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0142-9612/$ - se

doi:10.1016/j.bi

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Biomaterials 27 (2006) 3221–3229

www.elsevier.com/locate/biomaterials

Biocompatibility of cluster-assembled nanostructured TiO2 withprimary and cancer cells

Roberta Carbonea, Ida Marangia, Andrea Zanardia, Luca Giorgettia,b, Elisabetta Chiericib,Giuseppe Berlandab, Alessandro Podestab, Francesca Fiorentinib, Gero Bongiornob,

Paolo Piserib, Pier Giuseppe Peliccia, Paolo Milanib,�

aEuropean Institute of Oncology, Via Ripamonti 435, 20141 Milan, ItalybCentro Interdisciplinare Materiali e Interfacce Nanostrutturati (CIMAINA), Dipartimento di Fisica, Universita di Milano,

Via Celoria 16, 20133 Milan, Italy

Received 8 November 2005; accepted 27 January 2006

Abstract

We have characterized the biocompatibility of nanostructured TiO2 films produced by the deposition of a supersonic beam of TiOx

clusters. Physical analysis shows that these films possess, at the nanoscale, a granularity and porosity mimicking those of typical

extracellular matrix structures and adsorption properties that could allow surface functionalization with different macromolecules such

as DNA, proteins, and peptides. To explore the biocompatibility of this novel nanostructured surface, different cancer and primary cells

were analyzed in terms of morphological appearance (by bright field microscopy and immunofluorescence) and growth properties, with

the aim to evaluate cluster-assembled TiO2 films as substrates for cell-based and tissue-based applications. Our results strongly suggest

that this new biomaterial supports normal growth and adhesion of primary and cancer cells with no need for coating with ECM proteins;

we thus propose this new material as an optimal substrate for different applications in cell-based assays, biosensors or microfabricated

medical devices.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Titanium; Biocompatibility; Nanotopography; Biomimetic material; Cell adhesion; Extracellular matrix

1. Introduction

Cellular behavior in vivo and in vitro is influenced by themechanical, biochemical and topographical properties ofthe extracellular microenvironment where cells grow [1–3].In particular, the biochemical composition and themechanical behavior of the extracellular matrix (ECM)play an important role in many developmental phenomenaduring embryogenesis [4] or tumor-related conditions [5].Recently also stem cells were shown to be responsive to theextracellular environment with relevant consequences fortheir self-renewal or differentiation programs [6,7].

e front matter r 2006 Elsevier Ltd. All rights reserved.

omaterials.2006.01.056

ing author.

esses: roberta.carbone@ifom-ieo-campus.it (R. Carbone),

i.infn.it (P. Milani).

According to the most recent studies on biomaterials[8,9], cells can actively ‘sense’ and adapt to the surface ofadhesion and activate specific intracellular signals thatinfluence cell survival and behavior. In vivo, cell attach-ment is the consequence of the binding with specific celladhesion proteins in the ECM, and it is intrinsicallyinfluenced, besides by receptor–ligand specific interactions,by the physical and mechanical signals arising from thetopography of the external environment [1,2,10]. In vitro,on the other hand, cells set up a complex network ofinteractions both with the artificial surface and with thesecreted and serum ECM proteins. The possibility ofoptimizing cell-substrate interactions can open up newperspectives in the design of biomimetic supports [11,12].The topography of the ECMs is characterized by

features over different length scales ranging from the nano-to the mesoscale and it regulates the cellular behavior in a

ARTICLE IN PRESSR. Carbone et al. / Biomaterials 27 (2006) 3221–32293222

way that it is still far from a complete understanding[13–15]. The coexistence of ECM features at differentlength scales is probably one of the key factors, however itis not clear if there is a hierarchical organization ofdifferent structures and to what extent the various lengthscales can influence cellular response [16,17]

In order to elucidate the role of substrate topographyand to fabricate biocompatible interfaces capable ofmimicking the physiological conditions of the extracellularenvironment, a large number of studies have been devotedto the investigation of cell interactions with artificiallyproduced nanostructures such as pits, pillars, grooves, dotsor random structures obtained by chemically or physicallyetching metallic, semiconducting and polymeric surfaces[8,18–21].

Particular efforts have been devoted to the topographicalmodification of titanium and titanium dioxide surfacessince these materials are amongst the most studied andwell-characterized biomaterials [22]. Pure titanium andtitanium alloys are frequently used as dental and orthope-dic implants because of their excellent mechanical strength,chemical stability, and biocompatibility [23,24], whichultimately arise from the thin oxide layer that sponta-neously forms on the titanium surfaces [22].

The fabrication strategies employed to create syntheticsubstrates with tailored topography at the nano- andmicroscale are essentially top-down and in particular basedon hard and soft lithography for the fabrication of orderedstructures [25,26]. These approaches, when not based onnatural matrix-related proteins, despite the great improve-ments in miniaturization and accuracy, are not able toreproduce the morphology and the hierarchical organiza-tion typical of the ECMs [27].

Up to now, very few studies have been devoted to theelucidation of the interaction of cells with nanostructuredmaterials obtained by the bottom-up approach of nano-particle assembling [28–31]. This is quite surprising sincenanoparticle-assembled materials have an increasing role inthe fabrication of biocompatible devices as well asdiagnostic and therapeutic platforms; moreover, a bot-tom-up approach can offer more possibilities to organizethe structure on a multi-length scale similar to thatobserved in ECM.

In this paper we report the characterization of thebiocompatibility of nanostructured TiO2 films obtained bythe deposition of a supersonic beam of TiOx clusters. Thefilms, resulting from a random stacking of nanoparticles,are characterized, at the nanoscale, by a granularity andporosity mimicking those of recently observed ECMstructures [32,33]. Moreover titanium dioxide nanoparti-cles can form complexes with a variety of chemical groupsand immobilize functional peptides and macromolecules[34–39] which therefore could be employed to functionalizethe surface.

In view of the interesting opportunities of integrationwith microsystems offered by nanostructured TiO2 filmsassembled by supersonic cluster beam deposition (SCBD)

[40,41], we sought to investigate the biocompatibility ofthis new substrate with a wide range of cell lines incomparison with a surface which is commonly employedfor culturing cells (i.e., gelatin-coated glass coverslips). Toexplore the biocompatibility of this novel nanostructuredsurface, cells were analyzed in terms of morphologicalappearance and growth properties, with the aim to evaluateits possible use as a substrate for different cell-based andtissue-based applications [42–47].We have performed short- and long-term studies with

different cellular model systems using bright field micro-scopy for cell morphology, immunofluorescence for cytos-keletal analysis, BrdU incorporation and DAPI stainingfor cell cycle and cell growth analysis. Our results indicatethat cluster-assembled nanostructured TiO2 is a biocom-patible surface for cell culturing directly supporting normalgrowth and adhesion of different primary and cancer cells.

2. Materials and methods

2.1. Cluster-assembled nanostructured titanium substrates

Nanostructured TiO2 films (thickness of 50 nm) were grown on round

glass coverslips (15mm diameter, 0.13–0.16mm thickness, Electron

Microscopy Sciences) by depositing under high vacuum a supersonic

seeded beam of TiOx clusters produced by a pulsed microplasma cluster

source (PMCS). A detailed description of the PMCS and its principle of

operation and can be found in Refs. [40,48]. Briefly, the PMCS operation

principle is based on the ablation of a titanium rod by a helium plasma jet,

ignited by a pulsed electric discharge. After the ablation, TiOx ions

thermalize with helium and condense to form clusters. The mixture of

clusters and inert gas is then extracted in vacuum through a nozzle to form

a seeded supersonic beam, which is collected on a substrate located in the

beam trajectory. The clusters kinetic energy is low enough to avoid

fragmentation and hence a nanostructured film is grown. Further

oxidation of TiOx clusters takes place upon air inlet in the deposition

chamber [49].

By exploiting aerodynamic separation and focusing effects typical of

supersonic beams [50] it is possible to produce nanostructured TiOx films

with a controlled spread not only of the thickness and morphology but

also of the crystalline dimensions and the rutile/anatase ratio as described

in Refs. [50,51].

The surface morphology of cluster-assembled films was characterized

by atomic force microscopy (AFM) employing a Digital Instruments

Nanoscope multimode IV atomic force microscope in tapping mode. The

nanostructure of the films was characterized by transmission electron

microscopy (TEM), performed with a JEOL JEM-4000EX II operated at

400KeV.

2.2. Gelatin substrates

Round glass coverlips were coated with 0.2% gelatin PBS (phosphate

buffered saline solution—Gelatin, type B from bovine skin, tissue culture

grade, SIGMA) for 30min at R.T. After a brief wash with PBS 1X,

coverslips were ready for cell plating. Before AFM imaging, coverslips

were further gently rinsed in HPLC-grade water to remove salt residuals.

2.3. Cells and culture conditions

All cell lines were grown in tissue culture plates (Falcon, BD

Bioscience) at 37 1C in a humidified incubator with 5% CO2.

Immortalized Mouse Embryo Fibroblasts (kindly provided by Dr. G.

Scita, IFOM-IEO Campus, Milan, Italy) were cultured in Dulbecco’s

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Modified Eagle’s Medium (DMEM, BioWhittaker—Cambrex) supple-

mented with 10% Fetal Bovine Serum (FBS, Invitrogen corporation), 1%

L-Glutamine (BioWhittaker—Cambrex) and 1% Penicillin and Strepto-

mycin (P/S, BioWhittaker—Cambrex).

Human osteosarcoma U2OS (American Type Culture Collection—

ATCC) were cultured in DMEM supplemented with 10% FBS, 1%

L-Glutamine and 1% P/S.

Human fibroblasts Tig3-hTert (Tig3 fibroblasts immortalized with

Human telomerase reverse transcriptase, kindly provided by Dr. C.

Moroni, IFOM-IEO Campus, Milan, Italy) were cultured in DMEM

supplemented with 10% FBS (origin US), 1% L-Glutamine and 1% P/S.

Primary human melanocytes were isolated in our laboratory according

to the protocol developed by Meenhard Herlyn (www.wistar.upenn.edu/

herlyn/cellculture.htm). Cells were cultured in McCoy’s 5A Medium

(Invitrogen) supplemented with: 2% FBS (US origin), 5mg/ml recombi-

nant human Insulin, (Roche), 5 mg/ml human holo-Transferrin (SIGMA),

0.5 mg/ml Hydrocortisone (SIGMA), 20 pM Cholera Toxin from Vibrio

cholerae (SIGMA), 16 nM Phorbol 12-myristate 13-acetate (SIGMA),

10 nM Endothelin 1 human, porcine (SIGMA), 10 ng/ml Recombinant

human SCF (Peprotech), 1 ng/ml Recombinant human FGF-basic

(Peprotech).

2.4. Morphology

All cell lines were plated at day 1 at density described in Table 1 on

round glass coverslips, which were previously coated either with cluster-

assembled TiO2 or 0.2% of gelatin PBS, then placed in 12 wells TC plates.

At specific time points (MEFs, Tig3-hTert, U2OS 2–9–16 days, primary

melanocytes 2–9–20 days) cells were imaged in vivo by a bright field

inverted microscope (Olympus CK40). Images were acquired by Image

Grabber software using a color video 3CCD camera (JVC KY-F55B).

2.5. Immunofluorescence for cytoskeletal analysis

Immunofluorescence experiments were performed at 2 and 9 days after

plating in parallel on both types of coated glass coverslips. For

cytoskeletal analysis we used the following antibodies: anti human

vinculin (monoclonal, clone hVIN-1, SIGMA), phalloidin (Alexa fluor

488 phalloidin, Molecular Probes) and anti alpha tubulin (monoclonal,

clone B5-1-2, SIGMA). Briefly, cells were rinsed twice with PBS and fixed

with 4% paraformaldehyde (SIGMA) in Pipes Buffer solution (pipes

buffer: 80mM pipes pH 6.8, 5mM EGTA pH 8, 2mM MgCl2) for 15min;

after PBS washing cells were permeabilized with permeabilization buffer

(PBT) containing 0.2% BSA (Albumin Bovine Fraction, Biochemical),

0.1% Triton X-100 (SIGMA) in PBS for 10min, blocked with 2% BSA

for 1 h at room temperature, stained with primary antibodies in 2% BSA

for 45min, washed twice with PBS and stained with Alexa fluor 488

conjugated secondary Abs (Molecular Probes-Invitrogen) for 30min at

room temperature. Nuclei were counterstained with DAPI (40,6-Diamidi-

no-2-phenylindole, SIGMA), cells were rinsed with PBS and mounted

with Mowiol 4-88 (Calbiochem).

Images of different fields were acquired with a fluorescence microscope

(Olympus BX71) with a PLANAPO 60XOil (N.A. 1.40) magnification

objective. The images were acquired and elaborated with Adobe Photo-

shop 7.0 program (Adobe System Incorporated).

Table 1

Scheme of plating at day 1 (number of cells) and days of splitting. Splitting

was performed when cells reached 80% confluency

No. of plated cells Days of splitting

MEFs 6.5� 104 3–5–8–10–12–15

Tig3-hTert 8� 104 4–8–11–15

U2OS 8.5� 104 3–5–8–10–12–15

Human primary melanocytes 7� 104 6–12

2.6. DNA synthesis assay by BrdU incorporation

Cell cycle analysis was performed at specific time points by BrdU

(50Bromo 20deoxiuridine, SIGMA) incorporation.

Briefly, cells were pulsed with 33 mM BrdU for 30min at 37 1C in a

humidified incubator with 5% CO2; after PBS washing cells were fixed

with 4% paraformaldehyde in Pipes Buffer solution for 15min; after

washing with PBS, permeabilization with PBT and blocking with BSA

2%, cells were washed with PBS and stained with anti-BrdU (monoclonal,

BD Bioscience) diluted in 100U/ml DNase I RNase free (Roche), 3mM

MgCl2 and BSA 1% for 45min; after two washes with PBS cells were

stained with goat antimouse Alexa568 (Molecular Probes-Invitrogen);

cells were then washed with PBS, counterstained with DAPI and finally

mounted with Mowiol.

Cells were analyzed by fluorescence microscopy: five different fields (or

at least 500 cells) were acquired, and images were analyzed to quantify

BrdU positivity by ImageJ software (NIH, Bethesda, Washington).

3. Results and discussion

3.1. Morphological and chemical characterization of the

substrates

In Fig. 1 we present two AFM images of a cluster-assembled TiO2 film (A) and a gelatin-coated glass cover-slip (B). At the nanoscale, both films expose a granularsurface, with a grain diameter ranging from a fewnanometers up to 20 nm. The nanoscale texture of thetwo substrates is the result of the aggregation ofnanometer-sized building blocks: TiOx clusters, in the caseof cluster-assembled TiO2, and proteins in the case ofgelatin. Although similar in the nano-scale surface raster,the two surfaces are however different in morphology. Thegelatin coating is characterized by a close, uniformpackaging of proteins, resulting in a rather smooth surface(rms roughness ¼ 0.6 nm, specific area �1, average surfaceslope �31) irrespective of the preparation conditions(incubation time, gelatin concentration, mass-filtering ofprecursors; unpublished data). Cluster-assembled TiO2

films, on the other hand, result from the ballistic depositionof size-dispersed clusters, which impinge on the surface andstick, without diffusing [49,51]. This growth mechanismleads to a highly porous material, with a large specific areaand a small average surface slope [52] despite the largersurface roughness (in the case of the 30-nm-thick TiO2 filmshown in Fig. 1A, rms roughness ¼ 4.5 nm, specificarea ¼ 1.3, average surface slope �81). Large specific areaand small surface slope are likely to favor cell adhesion.Controlling the deposition time, cluster-assembled TiO2

films with increasing specific area and decreasing surfaceslope can be grown.In Fig. 2 we show a TEM micrograph of a section of a

typical cluster-assembled TiO2 film. The nanostructure ischaracterized by nanocrystalline regions embedded in anamorphous matrix. The size of the nanocrystals rangesfrom 50 to 100 nm to less than 10 nm. The nanograins arerandomly assembled to constitute a porous structure suchas those typical of the ballistic aggregation regime, thedensity of the film being roughly half of the corresponding

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Fig. 1. (A) AFM image of a cluster-assembled TiO2 film deposited on a glass coverslip (500� 500nm2, vertical scale 15 nm). The granular nature of the

film at the nanoscale is evident, which stems from the ballistic aggregation of precursor clusters stacking on the underlying glass surface during the

deposition from the supersonic beam. (B) AFM image of a gelatin-coated glass coverslip (500� 500 nm2, vertical scale 3 nm).

Fig. 2. TEM micrograph of a region of a cluster-assembled TiO2 film. The film is mainly amorphous, with nanocrystalline inclusions whose size ranges

from 50 to 100 nm to less than 10 nm. Both rutile and anatase nanocrystals are observed.

R. Carbone et al. / Biomaterials 27 (2006) 3221–32293224

bulk phase (2.5–2.7 g/cm3 against 3.9–4.3 g/cm3 for bulkTiO2, as obtained from optical methods [53]). Nanoparticlelattice spacing and diffraction patterns are consistent withnanocrystalline TiO2 where both anatase and rutile crystal-line phase are present [50].

The chemical and electronic properties of the surface ofcluster-assembled titanium oxide films have been pre-viously characterized by synchrotron-radiation X-rayPhotoelectron Spectroscopy (XPS) and Ultraviolet Photo-electron Spectroscopy (UPS) [53]. The XPS spectraindicated the presence of titanium, oxygen and carbononly, the latter arising from environmental contaminants.The surface was shown to be moderately hydrated, with asignificant fraction of the O1s photoelectron signal arisingfrom surface hydroxides. UPS showed a considerableamount of Ti 3d valence band states, related to under-coordinated titanium ions (Ti3+ point defects) arising fromoxygen vacancies and corner defects. These states areknown to play a crucial role in many adsorption processes,notably in the dissociative adsorption of water molecules[54,55], and thus contribute to the hydration and conse-

quently to the reactivity of the nanostructured surfacetowards organic moieties [56–58].Cluster-assembled nanostructured TiO2 films are opti-

cally transparent, with an estimated refraction index of1.7 [53].

3.2. Biocompatibility

To evaluate the degree of biocompatibility of the cluster-assembled TiO2 substrate, we compared different estab-lished human and mouse cell lines and a human primaryculture on 0.2% gelatin-coated coverslips and cluster-assembled TiO2-coated coverslips produced by SCBD. Thecomparison was carried out by bright field microscopy,immunofluorescence with cytoskeletal markers and BrdUincorporation for cell cycle analysis.We tested four different cellular models: human primary

fibroblasts immortalized with hTert (telomerase) (Tig3-hTert), spontaneously immortalized Mouse Embryo Fi-broblasts (MEFs), osteosarcoma cell line (U2OS) andhuman primary melanocytes.

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Fig. 3. Morphological analysis by bright field microscopy (20� magnification objective for MEFs, Tig3-hTert and U2OS, 10� magnification objective

for primary melanocytes) of cells at different time points on gelatin- and nanostructured (ns) TiO2-coated coverslips. (A) MEFs, (B) Tig3-hTert, (C) U2OS

were imaged at 2–9–16 days after plating in exponentially growing conditions, and (D) human primary melanocytes were imaged at 2–9 days in

exponentially growing conditions and at 20 days at confluency.

R. Carbone et al. / Biomaterials 27 (2006) 3221–3229 3225

The experiments were performed at short term (2 days)and long term (between 9 and 20 days) to fully explore thecellular interaction with the nanostructured surface and toreveal possible long-term effects of this new biomaterial oncellular behavior.

As shown in Fig. 3, cells were imaged by bright fieldmicroscopy at different time of culturing on gelatin andTiO2-coated coverslips following the scheme of splittingdescribed in Table 1. None of the cells examined showeddifferent morphology in terms of size, shape and orienta-tion on the TiO2 substrate. In addition, no sign of toxicitywith the appearance of cellular debris in the culturemedium was detected at any time during the experiment.

In particular, primary human melanocytes, which arevery ‘sensitive’ to stressful culture conditions in term ofmorphology and growth rate, showed a normal spindle-likemorphology without sign of premature senescence withmorphological changes after long-term culturing [59] onthe cluster-assembled TiO2 substrate (Fig. 3 panel D5, D6).

To further analyze cellular functions related to cytoske-leton and adhesions we performed immunostaining oftubulin, actin and vinculin at 2 and 9 days of cell culturing.As shown in Fig. 4 all cell lines tested showed similar

tubulin staining, no specific orientation of the centrosomeand similar extent of microtubules polymerization (Fig. 4panel A1 for MEFs, B1 for Tig3-hTert, C1 for U2OS andD1 for melanocytes), as well as similar actin structures interm of stress fibers, filopodia and lamellipodia (Fig. 4panel A2, B2, C2). Primary human melanocytes displayedrare cortical stress fibers on both substrates (Fig. 4 panelD2). We were particularly interested in evaluating adhesionthrough vinculin staining of adhesion plaques: anydifference related to number, morphology and position offocal adhesions should reflect the number of contacts of thecellular membrane with the substrate and consequentlyreveal the strength and the extent of molecular interaction(mainly trough integrin binding) with the different surfaces[60,61]. On both substrates, depending on the degree ofcellular confluence, we noticed heterogeneity in terms oflength, size and thickness of vinculin-stained focal adhe-sion. Particularly MEFs and Tig3-hTert showed thehighest degree of heterogeneity of focal adhesion morphol-ogy: as shown in Fig. 4 panel A3, B3, both cell linesdisplayed ‘long’ (small arrows) and ‘short’ (big arrows)vinculin-stained focal adhesions but both kind of structureswere comparably present on cells grown on the two

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Fig. 4. Immunofluorescence analysis of cytoskeletal and cellular adhesion markers on gelatin-and cluster-assembled nanostructured (ns) TiO2-coated

coverslips at short and long-term points. (A) MEFs, (B) Tig3-hTert, (C) U2OS, and (D) human primary melanocytes. At 2 and 9 days cells were fixed and

stained with anti–alpha tubulin Ab (1), Alexa488-Phalloidin (2) and antivinculin (3) to analyze focal adhesions. On panel A and B small arrows identify

‘‘long’’ focal adhesions and big arrows identify ‘‘short’’ focal adhesions.

R. Carbone et al. / Biomaterials 27 (2006) 3221–32293226

different substrates at both short- and long-term culturing.Panel C shows focal adhesion of U2OS cells with nosignificant differences. The distribution (cell body orperiphery) and the density of these structures wereundistinguishable on the two different substrates. Humanprimary melanocytes also showed small numbers ofvinculin-stained focal adhesions either on gelatin andcluster-assembled TiO2-coated coverslips.

To definitively assess the biocompatibility of this newsubstrate we analyzed by BrdU incorporation the cell cycleof all cell lines used in this study at 2 days and 10 days ofculturing. In particular for human primary melanocytes weanalyzed the growth inhibition effect due to contactinhibition on both substrates at 20 days. As shown inFig. 5, MEFs (panel A), Tig3-hTert (panel B) and U2OS(panel C) showed similar percentage of S phase at 2 daysand 10 days of culturing suggesting that our substrate doesnot inhibit or stimulate cell cycle progression: moreover thecell cycle analysis of melanocytes showed a similar profileof S phase in exponentially growing conditions (day 2:gelatin 20%, ns-TiO2 17%, day 5: gelatin 16.8%, ns-TiO2

20%) and a comparable growth inhibition at confluency(gelatin 3.5%, ns-TiO2 3.1%).

These results suggest that our new cluster-assemblednanostructured TiO2 surface is a biocompatible supportallowing normal cell adhesion and growth. No sign of

toxicity even after long-term culture toward cells ofdifferent origins (immortalized, tumor and primary cells)are evidenced. Most importantly, our analysis demonstratethat cells grown on this substrate are comparable either interms of morphology, cytoskeleton, focal adhesions andgrowth rate with cells cultured on a gelatin-coated surface.Recent work has specifically addressed the role of focaladhesions in relation with the functional activity ofdifferent biocompatible surfaces [61]. In our work, cellscultured even at long term on cluster-assembled TiO2

present high heterogeneity in focal adhesions localizationand no evident differences compared with cells grown ongelatin-coated coverslips.The relevance of our findings is underlined by the data

obtained on primary melanocytes. Primary cells undergospontaneous senescence in culture according to cumulativepopulation doublings [62], but this phenomenon is stronglyinfluenced by extrinsic factors (i.e. culture conditions) [63].If cells are grown in ‘‘non-appropriate’’ conditions (i.e.presence of stressing agents at subcytotoxic level), theyrapidly undergo premature senescence that is easilyrecognized by dramatic changes in cell morphology [59](i.e. flat, stellate phenotype). Our long-term studies onprimary cells do not show evidence of any gross alterationin cellular morphology (Fig. 3 panel D) suggesting that oursubstrate is suitable for optimal culturing of primary cells.

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% DNA synthesis of Mouse Embryo Fibroblasts

0.010.020.030.040.050.060.070.080.090.0

GEL 2 days TIT 2 days GEL 10 days TIT 10 days

% DNA synthesis of Tig3-hTert cells

0.0

10.0

20.0

30.0

40.0

GEL 2 days TIT 2 days GEL 10 days TIT 10 days

% DNA synthesis of U2OS Cells

0

10

20

30

40

50

60

70

GEL 2 days TIT 2 days GEL 10 days TIT 10 days

% Dna synthesis of Melanocytes

0.0

5.0

10.0

15.0

20.0

25.0

GEL 2days

TIT 2days

GEL 5days

TIT 5days

GEL 20days

TIT 20days

(A) (B)

(D)

(C)

Fig. 5. Cell cycle analysis by BrdU incorporation on cells at specific time points on gelatin- and cluster-assembled TiO2-coated coverslips. (A) MEFs, (B)

Tig3-hTert, (C) U2OS, and (D) primary human melanocytes. For each cell line at specific time points at least 500 cells were analyzed.

R. Carbone et al. / Biomaterials 27 (2006) 3221–3229 3227

We observed that cells adhere and grow on cluster-assembled TiO2 with similar modalities as on the referencebiocompatible surface, i.e. gelatin-coated coverslips.Although at this stage the specific roles of the varioustopographical, physical and chemical properties of thesurface in the cell/substrate interaction could not be ruledout, we are lead to make some hypotheses concerning theobserved phenomena. Besides the previously stressedsimilarities with the gelatin coating in the nano-scaleraster, the surface morphology of cluster-assembled TiO2

shows ‘local’ similarities with recently observed extracel-lular environments [32,33]. The size of the clusterscomposing the TiO2 film, as well the size of many ECMproteins, are of the order of a few tenths of nanometers; asan immediate implication, on a sufficiently small scale (i.e.few hundreds of nanometers) the surface morphology ofcluster-assembled TiO2 can ‘mimic’ a generic ECMenvironment. Given that the topographic features of thesubstrate play an essential role, the morphology of cluster-assembled TiO2 may favor the interaction of cells with itssurface.

Moreover, the high affinity of TiO2 nanoparticlestowards the adsorption of organic groups could also playan important role in promoting adhesion and growth oncluster-assembled TiO2 by favoring the adsorption ofserum proteins upon cell seeding and the deposition ofthe matrix eventually secreted by cells [30]. Small TiO2

nanoparticles (of size below 10 nm) have been shownrecently to promote the chemisorption of organic groups,and carboxylic acids in particular, by exploiting the

abundance of surface defect sites (undercoordinatedtitanium atoms) [64–67]. The latter are also known topromote the dissociative adsorption of water molecules[54,55]; the resulting adsorbed hydroxides are related to theacid/base properties of the surface in aqueous environment[57,58], which allow various acid/base reactions to occur atthe interface thus influencing its bioactivity. As anabundance of both Ti3+ defects and surface hydroxylspecies has been observed in cluster-assembled TiO2 films[53], we are led to speculate that protein adsorption, whichreadily takes place on various TiO2 surfaces [22,34–37],would be enhanced on cluster-assembled TiO2 by thechemisorption of acidic-sidechain carboxyl groups ontopmost nanoparticles. This same mechanism, besidesmaking the surface of cluster-assembled TiO2 a favorableenvironment for the adsorption of serum proteins andECM proteins secreted by cells, would offer interestingopportunities also for the adsorption of functional peptidesand proteins in the framework of cell-based assays.Whether the adsorption of proteins from serum andsecreted ECM is a major factor in promoting cell adhesionand growth, which are the leading adsorption mechanismsand how they are related with the physical and chemicalproperties of the surface, is matter of ongoing research(L. Giorgetti et al., unpublished data).

4. Conclusions

We have described the biocompatibility of nanostruc-tured TiO2 films produced by supersonic cluster beam

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deposition. The deposition of TiOx clusters on a substrateproduces low-density and highly porous films with grainsof few nanometers.

Once deposited, the films were not further functionalizedbefore being employed as culture substrates. Cells platedon this substrate adhered and grew with the samemorphology as cells grown on a gelatin-coated coverslip;the same cytoskeletal parameters (tubulin, actin and focaladhesion) were observed both for cells grown on cluster-assembled TiO2 and on gelatin. Cell cycle analysis by BrdUincorporation showed that our substrate does not inhibitnor stimulate cell growth since the rate of S phase is similarto cells grown on gelatin. These results strongly suggestthat cluster-assembled TiO2 is a biocompatible supportthat allows normal growth of tumor and primary cells.

The use of cluster-assembled TiO2 films as cell culturesubstrates is of particular interest for the coupling ofcultured cells on microfabricated devices, since SCBD isfully compatible with planar microfabrication technologiesand it allows the deposition of patterns with submicro-metric lateral resolution [50,68]. We thus propose that thismaterial can be a very interesting substrate for differentapplications requiring the integration of cell cultures onmicro- and nanodevices and arrays. SCBD can also be usedto coat bioactive orthopedic, dental, vascular implants.

Cluster-assembled TiO2 thin films are optically trans-parent and free of defects causing visible light scattering.Therefore, they are also particularly suited for high-resolution and confocal microscopy characterizations.

Acknowledgments

We thank C. Ducati for TEM characterization and G.Giardina and C. Spinelli for their help in melanocytescharacterization. This work has been supported by AIRCunder OGCG grant ‘‘Development and integration ofhigh-throughput technologies for the functional genomicsof cancer’’ and Fondazione CARIPLO under grant‘‘Sviluppo di sistemi micro— e nanostrutturati per analisifenotipiche di famiglie di geni’’.

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