osteoblast-like cell behavior on plasma deposited micro/nanopatterned coatings

Osteoblast-Like Cell Behavior on Plasma Deposited Micro/Nanopatterned Coatings

Francesca Intranuovo,*,† Pietro Favia,†,‡,§ Eloisa Sardella,‡ Chiara Ingrosso,|,⊥

Marina Nardulli,† Riccardo d’Agostino,†,‡,§ and Roberto Gristina‡

Department of Chemistry, University of Bari, Italy, Institute of Inorganic Methodologies and Plasmas,IMIP-CNR, Bari, Italy, Plasma Solution s.r.l., spin-off of the University of Bari, Italy, and Institute for

Chemical and Physical Processes, IPCF-CNR, Bari, Italy

Received September 22, 2010; Revised Manuscript Received November 12, 2010

The behavior of cells in terms of cell-substrate and cell-cell interaction is dramatically affected by topographicalcharacteristics as shape, height, and distance, encountered in their physiological environment. The combinationof chemistry and topography of a biomaterial surface influences in turns, important biological responses asinflammatory events at tissue-implant interface, angiogenesis, and differentiation of cells. By disentangling theeffect of material chemistry from the topographical one, the possibility of controlling the cell behavior can beprovided. In this paper, surfaces with different roughness and morphology were produced by radiofrequency (RF,13.56 MHz) glow discharges, fed with hexafluoropropylene oxide (C3F6O), in a single process. Coatings withdifferent micro/nanopatterns and the same uppermost chemical composition were produced by combining twoplasma deposition processes, with C3F6O and tetrafluoroethylene (C2F4), respectively. The behavior of osteoblast-like cells toward these substrates clearly shows a strict dependence of cell adhesion and proliferation on surfaceroughness and morphology.

Introduction

The in vitro study of cell behavior toward different materialsurface properties represents a necessary prerequisite in assessingthe biocompatibility of a material intended to be used in medicaldevices. In addition to mechanical properties, surgical require-ments, and ability to stand sterilization procedures, a biomaterialmust interact adequately with the biological environment throughits physical/chemical surface characteristics, because a strongdependence of cell adhesion/proliferation on substrate surfaceproperties exists. Cells can react, both in vivo and in vitro,differently to chemical1 and topographical stimuli.2 Roughness,wettability, surface mobility, chemical composition, crystallinity,and other material surface properties can direct biologicalreactions.3 Surface wettability influences cell-material interac-tions, because a poor spreading of eukaryotic cells is usuallyobserved on hydrophobic substrates and it increases on hydro-philic ones. As regards the roughening of the surface, it hasbeen widely demonstrated that microroughness contributes tocell attachment, spreading and differentiation.4 Moreover, thesuperimposing of a nanoroughness enhances local factor pro-duction. Indeed, many studies have demonstrated that thepresence of topographical micro/nanofeatures on a surfaceallows to control and manipulate two fundamental externalsignals: cell-substrate and cell-cell interactions. In this way,many cellular and biological processes are influenced, such ascell metabolism, phenotypic expression, and the inflammatoryresponse at tissue implant interface.5

The surface properties of a material can be tuned by creatingwell-defined topographical and chemical patterns on the surfaceto rapidly investigate the interaction between various cell typesand these materials by means of in vitro experiments.

Photolithography, reactive ion etching, and anisotropic etchingwere the first techniques developed to create surfaces with well-defined topography.6,7 Microcontact printing, inkjet printing, anddiamond cutting are usually suitable only for micropatterning.8

Sandblasting has also been employed to produce roughnessgradients on the substrate, allowing a systematic investigationof their effect on osteoblast and fibroblast cell behavior.9 Insitu polymerization, solvent casting, embossing, or melt moldingare used to obtain a replica of micro/nanopatterned surfaces withhigh fidelity.10 Anodic oxidation has been employed to modifytitanium surface oxides in both composition and topography,reaching an increase of osteoblast adhesion and proliferationon the anodic oxides.11 Most of the above-mentioned techniquesare expensive, time-consuming, and unable to independentlychange chemistry or topography of a material surface, thus,producing an effect on biological environment that can not beunivocally attributed only to a parameter. Among severalapproaches, used to modify material surfaces, plasma technolo-gies offer interesting benefits.12 Nonequilibrium, cold plasmaprocesses are energy efficient dry techniques that can bedeveloped in a wide pressure range and alter only the very toplayers of a material surface, preserving its bulk properties.13

By plasma processes it is possible to modify pre-existing (i.e.,commercialized) materials, improving their performances in thebiomedical field without affecting their mechanical properties.Basically, such processes can provide a variety of functionalizedsurfaces employed in biology and medicine. They involve thegrafting of chemical groups or the deposition of micro/nanocoatings to create surfaces characterized by differentroughness,14-16 or cell-adhesive films with well-definedchemistry,17-19 that can be eventually functionalized for bio-

* To whom correspondence should be addressed. Tel.: (39) 080 5443434.Fax: (39) 080 5443405. E-mail: [email protected].

† Department of Chemistry, University of Bari.‡ Institute of Inorganic Methodologies and Plasmas.§ Plasma Solution s.r.l.| Institute for Chemical and Physical Processes.⊥ Present address: Institute of Microelectronics and Microsystems IMM-

CNR Lecce, Italy.

Biomacromolecules 2011, 12, 380–387380

10.1021/bm101136n 2011 American Chemical SocietyPublished on Web 12/29/2010

molecule immobilization.20 Due to its high versatility and easyprocessing, in addition to obtaining coatings characterized bydifferent chemical/morphological properties in a single stageprocess, the plasma technology can also realize substrates whereonly a surface parameter varies by keeping constant the others.In this way, it is possible to produce coatings with variabletopography and constant chemical composition, or vice versa,as attested by previous literature studies.16,21 In turn, it couldbe possible to distinguish the role of a single surface parameteron cell behavior, irrespectively of the others. This is afundamental aspect in the assessment of cell guidance mecha-nism on materials, because they provide a variety of mechanical,chemical, and topographical stimuli whose effects on cells areusually difficult to disentangle.

Plasma deposition processes fed with fluorocarbon gases,especially when run in modulated power regime or in afterglow,can provide the deposition of Teflon-like and rough coatings,characterized by different topographical features like ribbonsor bumps,22-24 able to successfully tune the cell response.25,26

The deposition mechanism of such micro/nanostructured coat-ings was discussed by Milella et al.27

We have previously deposited fluorocarbon thin films byplasma enhanced chemical vapor deposition (PECVD) fed withhexafluoropropylene oxide (C3F6O).28 Their peculiar surfacecharacteristics (e.g., roughness, hydrophobicity) have beendemonstrated to depend on the experimental conditions, espe-cially on the distance of the substrate from the plasma regionin the reactor (glow vs afterglow areas). In particular, moreTeflon-like, hydrophobic and micro/nanostructured coatingswere deposited by increasing the distance from the glow region.

Among them, two thin film typologies, chosen for theirdifferent morphologies (i.e., petal-like vs spherical shapedcoatings), have been investigated in the present paper to studythe influence of the substrate topography and morphology onthe human osteoblast-like Saos-2 cells behavior, in terms of cellmorphology and proliferation.

In this light, plasma processes become a successful tool toproduce surfaces with different substrate morphologies in thesame plasma deposition experiment that give the opportunityto quickly study the cell behavior at different roughness degrees.We pursued our goal by looking at different techniques likeMTT, Coomassie Blue staining, SEM analysis, and cytoskeletonobservation to give a general view of the cell response.

Materials and Methods

Substrates and Plasma Deposition Conditions. Polyethyleneterephthalate (PET) substrates (Goodfellow, 0.5 mm thick) were coatedwith fluorocarbon films obtained by radiofrequency (RF, 13.56 MHz)PECVD processes. A RF parallel plate plasma reactor was used whosetechnical details have been described in a previous paper.28

First, PET samples were coated with films by plasmas fed withC3F6O, at the following experimental conditions: C3F6O (Fluorochem)40 sccm flow rate; 50 W power; 900 mTorr (0.120 kPa) pressure; 120min deposition time. Samples were positioned in the reactor chamberat 8 and 18 cm from the gas inlet. Then, these morphologically differentfilms and flat native PET substrates were further coated with afluorocarbon film, by a PECVD process fed with C2F4, at the followingplasma conditions: C2F4 (Fluorochem) 6 sccm flow rate; 100 W power;200 mTorr (0.027 kPa) pressure; 21 s deposition time.

Wettability Measurements. Static water contact angle (WCA)measures were carried out at room temperature (RT) by a CAM200digital goniometer (KSV instruments), equipped with a BASLER A60fcamera by sessile drop (2 µL) method. Five measures per sample wereperformed.

Chemical Characterization. X-ray photoelectron spectroscopy(XPS) analysis was performed by a Theta Probe Thermo VG Scientificinstrument (base pressure of 1 × 10-10 mbar), equipped with ahemispherical analyzer and a nonmonochromatic Al KR (hν 1486.6eV) X-ray source operating at 300 W. Photoelectrons were collectedat a takeoff angle of 53°, corresponding to a sampling depth of ∼10nm. High resolution spectra were shifted to their correct position bytaking the component centered at 292.0 eV (CF2) as reference.29,30 Thesoftware Thermo Avantage 3.28 (Thermo Electron Corporation) wasused either to determine the elemental composition from peak areaseither to peak fit the high resolution spectra. For C1s fitting, fivecomponents were considered, as shown in Figure 2: CF3 (294.5 ( 0.2eV, solid line), CF2 (292.0 ( 0.2 eV, dashed line), CF (290.0 ( 0.2eV, dotted line), C-CF (288.0 ( 0.2 eV, dash-dotted line), and C-C(285.0 ( 0.2 eV, solid line). The F/C ratio was calculated from thebest fitting of the C1s spectrum, according to the equation reported inthe Scheme 1, where CF3%, CF2%, and CF% are the contributions ofthe CFx (x ) 1, 2, and 3) components to the total C1s area.

The F/C ratio was allowed to probe the structure retention of themonomer and it provided an evaluation of cross-linking degree. Thelower the F/C ratio was, the higher the degree of cross-linking ofthe coating was. A F/C ratio close to 2 represented a chemical structuresimilar to conventional Teflon (high Teflon character).31

Morphological Characterization. A Stereoscan 360 Cambridgescanning electron microscope (SEM), operating at 20 KV, with a 50°tilt angle, was used to examine the surface morphology of the coatingsand evaluate its distribution on the whole sample surface. Because thesamples were nonconductive, they were sputter-coated with a 10 nmthick gold layer before SEM examination, using the Biorad PolaronDivision, SEM Coating System, E5100 Sputter Coater.

Height mode atomic force microscopy (AFM) investigations wereperformed in air, at RT, by means of a PSIA XE-100 SPM system,operating in tapping mode. A silicon SPM sensor for noncontact AFM(NanoWorld) was used, with a constant force of 42 N m-1 and aresonance frequency of 320 kHz. Topographic micrographs werecollected on six areas of each sample, with a scan size area of 10 × 10µm2, by sampling the surface at a scan rate of 0.8 Hz and a resolutionof 256 × 256 pixels. AFM images were processed by using a XEIProgram to flatten the topographic micrographs, to remove the slopeand curvature artifacts produced by the scanning process, and obtainstatistical data as surface root-mean-squared roughness (rms) and meanheight of sample features. The XEI software was also used to representa three-dimensional perspective of the sample surface with the originalpixel resolution of 256 × 256 pixels and to extract histogram panelsshowing the distribution of feature heights.

Cell Culture. Untreated and plasma-modified PET samples wereplaced in 24-well plates. Before cell seeding, the samples were soakedin ethanol for 15 min. Cell culture experiments were performed withthe human osteoblast Saos-2 cell line (ICLC, Italy). Cells were routinelygrown in Dulbecco’s Modified Eagle Medium (DMEM, Sigma Chemi-cal Co., Italy), supplemented with 10% heat-inactivated fetal bovineserum (FBS), 50 IU/mL penicillin, 50 IU/mL streptomycin, and 200mM glutamine, at 37 °C, in a saturated humid atmosphere containing95% air and 5% CO2, in 75 cm2 flasks (Barloworld Scientific, U.K.).For cell culture experiments, cells were detached with a Trypsin/EDTAsolution (Sigma, Italy), suspended in the correct medium, and seededat a concentration of 1 × 105 cells/mL on native cell culturedpolystyrene (CCPS) and on modified substrates for culture times up to96 h.

Mitochondrial Function Measurement. The mitochondrial activityof Saos-2 cells, seeded on the substrates at different culture times (24,

Scheme 1. Equation Used To Calculate the F/C Ratio

FC

)3CF3% + 2CF2% + CF%

100

Plasma Deposited Micro/Nanopatterned Coatings Biomacromolecules, Vol. 12, No. 2, 2011 381

48, 72, and 96 h), was determined with the MTT colorimetric assay.This test detects the conversion of 3-(4,5 dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (Sigma Co., St. Louis, MO, U.S.A.) toformazan. At each time point, the cells were incubated in a tenth of3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide medium,at 5% CO2 (37 °C, 3 h), to allow the formation of formazan crystals.32

They were then dissolved in 10% Triton X-100, with acidic isopropanol(containing 0.1 N HCl), acid-isopropanol (95 mL isopropanol with 5mL 3 N HCl). Finally, the optical density was read with a spectro-photometer (Jenway 6505, GB), at a wavelength of 570 nm, using 690nm as reference wavelength.33 Each experiment was performed intriplicate; data were presented as optical density (O.D.) values.

Cell Morphological Analysis. Saos-2 cells seeded on the substratesand analyzed at different cell culture times, were fixed in 4%Paraformaldehyde/PBS solution (15 min) and stained with a dyesolution composed by 0.2% Coomassie Brilliant Blue R250 (Sigma,Italy), 50% methanol and 10% acetic acid, for 3 min. Cells wereobserved on the samples at different magnifications, by means of aphase contrast microscope (Leica DM ILI). At least 15 images persample were acquired through a CCD camera (Leica DC100). Imageswere then analyzed with the Image J software (National Institute ofHealth, U.S.A.) to evaluate the substrate area covered by cells. Statisticalanalysis was performed by a two-way ANOVA test within groups,followed by a Bonferroni post-test, by using the GraphPad Prism version4.00 for Windows (GraphPad Software, San Diego, California, U.S.A.,www.graphpad.com). Differences were considered statistically signifi-cant for p < 0.01.

To observe the actin cytoskeleton, the cells were fixed in 4%formaldehyde/PBS solution, at RT for 20 min, permeated with PBScontaining 0.1% Triton X-100 and incubated with Alexa Fluor488phalloidin (Molecular Probes) at RT and for 20-30 min. Tubulin wasdetected with a monoclonal antibody raised in mouse (Sigma). Vinculinwas detected with a monoclonal antibody raised in mouse (Sigma).For both vinculin and tubulin staining, a secondary antibody againstmouse IgG conjugated with Alexa Fluor546 was used. After rinsing,samples were mounted in Vectorshield fluorescent mountant with DAPI(Vector Laboratories, U.K.) and then observed by means of anepifluorescence microscope (Axiomat, Zeiss, Germany).

For a SEM observation of cell structure and distribution on thesamples, the cells were fixed with 2.5% glutaraldehyde/0.1 M sodiumcacodylate solution and dehydrated using a series of ethanol/watersolutions (20, 40, 50, 70, 90, and 100%). Finally, the cell culturedsamples were air-dried under a biological hood and sputter-coated witha 10 nm thick gold layer for the SEM visualization.

Results and Discussion

Plasma Deposition of Micro/Nanostructured FluorocarbonThin Films. Fluorocarbon coatings, characterized by differentchemical composition and topography, were plasma depositedwith C3F6O feed at various distances from the glow to theafterglow region of the plasma reactor in the same singleprocess. In particular, by increasing the distance from the glowregion, more and more hydrophobic and Teflon-like coatingswere deposited, as described in a previous paper.28 Moreover,the branching/cross-linking degree of the coating decreased,while the fluorination degree and the monomer retentionincreased. This effect could be explained by a recombinationof radicals produced in the plasma phase, together with areduced extent of the fragmentation in the deposited film, dueto the reduced (absence) ion bombardment on the substrateplaced downstream.27 At the same time, a great change in bothsurface roughness and morphology was observed. Completelysmooth coatings in the glow, nanobumped films at a mediumdistance (8 cm) from the gas inlet and a “petal” building in theregion 11-18 cm from the glow were obtained. Here, singlelarge features seemed to be formed by the agglomeration of

several small nodules that tangled each other, showing tall petal-like micro/nanostructures.

In this study, substrates with different morphologies butidentical chemical composition were produced, with the aim ofunderstanding how the topography of the coatings, synthesizedat 8 and 18 cm from the gas inlet of the plasma reactor (namedAG8 and AG18, respectively) could influence the behavior ofosteoblast-like Saos-2 cells, in terms of adhesion, morphology,and proliferation.

To achieve this task, AG8 and AG18 coatings, characterizedby well distinct morphology and flat PET substrates were coatedwith the same thin fluorocarbon film, by means of a C2F4

PECVD. A scheme of the rationale of this work has beenpresented in Figure 1.

Wettability and Chemical and Morphological Characteriza-tion of the Fluorocarbon Thin Films. To ascertain any differencedue to the C2F4 coating, WCA, XPS, and AFM analyses wereperformed. The WCA for the flat C2F4 coating was 108 ( 3°(Table 1). The effect of C2F4 coating was to slightly lower theWCA for both AG8 and AG18 samples. Indeed, in the case ofAG8 samples, WCA values decreased from 135 ( 1° to 129 (3° and for AG18 from values greater than 170 to 165 ( 2°. Inthis last case a superhydrophobic material was obtained, due toa combination of chemical and topographical factors. Thechemical composition of AG8 and AG18 samples before C2F4

coating was close to that of Teflon, as attested by the F/Cpercentages shown in Table 1. On the other hand, after C2F4

plasma deposition, both AG8 and AG18 completely lost theTeflon character, with a lowering of F/C ratio, reaching valuessimilar to that associated with the flat coating, as indicated inTable 1. This evidence was correlated to an increase of CFx

components respect to CF2 in the C1s spectra, attesting a carbonchemical composition very close to the flat C2F4 coated sample,whose deconvoluted high resolution C1s spectrum has beenreported in Figure 2. These results demonstrated that substrateswith the same chemical composition were produced. Thisattested to a conformal deposited Teflon-like coating on theplasma micro/nanostructured surfaces.

The surface topography of AG8 and AG18 samples has beeninvestigated by AFM before and after the C2F4 plasma deposi-tion. Estimated values of the corresponding mean feature heightsand film rms roughness have been reported in Table 1. Theanalyses of surface topography showed that the AG8 coatinghad a nanoroughness (Table 1) and a morphology characterized

Figure 1. Scheme of the PECVD processes. The first C3F6O plasmadeposition (40 sccm flow rate; 50 W power; 900 mTorr pressure; 120min deposition time) allowed to produce two morphological differentthin films. The further C2F4 plasma deposition (6 sccm flow rate; 100W power; 200 mTorr pressure; 21 s deposition time) on these andon flat PET substrates, allowed to make their chemical compositionsimilar and keep their starting morphology different each other (namedflat, AG8 and AG18 samples).

382 Biomacromolecules, Vol. 12, No. 2, 2011 Intranuovo et al.

by a dense highly interconnected layer of uniform nodularstructures, as attested by the AFM micrograph and SEM imageof Figure 3a and e, respectively. The structures had nanosizedheights, distributed almost symmetrically around the mean value(Figure 3c). After the C2F4 plasma deposition, only a slightdecrease of both mean height and roughness was observed(Table 1), but the round-shaped morphology was preserved, asshown in the Supporting Information. On the other hand, theAG18 film had a different morphology respect to AG8,consisting of irregular and randomly distributed protrudingstructures as attested by the AFM micrograph and SEM imageof Figure 3b and f, respectively. Both mean heights androughness of the AG18 film were higher than those typical ofthe AG8 surface (Table 1). Such values for AG18 slightlydecreased after the C2F4 deposition (Table 1), preserving thepetal-like shaped morphology (shown in the Supporting Infor-

mation). It is worthwhile to notice that the feature heightdistribution of the AG18 sample evidenced the presence of bothmicro- and nanostructures, whose heights were asymmetricallydistributed around the mean value (Figure 3d). Such a resultwas observed both before and after C2F4 plasma deposition. Thissimultaneous presence of both micro- and nanostructures hasbeen demonstrated to be relevant on improving the cell affinity4

on the material surface.In our work, for both AG8 and AG18 films, the C2F4 plasma

deposition caused a slight decrease of mean height androughness, with a pronounced reduction of the Teflon character.Both these morphological and chemical changes have beenexpected to be responsible of the slight increase of surfacehydrophilicity.34-38

Cell Response to the Substrates Topography. The cellbehavior on flat PET, AG8, and AG18 films, having the samesurface chemistry and different roughness and morphology, wasinvestigated. For this purpose, the human cell line Saos-2 waschosen, as it is representative of a cell type that usually has agood affinity with biological surfaces characterized by a micro/nanotopography. Cell growth on different plasma-modified andCCPS substrates has been studied in terms of cell spreading,morphology, and cytoskeleton organization, at four culture times(24, 48, 72, and 96 h). CCPS surfaces were used as an internalcontrol because osteoblast cells are known to adhere and growvery well on them.39

Cell proliferation on substrates was quantified by MTT test.As expected, the cells grown on CCPS showed a higher adhesionand better proliferation than on the plasma-modified surfacesat all the four cell culture times, as the histogram in Figure 4clearly shows. This result was expected because it is well-knownthat hydrophobic surfaces discourage either protein and celladhesion with respect to hydrophilic surfaces as CCPS.

By comparing Saos-2 cell proliferation on the three plasmatreated surfaces, the only statistically significant difference wasobserved after 48 h of cell culture, when the MTT value on flatsubstrates was higher than that on the two micro/nanostructured

Table 1. WCA, F/C %, Mean Height, and Mean RMS Data for Flat, AG8, and AG18 Samples, before and after the C2F4 Deposition

flat (C2F4) AG8 (C3F6O) AG8 (C3F6O + C2F4) AG18 (C3F6O) AG18 (C3F6O + C2F4)

WCA (°) 108 ( 3 135 ( 1 129 ( 3 >170 165 ( 2F/C % 1.57 ( 0.03 1.91 ( 0.04 1.58 ( 0.03 1.97 ( 0.01 1.50 ( 0.03mean height (nm) 0.6 ( 0.2 247 ( 32 225 ( 55 1000 ( 100 800 ( 100mean rms (nm) 4.8 ( 1.0 74 ( 8 65 ( 11 386 ( 5 368 ( 2

Figure 2. Best fitting of C1s high resolution spectrum of flat, AG8and AG18 samples after the C2F4 deposition. In the fitting of thespectrum, the following components were considered: from the leftside, CF3 (294.5 ( 0.2 eV, solid line), CF2 (292.0 ( 0.2 eV, dashedline), CF (290.0 ( 0.2 eV, dotted line), C-CF (288.0 ( 0.2 eV, dash-dotted line), and C-C (285.0 ( 0.2 eV, solid line).

Figure 3. 3D view of AFM images, histogram panels of feature heightdistribution, and SEM images of AG8 (a, c, e) and AG18 (b, d, f)surfaces, both after the C2F4 deposition.

Figure 4. MTT activity of Saos-2 cells grown on flat, AG8, and AG18surfaces after the C2F4 deposition and on CCPS samples, at differentcell culture times (24, 48, 72, and 96 h). Optical density mean valueswere shown. Significant differences between the means were calcu-lated by the two-way ANOVA analysis and the Bonferroni post/test((0) p < 0.01 vs FLAT; (O) p < 0.01 vs AG8; (b) p < 0.01 vs AG18).


surfaces (p < 0.01). At the other cell culture times, no statisticallysignificant difference has been found among the three substrates.Cell proliferation data on flat surfaces linearly increased withthe culture time. Instead, for AG8 and AG18, the data werelow until 48 h and rapidly increased at 72 h, approaching thevalues obtained with flat samples (p > 0.05).

Because the MTT assay evaluates the metabolic activity ofthe cells, not giving information about the variation of theirnumber or shape, optical microscopy analysis has also beenperformed, after fixing and staining the cells with CoomassieBlue. The low magnification images in Figure 5 show the cellspreading and clustering after 96 h of culture, on the fourdifferent substrates. The cell behavior was evidently dependenton the substrate below. On CCPS substrates (Figure 5d), Saos-2cells spread and clustered to a larger extent than cells plated onfluorinated surfaces. Actually, the cells started clustering onCCPS in the first hours of culture, and this phenomenon wasevident at 24 h (Figure 6d) when it was hard to find cellsseparated from the others. On AG18 a similar cell clusteringwas observed (Figure 5c). The cells were less grouped with eachother on flat coatings (Figure 5a) and isolated more on AG8films (Figure 5b).

By observing Figure 6, the differences in size and shape ofsingle cells cultured for 24 h on the substrates have been betterappreciated. The cells on flat and AG8 substrates (Figure 6aand b, respectively) were rounded or elongated shaped. OnAG18 instead, the cells were elongated or polygonal shaped(Figure 6c), similar to the cell morphology on CCPS samples(Figure 6d).

To quantify the differences and changes in spreading of cells,the percentage of substrate area covered by cells on the surfaces,calculated from the Coomassie Blue stained images, wasmeasured by the Image J software. The graph in Figure 7summarizes the results as total area covered by cells, at fourcell culture times. After 72 h of cell culture, a difference in cellarea and spread was observed among the three differentlyfluorinated substrates.

These data confirmed the highest value of proliferation forcells grown on CCPS, observed with the MTT test. Instead,among the plasma-modified surfaces, the highest cell growthon AG18 substrates stood out. This could be due to the micro/

nanostructuring of its surface morphology. Indeed, it is knownthat the cell growth is favored at the surface discontinuities.Many recent papers have confirmed that adhesion, migrationarea, and extracellular matrix (ECM) production are higher onrough surfaces or with larger grain sizes.40,41 Besides, thesubstrates with grooves and cliffs of greater (micro-) sizestimulate the movement of a variety of cells.42 The microfeaturesof AG18 surfaces likely induced similar effects on osteoblastcell behavior.

Cytoskeleton analysis has provided another tool to study thedifferences in cell morphology on the substrates. It was firstfocused on tubulin and actin observation, since they representthe more abundant proteins of the cytoskeleton. The tubulinorganization (Supporting Information) merely reflected the cellshape, confirming the results observed with Coomassie Bluestaining in Figure 6.

In Figure 8, the Saos-2 cell cytoskeleton stained for actin(green) and nucleus (blue) for the four substrates, at 24 h culturetime, has been shown. The cells on CCPS (Figure 8f) presenteda well developed actin cytoskeleton with stress fibers throughoutthe cytoplasm. In regard to the plasma-modified surfaces, only

Figure 5. Optical images of Saos-2 cells cultured for 96 h on flat (a),AG8 (b), and AG18 (c) surfaces after the C2F4 deposition and onCCPS (d) substrates. Cells were fixed with a 4% paraformaldehyde/PBS solution and stained with Coomassie Blue. All images show theclustering of the cells, except for the AG8 sample (b), where most ofthe cells grew isolated without grouping each other.

Figure 6. Optical images of single cells after 24 h of culture time onflat (a), AG8 (b), and AG18 (c) surfaces after the C2F4 depositionand CCPS (d) substrates. Cells were fixed with a 4% paraformalde-hyde/PBS solution and stained with Coomassie Blue. Images showthe cell morphology change from rounded or elongated on flat (a)and AG8 (b) substrates to polygonal on AG18 (c) and CCPS (d)substrates.

Figure 7. Percentage of substrate’s area covered by Saos-2 osteo-blasts, grown on flat, AG8, and AG18 surfaces after the C2F4

deposition and CCPS samples, at different cell culture times (24, 48,72, and 96 h). The data represent mean values of cells area calculatedfrom 10 images with a 3 mm2 area, where the cells were previouslyfixed and Coomassie Blue stained. Significative differences betweenthe means were calculated by the Two-way ANOVA analysis andthe Bonferroni post/test ((0) p < 0.01 vs FLAT; (O) p < 0.01 vs AG8;(b) p < 0.01 vs AG18).


AG18 substrates induced a flattened polygonal shaped cellmorphology (Figure 8c), with a stress fibers organization verysimilar to CCPS. It was also present on flat (Figure 8a and c,clustered and single cells, respectively) and AG8 (Figure 8band d, clustered and single cells, respectively) surfaces, withoutany difference between single and clustered cells.

By considering the two rough surfaces (AG8 and AG18),another different actin staining was evident. Only on AG8,Saos-2 cells ended with spots of actin anchoring to thenanofeatures of the substrate below (Figure 8b,e).

Because these spots could be evidence of focal adhesion sites,we looked at the expression of vinculin, one of the majorcomponents of focal adhesions.

Saos 2 cells grown on the four surfaces, showed a perinuclearaccumulation of the protein and bean shaped spots of vinculin(Figure 9a), typical of focal adhesion structures at the end ofactin stress fibers. As previously described, Saos-2 cells on AG8surfaces presented spots of actin accumulated at the cellperiphery. When these spots were investigated with an actin/vinculin staining, the vinculin was present only at the end of

actin stress fibers (Figure 9b, white ellipse). Instead, on actinterminal spots, no colocalization of the two proteins was found(Figure 9b, white arrow). No consistent difference in the amountof vinculin focal adhesion sites was present on the foursubstrates (Supporting Information).

Further experiments could shed light on the presence of otherimportant cytoskeleton components such as paxillin and vimen-tin, in order to better understand the relationships betweensubstrate topography and Saos-2 cells, in terms of cytoskeletonrearrangement.

All surfaces were produced using the same plasma processprocedure to ensure the same surface chemistry, as previouslyreported. Therefore, we supposed that the differences observedin cell response were exclusively dependent on micro/nanoscaleroughness.

Visual observation of the cells, by both optical and fluores-cence microscopy, clearly showed how the cells interacted indifferent ways with the substrate. A more accurate study of thecell-substrates interactions on the three plasma-modifiedsurfaces was performed to better understand the role of the

Figure 8. Fluorescence microscopy images of Saos-2 cells grown for 24 h on flat (a, d), AG8 (b, e), and AG18 (c) surfaces after the C2F4

deposition and CCPS (f) substrates. The cells were fixed in 4% formaldehyde/PBS solution and incubated with Alexa Fluor488 phalloidin,allowing the observation of the actin (green fluorescence). The samples were then mounted in Vectorshield fluorescent mountant with DAPI,allowing the observation of the nucleus (blue). Red arrows indicate spots of actin at the end of the cells anchoring to the nanostructures of theAG8 substrates.

Figure 9. Fluorescence microscopy images of Saos-2 cells grown for 24 h on AG8 surfaces after the C2F4 deposition. Cells were fixed in 4%formaldehyde/PBS solution and incubated with Alexa Fluor488 phalloidin, allowing the observation of the actin (green fluorescence). Vinculinwas detected with a monoclonal antibody raised in mouse, followed by an incubation with a secondary antibody against mouse IgG, conjugatedwith Alexa Fluor546 (red). Finally, the samples were mounted in Vectorshield fluorescent mountant with DAPI, allowing the observation of thenucleus (blue). In (a) the vinculin staining shows the presence of a perinuclear accumulation of the protein and the spots of vinculin typical offocal adhesion structures. In (b) is shown a triple staining of actin (green), vinculin (red) and nuclei (blue). The white ellipse indicates thepresence of vinculin staining only at the end of actin stress fibers while no vinculin staining is shown on actin terminal spots (white arrow).


substrate micro/nanostructuring on the cell behavior. Figure 10shows how the Saos-2 cells attached in different ways accordingto the substrate roughening. Interactions by long filopodia wereobserved on flat coatings (Figure 10a2-5), while the adhesionto the nanodomes on AG8 surfaces was mainly exploited byboth lamellipodia (Figure 10b2-5) and filopodia (Figure 10b3).Instead, the tall microstructures on AG18 surfaces were mainlyexplored by lamellipodia (Figure 10c2-5) that were lessextended than those on AG8 and aided by very short filopodia(Figure 10c3-5). Besides, many cells had filopodia in contactwith the surface structures and their length seemed to becorrelated to the dimension of the micro/nanofeatures. In Figure10c4,5, the profile of the tall petal-like structures below the cellbody could be observed. Thus, the cells attached to the AG18substrate, even if very rough, by anchoring with thin lamelli-podia and short filopodia.

Thus, this SEM study clearly demonstrated that cells inter-acted directly with the micro/nanofeatures, confirming the strongdependence of the cell behavior on surface topography, con-sistent with the analyses previously illustrated.

It is likely that the different response of Saos-2 cells to surfaceroughness and morphology reflected differences in integrin-mediated signaling. Cells respond to biomaterial surfacesthrough interactions between the cell membrane receptorintegrins and the adsorbed ECM proteins including fibronectin.39,43

Protein adsorption on materials is highly influenced by surfacechemistry, hydrophilicity, and topography, and in particular,protein adsorption has been demonstrated to depend on the scaleof surface roughness. An example is represented by titaniumsurfaces, whose nanoscale surface texture seemed to have littleor no effect on protein adsorption and cell proliferation.However, microrough surfaces adsorb more fibronectin and theprotein orientation is different from that on flat surfaces, whichfurther alter integrin adhesion.

In light of the previous considerations, it could be deducedthat the differences observed in terms of major adhesion and

spreading on the micro-(AG18) in respect to nanoscale rough-ness (AG8) were due to the protein adsorption. The copresenceof micro/nanofeatures on AG18 surfaces seemed to providemore anchoring sites to the cells, facilitating their adhesion,according to previous studies.4

Conclusions

By plasma processes we have easily tuned the surfacetopography of PET substrates, affecting the ability of osteoblast-like cells behavior. A strict correlation between the materialroughness (feature height and micro/nanoscaling) and the celladhesion, proliferation, and morphology has been illustrated,demonstrating that among the properties of a surface, thetopography is a decisive factor in mediating the cell-materialinteractions. Moreover, because the chemical composition ofthese surfaces is fluorinated, known to be cell-repellent, the roleof topography has to be considered still more decisive on theosteoblast cell behavior. Particularly interesting are the highercell adhesion and spreading on the taller micro/nanoroughcoatings. Further studies, for example, on the different expres-sions of genes involved in osteoblast differentiation could betterexplain the mechanisms of cell-substrate interactions when thesubstrate roughening is varied.

Because this plasma modification process is independent fromthe material, these thin fluorocarbon coatings could be easilyapplied to any material to be used in the biomedical field. Forthe ease of producing different substrate morphologies in thesame deposition experiment, this fluorocarbon plasma depositioncan become a strong tool to quickly investigate the cell behaviorat different roughness degrees at any biological interface.

Acknowledgment. The authors thank the laboratory supportof Mr. S. Cosmai (IMIP-CNR Bari, Italy), Mrs. P. Rossini(Plasma Solution Srl, Italy), Ms. G. Genchi, and Mrs. R.Giordano. This work has been funded by the PRISMA INSTM

Figure 10. SEM images of Saos-2 cells cultured for 24 h on flat (a), AG8 (b), and AG18 (c) surfaces after the C2F4 deposition at differentmagnifications. The cells were fixed with 2.5% glutaraldehyde/0.1 M sodium cacodylate solution, dehydrated using a series of ethanol/watersolutions, and air-dried under a hood. High magnification pictures (a3,4,5; b3,4,5; c3,4,5) show the cell-material interactions, characteristic foreach kind of surface: long cell filopodia on flat, lamellipodia on AG8, short filopodia, and lamellipodia on AG18. Dotted arrows show highmagnification images of the cell protrusions on the substrates.


(PRISMA05MADA1 nanostructured surfaces having a specificbiological response) project and the INTERREG (I 2101003)Italy-Greece regional (Apulia and Acaia regions) project.

Supporting Information Available. Detailed informationabout the surface morphology of the plasma-modified substrates(AFM and SEM figures) and about the cell morphology(fluorescence and SEM figures) are provided. This material isavailable free of charge via the Internet at http://pubs.acs.org.

References and Notes(1) Matsuda, T.; Sugawara, T. J. Biomed. Mater. Res. 1996, 32 (2), 165–

73.(2) Dalton, B. A.; Walboomers, X. F.; Dziegielewski, M.; Evans,

M. D. M.; Taylor, S.; Jansen, J. A.; Steele, J. G. J. Biomed. Mater.Res. 2001, 56 (2), 195–207.

(3) Lee, J. H.; Khang, G.; Lee, J. W.; Lee, H. B. J. Biomed. Mater. Res.1998, 40, 180–86.

(4) Zhao, G.; Rainesa, A. L.; Wieland, M.; Schwartz, Z.; Boyan, B. D.Biomaterials 2007, 28, 2821–29.

(5) Singhvi, R.; Kumar, A.; Lopez, G. P.; Stephanopoulos, G. N.; Wang,D. I.; Whitesides, G. M.; Ingber, D. E. Science 1994, 264, 696–98.

(6) Wojciak-Stothard, B.; Madeja, Z.; Korohoda, W.; Curtis, A.; Wilkin-son, C. Cell Biol. Int. 1995, 19 (6), 485–90.

(7) Flemming, R. G.; Murphy, C. J.; Abrams, G. A.; Goodman, S. L.;Nealey, P. F. Biomaterials 1999, 20 (6), 573–88.

(8) Yim, E. K. F.; Reano, R. M.; Pang, S. W.; Yee, A. F.; Chen, C. S.;Leong, K. W. Biomaterials 2005, 26, 5405–13.

(9) Kunzler, T. P.; Drobek, T.; Schuler, M.; Spencer, N. D. Biomaterials2007, 28, 2175–82.

(10) Riehle, M.; Dalby, M.; Johnstone, H.; Gallagher, H.; Wood, M. A.;Casey, B.; McGhee, K.; Affrossman, S.; Wilkinson, C. D. W.; Curtis,A. S. G. Mater. Res. Soc. Symp. Proc. 2002, 705, 107–12.

(11) Zhu, X.; Chen, J.; Scheideler, L.; Reichl, R.; Geis-Gerstorfer, J.Biomaterials 2004, 25, 4087–103.

(12) Favia, P.; Sardella, E.; Lopez, L. C.; Laera, S.; Milella, A.; Pistillo,B. R.; Intranuovo, F.; Nardulli, M.; Gristina, R.; d’Agostino, R. InPlasma Assisted Decontamination of Biological and Chemical Agents;Guceri, S., Fridman, A., Eds.; NATO Science for Peace and SecuritySeries; NATO: Brussels, Belgium, 2008; pp 203-26.

(13) Denes, F. S.; Manolache, S. Prog. Polym. Sci. 2004, 29, 815–85.(14) Sardella, E.; Intranuovo, F.; Rossini, P.; Nardulli, M.; Gristina, R.;

d’Agostino, R.; Favia, P. Plasma Process. Polym. 2009, 6, S57–S60.(15) Di Mundo, R.; Gristina, R.; Sardella, E.; Intranuovo, F.; Nardulli, M.;

Milella, A.; Palumbo, F.; d’Agostino, R.; Favia, P. Plasma Process.Polym. 2010, 7, 212–23.

(16) Gristina, R.; D’Aloia, E.; Senesi, G. S.; Milella, A.; Nardulli, M.;Sardella, E.; Favia, P.; d’Agostino, R. J. Biomed. Mater. Res. 2009,88B, 139–49.

(17) Salerno, S.; Piscioneri, A.; Laera, S.; Morelli, S.; Favia, P.; Bader,A.; Drioli, E.; De Bartolo, L. Biomaterials 2009, 30, 4348–56.

(18) Pistillo, B. R.; Gristina, R.; Sardella, E.; Lovascio, S.; Favia, P.;Nardulli, M.; d’Agostino, R. Plasma Process. Polym. 2009, 6, S61–S64.

(19) Buttiglione, M.; Vitiello, F.; Sardella, E.; Petrone, L.; Nardulli, M.;Favia, P.; d’Agostino, R.; Gristina, R. Biomaterials 2007, 28, 2932–45.

(20) Jung, H. J.; Park, P.; Kim, J.-J.; Lee, J. H.; Han, K.-O.; Han, D. K.Artif. Organs 2008, 32 (12), 981–89.

(21) Miller, D. C.; Thapa, A.; Haberstroh, K. M.; Webster, T. J. Bioma-terials 2004, 25, 53–61.

(22) Labelle, C. B.; Gleason, K. K. J. Appl. Polym. Sci. 1999, 74, 2439–47.

(23) Martin, I. T.; Malkov, G. S.; Butoi, C. I.; Fisher, E. R. J. Vac. Sci.Technol., A 2004, 22 (2), 227–35.

(24) Lau, K. K. S.; Caulfield, J. A.; Gleason, K. K. Chem. Mater. 2000,12, 3032–37.

(25) Senesi, G. S.; D’Aloia, E.; Gristina, R.; Favia, P.; d’Agostino, R. Surf.Sci. 2007, 601, 1019–25.

(26) Rosso, F.; Marino, G.; Muscariello, L.; Cafiero, G.; Favia, P.; D’Aloia,E.; d’Agostino, R.; Barbarisi, A. J. Cell. Physiol. 2006, 207, 636–43.

(27) Milella, A.; Palumbo, F.; Favia, P.; Cicala, G.; d’Agostino, R. PureAppl. Chem. 2005, 77, 399–414.

(28) Intranuovo, F.; Sardella, E.; Rossini, P.; d’Agostino, R.; Favia, P.Chem. Vap. Deposition 2009, 15, 95–100.

(29) High Resolution XPS of Organic Polymers: The Scienta ESCA 300Database; Beamson, G., Briggs, D., Eds.; Wiley-VCH: Chichester,U.K., 1992.

(30) Handbook of X-ray Photoelectron Spectroscopy; Moulder, J., Stickle,W.-F., Sobol, P.-E., Bomben, K.-D. , Eds.; Perkin-Elmer Corp: EdenPrairie, MN, 1992.

(31) d’Agostino, R.; Cramarossa, F.; Fracassi, F.; Illuzzi, F. In PlasmaDeposition, Treatment and Etching of Polymers; d’Agostino, R., Ed.;Academic Press: New York, NY, 1990; pp 95-162.

(32) Mosmann, T. J. Immunol. Methods 1983, 65 (1-2), 55–63.(33) Yuanbin, L. Prog. Neuropsychopharmacol. Biol. Psychiatry 1999, 23,

377–95.(34) Wilkinson, C. D. W.; Riehle, M.; Wood, M.; Gallagher, J.; Curtis,

A. S. G. Mater. Sci. Eng., C 2002, 19, 263–69.(35) Barthlott, W.; Neinhuis, C. Planta 1997, 202, 1–8.(36) Zhu, M.; Zuo, W.; Yu, H.; Yang, W.; Chen, Y. J. Mater. Sci. 2006,

41, 3793–97.(37) Rupp, F.; Scheideler, L.; Rehbein, D.; Axmann, D.; Geis-Gerstorfer,

J. Biomaterials 2004, 25, 1429–38.(38) Liu, X.; Lim, J. Y.; Donahue, H. J.; Dhurjatic, R.; Mastro, A. M.;

Vogler, E. A. Biomaterials 2007, 28, 4535–50.(39) Biomaterials Science, 2nd ed.; Ratner, B. D., Hoffman, A. S., Schoen,

F. J., Lemons, J. E., Eds.; Elsevier Academic Press: San Diego, CA,2004.

(40) Martin, J. Y.; Schwartz, Z.; Hummert, T. W.; Schraub, D. M.; Simpson,J.; Lankford, J., Jr.; Dean, D. D.; Cochran, D. L.; Boyan, B. D.J. Biomed. Mater. Res. 1995, 29, 389–401.

(41) Lampin, M.; Warocquier-Clerout, R.; Legris, C.; Degrange, M.; Sigot-Luizard, M. F. J. Biomed. Mater. Res. 1997, 36, 99–108.

(42) Wojciak-Stothard, B.; Curtis, A.; Monaghan, W.; MacDonald, K.;Wilkinson, C. Exp. Cell Res. 1996, 223, 426–35.

(43) Garcıa, A. J. Biomaterials 2005, 26 (36), 7525–29.

BM101136N


osteoblast-like cell behavior on plasma deposited micro/nanopatterned coatings

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