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Langmuir 1999, 15, 4658-4663
Surface Studies on a Model Cell-Resistant System Marco Morra* and Clara Cassinelli Nobil Bio Ricerche, Str. S. Rocco 32, 14018 Villafranca d’Asti, Italy Received September 29, 1998. In Final Form: March 17, 1999 A model system involving a polyanionic alginic acid (AA) overlayer ionically immobilized on a polycationic polyethyleneimine underlayer, in turn linked to plasma oxidized polystyrene, was used to investigate the relationship between surface structure properties and resistance to cell adhesion. X-ray Photoelectron Spectroscopy (XPS) and water contact angle measurement were used to characterize the surfaces and to compare the results with data on resistance to adhesion of L-929 fibroblasts. A overlayer model was used to account for the XPS results in terms of fractional coverage by AA. Results show the strong effect of the surface density of AA chains on resistance to cell adhesion and underline the inadequacy of simple relationships between wettability by water and cell adhesion.
Introduction Cell-resistant surfaces, that is, surfaces that can resist cell adhesion, are of great interest in biomaterials science in general and tissue engineering in particular. The reason of this interest is 2-fold: from one side, it arises from the need to reduce and minimize the interaction between biological and synthetic phases in selected in vitro and in vivo applications.1 At a more fundamental level, it stems from the surface engineers quest to discover and master the basic working tools needed to design surfaces that behave like natural ones. Most biological structures can resist nonspecific interactions with surrounding molecules and biological species and, at the same time, they can establish very specific interactions with selected moieties.2 To be able to engineer synthetic surfaces endowed with specific molecular recognition properties the accomplishment of the first task, i.e., the control and prevention of nonspecific interactions, is mandatory. Which are the rules that can be used to engineer cellresistant surfaces? A poly(hydroxyethyl methacrylate) (PHEMA) hydrogel coating applied from alcoholic solutions is the most common tool used in routine cell culturing to prevent adhesion of cells to tissue culture plasticware.3 It is generally believed that acrylate hydrogel surfaces, either obtained by simple coating from solution or by grafting,4 resist cell adhesion in vitro because of their high water content and weak mechanical properties, do not allow “grip” of adhering cells to the substrate.3,5,6 Another popular class of cell-resistant surfaces exploits the unique properties of poly(ethylene oxide) (PEO). Steric or entropy loss effects of hydrated, freely fluctuating PEO chains are the physicochemical properties that are most frequently invoked to account for the properties of PEO coated surfaces7-11 even if results obtained by Lopez and * To whom correspondence may be addressed. E-mail: mmorra@ tin.it. (1) Vacanti, C. A. and Mikos, A. G., Tissue Eng. 1995, 1, 3. (2) Ratner, B. D. J. Biomed. Mater. Res. 1993, 27, 837. (3) Horbett, T. A.; Klumb, L. A. In Interfacial Phenomena and Bioproducts; Brash, J. L., Wojciechowski, P. W., Eds; Marcel Dekker: New York, 1996; p 351. (4) Tamada, Y.; Ikada, Y. J. Biomed. Mater. Res. 1994, 28, 783. (5) Lydon, M. J.; Minett, T. W.; Tighe, B. J. Biomaterials 1985, 6, 396. (6) Rollason, G.; Davies, J. E.; Sefton, M. V. Biomaterials 1993, 14, 153. (7) Desai, N. P.; Hubbell, J. A. J. Biomed. Mater. Res. 1991, 25, 829. (8) Nagaoka, S.; Mori., Y.; Tanzawa, H.; Kikuchi, Y.; Inagaki, F.; Yokota, Y.; Noishiki, Y. ASAIO J. 1987, 10, 76.
co-workers12 on plasma deposited PEO-like films and by Prime and Whiteside13 on self-assembled monolayers of short chains PEO have challenged this view. A further class of protein- and cell-resistant surfaces of great relevance for fundamental studies and practical applications is based on hydrophilic polysaccharides coatings.14-17 Contrary to the soft, slippery hydrogel-coated surfaces mentioned before, the thickness of the surface cell-resistant layer is of the order of nanometers14,15 or of a mono-(macro)molecular layer, while in the case of the quoted acrylate hydrogels, either obtained by coating from solution or grafting, the thickness is, at the very least, several hundred nanometers. Contrary to PEO surfaces there is no special need to resort to ingenious ways18,19 to achieve a high density of end-point-attached, freely fluctuating chains in order to obtain resistance to adhesion. Actually, in a comparison of terminally attached (or endon) versus multi-point attached (or side-on) dextran it was found that the latter configuration yielded the best resistance to protein adsorption.14 The intrinsic high hydrophilicity of these natural macromolecules, as well as steric and excluded volume effects are generally invoked to account for the properties of these surfaces,14 but a complete understanding of the surface structure-properties relationship in these systems is still lacking. The aim of this work is to try to gain further insights on the mechanisms that control the cell-resistance of polysaccharide coated surfaces. The inherently complex (9) Szleifer, I. Phys. A 1997, 244, 370. (10) Jeon, S. J.; Lee, J. H.; Andrade, J. D.; de Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149. (11) Jeon, S. J.; Andrade, J. D. J. Colloid Interface Sci. 1991, 142, 159. (12) Lopez, G. P.; Ratner, B. D.; Tidwell, C. D.; Haycox, C. L.; Rapoza, R. J.; Horbett, T. A. J. Biomed. Mater. Res. 1992, 26, 415. (13) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 23, 10714. (14) Osterberg, E.; Bergstrom, K.; Holmberg, K.; Schuman, T. P.; Riggs, J. A.; Burns, N. L.; Van Alstine, J. M.; Harris, J. M. J. Biomed. Mater. Res. 1995, 29, 741. (15) Brink, C.; Osterberg, E.; Holmberg, K.; Tiberg, F. Colloids Surf. 1992, 66, 149. (16) Morra, M.; Cassinelli, C.; Benedetti, L.; Callegaro, L.; Ambrosio, L.; Nicolais, L. Trans. 5th World Biomed. Congr. 1996, 171. (17) Pavesio, A.; Renier, D.; Cassinelli, C.; Morra, M. Med. Dev. Technol. 1997, 8, 20. (18) Golander, G. C.; Herron, J. N.; Lim., K.; Claesson, P.; Stenius, P.; Andrade, J. D. In Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications; Harris, J. M., Ed.; Plenum Press: New York, 1992; p 221. (19) Irvine, D. J.; Mayes, A. M.; Satija, S. K.; Barker, J. G.; SofiaAllgor, S. J.; Griffith, L. G. J. Biomed. Mater. Res. 1998, 40, 498.
10.1021/la981345m CCC: $18.00 © 1999 American Chemical Society Published on Web 05/15/1999
Model Cell-Resistant System
Langmuir, Vol. 15, No. 13, 1999 4659 Table 1. Surface Composition (% atom) Detected by XPS of the Samples sample
C
O
N
OPS PEIPS 0.0001AA 0.0005AA 0.05AA 0.5AA 1AA
84.0 75.0 70.9 67.9 66.7 63.2 60.5
16.0 15.3 19.6 23.7 25.4 30.7 34.5
9.7 9.0 8.4 7.9 6.1 4.8
a Untreated PS: 100% C. 0.05).
Figure 3. Plot of the calculated O/N ratio as a function of the AA fractional coverage γ. The intercept of the experimental values with the theoretical curve allows to account for the experimental data in terms of γ. The typical error bar is shown in one case.
where γ is the fraction of surface coverage (γ ) 1 continuous layer), t is the thickness of the polysaccharide layer, and COPS, CPEI, and Cpolysac are the carbon concentration in the oxidized PS, PEI, and polysaccharide layer. As a first approximation, a common l value for the three elements involved in this model was used. Because of the sampleelectron analyzer angle (as described in the Experimental Section) and of indications obtained from the measurement of samples with controlled surface overlayers, the value l ) 2.5 nm was used for calculations. Table 2 shows the surface composition data calculated from eq 2 as a function of the surface fractional coverage of the AA. It can be appreciated that, despite the oversimplifaction of the model and the assumptions made, for instance, the boundary between layers is not as sharp as shown in Figure 2 and the overall structure is probably much more disordered, both the qualitative trend of the O/C and N/C ratio and the quantitative values are in good agreement with the experimental data of Table 1. This allows us to plot the calculated O/N ratio as a function of the fractional coverage γ and, from the intercept of the experimental O/N ratio with the calculated curve, to express the XPS data of Table 1 in terms of the AA fractional coverage (Figure 3). Contact Angle Measurement. Results of water contact angle measurements are shown in Table 3. Only advancing angles are reported, the receding angle was
Table 4. Results of Cell Adhesion Measurements sample
cells/cm2 × 104
OPS PEIPS 0.0001AA 0.0005AA 0.05AA 0.5AA 1AA
33.0 ( 7.2a 37.1 ( 7.3a 30.4 ( 8.3a 26.3 ( 7.7a 8.2 ( 1.1b 3.4 ( 1.2c 0.2 ( 0.1d
Note: Differences between values characterized by different letters are statistically significant (p > 0.05).
always zero. All tested surfaces are in the hydrophilic portion of the wettability spectrum. AA coupling to a PEIPS surface leads to a decrease of the advancing water contact angle. The increase of the surface density of AA chains is apparently reflected in a further increase of wettability, as evaluated by the average advancing contact angle value, but differences among AA-coated samples are not statistically significant, as described in Table 3. L-929 Cell Adhesion to Substrate Surfaces. Results of cell adhesion experiments are reported in Table 4 and Figure 4. Cell-counting data, obtained as described in the Experimental Section, evaluation n. 1, are reported in Table 4. Data show that the number of cells on the surface decreases with increasing fractional coverage of AA. A nearly complete resistance to cell adhesion is obtained in the case of the 1AA surface (see the comments on the 1AA surface at the end of this section). The quantitative data of cell counting are clearly supported by SEM images of Figure 4. These figures show the density and morphology of cells after 3 days culturing on several of the tested surfaces. In the case of OPS (see Figure 4a. A similar result, not shown, was obtained with PEIPS.) a confluent monolayer of cells is observed. It is possible to see that cells are flat and well spread. Actually, plasma treatment of PS Petri dishes to produce so-called tissue culture polystyrene (TCPS) is well-known and commercially exploited since several decades.3 As soon as the surface density of AA increases, the density of attached cells decreases, in agreement with data of Table 4. Also cell morphology is affected and cells becomes more rounded. Finally, in the case of 1AA (Figure 4d), most of the surface is absolutely bare. Occasionally, some cluster of cells was observed in selected spots of the 1AA dishes. The very localized nature of this interaction suggests that it could be due to some spotty contamination of the AA surface or, more in general, to some local heterogenety or defect in the AA overlayer. The adventitious nature of this cell colonization is reflected in the considerable standard deviation obtained in this case (see Table 4). Discussion A model system was used to inquire about the surface structure-properties relationship of a scientifically and commercially important class of coatings. The system involves a polyanionic polysaccharide layer over a polycationic synthetic intermediate layer. The relationship between cell resistance in vitro and surface parameters was studied using two common chemico-physical surface characterization techniques (XPS and water contact angle). The long-term stability of the system under study and its dependence on the composition of the medium were not evaluated. The design of polysaccharide coated surfaces intended for long-term or in vivo applications actually involves some kind of covalent linking between the polysaccharide and the substrate surface.16,17,20 In the present experiments, a 3 day in vitro testing of cell
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Figure 4. SEM images showing the results of 3 days cell adhesion testing on (a) OPS (a identical result was obtained for PEIPS), (b) 0.05AA, (c) 0.5AA, (d) 1AA.
adhesion, this was purposely avoided in order to keep the surface structure as simple as possible. The resulting system is stable for the time span of the present experiments in the cell culture medium, as evaluated by XPS measurements and confirmed by the cell adhesion results. It must be stressed that cell adhesion is a complex phenomenon, mediated by the adsorption, from the culture medium, of cell-adhesive proteins.3 While, in this study, a “macroscopic” indicator, that is, the “number of adhered cells” was used to describe the outcome of the protein containing medium-cells-surface interaction, it is important to remember that this is actually a consequence of molecular events involving protein-surface interactions. In this respect, polysaccharide-coated surfaces have been shown to be protein resistant in conventional proteinadsorption experiments.14 Results shown in Table 3 and Figures 3 and 4 suggest some reflection, as follows. First of all, the surface density or surface fractional coverage of the hydrophilic polysaccharide, as captured by XPS data treated according to the overlayer model described in the Result section and in ref 23, seems directly related to resistance to cell adhesion. From a practical point of view this indicates that while XPS data of Tables 1 and 2 allow us to call all of the AA surfaces of Figure 3 “alginate-coated surfaces”, only 1AA is an “alginate-coated, cell resistant surface”. The successful immobilization of a molecule is not enough to
impart to the substrate surface all the properties expected from the molecular structure of the coating molecule, a more detailed tuning of the property-controlling-parameter(s) is mandatory. This is well-known in applications involving specific interactions, where the control of the structure of the immobilized molecule is a key issue, as nicely shown by heparin-coated surfaces.27 The present results indicate the importance of these issues in the prevention of nonspecific interactions and have obvious implications for process design and quality control. From a more basic point of view, it is interesting to elaborate on the structure-properties relationship underlined by the dependence of resistance to cell adhesion on the AA fractional coverage. AA is a polyanion, and the build up of a negative surface charge could cause an electrostatic repulsion between the surface and the negatively charged cells. However, cells adhere as well to the positively charged PEIPS as to the negatively charged OPS. Cell-resistant behavior is observed on a wide range of covalently linked hydrophilic polysaccharides, independent of the number of anionic groups along their chain, even when most carboxyl groups are consumed in the formation of covalent bonding with the substrate func(27) West, R. H.; Paul, A. J.; Hibbert, S.; Cahalan, P.; Cahalan, L.; Verhoeven, M.; Hendriks, M.; Fouache, B. J. Mater. Sci. Mater. Med. 1995, 6, 63.
Model Cell-Resistant System
tional groups.17 Moreover, due to the high density of amino groups introduced by PEI, most AA anionic sites are charge neutralized in ionic bonding. Steric repulsion effects are frequently invoked to account for the cell-resistant properties of PEO coated surfaces.9-11 In the present case, due (i) to the nature of the interfacial interaction leading to the surface immobilization of AA, that is, charge-to-charge attraction between the straight chain, high M, polyanionic AA, and polycationic PEI, and (ii) to the charge-to-charge repulsion between AA chains, it is highly unlikely that the AA chains will adopt the brushlike regime of freely fluctuating chains so pervasively pursued in the PEO way to cell resistance. In our view, the present results are closely reminiscent of those obtained by Prime and Whitesides13 on the adsorption of proteins onto surfaces containing endattached oligo(ethylene oxide) even if, in that paper, the use of the self-assembled-monolayers (SAM) technique allowed them to prepare much more ordered and welldefined surface structures. It was observed that very short chains containing the repeating unit of ethylene oxide (EO) can effectively resist protein adsorption, contrary to the generally accepted claims on the need for immobilization of long chains PEO molecules. Prime and Whitesides reported that the self-assembly process can prepare films with much greater numbers of EO chains per unit area than most chemical grafting methods. Accordingly, the principal criterion for protein resistance was the complete coverage by an EO film, i.e., the building up of a “shield” of EO units above the substrate surface. In the present case, increasing fractional coverage by AA chains results in the building up of a mannuronate-unit shield over the underlying substrate. The achievement of a high density of saccharide units is prompted by (i) the high number of amino groups introduced by PEI adsorption,26 (ii) the high frequency of anionic binding groups along the AA chains, and (iii) the cooperative mode of multipoint PEI-polysaccharide chains binding, as opposed to the competitive endpoint attachment of macromolecules and ensuing steric limitations effects.19 In our view, the saccharide shield of the present experiments (as the EO monolayers of Prime and Whitesides13 and the plasma-deposited PEO-like coatings of Lopez and co-workers12) exerts its cell-resistant effect through hydration forces, i.e., by the engagement of strong interactions with interfacial water molecules that compete with and prevent the adsorption of cell-adhesive proteins to the surface (this sentence is presented here as a hypothesis, it is presently being pursued in ongoing dedicated studies). Here we just remind that short-range hydration forces extending about 1 nm have been measured between stiff polysaccharides using the osmotic stress technique28 and that the conformational behavior of carbohydrates in aqueous solutions is deeply affected (28) Rau, D. C.; Parsegian, V. A. Science 1990, 249, 1278.
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by interactions with water molecules.29 The hypothesis on the nature of forces involved in the cell-resistant behavior of these systems allows one to introduce the last point of this paper, that is, the failure of the advancing water contact angle as a physicochemical descriptor of resistance to cell adhesion. Data show that the advancing water contact angle was unable to detect the different character of, for instance, 0.05AA or 0.5AA, which is not cell-resistant, and 1AA. In this respect it must be noted that, even if some relationship has been claimed between wettabilty by water and cell adhesion,3,4,30 this should only be considered as a first order approximation. Just to quote an example, the 46° water contact angle measured on OPS is the typical value of plasma treated and aged PS. If a freshly plasma treated PS is used, whose advancing water contact angle can be as low as 10° (or a freshly plasma-cleaned glass slide, whose contact angle is zero), the same cell adhesive behavior displayed by the present OPS is observed, showing the inconsistency of any simple relationship between the water contact angle and cell adhesion. This is not unexpected. Besseling31 has recently proposed an improved theoretical description of hydration forces. Van Oss32 calculated force-distance curves for interactions in aqueous media, showing the effect of short ranges hydration forces over and above the conventional DLVO curve. In both approaches the physicochemical mechanism at work is hydrogen bonding, prompted by the unique orientation dependent properties of water molecules, and the critical physicochemical surface parameters are the electron acceptor-electron donor or, Lewis acid-base, properties of the phases involved. The water contact angle alone cannot give a satisfactory description of these properties and, as a consequence, cannot be used as a reliable experimental variable. A more satisfactory analytical approach should involve the measurement of the Lewis acid-base properties of the AA surfaces by the Van Oss method,32 but this procedure is presently hindered by the well-known limitations of the quantitative aspects of this theory.33-35 The ongoing debate on this subject, and the new and improved method that are presently actively pursued in many laboratories,35,36 is expected to be of much help in the understanding and analytical description of these systems. LA981345M (29) Brady, J. W.; Schmidt, R. K. J. Phys. Chem. 1993, 97, 958. (30) Johnson, S. D.; Anderson, J. M.; Marchant, R. E. J. Biomed. Mater. Res. 1992, 26, 915. (31) Besseling, N. A. M. Langmuir 1997, 13, 2113. (32) Van Oss, C. J. Interfacial Forces in Aqueous Media; Dekker: New York, 1994. (33) Morra, M. J. Colloid Interface Sci. 1996, 182, 312. (34) Morra, M.; Cassinelli, C. J. Biomed. Sci., Polym. Ed. 1997, 9, 55. (35) Della Volpe, C.; Siboni, S. J. Colloid Interface Sci. 1997, 195, 121. (36) Lee, L. H. Langmuir 1996, 12, 1681.
