ARTICLE pubs.acs.org/Langmuir
Cell Adhesion on Nanotextured Slippery Superhydrophobic Substrates Rosa Di Mundo,† Marina Nardulli,† Antonella Milella,† Pietro Favia,†,‡,§ Riccardo d’Agostino,†,‡,§ and Roberto Gristina*,‡ †
Department of Chemistry, University of Bari, Via Orabona 4, 70126 Bari, Italy Institute of Inorganic Methodologies and Plasmas IMIP-CNR, c/o University of Bari, Via Orabona 4, 70126 Bari, Italy § Plasma Solution Srl, Spin Off of the University of Bari, Via Orabona 4, 70126 Bari, Italy ‡
ABSTRACT: In this work, the response of Saos2 cells to polymeric surfaces with different roughness/density of nanometric dots produced by a tailored plasma-etching process has been studied. Topographical features have been evaluated by atomic force microscopy, while wetting behavior, in terms of watersurface adhesion energy, has been evaluated by measurements of drop sliding angle. Saos2 cytocompatibility has been investigated by scanning electron microscopy, fluorescent microscopy, and optical microscopy. The similarity in outer chemical composition has allowed isolation of the impact of the topographical features on cellular behavior. The results indicate that Saos2 cells respond differently to surfaces with different nanoscale topographical features, clearly showing a certain inhibition in cell adhesion when the nanoscale is particularly small. This effect appears to be attenuated in surfaces with relatively bigger nanofeatures, though these express a more pronounced slippery/dry wetting character.
’ INTRODUCTION A basic understanding of cellmaterial surface interaction is of huge interest in tissue engineering and other biomedical applications. In the last 20 years, many investigations have shown that surface properties, namely, chemical composition, surface energy, wettability, roughness, and surface topography, significantly influence cell adhesion and growth at the surface of materials. Substrate engineering encompasses several of these factors in an attempt to create a favorable extracellular microenvironment, thus replicating as closely as possible the in vivo conditions. Cells in vivo produce complex chemical and topographical cues that might vary from macro- to nanosize dimension and that characterize important structures in the body like the extracellular matrix and the basement membrane.1 For this reason, there is a growing interest in producing materials for tissue regeneration, through the incorporation of micro- and nanoscale features, in order to guide cell response from initial attachment and migration to differentiation and new tissue formation. Recent developments in advanced micro- and nanofabrication techniques have enabled the fabrication of substrates that are able to resemble the structure and length scale of tissue architectures.2 Nanoscaled topography received increasing attention because of many studies reporting that mammalian cells respond in particular to nanoscale features on artificial materials.3,4 A possible explanation to these findings results from the fundamental knowledge of important biological structures, such as the basement membrane, representing in vivo a substrate for overlying cellular structures. In addition to biochemical and mechanical properties, basement membranes possess complex topographical r 2011 American Chemical Society
features having sizes in nanometric range produced by protein folding and banding, as for instance the 66 nm banding on collagen fibers among which many cells live. Therefore, the potential of using nanotopography to replicate the in vivo conditions makes this a promising tool for tissue engineering, since most structures in the body create a nanoenvironment. Indeed, the ultimate goal for biological surface science would be to provide an understanding of how the application of nanotechnologies to tissue may aid in our knowledge of cell biology, biochemical interactions, and how surface chemistry and structure of a material can be used to control cell response to that surface. There are a variety of advanced nanofabrication methods available for creating nanotopographic substrates.5 Many of these methods, reviewed in ref 6, are expensive and time-consuming, and they require access to complex equipment. Alternatively, one of the most important techniques relies on the use of low pressure plasma processes. Nonequilibrium, low-temperature plasma deposition, treatment, and etching processes are extremely popular in materials science technology for their ability to tune surface wettability, adhesivity, hardness, and chemical inertness in a controlled way, without altering their bulk.7 As well-known, the combination of a properly configured nanostructured topography with a low surface energy chemistry results in a superhydrophobic behavior.8 This is generally known Received: January 12, 2011 Revised: March 4, 2011 Published: March 17, 2011 4914
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to be not suitable for cell adhesion, since poor wetting leads to bad protein adsorption.9 Nevertheless, since surface roughness/ structuring can itself favor cell attachment, various and not fully expected results have been found onto textured hydrophobic or superhydrophobic materials. In a previous paper, we have shown that increasing the height, length, and density of ribbonlike features in Teflon-like films, though leading to superhydrophobicity, favored adhesion of osteoblasts.10 Latest advances in the field of plasma etching of commercial polymers have demonstrated that uniform expanses of nanostructures can be generated onto polymer surfaces with one-step maskless plasma processes in fluorine- and/or oxygen-containing feeds. Scale and, sometimes, the shape of these structures can be modulated by playing with plasma parameters.11,12 This technique has been exploited in the present work in order to get nanoscale-textured polystyrene (PS) surfaces to be used as cell culture substrates. Radio frequency low pressure plasmas in reactive ion etching (RIE) configuration with CF4 feed have been utilized. Differences in nanostructure scale have been obtained by varying the process duration. In order to achieve homogeneous fluorinated chemistry among differently textured substrates and a flat reference, a very thin and conformal film can be deposited on the top of the structured substrate, as previously reported.10 Wetting characterization of these surfaces has focused on water dynamics, which really can give indications about the adhesion between water and solid surface, which is particularly relevant when dealing with hydrophobic/superhydrophobic materials.13,14 Many different tests are validated to evaluate the biological compatibility of materials with cells; they can assess morphology, membrane integrity, proliferation, and specific cell functions. In this paper, we present how different topographic features on the two etched fluorinated surfaces, characterized by different roughness, and different hydrophobic behavior can alter the cell morphology of Saos2 cell lines. Cell response to surfaces has been mainly examined in terms of cell morphology using scanning electron microscopy (SEM) and in terms of cytoskeletal organization by means of fluorescent microscopy observation of actin cytoskeleton.
A CAM200 digital goniometer (KSV instruments) equipped with a BASLER A60f camera was used to evaluate the sliding angle of the samples. A volume resolved sliding angle evaluation was performed by measuring the sliding angle with water drops of different volume, ranging from 4 to 16 μL. A distilled water drop was deposited on the specimen fixed to a level tiltable plate and then the plate was inclined slowly, until the drop started to move. Angles have been digitally evaluated from the acquired image sequence through the instrument software (CAM200). All wetting characterization was performed within 1 day after the plasma modification experiments. Nevertheless, it is pointed out that no change in the wetting features has been observed for air-stored samples over a period of at least 2 months. X-ray Photoelectron Spectroscopy (XPS). XPS analyses were carried out by means of a Thermo Electron Corp. Theta Probe Spectrometer with a monochromatic Al KR X-ray source (1486.6 eV) at a spot size of 400 μm corresponding to a power of 100W at a take off angle of 37°. Survey (01200 eV) and high-resolution spectra (C1s, O1s, and F1s) were recorded at a pass energy of 200 and 150 eV, respectively. Atomic Force Microscopy (AFM). Film morphology was investigated by AFM (Auto Probe CP, Thermomicroscope). Images were acquired in noncontact mode using conical gold-coated silicon tips with high resonance frequency (280 kHz). Different surface areas (10 10, 5 5, and 2 2 μm2) were sampled on each sample. Root mean square (rms) roughness was evaluated as an average of five measurements carried out on different sample spots. Cell-Culture Experiments. Cell culture experiments on flat and nanoetched surfaces were performed with the human Saos2 osteoblastic cell line (ICLC). Cells were routinely grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma Chemical Co.) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 50 IU/mL penicillin, 50 IU/mL streptomycin, and 200 mM glutamine and maintained at 37 °C in a saturated humid atmosphere containing 95% air and 5% CO2 in 75 cm2 flasks (Barloworld Scientific, UK). For cell culture experiments, cells were detached with a trypsin/ EDTA solution (Sigma) and resuspended in the correct medium at a concentration of 1 105 cells/mL, and 1 mL was seeded on flat and nanostructured samples placed inside 3 cm Petri dishes and grown up to 8 days.
’ MATERIALS AND METHODS
analyzed at different cell culture times were fixed in 4% paraformaldehyde/PBS solution (15 min) and stained in a dye solution for 3 min in 0.2% Coomassie Brilliant Blue R250 (Sigma), 50% methanol, 10% acetic acid. Cells adhering to different substrata were observed at different magnifications with a phase contrast microscope (Leica DM ILI); at least 15 digital images per sample were acquired through a CCD camera (Leica DC100). Images acquired on different samples and at different times were analyzed with the Image J image analysis software (National Institute of Health, US) to evaluate the substratum area covered by cells upon varying the time. Statistical analyses were assessed by a two-way ANOVA test within groups (same time of cell culture), followed by a Bonferroni post-test, using the GraphPad Prism version 4.00 for Windows, GraphPad Software (San Diego, CA; www.graphpad.com). Differences were considered statistically significant for p < 0.01.
Nanodotted PS Surfaces. Disks 3 cm in diameter cut from polystyrene (PS) Petri dishes (Bibby Sterilin) were etched in a parallel plate reactor (upper electrode ground, 20 cm diameter, gas shower; lower RF electrode, 15 cm diameter, substrate holder; electrodes gap 5 cm). The following etching experimental conditions were used: CF4 flow rate 30 sccm; RF power 150 W; pressure 60 mTorr; two etching times, 5 and 10 min. A complete description of the etching/roughening process of PS can be found in refs 11 and 15. The PS nanostructured surfaces used in cell-culture experiments (along with the flat reference, untreated PS) were coated with a 25 ( 5 nm thin conformal CFx film deposited from a plasma fed with perfluorocyclobutane (C4F8) at a flow rate of 15 sccm, 80 mTorr, 100 W. Water Contact Angle (WCA). Water contact angle (WCA) in dynamic mode was measured by means of a Rame-Hart 100 goniometer. Advancing and receding angles were measured by depositing a droplet of 1 μL on the surface, increasing the volume to 4 μL, and finally decreasing it. Advancing angles are the maximum angles observed during the droplet growth. Receding contact angles are the ones just before the contact surface reduction. Each WCA value has been averaged from measurements of four drops with an estimated maximum error of 3°.
Cell Morphological Analysis: Coomassie Blue Staining and Image Analysis. Saos2 cells seeded on the substrata and
Cell Morphological Analysis: Indirect Immunofluorescence and Cytoskeleton Observation. In order to observe the actin cytoskeleton, cells were fixed in 4% formaldehyde/PBS solution, at room temperature (RT) for 20 min, permealized with PBS containing 0.1% Triton X-100, and incubated with Alexa Fluor488 phalloidin 4915
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Figure 1. AFM images of pristine (FLAT) and etched PS (5 MIN and 10 MIN) in CF4 plasma. All samples are coated with a 25 nm CFx film deposited by C4F8 plasma.
Table 1. XPS F/C Ratio, XPS O/C Ratio, Root Mean Squared Roughness (Rrms), Advancing/Receding (A/R) WCA Values, and Mean Structures Heights for PS Surfaces Nanotextured in CF4 Etching Discharges at Different Etching Time after a Conformal Coverage with a CFx Thin Film Utilized also for Covering the Flat Surface Reference (Untreated PS) etching time (min) XPS F/C XPS O/C
0 1.55 ( 0.05 0.02
5 1.55 ( 0.05 0.02
10 1.55 ( 0.05 0.02 16 ( 2
Rrms (nm)
6(2
10 ( 2
mean height (nm)
17 ( 3
32 ( 8
90 ( 25
WCA A/R (deg)
120/95
158/144
166/155
(Molecular Probes) at RT and for 2030 min. After rinsing, samples were mounted in Vectashield fluorescent mountant with DAPI (Vector Laboratories, UK) and then observed by means of an epifluorescence microscope (Axiomat, Zeiss, Germany).
Cell Morphological Analysis: Scanning Electron Microscopy (SEM). For SEM observation, cells were fixed with 2.5% glutaraldehyde/0.1 M sodium cacodylate solution, postfixed with a 1% osmium tetroxide/0.1 M sodium cacodylate solution, and dehydrated using a series of ethanol/water solutions (20, 40, 50, 70, 90, and 100%). Because the samples were nonconductive, they were coated with a thin layer of Au before SEM examination by using a plasma sputtering apparatus. A Stereoscan 360 Cambridge SEM operating at 20 kV was used to examine the coating surface morphology and evaluate structures and cells distribution on the entire sample surface.
’ RESULTS AND DISCUSSION Surface Modification and Characetrization. The experimental conditions used in this study were aimed at producing samples with different roughness at the nanometric scale and the same surface chemical composition by combining etching and postdeposition plasma processes. Figure 1 reports AFM images of PS samples after being first etched in CF4 plasma for 5 and 10 min and then coated with a 25 ( 5 nm CFx film in a C4F8 discharge, leading to the samples named 5 MIN and 10 MIN. The etching process, which is conducted by the plasma-produced fluorine atoms reacting with carbon and hydrogen at the polymer surface, leaves such surfaces quite uniformly covered with dot reliefs of nanometric size.15 Increasing the etching time means height, mean lateral size, and interclearance of structures increase. Scale of structures increases while shape basically does not vary. This effect, observed with increasing both treatment duration and power input, has been also reported for plasma
Figure 2. Advancing and receding water contact angle values of PS samples etched in CF4 plasmas at different etching time (0, 5, and 10 min) and coated with the same CFx film in C4F8 plasma. Adhesion energy for the same samples has been calculated as γlv(cos θr cos θa).9
roughening of other polymers12,16 and for PS with CF4/O2 mixtures under different experimental conditions.11 Surfaces resulting from the etching process are moderately fluorinated with an F/C ratio ranging from 0.4 to 0.6. After the CFx coverage, as assessed by XPS, the F/C ratio of the nanostructured surfaces become 1.55 ( 0.05, thus identical to that of the flat reference substrate (untreated PS), as reported in Table 1, showing also the same even distribution of CF, CCF, CF2, and CF3 groups, which is typical for Teflon-like films deposited from continuous plasmas.10 The assessment of chemistry homogeneity among flat reference and textured substrates, along with the observation that the shape of surface features was practically not altered by the coating, leads to retention of coating conformality sufficient for work purposes. The root-mean-square roughness (Rrms) values obtained from AFM images of the CFx coated surfaces becomes 6 ( 2 nm for untreated PS (FLAT), 10 ( 2 nm for the 5 MIN, and 16 ( 2 nm for the 10 MIN etched samples. Correspondingly, mean height values are 17 ( 3, 32 ( 8, and 90 ( 25 nm, respectively. In the same table, WCA advancing and receding values are reported. These values are also plotted in the diagram of Figure 2, along with the watersurface adhesion energy calculated with eq 1,13 with γlv being the liquid (water)vapor surface tension. E ¼ γlv ðcos θr cos θa Þ
ð1Þ
It can be there observed that the flat CFx surface is the most water adhesive and this character decreases as the scale of the 4916
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Figure 3. Sliding angle measurements at different drop volume [4 μL (a); 8 μL (b); 12 μL (c); 16 μL (d)] for the flat and 5 and 10 min etched PS surfaces coated with the CFx film. Arrows indicate the motion direction of the drop: no drop sliding is observed onto flat samples even at maximum tilting; 5 min surface allows water motion only at high drop volume (sliding angle of 72° and 34° for the 12 and 16 μL drops, respectively); for the 10 min surface, the drop cannot adhere or rolls off the surface at any volume.
nanotexture increases. This indicates that the wetting regime, within a generally hydrophobic domain, transits from a wet to a nonwet state uniquely as a consequence of texture variation.17 Such a behavior is better highlighted in Figure 3, where results of sliding measurements at different drop volume [4 μL (a); 8 μL (b); 12 μL (c); 16 μL (d)] are reported for the flat and both textured surfaces coated with the same CFx film: no drop sliding is observed onto flat samples, even at maximum inclination; 5 MIN surface allows water motion but only at high drop volume (72° tilting angle for the 12 μL drop and 34° for the 16 μL); the 10 MIN surface is the least adhesive, as the drop cannot adhere (low drop volume) or rolls it off (higher volume). The slippery behavior shown by textured surfaces (more pronounced for the 10 MIN), as aforementioned, derives from a nonwet (or dry) contact with water: this, for other kind of slippery superhydrophobic materials, has been proved to result in the formation of a layer of micro/nanobubbles at the interface between water and the superhydrophobic surface immersed in it.14 Cell Response Evaluation. Modification of cell shape gives important clues about how both surface chemistry and morphology can influence cellsubstrate interactions. For this reasons, before looking at cell adhesion results on the structured fluorocarbon surfaces of this work, it is worth considering how the shape of osteoblast cells looks like when they are grown on flat adhesive substrates with different chemistry. We used for this purpose scanning electron microscopy (SEM) analysis because this approach is able to discern substrate-dependent differences in the shape of individual cells in high-density cultures. SEM images in Figure 4 compare how cells adhere on a fluorinated flat substrate and on the substrate where most of the cell lines are usually grown, that is, cell culture polystyrene Petri dishes
(CCPS). This consists of PS grafted with oxygen/nitrogen polar groups by means of proper surface treatments (usually corona in air) to become wettable and cell/protein adhesive. When Saos2 cells are grown on CCPS they show a very spread morphology, while the same cells grown on flat fluorinated substrate present a decrease in both cell number and average area per single cell. Once we observed how cells can adjust their shape when interacting with substrates of different chemical surface composition, we could look at how their shape can be modulated by surfaces with surface features of different nanometric dimensions, but with the same surface CFx composition. When cell response to fluorinated substrates with different roughness at nanometric level is studied, SEM images show that cells grown for 24 h are well-spread on the flat surface while a nanometric increase in features height, as in the sample 5 MIN, determines a completely different cell shape (Figure 5a,b). When this difference in roughness is higher, as in the case of 10 MIN substrates, cells are more spread than the ones adhered on 5 MIN samples, although different from the ones grown on flat substrates (Figure 5ac). After 8 days of cell culture, when cells on CCPS substrates cover most of the surface (Figure 5d), cells on both nanostructured surfaces show a spread morphology, and no evident differences in cell morphology among fluorinated surfaces with increasing roughness is found except for a major cell density on 10 MIN surfaces (Figure 5e,f). When we look at single cell photos at higher magnification (Figure 5gi), cells on FLAT and 10 MIN samples showed a significantly greater spread than Saos2 cells adhered on 5 MIN samples. All these observations on cell shape bring us to speculate that a different way to interact with nanofeatures has to exist for Saos2 cells faced with 5 MIN and 10 MIN surfaces. 4917
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Figure 4. SEM microphotographs of Saos2 osteoblasts grown for 48 h on PSCC and fluorinated flat surfaces.
Figure 5. SEM images of Saos2 osteoblasts grown on FLAT (a, d, g), 5 MIN (b, e, h), and 10 MIN (c, f, i) surfaces for 24 h (a, b, c) and 8 days (d, e, f). Single cells at higher magnification (g, h, i) were grown on the three substrates for 24 h.
In order for cells to react to topography, they must be able to sense shape, and it is known that there are numerous cell structures responsible for initializing and transmitting the effect of surface topography throughout the cell to influence overall cell functions, such as stress fiber formation, lamellipodia, and filopodia. In particular, filopodia, or microspikes, presented by cells at their leading edges are thought to be involved in gathering special information. Filopodia, highly motile organelles involved in many cellular processes, including migration and sensing of local topography, are cytoplasmic protrusion that are extended by a cell migrating along a surface.18 They serve as topographical sensors, which are able to detect the immediate surrounding environment.19 SEM micrographs of the cells showed numerous filopodia that seemed to join cells to nanostructures, creating a direct link between cells and nanoscaled features. SEM images at high magnifications (Figure 6) can allow us to compare the differences in size among cell filopodia and the surface features of
the 5 MIN and 10 MIN samples. The mean diameter of the surface structures on the 5 MIN surfaces is 65 nm, while the 10 MIN surfaces present nanodomes of about 95 ( 10 nm diameters. These surface structures are even smaller than filopodia width, which is around 115 ( 10 nm. Our observation on cell shape in different substrates bring us to hypothesize that these difference in the ratio between nanostructures and filopodia width can be important in determining cell shape. Filopodia contain actin filaments cross-linked into bundles by actinbinding proteins, e.g., fimbrin.20 The filopodia attach to the substratum further down the migratory pathway and then contraction of actin stress fibers retracts the rear of the cell to move the cell forward. Since cells shape is modulated by the internal cytoskeleton, we directed our attention to study if any changes in the actin conformation within Saos2 cells grown on flat and nano/structured surfaces were observed. Fluorescence images in Figure 7 clearly show the presence of actin stress fibers on spread cells and not on 4918
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Figure 6. SEM images of Saos2 osteoblasts grown for 24 h on 5 MIN and 10 MIN surfaces showing in detail filopodia interacting with the surface nano/ structures.
Figure 7. Effect of nanotopography on actin filaments assembly in Saos 2 cells grown for 48 h on the three fluorinated surfaces: green, actin; blue, nuclei.
cells with a circle shape, a shape that is found in a lot of cells grown on 5 MIN surfaces, especially in the first days of cell culture. This technique allowed us to find a more remarkable difference in cell shape among cells grown on the three different fluorinated surfaces. Once it was observed how cells can stretch and adjust their shape when interacting with substrates of different roughness, our attention was devoted to the quantification of substrate area covered by cells after 24 and 72 h of culture. This has been done by the analysis of images of fixed and Coomassie Blue stained cells. The graph in Figure 8 clearly shows that also in the first 24 h of cell culture a statistically significant difference was present among cells grown on the two etched fluorinated surfaces (p < 0.001), while no differences were shown between cells adhered on FLAT and 10 MIN surfaces (p > 0.05); after 72 h cells cultured on the FLAT surfaces covered an area significantly larger than Saos2 on the two etched surfaces; cells grown on the 5 MIN nanofeatures covered significantly less substrate area with respect to the ones grown on 10 MIN
surfaces. We can hypothesize that the decrease of Saos2 proliferation rate observed on 5 MIN surfaces could be due to the decrease in cell spreading and change in mechanical force exerted by cells on the substrates. This hypothesis is confirmed by cytoskeleton organization analysis revealing a dramatic decrement in stress fibers formation for cells on 5 MIN substrates (Figure 7). As stated in the Introduction, it is important to remember that the interaction between cells and modified surfaces is mediated from a protein layer that comes both from the culture media and also from protein expressed by cells during cell culture. Many material surface factors can influence such interaction, ranging from charge to roughness.2124 The main clue that characterizes our surfaces is the presence of nanofeatures with different heights that produce in turn a different wettability ranging from hydrophobic to superhydrophobic surfaces. Protein adsorption on these surfaces is driven by surface wettability and surface topography. It has been stated that proteins adsorb according 4919
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Figure 8. Effect of nanotopography on the substrate area covered by Saos2 cells after 24 and 72 h on the four different surfaces. Histograms with mean values ((SD). For statistical analysis, two-way ANOVA with Bonferroni’s post-test was performed and different superscripts indicate p < 0.01: * vs FLAT, O vs 5 MIN, and þ vs 10 MIN.
to a “lock and key” mechanism that prefers surface features of a size very similar to the feature sizes of the protein.25 Our results indicate that Saos2 cells respond differently to surfaces with different nanoscale topographical features, clearly showing a certain inhibition in cell adhesion when the nanoscale is particularly small. Our explanation, on the basis of observed cell behavior, is that probably, when the nanoscale is particularly small, the amount and/or the conformation of the protein adsorbed on it do not permit cell adhesion and proliferation.
’ ACKNOWLEDGMENT The authors acknowledge the technical support of Mr. S. Cosmai and D. De Benedetti. Dr. V. De Benedictis, and Dr. F. Palumbo are acknowledged for their fruitful scientific discussions. Mrs. R. Giordano is acknowledged for her work in photo design. The financial support of the Public Laboratory of Apulian Industrial research of Plasmas “LIPP” is acknowledged.
’ CONCLUSIONS Substrate nanotopography, because of its resemblance to in vivo environment, can be utilized as a tool to study complex cell functions, such as adhesion, migration, and cytoskeleton reorganization. The results presented in this paper suggest that, at least in our combination of surface composition, surface morphology, and type of cells, when surface structuring is pushed toward nanometric dimension, the cellular response is driven toward a certain inhibition compared to the flat surface. Also superhydrophobicity of the textured surfaces, expressing a slippery/dry character, is believed to play an important role in inducing such an inhibition. The latter appears attenuated in the surface with higher roughness (i.e., relatively bigger structures), though still less adhesive to water compared to the flat reference. This could be ascribed to a more suitable topography scale to be sensed by cells, partly balancing the particularly hostile wetting effect. Within future investigations, surfaces with similar wetting behavior (slippery superhydrophobic), but provided with a nonfluorinated chemistry, e.g., with an organosilicon one like those very recently developed in our laboratory,26 could undergo the same investigation, in order to separate the role of chemistry from that of wettability in surface cytocompatibility. Further, the same surfaces may be tested with different cell typologies in order not only to make materials more or less adhesive but also to elicit specific cellular responses.
’ REFERENCES
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]. Tel: þ39-080-5443432. Fax. þ39-080-5443405.
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