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Preparation and characterization of TiO2 nanotube arrays on Ti6Al4V surface for enhancement of cell treatment
Accepted Manuscript Preparation and characterization of TiO2 nanotube arrays on Ti6Al4V surface for enhancement of cell treatment
M. Mansoorianfar, M. Tavoosi, R. Mozafarinia, A. Ghasemi, A. Doostmohammadi PII: DOI: Reference:
S0257-8972(17)30483-8 doi: 10.1016/j.surfcoat.2017.05.016 SCT 22335
To appear in:
Surface & Coatings Technology
Received date: Revised date: Accepted date:
27 March 2017 30 April 2017 5 May 2017
Please cite this article as: M. Mansoorianfar, M. Tavoosi, R. Mozafarinia, A. Ghasemi, A. Doostmohammadi , Preparation and characterization of TiO2 nanotube arrays on Ti6Al4V surface for enhancement of cell treatment, Surface & Coatings Technology (2017), doi: 10.1016/j.surfcoat.2017.05.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Preparation and Characterization of TiO2 Nanotube Arrays on Ti6Al4V Surface for Enhancement of Cell Treatment
M. Mansoorianfar1, M. Tavoosi1*, R. Mozafarinia, A. Ghasemi1, A. Doostmohammadi2
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1- Department of Materials Engineering, Malek-Ashtar University of Technology (MUT), Iran.
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* Corresponding author contact:
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2- Department of Materials Engineering, Shahrekord University (SKU), Iran.
Email: [email protected]
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Tel./Fax: +983134429844
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Abstract
Fabrication and characterization of TiO2 nanotube arrays on Ti6Al4V surface for enhancement
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of cell treatment in biomedical applications was the aim of this study. In this regard, the
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anodizing process has been carried out in an organic bath (ethylene glycol+ 0.5 wt.% NH4F+ 3 wt.% water) under applied voltages of 25 to 100 V. The anodized samples were characterized
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using scanning electron microscopy (SEM), hydrophilic/hydrophobic balance, cell attachment,
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ion release test and MTT assay. Based on achieved results, the nanotube arrays with good uniformity, high wettability and viability can successfully formed on Ti6Al4V surface in voltage
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range of about 50 to 75 V. The dimensional characteristics of formed nanotubes depend on applied voltage and play an essential role in viability and cell adhesion. In this work, the formed nanotubes array in applied voltage of 60 V (with average diameter and thickness of about 90-97 and 20-30, respectively) showed the best wettability, viability and cell response in comparison with other voltages. Keywords: Ti6Al4V; Anodizing; TiO2 nanotube; Viability.
ACCEPTED MANUSCRIPT 1. Introduction The Ti6Al4V alloy is widely used as implant material in orthopedics and dentistry due to its high biocompatibility, high mechanical resistance, low density, high corrosion resistance, high chemical inertness and toxicity. Although, this alloy is known as an ideal implant material, its
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viability must be modified during surface modification methods [1-2].
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biomedical characteristics such as roughness, composition, wettability, cell response and cellular
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Among different surface modification methods, surface roughening and formation of nano-scale topography on surface have been employed for increasing the capability of implants to integrate
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with osseous tissue. This kind of topography can affect on implant/tissue interface by providing
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more binding sites to cell membrane receptors and enhances biological events with indirect effect on cellular activity features [3]. In this regards, Xia et al. [4] showed that the nanostructure
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topologies on the Ti implant surface can improve the proliferation, differentiation and
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development of the osteoblastic phenotype and increase the bone-implant interfacial strength. Salou et al. [5] also studied the osseointegration of micro and nano-structured implants in rabbit
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femurs and showed that nano-structured implants have higher bone-to-implant contact and bone
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growth values in comparison with micro-structured implants. Moreover, similar results have been presented with Rosales-Leal [6] about the effect of nano-scale topography on cell attachment and
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bioactivity performance of Ti and its alloys. Anodizing processing is one of the most popular methods to obtaining nano-structured morphology in form of nanotubes on the surface of Ti and its alloys. This method was newly developed for the promising applications in biomedical and tissue engineering [7-11]. In this process, TiO2 nanotubes create from initial titanium oxide layer through anodizing in an
ACCEPTED MANUSCRIPT electrochemical cell including fluorine. The diameter and length of nanotubes can be controlled by the anodizing voltage, electrolyte composition and time duration of anodizing [8, 12]. In fact, numerous studies have been conducted about the formation of TiO2 nanotubes on Ti surface using anodizing process and the formation mechanism and kinetic of nanotubes structure
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were presented in details by different authors [3-12]. However, there is relatively little attention
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about the biological characterizations of TiO2 nanotubes array on Ti6Al4V alloy. Moreover,
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there is lack of information about the relation between the dimension of TiO2 nanotubes, wettability, viability and cell attachment behaviors in the literatures. So, this research study
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focuses on the fabrication and characterization of TiO2 nanotube arrays on Ti6Al4V surface for
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enhancement of cell treatment in biomedical applications.
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2. Materials and methods
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High purity Ti6Al4V sheet (Nilaco, Japan) with thickness of about 1 mm was used as substrate material. High purity ethylene glycol, NH4F, ethanol and acetone (Sigma-Aldrich, Australia)
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were also used as anodizing batch and cleaner solutions. Prior to anodizing process, Ti6Al4V
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sheets were brushed, polished and degreased with deionized water and acetone and finally dried at ambient temperature. In all testing, the anodizing solution (ethylene glycol+ 0.5 wt. % NH4F+
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3 wt. % water), anodizing temperature (25 °C) and time (60 min) were kept notably constant and the process was performed at different anodizing voltages within the range of 25 to 100 V. To modification of anodized nanotubes arrays as a uniform substrate for cell responses, the ultrasonic surface treatment has been performed and about 0.01 wt. % of Na2CO3 was added to the anodizing bath. Structural and morphological characterizations of anodized layer were carried out by field
ACCEPTED MANUSCRIPT emission scanning electron microscope (VEGA-TESCAN-XMU) at an accelerating voltage of 20 kV. The hydrophilic/hydrophobic balances of anodized samples were evaluated by means of contact angle measurements (Dataphysics, OCA 15 plus). Each contact angle value is the average of minimum 3 measurements. The investigation was carried out with an accuracy of ±1 °
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at a temperature of 25 °C. To evaluation of aluminum (Al) and vanadium (V) ion releasing, the
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anodized samples in optimum condition were immersed in PBS solution for different periods of
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time (1, 3, 7, 14, 21, 28 and 35 days) and the achieved solution were examined by inductively coupled plasma-atomic emission spectroscope (ICP-AES; ICPS-100IV, Shimadzu, Japan). The
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effects of nanotube structure on cell adhesion, cell proliferation and osteoblast expression,
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growth and differentiation of human osteoblast were also investigated by 3-(4,5-
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Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay.
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3. Results and discussion
Despite huge number of publications regarding different affecting elements in anodizing Ti and
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its alloys [11-13], there are a number of factors such as the diameter, wall thickness, length and
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the arrangement of nanotubes arrays which must be determined and modified. According to literature, substrate preparation, solution component and anodizing voltage, are the three main
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factors for modifying the nanotube arrays during anodizing process [14]. Based on TEM and XRD analysis in literatures [1-4], the formed TiO2 arrays on surface during anodizing process are in amorphous state. However, once amorphous titania is formed in low temperature environment, the strong Ti-O bond will suppress the atomic mobility necessary for rearrangement of the atomic structure. However, according to Shibata et al. [15] and Sul et al.
ACCEPTED MANUSCRIPT [16], the oxide layer produced by anodization is mainly in an amorphous form but can also contain some crystalline phases, depending on the anodization process parameters. In order to prepare uniform nanotube arrays, it is necessary to apply a template. The templateassisted technique promotes the growth of nanotubes. Considering this fact, we proposed the pre-
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anodizing plus ultrasound technique as a preparatory method before anodizing process [17]. In
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fact, in pre-anodizing stage, low-ordered nanotube array were formed (Fig. 1 (a)). During
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ultrasonic treatment, all the formed nanotubes were removed from the surface by sonic waves and an ideal surface (as a template) for anodizing process was gained (Fig. 1 (b)). Our findings
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indicated that the formed nanotube arrays on this modified surface (Fig. 1 (c)), has higher
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ordering than that of the mechanically and electrically polished samples.
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(a) (c) (b) Fig. 1. a) The morphology of low ordering nanotubes after Ti6Al4V pre-anodizing at voltage of 60 V for 15 min, b) as-prepared surface after pre-anodizing+ ultrasonic treatment, c) the morphology of high ordering nanotubes after second-anodizing for 60 min.
The principle of nanotube formation is the equilibrium between electrochemical (oxidation reaction in metal/oxide interface based on equation (1)) and chemical (etching reaction in oxide/solution interface based on equation (2)) reactions [13]. The rate of electrochemical reaction should not be very high or very low because it prevents the formation of nanotubes. It
ACCEPTED MANUSCRIPT should be mentioned that the electrochemical reaction is dependent on the potential, while the chemical reaction depends highly on pH and the fluorine ion concentration [18-21]. Ti + 2H2O → TiO2 + 4H+ + 4e-
(1)
TiO2 + 6F- + 4H+→ [TiF6]2- + 2H2O
(2)
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Based on the literature, adding carbonate compounds to the anodizing batch causes the formation
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rate and the internal diameter of nanotubes to be increased as a result of increasing the rate of
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chemical etch reaction. In fact, the carbonate compounds react with H+ based on equation (3): [CO3]2-+ 2H+→ CO2+ H2O
(3)
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Reducing the amount of H+ in the solution causes the pH to grow, therefore, the rate of chemical
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etch reaction increases. This event stimulates the tube formation and increase the number of preferred sites for nanotubes nucleation [18-21]. Regarding this fact, about 0.01 wt. % sodium
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carbonate was added to the anodizing bath as the catalyst and the nanotube modifier. As shown
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in Fig. 2, by addition of sodium carbonate to electrolyte the wall thickness and internal diameter
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of formed nanotubes decreases and increases, respectively.
(a) (b) Fig. 2. The morphology of formed nanotubes arrays on Ti6Al4V surface after anodizing at applied voltage of 60 V a) without and b) with carbonate.
ACCEPTED MANUSCRIPT The other important parameter influencing the morphology of the anodized layer is the voltage and the topological characteristics of nanotubes can be maintained by the applied anodizing voltage. In this regards, the surface morphology of Ti6Al4V surface after anodizing process under applied voltages of 25, 50, 60 and 75 V are presented in Figs. 3 to 6, respectively.
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Different topological characteristics of anodized surfaces contains of diameter, thickness and
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length of nanotubes are also presented in Table 1. Based on these results, several points can be
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concluded as:
1- At the voltage of 25 V (and lower voltages) there is not any evidence of nanotube generation
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on the Ti6Al4V surface. Under this condition, the voltage seems to be inadequate for continuous
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formation of TiO2 film; however the substrate was etched, revealing the grain boundaries of the Ti6Al4V. In this condition, the nucleation rate of nanotubes is so low and a thick barrier layer
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without nanotubes is formed on the substrate. By increasing the applied voltage up to 75 V, the
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rate of chemical etch reactions has been increases and a surface with nano pores can be formed. However, the lengths of the nanotubes in certain area are relatively low as compared to others.
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This finding is in conferment with to Saharudin et al [21] report about the formation of nanotube
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arrays on the surface of two-phase (α+β) Ti alloys such as Ti6Al4V. In fact, α phase is enriched with Al whereas β phase region enriched with V. Because of the different chemistries of these
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phases, the formation of nanotube array on Ti6Al4V surface is not uniform. However, the length of the nanotubes in β phase region is shorter than α phase as the solubility of V is faster than the Al [21]. 2- By increasing the applied voltage (beyond 75 V), the rate of the chemical etch reactions and the formation of [TiF6]2- enhances. This phenomenon leads to the formation of an unstable and
ACCEPTED MANUSCRIPT disordered porous morphology on surface. Finally, there remained some holes with larger diameters and a perforated substrate. 3- The optimum anodization voltage for the successful achievement of the nanotube arrays in Ti6Al4V alloys is in the range of 25 to 75 V. At the voltage ranges below 25 and higher than 75
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V, no nanotubes could be observed.
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4- The diameter and the length of the formed nanotubes as a function of anodizing voltage from
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50 to 75 V are presented in Table 1. As seen in this table, the diameter (form 75 to 120 nm) and length (from 2.5 to 3.4 µm) of the nanotubes increase with increasing the applied voltage. This
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result is in agreement with other similar studies in this field [20-22]. The effect of anodizing
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voltage on the diameter of the nanotubes can be related to the number of pits formed during the early stage of the anodizing process. It is inferred that samples anodized at high voltage will
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suffer from severe electric field dissolution forming more pits at an early stage of the process.
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These pits will be etched to form larger pores. As growth of the pores dominates, the resulting diameter of the nanotubes will be larger when anodizing is performed at higher voltage. At lower
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voltage, less electric field dissolution occurs forming TiO2 with smaller diameter pores [21].
Fig 3. The surface morphology of Ti6Al4V surface after anodizing process under applied voltage of 25 V.
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(b) (a) (c) Fig 4. The surface (a & b) and cross sectional (c) morphology of Ti6Al4V surface after anodizing process under applied voltage of 50 V.
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(a) (b) (c) Fig 5. The surface (a & b) and cross sectional (c) morphology of Ti6Al4V surface after anodizing process under applied voltage of 60 V.
(a) (c) (b) Fig 6. The surface (a & b) and cross sectional (c) morphology of Ti6Al4V surface after anodizing process under applied voltage of 75 V.
ACCEPTED MANUSCRIPT Table 1. Different characteristics of formed nanotubes on Ti6Al4V surface after anodizing process under different applied voltages. Nanotube
Voltage (V)
Contact
Thickness
Length
angle
(nm)
(nm)
(µm)
(θ0f)
50
74-80
25-28
2.5
36±10
60
90-97
20-30
Shape
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36±20
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3.2
75
100-120
24-29
3.4
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Diameter
36±20
ACCEPTED MANUSCRIPT Modification of Ti6Al4V surface with anodizing process can enhance the aforementioned properties required for the implants because such a layer serves as a passive layer, improves surface wettability and it enhances cellular response. Based on Table 1, the nano-tubular surfaces (with contact angle of about 36-39 o) show more hydrophilic than un-anodized surfaces (with
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contact angle of about 73.5 o). It is important to note that, the presented value of contact angle in
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this study is higher than the presented value with Yoriya et al. [23] (14 o) and Liu et al. (2 o) [24].
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This difference can be related to different morphology and chemical composition of formed nanotubes in different studies.
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To investigation of the cellular response of anodized samples, the adhesion of MG63 cells (after
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1 day of emersion) on anodized surfaces under applied voltages of 50, 60 and 75 V are presented in Fig. 7. As seen, the MG63 cells attached firmly on anodized surfaces with exaggerated
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filopodia induced inside the nanotubes. Based on this result, highly porous nanotube structures
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can increase the surface capability to integrate with osseous tissue and cellular activity and promote the growth of human osteosarcoma MG63 cells [19]. This result is in conferment with
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Saharudin et al. [21] report. In fact, nano topography affects the substrate/tissue interface by
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providing more binding sites to the cell membrane receptors. Consequently, the highest density and the best coverage of the attached cells in this work were achieved in anodized sample under
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applied voltage of 60 V.
To evaluate the biocompatibility of the anodized surfaces, the cell viability was investigated on the osteoblast-like cells because these cells are highly proliferative and less respecting the growth inhibition by cell-cell and cell-matrix contacts. Moreover, osteoblast tissues are in contact with the implant. MTT assay using human osteoblast-like cells depicted an improved cell viability of the anodized substrates (based on Fig. 8). Moreover, the highest value of cell viability is achieved in anodized samples under applied voltage of 60 V. In fact, the good cell
ACCEPTED MANUSCRIPT viability of Ti6Al4V anodized samples can also be related to the formation of highly porous nanotube structures on surface, increasing the surface wettability and the barriering effect of
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formed TiO2 layer against metal ions releasing.
(b)
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(a)
(d)
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(c)
(f) (e) Fig 7. The SEM images of MG63 bone marrow stromal cell cultured (after 1 day) on anodized surfaces under applied voltages of (a & b) 50, (c & d) 60 and (e & f) 75 V, at different magnifications.
ACCEPTED MANUSCRIPT Releasing the metal ions from implants can aeffects the lactate dehydrogenase and morphological changes in cells and the treated cells with these ions release lactate dehydrogenase into the physiological media [20]. By attention to this point, the amount of release ions from Ti6Al4V anodized samples is so important for biomedical applications. In this
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regards, the amount of release vanadium and aluminum ions form anodized surface (in applied
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voltage of 60 V) in PBS solution at 37 oC were examined. As seen from Fig. 9, the concentration
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of released vanadium and aluminum after 35 days of immersion reached to maximum values of about 19 and 7 ppm, respectively. These amounts of soluble ions are below the limits of
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International Agency for Research on Cancer (IARC). This result confirms the barriering effect
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of formed TiO2 nanotube structure against releasing the metallic ions.
Fig. 8. MTT assay (cell viability (%)) of Ti6Al4V substrate before (control sample) and after anodizing process under applied voltages of 50, 60, 75 V. This test has been done after 1, 3, 7 days of incubation in human osteoblast-like cells (MG63).
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Fig 9. The amount of released vanadium and aluminum ions from anodized surface in PBS solution after different periods of immersion times.
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4. Conclusion
Fabrication and characterization of TiO2 nanotube arrays on Ti6Al4V surface for enhancement
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of cell treatment in biomedical applications was the aim of this study. Based on achieved results, the nanotube arrays with good uniformity, high wettability and viability can successfully formed
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on Ti6Al4V surface in voltage range of about 50 to 75 V. The dimensional characteristics of
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formed nanotubes depend on applied voltage and play an essential role in viability and cell adhesion. In this work, the formed nanotubes array in applied voltage of 60 V (with average diameter and thickness of about 90-97 and 20-30, respectively) showed the best wettability, viability and cell response in comparison with other voltages.
Acknowledgements The authors acknowledge the financial support of the Nikceram Razi Co. Also I would like to
ACCEPTED MANUSCRIPT thank Mrs. Riahi for her corporation in the institute for nanoscience and nano technology of Sharif University of technology.
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ACCEPTED MANUSCRIPT [23] S. Yoriya, W. Kittimeteeworakul, N.i Punprasert, Effect of anodization parameters on morphologies of TiO2 nanotube arrays and their surface properties, Journal of Chemistry and Chemical Engineering 6 (2012) 686-691. [24] G. Liu, K. Du, K. Wang, Surface wettability of TiO2 nanotube arrays prepared by
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ACCEPTED MANUSCRIPT Highlights 1- This study focuses on the synthesis of TiO2 nanotube arrays on Ti6Al4V surface. 2- The wettability, viability and cell responce TiO2 nanotube arrays are investigated.
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3- The the relation between the dimension of nanotubes and cell attachment is proposed.
M. Mansoorianfar, M. Tavoosi, R. Mozafarinia, A. Ghasemi, A. Doostmohammadi PII: DOI: Reference:
S0257-8972(17)30483-8 doi: 10.1016/j.surfcoat.2017.05.016 SCT 22335
To appear in:
Surface & Coatings Technology
Received date: Revised date: Accepted date:
27 March 2017 30 April 2017 5 May 2017
Please cite this article as: M. Mansoorianfar, M. Tavoosi, R. Mozafarinia, A. Ghasemi, A. Doostmohammadi , Preparation and characterization of TiO2 nanotube arrays on Ti6Al4V surface for enhancement of cell treatment, Surface & Coatings Technology (2017), doi: 10.1016/j.surfcoat.2017.05.016
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Preparation and Characterization of TiO2 Nanotube Arrays on Ti6Al4V Surface for Enhancement of Cell Treatment
M. Mansoorianfar1, M. Tavoosi1*, R. Mozafarinia, A. Ghasemi1, A. Doostmohammadi2
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1- Department of Materials Engineering, Malek-Ashtar University of Technology (MUT), Iran.
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* Corresponding author contact:
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2- Department of Materials Engineering, Shahrekord University (SKU), Iran.
Email: [email protected]
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Tel./Fax: +983134429844
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Abstract
Fabrication and characterization of TiO2 nanotube arrays on Ti6Al4V surface for enhancement
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of cell treatment in biomedical applications was the aim of this study. In this regard, the
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anodizing process has been carried out in an organic bath (ethylene glycol+ 0.5 wt.% NH4F+ 3 wt.% water) under applied voltages of 25 to 100 V. The anodized samples were characterized
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using scanning electron microscopy (SEM), hydrophilic/hydrophobic balance, cell attachment,
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ion release test and MTT assay. Based on achieved results, the nanotube arrays with good uniformity, high wettability and viability can successfully formed on Ti6Al4V surface in voltage
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range of about 50 to 75 V. The dimensional characteristics of formed nanotubes depend on applied voltage and play an essential role in viability and cell adhesion. In this work, the formed nanotubes array in applied voltage of 60 V (with average diameter and thickness of about 90-97 and 20-30, respectively) showed the best wettability, viability and cell response in comparison with other voltages. Keywords: Ti6Al4V; Anodizing; TiO2 nanotube; Viability.
ACCEPTED MANUSCRIPT 1. Introduction The Ti6Al4V alloy is widely used as implant material in orthopedics and dentistry due to its high biocompatibility, high mechanical resistance, low density, high corrosion resistance, high chemical inertness and toxicity. Although, this alloy is known as an ideal implant material, its
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viability must be modified during surface modification methods [1-2].
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biomedical characteristics such as roughness, composition, wettability, cell response and cellular
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Among different surface modification methods, surface roughening and formation of nano-scale topography on surface have been employed for increasing the capability of implants to integrate
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with osseous tissue. This kind of topography can affect on implant/tissue interface by providing
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more binding sites to cell membrane receptors and enhances biological events with indirect effect on cellular activity features [3]. In this regards, Xia et al. [4] showed that the nanostructure
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topologies on the Ti implant surface can improve the proliferation, differentiation and
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development of the osteoblastic phenotype and increase the bone-implant interfacial strength. Salou et al. [5] also studied the osseointegration of micro and nano-structured implants in rabbit
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femurs and showed that nano-structured implants have higher bone-to-implant contact and bone
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growth values in comparison with micro-structured implants. Moreover, similar results have been presented with Rosales-Leal [6] about the effect of nano-scale topography on cell attachment and
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bioactivity performance of Ti and its alloys. Anodizing processing is one of the most popular methods to obtaining nano-structured morphology in form of nanotubes on the surface of Ti and its alloys. This method was newly developed for the promising applications in biomedical and tissue engineering [7-11]. In this process, TiO2 nanotubes create from initial titanium oxide layer through anodizing in an
ACCEPTED MANUSCRIPT electrochemical cell including fluorine. The diameter and length of nanotubes can be controlled by the anodizing voltage, electrolyte composition and time duration of anodizing [8, 12]. In fact, numerous studies have been conducted about the formation of TiO2 nanotubes on Ti surface using anodizing process and the formation mechanism and kinetic of nanotubes structure
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were presented in details by different authors [3-12]. However, there is relatively little attention
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about the biological characterizations of TiO2 nanotubes array on Ti6Al4V alloy. Moreover,
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there is lack of information about the relation between the dimension of TiO2 nanotubes, wettability, viability and cell attachment behaviors in the literatures. So, this research study
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focuses on the fabrication and characterization of TiO2 nanotube arrays on Ti6Al4V surface for
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enhancement of cell treatment in biomedical applications.
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2. Materials and methods
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High purity Ti6Al4V sheet (Nilaco, Japan) with thickness of about 1 mm was used as substrate material. High purity ethylene glycol, NH4F, ethanol and acetone (Sigma-Aldrich, Australia)
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were also used as anodizing batch and cleaner solutions. Prior to anodizing process, Ti6Al4V
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sheets were brushed, polished and degreased with deionized water and acetone and finally dried at ambient temperature. In all testing, the anodizing solution (ethylene glycol+ 0.5 wt. % NH4F+
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3 wt. % water), anodizing temperature (25 °C) and time (60 min) were kept notably constant and the process was performed at different anodizing voltages within the range of 25 to 100 V. To modification of anodized nanotubes arrays as a uniform substrate for cell responses, the ultrasonic surface treatment has been performed and about 0.01 wt. % of Na2CO3 was added to the anodizing bath. Structural and morphological characterizations of anodized layer were carried out by field
ACCEPTED MANUSCRIPT emission scanning electron microscope (VEGA-TESCAN-XMU) at an accelerating voltage of 20 kV. The hydrophilic/hydrophobic balances of anodized samples were evaluated by means of contact angle measurements (Dataphysics, OCA 15 plus). Each contact angle value is the average of minimum 3 measurements. The investigation was carried out with an accuracy of ±1 °
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at a temperature of 25 °C. To evaluation of aluminum (Al) and vanadium (V) ion releasing, the
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anodized samples in optimum condition were immersed in PBS solution for different periods of
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time (1, 3, 7, 14, 21, 28 and 35 days) and the achieved solution were examined by inductively coupled plasma-atomic emission spectroscope (ICP-AES; ICPS-100IV, Shimadzu, Japan). The
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effects of nanotube structure on cell adhesion, cell proliferation and osteoblast expression,
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growth and differentiation of human osteoblast were also investigated by 3-(4,5-
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Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay.
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3. Results and discussion
Despite huge number of publications regarding different affecting elements in anodizing Ti and
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its alloys [11-13], there are a number of factors such as the diameter, wall thickness, length and
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the arrangement of nanotubes arrays which must be determined and modified. According to literature, substrate preparation, solution component and anodizing voltage, are the three main
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factors for modifying the nanotube arrays during anodizing process [14]. Based on TEM and XRD analysis in literatures [1-4], the formed TiO2 arrays on surface during anodizing process are in amorphous state. However, once amorphous titania is formed in low temperature environment, the strong Ti-O bond will suppress the atomic mobility necessary for rearrangement of the atomic structure. However, according to Shibata et al. [15] and Sul et al.
ACCEPTED MANUSCRIPT [16], the oxide layer produced by anodization is mainly in an amorphous form but can also contain some crystalline phases, depending on the anodization process parameters. In order to prepare uniform nanotube arrays, it is necessary to apply a template. The templateassisted technique promotes the growth of nanotubes. Considering this fact, we proposed the pre-
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anodizing plus ultrasound technique as a preparatory method before anodizing process [17]. In
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fact, in pre-anodizing stage, low-ordered nanotube array were formed (Fig. 1 (a)). During
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ultrasonic treatment, all the formed nanotubes were removed from the surface by sonic waves and an ideal surface (as a template) for anodizing process was gained (Fig. 1 (b)). Our findings
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indicated that the formed nanotube arrays on this modified surface (Fig. 1 (c)), has higher
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ordering than that of the mechanically and electrically polished samples.
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(a) (c) (b) Fig. 1. a) The morphology of low ordering nanotubes after Ti6Al4V pre-anodizing at voltage of 60 V for 15 min, b) as-prepared surface after pre-anodizing+ ultrasonic treatment, c) the morphology of high ordering nanotubes after second-anodizing for 60 min.
The principle of nanotube formation is the equilibrium between electrochemical (oxidation reaction in metal/oxide interface based on equation (1)) and chemical (etching reaction in oxide/solution interface based on equation (2)) reactions [13]. The rate of electrochemical reaction should not be very high or very low because it prevents the formation of nanotubes. It
ACCEPTED MANUSCRIPT should be mentioned that the electrochemical reaction is dependent on the potential, while the chemical reaction depends highly on pH and the fluorine ion concentration [18-21]. Ti + 2H2O → TiO2 + 4H+ + 4e-
(1)
TiO2 + 6F- + 4H+→ [TiF6]2- + 2H2O
(2)
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Based on the literature, adding carbonate compounds to the anodizing batch causes the formation
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rate and the internal diameter of nanotubes to be increased as a result of increasing the rate of
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chemical etch reaction. In fact, the carbonate compounds react with H+ based on equation (3): [CO3]2-+ 2H+→ CO2+ H2O
(3)
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Reducing the amount of H+ in the solution causes the pH to grow, therefore, the rate of chemical
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etch reaction increases. This event stimulates the tube formation and increase the number of preferred sites for nanotubes nucleation [18-21]. Regarding this fact, about 0.01 wt. % sodium
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carbonate was added to the anodizing bath as the catalyst and the nanotube modifier. As shown
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in Fig. 2, by addition of sodium carbonate to electrolyte the wall thickness and internal diameter
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of formed nanotubes decreases and increases, respectively.
(a) (b) Fig. 2. The morphology of formed nanotubes arrays on Ti6Al4V surface after anodizing at applied voltage of 60 V a) without and b) with carbonate.
ACCEPTED MANUSCRIPT The other important parameter influencing the morphology of the anodized layer is the voltage and the topological characteristics of nanotubes can be maintained by the applied anodizing voltage. In this regards, the surface morphology of Ti6Al4V surface after anodizing process under applied voltages of 25, 50, 60 and 75 V are presented in Figs. 3 to 6, respectively.
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Different topological characteristics of anodized surfaces contains of diameter, thickness and
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length of nanotubes are also presented in Table 1. Based on these results, several points can be
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concluded as:
1- At the voltage of 25 V (and lower voltages) there is not any evidence of nanotube generation
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on the Ti6Al4V surface. Under this condition, the voltage seems to be inadequate for continuous
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formation of TiO2 film; however the substrate was etched, revealing the grain boundaries of the Ti6Al4V. In this condition, the nucleation rate of nanotubes is so low and a thick barrier layer
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without nanotubes is formed on the substrate. By increasing the applied voltage up to 75 V, the
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rate of chemical etch reactions has been increases and a surface with nano pores can be formed. However, the lengths of the nanotubes in certain area are relatively low as compared to others.
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This finding is in conferment with to Saharudin et al [21] report about the formation of nanotube
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arrays on the surface of two-phase (α+β) Ti alloys such as Ti6Al4V. In fact, α phase is enriched with Al whereas β phase region enriched with V. Because of the different chemistries of these
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phases, the formation of nanotube array on Ti6Al4V surface is not uniform. However, the length of the nanotubes in β phase region is shorter than α phase as the solubility of V is faster than the Al [21]. 2- By increasing the applied voltage (beyond 75 V), the rate of the chemical etch reactions and the formation of [TiF6]2- enhances. This phenomenon leads to the formation of an unstable and
ACCEPTED MANUSCRIPT disordered porous morphology on surface. Finally, there remained some holes with larger diameters and a perforated substrate. 3- The optimum anodization voltage for the successful achievement of the nanotube arrays in Ti6Al4V alloys is in the range of 25 to 75 V. At the voltage ranges below 25 and higher than 75
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V, no nanotubes could be observed.
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4- The diameter and the length of the formed nanotubes as a function of anodizing voltage from
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50 to 75 V are presented in Table 1. As seen in this table, the diameter (form 75 to 120 nm) and length (from 2.5 to 3.4 µm) of the nanotubes increase with increasing the applied voltage. This
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result is in agreement with other similar studies in this field [20-22]. The effect of anodizing
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voltage on the diameter of the nanotubes can be related to the number of pits formed during the early stage of the anodizing process. It is inferred that samples anodized at high voltage will
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suffer from severe electric field dissolution forming more pits at an early stage of the process.
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These pits will be etched to form larger pores. As growth of the pores dominates, the resulting diameter of the nanotubes will be larger when anodizing is performed at higher voltage. At lower
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voltage, less electric field dissolution occurs forming TiO2 with smaller diameter pores [21].
Fig 3. The surface morphology of Ti6Al4V surface after anodizing process under applied voltage of 25 V.
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(b) (a) (c) Fig 4. The surface (a & b) and cross sectional (c) morphology of Ti6Al4V surface after anodizing process under applied voltage of 50 V.
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(a) (b) (c) Fig 5. The surface (a & b) and cross sectional (c) morphology of Ti6Al4V surface after anodizing process under applied voltage of 60 V.
(a) (c) (b) Fig 6. The surface (a & b) and cross sectional (c) morphology of Ti6Al4V surface after anodizing process under applied voltage of 75 V.
ACCEPTED MANUSCRIPT Table 1. Different characteristics of formed nanotubes on Ti6Al4V surface after anodizing process under different applied voltages. Nanotube
Voltage (V)
Contact
Thickness
Length
angle
(nm)
(nm)
(µm)
(θ0f)
50
74-80
25-28
2.5
36±10
60
90-97
20-30
Shape
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36±20
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3.2
75
100-120
24-29
3.4
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Diameter
36±20
ACCEPTED MANUSCRIPT Modification of Ti6Al4V surface with anodizing process can enhance the aforementioned properties required for the implants because such a layer serves as a passive layer, improves surface wettability and it enhances cellular response. Based on Table 1, the nano-tubular surfaces (with contact angle of about 36-39 o) show more hydrophilic than un-anodized surfaces (with
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contact angle of about 73.5 o). It is important to note that, the presented value of contact angle in
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this study is higher than the presented value with Yoriya et al. [23] (14 o) and Liu et al. (2 o) [24].
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This difference can be related to different morphology and chemical composition of formed nanotubes in different studies.
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To investigation of the cellular response of anodized samples, the adhesion of MG63 cells (after
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1 day of emersion) on anodized surfaces under applied voltages of 50, 60 and 75 V are presented in Fig. 7. As seen, the MG63 cells attached firmly on anodized surfaces with exaggerated
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filopodia induced inside the nanotubes. Based on this result, highly porous nanotube structures
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can increase the surface capability to integrate with osseous tissue and cellular activity and promote the growth of human osteosarcoma MG63 cells [19]. This result is in conferment with
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Saharudin et al. [21] report. In fact, nano topography affects the substrate/tissue interface by
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providing more binding sites to the cell membrane receptors. Consequently, the highest density and the best coverage of the attached cells in this work were achieved in anodized sample under
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applied voltage of 60 V.
To evaluate the biocompatibility of the anodized surfaces, the cell viability was investigated on the osteoblast-like cells because these cells are highly proliferative and less respecting the growth inhibition by cell-cell and cell-matrix contacts. Moreover, osteoblast tissues are in contact with the implant. MTT assay using human osteoblast-like cells depicted an improved cell viability of the anodized substrates (based on Fig. 8). Moreover, the highest value of cell viability is achieved in anodized samples under applied voltage of 60 V. In fact, the good cell
ACCEPTED MANUSCRIPT viability of Ti6Al4V anodized samples can also be related to the formation of highly porous nanotube structures on surface, increasing the surface wettability and the barriering effect of
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formed TiO2 layer against metal ions releasing.
(b)
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(a)
(d)
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(f) (e) Fig 7. The SEM images of MG63 bone marrow stromal cell cultured (after 1 day) on anodized surfaces under applied voltages of (a & b) 50, (c & d) 60 and (e & f) 75 V, at different magnifications.
ACCEPTED MANUSCRIPT Releasing the metal ions from implants can aeffects the lactate dehydrogenase and morphological changes in cells and the treated cells with these ions release lactate dehydrogenase into the physiological media [20]. By attention to this point, the amount of release ions from Ti6Al4V anodized samples is so important for biomedical applications. In this
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regards, the amount of release vanadium and aluminum ions form anodized surface (in applied
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voltage of 60 V) in PBS solution at 37 oC were examined. As seen from Fig. 9, the concentration
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of released vanadium and aluminum after 35 days of immersion reached to maximum values of about 19 and 7 ppm, respectively. These amounts of soluble ions are below the limits of
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International Agency for Research on Cancer (IARC). This result confirms the barriering effect
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of formed TiO2 nanotube structure against releasing the metallic ions.
Fig. 8. MTT assay (cell viability (%)) of Ti6Al4V substrate before (control sample) and after anodizing process under applied voltages of 50, 60, 75 V. This test has been done after 1, 3, 7 days of incubation in human osteoblast-like cells (MG63).
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Fig 9. The amount of released vanadium and aluminum ions from anodized surface in PBS solution after different periods of immersion times.
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4. Conclusion
Fabrication and characterization of TiO2 nanotube arrays on Ti6Al4V surface for enhancement
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of cell treatment in biomedical applications was the aim of this study. Based on achieved results, the nanotube arrays with good uniformity, high wettability and viability can successfully formed
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on Ti6Al4V surface in voltage range of about 50 to 75 V. The dimensional characteristics of
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formed nanotubes depend on applied voltage and play an essential role in viability and cell adhesion. In this work, the formed nanotubes array in applied voltage of 60 V (with average diameter and thickness of about 90-97 and 20-30, respectively) showed the best wettability, viability and cell response in comparison with other voltages.
Acknowledgements The authors acknowledge the financial support of the Nikceram Razi Co. Also I would like to
ACCEPTED MANUSCRIPT thank Mrs. Riahi for her corporation in the institute for nanoscience and nano technology of Sharif University of technology.
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ACCEPTED MANUSCRIPT Highlights 1- This study focuses on the synthesis of TiO2 nanotube arrays on Ti6Al4V surface. 2- The wettability, viability and cell responce TiO2 nanotube arrays are investigated.
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3- The the relation between the dimension of nanotubes and cell attachment is proposed.