Osteoblast-Like Cell Behavior on Plasma Deposited Micro

380

Biomacromolecules 2011, 12, 380–387

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 topographical characteristics as shape, height, and distance, encountered in their physiological environment. The combination of chemistry and topography of a biomaterial surface influences in turns, important biological responses as inflammatory events at tissue-implant interface, angiogenesis, and differentiation of cells. By disentangling the effect of material chemistry from the topographical one, the possibility of controlling the cell behavior can be provided. 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 with different micro/nanopatterns and the same uppermost chemical composition were produced by combining two plasma deposition processes, with C3F6O and tetrafluoroethylene (C2F4), respectively. The behavior of osteoblastlike cells toward these substrates clearly shows a strict dependence of cell adhesion and proliferation on surface roughness and morphology.

Introduction The in vitro study of cell behavior toward different material surface properties represents a necessary prerequisite in assessing the biocompatibility of a material intended to be used in medical devices. In addition to mechanical properties, surgical requirements, and ability to stand sterilization procedures, a biomaterial must interact adequately with the biological environment through its physical/chemical surface characteristics, because a strong dependence of cell adhesion/proliferation on substrate surface properties 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 biological reactions.3 Surface wettability influences cell-material interactions, because a poor spreading of eukaryotic cells is usually observed on hydrophobic substrates and it increases on hydrophilic ones. As regards the roughening of the surface, it has been widely demonstrated that microroughness contributes to cell attachment, spreading and differentiation.4 Moreover, the superimposing of a nanoroughness enhances local factor production. Indeed, many studies have demonstrated that the presence of topographical micro/nanofeatures on a surface allows to control and manipulate two fundamental external signals: cell-substrate and cell-cell interactions. In this way, many cellular and biological processes are influenced, such as cell metabolism, phenotypic expression, and the inflammatory response at tissue implant interface.5 * 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 IMMCNR Lecce, Italy.

The surface properties of a material can be tuned by creating well-defined topographical and chemical patterns on the surface to rapidly investigate the interaction between various cell types and these materials by means of in vitro experiments. Photolithography, reactive ion etching, and anisotropic etching were the first techniques developed to create surfaces with welldefined topography.6,7 Microcontact printing, inkjet printing, and diamond cutting are usually suitable only for micropatterning.8 Sandblasting has also been employed to produce roughness gradients on the substrate, allowing a systematic investigation of their effect on osteoblast and fibroblast cell behavior.9 In situ polymerization, solvent casting, embossing, or melt molding are used to obtain a replica of micro/nanopatterned surfaces with high fidelity.10 Anodic oxidation has been employed to modify titanium surface oxides in both composition and topography, reaching an increase of osteoblast adhesion and proliferation on the anodic oxides.11 Most of the above-mentioned techniques are expensive, time-consuming, and unable to independently change chemistry or topography of a material surface, thus, producing an effect on biological environment that can not be univocally attributed only to a parameter. Among several approaches, used to modify material surfaces, plasma technologies offer interesting benefits.12 Nonequilibrium, cold plasma processes are energy efficient dry techniques that can be developed in a wide pressure range and alter only the very top layers 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 the biomedical field without affecting their mechanical properties. Basically, such processes can provide a variety of functionalized surfaces employed in biology and medicine. They involve the grafting of chemical groups or the deposition of micro/ nanocoatings to create surfaces characterized by different roughness,14-16 or cell-adhesive films with well-defined chemistry,17-19 that can be eventually functionalized for bio-

10.1021/bm101136n  2011 American Chemical Society Published on Web 12/29/2010

Plasma Deposited Micro/Nanopatterned Coatings

molecule immobilization.20 Due to its high versatility and easy processing, in addition to obtaining coatings characterized by different chemical/morphological properties in a single stage process, the plasma technology can also realize substrates where only a surface parameter varies by keeping constant the others. In this way, it is possible to produce coatings with variable topography and constant chemical composition, or vice versa, as attested by previous literature studies.16,21 In turn, it could be possible to distinguish the role of a single surface parameter on cell behavior, irrespectively of the others. This is a fundamental aspect in the assessment of cell guidance mechanism on materials, because they provide a variety of mechanical, chemical, and topographical stimuli whose effects on cells are usually 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 ribbons or bumps,22-24 able to successfully tune the cell response.25,26 The deposition mechanism of such micro/nanostructured coatings was discussed by Milella et al.27 We have previously deposited fluorocarbon thin films by plasma enhanced chemical vapor deposition (PECVD) fed with hexafluoropropylene oxide (C3F6O).28 Their peculiar surface characteristics (e.g., roughness, hydrophobicity) have been demonstrated to depend on the experimental conditions, especially on the distance of the substrate from the plasma region in the reactor (glow vs afterglow areas). In particular, more Teflon-like, hydrophobic and micro/nanostructured coatings were deposited by increasing the distance from the glow region. Among them, two thin film typologies, chosen for their different morphologies (i.e., petal-like vs spherical shaped coatings), have been investigated in the present paper to study the influence of the substrate topography and morphology on the human osteoblast-like Saos-2 cells behavior, in terms of cell morphology and proliferation. In this light, plasma processes become a successful tool to produce surfaces with different substrate morphologies in the same plasma deposition experiment that give the opportunity to quickly study the cell behavior at different roughness degrees. We pursued our goal by looking at different techniques like MTT, Coomassie Blue staining, SEM analysis, and cytoskeleton observation to give a general view of the cell response.

Materials and Methods Substrates and Plasma Deposition Conditions. Polyethylene terephthalate (PET) substrates (Goodfellow, 0.5 mm thick) were coated with fluorocarbon films obtained by radiofrequency (RF, 13.56 MHz) PECVD processes. A RF parallel plate plasma reactor was used whose technical details have been described in a previous paper.28 First, PET samples were coated with films by plasmas fed with C3F6O, at the following experimental conditions: C3F6O (Fluorochem) 40 sccm flow rate; 50 W power; 900 mTorr (0.120 kPa) pressure; 120 min deposition time. Samples were positioned in the reactor chamber at 8 and 18 cm from the gas inlet. Then, these morphologically different films and flat native PET substrates were further coated with a fluorocarbon film, by a PECVD process fed with C2F4, at the following plasma 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 CAM200 digital goniometer (KSV instruments), equipped with a BASLER A60f camera by sessile drop (2 µL) method. Five measures per sample were performed.

Biomacromolecules, Vol. 12, No. 2, 2011

381

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

3CF3% + 2CF2% + CF% F ) C 100

Chemical Characterization. X-ray photoelectron spectroscopy (XPS) analysis was performed by a Theta Probe Thermo VG Scientific instrument (base pressure of 1 × 10-10 mbar), equipped with a hemispherical analyzer and a nonmonochromatic Al KR (hν 1486.6 eV) X-ray source operating at 300 W. Photoelectrons were collected at a takeoff angle of 53°, corresponding to a sampling depth of ∼10 nm. High resolution spectra were shifted to their correct position by taking the component centered at 292.0 eV (CF2) as reference.29,30 The software Thermo Avantage 3.28 (Thermo Electron Corporation) was used either to determine the elemental composition from peak areas either to peak fit the high resolution spectra. For C1s fitting, five components were considered, as shown in Figure 2: CF3 (294.5 ( 0.2 eV, solid line), CF2 (292.0 ( 0.2 eV, dashed line), 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). The F/C ratio was calculated from the best fitting of the C1s spectrum, according to the equation reported in the Scheme 1, where CF3%, CF2%, and CF% are the contributions of the CFx (x ) 1, 2, and 3) components to the total C1s area. The F/C ratio was allowed to probe the structure retention of the monomer and it provided an evaluation of cross-linking degree. The lower the F/C ratio was, the higher the degree of cross-linking of the coating was. A F/C ratio close to 2 represented a chemical structure similar to conventional Teflon (high Teflon character).31 Morphological Characterization. A Stereoscan 360 Cambridge scanning electron microscope (SEM), operating at 20 KV, with a 50° tilt angle, was used to examine the surface morphology of the coatings and evaluate its distribution on the whole sample surface. Because the samples were nonconductive, they were sputter-coated with a 10 nm thick gold layer before SEM examination, using the Biorad Polaron Division, SEM Coating System, E5100 Sputter Coater. Height mode atomic force microscopy (AFM) investigations were performed 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 a resonance frequency of 320 kHz. Topographic micrographs were collected on six areas of each sample, with a scan size area of 10 × 10 µm2, by sampling the surface at a scan rate of 0.8 Hz and a resolution of 256 × 256 pixels. AFM images were processed by using a XEI Program to flatten the topographic micrographs, to remove the slope and curvature artifacts produced by the scanning process, and obtain statistical data as surface root-mean-squared roughness (rms) and mean height of sample features. The XEI software was also used to represent a three-dimensional perspective of the sample surface with the original pixel resolution of 256 × 256 pixels and to extract histogram panels showing the distribution of feature heights. Cell Culture. Untreated and plasma-modified PET samples were placed in 24-well plates. Before cell seeding, the samples were soaked in ethanol for 15 min. Cell culture experiments were performed with the human osteoblast Saos-2 cell line (ICLC, Italy). Cells were routinely grown in Dulbecco’s Modified Eagle Medium (DMEM, Sigma Chemical Co., Italy), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 IU/mL penicillin, 50 IU/mL streptomycin, and 200 mM glutamine, at 37 °C, in a saturated humid atmosphere containing 95% air and 5% CO2, in 75 cm2 flasks (Barloworld Scientific, U.K.). For cell culture experiments, cells were detached with a Trypsin/EDTA solution (Sigma, Italy), suspended in the correct medium, and seeded at a concentration of 1 × 105 cells/mL on native cell cultured polystyrene (CCPS) and on modified substrates for culture times up to 96 h. Mitochondrial Function Measurement. The mitochondrial activity of Saos-2 cells, seeded on the substrates at different culture times (24,

382


48, 72, and 96 h), was determined with the MTT colorimetric assay. This test detects the conversion of 3-(4,5 dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide (Sigma Co., St. Louis, MO, U.S.A.) to formazan. At each time point, the cells were incubated in a tenth of 3-(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 5 mL 3 N HCl). Finally, the optical density was read with a spectrophotometer (Jenway 6505, GB), at a wavelength of 570 nm, using 690 nm as reference wavelength.33 Each experiment was performed in triplicate; data were presented as optical density (O.D.) values. Cell Morphological Analysis. Saos-2 cells seeded on the substrates and analyzed at different cell culture times, were fixed in 4% Paraformaldehyde/PBS solution (15 min) and stained with a dye solution composed by 0.2% Coomassie Brilliant Blue R250 (Sigma, Italy), 50% methanol and 10% acetic acid, for 3 min. Cells were observed on the samples at different magnifications, by means of a phase contrast microscope (Leica DM ILI). At least 15 images per sample were acquired through a CCD camera (Leica DC100). Images were then analyzed with the Image J software (National Institute of Health, U.S.A.) to evaluate the substrate area covered by cells. Statistical analysis was performed by a two-way ANOVA test within groups, followed by a Bonferroni post-test, by using the GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, California, U.S.A., www.graphpad.com). Differences were considered statistically significant 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 PBS containing 0.1% Triton X-100 and incubated with Alexa Fluor488 phalloidin (Molecular Probes) at RT and for 20-30 min. Tubulin was detected with a monoclonal antibody raised in mouse (Sigma). Vinculin was detected with a monoclonal antibody raised in mouse (Sigma). For both vinculin and tubulin staining, a secondary antibody against mouse 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 an epifluorescence microscope (Axiomat, Zeiss, Germany). For a SEM observation of cell structure and distribution on the samples, the cells were fixed with 2.5% glutaraldehyde/0.1 M sodium cacodylate solution and dehydrated using a series of ethanol/water solutions (20, 40, 50, 70, 90, and 100%). Finally, the cell cultured samples were air-dried under a biological hood and sputter-coated with a 10 nm thick gold layer for the SEM visualization.

Results and Discussion Plasma Deposition of Micro/Nanostructured Fluorocarbon Thin Films. Fluorocarbon coatings, characterized by different chemical composition and topography, were plasma deposited with C3F6O feed at various distances from the glow to the afterglow region of the plasma reactor in the same single process. In particular, by increasing the distance from the glow region, more and more hydrophobic and Teflon-like coatings were 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 retention increased. This effect could be explained by a recombination of radicals produced in the plasma phase, together with a reduced extent of the fragmentation in the deposited film, due to the reduced (absence) ion bombardment on the substrate placed downstream.27 At the same time, a great change in both surface roughness and morphology was observed. Completely smooth coatings in the glow, nanobumped films at a medium distance (8 cm) from the gas inlet and a “petal” building in the region 11-18 cm from the glow were obtained. Here, single large features seemed to be formed by the agglomeration of

Intranuovo et al.

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

several small nodules that tangled each other, showing tall petallike micro/nanostructures. In this study, substrates with different morphologies but identical chemical composition were produced, with the aim of understanding how the topography of the coatings, synthesized at 8 and 18 cm from the gas inlet of the plasma reactor (named AG8 and AG18, respectively) could influence the behavior of osteoblast-like Saos-2 cells, in terms of adhesion, morphology, and proliferation. To achieve this task, AG8 and AG18 coatings, characterized by well distinct morphology and flat PET substrates were coated with the same thin fluorocarbon film, by means of a C2F4 PECVD. A scheme of the rationale of this work has been presented in Figure 1. Wettability and Chemical and Morphological Characterization of the Fluorocarbon Thin Films. To ascertain any difference due to the C2F4 coating, WCA, XPS, and AFM analyses were performed. The WCA for the flat C2F4 coating was 108 ( 3° (Table 1). The effect of C2F4 coating was to slightly lower the WCA for both AG8 and AG18 samples. Indeed, in the case of AG8 samples, WCA values decreased from 135 ( 1° to 129 ( 3° and for AG18 from values greater than 170 to 165 ( 2°. In this last case a superhydrophobic material was obtained, due to a combination of chemical and topographical factors. The chemical composition of AG8 and AG18 samples before C2F4 coating was close to that of Teflon, as attested by the F/C percentages shown in Table 1. On the other hand, after C2F4 plasma deposition, both AG8 and AG18 completely lost the Teflon character, with a lowering of F/C ratio, reaching values similar to that associated with the flat coating, as indicated in Table 1. This evidence was correlated to an increase of CFx components respect to CF2 in the C1s spectra, attesting a carbon chemical composition very close to the flat C2F4 coated sample, whose deconvoluted high resolution C1s spectrum has been reported in Figure 2. These results demonstrated that substrates with the same chemical composition were produced. This attested to a conformal deposited Teflon-like coating on the plasma micro/nanostructured surfaces. The surface topography of AG8 and AG18 samples has been investigated by AFM before and after the C2F4 plasma deposition. Estimated values of the corresponding mean feature heights and film rms roughness have been reported in Table 1. The analyses of surface topography showed that the AG8 coating had a nanoroughness (Table 1) and a morphology characterized



383

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

WCA (°) F/C % mean height (nm) mean rms (nm)

flat (C2F4)

AG8 (C3F6O)

AG8 (C3F6O + C2F4)

AG18 (C3F6O)

AG18 (C3F6O + C2F4)

108 ( 3 1.57 ( 0.03 0.6 ( 0.2 4.8 ( 1.0

135 ( 1 1.91 ( 0.04 247 ( 32 74 ( 8

129 ( 3 1.58 ( 0.03 225 ( 55 65 ( 11

>170 1.97 ( 0.01 1000 ( 100 386 ( 5

165 ( 2 1.50 ( 0.03 800 ( 100 368 ( 2

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

Figure 2. Best fitting of C1s high resolution spectrum of flat, AG8 and AG18 samples after the C2F4 deposition. In the fitting of the spectrum, the following components were considered: from the left side, CF3 (294.5 ( 0.2 eV, solid line), CF2 (292.0 ( 0.2 eV, dashed line), CF (290.0 ( 0.2 eV, dotted line), C-CF (288.0 ( 0.2 eV, dashdotted line), and C-C (285.0 ( 0.2 eV, solid line).

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

mation). It is worthwhile to notice that the feature height distribution of the AG18 sample evidenced the presence of both micro- and nanostructures, whose heights were asymmetrically distributed around the mean value (Figure 3d). Such a result was observed both before and after C2F4 plasma deposition. This simultaneous presence of both micro- and nanostructures has been 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 and roughness, with a pronounced reduction of the Teflon character. Both these morphological and chemical changes have been expected to be responsible of the slight increase of surface hydrophilicity.34-38 Cell Response to the Substrates Topography. The cell behavior on flat PET, AG8, and AG18 films, having the same surface chemistry and different roughness and morphology, was investigated. For this purpose, the human cell line Saos-2 was chosen, as it is representative of a cell type that usually has a good affinity with biological surfaces characterized by a micro/ nanotopography. Cell growth on different plasma-modified and CCPS 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 internal control because osteoblast cells are known to adhere and grow very well on them.39 Cell proliferation on substrates was quantified by MTT test. As expected, the cells grown on CCPS showed a higher adhesion and better proliferation than on the plasma-modified surfaces at all the four cell culture times, as the histogram in Figure 4 clearly shows. This result was expected because it is well-known that hydrophobic surfaces discourage either protein and cell adhesion with respect to hydrophilic surfaces as CCPS. By comparing Saos-2 cell proliferation on the three plasma treated surfaces, the only statistically significant difference was observed after 48 h of cell culture, when the MTT value on flat substrates was higher than that on the two micro/nanostructured

Figure 4. MTT activity of Saos-2 cells grown on flat, AG8, and AG18 surfaces after the C2F4 deposition and on CCPS samples, at different cell culture times (24, 48, 72, and 96 h). Optical density mean values were shown. Significant differences between the means were calculated 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).

384


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 on CCPS (d) substrates. Cells were fixed with a 4% paraformaldehyde/ PBS solution and stained with Coomassie Blue. All images show the clustering of the cells, except for the AG8 sample (b), where most of the cells grew isolated without grouping each other.

surfaces (p < 0.01). At the other cell culture times, no statistically significant difference has been found among the three substrates. Cell proliferation data on flat surfaces linearly increased with the culture time. Instead, for AG8 and AG18, the data were low until 48 h and rapidly increased at 72 h, approaching the values obtained with flat samples (p > 0.05). Because the MTT assay evaluates the metabolic activity of the cells, not giving information about the variation of their number or shape, optical microscopy analysis has also been performed, after fixing and staining the cells with Coomassie Blue. The low magnification images in Figure 5 show the cell spreading and clustering after 96 h of culture, on the four different substrates. The cell behavior was evidently dependent on the substrate below. On CCPS substrates (Figure 5d), Saos-2 cells spread and clustered to a larger extent than cells plated on fluorinated surfaces. Actually, the cells started clustering on CCPS in the first hours of culture, and this phenomenon was evident at 24 h (Figure 6d) when it was hard to find cells separated from the others. On AG18 a similar cell clustering was observed (Figure 5c). The cells were less grouped with each other on flat coatings (Figure 5a) and isolated more on AG8 films (Figure 5b). By observing Figure 6, the differences in size and shape of single cells cultured for 24 h on the substrates have been better appreciated. The cells on flat and AG8 substrates (Figure 6a and b, respectively) were rounded or elongated shaped. On AG18 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, was measured by the Image J software. The graph in Figure 7 summarizes the results as total area covered by cells, at four cell culture times. After 72 h of cell culture, a difference in cell area and spread was observed among the three differently fluorinated substrates. These data confirmed the highest value of proliferation for cells grown on CCPS, observed with the MTT test. Instead, among the plasma-modified surfaces, the highest cell growth on AG18 substrates stood out. This could be due to the micro/

Intranuovo et al.

Figure 6. Optical images of single cells after 24 h of culture time on flat (a), AG8 (b), and AG18 (c) surfaces after the C2F4 deposition and CCPS (d) substrates. Cells were fixed with a 4% paraformaldehyde/PBS solution and stained with Coomassie Blue. Images show the 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 osteoblasts, 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 calculated from 10 images with a 3 mm2 area, where the cells were previously fixed and Coomassie Blue stained. Significative differences between the means were calculated 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).

nanostructuring of its surface morphology. Indeed, it is known that the cell growth is favored at the surface discontinuities. Many recent papers have confirmed that adhesion, migration area, and extracellular matrix (ECM) production are higher on rough surfaces or with larger grain sizes.40,41 Besides, the substrates with grooves and cliffs of greater (micro-) size stimulate the movement of a variety of cells.42 The microfeatures of AG18 surfaces likely induced similar effects on osteoblast cell behavior. Cytoskeleton analysis has provided another tool to study the differences in cell morphology on the substrates. It was first focused on tubulin and actin observation, since they represent the more abundant proteins of the cytoskeleton. The tubulin organization (Supporting Information) merely reflected the cell shape, confirming the results observed with Coomassie Blue staining 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 culture time, has been shown. The cells on CCPS (Figure 8f) presented a well developed actin cytoskeleton with stress fibers throughout the cytoplasm. In regard to the plasma-modified surfaces, only



385

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 the AG8 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). Vinculin was detected with a monoclonal antibody raised in mouse, followed by an incubation with a secondary antibody against mouse IgG, conjugated with Alexa Fluor546 (red). Finally, the samples were mounted in Vectorshield fluorescent mountant with DAPI, allowing the observation of the nucleus (blue). In (a) the vinculin staining shows the presence of a perinuclear accumulation of the protein and the spots of vinculin typical of focal adhesion structures. In (b) is shown a triple staining of actin (green), vinculin (red) and nuclei (blue). The white ellipse indicates the presence of vinculin staining only at the end of actin stress fibers while no vinculin staining is shown on actin terminal spots (white arrow).

AG18 substrates induced a flattened polygonal shaped cell morphology (Figure 8c), with a stress fibers organization very similar to CCPS. It was also present on flat (Figure 8a and c, clustered and single cells, respectively) and AG8 (Figure 8b and d, clustered and single cells, respectively) surfaces, without any 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 the nanofeatures 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 major components of focal adhesions. Saos 2 cells grown on the four surfaces, showed a perinuclear accumulation of the protein and bean shaped spots of vinculin (Figure 9a), typical of focal adhesion structures at the end of actin stress fibers. As previously described, Saos-2 cells on AG8 surfaces presented spots of actin accumulated at the cell periphery. 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 actin terminal spots, no colocalization of the two proteins was found (Figure 9b, white arrow). No consistent difference in the amount of vinculin focal adhesion sites was present on the four substrates (Supporting Information). Further experiments could shed light on the presence of other important cytoskeleton components such as paxillin and vimentin, in order to better understand the relationships between substrate topography and Saos-2 cells, in terms of cytoskeleton rearrangement. All surfaces were produced using the same plasma process procedure to ensure the same surface chemistry, as previously reported. Therefore, we supposed that the differences observed in cell response were exclusively dependent on micro/nanoscale roughness. Visual observation of the cells, by both optical and fluorescence microscopy, clearly showed how the cells interacted in different ways with the substrate. A more accurate study of the cell-substrates interactions on the three plasma-modified surfaces was performed to better understand the role of the

386


Intranuovo et al.

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 different magnifications. The cells were fixed with 2.5% glutaraldehyde/0.1 M sodium cacodylate solution, dehydrated using a series of ethanol/water solutions, and air-dried under a hood. High magnification pictures (a3,4,5; b3,4,5; c3,4,5) show the cell-material interactions, characteristic for each kind of surface: long cell filopodia on flat, lamellipodia on AG8, short filopodia, and lamellipodia on AG18. Dotted arrows show high magnification images of the cell protrusions on the substrates.

substrate micro/nanostructuring on the cell behavior. Figure 10 shows how the Saos-2 cells attached in different ways according to the substrate roughening. Interactions by long filopodia were observed on flat coatings (Figure 10a2-5), while the adhesion to the nanodomes on AG8 surfaces was mainly exploited by both lamellipodia (Figure 10b2-5) and filopodia (Figure 10b3). Instead, the tall microstructures on AG18 surfaces were mainly explored by lamellipodia (Figure 10c2-5) that were less extended than those on AG8 and aided by very short filopodia (Figure 10c3-5). Besides, many cells had filopodia in contact with the surface structures and their length seemed to be correlated to the dimension of the micro/nanofeatures. In Figure 10c4,5, the profile of the tall petal-like structures below the cell body could be observed. Thus, the cells attached to the AG18 substrate, even if very rough, by anchoring with thin lamellipodia and short filopodia. Thus, this SEM study clearly demonstrated that cells interacted directly with the micro/nanofeatures, confirming the strong dependence of the cell behavior on surface topography, consistent with the analyses previously illustrated. It is likely that the different response of Saos-2 cells to surface roughness and morphology reflected differences in integrinmediated signaling. Cells respond to biomaterial surfaces through interactions between the cell membrane receptor integrins and the adsorbed ECM proteins including fibronectin.39,43 Protein adsorption on materials is highly influenced by surface chemistry, hydrophilicity, and topography, and in particular, protein adsorption has been demonstrated to depend on the scale of surface roughness. An example is represented by titanium surfaces, whose nanoscale surface texture seemed to have little or no effect on protein adsorption and cell proliferation. However, microrough surfaces adsorb more fibronectin and the protein orientation is different from that on flat surfaces, which further alter integrin adhesion. In light of the previous considerations, it could be deduced that the differences observed in terms of major adhesion and

spreading on the micro-(AG18) in respect to nanoscale roughness (AG8) were due to the protein adsorption. The copresence of micro/nanofeatures on AG18 surfaces seemed to provide more anchoring sites to the cells, facilitating their adhesion, according to previous studies.4

Conclusions By plasma processes we have easily tuned the surface topography of PET substrates, affecting the ability of osteoblastlike cells behavior. A strict correlation between the material roughness (feature height and micro/nanoscaling) and the cell adhesion, proliferation, and morphology has been illustrated, demonstrating that among the properties of a surface, the topography is a decisive factor in mediating the cell-material interactions. Moreover, because the chemical composition of these surfaces is fluorinated, known to be cell-repellent, the role of topography has to be considered still more decisive on the osteoblast cell behavior. Particularly interesting are the higher cell adhesion and spreading on the taller micro/nanorough coatings. Further studies, for example, on the different expressions of genes involved in osteoblast differentiation could better explain the mechanisms of cell-substrate interactions when the substrate roughening is varied. Because this plasma modification process is independent from the material, these thin fluorocarbon coatings could be easily applied to any material to be used in the biomedical field. For the ease of producing different substrate morphologies in the same deposition experiment, this fluorocarbon plasma deposition can become a strong tool to quickly investigate the cell behavior at different roughness degrees at any biological interface. Acknowledgment. The authors thank the laboratory support of 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


(PRISMA05MADA1 nanostructured surfaces having a specific biological response) project and the INTERREG (I 2101003) Italy-Greece regional (Apulia and Acaia regions) project. Supporting Information Available. Detailed information about 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 is available 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) Wo´jciak-Stothard, B.; Madeja, Z.; Korohoda, W.; Curtis, A.; Wilkinson, 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. Biomaterials 2007, 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. In Plasma Assisted Decontamination of Biological and Chemical Agents; Guceri, S., Fridman, A., Eds.; NATO Science for Peace and Security Series; 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.


387

(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. Biomaterials 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. Pure Appl. 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 300 Database; 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: Eden Prairie, MN, 1992. (31) d’Agostino, R.; Cramarossa, F.; Fracassi, F.; Illuzzi, F. In Plasma Deposition, 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.; SigotLuizard, M. F. J. Biomed. Mater. Res. 1997, 36, 99–108. (42) Wo´jciak-Stothard, B.; Curtis, A.; Monaghan, W.; MacDonald, K.; Wilkinson, C. Exp. Cell Res. 1996, 223, 426–35. (43) Garcı´a, A. J. Biomaterials 2005, 26 (36), 7525–29.

BM101136N

Osteoblast-Like Cell Behavior on Plasma Deposited Micro

Recommend Documents