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Photodeposition of Hydroxyapatite into a Titanium Dioxide Nanotubular Layer using Ca(EDTA) Complex Decomposition

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02 May 2023

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03 May 2023

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Abstract
A new photocatalytic hydroxyapatite (HA) synthesis method has been developed. This method is based on the unique ability of the TiO2 photocatalyst to decompose the Ca(EDTA) complex under UV illumination. As a result, released Ca2+ ions react with PO43- ions forming the HA particles. The photocatalytic formation of hydroxyapatite is found to have a fractional order, which may indicate the complex reaction mechanism and the presence of several limiting stages. The TNT-HA samples were studied by XRD, FTIR, SEM, GDOES, and biocompatibility study. High biocompatibility of the surfaces is proven by pre-osteoblast cell growth.
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Subject: Chemistry and Materials Science  -   Other

1. Introduction

The hydroxyapatite (HA, Ca10(PO4)6(OH)2) is the main inorganic constituent of bone tissue. Synthetic HA has been successfully used to fabricate biomaterials, osteogenic coatings, and other materials of biomedical interest [1,2,3,4,5]. There are different methodologies for fabrication of calcium phosphates, such as solid-state syntheses [6,7,8,9], hydrothermal syntheses [10,11,12,13,14,15], biomimetic systems [16,17,18], electrochemical process [19,20,21] and sol-gel methods [5,22,23,24]. However, it should be mentioned that synthetic methods involving high-temperature treatment, such as solid-state reaction or sol-gel, usually form large, agglomerated particles and do not lead to the anisotropic growth of the crystals.
The preparation of HA powder via decomposition of chemical complexes through chelating agents is a simple process, particularly associated with hydrothermal reactions. For example, the deposition of HA via the decomposition of Ca(EDTA) complexes under hydrothermal conditions is a simple way to produce HA whiskers [12], [15]. Chelates, including EDTA, citric acid, and lactic acid have been used to produce hydroxyapatite powders. Decomposition of Ca(EDTA) complex at temperatures below 140–150°C and in a range of pH can be utilized for biocompatible coating fabrication [25,26,27,28].
Another approach to fabricate hydroxyapatite is to use triethyl phosphate as a source of phosphate ions [29,30,31,32]. Triethyl phosphate (PO(C2H5O)3, TEP) is chosen as a source of PO43– because of its higher stability than triethyl phosphite. During the hydrolysis, TEP decomposes, resulting PO43– ions. However, this method requires long aging times (several days), and high solution temperatures are needed [33,34] because triethyl phosphate has a relatively low reactivity for hydrolysis [31].
Changing the synthesis from hydrothermal to electrochemical deposition allows hydroxyapatite to be produced at room temperature [35,36,37]. Electrochemical deposition of HA is based mainly on a pH jump effect due to reactions occurring near the cathode or anode. During electrode reactions of nitrate, peroxide, or H+ electroreduction [36,37,38], a local pH increases near the cathode surface, resulting in the deposition of calcium phosphates. However, electrolytes for hydroxyapatite deposition generally contain low concentrations of calcium and phosphate ions to avoid HA deposition in a volume cell. It is necessary to enlarge the concentration of Ca2+ and PO43–-precursors to increase the surface thickness. However, to prevent the precipitation of phosphates in the volume, it is necessary to reduce the electrolyte pH value from 6.0 to 3.5.
In this regard, photocatalytic [39] or photoelectrochemical [40] are very promising. Sultana et. al. [39] has shown the possibility of hydroxyapatite synthesis from eggshells adopting a UV-mediated solid-state method for the first time. This synthesis included a combination of ball milling of the initial reagents followed by UV-illumination. The UV-irradiation of the Ca and P precursors resulted in the HA formation under ambient temperature. In our previous work [40], we provided the photocatalytic deposition of HA onto photoactive substrates using triethyl phosphate decomposition in aqueous solutions.
In the present paper, we propose a new method of photocatalytic deposition of HA using Ca(EDTA) complexes photodecomposition. The nanotubular TiO2 (TNT) layer is chosen as photoactive substrate. The Ca(EDTA) complex is supposed to degrade under UV-illumination. We chose the chelate complexes because they are widely used in hydrothermal methods but have not yet been used for the photocatalytic precipitation of HA.
In this regard, our work aims to develop a photocatalytic method for the deposition of HA using chelate complexes. It should be noted that there are few papers in this area, which may be due to the relatively slow synthesis times compared to the most common sol-gel synthesis. At the same time, a long crystallization time is good for the formation of biomimetic hydroxyapatite [41]. So, this approach, despite its complexity, looks promising for obtaining biocompatible coatings.

2. Results and Discussion

In the present paper, we propose photocatalytic deposition of hydroxyapatite (HA) using titania nanotubular (TNT) layer and Ca(EDTA) as Ca2+ ions precursor. The photocatalytic synthesis of HA is based on the ability of photoholes generated in titanium dioxide under high-energy irradiation (the wavelength < 400 nm) to decompose completely various organic compounds in aqueous solutions. To prevent the precipitation of insoluble calcium phosphates directly in the volume of a solution containing calcium ions, Ca(EDTA) complex, which is stable in an alkaline medium, was used. Based on preliminary experiments, it is found that EDTA solutions absorb radiation in the UV region, and Ca(EDTA) complex has an absorption peak at 301 nm. Under UV radiation, the Ca(EDTA) complex is assumed to be destroyed resulting gradual Ca2+ release. Besides photodegradation, decomposition of the Ca(EDTA) complex could also occur with a local pH change [42].
The scanning vibrating electrode technique (SVET) was used to characterize the TNT photoelectrochemical activity upon UV-irradiation in aqueous solutions. The SVET method allows in-situ monitoring of ionic current density in an electrolyte close to the substrate surface.
Figure 1a shows the scheme of the experimental SVET setup. A TNT sample is attached by silver glue to the bottom of an electrochemical cell made of epoxy resin. The system under investigation is a working electrode (WE) platinum wire embedded in an epoxy resin to bring its round section into contact with a solution. The surface of annealed samples consists of TiO2 nanotubes with an average diameter of 25 nm. The thickness of TNT coating is 425–500 nm (Figure 1b).
A focused UV-irradiation wavelength locally irradiated the TNT surface is 365 nm, and the power is 5 mW/cm2. During SVET-analysis, a vibrating Pt-probe scans the surface, estimating its electrical potential in amplitude points of its vibration, and then recalculates it in ionic currents. The SVET maps of the TNT surface after 10 min of UV irradiation are presented in Figure 1c. The illuminated TNT surface exhibits a region of anodic activity, reflecting sites of localized photocurrent density, whereas there are no ionic fluxes without irradiation (Figure 1c). One peak of photocurrent activity is visible; consequently, this coating is photoactive and can destroy the Ca(EDTA) complex. Figure 1d shows the time dependence of the photocurrent generated under periodic illumination of the TNT bulk sample with a UV lamp. Switching off the lamp also leads to photocurrent fall, which proves the prepared samples' photoactivity.
Scanning ion-selective electrode technique (SIET) is based on potentiometric principles (Figure 2). This is the unique tool for the ion concentration gradient measurement [43,44,45,46]. Figure 2a shows the scheme of the SIET microelectrode. A glass capillary microelectrode with the ion-selective membrane in the tip scans the surface, measuring the concentration of a particular ion.
Previously, it was shown that electrons and holes photogenerated on the surface of titanium dioxide undergo secondary reactions with reactive oxygen species forming OH- and H+ ions [43,47,48]. Thus, it is reasonable to expect a local pH change near the TNT surface in regions with a flowing photocurrent. Figure 2c, demonstrates spatial pH-redistribution occurs self-consistently, accompanied by local electric potential redistribution during the free-energy minimization of the system. Under UV-irradiation, the pH of the TNT surface changes from 6.3 to 5.5. It should be noted that these pH values are also sufficient to destroy the Ca(EDTA) complex [42].
Having proved the photocatalytic activity of the obtained substrates, we preliminary studied the photodegradation of EDTA. The concentration of EDTA remaining in the solution was determined by back titration using a given sample of Ca2+ ions. The value of (C0 – CCa2+)/C0, which reflects the percentage of EDTA that has undergone degradation, was chosen to estimate the degradation value. At first, to determine the optimal concentration of Сa(EDTA) complex for the experiment, we study the ability of EDTA to decompose under UV-illumination in the presence of commercial photoactive titanium dioxide particles.
Despite different initial concentration, the relative percentage of EDTA is the same in the solution (Figure 3a). In this regard, we use EDTA at the concentration of 0.01 mol/L since it makes it easy to study changes in the reaction kinetics.
The decomposition kinetics of various Ca(EDTA) complexes were studied to find the optimal electrolyte composition. The EDTA concentration was taken as 0.01 mol/L, while the calcium ions concentration was varied from 0.005 to 0.02 mol/L so that the Ca2+:EDTA4– ratio was 1:2, 1:1 and 2:1. The complexes are irradiated in the presence of a TiO2 suspension (the particle concentration is 0.1 mg/mL) followed by Ca2+ release (Table 1). It is found that the Ca2+ : Na2EDTA2– ratio - 1:1 is the most stable and preferable for the photocatalytic precipitation of calcium phosphates.
The lnW–lnC dependence is used to determine the reaction order. Since the reaction order of Ca(EDTA) decomposition using TiO2 particles was fractional, the deposition time was increased up to 120 min. The order of Ca(EDTA) photocatalytic decomposition is 1.86.
Photocatalytic precipitation of calcium phosphates was conducted for 30, 60, and 120 min from an electrolyte containing 0.01 M Ca(EDTA) and 6 mM (NH4)2HPO4 at pH values within 7.9–8.3. It should be noted that when working with coatings, the degree of photodegradation of EDTA in the system is much lower than in the system where titanium dioxide powder was used instead of the coating.
The calculated reaction order of HA photocatalytic deposition on the TNT surface using Ca(EDTA) complex turned out to be 2.93. This value is much higher than in the case of suspension. This effect can be associated with a smaller amount of photocatalyst and its smaller specific surface area. This assumption is confirmed by the fact that after 30 and 60 min of UV-irradiation, the precipitation of calcium phosphates does not occur completely, which corresponds to the results obtained earlier in the study of the kinetics of EDTA photodegradation.
Fractional order is possible if: (i) the reaction mechanism has many stages; (ii) the mechanism includes several slow stages; (iii) the limiting stage will be reversible [49,50,51]. In addition, the fractional reaction order usually indicates the simultaneous undergoing of several reaction stages with similar rates [49]. Fractional order can also be a consequence of the participation of atoms in reactions along with molecules [51].
Considering the literature data described above [40,47,48,49,50,51,52], we assume the photodeposition of HA occur via following Equations:
TiO2+ hν → hvb+ + ecb–
H2O + hvb+ → OHad● + H+
О2 + H2O + ecb– → …→ OH––
О2 + ecb– → O2●–
O2●– + H+ → HO2●
Ca(EDTA) + OH●/ hvb+/O2–/HO2● → EDTA2– + Ca2+
HPO42– → PO43– + Н+
HPO42– + H2O → H2PO4– + ОН–
H2PO4– → HPO42– + Н+
HPO42– + ОН– → PO43– + H2O
5Ca2+ + 3PO43– + OH– = Ca5(PO4)3OH
The generation of active oxygen species on the titania surface has a radical nature (Equations 1-5). The occurrence of radical reactions and generation of H+ near the TNT surface is evident by SIET maps demonstrating a pH change from 6.8 to 5.0. The production of H+ occurs relatively quickly. After 10 min of UV-irradiation, a change in the near-surface pH is detected.
The Ca(EDTA) complex may be destroyed via reactions with any of the generated reactive oxygen species (Equation 6). However, we suggest that Ca(EDTA) oxidation will most likely undergo under the photoholes and OH● radicals’ action since these particles are produced via primary processes. While the generation of radical particles according to equations (3-5) can be slowed down due to intermediate oxidation stages.
In addition, the Ca(EDTA) decomposition process (Equation 6) can be complicated by the fact that it can proceed through the adsorption stage of the Ca(EDTA) complex on the TNT surface.
This assumption is supported by the fact that when we replaced the TNT surface with TiO2 particles, the order of photochemical formation of HA was also fractional but less than for the TNT surface. In both experiments, the conditions and the relative photocatalyst concentration were the same, while the specific surface area of TNT was significantly less than that of TiO2 particles.
Thus, we can assume that HA photochemical deposition on TiO2 particles surface proceeds with fewer limiting stages. Among these limiting steps there may be a step of Ca(EDTA) adsorption onto the photocatalytic surface and a step of phosphate ions diffusion towards titania surface, which can be faster in the case of a powder.
Another complex block of reactions is transformation of the HPO42– ion into the PO43– ion. We cannot add a PO43– ions directly to the electrolyte because of the rapidly proceeding hydrolysis, which makes the electrolyte pH strongly alkaline (pH > 10). At such pH values, Ca(EDTA) complexes are unstable.
The process of HPO42– dissociation (Equation 7) is unlikely, since the pKa3 of this process is 12.37 [53]. Calculating the hydrolysis constant for Equation 8:
K H = K w K a 2 = 10 − 14 6.3 − 8 = 1.58 ∙ 10 − 7
Comparing the constants for Equation 8 and 9, we see that the hydrolysis is more likely, since 1.58·10–7 > 6.3·10–8. However, the hydrolysis constant is only 2.5 times bigger than the dissociation constant (Ka = 6.3·10–8). It indicates that ion dissociation will also occur in the system, and the speed of these processes is not much different.
When the concentration of OH– increases, the process of HPO42– transition into PO43– can undergo according to Equation 10. In addition, OH– ions for the Equation 10 can be simultaneously produced in processes near TiO2 surface via Equations 1–5.
The HA formation reaction (Equation 11) is also not simple and requires the simultaneous participation of Ca2+, PO43– and OH– ions which have different ion mobility. All these processes can lead to the fractional order reaction of HA photochemical deposition.
Since the reaction of photocatalytic decomposition of the Ca(EDTA) complex proceeds directly on the titanium dioxide surface, it can be assumed that the formation of calcium phosphates also occurred near the TiO2 surface. There are reflections of titanium dioxide anatase modification on the X-ray pattern of the initial TNT sample (Figure 3d). After 120 min of UV-irradiation, characteristic HA peaks appear in the X-ray diffraction pattern of the sample in the region 2θ: 30–33° and 34.30° peaks characteristic of amorphous α-TCP and HA appear. In addition, at 2θ: 34.30°, a reflex appears corresponding to a 100% peak rhombohedral calcium phosphate. The IR spectra (not shown here). Also proved the presence of HA. There are bands at 1068, 958 and 600 cm - 1, corresponding to the internal vibrational modes of PO43 - groups, which indicates the presence of phosphates in the coating. The broad bands of OH-groups and water vibrations indicate hydrogen bonds presence.
Figure 4 shows SEM images and distribution of elements over the cross-section of TiO2 nanotube coatings after photocatalytic deposition of HA for 30, 60, and 120 min of UV-irradiation. After 30 min of HA deposition, the surface morphology retains the nanotube structure (Figure 4a). No calcium phosphate particles are found on the TNT surface, and there are also no HA reflections in the X-ray diffraction pattern of the obtained sample. However, the GDOES analysis of the element distribution along the cross section of TNT-HA coating shows the presence of calcium and phosphorus in the structural composition of the obtained coatings, and are evenly distributed along the surface of the entire nanotube (Figure 4b). According to this, we can assume that HA particles form inside titania nanotubes, and this deposition occurs unevenly. Since the deposition proceeds inside the nanotubes, the size of the resulting HA particles does not exceed 25 nm which is comparable to native bone crystallite size and promising for obtaining biocompatible coatings.
As seen in Figure 4d, the concentration of the complex in the solution gradually decreases and reaches a plateau at a time of 60 min, regardless of the ratio of Ca2+ and EDTA concentrations. At an irradiation time of 30 min, the photodegradation of EDTA almost does not proceed, while after 120 min of photocatalytic deposition, about half of the EDTA decomposes, and the released calcium cations bind to phosphate anions in solution to form calcium phosphates. The amount of formed calcium phosphates increases significantly on increasing the deposition time up to 60 min. The particles become coarser and deposit not only inside the nanotubes but also on the surface of the nanotubes (Figure 4c). The image shows that the coating begins to overgrow partially, but there are still areas on the surface free from the formed HA. The elements’ distribution along the profile confirms that Ca and P are distributed not only along the nanotube but are also concentrated in the near-surface region, which may also be due to the permeability of UV-irradiation.
Increasing the synthesis time to 120 min contributes to the complete overgrowth of the surface and a change in its morphology (Figure 4 e, f). The distribution of elements along the profile reflects a significant increase in calcium on the surface of nanotubes, which may indicate the growth of a loose thick coating layer on the surface of nanotubes. Increase the deposition time above 120 min does not make sense since the coating is completely covered with a layer of dielectric calcium phosphate, and photodecomposition does not proceed.
The biocompatibility of the samples has been examined using the MC3T3-E1 cell line (Figure 5). Figure 5f shows an example of cell attached to the TNT surface. The TNT nanotubes previously kept in an electrolyte solution for 120 min are used as a control.
As seen in Figure 5, all samples are biocompatible. The cell density on the TNT-HA samples increases by increasing the photodeposition time, which may indicate a biocompatibility increase in the obtained samples. Compared to the control, there is a slight cell density decrease on TNT-HA sample with 30 min of hydroxyapatite deposition. This effect may be due to the inhomogeneous deposition of hydroxyapatite on the sample surface.
Thus, the photocatalytic decomposition of the Ca(EDTA) complex can be used to precipitate calcium phosphates. Based on cell density, the optimal time for hydroxyapatite precipitation is 120 min since it allows obtaining samples with maximum bioactivity.

3. Materials and Methods

3.1. Materials

Ethylenediaminetetraacetic acid (EDTA, ≥99.8%), ammonium fluoride (≥99.99%, trace metals basis), calcium chloride (≥93.0%), calcium nitrate tetrahydrate (≥99.0%), ammonium nitrate (≥98%), potassium chloride (≥99.0%), NH3 (28 wt % in H2O, ≥99.99%), 1,2-ethanediol (anhydrous, 99.8%), sodium phosphate dibasic (anhydrous, ≥99.0%, ACS reagent) and hydrogen peroxide solution (30 wt % in H2O, ACS reagent), titanium (IV) oxide, anatase (nanopowder, <25 nm particle size, 99.7% trace metals basis) were supplied by Merck. Titanium plates (≥99.7%) purchased from Merck were used to prepare TNT layer. The Ti plates were previously chemically polished in a mixture HF/HNO3. The polishing solution was prepared by mixing concentrated HF (50 wt %) and HNO3 (65 wt %) at a volume ratio of 2:1. The Ti plates were rinsed with Milli-Q water (18 MΩ cm), and then dried with nitrogen.

3.2. Preparation of TNT

TNT layer was obtained by Ti plates anodizing in ethylene glycol electrolyte containing 0.75 wt % NH4F and 2 vol % H2O. The anodization was performed in two stages: (i) electrode polarization, which consisted of a voltage ramp from 0 to 40 V with a scan rate of 0.2 V s−1 , and (ii) oxidizing at a constant voltage of 40 V for 1 h. After the anodization, the electrodes were rinsed with water and then dried. The surface debris on the TNT was removed by ultrasonic treatment in Milli-Q water for 30 s. TNT layers were thermally treated at 450 °C for 3 h in the air for TiO2 crystallization to increase its photocatalytic activity.

3.3. Photodegradation of EDTA and Ca(EDTA) complexes study

Commercial TiO2 anatase (nanopowder, < 25 nm particle size, 99.7% trace metals basis) was used to study the decomposition of EDTA and Ca(EDTA) complexes. The concentration of TiO2 in the suspension was 0.1 mg /mL. The photodestruction time varied from 20 to 80 min. The 3 mL of TiO2 suspension was added in Petri dish and UV-irradiated (a wavelength of 365 nm, power of 5 W/cm2). For each 20 min, the aliquots were taken. The amount of released calcium ions was revealed by titration, and the presence of pure EDTA in solution was determined by back titration.

3.4. Photocatalytic hydroxyapatite deposition

Photocatalytic precipitation of calcium phosphates was based on the photodecomposition of Ca(EDTA) complexes under the action of UV-irradiation. The TNT coatings were placed in Petri dishes, and 3 mL of an electrolyte containing 0.01 M Ca(EDTA) and 6 mM (NH4)2HPO4 was added. The electrolyte pH is maintained within 7.9–8.3. The TNT was UV-irradiated using a UV-diode (a wavelength of 365 nm, power of 5 W/cm2), thermal radiation was filtered with a water filter. The distance between the lamp and the sample was 10 cm; exposure time varied from 30 to 120 min. The aliquots were taken at 30, 60, and 120 min of UV-illumination. After irradiation, the samples were kept in the dark at room temperature for 24 h and then washed and dried in air at 80°C for 5 h.

3.5. Sample characterization

The anodic and cathodic activity of electrode under polarization in water solution and photoactivity of illuminated TiO2 were studied by SVET and generated pH gradients by SIET. A system from Applicable Electronics (USA) modulated by an ASET program (Sciencewares, USA) was used to perform the SVET and SIET measurements. Anodized TiO2 (1.5 cm2) under low intensity light-emitted diode (365 nm) irradiation focused on a spot (~0.25 cm2) was used as working photoelectrode. An insulated Pt-Ir microprobe (Microprobe Inc., USA) with a platinum black spherical tip 30 μm in diameter was used as a vibrating probe for SVET experiments. The probe was made to vibrate both parallel and perpendicular to the specimen surface at a height of 150 μm. The amplitude of vibration was 30 μm, while the probe vibrated at frequencies of 136 Hz (perpendicular to surface) and 225 Hz (parallel to surface). The photocatalytic measurements were conducted in a three-electrode electrochemical cell. The TNT was connected as a working electrode, and the Pt wire and Ag/AgCl electrode were used as a counter and reference electrodes, accordingly.
The SIET method detected the local pH changes due to H+ activity. The glass-capillary microelectrodes were filled with Hydrogen Ionophore Cocktail I (Merck) to prepare the pH-selective membrane. The Ag/AgCl/KCl (sat) was used as the external reference electrode. The pH measurements were carried out near 25 μm above the surface.
SVET data was presented as obtained, and SIET data was recalculated according to the previous calibration. Mapping for each experimental condition is reproduced at least three times, one of typical maps is presented.
The XRD analysis was carried out using an Advance D8 diffractometer (Bruker, Germany) with Cu Kα radiation. All samples were examined in the range of 2θ from 10° to 70° at a scanning speed of 1°/min and a step size of 0.03°.
The TNT samples were measured using Hitachi S4100 (Hitachi, Japan). The operating voltage was 5 eV, and the TNT was sputtered with carbon.
The depth profile of the sample was analyzed using a HORIBA GD-Profiler 2 (Horiba, Japan). The glow discharge optical emission spectroscopy (GDOES) used operating pressure and power was 650 Pa and 30 W, respectively. The anode diameter was 4 mm.

3.5. Cell Culture and Immunofluorescent Staining

The pre-osteoblastic MC3T3-E1 cells were provided by the Ludwig Boltzmann Institute of Osteology (Vienna, Austria). The sample surface was seeded with cells at 103 cells/mL density.. The samples were cultured for 2 days in α modification of minimum essential medium (α-MEM, Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal calf serum (PAA Laboratories, Linz, Austria), 0.1% ascorbic acid (Sigma-Aldrich, St. Louis, MO), and 0.1% gentamicin (Sigma-Aldrich, Steinheim, Germany) in a humidified atmosphere with 5% CO2 at 37 °C.
On 2-d day, samples were washed with phosphate-buffered saline (PBS), fixed with 70% C2H5OH solution, and permeabilized with 0.1% Triton X-100 (Merck, Germany) for 15 min. The samples were stained using 1:20 solution of Alexa Fluor 488 phalloidin (Invitrogen, Eugene, OR) in the dark at 4°C for 1 h. After washing in PBS, the samples were incubated with 1:300 solution of TO-PRO-3 iodide (Invitrogen, Eugene, OR) solution for 5 min, followed by washing in PBS.
The cells were monitored using a confocal laser scanning microscope (Leica TCS SP, Germany).The excitation was performed at a wavelength range of 488–514 nm and 543–551 nm. The emission spectra were recorded at wavelength range of 512–542 nm and 565–605nm.

4. Conclusions

The photocatalytic deposition of HA on titanium dioxide nanotubes has been elaborated. The deposition mechanism is based on Ca(EDTA) complex photodegradation. The reaction order of the process is revealed to be 2.93, which can be concerned with multistage mechanism of these reaction, including several slow stages. The optimal electrolyte composition for enriched HA deposition (up to 80%) is 0.01 M Ca(EDTA) and 6 mM NH4H2PO4 (pH 8.0). The 60–120 min photocatalytic deposition time is optimal for biocompatible surfaces fabrication. Hydroxyapatite is found to deposit inside titania nanotubes. The size of the formed HA particles does not exceed 25 nm compared to native bone crystallite size. Thus, photocatalytic modification of TNT surface may find its application for fabrication of biocompatible coatings, which stimulates bone tissue growth.

Author Contributions

Conceptualization, E.V.S. and S.A.U.; methodology, formal analysis, V. Yu. Y., P.I.Z., P.V.N.; investigation, V. Yu. Y., P.I.Z., P.V.N., V.V. G.; writing—original draft preparation, V. Yu. Y. and P.I.Z. (equal contribution); writing—review and editing, E.V.S. and S.A.U.; visualization, V. Yu. Y. and P.I.Z..; supervision, S.A.U.; project administration, E.V.S. and S.A.U..; funding acquisition, S.A.U. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Russian Science Foundation grant no. 19-79-10244.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. (a) Scheme of the experimental setup used during scanning vibrating electrode (SVET) measurements. Insert shows the scheme of the vibration electrode; (b) SEM micrograph of the TNT surface used as a sample. The image shows the top-view and the cross-section view; (c) The SVET maps of the ionic current density above the initial TiO2 surface (the left one), under UV irradiation (the middle and the right ones). The wavelength is 365 nm, and the power is 5mW/cm2. Scale units: nA/cm2, spatial resolution is 15 mm. Solution: 0.1 M NaCl; (d) Time-dependence of the photocurrent generated under illumination of TiO2 nanotubes with a UV-lamp (365 nm, 5 mW/cm2). Letters “on” are regarded as switching the light on, whereas “off” – switching the light off.
Figure 1. (a) Scheme of the experimental setup used during scanning vibrating electrode (SVET) measurements. Insert shows the scheme of the vibration electrode; (b) SEM micrograph of the TNT surface used as a sample. The image shows the top-view and the cross-section view; (c) The SVET maps of the ionic current density above the initial TiO2 surface (the left one), under UV irradiation (the middle and the right ones). The wavelength is 365 nm, and the power is 5mW/cm2. Scale units: nA/cm2, spatial resolution is 15 mm. Solution: 0.1 M NaCl; (d) Time-dependence of the photocurrent generated under illumination of TiO2 nanotubes with a UV-lamp (365 nm, 5 mW/cm2). Letters “on” are regarded as switching the light on, whereas “off” – switching the light off.
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Figure 2. (a) Scheme of the scanning ion-selective microelectrode; (b) Scheme of the experimental setup used during scanning ion-selective microelectrode technique (SIET); (c) The pH distribution map of the initial TNT surface (left one) and under UV-irradiation (right one).
Figure 2. (a) Scheme of the scanning ion-selective microelectrode; (b) Scheme of the experimental setup used during scanning ion-selective microelectrode technique (SIET); (c) The pH distribution map of the initial TNT surface (left one) and under UV-irradiation (right one).
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Figure 3. (a) Time-dependence of EDTA concentration (C/C0) at initial EDTA concentrations of 0.005 mol/L(plot 1); 0.01 mol/L(plot 2); 0.1 mol/L(plot 3); (b) The lnW –ln(Ct)-dependence on photodegradation time of Ca(EDTA) complex taken at the concentration ratio of Ca2+ : EDTA4– of 1 : 2 (plot 1); 1 : 1 (plot 2); 2 : 1 (plot 3); (c) the reaction order determination of HA formation over titanium dioxide nanotubes upon photodecomposition of the Ca(EDTA) complex; (d) XRD pattern of initial TNT (plot 1) and TNT with HA photodeposited. TNT and HA-modified TNT samples were annealed at 450 °C for 3 h.
Figure 3. (a) Time-dependence of EDTA concentration (C/C0) at initial EDTA concentrations of 0.005 mol/L(plot 1); 0.01 mol/L(plot 2); 0.1 mol/L(plot 3); (b) The lnW –ln(Ct)-dependence on photodegradation time of Ca(EDTA) complex taken at the concentration ratio of Ca2+ : EDTA4– of 1 : 2 (plot 1); 1 : 1 (plot 2); 2 : 1 (plot 3); (c) the reaction order determination of HA formation over titanium dioxide nanotubes upon photodecomposition of the Ca(EDTA) complex; (d) XRD pattern of initial TNT (plot 1) and TNT with HA photodeposited. TNT and HA-modified TNT samples were annealed at 450 °C for 3 h.
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Figure 4. (a, c, e) SEM images of the TNT samples modified by photodeposition of HA during 30 min (a), 60 min (c), and 120 min (e); (b, d, f) Depth profile analysis of element distribution on the cross of TNT coating with photodeposited HA during 30 min (b), 60 min (d) and 120 min(f).
Figure 4. (a, c, e) SEM images of the TNT samples modified by photodeposition of HA during 30 min (a), 60 min (c), and 120 min (e); (b, d, f) Depth profile analysis of element distribution on the cross of TNT coating with photodeposited HA during 30 min (b), 60 min (d) and 120 min(f).
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Figure 5. (a–d) Pre-osteoblasts MC3T3-E1 cell growth on different substrates. Immunofluorescent analysis on cell density and morphology after 2 days of culturing on different substrates of the initial (a) TNT surface and (b–d) TNT with photodeposited HA: (b) after 30 min, (c) 60 min and (d) 120 min of deposition. Cells were stained for actin using phalloidin (green) and TO-PRO-3 iodide for nuclei (red); (e) Cell density on different surfaces after 30–120 min of photo deposition of HA. Insets depict cell morphologies. Scale bars represent means ± standard deviation (SD) from three replicates each from three independent experiments. A comparison was performed by analysis of variance (ANOVA). (∗) p < 0.05. TNT substrate with areas with and without photodeposited HA; (f) The MC3T3-E1 cell fragment on TiO2 nanotubes with photodeposited HA.
Figure 5. (a–d) Pre-osteoblasts MC3T3-E1 cell growth on different substrates. Immunofluorescent analysis on cell density and morphology after 2 days of culturing on different substrates of the initial (a) TNT surface and (b–d) TNT with photodeposited HA: (b) after 30 min, (c) 60 min and (d) 120 min of deposition. Cells were stained for actin using phalloidin (green) and TO-PRO-3 iodide for nuclei (red); (e) Cell density on different surfaces after 30–120 min of photo deposition of HA. Insets depict cell morphologies. Scale bars represent means ± standard deviation (SD) from three replicates each from three independent experiments. A comparison was performed by analysis of variance (ANOVA). (∗) p < 0.05. TNT substrate with areas with and without photodeposited HA; (f) The MC3T3-E1 cell fragment on TiO2 nanotubes with photodeposited HA.
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Table 1. The Ca(EDTA) complex destruction dependence on the initial EDTA concentration at different UV-irradiation time.
Table 1. The Ca(EDTA) complex destruction dependence on the initial EDTA concentration at different UV-irradiation time.
C(Ca2+)/C(EDTA) C0(Ca2+),mol/L C0(EDTA),mol/L Irradiation time, min Ct(Ca2+),
mol/L		 Ct(EDTA),
mol/L		 Ct/C0, %
1 : 1 0.01 0.01 20 0.00353 0.00642 64.7
0.01 0.01 40 0.00225 0.00769 77.5
0.01 0.01 60 0.00059 0.00931 94.1
0.01 0.01 80 0.00055 0.00936 94.5
1 : 2 0.005 0.01 20 0.00391 0,00302 60.9
0.005 0.01 40 0.00237 0.00380 76.3
0.005 0.01 60 0.00067 0.00458 93.3
0.005 0.01 80 0.00060 0.00465 94.0
2 : 1 0.02 0.01 20 0.00377 0.01241 62.3
0.02 0.01 40 0.00202 0.01528 79.8
0.02 0.01 60 0.00059 0.01862 94.1
0.02 0.01 80 0.00057 0.01876 94.3
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