Differential Cultured Fibroblast Behavior on Plasma and Ion-Beam

Boyan, B. D.; Hummert, T. W.; Dean, D. D.; Schwartz, Z. Biomaterials 1996, 17, ...... Spanos, C. G.; Ebbene, S. J.; Badyal, J. P. S.; Goodwin, A. J.; ...
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Langmuir 2002, 18, 9469-9475

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Differential Cultured Fibroblast Behavior on Plasma and Ion-Beam-Modified Polysiloxane Surfaces C. Satriano,† S. Carnazza,‡ S. Guglielmino,‡ and G. Marletta*,† Dipartimento di Scienze Chimiche, Universita` di Catania, Viale Andrea Doria, 6, 95125 Catania, Italy, and Dipartimento di Scienze Microbiologiche, Genetiche e Molecolari, Universita` di Messina, Salita Sperone, 31, Vill. S.Agata, 98166 Messina, Italy Received April 3, 2002. In Final Form: August 28, 2002 This paper reports evidence of the very unusual differential cell sensitivity toward compositionally similar surfaces obtained by irradiation treatments. In particular, we studied the adhesion, proliferation, and spreading of normal human dermal fibroblasts onto poly(hydroxymethylsiloxane) surfaces modified by either O2 plasma (aged in air or water) or 6 keV Ar+ beams. The cell response ranges from relatively low adhesion and scarce viability for O2-plasma-treated surfaces to a complete cell confluence and optimal spreading and proliferation in the case of 6-keV Ar+ beams. Two different types of altered layers have been identified for the two irradiation techniques, respectively consisting in an amorphous SiCxOy(Hz) phase, produced in the ion-mixing process in the collision cascades induced by 6-keV Ar+ beams, and an almost pure amorphous SiO2-like phase, produced by the highly selective removal of carbon-containing volatile species in the O2 plasma. The observed differential cell response in the case of various plasma-treated surfaces is related to the relative increase of surface free energy. The peculiar optimal cell response onto Ar+-irradiated surfaces is instead assigned to the peculiar electronic structure and related electrical properties of the ion mixed layer.

1. Introduction A growing interest is being seen in the study of the basic features of the process of cell interaction with solid organic and inorganic surfaces, in view of a number of relevant applications, including the fabrication of new prosthetic devices1,2 or the realization of microsystems aimed to manipulate cells, not to mention the application in the field of tissue engineering.3 All these applications demand very careful control of the cell-surface interaction process, including as a critical issue directionally guided cell growth.4-6 In general, the cell-surface interaction is accounted for in terms of the adhesion, proliferation, and spreading processes on surfaces having well-defined structural and chemical features. Thus, there is a large living debate about the nature of the critical factors for different types of cells and various surfaces, but a general consensus has been achieved about the importance of topography and chemical structure of the surfaces seen as “primary” factors, involving in turn a number of other more specific features. Accordingly, the importance of a number of critical surface parameters has been stressed, including the presence and concentration of specific functional groups and/or chemical and crystallographic “defects”, serving as “anchoring” sites,7-12 the surface free energy * Corresponding author. E-mail: [email protected]. † Universita ` di Catania. ‡ Universita ` di Messina. (1) Hutmacher, D. W. J. Biomat. Sci. Polym. Ed. 2001, 12, 107-124. (2) Howlett, C. R.; Evans, M. D. M.; Walsh, W. R.; Johnson, G.; Steele, J. G. Biomaterials 1994, 15, 213-222. (3) Desai, T. A.; Derek, J. H.; Ferrari, M. Biomol. Eng. 2000, 17, 23-36. (4) Boyan, B. D.; Hummert, T. W.; Dean, D. D.; Schwartz, Z. Biomaterials 1996, 17, 137-146. (5) Kam, L.; Shain, W.; Turner, J. N.; Bizios, R. Biomaterials 2001, 22, 1049-1054. (6) McFarland, C. D.; Thomas, C. H.; DeFilippis, C.; Steele, J. G.; Healy, K. E. J. Biomed. Mater. Res. 2000, 49, 200-210. (7) Nakao, A.; Kaibara, M.; Iwaki, M.; Suzuki, Y.; Kusakabe, M. Surf. Interface Anal. 1996, 24, 252-256.

and related properties, expressed in terms of the balance between hydrophilic and hydrophobic character,13-15 the topography,16-18 the electrical properties (charge state and electronic structure),19 and the adsorption of serum proteins and peptides.20 Also the interplay of the two “primary” factors has been extensively studied, i.e., topography and chemical structure, and particularly the chance that chemical modifications actually produce topographic features, on the nanometric scale, able to affect the cell behavior.21-23 Recently, a quite innovative research area has been open up, demonstrating that several types of cells exhibit a preferential adhesion to ion-irradiated surfaces.24-26 The (8) Tidwell, C. D.; Ertel, S. I.; Ratner, B. D. Langmuir 1997, 13, 3404-3413. (9) Cenni, E.; Granchi, D.; Arciola, C. R.; Ciapetti, G.; Savarino, L.; Stea, S.; Cavedagna, D.; Di Leo, A.; Pizzoferrato, A. Biomaterials 1995, 16, 1223-1227. (10) Acarturk, T. O.; Peel, M. M.; Petrosko, P.; LaFramboise, W.; Johnson, P. C.; DiMilla, P. A. J. Biomed. Mater. Res. 1999, 44, 355370. (11) Houseman, B. T.; Mrksich, M. Biomaterials 2001, 22, 943-955. (12) Webb, K.; Hlady, V.; Tresco, P. A. J. Biomed. Mater. Res. 2000, 49, 362-368. (13) Mack, D. R.; Sherman, P. M.Colloids Surf., B 1999, 15, 355363. (14) Bacˆa´kova´, L.; S ˆ vorcˆ´ık, V.; Rybka, V.; Micˆek, I.; Hnatowicz, V.; Lisa´, V.; Kocourek, F. Biomaterials 1996, 17, 1121-1126. (15) Ruardy, T. G.; Schakenraad, J. M.; van der Mei, H. C.; Busscher, H. J. Surf. Sci. Rep. 1997, 26, 1-30. (16) Domke, J.; Danno¨hl, S.; Pak, W. K.; Mu¨ller, O.; Aicher, W. K.; Radmacher, M. Colloids and Surfaces B: Biointerfaces 2000, 19, 367379. (17) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Science 1997, 276, 1425-1428. (18) Curtis, A.; Wilkinson, C. Biomaterials 1997, 18, 1573-1583. (19) Dewez, J. L.; Doren, A.; Schneider, Y. J.; Rouxhet, P. G. Biomaterials 1999, 20, 547-559. (20) Detrait, E.; Lhoest, J.-B.; Knoops, B.; Bertrand, P.; van der Bosch de Aguilar, Ph. J. Neurosci. Methods 1998, 84, 193-204. (21) Galli, C.; Collaud Coen, M.; Hauert, R.; Katanaev, V. L.; Wymann, M. P.; Gro¨ning, P.; Schlapbach, L. Surf. Sci. 2001, 474, L181-L184. (22) Craighead, H. G.; James, C. D.; Turner, A. M. P. Curr. Opin. Solid State Mater. Sci. 2001, 5, 177-184. (23) Desai, T. A. Med. Eng. Phys. 2000, 22, 595-606.

10.1021/la025800x CCC: $22.00 © 2002 American Chemical Society Published on Web 10/24/2002

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observed improvement of the cell response on the irradiated surfaces has been basically discussed in terms of the modification of contact angle (i.e., the surface free energy)27-29 and composition, put in relation to the formation of peculiar hydrogenated amorphous carbonor silica-like phases, depending on the original structure of the irradiated polymers.30,31 However, it is becoming more and more clear that a huge number of other factors may play an important role in stimulating the cell response. In fact, in the case of irradiated surfaces to the above-mentioned factors one must add also the role of the electronic defects, i.e., trapped charges or conducting domains within the altered layer at the surface and the effect of the long-living radicals or other metastable species, acting either as specific reacting sites or just as “messengers” to stimulate the cellular response. The aim of the present paper is to report the result of a detailed study of adhesion, proliferation, and spreading process for Normal Human Dermal Fibroblasts-Adult onto O2-plasma-treated and 6 keV Ar+-irradiated surfaces of poly(hydroxymethylsiloxane) thin films. In particular, the paper demonstrates that radiationinduced modification of the surfaces produces a non trivial differential cell response although the compositional modification and morphology are found to be very similar for all the treated surfaces. This fact, in turn, points toward a major role of the interplay of critical factors such as the surface free energy and the functional groups distribution at surfaces, with the electronic structure of the whole altered layer produced by the different types of treatment. 2. Materials and Methods 2.1. Substrates Preparation. Poly(hydroxymethylsiloxane) (PHMS) thin films (thickness of 500 ( 15 nm) were deposited on p-doped silicon (100) wafers from a commercial solution (Accuglass512B, purchased from Allied Signal) by means of the spin coating technique, without any further cure processing. Afterward the wafers were cut in pieces of 1 × 0.5 cm2 in area and modified at the surface by using either O2 plasma treatment or 6-keV Ar+ ion beams. Oxygen plasma treatments were carried out in a March Instrument solid-state Plasmod unit (Concord, CA) supplied with a RF generator with an excitation frequency of 13.56 MHz. Care was taken to pump down and purge the treatment chamber for at least 10 min prior to activating the RF field. The samples were placed on an aluminum plate inside a quartz chamber. The treatment conditions were as follows: 99.95% minimum purity oxygen; power, 100 W; pressure, 66.6 Pa; treatment time, 1 min. Two different sets of O2 plasma-treated samples were aged respectively in air (henceforth indicated as plasma/air samples) and in ultrapure MilliQ-water (henceforth indicated as plasma/ water samples) and used after reaching the steady state in composition and wettability in about 1 week. The effective thickness of the modified layer can be estimated to be higher than roughly 9 nm, because the XPS analysis of the fresh plasmamodified samples does not show any significant traces of the characteristic carbon signal coming from the original methyl groups of the polymer. (24) Suzuki, Y.; Kusakabe, M.; Iwaki, M. Nucl. Intr. Methods Phys. Res. 1991, B59/60, 1300-1303. (25) Lhoest, J. B.; Dewez, J. L.; Bertrand, P. Nucl. Intr. Methods Phys. Res. 1995, B105, 322-327. (26) Bacˆa´kova´, L.; Mare×f0, V.; Lisa´, V.; Sˆ vorcˆ´ık, V. Biomaterials 2000, 21, 1173-1179. (27) Tsuji, H.; Satoh, H.; Ikeda, S.; Gotoh, Y.; Ishikawa, J. Nucl. Intr. Methods Phys. Res. 1998, B141, 197-201. (28) Satriano, C.; Marletta, G.; Conte, E. Nucl. Intr. Methods Phys. Res. 1999, B148, 1079-1084. (29) Choi, S. C.; Choi, W. K.; Jung, H. J.; Park, J. G.; Chung, B. C.; Yoo, Y. S.; Koh, S. K. J. Appl. Polym. Sci. 1999, 73, 41-46. (30) Pignataro, B.; Conte, E.; Scandurra, A.; Marletta, G. Biomaterials 1997, 18, 1461-1470. (31) Satriano, C.; Marletta, G.; Conte, E. Langmuir 2001, 17, 22432250.

Satriano et al. Ion irradiation treatments were performed with 6-keV Ar+ at a fluence of 1 × 1015 ions/cm2 by using a VG EX05 ion gun. The irradiation was carried out at room temperature in a chamber at pressure of less than 10-5 Pa, and the current was kept at 1.5 µA/cm2, to avoid heating effects in the samples. By using the TRIM code,32 the projected range (i.e., the average penetration depth) of 6-keV Ar+ ions in PHMS was estimated to be 8.5 ( 3.0 nm, with a total deposited energy of about 700 eV/nm. 2.2. Substrates Characterization. The physicochemical surface characterization of the substrates both before and after the surface modification was performed by means of X-ray photoelectron spectroscopy (XPS), static contact angle (CA), and atomic force microscopy (AFM) measurements. 2.2.1. XPS Measurements. XPS analysis was carried out with a Kratos HX AXIS spectrometer equipped with a dual Al/Mg anode and a hemispherical analyzer. The spectra were obtained in fixed analyzer transmission mode (pass energy 40 eV) by using the Mg KR1,2 radiation. The estimated sampling depth is about 9 nm, according to an attenuation length of 3.0 nm for Si 2p peak in organic materials.33 Such a value is actually comparable to the estimated thickness of the ion- and plasma-modified layers (see above). XPS spectra were analyzed by using an iterative least-squares fitting routine based on Gaussian peaks and the Shirley background subtraction.34 Binding energies (BEs) of all the spectra were referenced to the intrinsic (before irradiation treatment) hydrocarbon-like C 1s peak set at 284.6 eV or to the adventitious one set at 285.0 eV (after the plasma treatments).35 2.2.2. Surface Free Energy Measurements. Measurements of surface free energy were performed by evaluating the static contact angle of three different liquids onto the untreated and treated surfaces. Contact angle measurements were performed at 25 °C and 65% relative humidity with the sessile drop method by using a manual goniometer (Kernco Inst.) in a horizontal position. Liquid drops, 5 µL, were applied by hand on different parts of the sample surfaces, and the static contact angles (θs) were read on both sides of the two-dimensional projection of the droplet. At least five measurements were made for each sample and then averaged. By using the three liquids tricresyl phosphate (apolar), pure deionized water, and glycerol (both polar liquids) and the Good-van Oss approach, the surface free energies were evaluated in terms of apolar Lifshitz-van der Waals (γLW) and polar Lewis acid (γ+) and basic (γ-) components.36,37 2.2.3. Atomic Force Microscopy (AFM). The surface microtopography and the morphology of the unirradiated and 6-keV Ar+-irradiated PHMS surfaces were measured with a multimode/ nanoscope IIIA atomic force microscope (Digital Instruments) working in tapping mode in air with a standard silicon tip. Data were acquired on square frames having edges of 10 µm, 1 µm, and 300 nm, with 256 × 256 data points and a scan rate of 1.989 Hz. Images were recorded by using both height and phase-shift channels. 2.3. Cell Culture. Normal human dermal fibroblasts-adult (henceforth NHDF) (Clonetics, Bio-Whittaker) cell line was used to test the cell adhesion on the various modified surfaces. The cells were routinely maintained in Dulbecco’s modified Eagle medium (DMEM, Euroclone) supplemented with 10% fetal bovine serum (FBS), penicillin (100 milion units/mL), streptomycin (0.1 mg/mL), and l-glutamine (2 mM) (all Euroclone), at 70-80% confluence in 25 cm2 polystyrene flasks (Nunc), at 37 °C in a humidified 5% CO2 atmosphere. The PHMS samples were placed in the bottom of a six-well polystyrene tissue culture plate (Nunc). A reference untreated PHMS sample was used to compare the NHDF behavior on plasma-treated and irradiated PHMS substrates. Fibroblasts were trypsinized with 0.25% trypsin and 0.02% EDTA for 5 min, (32) Ziegler, J. F.; Biersack, J. P. In The Stopping and Range of Ions in Solids; Littmark, U., Eds.; Pergamon Press: New York, 1999. (33) Suzuki, N.; Iimura, K.; Satoh, S.; Saito, Y.; Kato, T.; Tanaka, A. Surf. Interface Anal. 1997, 25, 650-659. (34) Tougaard, S.; Jansson, C. Surf. Interface Anal. 1993, 20, 10131046. (35) Suzuki, Y.; Kusakabe, M.; Iwaki, M.; Suzuki, M. Nucl. Instr. Methods 1988, B32, 120-124. (36) Kwok, D. Y.; Neumann, A. W. Adv. Colloid Interface Sci. 1999, 81, 167-249. (37) Good, R. J. J. Adhesion Sci. Technol. 1992, 6, 1269-1302.

Differential Cultured Fibroblast Behavior

Figure 1. Adhered NHDF cells on the various PHMS surfaces after 5 h of incubation. centrifuged at 5403g for 5 min, and resuspended in the same culture medium. Cell suspension, 3 mL, with a cell concentration of about 1.5 × 105 cell/ml was added to each well. The cell adhesion as well as proliferation and tendency to spreading were evaluated respectively after 5 and 48 h of incubation at 37 °C in a humidified 5% CO2 atmosphere. On each well a reference untreated PHMS was placed, to allow a direct comparison of the two different treatments. Any external influence on the culture procedure had the same effect on the cells in all the samples and thus provides an experimental area with an internal control. At the end of the culture periods, samples were washed with PBS (phosphate buffer saline solution), fixed with 4% pformaldehyde for 30 min at room temperature, permeabilized with Triton X-100 (0.1% in PBS), and stained with Hoechst (50 µg/mL) and Blue Evans (10 µg/mL) for 30 min at 37 °C. The use of these two fluorochromes allowed both visualization of adhered cells on polymer surfaces without optical interferences and evaluation of cell vitality and spreading by differential staining of the nucleus and cytoplasm. The samples were washed twice in magnesium- and calciumdeprived PBS, dried in air, and mounted with mounting medium (SIGMA) for observation by epifluorescence microscopy. At least 10 microscopic fields per sample were randomly acquired with x20 magnification by a COHU high performance CCD camera and Leica Qwin software. The images obtained were elaborated in luminosity and contrast through an interactive image analyzer for analysis of cell morphology, spreading, and distribution on the investigated substrates. Quantitative evaluation of adhered cells was performed by using the Scion image software (Windows version of NIH image software), which allowed the evaluation of the cell coverage in terms of integrated density (ID ) N[M - B], where N is the number of pixels in the selection, M is the average gray value of the pixels, and B is the most common pixel value). Results of the image analysis are expressed as mean-standard deviation for each group of treated samples. Differences among groups were established by T-student test analysis by a twopopulation comparison. Statistical significance was considered at a probability P < 0.05.

Results 1. Cell-Surfaces Interaction. NHDF cells were seeded on the different surfaces and cell adhesion was determined after 5 h of incubation (short-term effect), while morphology and spreading were determined after 48 h (longer term effect). The quantitative estimation of the cell coverage on the various PHMS surfaces after 5 h of incubation is reported in Figure 1 in terms of average number of adhered NHDF cells per centimeters squared, as derived from experimental integrated density values (see Materials and Methods). This specific incubation time was chosen in such a way that the observed cell response is only due to the primary cell-surface interaction, as far as it was established that the spreading effect remains negligible up to

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Figure 2. Light micrograph of the NHDF cells adhered and spread on untreated PHMS surfaces.

12 h in the present culture conditions (not shown here). The results show clearly that while no significant adhesion is found on the untreated PHMS, a significant but treatment-dependent increase of cell adhesivity is observed both for plasma and ion beam irradiated surfaces. In particular, the highest cell coverage was found for the ion-irradiated and plasma/water treated samples, while in the case of plasma/air treated surfaces the adhesion is reduced by about a factor 2. This fact suggests that the different treatments and aging conditions produce different surfaces and then different adhesion levels. The cell viability and the tendency to spreading have been studied after an incubation time of 48 h also if the used cell line needs an average incubation time of 6-9 days for complete spreading. The first result at this incubation time is that the number of adhered cells is actually almost the same as the one observed at only 5 h of incubation, as derived by the measured ID mean values (not reported here). Thus, we do not observe any significant proliferation but only the effect of the onset of the spreading process, which is responsible for the cell viability and morphology at longer incubation time. It is stressed that already at this stage dramatic differences in the cell morphology and viability have been found for the different treated and untreated PHMS surfaces. In particular, Figures 2-4 report the light micrographs of the NHDF cells adhered and spread on the various untreated and treated PHMS surfaces. It can be seen that on untreated PHMS surfaces (Figure 2) only sporadic cells, after careful washing, are adhered and maintain a globular shape, with thickened chromatin and little evidence of spreading. These three peculiar morphology features clearly indicate the low viability of these cells. The cell appearance in terms of morphology and endocellular structure is strongly dependent on the type of surface treatment. In particular, for the plasma/air samples (Figure 3a), also if the number of adhered cells is very high, the cells are less spread, frequently exhibiting a globular shape and a general tendency to form clusters by a preferred attachment to each other rather than on the surfaces. Such a behavior seems critically related to a strongly reduced surface adhesion. At variance with this, in the case of plasma/water surfaces (Figure 3b), the number of adhered cells is higher (about a factor 2) than in the case of plasma/air samples and the cells look in general healthy, with a characteristic elongated and wellshaped morphology, without major clustering effects. However, about 15-16% of the cellular population still shows a globular shape, indicating in turn that the plasma/ air surfaces still have partial cytocompatibility. Finally, the cell adhered onto the 6-keV Ar+ irradiated surfaces looked tapered and evenly distributed throughout

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Figure 3. Light micrographs of the NHDF cells adhered and spread on O2-plasma-treated PHMS surfaces: (a) plasma/air samples; (b) plasma/water samples.

Figure 4. Light micrograph of the NHDF cells adhered and spread on 6-keV Ar+-irradiated PHMS surfaces.

Figure 5. Light micrograph of the NHDF cells adhered and spread around the borderline between untreated (left area of the photo) and 6-keV Ar+-irradiated (right area of the photo) PHMS surfaces.

the irradiated surface (Figure 4). The cells in fact are completely spread and confluent, showing a regular mitotic activity. Furthermore, the cells exhibit an ordered disposition closely related to the progress of the proliferation activity, occurring according to the islandlike model of growth, peculiar to the considered cell line. As a final point, it is stressed that the irradiation treatment is so effective that an evident borderline in cell adhesion and spreading is clearly formed between the irradiated area, filled with spread cells, and the contiguous untreated region, where the cells are completely absent (Figure 5). This effect clearly indicates that the cells feel in a very efficient way the competition between unirradiated and irradiation-modified regions with a high spatial resolution, of the order of micrometers. 2. Physico-chemical Characterization. The biological response for a given surface may be determined by several factors, including the chemical composition, the presence of specific functional groups on the surface, the surface free energy and related properties, the morphology factors and in particular the roughness, the charge state,

Figure 6. Average surface atomic composition from XPS analysis for the various untreated and surface-modified PHMS samples.

etc...38 Both the plasma and ion-based treatments of polymeric surfaces are known to induce severe modification of some or all these factors. In the present case, we tried to determine which are the most relevant treatmentinduced modifications, able to explain in particular the strikingly different biological response observed for similar treatments. 2a. Surface Chemical Structure. The average surface atomic composition of the polymer is not very different for all the employed modification techniques (see Figure 6). The only relevant difference with respect to the untreated PHMS surfaces is the decrease of the carbon content from the original value of ∼23% to about 7%, 6%, and 12% respectively for plasma/air, plasma/water, and 6-keV Ar+irradiated samples. It should be noted that in the present experiment the freshly plasma-treated surfaces are completely carbon-depleted within the XPS sampling depth (results not shown here). Thus, the observed carbon species on the aged samples may well derive either from extensive recovering of the plasma-treated layers or from the selective attachment of contaminants on the plasma-activated surfaces. In any case, as far as NHDF cells interact with these “real” surfaces, in the following we will take it into account. The detailed analysis of the photoelectron peak shape and energy shows significant differences among the various samples. In fact, while the Si 2p peak in the untreated PHMS is fitted by a single Gaussian component centered at 102.2 ( 0.2 eV of BE, assigned in agreement with literature to SiO3C clusters,39-41 for both plasmatreated and 6 keV Ar+-irradiated surfaces, the Si 2p peak is found at 103.6 ( 0.2 eV, which is characteristic of the (38) Horbett, T. A.; Klumb, L. A. In Interfacial Phenomena and Bioproducts; Brash, J., Wojciechowski, P. W., Eds.; Dekker: New York, 1996; p 351.

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Figure 8. Liftshiz van der Waals (γLW) and Lewis acid (γ+) and base (γ-) components of the surface free energies for untreated PHMS, the various surface-modified PHMS samples, and tissue culture polystyrene (PS) control. Figure 7. C 1s photoelectron peaks for untreated PHMS and the various surface-modified PHMS samples.

formation of a SiO2-like phase, predominantly formed by SiO4 clusters.42 At variance of this, the O 1s peaks do not show any significant difference in both width and peak position. Finally, the C 1s peak shows a quite complex modification after the various treatments and aging (see Figure 7). In particular, for the untreated PHMS surfaces the C 1s peak can be fitted by using only two basic components: the main one (C1 in Figure 7) is positioned at 284.6 eV of BE and assigned to >C-Si bonds, in agreement with literature data,42 the second peak component (C2) at 286.6 eV of BE, assigned to >C-OH and >C-O-C groups belonging either to the terminal polymer groups or to the solvent residues. For the plasma-treated PHMS surfaces, i.e., both plasma/air and plasma/water samples, one observes that while the C1 component is dramatically reduced with respect the C2 one, these components remain basically the same as in original PHMS. This observation may support the hypothesis that the carbon species after aging derive from recovery processes involving the polymer bulk. Furthermore, a new quite small component (C3) appears centered around 289.0 eV of BE, being attributed to >C(dO)sO bonds.43 A different situation is found for the ion-irradiated samples. In fact, two new components appear at 286.1 eV (C4) and 287.2 eV (C5) respectively assigned to the formation of C-O-Si moieties (for the C4 component) and to the formation of >CdO and >C(O)2 groups (for the C5 component).44 These new components completely replace the pristine C2 component. Finally, the C3 component, due to >C(dO)sO bonds, is present also in this case, roughly with the same relative weight. It has to be stressed that the relative weight of the oxidized carbon species with respect to the total carbon content is drastically (39) Alfonsetti, R.; Lozzi, L.; Passacantando, M.; Picozzi, P.; Santucci, S. Appl. Surf. Sci. 1993, 70/71, 222-225. (40) Alfonsetti, R.; de Simone, G.; Lozzi, L.; Passacantando, M.; Picozzi, P.; Santucci, S. Surf. Interface Anal. 1994, 22, 89-92. (41) Iacona, F.; Kelly, R.; Marletta, G. J. Vacuum Sci. Technol. B 1999, 17, 2771-2778. (42) To´th, A.; Berto´ti, I.; Marletta, G.; Ferenczy, G.; Mohai, M. Nucl. Instr. Methods Phys. Res. 1996, B116, 299-304. (43) Moulder, J. F.; Strickle, W. F.; Sobol, P. E.; Bomben, K. D. In Handbook of X-ray Photoelectron Spectroscopy; Chastain, J., Ed.; PerkinElmer Corporation: Eden Praie, MN, 1998. (44) Spanos, C. G.; Ebbene, S. J.; Badyal, J. P. S.; Goodwin, A. J.; Merlin, P. J. Macromolecules 2001, 34, 8149-8155.

different in the plasma versus ion-irradiated samples. In fact, the oxidized species constitute roughly 2.2% and 2.4% of the surface atomic concentration of plasma/air and plasma/water samples, while these are about 4.1% of the Ar+-irradiated surfaces. Thus, the intrinsic structure of the altered layers produced respectively by plasma and ion irradiation seems very different, as far as the Ar-irradiated samples exhibit the formation of a peculiar amorphous SiCxOy(Hz) phase, with a high content of carbonyl-containing species, and the plasma-treated samples rather have the structure of a SiO2-like phase, i.e., without Si-C linkages, and with a smaller amount of carbonyl-containing species. 2b. Surface Free Energy. To determine the relative importance of the various components of the surface free energy, we performed the three-liquid analysis for the untreated and plasma- and ion-treated PHMS surfaces. In particular, the surface free energy of each sample was evaluated in terms of the apolar or Lifshitz-van der Waals component γiLW, which includes the dispersion as well as the induction and orientation contributions to the van der Waals interactions and acid (γi+, the electron-acceptor parameter) and basic (γi-, the electron-donor parameter) Lewis polar components, such that

γiLW + 2xγi +γi - ) γiTOT

(1)

2xγi +γi- ) γiAB

(2)

and

with γiTOT being the total surface free energy and γiAB the polar Lewis acid-base component for the i solid. Figure 8 reports the Lifshitz-van der Waals (γLW) and the Lewis acid (γ+) and basic (γ-) components for the various samples as well as polystyrene, assumed as general reference material for cell culture. One can see that in general, after the irradiation treatments, the relative increase of the polar γ- and γ+ components is much higher than that of the corresponding apolar γLW term. Furthermore, the Lewis basic term γ- largely predominates with respect to the corresponding acid γ+ term, indicating that both plasma treatment and ion irradiation substantially produce a monopolar surface, characterized by an electron-donor behavior (see inset in Figure 8). It is interesting to note that polystyrene does not show a significant value of the polar acid and basic components.

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Accordingly, we attribute the observed differences in the surface free energy for the various samples to the change of the chemical structure of the outermost surfaces. Discussion and Conclusions

where w stands for water. When ∆Giwi > 0, the surface is hydrophilic, and when ∆Giwi< 0, the surface is hydrophobic.45,46 The change of ∆Giwi induced in PHMS is reported in Figure 9, showing that the water wettability of PHMS surfaces is increased in every case by the surface modification. In particular, the original very hydrophobic PHMS surface (i.e., large negative value of ∆Giwi, corresponding to a water contact angle value of θs ∼ 90°) becomes mildly hydrophilic upon O2 plasma/air aging treatment (i.e., small positive value of ∆Giwi, corresponding to θs ∼ 34°), and it is definitely converted into a very hydrophilic surface upon O2 plasma/water aging (large positive value of ∆Giwi, with θs ∼ 12°).47 At variance of this, notwithstanding the very high polar character of the 6-keV Ar+-irradiated PHMS surfaces, these exhibit a moderate hydrophobic character, with a small negative value of ∆Giwi, which corresponds to a water contact angle θs ∼ 53°, intermediate between untreated PHMS and plasma-treated surfaces. It is to be noted that the contact angle modification for the various irradiated surfaces could in principle be due to radiation-induced morphology changes. Accordingly, the average roughness of all the investigated surfaces has been measured by atomic force microscopy (AFM). Table 1 reports the results in terms of root-mean-square (Rrms) and mean (Ra) roughness. It appears that the various treatments do not induce any significant change in roughness for either O2-plasma-treated and 6-keV Ar+irradiated samples.

It is commonly claimed that cell adhesion and spreading processes are basically related to several factors, including the substrate hydrophilic/hydrophobic character or its surface free energy, the polar character of the surfaces, the morphology on a nanometric or micrometric scale, the presence of specific chemical functionalities, presumably acting through their effect on the selective adsorption of serum proteins, etc.38 However, the relative weight and the possible interference of the various factors in a real system is still a matter of large and often contradicting debate, also involving sometimes puzzling experimental findings. In fact, throughout this paper we have shown that the best cell adhesion results are found for plasma/water and 6-keV Ar+-irradiated PHMS samples, which have indeed very different contact angles (respectively θs ) 12° ( 3° for plasma/water samples and θs ) 52° ( 3° for 6-keV Ar+irradiated surfaces), suggesting that mere wettability cannot explain the effect. Nor can the relative acid-base term be considered as a determining factor, as far as very different cell spreading behavior was observed respectively for plasma/water and plasma/air surfaces, having very similar acid-base components. Also, we observe again very different biological response for surfaces of almost identical roughness and very similar compositional structure. Finally, a certain number of factors seems to play a somewhat unclear role. As an example, let us recall the possible role of the monopolar character of the irradiated surfaces, indicated by the ratio between the Lewis basic γ- and acid γ+ components. This factor indeed increases in a parallel way with the observed enhancement of cell adhesion and spreading, suggesting that the monopolar Lewis basic character could influence the cell behavior, but it is hard to assign such a critical importance to an effect of such small magnitude (Figure 8 above). In view of the above considerations, we rather propose that the observed selective cell response of the different irradiated surfaces is due to the competition of two basic factors, i.e., wettability and electronic structure, which depend critically on the specific modification process. In fact, the chemical structure and related electronic properties of plasma and Ar-irradiated PHMS surfaces are expected to be intrinsically different due to the diversity in the primary modification processes. In particular, the basic energy deposition process for low-energy ions is based on concerted series of impulse-transfer events, globally indicated as collision cascades.48 This mechanism has been shown to induce characteristic primary “nonthermodynamic” processes, mostly consisting in the random breaking of the bonds present in the collision cascade volume and leading to a huge number of secondary “thermodynamically driven” rearrangement processes.49 The net result of the whole process in our case is that carbon is only partially removed from the irradiated layer, as a result of physical sputtering and chemical removal, producing the emission of COx and CHy species, while a

(45) van Oss, C. J. In Polymer Surfaces and Interfaces II; Feast, W. J., Munro, H. S., Richards, R. W., Eds; Wiley: Chichester, 1993; p 267. (46) van Oss, C. J. Colloids Surf., B 1995, 5, 91-110. (47) Satriano, C.; Kasemo, B.; Marletta, G. Chem. Mater., submitted for publication.

(48) Stoneham, A. M. Nucl Instr. Methods 1990, B48, 389-398. (49) Marletta, G.; Iacona, F. In Materials and Processes for Surface and Interface Engineering; NATO-ASI Series, Serie E: Applied Sciences; Pauleau, Y., Ed.; Kluwer Academic Publishers: Dordrecht, 1995; Vol. 290, p 597.

Figure 9. Free energy of interaction in water ∆Giwi for untreated and the various surface-treated PHMS samples. Table 1. Values of Root Mean Square Roughness (Rrms) and Mean Roughness (Ra) Obtained by AFM Measurements untreated plasma/air plasma/H2O 6-keV Ar+ irr.

Rrms, nm

Ra, nm

0.55 ((0.05) 0.48 ((0.05) 0.49 ((0.05) 0.55 ((0.05)

0.45 ((0.05) 0.38 ((0.05) 0.39 ((0.05) 0.44 ((0.05)

The relative hydrophilic/hydrophobic character of the surfaces was evaluated, by using the above derived polar and apolar contributions, in terms of the free energies of interaction in water ∆Giwi, given by

∆Giwi ) -2γiw ) -2(xγiLW - xγwLW)2 - 2(γiAB +

γwAB - 2xγi +γw- - 2xγi -γw+) (3)

Differential Cultured Fibroblast Behavior

quite relevant part of carbon is just “mixed” with Si, O, and H atoms in the altered layer, forming a complex SiCxOy(Hz) “oxycarbide” phase. In fact, according to the XPS evidence, the original >COH and >COC groups are almost completely destroyed, while new >COSi moieties (at 286.1 eV) (not seen with plasma-treated samples) are formed. These ion-induced phases exhibit a characteristic value of contact angle θs roughly between 50° and 30°, approximately following the residual carbon concentration inside the altered layer.31 Furthermore, this mixed oxycarbide matrix has mechanical, optical, and electrical properties (including band gap and electronic structure) which are very different with respect to those of SiO2-like phases.50,51 To explain the possible influence of the electronic structure of the ion mixed phase on the surfacebiosystem interaction, let us recall that recently it has been demonstrated that the whole electronic structure of metal or semiconductor surfacial layers (i.e., about 5-nm layers) may strongly drive the selective adsorption processes of dipole- or monopole-containing biomolecules and is in turn affected by their adsorption.52 The preferential adsorption of selected biomolecules is obviously the key, determining the overall differential cell interaction with surfaces of different electronic structure. At variance of this, the plasma treatments basically induce the formation of an almost completely carbon(50) Marletta, G.; Bertoti, I.; To´th, A.; Tran Minh Duc, Sommers, F.; Ferenc, K. Nucl. Instr. Methods 1998, B141, 684-692. (51) Pivin, J.-C.; Colombo, P. J. Mater. Sci. 1997, 32, 6163-6168. (52) Vilan, A.; Cahen, D. Trends Biotechnol. 2002, 20, 22-29.

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depleted layer, due to the predominant effect of chemical sputtering processes, basically involving carbon oxidation reactions and outgassing of volatile species. This process is responsible for the formation of an amorphous SiO2like altered layer, with a small amount of surfacial oxidized carbon species and Si-OH upon aging.53 Accordingly, the highly hydrophilic character exhibited by these surfaces may be accounted for mostly in terms of the combined effect of the polar bonds of the exposed Si-O-Si, Si-OH groups and the surface-anchored oxidized carbon species. In view of the described differences, the cell response to the plasma-treated surfaces seems to be mostly related to the wettability change, while for ion-irradiated samples it may be basically related to the peculiar electronic structure of ion-mixed SiCxOy(Hz) phase. The present results open the way to further studies aimed to expanding the basic knowledge on this very complex matter, by prompting more specific investigations on the role of the serum proteins in both short- and longterm cell-surface interactions. Acknowledgment. We are grateful to Prof. G. Strazzulla (Osservatorio Astrofisico of Catania) for providing access to the ion-beam irradiation facility. Financial support from PRIN Program 2001 (MIUR-Rome) is gratefully acknowledged. LA025800X (53) Licciardello, A.; Satriano, C.; Marletta, G. In Secondary Ion Mass Spectrometry (SIMS XII); Benninghoven, A., Bertrand, P., Migeon, H. N., Eds.; Wiley: Sept. 2000, p 889.