Materials Science and Engineering C 37 (2014) 390–398

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Materials Science and Engineering C

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Photoinduced properties of nanocrystalline TiO2-anatase coating onTi-based bone implants

Martina Lorenzetti a,b, Daniele Biglino a, Saša Novak a,b,⁎, Spomenka Kobe a,b

a Jožef Stefan Institute, Ljubljana, Sloveniab Jožef Stefan International Postgraduate School, Ljubljana, Slovenia

⁎ Corresponding author.E-mail address: sasa.novak@ijs.si (S. Novak).

0928-4931/$ – see front matter © 2014 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.msec.2014.01.029

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 July 2013Received in revised form 14 November 2013Accepted 18 January 2014Available online 25 January 2014

Keywords:Antibacterial effectTiO2 nanocrystalsHydrothermal treatmentPhotocatalytic activityPhoto-induced wettability

The paper reports on the photoinduced properties of hydrothermally treated (HT) titanium used for boneimplants. The anatase coatings composed of 30–100 nm anatase crystals exhibited high photocatalytic activityand good photo-induced wettability, reaching a superhydrophilic state, despite the larger crystal dimensionsthan the previously reported optimal ones. These properties are due to a suitable combination of surface texture,roughness, thickness, crystal morphology and particle size, which allowed the two independent photo-inducedphenomena to occur simultaneously. The results on caffeine degradation by photocatalysis and the prolongedeffect (up to twoweeks) of photo-inducedwettability in dark suggested a possible applicability of the HT anatasecoatings as bacteria-repelling surfaces for body implants, in favor of a better osseointegration in vivo.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Demand for hard tissue replacement is growing rapidly, caused bythe raise of aged people ratio and the increase of crippling accidentnumber. Despite the great amount of metals and alloys available, onlyfew of them are suitable candidates as biomaterials for implants. Thesuccess of titanium and its alloys is due to their intrinsic characteristics.First of all, from the mechanical point of view they have a good specificstrength and acceptable corrosion resistance; then, the evidence ofallergies is relatively low; finally, they present the best biocompatibilityamong metallic materials, thanks to a natural, amorphous passivetitanium oxide layer on the surface [1]. However, several problemsconcerned to surface properties have still to be solved, such as a poorbioactivity, insufficient biocompatibility on long term (e.g. release oftoxic ions) and development of infections. Infections in particular arecommon serious complications after implant surgery. It has beenestimated that implant-associated infections occur in 1–5% of theapproximately 606,000 total knee arthroplasties and 423,000 total hiparthroplasties in the US, which usually have to be solved by furtherrevision surgeries [2]. In addition, it is proven that the presence of animplant itself in the human body increases the predisposition toinfections, activating the host immune system, up to the failure of theimplant itself by rejection [3]. Therefore, it is clearly understandablehow the growing demand of sterility control in body implants hasgiven the input to realize devices with peculiar modified surfaces.One of the strategies is to improve the characteristics of the naturally

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formed amorphous TiO2 layer, by producing a more stable and thickercrystalline titania layer by various surface modification techniques,based on mechanical, chemical, physical or electrochemical methods.Among them, hydrothermal synthesis (HT) is a well-known methodfor crystal growing. Recently, this technique was used in order tosynthesize titania coatings on various substrates, such as metals [4–6].Hydrothermal treatment appeared as a simple and cost-effective tech-nique to produce thin firmly attached layers of anatase of well-definedmorphology and crystallography.

Moreover, as semiconductor, anatase is a well-known photocatalystand has been used in various applications, among which self-cleaningand self-sterilizing, air and water purification, deodorization, etc., thankthe occurrence of two photo-induced phenomena, heterogeneousphotocatalysis (expressed as photocatalytic activity) and photo-inducedwettability (super-hydrophilicity). In addition, the same phenomenamay be potentially employed in prevention of bacteria attachment ontitanium bone implants. The photocatalytic activity helps to degradeorganics that come in contactwith the surface, while a good hydrophilic-ity makes cleaning more efficient. The two photo-induced phenomenacan occur simultaneously on the same TiO2 surface and act mutually toeach other, even if they are theoretically different [7,8]. Heterogeneousphotocatalysis happens when two or more phases are involved inthe photocatalytic reaction; in the case of titanium dioxide, used asphotocatalyst, it is present in solid form, while the second phase wouldbe either liquid (e.g. in water suspension) or gas (in air). A light sourceis used to initiate the photoreaction by activation of the semiconductormaterial (photocatalyst), giving raise to photomineralization or hetero-geneous photocatalytic oxidation (PCO). PCO mechanism has beenintensively studied, so that today the mechanism is elucidated [7]. On

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the other hand, following another electronic pathway, titania coatingirradiation can result in another process, causing photo-induced wetta-bility on the surface and resulting in a superhydrophilic state.

As already known, dimensions and morphology of anatase crystalshave an influence on the photoactivity of the coatings. It was shownthat very small anatase nanocrystals (range: 10–20 nm) and/or texturedanatase nanocrystals, in particular presenting well developed (001)planes, have the highest performances in terms of photocatalytic activity,due to the high amount of very reactive surface oxygens O2c and surfaceunder-coordinated Ti atoms [9,10]. Thus, lots of efforts have been donein order to optimize the production of anatase nanoparticles withexposed (001) facets, since firstly Yang et al. [11] discovered an HTsynthetic route, using HF to stabilize and decrease the high surfaceenergy of (001) plane. Recently, anatase coatings with exposed (001)crystal facets were obtained by HT using HF solution with differentpHs by adding NaOH [12]; the study assessed that at pH N 5.8 thecontent of F− is insufficient to preserve (001) facets formation, becausethe surface fluorination absorption of Na+F− is thermodynamicallyprohibited.

Based on the calculations done by Barnard et al. [13] and on recentresults assessed in our research group [6], a simple method has beendeveloped to synthesize anatase polycrystalline coatingswith a texturedsurface structure by different hydrothermal treatment on Ti-substratesin weakly-acidic and basic pH (up to pH = 10) conditions, avoidingthe use of toxic and corrosive precursors such as HF, HCl or fluorine/chlorine complexes.

Taking into account the aforementioned statements, we believe thata TiO2-anatase coating, due to its nature, offers the possibility to beemployed not only as a barrier to hinder alloying elements release[14] and to enhance bioactivity of the implant [15,16], but also as aphotosensitive coating to prevent bacterial infections before and duringthe implantation. In this preliminary stage, preceding bacterial tests,photo-induced wettability and photocatalytic activity of the HT TiO2

coatings were thoroughly studied with the aim to maximize theirpotential effect. Thus, the aim of this paper was to connect physico-chemical characteristics of the coatings with their photo-inducedproperties and to identify the most promising coating structure tobe applied on bone prosthesis.

2. Materials and methods

2.1. Materials

The growth of TiO2-anatase nanostructured coatings was performedon commercially pure titanium (cp Ti grade 2, ASTM F67, Pro-titanium,China) in the formof discswith a diameter of 15mm, thickness of 2mm,and grooves of 0.03 mm width after machining and on cpTi electropolished plates of 15 mm × 10 mm and thickness of 1 mm(Morphoplant GmbH, Germany).

Titanium(IV) isopropoxide (Ti(iOPr)4, Acros Organics, Belgium),TiO2 powder Aeroxid P25 (Degussa/Evonic GmbH, Germany), absoluteethanol (EtOH, Carlo Erba, Italy), potassium hydroxide (KOH, CarloErba, Italy), tetramethylammonium hydroxide (TMAH, Sigma-AldrichChemie GmbH, Germany), caffeine (Sigma-Aldrich Chemie GmbH,Germany), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide(DEPMPO, C9H18NO4P, Vinci Biochemicals), silicon carbide paper(SiC paper, # 4000, Struers, Denmark). All chemicals were used asreceived without any further purification.

2.2. Experimental

2.2.1. SynthesisBefore hydrothermal treatment, the substrates were cleaned twice

in de-ionized water and once in EtOH for 10 min each in ultrasonicbath (Sonorex, Bandelin Electronic, Germany). The aqueous suspen-sions were prepared by using Ti(iOPr)4, thus in acidic pH, and,

eventually, adjusting the pH up to 10, adding TMAH. Two sampleswere prepared by using the Ti(iOPr)4 suspension but on the two differ-ent substrates (sample A on electropolished cp Ti, sample B on ma-chined (M) cp Ti substrate, respectively); the third sample wasobtained by using the suspension with Ti(iOPr)4 and TMAH (sampleC). The half-filled Teflon vessels, containing also the cleaned substrates,were then placed in steel autoclaves and heated to 200 °C for 24 h(APT.line, Binder GmbH, Germany). The hydrothermally treated sam-ples were cleaned as mentioned above and dried in air.

Non-treated substrates of cp Tiwere used as reference for comparingTiO2 crystal morphology and wetting angles with the HT samples. Inaddition, substrates of cp Ti were spin-coated for 10 consecutive timeswith a 0.1 M suspension of P25 powder in absolute ethanol at3000 rpm for 35 s and then dried for 150 min at 200 °C; such preparedsamples (TiP25) were used for comparison during experiments ofphotocatalysis, since P25 is known to be one of the most photo-activepowders among the commercial ones.

2.2.2. Coatings characterizationThe morphology of the TiO2-anatase crystals was checked by field-

emission scanning electron microscopy (FEG-SEM, Zeiss SUPRA 35VP,Carl Zeiss SMT, Germany and JEOL JSM 7600 F, Japan), while the crystalstructure was analyzed by Raman spectroscopy (T64000 Jobin Yvonspectrometer, Horiba Scientific, Japan), with frequencies scannedbetween 100 cm−1 and 700 cm−1 using a 532 nm laser with a powerof 20mW, focused through an100×microscope objective, an acquisitiontime of 60 s on 5 different spots per each sample.

Surface roughnesswas examined by optical interferometer (CountorGT, Bruker Corporation, Germany) on larger areas (2.0 × 1.40mm2, 240× 180 μm2), and by atomic forcemicroscope (AFM, Solver Pro, NT-MDT,Russia) on a smaller area (1 × 1 μm2). Profile mean roughness (Ra), sur-facemean roughness (Sa) and rootmean square (Sq) were calculated on3 different areas per each magnification, so that the values areexpressed as average; Ra was evaluated on the largest area, inaccordance with the specifications contained in the standard guidanceISO 4288:1996.

2.2.3. Photo-induced wettability and photocatalytic activity measurementsSessile drop contact angles (CA) of the discs were measured using

Theta Lite T101 optical tensiometer (Attension, Biolin Scientific,Finland). Five droplets of distilled water were placed at differentpositions on each sample and the average value was taken as contactangle measure, with a standard deviation (σ) calculated as ±5°. Themeasurements were performed before and after 15 h of irradiation ofthe samples; the recovery was measured on pre-irradiated samples,after storing them in dark for 7 and 14 days.

The pre-irradiation of samples to check the photo-induced hydro-philicity, as well as photocatalytic activity experiments on HT treatedsubstrates for the decomposition of caffeine, were performed at 20 °Cin a climatic chamber equipped with cooling system (I-265 CK UV,Kambič Laboratory Equipment, Slovenia), using Osram's Ultra Vitaluxbulb with simulated solar spectrum (white light, UVA, UVB) withoutUVC and total integrated irradiance intensity of 3 mW/cm2.

During the photocatalytic activity experiments, each sample wasplaced in a 8 ml quartz reactor containing 2 ml of caffeine solution at10 mg/l concentration, covered by a quartz cap. In the first 30 min thereactor was left in dark, to achieve absorption–desorption equilibrium.Afterwards, the reactor was exposed to illumination (t = 0 min forreaction) and samples were taken out at 0, 60, 120 and 180 min.Quantities of degraded caffeine and absorption spectra were mea-sured by high-precision UV/Vis/NIR spectrophotometer Lambda950 (PerkinElmer, US).

The photocatalytic activity of the TiO2 surface coating was addition-ally estimated using spin-trapping electron paramagnetic resonance(EPR) spectroscopy. EPR spectra were recorded at RT employinga Bruker Elexsys SuperX E500 continuous wave X-band (9–10 GHz)

Table 1Summary of synthesis conditions and titania grown crystal information for samples A, B and C.

Sample Substrate pH condition Prevalent TiO2 phase Estimate crystal size (nm)

A Electropolished (EP) Acidic (pH≈ 5) Anatase 50–100B Machined (M) Acidic (pH≈ 5) Anatase 30–70C Machined (M) Basic (pH≈ 10) Anatase 20–30

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spectrometer, equipped with standard Super-High-Q cavity. A dropletof a solution containing 0.05 M DEPMPO spin-trap in 30% ethanol(adding KOH to obtain a final pH ≈ 7.5) was placed on the coateddiscs, as well as on cp Ti and TiP25. Each disc was irradiated withdifferent wavelength (290 nm, 320 nm) diodes for 5 min. The solutionwas taken up into an extra-pure quartz capillary tube and placed inthe spectrometer cavity to record the EPR spectra. For comparison, thesame procedure was performed on not irradiated samples.

EPR spectra were simulated and fitted using EasySpin MatlabMathwork tool [17]. Isotropic and fast-motion cw EPR spectra functionwas used to calculate the simulated spectra, genetic algorithm wasemployed to obtain the best fitting.

The absorbance spectra were recorded by UV/Vis/NIR spectropho-tometer Lambda 950, equipped with an integrating sphere module(PerkinElmer, 150 mm) for diffusive reflectance measurements; thespectra were collected between 250 nm and 800 nm by two differentmodes: firstly acquiring diffuse reflectance curves directly from thecoated samples; secondly, measuring light absorption on suspensions

Fig. 1. SEM micrographs at different magnifications of:

obtained by mixing distilled water with scratched crystals from thecoatings. The scratching was achieved by using SiC paper. The bandgap values were estimated by direct extrapolation of the absorptionedge at the intersection point between its tangent and the flat baselineof the spectrum.

3. Results and discussion

The results are presented as a comparison in surface propertiesbetween samples synthesized by the same hydrothermal (HT) treatmentbut on different substrates (sample A vs. sample B) and among samplessynthesized by different HT treatments but on the same substrate(sample B vs. sample C), as shown in Table 1.

3.1. Morphological characteristics of the coatings

After synthesis, each titania coating appeared differently colored,depending on coating thickness [18,19], changing from the light gray

a, b) sample A; c, d) sample B; and d, e) sample C.

Fig. 2. Raman spectra of samples A, B and C, showing characteristic anatase peaks atfrequencies of 148 cm−1, 203 cm−1, 401 cm−1, 522 cm−1 and 639 cm−1.

Table 2Profile mean roughness (Ra), surface mean roughness (Sa) and root mean square(Sq) values for samples A, B and C, obtained by optical interferometer (scanned area:240 × 180 μm2) and AFM (scanned area:1 × 1 μm2).

Sample Area: 240 × 180 μm2 Area: 1 × 1 μm2 (AFM)

Sa (nm) Sq (nm) Sa (nm) Sq (nm)

A 189 ± 15 257 ± 21 3.1 ± 0.7 3.9 ± 0.8B 324 ± 8 397 ± 4 8.9 ± 1.8 11.0 ± 2.1C 540 ± 34 647 ± 37 9.8 ± 0.3 12.7 ± 0.1

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color of the pure titanium substrates to the light gold (sample A), darkgold (sample B) and dark gray-violet (sample C) colors. FEG-SEMmicrographs (Fig. 1) showed that the coatings were homogeneousand covered the samples surface, following the grooves of machining(original surface roughness) for samples B and C. Estimated TiO2

crystal dimensions were in the range of 50–100 nm for the sample A,synthesized by using acidic solution on a flat surface, 30–70 nm for thesample B, synthesized by using acidic solution on a machined surface,down to 20–30 nm for the sample C prepared from alkaline solution.In addition, sample C presented a multi-layered structure with thepresence of pores, as evident from Fig. 1e.

The variety in TiO2 crystal dimensions observed by SEM imaging canbe associated with different amounts of formed nuclei and growth rate.Nucleation can be influenced by several factors; first of all, surfaceroughness played a role in the nucleation of sample B in comparisonwith sample A. The grooves obtained by surface machining providededges and surface defects which acted as active sites of higher bindingenergy and drove the further incorporation of ions from the solution.Thus, a higher amount of nuclei occurred on sample B than sample A,with a consequent growth of more numerous but smaller crystals.Secondly, nucleation was increased by using TMAH as pH modifier incase of sample C; it was already demonstrated [20] that TMAH, asorganic alkali, is able to form hydrogen bonds with titanium, whichis then released during HT, providing further Ti sources for crystaldevelopment; in our case, higher concentration of free Ti ions resultedin a higher amount of nanocrystalswith smaller dimensions (sample C).

Among the characteristics of bone implants, surface roughness andtopography are also very important, as they affect the bacteria adhesion[21] as well as photoinduced properties of a catalyst. To describe thesurface topography of the samples, surface mean roughness (Sa) androot mean square (Sq) were taken into account; the calculated valuesare reported in Table 2. The presence of crystals on the surface increasedthe surface roughness in comparison with the not HT substrates (Ti NTelectropolished vs. sample A, Ti NT machined vs. samples B and C).As presented in Fig. 2a, c, e, on the large scale (Area: 240 × 180 μm2)topography of sample A appeared smoother than samples B and C,despite the polycrystalline coatings, due to the different starting substrateroughness, polished or machined respectively. Moving to smallerscanned surface areas, crystal size played also a role, with a prominentincrease in roughness for sample C rather than for sample B (both frommachined substrates). This effect was even more accentuated at thehighest scanning magnification used (Fig. 3b, d, f); on scanned areasof 1 × 1 μm2 in fact, where the influence of the substrate waviness(machined grooves) was avoided, roughness became a function oftexture and crystal size (Table 1), following the sequence of increasingroughness as: sample A N sample B N sample C.

3.2. Crystal structure

Raman spectroscopy spectra (Fig. 3) on the titania coatings revealedpeaks at frequencies corresponding to titanium dioxide in the polymor-phic phase of anatase, at frequencies of 148 cm−1 (B1g mode, veryintense), 203 cm−1 (Egmode, veryweak), 401 cm−1 (B1gmode, intense),522 cm−1 (A1g, B1g modes, less intense) and 639 cm−1 (Eg mode, less

intense) [22–24]. No other peaks were detected, excluding the presenceof other crystalline phases.

3.3. Photo-induced wettability

In order to characterize the photo-induced phenomena, whichcan occur on photo-activated TiO2 nanocrystals, the photo-inducedwettability and photocatalytic activity of the polycrystalline anatasecoatings were explored. The photo-inducedwettability of the examinedsurface coatings was observed by evaluating surface contact angleby sessile drop measurement of distilled water before and after 15 hunder UV–vis irradiation on freshly cleaned surfaces. The resultsare summarized in Fig. 4. The comparison between the not treatedsubstrates revealed that the contact angle was slightly higher for thepolished (CA ≈ 70°) than for the machined surface (CA ≈ 62°). Thepresence of HT TiO2 layers on the surface was sufficient to reduce thecontact angle with respect to the bare Ti substrate, lowering the valuesto 53° (−25%), 50° (−20%) and 12° (−80%) for samples A, B and Crespectively. The observed differences among the samples A, B and Ccan be ascribed to their various surface roughness: larger effect ofcoating was observed with increasing roughness. In addition, sample Callows a capillary effect, due to a higher surface porosity (Fig. 1e) thansamples A and B, which enhances surface water wettability [25].

Hence, the behavior shown by the examined HT samples revealedthe capability of the titania coatings to render the surface morehydrophilic. As expected, further reduction of surface contact anglevalues on the coatings occurred after UV–vis irradiation, resulting ina fully-hydrophilic behavior in case of samples A (CA ≈ 48°) and B(CA ≈ 37°) and a super-hydrophilic behavior in case of sample C (CA≈ 4°), as visible in Fig. 4. Finally, the recovery of the initial wettingstate occurred only within 1 week for sample A (CA≈ 55°) and within2 weeks for samples B (CA ≈ 46°) and C (CA ≈ 10°). Given thatthe photogenerated hydroxyl groups absorbed on the surface afterirradiation are thermodynamically metastable, the photoinducedwettability is known to be reversible in dark. Even if the debate overthe mechanism of CA recovery in dark is still open, the most believedtheory assesses that, in presence of oxygen, the chemisorbed hydroxylgroups gradually desorb and the surface defect sites are graduallyreplaced by oxygen atoms; however, the reverse conversion happenswith a rate much slower than that of the photo-induced hydrophilicprocess [26,27]. Another possible explanation for the observed recoveryis the gradual re-adsorption of nonpolar impurities from surroundings.The obtained results hence suggest enhanced lifetime of the metastablephoto-induced hydrophilic state, which is supposed to be beneficial forthe implant behavior in the early stage of implantation [21].

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On the other hand, no CA variation was observed for both the nottreated substrates after irradiation, confirming that the photo-inducedeffect can occur only in the presence of titania crystals, which actas photocatalyst.

3.4. Photocatalytic activity: organics degradation

Spectrophotometric analysis of organic compound degradation isone of the most used methods for studying TiO2 photoactivity. Caffeinewas chosen as appropriate degradation molecule model for differentreasons: its absorption peak does not interfere with TiO2 absorptionone, as well as caffeine degradation products, it is not sensitive to pHchanges and it is easily quantifiable by spectrophotometer [28,29].

Degradation of caffeine molecule by photocatalytic oxidation (PCO)process of the titania coating was monitored over 180 min of exposureto UV–vis irradiation and is illustrated in Fig. 5. As expected, no changein caffeine concentration occurred on the non-coated Ti, as well as onthe non-irradiated HT samples. Irradiation of the samples A, B and Cled to a drop in caffeine concentration, which occurred with differentrates, as evidenced by the reaction rate constants K reported in

Fig. 3. Images of surface roughness obtained by optical interferometer (left, scanned a(left, palette bar: −1.95/+2.94 μm, right, palette bar: 0/+20 nm); c, d) sample B (left(left, palette bar: −2.52/+3.20 μm, right, palette bar: +10/+110 nm).

Table 3; K values were calculated by formula (1) assuming an apparentfirst-order reaction:

−ln C=C0ð Þ ¼ K � t ð1Þ

where C is the caffeine concentration at a certain time t and C0 is theinitial caffeine concentration [23].

For comparison, degradation of caffeine by spin-coated P25 powderon Ti substrate (TiP25) was also measured.

Caffeine degradation experiments revealed that all the preparedcoatings possess a high ability to destroy the caffeine moleculescontained in an aqueous solution of 10 mg/l in concentration, reachingthe total (100%) and quasi-total (92% and 79%) caffeine degradationin case of sample A and samples B and C respectively, within 3 h ofexposure to UV–vis light excitation (Fig. 5). As revealed by comparingthe reaction rate constants, the HT coatings exhibited only slightlylower photoactivity than the deposited TiO2 P25 powder (particlesize ~20 nm), although they were much thinner and the crystal sizewas larger. While the coating on the sample A decomposed caffeinewithin 3 h of irradiation, in the case of TiP25 only 2 h was needed for

rea: 240 × 180 μm2) and AFM (right, scanned area: 1 × 1 μm2) of: a, b) sample A, palette bar: −2.70/+3.54 μm, right, palette bar: 0/+70 nm); and d, e) sample C

Table 3Calculated reaction rate constants for caffeine degradation of samples TiP25, A, B, C.

Reaction rate constant k (×10−3 s−1)

TiP25 0.120A 0.105B 0.058C 0.028

Fig. 4. Contact angle values measured before UV–vis irradiation (NO IRR), after 15 h ofirradiation (15 h IRR), after 1 week of storage in dark (1 week recovery) and after2 weeks of storage in dark (2 weeks recovery) on the not-treated substrates (Ti NTelectropolished and Ti NT machined) and on samples A, B and C.

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the same effect. However, this result has to be carefully evaluated, sincethe P25 particles were just spin-coated on the surface, thus, not stablyattached; this means that part of them could have been detached andsuspended into the caffeine solution, with an increase of exposed sur-face area and, consequently, a higher organic degradation.

It is known that crystal dimensions are crucial for a good e−-holepair formation: the smaller the diameter, the higher the probability toexcite electrons before they recombine with free holes at the surface.It has been reported for several years [30] that anatase powdersconsisting of crystals smaller than 20 nm possess the ideal dimensionfor the best photocatalytic activity. However, the optimum crystallitesize significantly increases in thin films, due to a different charge carriermigration which proceeds through multiple inter-granular transferstoward the surface [8]. Surprisingly, according to our results, HTcoatings with crystals in the range of 50–100 nm (samples A and B)appeared the most effective in caffeine degradation, reaching evencomplete disruption of the pollutant. This optimum size is twice higherthan optimum crystal dimensions previously reported for immobilizedpowders on supports (30–50 nm) [8].

Additionally, other parameters can influence photocatalytic activity ofthin coatings. For instance, the effective grain size (secondary aggregation

Fig. 5. Caffeine degradation occurred in 3 h of UV–vis irradiation of the solution in contactwith the not treated substrate and with samples TiP25, A, B and C.

withinpolycrystalline particles) largely affects thequality of photoactivityof a film; thus, even if an apparent optimal size was reached in sample C,smaller grainsmay allow a faster e−-hole recombination rate and, conse-quently, a slightly slower photoactivity [23]. Besides, it was reported thatphotocatalytic activity depends also on coating thickness, and a criticalsaturation thickness exists. It was also suggested that thewhole thicknesscontributes to the phenomenon (from ≈100 nm up to hundreds nm,depending on the synthesis techniques) [8,31]; by both Auger Elec-tron Spectroscopy and Focused Ion Beam analysis (data not shown),sample C appeared 4 times thicker than sample B (120 ± 40 nm vs.36 ± 10 nm). The grain size effect in combination with the higherthickness could be two factors that strongly altered the photocata-lytic activity of sample C, enhancing charge recombination probabil-ity and giving the reasons why this coating is less photoactive,despite the smaller nanocrystals.

The observed photo-activity of the HT samples was just reversecompared to the CA value trend; for instance the superhydrophilicsample C is the least active in caffeine degradation. This supports thestatement that the two photo-induced phenomena are not connected,since they are caused by different electronic pathways.

3.5. UV–vis absorption spectra

The coatings were further analyzed by UV–vis spectroscopy. First,absorption spectra acquisition was performed directly on titania layersof samples A, B, C and TiP25 by diffusive reflectance measurements.As shown in Fig. 6a, TiP25 exhibited peaks in the visible region, givenby the substrate absorption contribution, and also an intense peak inUV-A, corresponding to P25 titania absorption; on the other hand, HTsamples presented just peaks in the visible spectrum, but not in theUV-A region, where titaniumdioxide absorbs. The comparison betweenthe spectra of samples A, B and C with TiP25, obtained directly from thecoatings, confirmed that the titania crystal absorption peak of the HTsamples was hidden below the more intense peaks coming from theTi-substrate; just in the case of the spin-coated TiP25 sample, wherethe layer thickness was up to 1 μm, it was possible to recognized theP25 peak in the UV-A region and also to calculate its band gap value(Table 4).

Thus, in the next step the titania crystals were scratched from thethree coatings (A, B, C) and suspended in de-ionized water, in order toget information selectively from the synthesized titania layers. In thiscase (Fig. 6b), the obtained UV–vis absorption spectra (absorbance)revealed a very similar peak in UV-A region, with a clear jump centeredat around 320 nm assignable to anatase phase, without any otherfeature in the visible part of the spectrum. Band gap wavelengths andestimated band gap energies are shown in Table 4. The exact positionof the peak and, consequently, the band gap change with the materialand with the aggregation state. Anatase in powder state possesses avery peculiar spectrum of absorption with the shape of a reverse-Scurve and a very specific band gap (3.20 eV); in contrast, the spectrumof adsorption shifts in case of thin films. The spectra appeared verysimilar in intensities and shape, but they presented slightly differentband gap values (still within the error bar) in the range from 3.39 eVto 3.43 eV (Table 4). This is in accordance with the trend obtained byTanemura et al. [32]: titania coatings present a broader band gap thantitania powders (bulk), resulting in 3.39 eV for polycrystalline anatase

Fig. 6. Absorption spectra a) collected by diffuse reflectance mode on samples TiP25, A, B and C and b) on aqueous suspensions of scratched crystals from coatings of samples A, B and C.

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films. In addition, band gap is influenced by crystal structure, grain sizeand morphology [33], which may explain the shift in value of ourcoatings.

Moreover, the optical absorbance reached higher intensities, movingtoward higher energies (lowerwavelengths); for instance the absorbancewas higher at thewavelength of 290 nm than the one of 320 nm. It can besupposed that higher amount of absorbed photons will induce higheramount of electrons in excited states, resulting in an expected higherradical formation at 290 nm rather than at 320 nm.

3.6. Photocatalytic activity: radical formation

In order to confirm the radical production associated to caffeinedegradation experiments, EPR measurements were also carried out,using emitting diodes at 320 nm and 290 nm for the photoactivationof the coatings. The wavelengths were selected in accordance with theabsorption spectra (Fig. 6), in order to ensure the sufficient amount ofenergy (3.87 eV at 320 nm, 4.27 eV at 290 nm) to produce surfaceelectron excitation, being higher than the theoretical band gap energyof bulk anatase (3.20 eV at 387 nm).

EPR has the important characteristic to detect exclusively paramag-netic species, such as free radicals; the region of the electromagneticspectrum (microwave) used to detect the radicals is far from the oneused to irradiate the sample (UV–vis), therefore no interferencebetween the generation and detection of radicals occurs. Primaryradicals (•OH, O2•

−) generated during PCO on TiO2 are short life species,therefore they are not easily detectable. Ethanol is a scavenger of thefree radicals, particularly effectivewith hydroxyl radicals, by generationof new, more stable EtOH• radicals; these subsequently react with spin-trap DEPMPO, generating a stable spin adduct, DEPMPO–CH(CH3)OH•,that can be easier detected by EPR [34].

In Fig. 7 the experimental (Fig. 7a) and simulated (Fig. 7b) EPRspectra of sample A were compared (similar spectra were obtained forthe samples B, C, and TiP25 as well). The analysis of the simulationrevealed the presence of two species. By comparing the hyperfine cou-plings obtained by the simulation with the ones in literature (Table 5),itwaspossible to assign thefirst signal to theDEPMPO–CH(CH3)OH• ad-duct. The hyperfine couplings of the second species are similar to the

Table 4Band gap absorption edgewavelengths and estimated band gap energies from absorptionspectra of samples TiP25, A, B and C.

Sample Absorption edge (nm) Band gap energy (eV)

A 362 ± 5 3.43 ± 0.04B 366 ± 5 3.39 ± 0.04C 361 ± 5 3.43 ± 0.04

ones for DEPMPO–CH3•, therefore it is reasonable to be assigned to anorganic radical species, probably a by-product of the decomposition ofthe spin-trap itself. The second species is present in all spectra, includingthe ones recorded on cp Ti (Fig. 7c), suggesting that the organic speciesis not photo-catalytically generated. EPR experiments were also carriedout on both not irradiated and overnight pre-irradiated samples (datanot shown); in both cases the recorded spectra showed only the signalof the by-product radical, confirming that samples are not photo-activeif not illuminated or just pre-irradiated. Indeed, the photo-excited elec-trons, induced during the overnight pre-irradiation (in air), could notbe stabilized into this excited state longer enough to show a prolongedphotocatalytic effect in dark before their recombination; thus, pre-irradiation of the coatings had no effect on photocatalytic activity, butit had effect on wettability, as mentioned above.

In Fig. 8 the peak-to-peak intensities of the first line of EPR spectraare compared both for each sample and at different irradiationwavelengths. The activity, obtained from the analysis of peak-to-peakintensity of the first spectrum line, did not show any significantdifference between samples A, B, and C (Fig. 8): it is comparable at thesame wavelength (within the error) and it is about 80–100% higherwhen irradiated at 290 nm than at 320 nm, in agreement with thetrend revealed by the absorption spectra. On the other hand, TiP25 pre-sented a higher activity at 320 nm rather than at 290 nm, due to its shiftin absorption (Fig. 6); however, the TiP25 activity was in the samemag-nitude order of the TiO2 coated samples ones.

Fig. 7. EPR spectra of: a) sample A (experimental); b) sample A (simulated); and c) cp Ti(experimental).

Table 5Hyperfine coupling (in mT) of P, H, and N obtained from the EPR simulated spectra.

A(P) A(H) A(N) Ref.

Hydroxyl radical 4.90848 1.46263 2.13060 This workDEPMPO–CH(CH3)OH 4.90 1.45 2.14 [34]By-product radical 4.67369 1.43618 2.26213 This work

4.74932 1.44933 2.03530DEPMPO–CH3 4.77 1.52 2.23 [35]

397M. Lorenzetti et al. / Materials Science and Engineering C 37 (2014) 390–398

The EPR experiments confirmed that a well-defined morphology ofthe TiO2 coatings is essential for the photocatalytic activity; however, nosignificant variation in the amount of produced radicals, due to differ-ences inmorphology of the as-prepared hydrothermally treated coatings,was revealed. Comparing this statement with the results of caffeine deg-radation, it can be assessed that crystal morphology and coating rough-ness play an important role during the surface-pollutant interaction. Infact, a better absorption and, accordingly, a better contact between caf-feine molecules and titania crystals on samples A and B (largernanocrystals and lower roughness), rather than on sample C (smallernanocrystals and higher roughness),may lead to a higher andmore directattack of OH radicals; consequently, it can explain the higher caffeine deg-radation on the former coatings.

A combination of the spectrophotometric and EPR techniques gives adeeper understandingof titania coating behavior during PCOprocess: EPRhighlights the OH radicals production, while caffeine degradation revealsthe role of photocatalyst surfacemorphology duringOH radicals–organicsreaction.

Hence, it is proposed that a combination of the two photo-inducedphenomena could be a helpful and new approach in order to providegood conditions for enhancing cell attachment and proliferation and, ontheother hand, obtaining an antibacterial surface by inhibition of bacterialadhesion on it, with a consequent prevention of biofilm formation.

4. Conclusion

The growing necessity for metallic bone implants has led variousgroups to study in detail the material surface properties and reducingbiomaterials associated infections with the aim to improve their firmand durable integration with bone. In this study, hydrothermal processwas used to synthesize a crystalline anatase coating that would hypo-thetically have photoinduced antibacterial effect. To prepare the mostactive coatings for further bacterial tests, in this first stage of thestudy, surface properties of the coatings were examined; therefore,

Fig. 8. Comparison of peak-to-peak EPR intensity of samples A, B, C and TiP25 at 290 nmand 320 nm.

here we report on photo-induced phenomena, namely photo-inducedwettability and photocatalysis, of HT prepared titania coatings, support-ed by photo-induced radical formation studies.

The HT coatings were composed predominantly of anatase crystals inthe range of 20–100 nm. It was found that initial surface roughness of thesubstrate andpreparation conditions influenced size and shape of anatasecrystals, as well as the coating thickness. The presence of hydrothermallytreated coatings influencedwettability, leading tomore hydrophilic char-acter than bare substrates and reaching values of contact angles down12°. Themost pronounced change inwettingwasobserved for the sampleC, which was characterized with the smallest particles. The wetting wasfurther affected by UV–vis irradiation of HT samples. The coating withthe smallest nanocrystals (sample C) disclosed a superhydrophilic charac-terwithCAvalue of 4°.Moreover, thepowerful photo-inducedwettabilityeffect shown by the HT coatings was furthermore demonstrated by theslow (1–2 weeks) recovery in dark.

All the prepared coatings showed a high photocatalytic activity, prov-en by photo-induced caffeine degradation and EPR detection of photo-induced radical formation. The coating composed of firmly bonded crys-tals in the range of 50–100 nm revealed higher photocatalytic activityand reaction rate constant than the one with finer crystals. The photo-activity was similar to that observed for the deposited P25 powder,despite the larger crystals and thinner layer. EPR experiments revealedthat the activity of the HT-coatings was comparable among the samplesbut it was higher at lower wavelengths (290 nm rather than 320 nm),in agreement with absorption spectra.

Even though photocatalysis and superhydrophilicity are governed bydissimilar photo-induced pathways, when occurring on anatase TiO2

coatings, they seem to be affected by the same parameters (surface tex-ture, roughness, thickness, crystal morphology and particle size, particleagglomeration) and to mutually influence each other. For these reasons,a compromise between surface parameters, including roughness, isrequired for reducing the bacterial adhesion in favor of a betterosseointegration. Thus, the two coatings grown on machined surfacesowned themost promising combination of surface properties; in particu-lar, one of the coatings resulted in a higher photocatalytic activity, whilethe other showed surface photo-induced super-hydrophilicity.Since both phenomena are fundamental for further proving the ad-vantageous influence in antimicrobial applications, the twoabovementioned coatings will be further examined for their expect-ed photo-induced antibacterial effects. In conclusion, the resultsobtained so far can be taken as stepping stone for the fabricationof bacteria-repelling surfaces.

Acknowledgment

This work was supported within the EU 7th Framework Programunder Marie-Curie ITN project BioTiNet (grant No. 264635). The authorswish to thank Alma Mehle for technical support with EPR and MatejaPaščinski for technical assistance, Maša Zalaznik (University of Ljubljana,Ljubljana) for optical interferometer results and Milivoj Plodinec forRaman spectra (Ruder Bošković Institute, Zagreb). Asst. prof. GoranDražićand Matic Krivec are acknowledged for fruitful discussion.

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