Using Applied Electrochemistry to Obtain Nanoporous TiO 2 Films on Ti6Al4V Implant Alloys and Their Preclinical In Vitro Characterization in Biological Solutions

: Nanoporous TiO 2 ﬁlm is deposited on grade 5 Ti6Al4V implant alloy by electrochemical oxidation. The nanopores of the ﬁlm, as highlighted by electron microscopy, have a mean diameter of 58.6 nm, which is measured and calculated from an average value of 10 measurements. The increase in oxygen concentration compared to the untreated alloy, which indicates the oxidation of the titanium alloy surface, is visualized using X-ray spectroscopy coupled to an electron microscope. The beneﬁcial effect of the oxidation and controlled formation of the TiO 2 ﬁlm on the implant alloy is proven by the comparative evaluation of degradation over time through the corrosion of both the untreated alloy and the alloy with an electrochemically formed and controlled TiO 2 ﬁlm in Hank’s solution, which simulates the most corrosive biological ﬂuid, blood. The results show that the electrochemical modiﬁcation of the grade 5 titanium alloy to form a nanoporous TiO 2 surface ﬁlm using the electrochemical oxidation method conﬁrms the potential of improving the anticorrosive properties of titanium alloys used in implant applications.


Introduction
In recent years, a great emphasis has been placed on the applications of materials in biomedical fields [1,2].Metal and metal oxide biomaterials have been, and are mainly used, to manufacture medical devices for replacing rough tissues such as artificial hip joints, bone plates, and dental implants because they are very reliable in terms of mechanical performance [3,4].Titanium and its alloys are of high interest as implantable materials due to their superior resistance to corrosion, good mechanical properties, remarkably high specific resistance, low elasticity modulus, and excellent biocompatibility in comparison with other competing biomaterials, such as stainless steel, CoCr alloys, and even nitinol alloys [5][6][7].The high corrosion resistance of titanium and its alloys is due to the formation of a stable, protective, and adherent passive oxide film on their surface.This film forms instantly when the titanium surface is exposed to air or humidity [8].The nature, composition, and thickness of the protective oxide layers formed on titanium and titanium alloys depend on the environmental conditions.Usually, the composition of the protective oxide film is based on TiO 2 , Ti 2 O 3, or TiO [9][10][11][12], the thicknesses of the passive films formed on these materials are about 3-10 nm [9][10][11][12][13][14][15][16], and are made of metal oxides (ceramic films).However, the titanium's passive state is not completely stable, and in certain corrosive environments, such as human body fluids, it was found that localized breakdown of the passive oxide film occurs at a microscopic scale.Consequently, a strong destructive attack can occur at the surface of the material due to its reaction to the hostile operating environment [17,18].Any Coatings 2023, 13, 614 2 of 15 deficiency in the performance of the TiO 2 film on the titanium surface is very detrimental to the corrosion behavior as well as other characteristics required for implantation and biocompatibility [17,18].More than that, the native TiO 2 film spontaneously grown on the surface of the titanium and its alloys, does not have strong bioactivity for the osseointegration of the bone, making it necessary to modify the surface of the titanium and its alloys.Therefore, the surface modification of titanium and its alloys is often used to improve their mechanical, biological, and chemical properties [19].
Thus, engineers and scientists searched for various techniques to increase the biocompatibility and the corrosion behavior of metallic implants to better adapt them to the implantation environment [19].Therefore, surface modification became one of the most attractive branches in the field of biomaterials research.Surface modification techniques are the most viable alternatives to increase corrosion resistance and the properties associated with biomaterial surfaces [18].Superior behavior to corrosion, improved resistance to wear, better osseointegration, high biocompatibility, and perfect aesthetics are the most important characteristics that can be obtained through surface modification [19].Anodic oxidation of titanium is considered an effective coating technology for bioimplant surfaces.The creation of biofunctional films of titanium oxide through dielectric breakdown provides improved adhesion of the film to the substrate and helps in obtaining high-quality films with porous or nontubular structures by varying electrolytes, temperature, alloying elements, voltage, current density, and time.Furthermore, the anodic oxidation process can increase the thickness of the native oxide layer on the surfaces of the titanium materials to develop corrosion resistance and decrease the release of metal ions with toxic properties [20][21][22][23][24][25][26][27][28][29][30].Various studies are reported in the specialized literature that deal with the subject of modifying the surfaces of titanium alloys by various methods to increase corrosion resistance in different environments that simulate the fluids of the human body [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34].In-depth research to study the effect of time on the degradation by the electrochemical corrosion of untreated grade 5 Ti6Al4V alloy compared to electrochemically oxidized grade 5 Ti6Al4V alloy has not been reported in specialized literature.Additionally, the effect of electrochemically obtained nanoporous TiO 2 films on the corrosion resistance of titanium alloys in biological solutions has also not been studied.
The purpose of this research was to produce a nanoporous layer of TiO 2 on the surface of grade 5 Ti6Al4V alloy using the electrochemical oxidation method, which imposes a high voltage for a short period of time to make the process of forming the nanoporous titanium oxide film more efficient.The beneficial effect of the oxidation and controlled formation of the TiO 2 film on the implant alloy is proven by the comparative evaluation of the degradation over time through the corrosion of both the untreated alloy and the alloy with an electrochemically formed and controlled TiO 2 film in Hank's solution, which simulates the most corrosive biological fluid, blood.

The Electrochemical Formation of the Nanoporous TiO 2 Surface Layer on Ti6Al4V Grade 5 Alloy
Ti6Al4V grade 5 alloy (Goodfellow SARL, Lille, France) has a chemical composition of N-0.003%, Al-6.01%,C-0.008%, V-3.83%, H-0.002%, Fe-0.083%,O-0.088%, and Ti-balance [26] and is used as a substrate for the growth of the nanoporous film of titanium oxide (TiO 2 ) through the electrochemical oxidation method.To obtain nanoporous layers of titanium oxide on the surface of the grade 5 Ti6Al4V alloy, different electrolytes based on sulfuric acid were tested.The most reproducible results were obtained using a 1 M H 2 SO 4 electrolyte.The electrochemical oxidation process takes place in a classical electrochemical cell composed of two electrodes, where the grade 5 Ti6Al4V plate with an active surface of 10 cm 2 is used as the anode and the cathode is a grade 5 Ti6Al4V plate with an active surface of 54 cm 2 .Before each experiment, the anode and cathode were cleaned with alcohol for 5 min in an ultrasonic bath, then rinsed with distilled water.The electrochemical oxidation process to obtain nanoporous TiO 2 film on Ti6Al4V grad 5 alloy take place in an electrochemical cell with specific electrodes as: (i) anode -the titanium alloy to be oxidized, (ii) cathode an inert material that could be another plate of the same titanium alloy having a higher surface area and the oxidizing electrolyte.The two Ti6Al4V grade 5 plates are immersed in 450 mL of H 2 SO 4 1 M electrolyte solution with a pH of 0.41, at a distance of 5 cm from each other.With the help of two electrical contacts, the electrodes are connected to a direct current voltage source TDK LAMBDA GEN 300-8 (0-300V).Several values of the potential or voltage (from 150, 200 and 250 V) were imposed on the electrochemical oxidation process and several oxidation times (0.5 to 3 min) were experimented.The most effective parameters were found to be the voltage of 200 V and the time of 3 min.All electrochemical oxidation tests are done at room temperature 22 ± 1 • C and repeated 6 times to check data reproducibility.Figure 1 presents the electrochemical oxidation process to obtain nanoporous TiO 2 film on grade 5 Ti6Al4V alloy showing the electrochemical cell with specific electrodes, the schematic nanoporous TiO 2 film obtained on titanium alloy, the measured roughness of the nanoporous TiO 2 film approximated as nanopores depth, and the SEM subphase morphology proving the nanoporous surface of TiO 2 film obtained and some measurement of nanopore diameter sizes.each experiment, the anode and cathode were cleaned with alcohol for 5 min in an ultrasonic bath, then rinsed with distilled water.The electrochemical oxidation process to obtain nanoporous TiO2 film on Ti6Al4V grad 5 alloy take place in an electrochemical cell with specific electrodes as: (i) anode -the titanium alloy to be oxidized, (ii) cathode an inert material that could be another plate of the same titanium alloy having a higher surface area and the oxidizing electrolyte.The two Ti6Al4V grade 5 plates are immersed in 450 mL of H2SO4 1 M electrolyte solution with a pH of 0.41, at a distance of 5 cm from each other.With the help of two electrical contacts, the electrodes are connected to a direct current voltage source TDK LAMBDA GEN 300-8 (0-300V).Several values of the potential or voltage (from 150, 200 and 250 V) were imposed on the electrochemical oxidation process and several oxidation times (0.5 to 3 min) were experimented.The most effective parameters were found to be the voltage of 200 V and the time of 3 min.All electrochemical oxidation tests are done at room temperature 22 ± 1 °C and repeated 6 times to check data reproducibility.Figure 1 presents the electrochemical oxidation process to obtain nanoporous TiO2 film on grade 5 Ti6Al4V alloy showing the electrochemical cell with specific electrodes, the schematic nanoporous TiO2 film obtained on titanium alloy, the measured roughness of the nanoporous TiO2 film approximated as nanopores depth, and the SEM subphase morphology proving the nanoporous surface of TiO2 film obtained and some measurement of nanopore diameter sizes.

Evaluation of the Corrosion Resistance of the Untreated Ti6Al4V Grade 5 Alloy and the Treated Alloy with the Electrochemically Formed Nanoporous TiO2 Surface Film
A PGZ 100 electrochemical station connected to a PC equipped with VoltaMaster4 software was used to study the corrosion behavior of the untreated grade 5 Ti6Al4V alloy and the electrochemically formed nanoporous TiO2 film.The experimental setup consisted of an electrochemical cell especially made from double-walled glass to maintain the electrolyte at a constant temperature.The cell is composed of three electrodes: (i) the working electrode-WE, which is the untreated titanium alloy or the nanoporous oxide film, (ii) the reference electrode-RE, which is of Ag/AgCl with saturated KCl solution and with a potential constant of +199 mV vs. SHE, and (iii) the auxiliary electrode-AE made of a network of electrochemically inert Pt-Rh material.To carry out the corrosion tests, the untreated grade 5 Ti6Al4V alloy and electrochemically oxidized Ti6Al4V alloy are connected to a

Evaluation of the Corrosion Resistance of the Untreated Ti6Al4V Grade 5 Alloy and the Treated Alloy with the Electrochemically Formed Nanoporous TiO 2 Surface Film
A PGZ 100 electrochemical station connected to a PC equipped with VoltaMaster4 software was used to study the corrosion behavior of the untreated grade 5 Ti6Al4V alloy and the electrochemically formed nanoporous TiO 2 film.The experimental setup consisted of an electrochemical cell especially made from double-walled glass to maintain the electrolyte at a constant temperature.The cell is composed of three electrodes: (i) the working electrode-WE, which is the untreated titanium alloy or the nanoporous oxide film, (ii) the reference electrode-RE, which is of Ag/AgCl with saturated KCl solution and with a potential constant of +199 mV vs. SHE, and (iii) the auxiliary electrode-AE made of a network of electrochemically inert Pt-Rh material.To carry out the corrosion tests, the untreated grade 5 Ti6Al4V alloy and electrochemically oxidized Ti6Al4V alloy are connected to a copper wire and then insulated with epoxy resin to obtain a well-defined active surface of 4 cm 2 .Before each corrosion evaluation experiment, both the analyzed sample and the counter electrode were washed with distilled water.The volume of electrolyte, that is Hank's biological solution, which simulates blood and is used for each experiment, was kept at a constant 250 mL.Hank's biological solution was prepared with distilled water by dissolving the following reagents: NaCl-8.8g and C 6 H 12 O 6 -1 g•L −1 .The physic- ochemical parameters of Hank's biological solution were measured using a Sension+ multiparameter.The resulting values were pH = 7.41, conductivity = 14.6 mS/cm, and salinity = 8.4 ppt.The tests were performed at human body temperature, 37 ± 1 • C, with the help of a thermostatic solution system.All studied samples were repeated six times to verify the reproducibility of the experimental data.From the moment the sample was immersed in Hank's biological solution, sequences of electrochemical measurements of open circuit potential (OCP), polarization resistance (R p ), and corrosion rate (V corr ) were performed over 81 h, to highlight the effect of immersion time on the resistance of the respective surfaces in the biological solution.The experimental protocol for the comparative corrosion investigation is schematized in Figure 2.
Coatings 2023, 13, x FOR PEER REVIEW 4 of 16 copper wire and then insulated with epoxy resin to obtain a well-defined active surface of 4 cm 2 .Before each corrosion evaluation experiment, both the analyzed sample and the counter electrode were washed with distilled water.The volume of electrolyte, that is Hank's biological solution, which simulates blood and is used for each experiment, was kept at a constant 250 mL.Hank's biological solution was prepared with distilled water by dissolving the following reagents: NaCl-8.The electrochemical measurements were carried out in the following steps: (1) At t1 = 0 (immersion): OCP1 lasting 360 min.
(3) At 709 min: OCP2, with a duration of 720 min (12 h).The total duration of sample immersion in the biological solution was selected as the average duration of a perturbation process in the human body, which is considered to be approximately 3 days.Each Rp-Vcorr measurement sequence contains 100 value points calculated from as many linear polarization curves, using the Stern Geary equation and Tafel slopes, as shown in Equations ( 1)-(3): The electrochemical measurements were carried out in the following steps: (1) At t1 = 0 (immersion): OCP 1 lasting 360 min.
(5) At 1578 min: OCP 3 , with a duration of 720 min (12 h). ( 6) At 2298 min: R p3 -V corr3 , with a duration of 149 min.The total duration of sample immersion in the biological solution was selected as the average duration of a perturbation process in the human body, which is considered to be approximately 3 days.Each R p -V corr measurement sequence contains 100 value points calculated from as many linear polarization curves, using the Stern Geary equation and Tafel slopes, as shown in Equations ( 1)-( 3): Coatings 2023, 13, 614 where i corr is the corrosion current density, B is the specific constant for each system-material/environment, b a is the anodic Tafel slope, and b c is the cathodic Tafel slope.The morphological characterization of the untreated grade 5 Ti6Al4V alloy and the electrochemically formed nanoporous TiO 2 film was carried out using an FEI QUANTA 200 scanning electron microscope (ThermoFisscher, Carston, UK).The connection of a dispersed X-ray analyzer to the SEM allowed an elemental (compositional) analysis of the samples to be made, which was studied with the help of the EDAX GENESIS program.Because nanoporous films of titanium oxide are electrical insulators, they were coated with a 5 nm thick layer of gold to prevent them from being charged with electricity.The diameters of the formed nanopores were measured directly from the SEM micrographs.The morphological and compositional analysis (SEM-EDX) was carried out using the equipment described in chapter 2.3 of this research work.Figure 3a-c shows the EDX spectrum in layer (a), the EDX elemental analysis in weight percent in layer (b), and the SEM surface morphology of the untreated grade 5 Ti6Al4V alloy in layer (c).
where icorr is the corrosion current density, B is the specific constant for each system-material/environment, ba is the anodic Tafel slope, and bc is the cathodic Tafel slope.

Characterization of the Untreated Grade 5 Ti6Al4V Alloy and the Treated Alloy with the Electrochemically Formed Nanoporous TiO2 Surface Film
The morphological characterization of the untreated grade 5 Ti6Al4V alloy and the electrochemically formed nanoporous TiO2 film was carried out using an FEI QUANTA 200 scanning electron microscope (ThermoFisscher, Carston, UK).The connection of a dispersed X-ray analyzer to the SEM allowed an elemental (compositional) analysis of the samples to be made, which was studied with the help of the EDAX GENESIS program.Because nanoporous films of titanium oxide are electrical insulators, they were coated with a 5 nm thick layer of gold to prevent them from being charged with electricity.The diameters of the formed nanopores were measured directly from the SEM micrographs.

Untreated Ti6Al4V Grade 5 Alloy
The morphological and compositional analysis (SEM-EDX) was carried out using the equipment described in chapter 2.3 of this research work.Figure 3a-c   For the EDX analysis of the untreated grade 5 Ti6Al4V alloy implant, Figure 3a,b indicates the presence of the main elements of the studied alloy: Ti, Al, and V, with their mass For the EDX analysis of the untreated grade 5 Ti6Al4V alloy implant, Figure 3a,b indicates the presence of the main elements of the studied alloy: Ti, Al, and V, with their mass percentage values Ti = 90.26%,Al = 5.36%, and V = 3.48%.Oxygen, in a percentage of about 0.90 wt%, is present because a thin layer of titanium oxide (TiO 2 ) is natively formed on the surface of the untreated alloy, which is calculated at a percentage of 2.24 wt% TiO 2 .The percentage of titanium oxide (TiO 2 ) formed on the surface of the grade 5 Ti6Al4V alloy was determined from the transformation of the mass percentage of oxygen from the general analysis of the molecular mass of TiO 2 (79.866 g/mol).In Figure 3c, it can be seen from the surface morphology that the surface of the untreated grade 5 Ti6Al4V alloy is relatively smooth and has no surface defects.surface of the untreated alloy, which is calculated at a percentage of 2.24 wt% TiO2.The per-centage of titanium oxide (TiO2) formed on the surface of the grade 5 Ti6Al4V alloy was determined from the transformation of the mass percentage of oxygen from the general analysis of the molecular mass of TiO2 (79.866 g/mol).In Figure 3c, it can be seen from the surface morphology that the surface of the untreated grade 5 Ti6Al4V alloy is relatively smooth and has no surface defects.The EDX analysis results shown in Figure 4a show the presence of the main elements observed in the electrochemically oxidized biomaterial studied, i.e., Ti, Al, and V; their mass percentage values are shown in Figure 4b, which are different from the untreated alloy, Ti = 59.64 wt%, Al = 3.95 wt%, V = 1.32 wt%, and oxygen, which increased to about O = 35.00wt%.

Titanium Oxide Film Electrochemically Formed on the Surface of the Ti6Al4V Alloy
At the same time, it was observed that at the oxidation time of 3 min, there was an increase in the percentage of oxygen to about O = 35.00wt% compared to the untreated grade 5 Ti6Al4V alloy, which confirms the increase in the titanium oxide film to about TiO2 = 87.35wt% on the sample's surface.
From the analysis results shown in Figures 3 and 4, it can be observed that the surface morphology of the grade 5 Ti6Al4V alloy electrochemically oxidized at 200 V for 3 min changes compared to the surface of the untreated grade 5 Ti6Al4V alloy.Before the anodic oxidation process, the surface of the untreated grade 5 Ti6Al4V alloy shows a thin native oxide layer that instantly forms on the surface of the untreated alloy in air.On the other hand, the electrochemically oxidized samples at 200 V show the growth of the titanium oxide film on the surface of the electrochemically oxidized grade 5 Ti6Al4V alloy.
The formation of nanoporous titanium oxide (TiO2) depends on the electrochemical oxidation time of the grade 5 titanium alloy and is exemplified in Figure 5.To support this The EDX analysis results shown in Figure 4a show the presence of the main elements observed in the electrochemically oxidized biomaterial studied, i.e., Ti, Al, and V; their mass percentage values are shown in Figure 4b, which are different from the untreated alloy, Ti = 59.64 wt%, Al = 3.95 wt%, V = 1.32 wt%, and oxygen, which increased to about O = 35.00wt%.
At the same time, it was observed that at the oxidation time of 3 min, there was an increase in the percentage of oxygen to about O = 35.00wt% compared to the untreated grade 5 Ti6Al4V alloy, which confirms the increase in the titanium oxide film to about TiO 2 = 87.35wt% on the sample's surface.
From the analysis results shown in Figures 3 and 4, it can be observed that the surface morphology of the grade 5 Ti6Al4V alloy electrochemically oxidized at 200 V for 3 min changes compared to the surface of the untreated grade 5 Ti6Al4V alloy.Before the anodic oxidation process, the surface of the untreated grade 5 Ti6Al4V alloy shows a thin native oxide layer that instantly forms on the surface of the untreated alloy in air.On the other hand, the electrochemically oxidized samples at 200 V show the growth of the titanium oxide film on the surface of the electrochemically oxidized grade 5 Ti6Al4V alloy.
The formation of nanoporous titanium oxide (TiO 2 ) depends on the electrochemical oxidation time of the grade 5 titanium alloy and is exemplified in Figure 5.To support this statement, Figure 5 shows the surface morphologies of the electrochemically oxidized grade 5 Ti6Al4V samples: (a) at 200 V for 1 min, (b) at 200 V for 2 min, and (c) at 200 V for 3 min at a magnitude of 50,000×, with the measurement of the pore diameter size directly from the SEM results.
From the analysis of Figure 5, a decrease in the size of the nanopores can be observed with an increase in the electrochemical oxidation process time.Thus, the average size of the nanopores at 1 min decreases from 80.43 to 58.6 nm at the oxidation time of 3 min.
To better highlight the abovementioned results, Figure 6 presents the comparative evolution of the obtained average TiO 2 nanopore diameter values corresponding to grade 5 Ti6Al4V samples electrochemically oxidized at 200 V for 1, 2, and 3 min.From the analysis of Figure 5, a decrease in the size of the nanopores can be observed with an increase in the electrochemical oxidation process time.Thus, the average size of the nanopores at 1 min decreases from 80.43 to 58.6 nm at the oxidation time of 3 min.
To better highlight the abovementioned results, Figure 6 presents the comparative evolution of the obtained average TiO2 nanopore diameter values corresponding to grade 5 Ti6Al4V samples electrochemically oxidized at 200 V for 1, 2, and 3 min.A decrease in the size of the nanopores formed through the anodic oxidation process with increasing time on another alloy has been also reported in the specialized literature by other authors [25].
The size of the nanopores and their uniformity for the three-minute electrochemical oxidation parameters led to the comparative evaluation of the resistance to corrosion degradation of the untreated titanium alloy with titanium oxide (TiO2) film in Hank's A decrease in the size of the nanopores formed through the anodic oxidation process with increasing time on another alloy has been also reported in the specialized literature by other authors [25].The size of the nanopores and their uniformity for the three-minute electrochemical oxidation parameters led to the comparative evaluation of the resistance to corrosion degradation of the untreated titanium alloy with titanium oxide (TiO 2 ) film in Hank's solution, which simulates the corrosive aggressiveness of biological fluids from the human body.The open circuit potential (OCP) was measured at different time intervals for the treated and untreated Ti6Al4V alloy in Hank's solution over an 80 h period.This was carried out to especially monitor the time necessary for the tested surfaces to reach a stationary state since they are intended to be used as implants.The occurrence of passivity is generally due to the formation of a spontaneous passive film on metal surfaces that acts as a kinetic barrier against corrosion, thereby significantly reducing the corrosion rate.Another reason for monitoring the open circuit potential over a longer period was to evaluate the stability and the reactivity of the tested surfaces over time.The chemical properties of the oxide film play an important role in the biocompatibility of implants with surrounding tissues.The chemical interaction of a metallic material with bodily fluids is important for the stability of an implant in the human body [21][22][23][24][25][26][27].To simulate human body conditions, the corrosion tests were performed in Hank's physiological solution.The open circuit potential during the immersion period in Hank's biological solution for the untreated Ti6Al4V alloy (1) and the titanium oxide film electrochemically formed on it (2) for 75.5 h are presented in Figure 7.The open circuit potential difference between the two surfaces at the end of monitoring is even greater, ∆E = 390 mV.This trend confirms the improvement of the grade 5 Ti6Al4V alloy surface through controlled electrochemical oxidation, which gives it this fine film of nanoporous titanium oxide (TiO 2 ) with qualities of nobler behavior in biological solutions.
For the untreated alloy, it was observed that the value of the open circuit potential shows a continual increase for 30 h after which the value stabilizes at around 424 mV vs. Ag/AgCl.The difference in OCP for the untreated titanium alloy from the beginning of immersion to the end of the 75.6 h immersion period was 646 mV, while for the electrochemically oxidized titanium alloy, the difference between immersion start and finish OCP value, ∆E, was 701 mV.This behavior can be explained by the formation of a more stable and protective thin oxide film on the surface of the electrochemically oxidized alloy, which acts as a protective film to prevent the release of metal ions into the biological environment.The trend toward higher values in the case of different types of materials obtained by electrochemical oxidation compared to different untreated alloys has also been observed by other authors in the specialized literature [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35].The comparative evolution of the polarization resistance for the untreated grade 5 titanium alloy and for the electrochemically oxidized titanium alloy with the nanoporous film of titanium oxide (TiO 2 ) is presented in Figure 8a-c at three time periods in the total sample immersion period in Hank's biological solution.

Evolution of the Polarization Resistance (Rp) vs. Time in Hank's Biological Solution for the Untreated Alloy and the Treated Alloy with the Electrochemically Formed Nanoporous TiO2 Surface Film
After immersing the untreated and electrochemically oxidized grade 5 titanium alloy samples to form the protective nanoporous film of titanium oxide ( TiO2), the open circuit potential was recorded in several sequences for 75.6 h.Between the open circuit potential measurement sequences at the start and end of the experiment, a total of seven recordings of 100 points of polarization resistance were made and calculated from as many linear polarization curves by using the Stern Geary equation and Tafel slopes (as explained in chapter 2, Materials and Methods).
The comparative evolution of the polarization resistance for the untreated grade 5 titanium alloy and for the electrochemically oxidized titanium alloy with the nanoporous film of titanium oxide (TiO2) is presented in Figure 8a-c   From the analysis of Figure 8, it can be seen that the values of the polarization resistance for the untreated grade 5 Ti6Al4V alloy immersed in Hank's biological solution (Figure 8(1)) are lower than the values of the polarization resistance recorded for the treated From the analysis of Figure 8, it can be seen that the values of the polarization resistance for the untreated grade 5 Ti6Al4V alloy immersed in Hank's biological solution (Figure 8(1)) are lower than the values of the polarization resistance recorded for the treated electrochemically controlled oxidized Ti6Al4V alloy (Figure 8(2)) for all measurement periods during immersion.After 6 h of immersion, the polarization resistance (R p ) of the untreated titanium alloy showed a slight increase from immersion and stabilized at a value of 1.95 Mohm•cm 2 , while the polarization resistance of the electrochemically oxidized titanium alloy showed constant values with a stabilization value of 3.67 Mohm•cm 2 at the end of the first measurement period.This is higher than the polarization resistance of the untreated alloy.
After 38 h of immersion, the values of the polarization resistance of the untreated grade 5 titanium alloy show a slight decrease and oscillations in Figure 8b, so that at the end of this measurement period, the R p value was 1.78 Mohm•cm 2 , lower than in the first period measurement.The values of the polarization resistance of the electrochemically oxidized grade 5 titanium alloy after 38 h were slightly higher than those in the first measurement period, with an R p value of 4.25 Mohm•cm 2 toward the end of the measurement period.Thus, even after 38 h, the R p for the electrochemically oxidized titanium alloy is higher than the R p for the untreated titanium alloy by approximately three times, which confirms the effectiveness of the electrochemical oxidation process in improving the resistance of the implant to the aggressive action of biological fluids in the human body.
After 75.5 h of immersion in Hank's biological solution, the same trend of the polarization resistance values was maintained for the two surfaces of the immersed untreated grade 5 Ti6Al4V alloy and the electrochemically oxidized grade 5 Ti6Al4V alloy.The value of the polarization resistance (R p ) of the untreated alloy slightly decreases toward the end of the immersion period to R p = 1.59 Mohm•cm 2 , while the value of the polarization resistance of the electrochemically oxidized Ti6Al4V alloy slightly increases, reaching a value of R p = 7.56 Mohm•cm 2 at the end of the immersion period, a value that was approximately five times higher than that of the untreated alloy.To better highlight the obtained results, Figure 9 shows the average values of all of the polarization resistance values recorded at different immersion periods for the untreated grade 5 Ti6Al4V alloy compared to the electrochemically oxidized grade 5 Ti6Al4V alloy.for the untreated titanium alloy by approximately three times, which confirms the effectiveness of the electrochemical oxidation process in improving the resistance of the implant to the aggressive action of biological fluids in the human body.After 75.5 h of immersion in Hank's biological solution, the same trend of the polarization resistance values was maintained for the two surfaces of the immersed untreated grade 5 Ti6Al4V alloy and the electrochemically oxidized grade 5 Ti6Al4V alloy.The value of the polarization resistance (Rp) of the untreated alloy slightly decreases toward the end of the immersion period to Rp = 1.59 Mohm•cm 2 , while the value of the polarization resistance of the electrochemically oxidized Ti6Al4V alloy slightly increases, reaching a value of Rp = 7.56 Mohm•cm 2 at the end of the immersion period, a value that was approximately five times higher than that of the untreated alloy.To better highlight the obtained results, Figure 9 shows the average values of all of the polarization resistance values recorded at different immersion periods for the untreated grade 5 Ti6Al4V alloy compared to the electrochemically oxidized grade 5 Ti6Al4V alloy.From the analysis of Figure 9, it can be observed that in the case of the electrochemically oxidized grade 5 Ti6Al4V alloy immersed in Hank's solution, the values of the polarization resistance were higher compared to the untreated grade 5 Ti6Al4V alloy at all studied times.Additionally, in the case of the electrochemically oxidized grade 5 Ti6Al4V alloy, an increase in the values of the polarization resistance was observed with increasing immersion time.At the first measurement, the electrochemically oxidized alloy had an Rp1 value of 3.70 Mohm•cm 2 ; at the end of the 75.5 h of monitoring, the polarization resistance had reached an Rp7 value of 7.15 Mohm•cm 2 , a behavior that demonstrates the effectiveness of From the analysis of Figure 9, it can be observed that in the case of the electrochemically oxidized grade 5 Ti6Al4V alloy immersed in Hank's solution, the values of the polarization resistance were higher compared to the untreated grade 5 Ti6Al4V alloy at all studied times.Additionally, in the case of the electrochemically oxidized grade 5 Ti6Al4V alloy, an increase in the values of the polarization resistance was observed with increasing immersion time.At the first measurement, the electrochemically oxidized alloy had an R p1 value of 3.70 Mohm•cm 2 ; at the end of the 75.5 h of monitoring, the polarization resistance had reached an R p7 value of 7.15 Mohm•cm 2 , a behavior that demonstrates the effectiveness of the electrochemical oxidation process of the titanium alloy for improving the resistance of the implant to the aggressive action of biological fluids in the human body.
The increase in the polarization resistance values in the case of the nanoporous TiO 2 film electrochemically formed on titanium alloy was due to the presence of the TiO 2 layer that provided superior insulating properties through its electrochemically controlled formation.This behavior has also been observed in the specialized literature by other authors with other biomaterials [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34].For the electrochemically oxidized grade 5 Ti6Al4V alloy, an increase in the polarization resistance values was observed with increasing immersion time; for the untreated grade 5 Ti6Al4V alloy, it was observed that the polarization resistance values showed a slight decrease after 38 h of immersion in comparison to the value obtained after 360 min, and a slight increase at the end of the immersion period.This behavior was due to the nonuniformity of the natively formed titanium oxide film on the surface of the untreated alloy, which leads to a much faster degradation process of the material in comparison to the electrochemically oxidized titanium alloy having the nanoporous TiO 2 film.

Time Evolution of the Corrosion Rate (V corr ) in Hank's Biological Solution of the Untreated Alloy and the Treated Alloy with the Electrochemically Formed Nanoporous TiO 2 Surface Film
Polarization resistance is the only corrosion monitoring method that makes it possible to measure corrosion rates (expressed as thickness loss over time) directly, in real time.The corrosion current determined using this method represents the current that occurs at the corrosive metal/medium interface when the metal is immersed in the solution.
The variation in the corrosion rate expressed as a penetration index for the untreated alloy and for the alloy oxidized with titanium oxide (TiO 2 ) film is shown in Figure 10a-c at three times during the sample immersion period in Hank's biological solution, and in Figure 11 as the mean value across all studied times.
From the analysis of Figure 10, it can be seen that the corrosion rate values (V corr ) for the untreated grade 5 Ti6Al4V alloy immersed in Hank's biological solution, Figure 10(1), are higher than the corrosion rate values recorded for the treated grade 5 Ti6Al4V alloy electrochemically oxidized Ti6Al4V alloy, Figure 10(2), for all measurement periods during immersion.Thus, after 6 h of immersion, in Figure 10a, the corrosion rate (V corr ) of the untreated titanium alloy shows a slight decrease from immersion with stabilization at a value of 0.032 µm/year, while the corrosion rate value for the treated alloy shows constant values with a stabilization value of 0.023 µm/year at the end of the first measurement period, a corrosion rate that was approximately two times lower than for the untreated alloy.
After 38 h of immersion, Figure 10b, the corrosion rate for the untreated grade 5 Ti6Al4V alloy showed an overall slight increase at the end of this measurement period, with a V corr value of 0.036 µm/year, higher than at the end of the first measurement period.The corrosion rate value for the electrochemically oxidized grade 5 Ti6Al4V alloy after 38 h was slightly lower than at the end of the first measurement period, with a V corr value of 0.018 µm/year.Thus, even after 38 h, the V corr for the electrochemically oxidized titanium alloy was lower than the V corr for the untreated titanium alloy by approximately two times, which confirms the efficiency of the electrochemical oxidation process of the titanium alloy for improving the resistance of the implant to the aggressive action of biological fluids in the human body.
After 75.5 h of immersion in Hank's biological solution, the same trend for the corrosion rate values was maintained for the treated and untreated alloy surfaces.The value of the corrosion rate, V corr , for the untreated alloy decreased slightly toward the end of the immersion period to V corr = 0.030 µm/year, while the value of the corrosion rate of the electrochemically oxidized grade 5 Ti6Al4V alloy slightly decreased, reaching the end of the immersion period with a V corr value of 0.001 µm/year, thus being approximately 30 times lower than the untreated V corr level.

Alloy and the Treated Alloy with the Electrochemically Formed Nanoporous TiO2 Surface Film
Polarization resistance is the only corrosion monitoring method that makes it possible to measure corrosion rates (expressed as thickness loss over time) directly, in real time.The corrosion current determined using this method represents the current that occurs at the corrosive metal/medium interface when the metal is immersed in the solution.
The variation in the corrosion rate expressed as a penetration index for the untreated alloy and for the alloy oxidized with titanium oxide (TiO2) film is shown in Figure 10a-c at three times during the sample immersion period in Hank's biological solution, and in Figure 11 as the mean value across all studied times.From the analysis of Figure 10, it can be seen that the corrosion rate values (Vcorr) for the untreated grade 5 Ti6Al4V alloy immersed in Hank's biological solution, Figure 10(1), are higher than the corrosion rate values recorded for the treated grade 5 Ti6Al4V alloy electrochemically oxidized Ti6Al4V alloy, Figure 10(2), for all measurement periods during immersion.Thus, after 6 h of immersion, in Figure 10a, the corrosion rate (Vcorr) of the untreated titanium alloy shows a slight decrease from immersion with stabilization at a value of 0.032 µm/year, while the corrosion rate value for the treated alloy shows constant values with a stabilization value of 0.023 µm/year at the end of the first measurement period, a corrosion rate that was approximately two times lower than for the untreated alloy.
After 38 h of immersion, Figure 10b, the corrosion rate for the untreated grade 5 Ti6Al4V alloy showed an overall slight increase at the end of this measurement period, with a Vcorr value of 0.036 µm/year, higher than at the end of the first measurement period.The corrosion rate value for the electrochemically oxidized grade 5 Ti6Al4V alloy after 38 h was slightly lower than at the end of the first measurement period, with a Vcorr value of 0.018 µm/year.Thus, even after 38 h, the Vcorr for the electrochemically oxidized titanium alloy was lower than the Vcorr for the untreated titanium alloy by approximately two times, which confirms the efficiency of the electrochemical oxidation process of the titanium alloy for improving the resistance of the implant to the aggressive action of biological fluids in the human body.After 75.5 h of immersion in Hank's biological solution, the same trend for the corrosion rate values was maintained for the treated and untreated alloy surfaces.The value of the corrosion rate, Vcorr, for the untreated alloy decreased slightly toward the end of the immersion period to Vcorr = 0.030 µm/year, while the value of the corrosion rate of the electrochemically oxidized grade 5 Ti6Al4V alloy slightly decreased, reaching the end of the immersion period with a Vcorr value of 0.001 µm/year, thus being approximately 30 times lower than the untreated Vcorr level.
From the analysis of Figure 11, it can be seen that in the case of the electrochemically oxidized nanoporous TiO2 film-treated alloy immersed in Hank's blood-simulating solution, the corrosion rate values were lower compared to the untreated grade 5 Ti6Al4V alloy at all studied times.High polarization resistance means a low corrosion rate, a behavior that is desired for all types of biomaterials that are to be inserted into the human body.For the electrochemically oxidized grade 5 Ti6Al4V alloy, a decrease in the corrosion rate values was observed with increasing immersion time.
If at the first measurement, the electrochemically oxidized alloy has an average value of Vcorr1 = 0.023 µm/year; at the end of the 80 h monitoring period, Vcorr reached an average value of Vcorr7 = 0.005 µm/year.This is a behavior that demonstrates the efficiency of the electrochemical oxidation process of the titanium alloy for improving the resistance of the implant against the aggressive action of the biological fluids in the human body.For the untreated grade 5 Ti6Al4V alloy, it was observed that the average values of the corrosion rate show a slight increase after 38 h of immersion compared to the value obtained after 360 min, and a slight decrease at the end of the immersion period (80 h).From the analysis of Figure 11, it can be seen that in the case of the electrochemically oxidized nanoporous TiO 2 film-treated alloy immersed in Hank's blood-simulating solution, the corrosion rate values were lower compared to the untreated grade 5 Ti6Al4V alloy at all studied times.High polarization resistance means a low corrosion rate, a behavior that is desired for all types of biomaterials that are to be inserted into the human body.
For the electrochemically oxidized grade 5 Ti6Al4V alloy, a decrease in the corrosion rate values was observed with increasing immersion time.

Figure 1 .
Figure 1.Presentation of the electrochemical oxidation process to obtain the nanoporous TiO2 film on grade 5 Ti6Al4V alloy: (a) electrochemical cell showing the electrodes and reactions at the electrodes in the anodizing electrolyte; (b) a schematic of nanoporous TiO2 on a titanium alloy surface; (c) the Ti6Al4V bulk alloy; (d) the measured roughness of TiO2 nanoporous film approximated as nanopore depth; and (e) SEM surface morphology of TiO2 nanoporous film showing the measurements of nanopore diameters.

Figure 1 .
Figure 1.Presentation of the electrochemical oxidation process to obtain the nanoporous TiO 2 film on grade 5 Ti6Al4V alloy: (a) electrochemical cell showing the electrodes and reactions at the electrodes in the anodizing electrolyte; (b) a schematic of nanoporous TiO 2 on a titanium alloy surface; (c) the Ti6Al4V bulk alloy; (d) the measured roughness of TiO 2 nanoporous film approximated as nanopore depth; and (e) SEM surface morphology of TiO 2 nanoporous film showing the measurements of nanopore diameters.
and C6H12O6-1 g•L −1 .The physicochemical parameters of Hank's biological solution were measured using a Sension+ multiparameter.The resulting values were pH = 7.41, conductivity = 14.6 mS/cm, and salinity = 8.4 ppt.The tests were performed at human body temperature, 37 ± 1 °C, with the help of a thermostatic solution system.All studied samples were repeated six times to verify the reproducibility of the experimental data.From the moment the sample was immersed in Hank's biological solution, sequences of electrochemical measurements of open circuit potential (OCP), polarization resistance (Rp), and corrosion rate (Vcorr) were performed over 81 h, to highlight the effect of immersion time on the resistance of the respective surfaces in the biological solution.The experimental protocol for the comparative corrosion investigation is schematized in Figure2.

Figure 2 .
Figure 2. Experimental protocol summarizing the sequential electrochemical measurements during the total immersion period in biological solution.

Figure 2 .
Figure 2. Experimental protocol summarizing the sequential electrochemical measurements during the total immersion period in biological solution.

2. 3 .
Characterization of the Untreated Grade 5 Ti6Al4V Alloy and the Treated Alloy with the Electrochemically Formed Nanoporous TiO 2 Surface Film 3. Results 3.1.Morphological and Compositional Analysis of the Untreated Titanium Alloy Surfaces and Electrochemically Formed TiO 2 Films through Electron Microscopy (SEM-EDX) 3.1.1.Untreated Ti6Al4V Grade 5 Alloy shows the EDX spectrum in layer (a), the EDX elemental analysis in weight percent in layer (b), and the SEM surface morphology of the untreated grade 5 Ti6Al4V alloy in layer (c).
Figure 4a-d shows the EDX spectrum in layer (a), the EDX elemental analysis in weight percent in layer (b), and the SEM surface morphology of the grade 5 Ti6Al4V implant alloy electrochemically oxidized at 200 V and at a time of 3 min in layers (c) and (d).As specified in chapter 2, Materials and Methods, the most effective and relevant values for obtaining titanium oxide films on this type of implant alloy are 200 V for the potential and 3 min for the time, under the conditions of the specified electrochemical cell.

Figure
Figure 4a-d shows the EDX spectrum in layer (a), the EDX elemental analysis in weight percent in layer (b), and the SEM surface morphology of the grade 5 Ti6Al4V implant alloy electrochemically oxidized at 200 V and at a time of 3 min in layers (c) and (d).As specified in chapter 2, Materials and Methods, the most effective and relevant values for obtaining titanium oxide films on this type of implant alloy are 200 V for the potential and 3 min for the time, under the conditions of the specified electrochemical cell.

Figure 4 .
Figure 4. SEM surface micrograph and EDX analysis of the treated Ti6Al4V grade 5 implant alloy electrochemically oxidized at a potential of 200 V for 3 min: (a) EDX spectrum; (b) EDX elemental analysis; and (c,d) SEM surface morphology at different magnifications.

Figure 4 .
Figure 4. SEM surface micrograph and EDX analysis of the treated Ti6Al4V grade 5 implant alloy electrochemically oxidized at a potential of 200 V for 3 min: (a) EDX spectrum; (b) EDX elemental analysis; and (c,d) SEM surface morphology at different magnifications.
5 shows the surface morphologies of the electrochemically oxidized grade 5 Ti6Al4V samples: (a) at 200 V for 1 min, (b) at 200 V for 2 min, and (c) at 200 V for 3 min at a magnitude of 50,000×, with the measurement of the pore diameter size directly from the SEM results.

Figure 5 .
Figure 5. Morphological and dimensional analysis of the titanium oxide (TiO2) film nanopores formed on the surface of the titanium alloy depending on the oxidation time at a potential of 200 V: (a) nanopores formed on Ti6Al4V alloy with an oxidation time of 1 min, (b) nanopores formed on Ti6Al4V alloy with an oxidation time of 2 min, and (c) nanopores formed on Ti6Al4V alloy with an oxidation time of 3 min.

Figure 5 . 17 Figure 6 .
Figure 5. Morphological and dimensional analysis of the titanium oxide (TiO 2 ) film nanopores formed on the surface of the titanium alloy depending on the oxidation time at a potential of 200 V: (a) nanopores formed on Ti6Al4V alloy with an oxidation time of 1 min, (b) nanopores formed on Ti6Al4V alloy with an oxidation time of 2 min, and (c) nanopores formed on Ti6Al4V alloy with an oxidation time of 3 min.

3. 2 .
Evolution of the Open Circuit Potential (OCP) vs.Time in Hank's Biological Solution for the Untreated Alloy and the Treated Alloy with the Electrochemically Formed Nanoporous TiO 2 Surface Film

16 Figure 7 .
Figure 7. Comparative evolution of the open circuit potential during immersion in Hank's biological solution for 80 h: (1) the untreated grade 5 Ti6Al4V alloy and (2) the treated grade 5 Ti6Al4V alloy with electrochemically formed titanium oxide surface film.From the analysis of Figure 7, it can be observed that in the case of both studied surfaces, the open circuit potential values move toward nobler (more positive) values with increasing immersion period, and the open circuit potential of the electrochemically oxidized surface registers more positive values (nobler) compared with the untreated titanium alloy throughout the whole immersion period.A shift in the open circuit toward more positive values indicates the formation of a passive film, while a decrease in the open circuit toward more negative values indicates breaks in the passive film, the dissolution of the film, or the absence of film formation.Comparatively, the values of the electrochemically oxidized titanium alloy immersed in Hank's solution were more positive than those of the untreated alloy throughout the whole immersion period.At the beginning of the immersion period, the value of the open circuit potential for the untreated alloy was E = −222 mV vs. Ag/AgCl, and for the electrochemically oxidized titanium alloy, this value was more positive, E = 113 mV vs. Ag/AgCl.The difference in the open circuit potential between the two surfaces at the start of immersion was ΔE = 335 mV.At the end of the monitoring process, it was observed that the electrochemically oxidized alloy reaches an open circuit potential value of E = 814 mV vs. Ag/AgCl, while the value of the open circuit potential for the untreated alloy is more negative, E = 424 mV vs. Ag/AgCl.The open circuit potential difference between the two surfaces at the end of monitoring is even greater, ΔE = 390 mV.This trend confirms the improvement of the grade 5 Ti6Al4V

Figure 7 .
Figure 7. Comparative evolution of the open circuit potential during immersion in Hank's biological solution for 80 h: (1) the untreated grade 5 Ti6Al4V alloy and (2) the treated grade 5 Ti6Al4V alloy with electrochemically formed titanium oxide surface film.From the analysis of Figure 7, it can be observed that in the case of both studied surfaces, the open circuit potential values move toward nobler (more positive) values with increasing immersion period, and the open circuit potential of the electrochemically oxidized surface registers more positive values (nobler) compared with the untreated titanium alloy throughout the whole immersion period.A shift in the open circuit toward more positive values indicates the formation of a passive film, while a decrease in the open circuit toward more negative values indicates breaks in the passive film, the dissolution of the film, or the absence of film formation.Comparatively, the values of the electrochemically oxidized titanium alloy immersed in Hank's solution were more positive than those of the untreated alloy throughout the whole immersion period.At the beginning of the immersion period, the value of the open circuit potential for the untreated alloy was E = −222 mV vs. Ag/AgCl, and for the electrochemically oxidized titanium alloy, this value was more positive, E = 113 mV vs. Ag/AgCl.The difference in the

3. 3 .
Evolution of the Polarization Resistance (R p ) vs. Time in Hank's Biological Solution for the Untreated Alloy and the Treated Alloy with the Electrochemically Formed Nanoporous TiO 2 Surface Film After immersing the untreated and electrochemically oxidized grade 5 titanium alloy samples to form the protective nanoporous film of titanium oxide (TiO 2 ), the open circuit potential was recorded in several sequences for 75.6 h.Between the open circuit potential measurement sequences at the start and end of the experiment, a total of seven recordings of 100 points of polarization resistance were made and calculated from as many linear polarization curves by using the Stern Geary equation and Tafel slopes (as explained in chapter 2, Materials and Methods).
at three time periods in the total sample immersion period in Hank's biological solution.

Figure 9 .
Figure 9. Comparative evolution of the average values of polarization resistance during the 75.5 h immersion period in Hank's biological solution: (1) untreated Ti6Al4V grade 5 alloy and (2) the treated Ti6Al4V grade 5 alloy with an electrochemically nanoporous titanium oxide surface film.

Figure 9 .
Figure 9. Comparative evolution of the average values of polarization resistance during the 75.5 h immersion period in Hank's biological solution: (1) untreated Ti6Al4V grade 5 alloy and (2) the treated Ti6Al4V grade 5 alloy with an electrochemically nanoporous titanium oxide surface film.

Figure 10 .
Figure 10.Comparative evolution of the average values for the corrosion rate (V corr ) during the immersion period in Hank's biological solution: (a) after 6 h (360 min); (b) after 38 h (2298 min); (c) after 75.5 h (4534 min).(1) the untreated Ti6Al4V alloy and (2) the treated Ti6Al4V alloy with the electrochemically formed nanoporous titanium oxide film.

Figure 11 .
Figure 11.shows the evolution of the average values for the corrosion rate of the two alloys immersed in Hank's biological solution for the entire immersion period, measured at different times after immersion.

Figure 11 .
Figure 11.Shows the evolution of the average values for the corrosion rate of the two alloys immersed in Hank's biological solution for the entire immersion period, measured at different times after immersion.