Effect of current density on the morphology and electrochemical properties of nanotubular TiO2 for implant applications

This study focuses on investigating the influence of current density (i) (A/dm2) at values of 0.5 A dm−2, 1.0 A dm−2 1.5 A dm−2, and 2.0 A/dm2 on the surface structure of nanotubular titanium dioxide (TiO2) in an ethylene glycol solvent containing a certain amount of fluoride salt and water. The surface structure observed via FESEM images reveals that different current densities yield different nanotubular TiO2 structures, predominantly in the form of anatase TiO2 crystals. EIS and CV measurements indicate that at a current density of i = 1.5 A dm−2, the nanotubular TiO2 layer exhibits corrosion resistance performance up to 90.06% compared to the bare titanium (Ti) samples. Confocal laser scanning microscopy (CLSM) demonstrates enhanced attachment of BHK cells on anodized titanium surfaces compared to unmodified controls. These findings suggest that nanotubular TiO2 presents a biocompatible material with promising potential for biomedical implant applications.


Introduction
Titanium (Ti) is a material known for its high mechanical strength and excellent biocompatibility [1,2].The biocompatibility of titanium is attributed to the formation of a TiO 2 oxide layer on its surface [3].Under natural conditions, the TiO 2 layer naturally forms on the titanium substrate due to temperature, humidity in the air, and the surrounding environment.This self-generated layer is extremely thin, ranging from 2 to 6 nm [4].Due to its low wettability, the natural titanium material has limited applications in the biomedical field [5].Therefore, the previous researches have been focused on improving the corrosion resistance and the biocompatibility of titanium by coatings it's with relatively thick TiO 2 layers [6][7][8].
Various methods for creating titanium dioxide coatings on titanium substrates have been investigated, including physical vapor deposition (PVD) [9], sol-gel method [9,10], microarc oxidization (MAO) [11][12][13], chemical vapor deposition (CVD) [14], and electrochemical method [15], et al The anodization method for titanium involves creating a titanium dioxide coating based on the oxidation-reduction process at the surface of electrodes placed in an electrolyte solution under the influence of direct current.The anodization method offers several advantages as it allows for the adjustment of chemical composition [16] and electrochemical properties [17] of the coating by altering the chemical composition of the solution [18].Additionally, variations in anodization parameters such as voltage [19], current density [20], time [21], temperature [22], et al result in coatings with different surface morphologies [16][17][18][19][20][21][22], electrochemical properties [21], and biological compatibility [20].Currently, the anodization method has been widely researched to enhance surface characteristics of various materials like titanium, aluminum, niobium, and magnesium, by forming a thick oxide layer ranging from 1-100 μm, exhibiting improved electrochemical properties [23], wettability, and biological compatibility compared to the native titanium substrates.
In the synthesis of titanium dioxide on a titanium substrate using the anodization method, the titanium electrode needs to be connected to the positive terminal of a direct current power source.The negative terminal of the direct current power source is connected to the platinum electrode.Both electrodes are immersed in the same electrolyte solution during the anodization process (figure 1).The electrolyte solution commonly used for the anodization titanium includes inorganic solutions, organic solutions, or hydride-based solutions.
Many research papers have clearly described the mechanism of synthesizing TiO 2 by electrochemical methods on titanium substrates with a focus on biomedical applications.The most used solution system is a mixture of ethylene glycol, ammonium fluoride, and water [24][25][26].The research also clearly describes that the creation of a porous TiO 2 layer on the titanium substrate is achieved through the processes of TiO 2 layer formation and the dissolution of the TiO 2 layer by fluoride ions in the solution.
When implanting artificial materials in biomedical applications, evaluating the corrosion characteristics of the implant material is extremely important.Human body fluids contain ions such as Cl − , Na + , Ca 2+ , K + , HPO 4 2− , etc. Therefore, titanium and titanium alloys often undergo corrosion when implanted in the human body, which reduces the mechanical strength of the implant, releases metal ions that can cause toxic reactions, and decreases the ability for bone integration [27,28].
In environments containing Cl − ions with dissolved oxygen, the most common form of corrosion for titanium and titanium alloys is localized corrosion [29].Due to the small size of Cl − ions, they can penetrate the natural oxide barrier layer through cracks.Furthermore, because titanium is a multivalent metal, in addition to the most stable TiO 2 compound, several less stable oxides such as TiO, Ti 2 O 3 , Ti 3 O 5 and TiO 3 , can form on the surface of titanium.These oxides reduce the passivity of the oxide layer.The anodizing method demonstrates significant advantages as they easily produce uniform coatings with well-controlled thickness and structure of the titanium dioxide layer, enhancing the corrosion resistance of the coating [28].
Many previous studies have shown that the surface of titanium after anodization has better biocompatibility compared to untreated titanium.The authors explain that after anodization, the surface of titanium forms oxide layers with a hexagonal structure [30], providing better adhesion for cells.Additionally, the thick compound layer on the titanium surface after anodization prevents corrosive ions from penetrating and damaging the inherent barrier layer of titanium.
This study demonstrates a significant correlation between current density with the microstructure, corrosion resistance of TiO 2 nanotubes and proposing a suitable surface for biocompatibility test.These findings offer valuable insights into the potential applications of nanotubular TiO 2 in the medical field.

Titanium anodization
The Ti in the study had dimensions of 10 × 10 × 1 mm 3 (Merk, 99.9%).Prior to anodization, bare Ti was mechanically polished using SiC sandpaper up to a 2000 grit (Foxwoll, Korea), followed by ultrasonic vibration in ethanol for approximately 10 min at room temperature.Subsequently, the test samples were rinsed with deionized water and dried in hot air about 80 °C until completely dry.
The test samples were anodized in a solution of ethylene glycol (Merck, 99.9%) supplemented with NH 4 F (Sigma, 99.9%) and water with concentrations of 0.5 wt% and 2 vol.%, respectively.The electrochemical system was set up as shown in figure 1, with the positive electrode of the DC power (B&K Precision, 9206B, USA) supply connected to the Ti considered as the working electrode, and the negative electrode of the power supply connected to a platinum electrode, considered as the counter electrode.Anodization process with a stirring rate of 450 rpm, the current density (i) (A/dm 2 ) was investigated at different values including 0.5 A dm −2 1.0 A dm −2 1.5 A dm −2 and 2.0 A dm −2 for a duration of 60 min at room temperature.After the anodization process, the test samples were thoroughly rinsed with deionized water to completely remove any residual solution on the surface, then dried in hot air.

Properties and electrochemical characteristics
The surface structure of the test samples was observed using FE-SEM, JEOL with the JSM-7600F model at a voltage of 20 kV.The phase composition of the surface coating of the test samples was determined using XRD method at voltages and currents of 40 kV and 30 mA, respectively.The bonding strength of the TiO 2 coating to the titanium substrate was measured using the TABER Multi-Finger Scratch/Mar Tester-Model 710, employing a tungsten carbide scratch tip with a diameter of 0.25 mm and a maximum load of 6 N. Following scratching, SEM was utilized to evaluate the thickness of the TiO 2 nanotubes coating.The corrosion resistance properties of the test samples compared to the Ti samples were evaluated using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) in simulated body fluid (SBF) with a pH value of approximately 7.During EIS and CV measurements, the solution temperature was maintained at approximately 25 °C, the test sample served as the working electrode and platinum functioned as the counter electrode immersed in SBF.The CV measurements were scanned in the potential range from −100 mV to 100 mV at a scan rate of 10 mV.s −1 , and the EIS measurements were scanned in the frequency range from 0.1 Hz to 10 5 Hz.EIS spectra were analyzed to obtain electrochemical characteristic parameters of the test samples such as corrosion resistance (R p ), corrosion current (I corr ), and corrosion potential (E corr ).

Cell attachment and quantitative analysis
Prior to cell attachment experiments, bare Ti and nanotubular TiO 2 were sterilized by autoclaving at 121 °C and run on for 60 min with dry cycle.Baby hamster kidney (BHK) cells were cultured in DMEM medium at 37 °C, 5% CO 2 , and humidified conditions.Cell suspensions with a uniform concentration and volume of 2 × 10 4 cells/ml were seeded onto bare Ti and nanotubular TiO 2 .Cell attachment was visualized using a CLSM microscope (FV3000RS, Olympus).After 72 h of culture, BHK cells on bare Ti and nanotubular TiO 2 were fixed with 4% paraformaldehyde in PBS for 10 min, washed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 min, stained with fluorescent phalloidin 555 for 45 min, and stained with DAPI for 5 min to visualize the cell nuclei.Stained cells attached to bare Ti and nanotubular TiO 2 were placed on glass coverslips, and cell attachment was observed.MTS assay (MTS, Promega, Madison, WI, USA) was also used for quantitative analysis of biocompatibility.In this assay, the formazan product was measured by a microreader (Chromate 4300 Microplate Reader) using absorbance of 490 nm.

Surface morphology analysis
The surface morphology of machined titanium is completely flat.When performing the anodization process in a mixture of ethylene glycol and ammonium fluoride, the titanium surface always involves two processes: tube formation and tube dissolution.At low current densities, areas on the surface with F − ions unable to penetrate through the TiO 2 barrier layer result in either incomplete TiO 2 coverage or the presence of rough, irregularly shaped regions.As the current density increases, the processes of tube formation and dissolution reach equilibrium, leading to uniform nanotubular TiO 2 across the entire surface.However, at high current densities, increased F − ion penetration on the coating layer enhances tube dissolution, resulting in thinner nanotubular with increased inner diameters.The surface morphology of anodized titanium samples under different current densities is illustrated in figure 2. At i = 0.5 A/dm 2 in figure 2(a), the surface coating exhibits uneven formation, with areas on the titanium substrate where thinner nanotubular TiO 2 are not fully developed.The nanotubes on the surface have an inner diameter of approximately 53 nm, with some regions showing rough surface morphology.At low current densities, the dissolution of the barrier layer on the bare Ti surface encounters difficulty, thereby impeding the formation process of nanotubular TiO 2 .At i = 1.0 A/dm 2 in figure 2(b) and i = 1.5 A dm −2 in figure 2(c), both samples exhibit relatively flat surfaces, with nanotubular TiO 2 having relatively uniform diameters of around 62 nm, wall thickness about 50-60 nm.However, at a current density of i = 1.5 A/dm 2 as shown in figure 2(c), the nanotubular have significantly thicker walls due to the increased interaction between O 2− and Ti 4+ ions in the electrolyte solution, and the openings of the tubes are much flatter compared to the i = 1.0A dm −2 as shown in figure 2(b).At a current density of i = 2.0 A dm −2 , the inner diameter of the nanotubular TiO 2 is approximately 93 nm, with reduced tube openings and the appearance of dissolution sites resulting in fragmented fibers at the tube openings.
The result is consistent with previous studies, indicating that as the current density increases, it will dominate the formation process as well as the dissolution process of thinner nanotubular TiO 2 under the influence of F − ions [31][32][33].Therefore, selecting the appropriate current density in the titanium dioxide anodization process is crucial.
In addition to the biological compatibility, the adhesion capability of titanium dioxide to the substrate is one of the mechanical requirements for implant materials.Samples synthesized at current densities of i = 0.5 A/dm 2 and i = 2.0 A/dm 2 exhibit easily detachable surface coatings as shown in figures 3(a) and (b).Therefore, subsequent testing procedures in this paper do not include experiments on these two samples synthesized at current densities of i = 0.5 A/dm 2 and i = 2.0 A dm −2 .
Figure 4 illustrates the scratch and SEM images of the sample post-scratch test conducted to assess the bonding strength of the titanium sample anodized at a current density of 1.5 A dm −2 .Typically, the critical scratch force for titanium dioxide coatings falls within the range of 3-10 N, depending on the synthesis method and conditions.For the anodization method, the titanium dioxide layer typically exhibits a critical scratch force of around 3-8 N, varying based on the coating parameters and thickness [34].In figure 4(a), the average critical scratch force is 4.69 ± 0.16 N. Additionally, in figure 4(b), the scratch depth is approximately 10 μm, indicating that the coating has relatively good scratch resistance and high consistency during the testing process.However, this value may need to be further improved to meet more stringent durability requirements for certain specialized applications.

X-ray diffraction pattern
The samples of titanium after anodization at different current densities were heat-treated at 550 °C to transform the TiO 2 product from amorphous to crystalline form [35]. Subsequently, the x-ray diffraction (XRD) patterns were utilized to determine the structure and phase composition of the anodized coatings.Figures 5(b)-(c) illustrates the structure and phase composition of the anodized coatings fabricated under different current density conditions, while figure 5(a) shows the structure and phase composition of the coating on the bare Ti sample.The XRD spectrum of bare Ti sample after anodizing process at i = 1.5 A/dm 2 clearly exhibits sharp    figure 5(b) XRD spectrum of bare Ti samples after anodizing process at i = 1.0A dm −2 , the intensity of these phases remains mostly unchanged, with the addition of enhanced diffraction peak (220) of TiO 2 material with a rutile phase structure at an angle of 2θ approximately 56.4°, following the JCPDS standard card 21-1276 [36].

Electrochemical properties 3.3.1. Corrosion resistance
The polarization curves (I-E) of the bare Ti and nanotubular TiO 2 are presented in figure 6(a).Corrosion parameters based on Tafel curve measurements have been processed and summarized in table 1.
Compared to the bare Ti, the nanotubular TiO 2 exhibits changes in the polarization curve and corrosion potential (E corr ).From figure 6(a), the corrosion potential of the nanotubular TiO 2 is higher than that of the bare Ti.The specific values for corrosion potential, corrosion resistance, and corrosion rate of the bare Ti sample and the nanotubular TiO 2 at current densities of i = 1.0 A/dm 2 and i = 2.0 A/dm 2 are presented in table 1.It can be observed that in terms of material properties, Ti is a material with high corrosion resistance, with a corrosion rate of only 5.73 (mg/m 2 h).When the bare Ti sample is anodized, the corrosion current rate continues to decrease to 0.70 mg m −2 h and 0.34 mg m −2 h at current densities of i = 1.0 A/dm 2 and i = 1.5 A dm −2 , respectively, contributing to an increase in corrosion protection efficiency to 87.78% and 90.06% compared to the Ti.samples at various current densities are larger than those for the bare Ti, indicating that the corrosion resistance of the anodized titanium samples is higher compared to the bare Ti.Furthermore, the anodized titanium samples with current densities of 1.0 A dm −2 and 1.5 A dm −2 on the Nyquist plot, the linear phase angle with frequencies below 45 degrees indicates the presence of the Warburg resistance.Figure 6(d) depicts the Bode phase spectra of the anodized titanium samples compared to Ti.It shows that the Bode plot of the nanotubular TiO 2 has a similar shape to that of the Ti.The maximum phase angle of the bare Ti at a frequency around 10 Hz is lower, indicating a tighter surface than the nanotubular TiO 2 with the maximum phase angle in the frequency range of 100 Hz.However, the maximum phase angle of anodized titanium is approximately −82°which is higher than the −78°of bare Ti, indicating a higher corrosion protection ability of the anodized titanium sample compared to Ti.

Electrochemical impedance spectroscopy
Figure 7 illustrates the equivalent layers and circuits for both the bare Ti and anodized titanium samples.The bare Ti sample surface features a tight barrier layer with a very high resistance, R B , approximately 1.1 × 10 5 Ωcm 2 , as depicted in figure 7(a).Meanwhile, in figure 7(b), the anodized titanium sample forms a porous layer indicated by the Warburg resistance.The components in the equivalent circuit include the resistance of the electrolyte solution (R S ), the resistance of the native barrier layer (R B ), and the double-layer capacitance of the barrier layer with the outer environment/layer (CPE B ).The Warburg resistance (W P ) component is characteristic of the ion diffusion process within the titanium dioxide nanotube layer.These parameters are specified in table 2. To provide a more accurate description of the components in figure 7(b), the layer of nanotubular TiO 2 formed after the anodization process is described by the parallel combination of CPE P and R P .
The values of n for the coating on the anodized titanium sample at two current densities, i = 1.0A dm −2 and i = 1.5 A dm −2 , are 0.9431 and 0.9471, respectively.Meanwhile, the results on the Bode phase plot in the different graphs are not significantly different.It can be inferred that the physical properties of the film on the anodized titanium sample at both current densities of i = 1.0A dm −2 and i = 1.5 A dm −2 are tight, with a relatively even surface.CPE 1 for both samples are equivalent to a capacitor.
The results obtained from Nyquist plots and Bode phase plots consistently indicate improved corrosion protection effectiveness for the anodized titanium samples compared to the Ti.This improvement is attributed to the uniform formation of nanotubular TiO 2 layer on the titanium surface during the anodization process, resulting in a more tightly packed structure and an increased corrosion potential for these samples.
Based on the consistent results achieved in terms of structure uniformity and highest corrosion protection efficiency, we have selected the nanostructured TiO 2 nanotube sample fabricated at a current density of 1.5 A/ dm 2 for further investigation into its biocompatibility.shown in figure 8(b) and nanotubular TiO 2 as shown in figure 8(d).Notably, cells on nanotubular TiO 2 display better connectivity with distinct polygonal morphology, suggesting good biocompatibility of the nanotubular TiO 2 .This is entirely consistent with previous studies, as the anodization of titanium surfaces results in the formation of a highly porous titanium oxide layer, facilitating strong cell adhesion and robust cell growth on the surface [37].The cell functions based on nuclear mechanosensing are crucial information in cell studies [38], which includes assessing the interaction between materials and cells, as demonstrated in figure 9.

Cell attachment and quantitative analysis
The cell growth on TiO 2 at a current density of 1.5 A dm −2 was also compared with the bare Ti substrate and further examined using the MTS assay, one of the most important parameters for evaluating the biocompatibility of artificial materials.Figure 9 shows cell growth on bare Ti and TiO 2 at 1.5 A dm −2 .The rate of cell growth on  TiO 2 at 1.5 A dm −2 was significantly higher than that of the bare Ti substrate (p < 0.05).This suggests that TiO 2 at 1.5 A/dm 2 can promote the growth and proliferation of BHK cells without causing cytotoxic effects, due to the superior corrosion resistance and nanotubular architecture.Consequently, the TiO 2 layer functions as a cellular binding site within the culture medium, promoting better cell growth [38].

Conclusion
The influence of current density on the surface properties and corrosion resistance of nanotubular TiO 2 grown on Ti substrates is a well-established area of research.This study demonstrates a significant correlation between current density, the size and morphology of the resulting nanotubular TiO 2 .Specifically, increasing current density leads to a corresponding increase in the inner diameter of the nanotube openings.The employed fabrication method primarily yields nanotubular TiO 2 with anatase crystal structure.Notably, at a current density of 1.5 A dm −2 , the formed nanotubular TiO 2 layer exhibits exceptional properties, including up to 90.06% corrosion protection efficiency, good biocompatibility, and high biological safety.These findings suggest promising applications for such tailored nanotubular TiO 2 in the field of implantology.

Figure 1 .
Figure 1.Electrochemical diagram of the titanium anodizing process.

Figure 4 .
Figure 4. Scratch test of anodized titanium sample at 1.5 A dm −2 (a) critical scratch force; (b) Scratch depth.

Figure 6 (
b) represents the Nyquist impedance spectra for the bare Ti substrate and the nanotubular TiO 2 at different current densities in an SBF solution.It can be observed that the circles for the anodized titanium

Figure 8
compares cell development on Ti and nanotubular TiO 2 after 72 h of growth.Both surfaces show high cell density at 10X magnification in figure 8(a) and figure 8(c), with thinner nanotubular TiO 2 exhibiting more uniform growth.Higher magnification (20X) reveals clear cell features (nuclei and membranes) on both Ti as

Table 1 .
Results of the polarization curve I-E.