Thin Solid Films 518 (2009) 160–170

Contents lists available at ScienceDirect

Thin Solid Films

j ourna l homepage: www.e lsev ie r.com/ locate / ts f

Neural cell growth on TiO2 anatase nanostructured surfaces

Jorge E. Collazos-Castro c, Ana M. Cruz a, Mónica Carballo-Vila c, Mónica Lira-Cantú b, Llibertat Abad a,Ángel Pérez del Pino a, Jordi Fraxedas b, Aurélie San Juan d, Carlos Fonseca d,Ana P. Pêgo d, Nieves Casañ-Pastor a,⁎a Instituto de Ciencias de Materiales de Barcelona, CSIC, Campus de la Universidad Autónoma de Barcelona, E-08193 Barcelona, Spainb Centre d' Investigació en Nanociència i Nanotecnologia, CIN2-CSIC-ICN, Campus de la Universidad Autónoma de Barcelona, E-08193 Barcelona, Spainc Neural Repair Laboratory, Hospital Nacional de Parapléjicos (SESCAM), Unidad Asociada al CSIC, Finca La Peraleda s/n. 45071 Toledo, Spaind INEB — Instituto de Engenharia Biomédica, Divisão de Biomateriais — Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal 4150-180 Porto, Portugal

⁎ Corresponding author. Tel.: +34 935801853x275; faE-mail address: nieves@icmab.es (N. Casañ-Pastor).

0040-6090/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.tsf.2009.06.048

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 January 2009Received in revised form 23 June 2009Accepted 24 June 2009Available online 2 July 2009

Keywords:Titanium oxideCoatingsAnataseNeural cellCell cultureNeurite growth

Titanium oxides have anti-inflammatory activity and tunable electrochemical properties that make themattractive materials for biomedical applications. This work investigated the compatibility of nanometriccoatings of low-temperature phases of TiO2 with neurons in 4-day and 10-day cultures, using different celldensities to quantify cell survival and neurite extension. TiO2 films were prepared by sol–gel and thermaltreatment (250–450 °C) of hydrolyzed titanium tetra-isopropoxide on electrically conducting or insulatingslides. The conducting substrates were not passivated by the nanometric oxide layer and could be used aselectrodes. Characterization of the films showed nano-structured TiO2 containing exclusively Ti+4 valence inanatase and amorphous phases. When coated with polylysine, all films permitted good neuron attachmentand survival for at least ten days in culture. However, they generally reduced neurite growth compared to cellculture borosilicate glass, with dendrites more affected than axons. The analyses of surface topography,hydrophilicity, charge and chemical composition revealed that TiO2 chemistry was the main factorresponsible for neurite inhibition.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Implantable neuroprostheses and electro-stimulators demandalloplastic materials with improved biocompatibility and electroche-mical properties to carry electrical signals to the nervous tissue.Titaniumoxides (TiOx), traditionally used in non-electrical prostheses,are of interest for neurological applications since they have an anti-inflammatory activity [1,2], can behave either as electrical insulators[3] or conductors [4,5], and have recently shown to intercalate ionsreversibly [6]. Titanium implants are widely used and successfullytolerated by the human body [7]. Its biocompatibility is largely due tothe oxide layer (mainly TiO2) of around 5 nm that forms its surfacewhen exposed to air or oxygen-containing solutions [8] and thatinhibits the inflammatory response by breaking down reactive oxygenspecies [1,2], at physiological pH and in the presence of proteins. Thechemical reactions that neutralize oxygen radicals involve reversiblechanges in Ti valence, and allow to synthesize oxides with enhancedanti-inflammatory properties [9] for the fabrication of implantabledevices. Most importantly, changes in Ti valence are accompanied bymodifications in the structure and electrical behaviour of the oxide [10]as a function of Ti/O ratio, that could be exploited to achieve dynamic

x: +34 935805729.

ll rights reserved.

functional biomaterials [11]. The reduced phases, TiOx , also have ionicconductivity [12] and are currently applied in non-biological electro-chemical systems [13,15]. Interestingly, the nanometric films of TiO2

formed on electrically conductive substrates display electrochemicalactivity that permits to reduce or to increase the adsorption of proteins[14] and to modulate reversibly their redox state [4] by application ofan electrical bias potential.

The three major crystalline phases of TiO2 (anatase, rutile andbrookite) differ in their physicochemical behaviour, rutile being themost stable thermodynamically [11]. We have recently reported thatTiO2-rutile in bulk forms sintered at high-temperatures (1300 °C to1600 °C) permits good adherence and axonal growth of culturedcerebral cortex neurons though it inhibits dendrite development [16].The high annealing temperatures needed to prepare such phase,besides limiting its use for the fabrication of implantable devices, favorrandomroughness in themicron range thatmakes it difficult to discernbetween topography and surface chemistry effects on the extension ofneural processes. Therefore, studying neural growth on TiO2 withalternative crystalline forms and topographies may help to elucidatethe cause of neurite inhibition. This work shows the initial studies ofneuronal development on nanostructured TiO2 coatings with homo-geneous surface topographies. Here, amorphous and anatase TiO2

films were obtained on transparent supporting substrates in order tofollowup the evolution of living cellswith the invertedmicroscope andto evaluate the electrochemical behaviour of the films. The surfaces

161J.E. Collazos-Castro et al. / Thin Solid Films 518 (2009) 160–170

were extensively characterized and evaluated as substrates forculturing primary cerebral cortex neurons, cell survival and growthafter four and ten days in vitro (DIV). The conditions chosenwhere theideal ones tomaximize the detection of any biological adverse effect ofthe oxides on the neurons including very low-density cell cultures thatallowed measurement of the extension of dendrites and axons inindividual cells.

2. Materials and methods

2.1. Preparation and characterization of TiO2 films

ITO (indium tin oxide) coated glass slides (300 nm ITO layer on26×76 mm soda-lime slides from Solemns) and quartz slides (VWRInternational) were used as substrates. The TiO2 coatings wereobtained by spin coating of ethanol solutions of titanium isopropoxide(Ti(OiPr)4 99.9%, Aldrich 0.7 mol) and acetylacetone (99%, Aldrich1.4 mol), aged during 1 day and filtered (0.2 µm filter). Spin coating ofthe solution was performed using a spin processor (Laurell Technol-ogies Corporation, model WS-400B-6NPP-LITE/8K) at 3000 rpm, witha careful control of the injection speed and angle of the solution atatmospheric humidity below 40%. The films were later annealed at250, 350 or 450 °C. Up to 40 replicas of coatings composed of each 1, 2and 3 layers were prepared with the corresponding thermal annealingfor each and were later used for cell culture and material character-ization. Reference hydrolytic grade 1 borosilicate coverglass wasMicroscope Cover Glass N°1 (Marienfeld GmbH & Co. KG Lauda-Königshofen, Germany, treated under SO2 for sodium surface deple-tion) and will be called Borosilicate H hereafter.

Chemistry, thickness and degradation of the films were studied byX-ray grazing angle diffraction (GIXRD), X-ray photoelectron (XPS)and Raman spectroscopies. GIXRD was carried out at 0.3° and 0.5°incidence on a Siemens D500 Diffractometer, equipped with parallelbeam attachment (parallel beam collimator and flat LiF(100) mono-chromator in parallel arrangement) in the diffracted beam using 0.05°step. Cu Kα radiation (λ=1.5418 Å) was selected. The sameinstrument allowed us to determine the total thickness of the filmby X-ray reflectometry, using the interference pattern of the diffractedbeam. XPS measurements of as-prepared samples were performed atroom temperature with a SPECS EA10P hemispherical analyzer usingnon-monochromatic Al Kα (1486.6 eV) radiation as excitation sourcein a base pressure of ca.10–9 mbar. The reported Ti 2p and O1s bindingenergies are in coincidence with previous results from reference TiO2

samples measured with the same instrument under the sameconditions (see results). The estimated error is± 0.3 eV. Micro-Raman measurements in backscattering geometry were carried out atroom temperature using the 5145 Å line of an Argon-ion laser with aRaman spectrometer (Jovin-Yvon T-64000) attached to a microscope(Olympus) and equipped with a liquid-nitrogen-cooled CCD detector.The degradation of the films in aqueous solutions and possible releaseof residues were tested by keeping them in 10 ml of distilled and de-ionized water at 37 °C for up to three months and analyzing thesolution in contact by inductive coupled plasma spectrometry, atomicforce microscopy (AFM; Agilent Technologies, model 5400 SPM) indynamic mode was used to evaluate the topography of the films in 10randomly selected areas, using Si tips (10 and 4 nm). (Cantilever forceconstant approx. 40 N/m, resonance frequency about 170 kHz). Theimages were processed with Mountains software (Dig. Surf).

Contact angle measurements were performed with a goniometer(Pocket, model PG2) using 1 µl droplets of distilled water orNeurobasal™ culture medium supplemented with L-glutamine andB27 (all from Invitrogen/Gibco, Carlsbad, CA), immediately after placingthe drop on either polylysine (PLL) coated or uncoated surfaces.Cleaning and PLL deposition was perfomed as for the cell cultures.B27-supplemented Neurobasal™ is a serum-free cell culture mediumoptimized for long-term growth of central nervous systemneurons [17].

Neurobasal™ contains inorganic salts, aminoacids, vitamins, glucose,phenol red, buffers and sodium pyruvate, whereas the B27 supplementis composed of several vitamins, hormones and proteins (albumin,catalase, insulin, superoxide dismutase and transferrin).

2.2. Electrical and electrochemical response

The electrical conductivity of the films was evaluated in the drystate by current sensing AFM (0–6 V) and in the wet state byelectrochemical impedance spectroscopy (EIS) (10 Hz to 1 MHz usingan impedance/gain-phase analyzer (Solartron 1260), and 5 mVamplitude sine waves with a three electrode configuration in 0.15 MNaCl aqueous solution). The counter and reference electrodes wereplaced close to the coating (b1 mm).

The electrochemical activity of the films was studied by cyclicvoltammetry (CV) using a potentiostat/galvanostat (Bio-logic VMP3)in 0.15 M NaCl aqueous solution, 0.1 M sodium phosphate buffer pH7.2, 0.1 M sodium acetate buffer pH 5.0, or cell culture media(Neurobasal+B27), using a three-electrode cell configuration, with Ptsheet as counter electrode and Pt wire as quasi-reference electrode.The ζ-potential was measured with a commercial electrokineticanalyzer (EKA, brand name) streaming potential apparatus (AntonPaar GmbH, Austria), using a clamping cell, with the surface of interestpushed toward a polymethyl methacrylate potential reference insertwith channels. The electrolyte (0.01 M phosphate buffer saline (PBS),pH 7.0) flowed through the measuring cell with alternating flowdirection and the streaming potential was measured during eachpressure ramp (4 measurements in each direction). The potential wascalculated from the streaming potential with VisioLab software.

2.3. Cell culture

Prior to cell plating, both TiO2 films and Borosilicate H coverglasseswere cleaned by sonication in sterile distilled water, further cleaned byone hour-immersion in distilled water at 70–80 °C and finally coveredwith a solution of poly-L-lysine (PLL, 4 µg/cm2, Sigma-Aldrich) for 30minat 37 °C. PLL was used in the study of TiO2-neuron interactions for threereasons: 1) it promotes cell attachment by enhancing electrostaticinteractions between the cell membrane and the substrate, whichwouldhelp to discern the role that the electrical charge of the surface,topography and the coating have in promoting cell growth [18], 2)because cell attachment to PLL is mediated by charge in an unspecificmanner and does not involve integrins [19], the development of cells onPLL-coated substrates is less likely to be altered by the orientation ofmolecules in the coating layer; and 3) we have used PLL previously topromote neuron adhesion and differentiation on rutile surfaces [16],which allows comparison of the present results. In the absence of PLLneurons did not attach at all to either control or TiO2 surfaces for 2 hplating. Neural cellswere obtained from the cerebral cortexof E14Wistarrat embryos. The isolated cortices were dissociated for 30min at 37 °C inHBSS supplemented with pyruvate, albumin, trypsin and DNAase (allfrom Sigma-Aldrich), followed by trituration with fire polished Pasteurpipettes. The cells were seeded at high density (25000 cells/cm2) forquantification of survival at four and ten days in vitro (DIV) and at lowdensity (500 cells/cm2) for measurement of axon and dendrite lengths.The cells were plated in DMEM-F12 medium containing 10% fetal calfserum (Invitrogen/Gibco, DMEM: Dubelcco's modified Eagles medium)andafter 2 h themediumwas changed toNeurobasal supplementedwithL-glutamine, B27 supplement, penicillin–streptomycin (all from Gibco)and gentamicin (Normon, Madrid). The replacement of the platingmediumwith serum-freeNeurobasal intends to prevent glial growth thatcould increase neuron survival and hide the adverse effects of the testedmaterials. Rat brain neurons remain viable for more than two weekswhen cultured in serum-free Neurobasal [20], making of this mediumformulation a useful tool for the study of neuron-biomaterial interactionsalthough addition of growth factors would be necessary to elicit a full

162 J.E. Collazos-Castro et al. / Thin Solid Films 518 (2009) 160–170

development of the electrophysiological properties of neurons [21].Cultureswere kept at 37 °C inhumid atmosphere (5%CO2) andhalf of thecell culture mediumwas replaced every 4 days.

2.4. Immunocytochemistry and cell quantification

Four or ten days after plating, the cell cultures were fixed inNeurobasal +2% paraformaldehyde at room temperature for 12 min.Indirect double-immunofluorescent labeling combined with Hoechst33342 (Molecular Probes, 2 ml/ml in PBS applied for 50 min afterfixing the cells) nuclear staining was used for definition of cellphenotype and identification of condensed and fragmented nuclei.Briefly, fixed cultures were incubated for 30 min in 0.1 M salinephosphate buffer (PBS) pH 7.4 containing 0.2% Triton and 5% normalgoat serum, rinsedwith PBS and then incubated overnight at 4 °C withcombinations of the following primary antibodies: rabbit policlonalanti-TAU (Sigma T-6402, 1:100), mouse monoclonal anti-GFAP (DakoZ-0334, 1:500), rabbit policlonal anti-NF (Affinity NA-1297, 1:750),mouse anti-vimentin (Neomarkers, MS-129, clone V9, 1:1000). Alexa-488 anti-rabbit (Molecular Probes, 1:500) and Alexa-594 anti-mouse(Molecular Probes,1:1000) were used as secondary antibodies. Mouseanti-MAP-2 (Sigma M-4403, 1:500) and mouse anti-b3-Tubulin(Sigma T-8660, 1:400) antibodies were used to differentiate dendritesfrom axons. However, at 4 DIVmore than 60% of the neurons were stillin developmental stages 2–3 (morphological classification [22]),limiting the use of MAP-2 and b3-Tubulin staining for classifyingneural processes because of the lack of specificity [23,24]. Therefore,processes were distinguished by morphological criteria and neuronswere classified from stages 1 to 5 according to their morphology [22].In stages 1 and 2, neurites were defined as cell processes arising fromthe soma with lengths over 10 µm. In more advanced stages, axonswere defined as neurites with a length of N50 µm andmore than twicethe length of other neurites, which if longer than 10 µmwere regardedas dendrites. For quantification of cell survival, pictures at highresolution (2776×2074 pixels) were taken using a digital microscopesystem (Olympus DP50) and the fluorescent images of both Hoechstand antibody staining were combined. Sixteen radial fields(563×401 µm each) were systematically photographed in eachsample and cell counts were extrapolated to the total surface area.Axon and dendrites were counted and measured four and ten daysafter culturing the cells at low density on the different substrates.Averages from at least 100 neurons in stages 1–2 and 3–4 wereobtained for each sample type. One-way analysis of variance (ANOVA)and Holm-Sidak posttest (0.050 of alpha level) were used to comparethe data utilizing commercial software (SigmaStat 3.11, Systat).Logarithmic transformation was performed before statistics whendata did not follow a normal distribution.

Fig. 1. Grazing angle X-ray diffraction of three-layered TiO2 coatings on ITO-glass taken at dif350 °C annealed TiO2 coatings. B) Diffraction pattern for 450 °C annealed samples.

3. Results

3.1. General characteristics of titanium dioxide films

Nanometric TiO2 films stable in aqueous media were successfullyobtained by spin coating of hydrolyzing solutions of the isopropoxidesalt and further annealing between 250 °C and 450 °C in air. Coatingscomposed of 1, 2 and 3 layers were prepared for a better differentiationof the events caused by TiO2 itself from those due to the underlyingsubstrates. X-ray reflection estimated a reproducible thickness ofapproximately 30 nm per layer irrespective of the thermal treatment;consequently, the 3-layered coatings were the thickest (about 90 nm)and had the smallest probability that the PLL, neurons or components ofthe culture medium interacted with the ITO or quartz beneath the TiO2

surface. As TiO2 nanoparticles are toxic to several cell types [25,26], TiO2

film degradation and release of residues were studied by keeping themin aqueous solutions at 37 °C up to 3months and analyzing the chemicalcomposition of the solutions. Neither Ti ions nor TiO2 particles orNa ionswere detected in the solutions at any time.

3.2. Chemistry and structure of the films

GIXRD at 0.3° incidence on 3-layered coatings showed that thoseannealed at 250 °C and 350 °C were amorphous, only peaks for the ITOsubstrate being were observed in their diffraction pattern irrespectiveof the incidence angle (Fig. 1A). In contrast, samples treated at 450 °Cshowed additional TiO2–anatase peaks resulting from crystallizationonset, without evidence of other crystallized phases (Fig. 1B). In thelatter, the peakwidth indicated a small particle size and/or low degreeof crystallinity, as expected from the low-temperature treatment.RAMAN spectra were consistent with diffraction analyses, showingcoherence only in the 450 °C sample and a spectrum typical of TiO2

anatase (Fig. 2) [27]. XPS data (Figs. 3 and 4) showed in all samples theexpected Ti 2p3/2 and 2p1/2 peaks at 458.6 and 464.3 eV correspondingto the Ti+4 oxidation state [28,29] with no other peaks or shouldersthat could evidence lower oxidation states. Oxygen O1s peaks (Fig. 5)also corresponded to those expected for the TiO2 phase, with themajor peak at 530.0 eV indicating a main contribution of latticeoxygen. It was evidenced however, a shoulder around 533 eVindicative of OH groups at the surface of 1 and 3-layered coatingstreated at 250 °C or 350 °C, suggesting some degree of surfacehydration at those temperatures. The hydroxyl groups disappeared inthe samples treated at 450 °C.

A significant featurewas the presence of Na in the films deposited onITO-glass and treated at 450 °C. The absence of this element in the filmsdeposited on Quartz slides, and the fact that Na mean free path is 1 nm,strongly suggests that sodium migrated from the glass substrate to the

ferent incidence angles and measured at 0.05o step. A) Diffraction pattern for 250 °C and

Fig. 2. Raman spectra for three-layered TiO2 coatings annealed at various temperatures.All the labelled peaks in the 450 °C treated sample correspond to anatase and were notobserved for other annealing temperatures.

Fig. 3. Survey XPS spectra for TiO2 coatings on ITO-glass and quartz substrates annealed at vasodium peaks in the oxide coatings deposited on glass, but not on quartz substrates, when

Fig. 4. XPS spectra for TiO2 coatings on ITO-glass and quartz substrates annealed at differentspectra are normalized to their maxima. The peak shifts are within experimental error.

163J.E. Collazos-Castro et al. / Thin Solid Films 518 (2009) 160–170

oxide surface, a phenomenon enhanced by higher temperature treat-ments. The identity of theminor phase containing sodium at the surfacecould not be established. On the other hand, XPS spectra from all filmtypes showed an enlarged C 1s peak at 284.8 eVwith a component near289 eV that would correspond to carbonate groups that may be theexpected endemic peaks due to adsorption of CO2 and other carboncompounds from the atmosphere. However, the general survey spectrashowed that the relative amount of carbon diminished as the treatmenttemperature increased, suggesting that the films treated at 250 °C hadsome additional C content resulting from the incomplete hydrolysis ofthe isopropoxide at this temperature. Thus, the films treated at 350 °Chad the purest TiO2 composition at the surface, without Na migrationfrom the glass substrate or significant amount of C from organicprecursors. Nevertheless, they still retained hydroxyl groups that couldinfluence peptide adsorption and cellular responses.

3.3. Surface topography and hydrophilicity

According to AFMdynamicmodemeasurements, TiO2 films showahomogeneous distribution of coalescent grains of nanometric size

rious temperatures. The spectra were normalized to their maxima. Note the presence ofannealed at the highest temperature.

temperatures showing the Ti 2p3/2 and Ti 2p1/2 peaks, at values expected for Ti+4. The

Fig. 5. XPS spectra for TiO2 coatings on ITO-glass and quartz substrates annealed at various temperatures showing the O 1s lines. Mainly oxide type oxygen and OH groups areobserved, the latter decreasing with temperature. The spectra are normalized to their maxima.

164 J.E. Collazos-Castro et al. / Thin Solid Films 518 (2009) 160–170

(b30 nm) (Figs. 6 and 7). The ITO substrate also had nanometricgranular structure and the Quartz slides were virtually flat. The size ofTiO2 grains increased with the temperature of annealing and numberof layers in the film, with small change in surface root mean square(RMS) roughness (b1 nm). The smallest roughness was found for the350 °C treated samples (even below resolution, b1 nm, see Fig. 7).

Water contact angle measurements showed a clear increase in thehydrophilicity of the films as the temperature of annealing increasedboth in 1 and 3-layered films, with the films composed of three layersbeing always more hydrophilic than those composed of a single layer(Fig. 8). As mentioned above, exactly the same trend was observed for

Fig. 6. Representative AFM surveys of titanium dioxide coatings on different substrat

grain size, suggesting that the increase in hydrophilicity with tempera-ture was caused by the increase in grain size, although the possibilityalso exists that hydrophobic precursors traces in the 250 °C treatedfilmsand hydrophilic Na phases in the 450 °C films contributed to the valuesof water contact angles. Borosilicate Hwas very hydrophilic, muchmorethan TiO2 films, giving a negligible contact angle.

Contact angle measurements using culture medium showed asignificant difference. (Fig. 9) The lineal tendency observed withwater droplets was broken, the 350 °C samples becoming the leasthydrophilic. This implies that the composition of the culture mediuminfluences the interactions between the liquid phase and the surface,

es and annealed at various temperatures. Colour scale is the same for all images.

Fig. 7. A) Root mean square (RMS) roughness and B) grain size measurements from AFM topographic analyses of TiO2 films composed of one or three layers and annealed at differenttemperatures. The data represent the mean±one standard deviation of at least 6 measurements taken from different images.

165J.E. Collazos-Castro et al. / Thin Solid Films 518 (2009) 160–170

an important finding as water contact angle is frequently used topredict the interaction of material surfaces with physiological elec-trolytes. PLL effect on contact angles for water and culture mediumwere also measured. PLL readily adsorbs to TiO2 from aqueous solu-tions at pH 7.4 through electrostatic interactions [30] and, however,the results were very different for both liquids. PLL shifts in oppositedirections the contact angle for water droplets on the 250 °C and450 °C treated oxide films, without effect in the 350 °C treated films(Fig. 9 B), levelling the values among different types of samples.However, when cell culture medium is used, the “hydrophilicity” of250 °C and 350 °C PLL-coated films is increased without changingsignificantly the behaviour of 450 °C treated films. The samples

Fig. 8. A) Contact angle images of TiO2 coatings on ITO-glass annealed at various temperatulayers. C) Water contact angle measurements for TiO2 coatings composed of one layer with

treated at 350 °C were still the less hydrophilic. This behaviour reflectsa different interaction of cell culturemediawith each surface either onraw material or PLL-coated material and imply a significant variationin PLL configuration that lead to different surface ensembles in eachcase.

3.4. Electrical properties of the films

Gross macroscopic two-point resistance measurements showedelectrical conductivity in dry ITO–TiO2 samples. Since this could bedue to the disruption of the fine oxide coating by the pressure of theelectrode, nanometric scale measurements of conductivity using

res. B) Water contact angle measurements for TiO2 coatings composed of one or threeor without polylysine coating.

Fig. 9. Contact angle measurements for TiO2 coatings and control surfaces using a Neurobasal culture medium drop and testing the surfaces with and without polylysine coating.A) Single-layer TiO2 films, B) three-layered TiO2 films. The data represent the mean±one standard deviation. Note that error bars are in some cases smaller than the symbols. Valuesfor borosilicate with and without PLL are coincident and very small.

Fig. 10. Representative cyclic voltammetries (2 mV/s) of three-layered TiO2 coatings invarious media: A) 0.1 M acetate buffer pH 5.0 (sodium salt), B) 0.1 M phosphate bufferpH 7.1 (sodium salt) and C) culture media (Neurobasal with B27 supplement). Pt wasused as a quasi-reference electrode. Scans start at rest potentials and go towardsreduction potentials in the same range.

166 J.E. Collazos-Castro et al. / Thin Solid Films 518 (2009) 160–170

current sensing AFM were performed and it was found that thecoating is insulating, with a threshold for conductivity with the tip-substrate bias near 6 V.

Because the films are intended to be used in physiologicalelectrolytes, their electrochemical behaviour was studied whenimmersed in different solutions. EIS preliminary measurements (5 mVamplitude) through the interface showed a resistance in the order of200Ω, close to the expected value for the electrolyte. Since tunnelling isnot considered a possibility through 30–90 nm films, the electricaltransport through the TiO2-interfacemust be enabled by ionic transportanddiffusionof the liquid electrolyte through thenanoporousoxide. Thefact that TiO2 coatings did not passivate the ITO conducting substrateallowed us also to evaluate the electrochemical behaviour of the ITO–TiO2 system by cyclic voltammetry (CV) with the films immersed inliquid electrolytes (Fig. 10). A similar behaviour was observed for allfilms independent of the number of layers. Water oxidation andreduction occurred with low capacitance values despite the insulatingcharacter of TiO2, a fact that makes possible the use of these coatings inany electroactive device. The safe electrochemical window where noreaction occurred at physiological pHwas from−0.5 V to+0.2V versusPt (idem versus Ag/AgCl). No evidence of sodium intercalation in theoxide film was found when the CV was performed in NaCl aqueoussolution. In acetate, a peak near −0.9 V observed for the ITO substratedecreased considerably when it was covered with TiO2, indicating thatthe contact of the ITO surfacewith the electrolytewas restricted to smallareas devoid of TiO2 and providing indirect evidence of the presence ofnano-pores in the oxide film in a small fraction of the area. Films treatedat 450 °C displayed less redox activity between oxygen or hydrogenevolution, probably due to the limited reactivity of anatase. On thecontrary, in phosphate and acetate media, the amorphous TiO2 phases(treated at 250 °C and 350 °C) showed distinctive reduction peaksprobably relatedwith the existence of precursors. Identification of thosepeaks is complex and will be investigated in future work. In cell culturemedia, however, the most inert electrode is the 350 °C sample, which isalso the most hydrophobic.

The ζ- potential showed that all TiO2 surfaces were negativelycharged at neutral pH, in agreement with reported values [31], andsignificantly less negatively charged than the borosilicate glass, withmean values for ζ-potentials of −43.5±2.0 mV for Borosilicate H,−33.0±2.3 mV for 250 °C treated films, −32.5±1.8 mV for 350 °Ctreated films, and−24.3±2.5 mV for 450 °C treated films (pb0.001).In turn, the oxide films treated at 450 °C were less negatively chargedthan those treated at 250 °C and 350 °C (pb0.05). This may beexplained by the removal of hydroxyl groups from the surfacewith thethermal treatment. No difference was observed between one or threelayer coatings, and therefore the films are similar in terms of charge.

167J.E. Collazos-Castro et al. / Thin Solid Films 518 (2009) 160–170

3.5. Neuron adhesion and survival on TiO2 films

The films were tested as substrate for neurons in 4-day and 10-daycultures (25000 cells/cm2) obtained fromembryonic rat cerebral cortex.Immunocytochemical studies in combination with Hoechst nuclearstaining revealed that at 4 DIV the cultures contained almost exclusivelyneurons, other cells being less than 0.1%of the total. TiO2filmsdepositedon ITO-glass and annealed for 2 h performed very well in terms ofneuron adhesion and survival, at four days the cells exhibiting longcellular processes and normal nuclei appearance (Fig. 11), although theone layer films treated at 350 °C and three layers treated at 250 °C hadonly 50% the amount of neurons found on control surfaces (Fig. 12A).One layerfilms showedworse results than three layerfilms, possibly dueto interactions of biological components with the ITO substrateunderneath the oxide, enabled by the diffusion of the culture mediumthrough the nanoporous oxide layer. On the other hand, the three-layered TiO2 coatings treated at 350 °C for 12 or 24 h performedconsistently similar to the glass control surfaces in terms of cell survivalat 4 DIV (Fig. 12 B), confirming that this method of film preparationworks reproducibly well in the production of good cell substrates. 10-day cultures were done to evaluate whether TiO2 films are able tointerface neurons for longer times. In this case, three-layered filmsannealed at 350 °C or 450 °Cwere used, deposited not only on ITO-glassbut also on Quartzwith the aim of evaluating possible differences in cellbehaviour caused by different underlying substrates. No significantdifferences were found between oxide coatings and borosilicate glass intermsof cell number (Fig.12 C), demonstrating that the nanometric TiO2

films support neuron survival for several days without producing acutecytotoxicity. The excellent growth of neurons on TiO2 coated ITOsurfaces contrasted with that observed on ITO itself. In our serum-freeculture conditions, neurons barely attached to ITO and when attachedthey developed only a few cell processes, finally clustering and dying ordetaching from the surface between the third and fourth day in culture.

Fig. 11. Representative microphotographs of cerebral cortex neurons seeded at 25000 cellscoatings treated at 350 °C for 2 h (C and D). A) And C), Tau immunocytochemical staininrespectively. Asterisks in A) show the position of two dead cells and in B) the fragmented andarrowheads signal dendrites. Scale bar=15 µm.

Since the ITO surfaces were significantly more positive than borosilicateglass, as indicated by the ζ-potential value (−21.8±2.3mV, vs−43.5±2.0mV; pb0.001), a low PLL adsorption on the ITO surface could explainthe poor cellular outcomes in this case.

On the other hand, glial cells were frequently detected on bothcontrol borosilicate coverglass and TiO2 coatings after 10 DIV, but theyalways represented less than 5% of all cells. Glial proliferation and thetendency of neurons to clustering probably contributed to thevariability obtained in neuron counts on samples prepared in thesame conditions, but did not prevent estimation of neuronal numbers.

3.6. Development of neuronal processes on TiO2

Neurons seeded at 25000 cells/cm2 on TiO2 films had goodappearance and numerous cell processes both at 4 DIV (Fig. 13) and 10DIV (not shown). However, at this density the cells exert reciprocaltrophic influences that may hide subtle adverse effects of the substrate,and the neurites of different cells overlap extensively making verydifficult their measurement. Therefore, low-density (500 cells/cm2)cultures were done for assessing neuron-substrate interactions withoutthe interference of trophic influences from neighboring cells, and forquantifying the extension of dendrites and axons in individual neurons.In this case, we used the coatings that resulted worst (one layerannealed at 350 °C) and best (three layers annealed at 350 °C or 450 °C)in 4 DIV. After 4 days in culture about half the neurons on controlsurfaces were in developmental stages 1–2 (growth of lamellipodia andminor neurites) and the remaining was in stages 3–4 (growth of axonsand dendrites) with no cells in stage 5 (mature). The neurons in stages1–2 had less neurites in oxide films than on control surfaces (pb0.001)but this neuritogenesis inhibition did not prevent differentiation ofaxons and dendrites. The numberof neurons that developed to stages 3–4 after 4 days on 350 °C-oxide 3 layerfilmswas similar to control values,but was only 14% on 450 °C-films (pb0.001). However, at 10 DIV this

/cm2 and cultured 4 days on control borosilicate glass (A and B) or three-layered TiO2

g; B) and D), Hoescht staining of the nuclei corresponding to the cells in A) and C),condensed appearance of their nuclei. The arrows signal long processes (axons) and the

Fig. 12. A) Neuron survival in high-density (25000 cells/cm2) cultures after 4 days invitro (DIV) on TiO2 films annealed for 2 h. B) Neuron survival in high-density culturesafter 4 DIV on three-layered TiO2 films treated at 350 °C for 12 or 24 h. C) Neuronsurvival in high-density cultures after 10 DIV on TiO2 films deposited on ITO or Quartzand annealed for 2 h. The data represent the mean±statistical significant difference atpb0.05 compared to controls.

Fig. 13. Growth of neural processes in low cell density (500 cells/cm2) cultures on TiO2

films and control surfaces. The data represent the mean±statistical significantdifference at pb0.05 of: A) length of the primary axon, B) length of the primarydendrites, and C) number of primary dendrites per cell.

168 J.E. Collazos-Castro et al. / Thin Solid Films 518 (2009) 160–170

developmental difference disappeared. The study of dendrites andaxons (neurons in stages 3–4) also revealed significant features. It iswellknown that axons from isolated rat cerebral neurons growabout 150µmin the first 4 days of culture [32]. In our study, that valuewas even betterfor control and 350 °C-oxide 3 layer films (Fig. 13), even though axonswere about 20% shorter on 350 °C-oxide films than on control surfaces(Fig. 9A), and dendrite length was reduced in the same proportion(Fig. 13 B). Three-layered 450 °C oxide coatings impaired to a greater

extent axonal and dendrite growth, reaching only 50% of control valuesat4DIV (Fig.13A, B). Axons grewanadditional 15% fromdays4 to10 anddeveloped collaterals on control surfaces but not on oxide films,indicating that the oxides interrupted neuron maturation. Howeverthe effectwasmore pronounced on dendrites, as the neurons on 350 °C-films had less than 40% dendrites than on control surfaces at 4 DIV, andtheneuronson450 °Cfilmswere almost devoid of them(Fig.13 C). Fromday 4 to 10, neurons on the 350 °C films even retracted some of the fewdendrites that had developed (Fig. 14).

Fig. 14. Representative photomicrographs of cerebral cortex neurons (500 cells/cm2)processed for Tau immunocytochemistry after 10 days of culture on borosilicate glass(A) or three-layered TiO2 coatings treated at 350 °C for 2 h (B). The long arrows signalaxons and the short arrows signal dendrites. Scale bar=25 µm.

169J.E. Collazos-Castro et al. / Thin Solid Films 518 (2009) 160–170

In summary, the results of low-density cultures showed significantdifferences in the development of neurons on TiO2 and controlsurfaces, with particular effects in each cell stage. The films permittedneuronal adhesion and axonal differentiation and growth in initialstages, but inhibited dendrite development and axonal maturation incomparison to control surfaces. This cell behaviour was detected ondifferent types of TiO2 coatings, suggesting that it arises mainly fromthe physicochemical properties of the oxide.

4. Discussion

The aims of this study were to prepare nanometric TiO2 films onelectrically conductive or insulating substrates and to study theirsuitability as support for the growth and development of primarycerebral cortex neurons, as an initial step in assessing the potential useof low-temperature phases in the form of coatings for neuroprostheticapplications. Low temperature annealing (250–450 °C) yieldedamorphous or anatase TiO2 surfaces with grain sizes below 30 nm,crystallinity and grain size increasing with temperature. Annealing at350 °C rendered amorphous films with the purest chemical composi-tion. Three-layered TiO2 coatings (90 nm thick) annealed at 350 °Calso performed reproducible well as neuronal substrate. Significantly,the oxide coatings did not passivate the conducting substrates, allowingits use as coatings on conducting devices addressed to electrical stimu-lation of neural cells.

When coated with PLL, three-layered TiO2 films annealed at 350 or450 °C permitted very good neuronal attachment without evidence ofcytotoxicity, performing better than several alloplastic materialsreported [33,34]. However, the extension of neural processes is smallerin comparisonwith the best observed case, standard cell culture glass. Inparticular, the initiation of undifferentiated neurites and dendrites wasstrongly inhibited by either amorphous (350 °C) or crystallized (450 °C,

anatase) TiO2 surfaces, while early axonal growth on the amorphousoxide films was only slightly reduced in comparison to controlborosilicate, but axons eventually failed in maturation. Film treatmentat 450 °C impaired the growth of axons and dendrites in early stages ofcell development to a greater extent than treatment at 350 °C, probablydue to the migration of sodium ions to the surface. Those differenceswere not evident in neurons cultured for longer times, when theyappeared equally inhibited on both types of oxide films. This suggeststhat oxide crystallinity and larger grain size occurring at highertemperatures is not a key factor for neuronal attachment or neuritedevelopment on TiO2 surfaces. In fact, neuronal growth on nanostruc-tured amorphous and anatase TiO2 was quite similar to that wedescribed recently on microstructured rutile TiO2 [16], indicating thatdevelopmental impairment is caused by the surface chemistry of TiO2

and not by its topography. Surface topography in nanometric range hasbeen shown to influence mesenchymal stem cell attachment andspreading on TiO2 thin films [35] but had no effects on osteoblastproliferation and viability [36]. Those studies along with the presentresults indicate that the effects of nanotopography on cell behaviour arehighly dependent on the cell type and function.

The exact reasons for which all TiO2 surfaces allow cell growth andattachment but impair neuronal development compared to borosilicateglass remain unknown. Although several surface properties are knownto influence cell attachment and growth on biomaterials [37], theirhierarchy and mechanisms are far from being completely understoodand there are few studies concerning the influence of materialproperties on the differentiation of axons and dendrites. The physico-chemical properties of TiO2 could influence cell growth either directly orthrough alterations in the adsorption and/or orientation of the peptidemolecules (PLL) used to promote cell adhesion, or through interactionwith cell culture media. We have observed different final properties foreach coated surfaces andwe have found that PLL adsorption occurs in allTiO2films reported in this study (Casañ-Pastor et al., unpublished), but aprecise determination of the amount of peptide adsorbed, its orientationand interaction with the surface is still elusive because of the smalldimensions of the layer (about 1 nmas determined by AFM). It has beenreported also that PLL adsorbed at 37 °C forms a layer only 1 nm thick ongold and platinum substrates, whereas laminin forms a layer of about80 nm in the same conditions [38]. In our case, the neuronswere in veryclose contact with the oxide surface and could be directly influenced byits properties. Surface hydrophilicitymay also play an important role, asthewater contact angles of TiO2 surfaces consistently indicated that theoxide is hydrophilic though much less than control borosilicate glass,which always displayed a negligiblewater contact angle. PLL leveled thehydrophilicity for the different TiO2 films, even though somedifferencesremained among them probably as consequence of a different orienta-tion of the peptide depending on the original surface charge. This hasalso been reported to occur with laminin coatings on silicon surfaces[21] and amphiphilic peptides on graphite and mica surfaces [39]. Ifsurface polarity is a major determinant of water contact angle assuggested [40], the leveling effect of the PLL coating implies that thedifferent oxide films became charge balanced, and a similar neuronalgrowth would be expected. The differences in contact angle for waterand cell culture medium are significant irrespective of the presence ofPLL on the surface. Since the culture medium is such a complex mixtureof organic, inorganic and colloidal systems, that can interact in variousmannerswith the surface, the possibility exists that TiO2 surfaces adsorbpreferentially some of its components, creating an enrichment ordepletion of them in the immediate cell environment. Then, althoughwater contact angle measurements are useful indicators of surfaceinteraction with aqueous media, contact angles with the actual culturemedia are better indicators of the interface exposed to the cells.

A plausible explanation for the developmental impairment of neu-rons on TiO2 could be that a smaller amount of PLL is adsorbed on theoxide or that the peptide conformation is not ideal for cell attachment.Since the amount of PLL estimated to adsorb from aqueous solutions at

170 J.E. Collazos-Castro et al. / Thin Solid Films 518 (2009) 160–170

37 °Congold andplatinumsubstrates is only about 0.15ng/cm2 [38], anypeptide vacancies or alterations in such thin layer could have profoundeffects on the cells growing on the surface. ζ-potentialmeasurements ofoxide and control surfaces in neutral buffer showed that TiO2 coatingsare less negatively charged than cell culture borosilicate glass. At neutralpH PLL is positively charged and adsorbs on TiO2 through electrostaticinteractions [30]. Consequently, the less negative charge of TiO2 couldreduce the amount of adsorbed PLL affecting secondarily neuronalgrowth. An analogous mechanism would explain the neuronal impair-ment for developing voltage-dependent currents that was observed onsilicon wafers insulated with Si3N4 [21]. The silicon wafers also had areduced surface negativity compared to coverglass, and itwas suggestedthat this factor affected cellulardevelopment indirectly bymodifying thebinding and/or orientation of the protein (laminin) on the surface.

In summary, reproducible nanostructured TiO2 films were preparedby spin coating and thermal annealing of titanium tetra-isopropoxideprecursors. The films are stable in aqueous solutions and when coatedwith polylysine they permit good adhesion and survival of neurons andalso the growth of their axons. However, dendrites development isimpaired onTiO2 irrespective of the crystallochemical and topographicalstructure of the surface, leading eventually to a general cell dysfunction.It remains to be determined whether the same inhibitory effect occurswhen the surface is coated with other cell adhesion proteins, andwhether it is mediated by signaling mechanisms taking place at theneuron soma or locally at the neurite/dendrite. Although additionalstudies are needed to better characterize and control the neuron–oxideinteractions so that full extension of neuronal processes is obtained, thepresent results, together with the known biocompatibility of TiO2

[41,42] and the fact that it can be manufactured using a wide range oftechniques and with varied structures, open the possibility of a newapplication for this material as a component of implantable devices forthe nervous system, as coating of electrically conducting or insulatingparts.

Acknowledgements

This work was supported by grants from the Spanish Ministry ofScience and Education (MAT2005-07683, MAT2007-29316-E,MAT2008-06643), CSIC (PIF06-021) and the European CommissionFP6 NEST Program (Contract 028473). The aid of Dr. N. Mestres(ICMAB-CSIC) and J. Bassas (ICT-UB) in Raman andGrazing angle X-raydiffraction, and of Vanesa Padial (HNP-SESCAM) in cell cultures is alsogreatly acknowledged.

References

[1] R. Suzuki, J.Á. Frangos, Clin. Orthop. Relat. Res. 372 (2000) 280.[2] R. Suzuki, J. Muyco, J. McKittrick, J.A. Frangos, J. Biomed. Mater. Res. A 66 (2003)

396.

[3] M.H. Ulbrich, P. Fromherz, Appl. Phys. A. 81 (2005) 887.[4] E. Topoglidis, T. Lutz, R.L. Willis, C.J. Barnett, A.E Cass, J.R Durrant, Faraday Discuss.

116 (2000) 35.[5] J.R. Smith, F.C. Walsh, R.L. Clarke, J. Appl. Electrochem. 28 (1998) 1021.[6] N.N. Dinh, N.T. Oan, P.D. Long, M.N. Bernard, A. Hugot-Le Goffb, Thin Solid Films

423 (2003) 70.[7] D.M. Brunette, P. Tengvall, M. Textor, P. Thomsen (Eds.), Titanium in Medicine,

Springer-Verlag, Berlin, 2001.[8] M. Textor, C. Sittig, V. Frauchiger, S. Tosatti, D.M. Brunette, in: D.M. Brunette, P.

Tengvall, M. Textor, P. Thomsen (Eds.), Titanium in Medicine, 171, Springer-Verlag,Berlin, 2001.

[9] R. Contreras, H. Sahlin, J.Á. Frangos, J. Biomed. Mater. Res. A 80 (2007) 480.[10] K. Kolbrecka, J. Przyluski, Electrochim. Acta 39 (1994) 1591.[11] U. Diebold, Surf. Sci. Rep. 48 (2003) 53.[12] B.S. Jeong, D.P. Norton, J.D. Budai, Solid-State Electron. 47 (2003) 2275.[13] S.E. Moulton, J.N. Barisci, A. Bath, R. Stella, G.C. Wallace, Langmuir 21 (2005) 316.[14] D.R. Jackson, S. Omanovic, S.G. Roscoe, Langmuir 16 (2005) 5449.[15] V. Subramanian, A. Karki, K.I. Gnanasekar, FP. Eddy, B. Rambabu, J. Power Sources

159 (2006) 186.[16] M. Carballo-Vila, B. Moreno-Burriel, J.R. Jurado, E. Chinarro, N. Casañ-Pastor, J.E.

Collazos-Castro, J. Biomed. Mater. Res. A 90 (2009) 94.[17] G.J. Brewer, J.R. Torricelli, E.K. Evege, P.J. Price, J. Neurosci. Res. 35 (1993) 567.[18] A.M. Rajnicek, S. Britland, C.D. McCaig, J. Cell Sci. 110 (1997) 2905.[19] W. Bao, M. Thullberg, H. Zhang, A. Onischenko, S. Strömblad, Mol. Cell. Biol. 22

(2002) 4587.[20] N.E. Sanjana, S.B. Fuller, J. Neurosci. Methods 136 (2004) 151.[21] W.J. Parak, M. George, M. Kudera, H.E. Gaub, J.C. Behrends, Eur. Biophys. J. 29

(2001) 607.[22] C.G. Dotti, C.A. Sullivan, G.A. Banker, J. Neurosci. 8 (1988) 1454.[23] R.W. Davenport, C.D. McCaig, J. Neurobiology 24 (1993) 89.[24] J.A. Chuckowree, J.C. Vickers, J. Neurosci. 23 (2003) 3715.[25] T.C. Long, N. Saleh, R.D. Tilton, G.V. Lowry, B. Veronesi, Environ. Sci. Technol. 40

(2006) 4346.[26] L. Zhao, J. Chang, W. Zhai, J. Biomater. Appl. 19 (2005) 237.[27] V. Swamy, A. Kusnetzov, L.S. Dubrovinsky, R.A. Caruso, D.G. Shchukin, B.C. Muddle,

Phys. Rev., B 71 (2005) 1.[28] M.P. Lozano, J. Fraxedas, Surf. Interface Anal. 30 (2000) 623.[29] E. Martínez-Ferrero, Y. Sakatani, C. Boissière, D. David Grosso, A. Fuertes, J.

Fraxedas, C. Sánchez, Adv. Funct. Mater. 17 (2007) 3348.[30] A.D. Roddick-Lanzilotta, A.J. McQuillan, J. Colloid Interface Sci. 217 (1999) 194.[31] M. Kosmulski, Adv. Colloid Interface Sci. 99 (2002) 255.[32] Y. Ikegaya, Y. Itsukaichi-Nishida, M. Ishihara, D. Tanaka, N. Matsuki, Neuroscience

97 (2000) 215.[33] L.A. Cyster, K.G. Parker, T.L. Parker, D.M. Grant, Biomaterials 25 (2004) 97.[34] A. Blau, C. Weinl, J. Mack, S. Kienle, G. Jung, C. Ziegler, J. Neurosci. Methods 112

(2001) 65.[35] D.S. Kommireddy, S.M. Sriram, Y.M. Lvov, D.K. Mills, Biomaterials 27 (2006) 4296.[36] K. Cai, J. Bossert, K.D. Jandt, Colloids Surf., B 49 (2006) 136.[37] Y-XWang, J.L. Robertson, W.B. Spillman Jr., R.O. Claus, Pharm. Res. 21 (2004) 1362.[38] F. Greve, S. Frerker, A.G. Bittermann, C. Burkhardt, A. Hierlemann, H. Hall,

Biomaterials 28 (2007) 5246.[39] H. Yang, S-Y. Fung, M. Pritzker, P. Chen, PLoS ONE 2 (2007) e1325.[40] N. Giovambattista, P.G. Debenedetti, P.J. Rosky, J. Phys. Chem. B 111 (2007) 9581.[41] M.A. Onur, Z. Taş, A. Gürpιnar, A. Şahin, M.C. Çehreli, Clin. Oral Implants Res. 15

(2004) 513.[42] T. Lopez, E. Ortiz-Islas, J. Manjarrez, F. Rodríguez-Reinoso, A. Sepúlveda, R.D.

Gonzalez, Opt. Mater. 29 (2006) 70.