfilm: (a) TEM image showing a general overviewof thefilm; (b) upper region of thefilm. In (c) the STEM-EDS spectrum obtained from the area correspondent to the inset red square A in (b) is shown. The inset spectrum in (c), with energy values comprised between 2 and 5 keV, intends to show in more detail the detected peak for Ca K. Fig. 5. STEM-DF images of the FIB cross-section of NT-Ca/P/Zn film: (a) general overview of the film; (b) upper region of the film. In (c) the STEM-EDS spectrum obtained from the area correspondent to the inset red square A in (b) is depicted. The inset EDS spectrum in (c), with energy values comprised between 3 and 10 keV, intends to show inmore detail the detected peaks for Ca K and Zn K elements. 158 S.A. Alves et al. / Surface & Coatings Technology 324 (2017) 153–166 Fig. 6. STEM-DF images of the FIB cross-section of TiO2 nanotubular films in the interface region at (a) lower and (b) higher magnifications. 159S.A. Alves et al. / Surface & Coatings Technology 324 (2017) 153–166 in Fig. 8b it is observed the evolution of the current during the anodiza- tion step carried out at 100 V for 30min. As observed, as soon as a volt- age of 100 V is applied, the current reaches the limiting value of 2.5 A (0.5 A/cm2) for a few milliseconds, followed by a sudden decrease until 9.2 mA (2 mA/cm2), a value that was kept constant until the end of the anodization period. The current vs. time evolution recorded dur- ing the synthesis of NT-Ca/P/Zn samples, is shown in Fig. 8c. In this case, Fig. 7. STEM-DF images of the FIB cross-sections of (a) NT-Ca/P and (b) NT-Ca/P/Zn films in t highlighted by inset red squares in (a) and (b), respectively. The white arrows show the inter and TiO2 nanotubes. during anodization, the current was kept at 2.5 A for a longer period (a few seconds), before it reaches values of 9.2 mA (2 mA/cm2). The chemical features of the interface of bio-functionalized films were studied aiming a better comprehension of the relation between the current vs. time evolution recorded during anodization, with their characteristics. For this purpose, line profile STEM-EDS analyses were carried out at the interface of NT-Ca/P films (Fig. 9a). The spectrum in he interface region. Higher magnification images are shown in (c) and (d) for the region face between the nano-thick oxide films (grown during bio-functionalization processes) Fig. 8. (a) Current vs. time evolution during the second anodization step of nanotextured Ti for TiO2 nanotube synthesis. The current evolutions during 30 s of reverse polarization (30 s RP) and 30min of anodization are shown for treatments carried out in the (b) Ca/P and (c) Ca/P/Zn-based electrolytes. The inset graphs in (b) and (c) intend to show inmore detail the current evolution during the initial stage of anodization processes. 160 S.A. Alves et al. / Surface & Coatings Technology 324 (2017) 153–166 Fig. 9b was extracted from the inset white square A in Fig. 9a, and it shows the presence of Ca and P, jointly with other elements such as Ti, O, F, Ga and Si. The STEM-EDS elementalmaps showing the distribution of Ti, O and F at the interface region are shown in Supplementary mate- rial. The STEM-EDS spectra acquired from line scan analyses performed in three different spots at the interface of NT-Ca/P films is shown in Fig. 9c. These analyses were carried out along the uppermost part of the nano-thick film formed by anodization, as indicated by a, b and c white spots inserted in Fig. 9a. As it is observed, Ti, O and F were found along this line, however, there is a peak of Ca, only detected in point b. To check the Ca distribution outside and inside this interface, additional STEM-EDS analyses were carried along the points 1, 2 and 3 (shown in Fig. 9a), whose spectra are shown in Fig. 9d. The expected el- ements were found, namely Ti, O and F. Interestingly, the peak for Ca was also found only in point 2, evidencing the entrapment of this ele- ment at that place. Finally, the chemical features of the interface of NT-Ca/P/Zn films (Fig. 10a)were investigated. The general spectrumac- quired from the area delimited by the inset white square in Fig. 10a is shown in Fig. 10b. The presence of the elements, such as Ti, O, F, Si, P, Ca and Zn was detected. The STEM-EDS elemental maps showing the distribution of Ti, O and F at the interface region of NT-Ca/P/Zn film are shown in Supplementary material. Line scan STEM-EDS analyses were carried out at 7 specific points across the nano-thick oxide film formed by anodization (shown in Fig. 10a), and the correspondent ac- quired spectra are shown in Fig. 10c. The STEM-EDS analysis for each point, shows clearly the presence of P and Zn elements non-uniformly distributed across the interface. The presence of P appeared more pro- nounced in the intermediate zone of the film, as shown by the peaks of P found in the spots numbered from 2 to 6. Moreover, the presence of Zn was found more prominent in the spots 3 and 5. It is noteworthy to highlight that in some of the presented spectra part of the elemental peaks are not being depicted in full, once a more detailed view of the less counted peaks is aimed. 4. Discussion 4.1. Morphological and chemical features of bio-functionalized TiO2 nanotubes Multi-step anodization of Ti in a fluoride-containing electrolyte has been extensively employed to synthesize TiO2 nanotubes with im- proved self-ordering [18,19,23,28,32,35,36,54–57] .To achieve the de- sired bone-inspired surface morphology observed in Fig. 1a, TiO2 nanotubes were synthesized by anodization of a nano-patterned Ti sur- face with the nanotube bottom hemispherical morphology, resulted from a first anodization step of a Ti smooth surface, in which TiO2 nano- tubeswere grown drilling their rounded bottom, and afterwards, inten- tionally removed [54]. During anodization, it is believed that nanotubes growth is based on electric field assisted oxidation and dissolution pro- cesses, which rely on the formation of a passive Ti oxide film through the recombination of Ti4+, O2– and OH– ions and the local chemical dis- solution of the growing oxide by F− ions [54,58]. NT surface morphology is characterized by the presence of bigger and smaller pores, and in some cases, the formation of multiple pores inside a bigger pore is observed (Fig. 1a). Macak et al. [59] observed that, in the very first stage of TiO2 nanotube growth, a thin and non- Fig. 9. STEM-EDS analyses in the interface region of NT-Ca/P film: (a) STEM-DF image showing the NT-Ca/P film interface, with the white insets indicating where the elemental analyses were performed; (b) STEM-EDS spectrumobtained from the region comprised in the inset red square A in (a); (c) line scan STEM-EDS analyses along the uppermost part of the nano-thick oxide film formed by anodization, as indicated by a, b and c white spots inserted in (a); (d) line scan STEM-EDS analyses across the uppermost part of the nano-thick oxide film, as indicated by the inset white number 1, 2 and 3 in (a). 161S.A. Alves et al. / Surface & Coatings Technology 324 (2017) 153–166 porous layer was formed where localized accelerated dissolution oc- curred under the action of the electric field. From two-step anodization, it is expected that the nano-imprints generated from the first anodizing step act as nucleation sites for initial pore formation and thus, behave as an intentionally designed template to trigger localized dissolution and obtain the desired final morphology [58]. The formation of multiple pores inside a main one is probably related with the events taking place in the beginning of the second anodization step. Possibly, the pri- mary localized dissolution occurred in multiple sites inside the same nano-dimple, resulted from the first anodization step. Macak et al. [59] observed that the surface morphology is maintained after 10 min of anodization. Thus, it is expected that the surface morphology resulting from the primary localized dissolution is preserved over time, since during anodization, pronounced dissolution takes place at the bottom of the pores, where the electric filed is stronger, making them significantly deeper over time [58,59]. The illustration of the dif- ferent growth stages of TiO2 nanotubes from nano-patterned Ti sub- strates is schematically shown in Fig. 11a – c. The bone-inspired TiO2 nanotubeswere dopedwith Ca and P, whose morphology and composition are shown in Fig. 1c and d, respectively. Reverse polarization anodization in a Ca/P-based electrolyte appeared as a very promisingway tomodify the chemistry of the nanotubeswith- out compromising their morphological features. In accordancewith our previous study, Ca and P elements are possibly assigned to the presence of Ca3(PO4)2/CaHPO4, CaF2, CaCO3 and CaO species [48]. Beyond bone- constituting Ca and P, TiO2 nanotubes were also bio-functionalized with Zn, which plays a major role in bone remodeling while exhibiting antibacterial properties [60]. The incorporation of Ca, P and Zn was suc- cessfully achieved by reverse polarization anodization of TiO2 nano- tubes in the Ca/P/Zn-based electrolyte (Fig. 1f), with no differences observed on the acquired morphology (Fig. 1e), when compared to NT and NT-Ca/P samples. In general, the bioactive elements (i.e. Ca, P and Zn) are uniformly distributed along the TiO2 nanotubular samples, as observed in EDS elemental maps shown in Fig. 2b and c. TiO2 nanotubes were grown perpendicularly oriented to the Ti sub- strate (Fig. 3a) with high self-ordering level, as observed through single nanotubes depicted in the STEM-DF image of the upper part of the film (Fig. 3b). STEM-EDS elemental maps taken in the upper part and at the interface region of the film show the presence of Ti, O and F along the nanotubes length (Fig. 3c). Berger et al. [61] proved for the first time that TiO2 nanotubes grown in fluoride-based ethylene glycol electro- lytes form a fluoride rich layer (few nm thick) between the individual nanotubes, and this might be the reason for the detection of fluoride species all over the nanotubular layer through STEM-EDS analysis. After bio-functionalization of TiO2 nanotubes in both electrolytes, the high level of ordering and integrity of the tubes is maintained along their length, and single nanotubes are clearly seen without any aggregates along the tube walls (Fig. 4 and Fig.5). As regards the thick- ness of TiO2 nanotubes, this was reduced after bio-functionalization treatments. In accordance with our previous work [48], the thinning phenomenon of the film is related with the reverse polarization step, during which the reduction of TiO2 to TiO2 − x(OH)x (e.g. TiOOH) is ex- pected to take place, followed by its chemical dissolution in the electro- lyte [62–65]. Furthermore, one must be considered that the dissolution of the oxide film may also occur right after the period of reverse polar- ization, i.e. when the anodic polarization step starts. During anodic po- larization of TiO2 nanotubes in the Ca/P and Ca/P/Zn-based electrolytes, a high peak of current is observed in both cases, during which the creation of very acidic conditions inside the tubes is expected to take place and consequently induce to the dissolution of the oxide [66,67]. This may further explain the lower thickness of NT-Ca/P/Zn when compared to NT-Ca/P films, since the pH of the Ca/P/Zn-contain- ing electrolyte is lower and the duration of the high current period is longer for the anodization process carried out in this solution (Fig. 8b Fig. 10. STEM-EDS analyses in the interface region of NT-Ca/P/Zn film: (a) STEM-DF image showing the NT-Ca/P/Zn film interface, with the white insets indicating where the elemental analyseswere performed; (b) STEM-EDS spectrumobtained from the region comprised by the insetwhite square A in (a); (c) line scan STEM-EDS analyses along the nano-thick oxidefilm formed by anodization, as indicated the white spots numbered in (a) from 1 to 7. 162 S.A. Alves et al. / Surface & Coatings Technology 324 (2017) 153–166 and Fig. 8c). From the STEM-EDS spectra in Fig. 4c and Fig. 5c it can be observed that Ti and O were detected, related with the presence of Ti oxide composing the nanotubes. The uniform distribution of Ti, O and F elements was also observed along the length of NT-Ca/P and NT-Ca/ P/Zn films, through STEM-EDS elemental maps taken in the upper, mid- dle and interface region of these films (Supplementary material). These results indicate that bio-functionalization processes have not influenced the composition of the nanotubes regarding its F content. Beyond other techniques have been used to modify the chemical properties of Ti, in particular, reverse polarization has been revealed as a versatile ap- proach to develop bio-functional implant surfaces for biomedical appli- cations [25,68–70]. 4.2. Characterization of Ti/TiO2 nanotubes interface Beyond the wide range of studies reported in literature showing the promising features of TiO2 nanotubes for the design of new implant sys- tems, little knowledge still exists on their adhesion properties to the Ti substrate, and therefore on their ability to ensure an appropriate long term biomechanical stability. Recently, a few studies have shown that TiO2 nanotubes are susceptible to peeling off from the underlying sub- strate, while rinsing with water or drying, because of the poor adhesion strength of TiO2 nanotubes to Ti [37,46]. For this reason, the interfacial features of the TiO2 nanotubular films produced by two-step anodiza- tion of Ti were investigated. After the second anodization step for TiO2 nanotube synthesis, the presence of a non-continuous interface was found between the nanotubular film and the Ti substrate, as shown in Fig. 6. This interface is characterized by a hollow space extended over the film width. Aiming to understand the mechanisms underlying the TiO2 nano- tube film detachment, researchers have recently made interesting find- ings. Different mechanisms have been proposed for nanotube detachment, namely the water assisted dissolution of the fluoride-rich layer existing beneath the nanotubes [37,46]. It has been generally ac- cepted that a layer enriched with fluoride is formed at the oxide/metal interface during anodization. This layer can be ascribed to the twice fast migration rate of F− compared to O2– ions, resulting in a fluoride rich layer which has a thickness of a few tens of nanometers [37,61]. The existence of this fluoride rich layerwas already proved by XPS sput- ter profiles taken from the tube bottom side of the tubes [71]. In accor- dance with Miraghaei et al. [46], TiO2 nanotubes are detached from the substrate after immersion in aqueous solutionsdue to dissolution of TiF4 layer existing between the tubes and Ti. Therefore, the existence of an interfacial hollow space after anodization of Ti might be related to water assisted dissolution of a fluoride-rich layer formed underneath the nanotubes. The hollow space existing between Ti substrate and TiO2 nanotubes bottom is of a few tens of nanometers (Fig. 6b), similar to the thickness reported for the fluoride-rich layer [61]. Notwithstand- ing, as reported by Zhao et al. [47], the existence of this non-continuous interfacemight be also related to a hydrogen-assisted crackmechanism. Researchers have recently made some efforts to improve the adhe- sion between TiO2 nanotubes and Ti substrate. Zhao et al. [47] reported a novel method to control the detachment of TiO2 nanotubes by their post-treatment in solvents with different polarities. The beneficial effect of anodization of TiO2 nanotubes has been reported by Miraghaei et al. [46] and Yu et al. [37] due to the formation of a new barrier layer be- neath them. Recently, annealing was also a method used by Roguska et al. [72] to stabilize the interfacial region between Ti substrate and Fig. 11. Illustration of the different growth stages of TiO2 nanotubes fromnano-patterned Ti substrates. In (a) it is depicted thefirst stage duringwhich the local chemical dissolution of the growing anodic oxide film by F− ions takes place, with the nano-imprinted dimples acting as single or multiple nucleation sites; in (b) is shown a second stage in which the pronounced dissolution takes place at the bottom of the pores, where the electric filed is stronger; finally in (c) is depicted a later stage achieved after a long period of anodization (i.e. 30 min), after which the initial structure remains in the top region of the filmwhile ordered tubes are underneath. The schematic illustration of the bio-functionalizationmechanisms of TiO2 nanotubes by reverse polarization and anodization are shown in (d) and (e), respectively. During anodization, Ti4+ ions are generated as a consequence of the polarization of Ti and they migrate across the hollow space and the bottom part of the tubes reacting with O2– ions, leading to the formation of a Ti oxide film with thickness comprised between 230 and 250 nm. 163S.A. Alves et al. / Surface & Coatings Technology 324 (2017) 153–166 TiO2 nanotubes. However, beyond the potential of the previously re- ported methodologies to improve the adhesion strength of TiO2 nano- tubes, the morphological and chemical characterization of the Ti/TiO2 interface is still missing. The creation of new knowledge on this field is of upmost relevance since it will provide a better comprehension of the adhesion phenomenon, before and after functionalization treat- ments, therefore providing new insights for further improvements. 4.3. Understanding the bio-functionalization mechanisms of TiO2 nano- tubes by reverse polarization and anodization After bio-functionalization of TiO2 nanotubes it is observed an inter- face with different features when compared to conventional TiO2 nano- tubes, as a consequence of the formation of a nano-thick oxide film at the interface region of NT-Ca/P and NT-Ca/P/Zn films, which presents a nanoporous morphology (Fig. 7). To explain this observation, current vs. time evolution recorded during bio-functionalization processes in the Ca/P and Ca/P/Zn-based electrolytes were considered (Fig. 8b and c). During anodization processes, three main stages can be identified. Firstly, once 100 V is applied, there is an increase in current values to its limiting value of 2.5 A (0.5 A/cm2, first stage), followed by a period during which the current drops very quickly to values of 9.2 mA (2 mA/cm2, second stage), a stage that is kept constant until the end of the anodization process (third stage). This is the typical current vs. time evolution characteristic of the growth behavior of a compact oxide film on Ti by anodization in a fluoride free electrolyte [54], whose growth mechanisms have been previously studied [73–75]. A similar behavior was reported by Oliveira et al. [51] during anodic oxi- dation of Ti in an electrolyte with composition similar to the one used in this study. The authors explained that the first stage of the graph (with limiting current of 2.5 A), is related to the time during which a high current contributes to the fast growth of the oxide film. As the oxide becomes thicker, its resistivity increases resulting in the decrease of the current to lower values, most likely due to the high resistivity of the newly oxide film formed that is kept constant with time. Dyer and Leach [73] also proposed that the non-linear voltage vs. time behavior observed during the galvanostatic anodic film growth on Ti, might be explained by changes in the ionic conductivity of the oxide. The behav- ior observed in Fig. 8b and c explains the nano-thick oxide film forma- tion observed in Fig. 7, independently of the electrolyte composition. A longer duration for the first stage was achieved for anodization in the Ca/P/Zn electrolyte, probably related with its higher conductivity (Fig. 8c). Oliveira et al. [51] reported that as higher this period, higher the total charge in the system is. The authors pointed out that 164 S.A. Alves et al. / Surface & Coatings Technology 324 (2017) 153–166 theoretically, this would influence the total amount of oxide grown dur- ing the process, with effects on the measured thickness and compact- ness of the film. This may be related with the more pronounced points of contact existing between the Ti substrate and NT-Ca/P/Zn film, when compared to the ones exiting at NT-Ca/P film interface, as ob- served in Fig. 7a and Fig. 7b. Nonetheless, no significant differences were found in the thickness of the newly formed interfacial films, as in both cases, thickness ranges between 230 and 250 nm. The study of the chemical composition of the nano-thick films formed by anodization showed that these are composed of O and Ti, in- cluding in the inter pore-areas, evidencing the growth of a Ti oxide (Fig. 7c, Fig. 7d and Supplementary material). Furthermore, the lighter con- trast at the nano-thick oxide film region observed in STEM-DF images (Fig. 7), evidences the presence of a film with a higher density com- pared to the one in a darker area. The existence of the bioactive ele- ments (i.e. Ca, P and Zn) at the interface region (Fig. 9 and Fig. 10) shows that the electrolyte penetrated along the film length during functionalization processes. Line scan STEM-EDS analyses along and across the uppermost region of the nano-thick film formed in NT-Ca/P interface (Fig. 9a), shows that Ca was not homogeneously distributed along the film (Fig. 9c) and interestingly, appeared to be entrapped on it (Fig. 9d). Ca entrapment at the interface is an additional indicator that the oxide film was formed during bio-functionalization processes. Line scan STEM-EDS analysis along NT-Ca/P/Zn interface evidenced a non-uniform distribution of Zn and P across the nano-thick oxide film formed. Beyond Ca has been detected in this interface (Fig. 10b), its presence was not identified along the line scan analyses, and this might be related with its non-uniform distribution along the film length, as discussed previously. As previously reported by Chen et al. [25], by cathodic polarization of TiO2 nanotubes in a Ca- and P-containing electrolyte, nanoscale calcium phosphate was successfully deposited on the inner and outer walls of the nanotubes. Furthermore, Huang et al. [76] observed the deposition of a hydroxyapatite coating composed of Cu and Zn on Ti surfaces, which was synthesized by electrodeposition method in an electrolyte containing Ca, Cu, Zn and P. In accordance with these previous studies, during reverse polarization, it is believed that positively charged ions in solution, namely Ca2+ and Zn2+ ions, are directed towards TiO2 nanotube surface and penetrate the nanotubes towards its bottom part, as schematically illustrated in Fig. 11d. It is believed that during this stage, part of the Ca2+ and Zn2+ ions are adsorbed to TiO2, and pos- sibly Ca2+ ions reactwith F− ions. It is noteworthy that F− ions are pres- ent on TiO2 nanotube wall as a result of the nanotube synthesis process. As soon as the anodization process starts, it is expected that an inversion in the electrode polarity leads to an inversion on the ions movement: those positively charged still remaining in solution, such as Ca2+ and Zn2+, tend to move away the bottom part of the film and, on the other hand, negatively charged ions such as phosphate (PO4 3−), are di- rected towards the interface as illustrated in Fig. 11e. Simultaneously, it is expected that Ti4+ ions are generated at Ti surface, and under the action of the electric filed, migrate through the hollow interface and the bottom part of the tubes towards the oxide-electrolyte interface, reacting with O2– ions moving in opposite direction and leading to the formation of the Ti oxide film [54,66,77,78]. As explained in previous studies, anodization induces to the field-enhanced oxidation of Ti ac- companied by the release of Ti4+ ions, which reactwithO2– ions created by field-assisted deprotonation of H2O or OH– ions present in the electrolyte, a reaction that takes place at the oxide-electrolyte inter- face [54,66]. Khalil and Leach [74] also explained that the oxide growth under the influence of the electric field involves the diffusion of both metal and oxygen, and is dependent on the relative move- ment of the two species through the oxide. A possible mechanism for Ca entrapment at the interface region can be ascribed to the growing of the oxide film during anodization and the simultaneous movement of positively charged Ca2+ ions in direction to the upper- most part of the nanotubes. In our previous work [48], TiO2 nanotubes displayed a significantly lower passive current in artificial saliva after bio-functionalization treat- ments, independently of reverse polarization step. From the findings achieved in this investigation, the formation of a nano-thick oxide film as a consequence of anodization might be the reason for the significant improvement on the electrochemical behavior of nanotubular films. These results indicate that the nano-thick oxide film display the ability to protect the Ti substrate against corrosion, thus guaranteeing good prospects for their application for osseointegrated implants. The formation of an oxide film as a consequence of anodization in an aqueous electrolyte might influence the adhesion of the film to Ti sub- strate. This is an issue of main importance and therefore further adhe- sion tests should be conducted. Furthermore, the investigation of the mechanical properties of the bio-functionalized TiO2 nanotubes would be also of valuable interest. The mechanical properties of the films dic- tate their ability to withstand to mechanical stress, and thus to resist to degradation. The investigation of the degradation behavior of TiO2 nanotubular samples before and after bio-functionalization, should be addressed under the simultaneous action of wear and corrosion (tribocorrosion), aiming to simulate the harsh and real conditions that osseointegrated implants are submitted to. Lastly, but not the least, a deepest investigation on the biological responses to these bio-function- alized TiO2 nanotubes is of upmost importance. More specific biological assaysmust be accomplished to ensure that the surface features of these structures do not compromise the cellular functions. 5. Conclusions Bioactive elements of Zn, Ca, and Pwere successfully incorporated in TiO2 nanotubular structures through reverse polarization and anodiza- tion processes. Cross-sectioned bio-functionalized nanotubular films were characterized regardingmorphology and chemistry with a special focus given to Ti/TiO2 nanotubes interface. Hereafter themain outcomes of this research are highlighted: - The incorporation of Ca, P and Zn elements in TiO2 nanotubes was successfully achieved by reverse polarization and anodization pro- cesses, with the bioactive elements uniformly distributed along the topmost regions of the films as well along their length. - Bio-functionalization treatments do not compromise the bone-in- spired morphology of TiO2 nanotubes, neither their high self-order- ing nor integrity. - The anodization of TiO2 nanotubes in aqueous electrolytes induces the growth of a nano-thick protective oxide film (230–250 nm) at the Ti/TiO2 nanotube interface region, which appears to improve the interfacial features, suggesting a better adhesion property. This interfacial nano-thick oxide film is constituted by Ca, P and Zn, how- ever, these elements appeared to be non-uniformly distributed across the film length, with Ca found to be entrapped on its superfi- cial region. Reverse polarization arises as a fundamental step to provide biocom- patibility to TiO2 nanotubeswhile anodization promotes the growth of a nano-thick oxide film at the Ti/TiO2 interface, enhancing the corrosion behavior of TiO2 nanotubes. Additionally, the growth of the nano-thick oxide film improves the bonding strength of the nanotubular film to the Ti substrate, a critical factor determining the biomechanical stability of an implant, and so its long-term success. This work brings up a first insight on the bio-functionalization mechanisms of TiO2 nanotubular films by reverse polarization and anodization processes. This novel methodology may inspire the emergence of novel surface treatment strategies seeking the long-term performance of metallic-modified implants. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.surfcoat.2017.05.073. http://dx.doi.org/10.1016/j.surfcoat.2017.05.073 http://dx.doi.org/10.1016/j.surfcoat.2017.05.073 165S.A. 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Introduction 2. Materials and methods 2.1. Surface pre-treatment 2.2. Synthesis of TiO2 nanotubes by two-step anodization 2.3. Bio-functionalization of TiO2 nanotubes with calcium, phosphorous and zinc by reverse polarization and anodization 2.4. Characterization of TiO2 nanotubular films 3. Results 3.1. Surface characterization 3.2. Cross-sectional characterization of nanotubular films 4. Discussion 4.1. Morphological and chemical features of bio-functionalized TiO2 nanotubes 4.2. Characterization of Ti/TiO2 nanotubes interface 4.3. Understanding the bio-functionalization mechanisms of TiO2 nanotubes by reverse polarization and anodization 5. Conclusions Acknowledgements References