Surface Chemical Structure and Cell Adhesion onto Ion Beam

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Surface Chemical Structure and Cell Adhesion onto Ion Beam Modified Polysiloxane C. Satriano, E. Conte, and G. Marletta* Dipartimento di Scienze Chimiche, Universita` di Catania, Viale A. Doria 6-95125 Catania-Italia Received September 14, 2000. In Final Form: December 27, 2000 The modification of the adhesion and spreading of BHK21 fibroblast cells has been studied for new biocompatible surfaces obtained by irradiating polyhydroxymethylsiloxane thin films with increasing doses of 5 keV Ar+ ion beams. The irradiated surfaces showed a dose-dependent increase of cytocompatibility, with an observed onset of the effect at about 5 × 1014 ions/cm2. At this dose, in fact, we found both the enhancement of cell adhesion, for an incubation time of 2 h, and complete cell confluence after an incubation time of 48 h. The observed fluence-dependent trends in cell adhesion and spreading have been correlated with the irradiation-induced modifications of the polymer surface composition and the related change in surface energy, obtained by using X-ray photoelectron spectroscopy (XPS) and contact angle measurements of three liquids. XPS data showed that ion irradiation induced a progressive compositional modification of the polymer toward a SiOx-rich phase, because of the irradiation-induced formation of [SiO4] clusters and decrease of the original [SiO3-C] ones, involving the loss of more than 50% of the original methyl groups and the transformation of the residual carbon-containing groups in a dispersed hydrogenated amorphous carbon phase of nanometric size. The surface free energy measurements, performed with the static contact angle technique, showed that ion irradiation transforms the initially hydrophobic surfaces, with θ ) 77.6° ( 1.5°, into much more hydrophilic ones, with θ ) 31.4° ( 1.7°. Furthermore, the contact angle is found to undergo an abrupt decrease just at an ion dose of 5 × 1014 ions/cm2, that is, where the onset of cell adhesion and confluence is observed. The analysis of the observed changes in the total surface energy in terms of the relative polar and dispersive force contributions showed that the strong enhancement of the hydrophilic character of the irradiated surfaces is mainly due to the raising of the polar acid-base force components, this effect being due to the enrichment of the irradiated surfaces with the permanent dipoles of the [SiO4]-based network and the elimination of the original pendant methyl groups.

Introduction There is an increasing interest in developing new methods to induce controlled cell adhesion onto polymeric materials, in view of their application as biomaterials employed both in strategies of tissue engineering and in fabrication of various types of implant devices. The critical step in all these methods obviously involves the modification of a few surface layers at the surface of the materials, as the cellular behavior on biomaterial surfaces, including their adhesion, spreading, and growth, depends on the surface properties and the processes localized within a nanometer-wide interfacial region.1 Many physical and chemical techniques of surface modification have been applied to achieve more or less extended changes in polymer wettability, charge, and morphology, which have been correlated, in a quite heuristic way, to enhanced cell adhesion. Among the various techniques, those based on the use of a very high energy density, for instance, plasma treatments and ion irradiation, are particularly attractive, because of their peculiar characteristic of modifying a well-controlled thickness of the material of interest, without affecting the bulk structure and properties. Thus, for instance, although plasma treatments are well-established methods to change typical properties of the very outer surface in polymers, like wettability and the relative hydrophilic/ * Corresponding author. Prof. G. Marletta, Dipartimento di Scienze Chimiche, University of Catania, Viale A. Doria, 6-95125 Catania, Italy. Telefax: ++39-095-33.64.22. E-mail: gmarletta@ dipchi.unict.it. (1) Descouts, P. A. R. In Nanoscale Probes of the Solid/Liquid Interface; Gewirth, A. A., Siegenthaler, H., Eds.; Kluwer Academic Publishers: Dordrecht, 1995; pp 317-331.

hydrophobic character of surfaces,2-3 ion irradiation has been demonstrated to be able to induce durable modifications of the composition and morphology of thin layers of variable thickness at the polymer surfaces.4-7 In turn, the ion beam induced modifications have been demonstrated to affect dramatically various properties of the modified layers, including the surface energy,8-9 the optical10 and electrical behavior,11,12 and the hardness.13 (2) Owen, M. J.; Smith, P. J In Polymer Surface Modification: Relevance to Adhesion; Mittal, K. L., Ed.; VSP: Utrecht, The Netherlands, 1995; pp 3-5. (3) Fakes, D. W.; Davies, M. C.; Brown, A.; Newton, J. M. Surf. Interface Anal. 1988, 13, 233. (4) Venkatesan, T.; Calcagno, L.; Elman, B. S.; Foti, G. In Ion Beam Modification of Insulators; Mazzoldi, P., Arnold, G., Eds.; Elsevier: Amsterdam, 1987; p 301. (5) Marletta, G. Nucl. Instrum. Methods Phys. Res., Sect. B 1990, 46, 295. (6) Pignataro, S.; Marletta, G. In Metallized Plastics 2 - Fundamentals and Applied Aspects; Mittal, K. L., Ed.; Plenum Press: New York, 1991; p 269. (7) Marletta, G.; Iacona, F. In Materials and Processes for Surface and Interface Engineering; NATO-ASI Series, Series E: Applied Sciences; Pauleau, Y., Ed.; Kluwer Academic Publishers: Dordrecht, 1995; Vol. 290, p 597. (8) Suzuki, Y.; Swapp, C.; Kusakabe, K.; Iwaki, M. Nucl. Instrum. Methods Phys. Res., Sect. B 1990, 46, 354. (9) Suzuki, Y.; Kusakabe, M.; Iwaki, M. Nucl. Instrum. Methods Phys. Res., Sect. B 1993, 80/81, 1067. (10) Giedd, R. E.; Dennis, D. L.; Plumoff, J. A.; Wang, Y. In Materials Modification by Ion Irradiation; Proceedings of SPIE, Vol. 3413; International Society for Optical Engineering: Bellingham, WA, 1999; p 27. (11) Wang, Y. Q.; Giedd, R. E.; Bridwell, L. B. Nucl. Instrum. Methods Phys. Res., Sect. B 1993, 37/38, 659. (12) De Bonis, A.; Bearzotti, A.; Marletta, G. Nucl. Instrum. Methods Phys. Res., Sect. B 1999, 151, 101. (13) Lee, E. H.; Lewis, M. B.; Blau, P. J.; Mansur, L. K. J. Mater. Res. 1991, 6, 610.

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In particular, ion induced modification of polymer surfaces resulted in a significant improvement of biocompatibility in terms of both increased blood compatibility and enhanced cell adhesion and spreading onto the irradiated polymer surfaces.8,9,14-20 All the observed modification effects are basically related to the energy deposition mechanisms and to the total deposited energy, whereas the nature of the implanting ions, if a reactive one is used, plays only a very minor role.5-7 In the recent literature, the beam-induced cell adhesion enhancement has been basically related to two basic observations, that is, the generalized lowering of the water contact angle on the irradiated surfaces and the observed formation of carbon-rich layers at the irradiated polymer surfaces. As to the first observation, we just recall that there is a general consensus about the important role played by the surface wettability, as a key factor to control the cell adhesion process.21-25 As to the second point, recent experiments showed that cell adhesion occurs in a very efficient way on pyrolitic carbon phases26 and it has been observed that quite peculiar amorphous carbon species are formed at the ion fluence corresponding to the observation of enhanced cell adhesion. It should be stressed that these observations are mostly derived from experiments performed on polymeric materials based on carbonaceous backbones, including polystyrene, polyurethane, and polyethersulfone, which under ion irradiation have been shown to evolve mostly toward carbon-rich phases.7,14 At the present stage of knowledge, the understanding of the cell adhesion mechanisms needs clarification of the relative importance of the two factors indicated above. The present paper studies the relationship among beaminduced chemical modifications, as determined in great detail by using X-ray photoelectron spectroscopy (XPS) measurements, the related changes in surface energy, measured by performing contact angle measurements with the three-liquids technique, and the cell adhesion and spreading behavior for polyhydroxymethylsiloxane (PHMS), that is, a polymeric material characterized by a silicon-based backbone. The underlying idea is to operate in conditions in which the formation of amorphous carbon phases is prevented by the specific behavior of this polymer under ion irradiation, which has been shown to evolve mainly toward the formation of SiOx-rich phases,20,27 allowing a test of the connection between beam-induced (14) Suzuki, Y.; Kusakabe, M.; Iwaki, M. Nucl. Instrum. Methods Phys. Res. 1991, B59/60, 1300. (15) Lee, J. S.; Kaibara, M.; Iwaki, M.; Sasabe, H.; Suzuki, Y.; Kusakabe, M. Biomaterials 1993, 14, 958. (16) Lhoest, J. B.; Dewez, J.-L.; Bertrand, P. Nucl. Instrum. Methods Phys. Res., Sect. B 1995, 105, 322. (17) Baca´kova´, L.; Svorcı´k, V.; Rybka, V.; Misek, I.; Hnatowicz, V.; Lisa´, V.; Kocourek, F. Biomaterials 1996, 17, 1121. (18) Nakao, A.; Kaibara, M.; Iwaki, M.; Suzuki, Y.; Kusakabe, M. Surf. Interface Anal. 1996, 24, 252. (19) Pignataro, B.; Conte, E.; Scandurra, A.; Marletta, G. Biomaterials 1997, 18, 1461. (20) Satriano, C.; Marletta, G.; Conte, E. Nucl. Instrum. Methods Phys. Res., Sect. B 1999, 148, 1079. (21) Lee, J. H.; Kim, H. G.; Khang, G. S.; Lee, H. B.; Jhon, M. S. J. Colloid Interface Sci. 1992, 151, 563. (22) Ruardy, T. G.; van der Mei, H. C.; Busscher, H. J.; Schakenraad, J. M. Surf. Sci. Rep. 1997, 29, 1. (23) Garbassi, F.; Morra, M.; Occhiello, E. Polymer Surfaces From Physics to Technology, 1st ed.; Wiley: Chichester, 1994; p 395. (24) Tsuruta, T. Adv. Polym. Sci. 1996, 126, 1. (25) Facchini, P. J.; Neumann, A. W.; Di Cosmo, F. Appl. Microbiol. Biotechnol. 1988, 29, 346. (26) Cenni, E.; Granchi, D.; Arciola, C. R.; Ciapetti, G.; Savarino, L.; Stea, S.; Cavedagna, D.; Di Leo, A.; Pizzoferrato, A. Biomaterials 1995, 16, 1223. (27) To´th, A.; Berto´ti, I.; Marletta, G.; Ferenczy, G. G.; Mohai, M. Nucl. Instrum. Methods Phys. Res., Sect. B 1996, 116, 299.

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chemical modification and surface biocompatibility, in a situation in which an amorphous carbon phase is hardly being formed. Last but not least, although most of the previous experiments have been performed by using relatively high energy ion beams (30-150 keV), to obtain a “surface-confined” modification we have used low-energy ion beams, that is, 5 keV Ar+, typically involving the formation of an altered layer about 10 nm thick.28 Experimental Section Materials. A commercial poly(methyl-siloxane) (Accuglass 512 purchased from Allied Signal, CA), having a mass-averaged molecular weight of 10 000 and a polydispersion index of about 1.1, was used. The solution was electronic grade, that is, with a content of metal impurities C-Si bonds, in agreement with literature data.27 The second peak component (CII) at BE ) 286.6 eV is assigned to >C-OH and >C-O-C groups belonging either to the terminal polymer groups or to the solvent residues. Also, the O 1s peak before irradiation (Figure 3b) is formed by two components; the most intense one (OI), assigned to the Si-bonded oxygen, is centered at BE ) (532.1 ( 0.2) eV, and the other one (OII), resulting from the C-O bonds and/or >OH groups, is centered at BE ) (533.5 ( 0.2) eV. The peak fitting shows that the experimental ratio between the two components OI-OII is ∼10:1, whereas the expected one is 1:1. This observation can be explained by the possible condensation of -OH groups at the polymer surface because of the UHV environment or a very fast modification induced by X-ray irradiation during the acquisition of the spectra. The fact that this effect is confined to the sample surfaces is supported by the Fourier transform infrared results, showing a broad peak at about 3400 cm-1 diagnostic of -OH stretching, mildly shifted by the H-bonding, and two more peaks at ∼1270 and ∼1100 cm-1, assigned respectively to -OH deformation and C-O stretching modes characteristic of aliphatic alcohols.35 The presence of >C-O-C and/or >OH groups is also confirmed by the observation that the CII and OII components have comparable intensities (∼4.0% and 5.0%, respectively), suggesting that the OII component is indeed due to alcohollike groups at the polymer surfaces. Finally, the XPS Si 2p peak before irradiation (Figure 3c) is fitted by using a single Gaussian (SiI) component centered at (102.6 ( 0.2) eV of BE, assigned to Sicontaining groups linked to two effective O atoms, in agreement with the literature.36,37 (35) Lin-Vien, D.; Colthup, N. B.; Fateley, W. G.; Grasselli, J. G. The Handbook of Infrared and Raman Characteristic Frequencies of Organic Molecules; Academic Press: San Diego, CA, 1991. (36) Alfonsetti, R.; Lozzi, L.; Passacantando, M.; Picozzi, P.; Santucci, S. Appl. Surf. Sci. 1993, 70/71, 222. (37) Alfonsetti, R.; De Simone, G.; Lozzi, L.; Passacantando, M.; Picozzi, P.; Santucci, S. Surf. Interface Anal. 1994, 22, 89.

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Table 1. Atomic Composition (Atom %) As Derived from XPS Analysis in Situ and after 48 Hours of Aging in Air of the PHMS Samples Irradiated with 5 keV Ar+ Ions PHMS Unirradiated C 1s (%)

O 1s (%)

Si 2p (%)

22.8a

53.3

23.9

PHMS Irradiated C 1s (%)

O 1s (%)

Si 2p (%)

fluence (cm-2)

in situ

48 h aging

in situ

48 h aging

in situ

48 h aging

F ) 1 × 1013 F ) 1 × 1014 F ) 5 × 1014 F ) 1 × 1015 F ) 5 × 1015

20.7 18.2 14.6 13.4 10.2

23.4 23.9 17.6 15.0 16.6

54.8 56.2 59.6 60.6 60.9

51.9 53.1 59.8 62.2 59.9

24.5 25.6 25.7 26.1 28.9

22.5 23.0 22.6 22.8 23.5

a

The typical error bars for all the values are about (2%.

Figure 5. (a) Si 2p photoelectron peak of PHMS irradiated with Ar+ 5 keV with the fluence of 5 × 1014 ions/cm2. (b) Evolution with the irradiation fluence of the two components of the Si 2p peak.

Figure 4. O/Si and C/Si atomic ratios for PHMS surfaces irradiated with Ar+ 5 keV and analyzed in situ (solid symbols) and after 48 h of aging (open symbols).

Let us discuss now the compositional effects induced by the ion bombardment for samples irradiated and analyzed in situ and after 48 h of aging in atmosphere. The main quantitative compositional effects are reported in Table 1. The data reveal that for the samples analyzed in situ (i.e., without aging) the basic compositional modification involves the drastic decrease of C concentration up to about 50% of the original value, and O and Si increase in a parallel way. At variance with this, the samples analyzed 48 h after irradiation (i.e., aged in atmosphere) show a relatively mild recovery in the carbon concentration, and the oxygen and silicon content seemingly does not undergo a significant variation. In fact, the compositional trends of modifications remain substantially unaltered, as shown in Figure 4, which reports the O/Si and C/Si atomic ratios in situ and after 48 h of aging. We stress the fact that the polymer after irradiation, even upon exposure to atmosphere, is substantially compositionally stable, probably owing to the formation of a thick and highly reticulated SiOxCyHz network preventing drastic rearrangement.

Figure 6. C 1s peaks of PHMS samples: pristine (a) and Ar+bombarded with fluences of 1 × 1014 (b), 5 × 1014 (c), and 5 × 1015 (d) ions/cm2.

Let us discuss in more detail the XPS peak shapes. Ion irradiation induces a small shift of the Si 2p peak position with a negligible peak broadening (full width at halfmaximum is increased by about 0.3 eV). This effect can be accounted for in terms of the formation of a new component, centered at about 103.6 ( 0.2 eV (see Figure 5a). This new component (SiII) becomes predominant with respect to the pristine [SiCO2OH] at the fluence of 5 × 1014 ions/cm2 (Figure 5b). Figure 6 reports the C 1s spectra of PHMS before and after irradiation at increasing ion fluences. One can observe that the main peak at BE ) 284.6 eV remains practically unaffected, whereas the original CII component at 286.6 eV (assigned to solvent residues or to the oxygen-

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containing chain terminal groups) after ion irradiation shifts to a lower BE (∼286.3 eV) and increases in intensity almost linearly with the dose (see Figure 6b-d). This fact can be interpreted by assuming that the irradiationinduced component is due to the formation of C-O-Si moieties because of the effects of recoiling oxygen atom reactions with the backbone C and Si atoms in the ioninduced collision cascade, as demonstrated for ion-induced modification of polyimides and polysulfones.7 According to the described results, the beam-induced modification process can be understood in terms of the elimination of -CH3 groups and the simultaneous formation of new Si-O-Si and Si-O-C interchain bonds, corresponding to the transformation of the original organic material into an inorganic-like one. Contact Angle Measurements. The modification of the surface energy with beam irradiation has been studied by using contact angle measurements. In particular, as a first investigation we employed the sessile drop method38 with deionized water to determine the general behavior of the surfaces with ion irradiation. The measured value of the advancing contact angle for unirradiated PHMS surfaces was θadv ) 90.0° ( 1.5°, and for the receding angle we found θrec ) 77.6° ( 1.5°. The value obtained for θadv can be compared with the value of θ ) 101° for the static contact angle measurement obtained for poly(dimethylsiloxane) (Owens et al.39) and the value of θ ) 98.8° for silicone rubber,40 whereas for typical oxygen-containing polymers such as poly(ethylene terephthalate) and poly(methyl methacrylate) the measured contact angles are of the order of θ ) 80-81°,39 hence remaining in the field of hydrophobic surfaces. To clarify the real chemical structure of the top surface layer, we have performed angular-dependent XPS (ADXPS) experiments, consisting of the variation of the takeoff angle R with respect to the sample normal, in such a way that the sampling depth follows the equation30

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Figure 7. Change in the C 1s, O 1s, and Si 2p atomic percentage by modifying the takeoff angle (R) values.

d ) 3λ cos R These measurements allowed us to obtain the nondestructive compositional profile of the first 6 nm of the PHMS surface. The results are reported in Figure 7. It can be observed that the carbon concentration is highest in the surface-enhanced mode. Indeed, the C concentration in the surface-enhanced mode (R ) 75°, sampling depth ∼ 2 nm) roughly corresponds to a Si1.3CO2.0 composition, whereas in the “bulklike” mode (R ) 0°, sampling depth of ∼ 9 nm) a composition of Si1.5CO2.5 is found. This result supports the picture that the most probable orientation of the -OH groups in PHMS is toward the bulk of the polymer, so that the hydrophobic character of the virgin PHMS surfaces is due to the orientation of -CH3 groups toward the external side. Figure 8 shows the average advancing and receding contact angles for deionized water on the unirradiated and irradiated PHMS thin films at several fluences. It can be seen that the irradiated surfaces undergo a dramatic decrease of θadv and θrec with increasing ion fluence. The change is particularly relevant at the fluence of 5 × 1014 ions/cm2, that is, at the same fluence observed for the onset of massive cell adhesion, where the advancing (38) Garbassi, F.; Morra, M.; Occhiello, E. In Polymer Surfaces From Physics to Technology; Wiley: Chichester, U.K., 1994; p 161. (39) Owens, D. K.; Wendt, R. C. J. Appl. Polym. Sci. 1969, 13, 1741. (40) Suzuki, Y.; Kusakabe, M.; Iwaki, M.; Suzuki, M. Nucl. Instrum. Methods Phys. Res., Sect. B 1988, 32, 120.

Figure 8. Advancing and receding contact angles of deionized water on the unirradiated and irradiated PHMS thin films in the fluence range of 5 × 1013 to 5 × 1015 ions/cm2.

contact angle drops to about 55° and the receding one drops to 43°. At a higher fluence, the θ value remains practically constant. Measurements with AFM (atomic force microscopy) of the irradiated surfaces indicated that the surface roughness does not appreciably change with Ar+ 5 keV bombardment.41 This fact suggests that the irradiation of PHMS with Ar+ 5 keV ions in the range of fluence used is insufficient to produce changes in surface topography which could account for the enhanced wetting performance in the order of magnitude reported here. The nature of the forces effectively acting on the substrate surfaces used for cell-compatibility experiments can be understood by calculating the surface free energies (γStot) and their components from the raw contact angle data by means of the three-liquids technique, interpreting the data in terms of the surface tension component (STC) theory for interfacial tension.42 (41) Marletta, G.; Bertoti, I.; To´th, A.; Tran Minh Duc; Sommers, F.; Ferenc, K. Nucl Instrum. Methods Phys. Res., Sect. B 1998, 141, 684. (42) Lee, L.-H. J. Adhes. Sci. Technol. 1993, 7, 583.

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Table 2. Surface Free Energy Component of the Liquids (in mJ/m2) Based on γwater(+) ) γwater(-) Reference Conventiona

waterb glycerolb TCPc a

γLLW (mJ/m2)

γL(+) (mJ/m2)

γL(-) (mJ/m2)

γLtot (mJ/m2)

21.8 34.0 40.9

25.5 3.92

25.5 57.4

72.8 64.0 40.9

Reference 44. b Reference 42. c Reference 37.

The thermodynamic wettability (or work of adhesion WA) of a solid surface is given by the equation of YoungDupre´:

WA ) γLV(1 + cos θ)

(1)

where γLV is the free energy of the liquid against its saturated vapor, given that

(cos θ)γLV ) γSV - γSL - πe

(2)

where γSV and γSL are the free energy of the solid against its saturated vapor and of the interface between solid and liquid, respectively, and πe is the equilibrium pressure of adsorbed vapor of the liquid on the solid.38 On the other hand, the total surface free energy of the solid (γSV or γStot) can be derived by considering the Lifshitz-van der Waals (LW) and the short acid-base (AB) or donor-acceptor interactions as proposed by van Oss et al.:43

γStot ) γSLW + γSAB

(3)

γSAB ) 2(γS(+)γS(-))1/2

(4)

where γS(+) stands for the electron acceptor (Lewis acid) part of the surface free energy of the solid and γS(-) stands for its electron donor (Lewis base) part. From eqs 3 and 4, Young’s equation in terms of the LW-AB model can now be established:44

(1 + cos θ)γSV ) 2[(γSLWγiLW)1/2 + (γS(+)γi(-))1/2 + (γi(+)γS(-))1/2] (5) By measuring the contact angle of three liquids with known parameters γiLW, γi(+), and γi(-) (see Table 2), we were able to calculate the three unknown parameters γSLW, γS(+), and γS(-). The results of such calculations for the experimental data are shown in Figure 9, where we can observe that both γSLW and γSAB increase with the dose of bombardment, but whereas the dispersive component reaches a plateau above the fluence of 1 × 1014 ions/cm2, the polar component shows an abrupt rise from 1 × 1014 ions/cm2 to 5 × 1014 and 1 × 1015 ions/cm2. This remarkable increase in the polarity of the irradiated surfaces is in nice agreement with all the abovereported data showing that the compositional modification of PHMS basically involves the formation at the surface of Si-O-Si and Si-O-C linkages. Discussion and Conclusions Let us discuss the correlation between the biological effects and the physicochemical modifications induced by ion irradiation. To this purpose, we will compare the real (43) 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. (44) Good, R. J. J. Adhes. Sci. Technol. 1992, 6, 1769.

Figure 9. Modification of the surface free energy components of PHMS with Ar+ irradiation.

onset of the cell adhesion enhancement with the typical fluence dependence of the various specific modifications. Let us first consider the modification of the surface chemical composition, as determined from XPS spectra. The irradiation effects have been seen to involve the formation of an altered layer formed by SiOxCy phases, whose detailed composition and chemical structure depend on the ion dose. In fact, the analysis of the Si 2p XPS peak shows the evolution with increasing irradiation fluence of the Si component assigned to [SiO4] clusters, with respect to the initial ones due to [SiO3-C], which closely follows the C decrease. This beam-induced change in the surface chemical composition obviously corresponds to the strong decrease in the hydrophobic character of the PHMS after irradiation. On the other hand, the above-discussed XPS data strongly support the hypothesis that the increased hydrophilicity of the PHMS after ion irradiation is basically due to the formation of more polar chemical species, as is confirmed by the above-reported calculation of the surface free energy components, showing that the polar acidbase component is mainly responsible for the total surface free energy increase. In Figure 10 a,b are reported the increases in both the [SiO4] component (normalized to the total Si) and cell coverage with respect the contact angle values at increasing irradiation doses. It is evident that both the modifications of the surface chemical composition and the biological response are linearly correlated to the increase of surface energy with ion bombardment. In particular, Figure 10a clearly demonstrates that the decrease in the θ values linearly follows the evolution of the irradiated PHMS surfaces toward the formation of a SiOx-like surface. Indeed, at the highest irradiation fluence reported in this graph (1 × 1015 ions/cm2) we found a [SiO4] content corresponding to 90% of the total silicon at the surface. Accordingly, the related contact angle θ is ∼40°, very close to that measured for pyrolitic SiO2 (θ ) 41° ( 2°). On the other hand, the very good correlation of adhered cells with contact angle decrease, reported in Figure 10b, for both short (2 h) and long (48 h) cell incubation times, suggests that the surface free energy is a critical factor for both cell adhesion and proliferation. To this purpose, it has to be recalled that fibroblast adhesion to surfaces has been shown not to be selective with respect to the

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We can conclude that the fundamental factor inducing the increase of cell adhesion and spreading onto ionirradiated polysiloxane surfaces consists of the formation of a more polar surface, because of its enrichment with the permanent dipoles of the Si-O-Si network, inducing in turn the observed cell adhesion and spreading phenomena. It should be stressed that the real mechanism of the enhanced cell adhesion and spreading on hydrophilic surfaces remains to be clarified, as it is well-known that the cell-surface interaction in the presence of proteins strongly depends on the first step of the protein adsorption, which dramatically affects the subsequent interaction of cells with the adsorbed protein layer. In this context, there is the possibility that a relevant role is played by the presence of residual CxHy groups, which can be thought to act on the protein adsorption process either by prompting the formation of linkage through C-located radical sites or by modifying the surface energy as a result of the irradiation-induced surface segregation of methyl or methylenic groups. In any case, the results presented in this paper support the view that ion irradiation promotes the formation of biocompatible surfaces through the activation of specific chemical groups. The wettability properties are just a consequence of this huge surface chemical modification. Figure 10. [Si(-O)4] content (a) and cell coverage values (b) versus the static contact angle values for unirradiated and irradiated surfaces.

hydrophobic or hydrophilic nature of the involved surfaces.45 Furthermore, it has been also reported that human skin fibroblasts on dichlorodimethylsilane surfaces on glass preferentially adhered to surfaces having a contact angle value of about 50°, while the spreading was increasing with hydrophilicity.46 The results presented in this paper support these previous reports, as the fibroblast adhesion (open symbols in Figure 10b) has a significant increase exactly for the same contact angle values obtained in refs 45 and 46. However, we also find that a significant improvement of the cell spreading, leading to a fully confluent cell layer, is occurring for the same contact angle range. (45) Ruardy, T. G.; Moorlag, H. E.; Schakenraad, J. M.; Van der Mei, H. C.; Busscher, H. J. J. Colloid Interface Sci. 1997, 188, 209. (46) Ruardy, T. G.; Schakenraad, J. M.; Van der Mei, H. C.; Busscher, H. J. J. Biomed. Mater. Res. 1995, 29, 1415.

It is clear that to fully understand the mechanism for the modification of cell-surface interaction resulting from irradiation effects, the research effort has to be directed to the effect of ion-induced surface modification on the protein adsorption process. Such a further research line is currently in progress in our laboratory. Acknowledgment. The authors are indebted to Dr. F. Sinatra (Istituto di Biologia Generale, Universita` di Catania) for the electron microscopy photos. The Italian Ministry for University and Technological Research (MURST-Rome) is acknowledged for a partial grant for the research under the National Program “Cofinanziamento 1997” (Project on “Crescita, struttura e reattivita` di superfici di materiali e di film superficiali”). The Targeted Project “Special Materials for Innovative Technologies II” of CNR-Rome is also gratefully acknowledged for financial support. LA001321R