Photocatalytic Deposition of Hydroxyapatite onto a Titanium Dioxide

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Photocatalytic deposition of hydroxyapatite onto titanium dioxide nanotubular layer with fine tuning of layer nanoarchitecture Sviatlana Alexandrovna Ulasevich, Sergey K. Poznyak, Anatoly I. Kulak, Aleksey D Lisenkov, Maksim Starykevich, and Ekaterina V. Skorb Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b00297 • Publication Date (Web): 18 Mar 2016 Downloaded from http://pubs.acs.org on March 21, 2016

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Photocatalytic deposition of hydroxyapatite onto titanium dioxide nanotubular layer with fine tuning of layer nanoarchitecture Sviatlana A. Ulasevich,*a,b Sergey K. Poznyakc, Anatoly I. Kulak,b Aleksey D. Lisenkovd, Maksim Starykevich,d and Ekaterina V. Skorba a

Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14424 Potsdam, Germany. E-mail: [email protected] b

Institute of General and Inorganic Chemistry of National Academy of Sciences of Belarus, 220072 Minsk, Belarus.

c

The Research Institute for Physical Chemical Problems of the Belarusian State University, 220030 Minsk, Belarus.

d

Department of Materials and Ceramics Engineering, CICECO, University of Aveiro, 3810-193 Aveiro, Portugal

KEYWORDS: titanium dioxide, nanotubes, hydroxyapatite, photocatalysis, biocompatibility.

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ABSTRACT

A new effective method of photocatalytic deposition of hydroxyapatite (HA) onto semiconductor substrates is proposed. Highly ordered nanotubular TiO2 (TNT) layer formed on titanium via its anodization is chosen as the photoactive substrate. The method is based on photodecomposition of phosphate anion precursor, triethylphosphate (TEP), on the semiconductor surface with following reaction of formed phosphate anions with calcium cations presented in the solution. HA can be deposited only on irradiated areas, providing the possibility of photoresist free HA patterning. It is shown that HA deposition can be controlled via pH, light intensity and duration of the process. Energy-dispersive X-ray spectroscope profile analysis and glow discharge optical emission spectroscopy of HA modified TNT proves that HA deposits over the entire TNT depth. High biocompatibility of the surfaces is proven by protein adsorption and pre-osteoblast cell growth.

1. INTRODUCTION Different methods are used to create hydroxyapatite (HA) coatings on bio-important substrate such as titanium, widely applied for dental and orthopedic implants.1–3 Plasma spraying process,4 thermal spraying,5 sputter coating,6 pulsed laser deposition,7 chemical8–11 and electrochemical deposition12–17 can be mentioned. Among them, “wet” methods,14 especially electrochemical deposition,18 are preferable as they allow the coating deposition onto large, irregularly shaped surfaces.19.20 Electrochemical deposition of HA is based on cathode electrode reactions of nitrate, peroxide or H+ electroreduction with pH jump effect.14 During these reactions a local pH increase is created near the electrode surface, which leads to deposition of calcium phosphates on


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cathode.14–16 Generally solutions with pH 3.5–6.0 containing low concentrations of calcium and phosphate ions are used in this method to prevent the formation of calcium phosphates in a bulk of electrolyte. However, coatings formed from acidic solutions are brushite17 or octacalcium phosphate,21 not HA, and contain acidic phosphate groups. Conversion of these coatings into HA ones requires additional procedures, e.g. long-term soaking in NaOH.17 In the present paper, we propose a new method of photocatalytic deposition of HA onto photoactive substrates. The method is based on photodecomposition of phosphate anion precursors on the semiconductor surface. Nanotubular TiO2 (TNT) layer is chosen as a substrate to demonstrate the method prospects because it is known to possess a high photocatalytic activity when irradiated with ultraviolet (UV) light in aqueous solutions.22–24 TNT photoactivity can be used for the decomposition of organophosphorus compounds that are stable in alkaline solutions with initially formation of HA, not brushite or octacalcium phosphate. In our research we use anodic polarization for better charge separation since electrochemically assisted photocatalysis24 is known as an effective method to increase a charge separation in comparison with not-assisted photocatalysis. If charges are separated efficient, e.g. during electrochemically assisted photocatalysis, all generated photoholes can participate in oxidation reactions. Compared to the existing methods electrochemically assisted photocatalytic deposition of HA looks to be similar to electrochemical deposition. Both of these methods use electrolytes, cathode and anode. But these methods have significant differences: - Photocatalytic deposition of HA is based on photodecomposition of organophosphorus compound while electrochemical deposition of HA is based on pH jump effect. - Photocatalytic deposition of HA can be conducted without polarization while electrochemical deposition does not occur without polarization.


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- Hydroxyapatite deposits on the anode during photocatalytic method while it deposits on the cathode during electrochemical method. - As it is mentioned above, solutions for electrochemical deposition of HA are generally acidic (pH 3.5–6.0) while for photocatalytic deposition of HA we use alkaline solutions (pH 8.0–12.0). In addition, HA coating on TNT can result in further improvement of TNT biocompatibility which is interesting due to unique nanotubular architecture to trigger bone formation.25 Moreover, unique nanotubular porous architecture is prospective for surface capsules development and can be used, in future, to build drug delivery implants.26

2. METHODS Materials. Triethylphosphate (TEP, ≥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%), hydrogen peroxide solution (30 wt. % in H2O, ACS reagent) were supplied by Sigma-Aldrich. Titanium plates (≥99.7%) purchased from Sigma-Aldrich was used as a substrate material. Before anodic oxidation the plates was chemically polished in a mixture (2:1 by volume of HF: HNO3) of concentrated HF (50 wt. %) and HNO3 (65 wt. %) at 80°C for 3 – 5 s, thoroughly rinsed with deionized water, and then dried with nitrogen. Milli-Q-water (18 MΩ·cm) was used for preparing all aqueous solutions. Preparation of TNT. Ti plates were anodized in ethylene glycol electrolyte containing 0.75 wt. % NH4F and 2 vol. % H2O to prepare TNT layer on the Ti surface. The electrochemical anodization consisted of a voltage ramp from 0 V to 40 V with a scan rate of 0.2 V·s–1 followed by holding at 40 V for 1 h. After the electrochemical deposition, the electrodes were rinsed with


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water and then dried. Then the as-anodized samples were ultrasonically cleaned in Milli-Q-water for 30 s to remove surface debris on the TNT surface. TNT layers were annealed at 450°C for 3 h in air for TiO2 crystallization increasing its photocatalytic activity. SEM analysis reveals a thickness of TNT layer is ca. 6 µm after 1 h of electrochemical oxidation. Electrochemical and photoelectrochemical measurements. An electrochemical cell with a three-electrode arrangement was used for photoelectrochemical experiments. A platinum wire served as the counter electrode and an Ag/AgCl (saturated KCl solution) electrode as the reference electrode. Titanium plates with the grown TNT layer were used as working electrode. The reference electrode was placed close to the working electrode using a Luggin probe capillary. The cell was provided with a quartz window for UV irradiation of the working electrode. An Autolab PGSTAT 302N potentiostat (Metrohm Autolab b.v., the Netherlands) was used in these experiments. The working electrode surface was irradiated by a focused UV light beam from a high-pressure mercury lamp (250 W). Thermal radiation was filtered by a water filter equipped by high-quality quartz windows. Prior and during photocatalytic HA deposition an electrolyte in the cell was bubbling with Ar during 30 – 60 min to remove CO2 diluted in water and to prevent carbonate hydroxyapatite formation. X-ray diffraction (XRD) analysis was performed using an Advance D8 diffractometer (Bruker, Germany) with CoKα 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° . Raman spectra were taken at room temperature using a Nanofinder High End confocal microscope (Leica TCS SP, Germany) with a 573 nm solid-state exciting laser.


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Scanning electron microscopy (SEM) was performed using Hitachi S4100 and Hitachi SU70 microscopes coupled with an energy-dispersive X-ray spectroscope (EDS) for morphological characterization of the sample surface. Samples were sputtered with a carbon. Glow Discharge Optical Emission Spectroscopy (GDOES) depth profile analysis of the coatings was completed using a HORIBA GD-Profiler 2 operating at a pressure of 650 Pa and a power of 30 W. The anode size was of 4 mm in diameter. Analysis of free phosphate ions in the electrolyte was carried out colorimetrically by a spectrophotometer Shimadzu UV 2550 with the use of vanadium molybdenum complex.27 The biocompatibility of step-wise HA-modified TNT coating was demonstrated by adsorption of proteins at its surface. Mixed proteins (Alice - ProteoGenix) labeled with the fluorescent dye were used as an adsorption reagent. The proteins consist of phospholyrase b, bovine serum albumin, ovalbumin, carbonic anhydrase II, soybean trypsin inhibitor A, and lysozyme, each of which was labeled with the fluorescent dye, 2-(4-Amidinophenyl)-1H-indole6-carboxamidine (DAPI). Each protein was prepared at a concentration of 7–16 mg/ml. Confocal fluorescence microscope images were analyzed after the samples being in contact with proteins solution for 5 and 20 min. Cell culture. Murine pre-osteoblastic cells MC3T3-E1 (provided by the Ludwig Boltzmann Institute of Osteology, Vienna, Austria) were seeded with a density of 6·103 cells/cm3 on the surface and cultured for 2 days in a-MEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10% foetal 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.


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Immunofluorescent staining. Samples with were washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton-X100 (SigmaAldrich, Steinheim, Germany) for 15 min. Then samples were incubated for 1 h in a 1:20 solution of Alexa Fluor ® 488 phalloidin (Invitrogen, Eugene, Oregon, USA) in a dark at 4°C. After washing in PBS the samples were stained for nuclei with a 1:300 solution of TO-PRO-3 iodide (Invitrogen, Oregon, USA) for 5 min. Confocal microscopy. Images of cell nucleus and actin cytoskeleton cells were obtained using a confocal laser scanning microscope (Leica TCS SP, Germany). An Ar-ion (488 nm, 514 nm) and a He-Ne laser (543 nm) were used as an excitation source.

3. RESULTS AND DISCUSSION Our method of HA photocatalytic deposition is based on photodecomposition of phosphate anion precursors on the titania surface under UV illumination and anodic polarization. Triethylphosphate (TEP) is used in the present work as PO43– precursor because this organic compound is highly soluble in water, non-toxic, chemically stable in alkaline solutions and absorbs weakly UV light up to λ = 200 nm, i.e photochemically inactive. It was shown previously28,29 TEP can be photocatalytically decomposed on the surface of TiO2 particles in aqueous solutions. We use this effect to synthesize HA directly on the TiO2 surface. During photocatalytic decomposition of TEP phosphate ions are formed on the titania surface and can react with Ca2+ and OH– ions, producing HA film on the surface. The mechanism of HA formation during photocatalytic deposition can be described by following steps: - TiO2 photoactivation with the formation of photoholes (h+vb) and photoelectrons (e-cb):30


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TiO2 + hν → h+vb + e–cb ,

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(1)

- radical generation on the TiO2 surface: H2O + h+vb → TiO2 + OH●ad + H+,

(2)

- photodecomposition reactions: TEP + OH●/h+vb → PO43– and other degraded/mineralized products.28

(3)

- interaction of phosphate anions (product of TEP decomposition) with Ca2+ and OH– ions presented in the electrolyte with formation of HA: 5Ca2+ + 3PO43– + OH– = Ca5(PO4)3OH

(4)

Photocatalytic process on the surface of TiO2 electrode according to the reaction (2) can lead to a local acidification of the near-surface region of the solution. This fact is confirmed by the change of the pH of electrolyte during photocatalytic deposition of HA. When initial pH is 10.0, the resultant pH is 9.8 after 1 h of the photocatalytic deposition; initial pH of 9.2 changes to 8.8 while initial pH of 8.4 drops till 5.2. To smooth over this undesirable effect we used ammonium buffer solution with pH 10.0 – 11.0 as a background electrolyte. High values of the pH are more preferable for HA synthesis. However, at pH values more than 12 electrolyte becomes opaque due to the formation of Ca(OH)2 in a bulk of electrolyte. Figure 1a shows typical photocurrent-potential plots measured under chopped UV illumination of the TNT electrodes in deaerated 0.03 M NH4Cl solution with pH 10.0 Photocurrent appears at potentials –0.8 ÷ –0.9 V, then grows sharply with potential and finally levels off at E ≥ 0.4 V. Addition of the TEP to the electrolyte does not influence appreciably both the shape of the iph – E curves and the photocurrent values. This effect may be concerned with high efficiency of photoholes (h+vb) and photoelectrons (e–vb) separation in a bulk of TNT electrode. However,


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chemical analysis showed that phosphate ions appeared in the TEP-containing electrolyte as a result of TEP photodecomposition on the TiO2 electrode. Approximately 9·10–4 mol·L–1 of PO43– ions are produced during UV illumination of 0.02 M TEP solution for 2 h. The average rate of PO43– ions supply in the bulk of electrolyte is ca. 7·10–6 mol·L–1·min–1. This relatively slow rate of PO43– ions generation can be explained by complicated mechanism of TEP photodegradation leading to a large number of products and intermediates.28,29 Such gradual formation of PO43– in electrolyte during TEP photolysis is very preferable since HA synthesis requires long time.31,32 Figure 1b presents temporal evolution of the photocurrent at a fixed potential (+0.5 V) of the TNT electrode in solution containing 0.02 M TEP and 0.03 M NH4Cl (pH 10). After initial insignificant drop photocurrent varies insignificantly with time during a long period. Addition of Ca2+ ions into the electrolyte leads to about two times decrease of the photocurrent during 2 – 10 min. This photocurrent drop can be associated with a partial blocking of the TNT surface with growing calcium phosphate layer. There is no formation of HA in the bulk of the electrolyte. SEM images reveal an average inner diameter of as-prepared titania nanotubes is ca. 60 nm (Figure 2a). This diameter decreases gradually during photocatalytic deposition of HA, depending on the irradiation time (Figure 2b–j). Even three times decrease in the nanotube diameter remains the unique ordered morphology of the HA-modified TNT coating. UV irradiation longer than 120 min leads to partial closing of the pores. Figure 2 h shows that HA deposits uniformly on the TNT layer and there are not uncovered areas. The EDS analysis of cross-section of HA-modified TNT coating, shows the uniform distribution of the calcium and phosphorous elements in the thickness of TNT nanotubes layer (Figure 3a,b). The EDS analysis has also revealed that content of Ca and P elements grows by


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1.3 times when increasing the time of photocatalytic deposition of HA from 1 to 2 h, indicating that the formation of HA occurs mainly during the first hour of photocatalytic deposition. Depth profile analysis of the coatings additionally confirms the uniform distribution of Ca and P through the TNT layer (Figure 3 c,d). The data obtained prove that the titania nanotubes are photocatalytically covered with HA through whole their length as shown schematically in Figure 2j (insert). XRD pattern of the HA-modified TNT coating is shown in Figure 4a. It should be noted that as-deposited HA is not well detectable in XRD spectra. Only peaks characteristic of anatase from the TNT coating are observed at 2θ: 29.4, 44.9, 47.0, 55.9 and 63.1°. After annealing the sample, HA signals can be seen in the XRD spectrum as crystalline phase (Figure 4a). The most intensive broad peak of HA is observed at 37° – 39°. Broadened peaks may indicate small size of HA crystallites. No other phases except TiO2 and HA have been detected. Raman spectroscopy was applied for further identification. The TNT has a weak signal at 800 cm–1 characteristic to anatase in this part of spectrum (Figure 4b, curve 1).21 After photocatalytic deposition of HA an additional band at 949 cm–1 and a broad band in the range of 1030 – 1100 cm–1 are seen in Figure 4b, curve 2, which could be a superposition of two narrower bands. Raman spectrum of polycrystalline HA as a standard is also measured and demonstrates the modes at 961, 1047 and 1074 cm–1 (Figure 4b, curve 3). Thus the additional Raman modes of the photocatalytically treated TNT electrodes can be assigned to HA. The main peak is shifted from 961 cm–1 to 949 cm–1 and widened The full width at half-maximum (FWHM) of this peak is 30 cm–1 while FWHM of standard peak is 10 cm–1. Its shift and broadening may be associated with nanocrystalline structure of the deposited HA.33


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The photocatalytical deposition can be used for HA patterning using the local illumination of the TNT surface. TNT coating has been illuminated locally remaining some part in the dark. HA can be formed on light-exposed areas, while not irradiated areas will be HA free. The biocompatibility of obtained HA-TNT coatings is demonstrated by adsorption of mixed proteins (Alice - ProteoGenix) labeled with the fluorescent dye (Figure 5a–c). The confocal fluorescence microscope images are analyzed in different time periods after the samples being in contact with protein solution. The protein adsorption occurs faster on HA-lined TNT regions (Figure 5 a,b) as compared to those without HA. In 5 min after contact with the proteins solution, the proteins adsorb directly on the areas with HA (Figure 5a,b). In comparison with HA neat TNT surface can also adsorb proteins but not so fast as HA. Thus in 20 min after contact with the proteins solution, the adsorption starts everywhere: on the TNT coating with and without HA (Figure 5c). Faster proteins adsorption onto HA side in comparison with TNT relies on several factors: (i) protein–material interactions such as specific binding at surface, non-specific binding through hydrogen bonding, electrostatic interactions which are faster to HA side,34 and (ii) surface characteristics such as energy and hydrophobicity, degree of crystallinity of HA preferable for the proteins, ability of charged

HA surface to adsorb biological molecules

containing carboxyl groups, phosphate groups and amino groups.34 Generally, the faster is protein adsorption the higher is biocompatibility, which is characteristic, in our case, for the side modified with HA. Thus it is expected higher biocompatibility of the surface coated with HA in comparison with TNT due to faster protein adsorption. Indeed cell density is significantly higher on TNT with HA coating (Figure 6).


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TNT surface with illuminated and non-illuminated areas, with HA and without HA, correspondly, are examined using cell culture (Figure 6c). We observe a dramatically lower cell number on the half with a TNT surface as compared to TNT-HA. It has been demonstrated in cell culture experiments on TNT of different sizes that adhesion, proliferation, and migration of mesenchymal stem cells are optimal on ordered nano-pore arrays with spacing in the range of 15 – 40 nm; these length scales also lead to significantly less apoptosis than on 100 nm structures.35 As it is mentioned above deposition of HA decreases pore size from ca. 60 nm to ca. 30 nm. Thus higher biocompatibility of the surface coated with HA in comparison with TNT can be also associated with smaller pore size.36. It is interesting that together with step like gradient in cell response to nanotopology, cells also have the same tendency with respect to shape, being spindle-like on TNT (Figure 6d, left inset (ca. 80% are spindle-like)). 4. CONCLUSIONS In summary, the photocatalytic deposition of hydroxyapatite onto photoactive titanium dioxide nanotube layer remaining TNT unique nanomorphology is very promising for the formation of HA layers with a high biocompatibility. In comparison to other known methods, e.g. chemical and electrochemical methods, the proposed method is attractive due to the following advantages. 1)

High pH of the solution is favours for HA formation.31

2)

HA is formed onto TNT coating during photocatalytic deposition in one-step procedure. Therefore there is no need in following hydrothermal treatment13 or soaking in NaOH,17 which are required in some chemical13,36 and electrochemical methods.12,17,21

3)

HA is deposited only on irradiated areas, providing the possibility of photoresist free HA patterning.


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Figure capture: Figure 1. a) Voltage–photocurrent dependence at +0.5 V (vs Ag/AgCl) of TNT coating in an electrolyte of 0.03 M NH4Cl, 0.02 M TEP at pH 10 under UV irradiation; b) Photocurrent–time dependence of TNT coating under anodic polarization at +0.5 V (vs Ag/AgCl) in an electrolyte of 0.03 M NH4Cl, 0.02 M TEP without 0.03 M CaCl2 (1) and with 0.03 M CaCl2 (2) at pH 10.

Figure 2. a–f, h) SEM images of TNT coating after HA photocatalytic deposition during: 0 min (a), 30 min (b), 60 min (c), 100 min (d), 120 min (e, h) and 150 min (f); j) Dependence of TNT inner pore diameter on time of photocatalytic deposition. Insert points the advantage of the method as a possibility to remain unique morphology of TNT coating after HA deposition.

Figure 3. a) SEM images of TNT coating with photocatalytically deposited HA (cross section, length of the nanotubes is marked by arrow); b) element distribution on the cross of TNT coating with photocatalytically deposited HA, image obtained by EDS analysis; c–d) depth profile analysis of the TNT coatings (c) and TNT coating with photocatalytically deposited HA (d).

Figure 4. a) X-ray diffraction pattern of TNT nanotubes with HA deposited photocatalytically. HA-modified TNT samples were annealed at 450°C for 3 h; b) Raman spectra of TNT coating (1), photocatalytically deposited HA on TNT coating (2), and polycrystalline HA powder, as a control (3).

Figure 5. Confocal fluorescence microscope images of TNT substrate with areas with (signed as TNT) and without (signed as TNT/HA) photocatalytically deposited HA: a) after 5 min being in


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contact with proteins solution; b) increased resolution image of (a); and c) after 20 min of being in contact with proteins solution; d) The mean fluorescence intensity of mixed proteins labeled with DAPI on TNT substrate with areas with and without photocatalytically deposited HA. Hydroxyapatite side adsorbs proteins faster in comparison with TNT side.

Figure 6. Pre-osteoblasts MC3T3-E1 cell growth on different substrates. Immunofluorescent analysis on cell density and morphology after 2 days of culturing on different substrate: a) TNT; b) TNT-HA; and c) TNT substrate with areas with and without photocatalytically deposited HA. Cells were stained for actin using phalloidin and TO-PRO-3 iodide for nuclei; d) Cell density on different surfaces as culture time prolonged. Insets depict cell morphologies on TNT (left) and TNT-HA (right) pointed percentage of elongated-cells on corresponding substrate. Bar charts represent means ±SD from 3 replicates each from 3 independent experiments. Comparison was done by ANOVA *p < 0.05.


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Figure 1.


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Figure 2.


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Figure 3.


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Figure 4.


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Figure 5.


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Figure 6.


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AUTHOR INFORMATION Corresponding Author * Corresponding author. E-mail: [email protected]. Present Addresses † Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14424 Potsdam, Germany. E-mail: [email protected] ACKNOWLEDGMENT Authors thank Cristina Pilz (MPIKG), Rona Pitschke and Heike Runge (MPIKG) for help in the laboratory, providing protocols and all advices.

REFERENCES (1) Akin, F. A.; Zreiqat, H.; Jordan, S.; Wijesundara, M. B. J.; Hanley, L. Preparation and analysis of macroporous TiO2 films on Ti surfaces for bone–tissue implants. J. Biomed. Mater. Res. 2001, 57, 588–596. (2) Sul, Y. Electrochemical growth behavior, surface properties, and enhanced in vivo bone response of TiO2 nanotubes on microstructured surfaces of blasted, screw-shaped titanium implants. Int. J. Nanomedicine. 2010, 5, 87. (3) Tsuchiya, H.; Macak, J. M.; Müller, L.; Kunze, J.; Müller, J.; Greil, P.; Virtanen, S.; Schmuki, P. Hydroxyapatite growth on anodic TiO2 nanotubes. J. Biomed. Mater. Res. 2006, 77A, 534–541. (4) Groot, K. De.; Geesink, R.; Klein, C.; Serekian, P. hydroxylapatite. J. Biomed. Mater. Res. 2004, 21, 1375–1381.

Plasma sprayed coatings of

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Photocatalytic Deposition of Hydroxyapatite onto a Titanium Dioxide

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