Langmuir 1995,11, 4135-4140
4135
Cellular Responses on a Wettability Gradient Surface with Continuous Variations in Surface Compositions of Carbonate and Hydroxyl Groups Tomoko Ueda-Yukoshi and Takehisa Matsuda" Department of Bioengineering, National Cardiovascular Center Research Institute, 5-7-1Fujishirodai, Suita, Osaka 565, Japan Received December 9, 1994. In Final Form: June 30, 1995@ This article reports adhesion, spreading, migration, and proliferation responses of bovine endothelial cells (ECs) on the surface with a unidirectional wettability gradient. The surface chemical gradient, in which molar fractions of cyclic carbonate and hydroxyl groups were inversely varied, was prepared by continuousimmersionof a poly(viny1ene carbonate)(PVCa)film into an aqueous solutionof sodiumhydroxide at a constant speed. Advancing water contact angles of the surface,which ranged from 60"for an untreated PVCa surface to around 20" for a well-treated surface, gradually decreased with the distance from the untreated end of the film. Quantitative analyses of cellular characteristics of adhered cells on the gradient surface, which included the initial adhesion rate and the morphological indices of adhered cells such as spreading area, peripheral length, and circular coefficient, showed that these cells gave the highest values on the untreated PVCa surface and rapidly decreased on the surface regions with shorter periods of hydrolysis,followed by a slower decrease on surface regions with longer periods of hydrolysiswith increasing distance from the untreated end. Little adhesion occurred in a well-hydrolyzed,very hydrophilic surface region. The highest migration rate of adhered cells occurred on the slightly hydrophobic surface region (advancing contact angle, 35")where adhered cells tended to delaminate as tissue formation proceeded. These results indicate that each cellular potential of ECs is dependent on the wettability of the gradient surface, producing polymorphorous states of adhered cells. Thus, the wettability gradient surface served as an excellent substrate to simultaneously study cellular response dependence on surface chemical composition and wettability.
Introduction Considerable importance has been given to the understanding of cell-material interactions which provide the fundamental basis of surface design of biocompatible artificial organs. When sequential cellular events such as adhesion, spreading, migration, and proliferation occur well on artificial substrates, a tissue will be formed on a material surface in vitro as well as in vivo. When cellular adhesion is greatly suppressed, such a nonadherent material typified by a very hydrophilic surface will be suited for extracorporeal devices subject to short-term contact with blood. These cellular potentials largely depend on the nature and strength of cell-material interactions, which are determined by the surface nature and composition of the outermost layers of a mate~ial.l-~ Therefore, the surface control of these cellular potentials is crucial to tissue-compatible and blood-compatible surface designs. For example, in the fabrication of hybrid blood vessels, of which inner surfaces were completely covered by monolayered endothelial cells (ECs) as was found for a luminal surface of natural vessels, high adhesion, migration, and proliferation potentials of ECs on a luminal surface of artificial prostheses are required. In general, the cellular behavior dependence on material surfaces has been studied on individual samples, which is time and labor consuming. There has been considerable interest in the preparation of surfaces whose properties
* To whom correspondence should be addressed. Telephone: 816-833-5012.Fax: 81-6-872-7485. Abstract published in Advance A C S Abstracts, September 15, 1995. (1)Andrade, J. D. In Surface and interfacial aspects of biomedical polymers; Andrade, J. D., Ed.; Plenum Press: New York, 1985;Vol. 1, P 1. (2) Ratner, B. D. J . Biomed. Mater. Sci. 1993,27,837. (3)Horbett, T.A.; Brash, J. L. In Proteins ut biochemical physicochemical and biochemical studies; Horbett, T.A., Brash, J. L., Eds.; ACS SymposiumSeries 343;American Chemical Society: Washington, DC, 1987;p 1. @
and chemical compositions change continuously in one dimension (gradient surface) in conjunction with biological responses at interfaces since a continuum of a selected and controlled physicochemical property can be studied in one experiment on one ~ u r f a c e . ~In- ~ addition to protein adsorption characteristics on gradient surfaces, cell adhesion response was briefly r e p ~ r t e d . ~ Our previous study showed how a poly(viny1ene carbonate) (PVCa) surface was hydrolyzed to produce poly(hydroxymethylene) (PHM) in an alkaline solution as shown in Figure 1.l0 The untreated polymer surface is relatively hydrophobic and polar due to a cyclic carbonate linkage, and the PHM surface in which one hydroxyl group is attached to the repeatingmethine group of a main chain has been characterized as the polymer with the highest hydroxyl content per repeating monomer unit among existing vinyl-type polymers. Both polymers were insoluble in water. Extensive intermolecular and intramolecular hydrogen bonds could be responsible for the insolubility of PHM in water. The extent of surface hydroxylation depended on the experimental variations such as concentration ofhydroxide ion, reaction time, and temperature. Therefore, at an intermediate degree of hydrolysis, cyclic carbonate and hydroxyl groups coexist on surfaces, and their populations were inversely changed as the hydrolysis proceeded further. In this study, we prepared a wettability gradient surface in which the surface carbonate group and hydroxyl group are inversely populated along one direction. The cellular (4)Elwing, H.; Welin, S.; Askendal, A.; Nilsson, U.; Lundstrom, I. J . Colloid Interface Sci. 1987,119,203. (5) Elwing, H.; Askendal, A.; Lundstrom, I. J . Biomed. Mater. Res. 1987,21,1023. ( 6 )Hlady, V.; Golander, C.; Andrade, J. D. Colloids Sure 1987,25, 185. (7)Golander, C.-G.;Pitt, W. G. Biomaterials 1990,11, 32. (8) Lin, Y. S.; Hlady, V.; Janatova, J. Biomaterials 1992,13,497. (9)Lee, J. H.; Lee, H. B. J . Biontater. Sci., Polym. Ed. 1993,4,467. (10)Kishida, A,; Matsuda, T.Polym. Prep., Jpn. 1991,40, 1442.
0743-746319512411-4135$09.0010 0 1995 American Chemical Society
Ueda-Yukoshi and Matsuda
4136 Langmuir, Vol. 11, No. 10, 1995 50 n
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characteristics of bovine ECs, such as adhesion, spreading, migration, and proliferation on the wettability gradient surface thus prepared, were quantitatively studied. Such a study is expected to provide us with fundamental information on cell-material interactions at the same time on the same sample.
Materials and Methods Materials. Vinylene carbonate was obtained from Tokyo Kasei Organic Chemicals Co., Ltd. (Tokyo, Japan). NJVDimethylformamide(DMF),2,2'-azobisisobutyronitrile (AIBN), and the aqueous solution of sodium hydroxide (NaOH, 1.0 x M) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Vinylene carbonate and DMF were purified by distillation before use. AIBN was used without further purification. Glass plates (24 x 60 x 0.15 mm3)obtained from Matsunami Co., Ltd. (Tokyo, Japan) were washed with ethanol under sonification for 5 min and dried in air before use. Polymerization of Vinylene Carbonate. A mixture of vinylene carbonate (11g, 0.13 mol) and AIBN (34 mg, 0.21 mmol; [monomerl/[initiatorl = 6.2 x lo2) was sealed under reduced pressure after three freeze-thaw cycles, and the polymerization was carried out with shaking a t 60 "C for 14 h. The reaction mixture was dissolved in DMF, and the solution was poured into a large amount of methanol. PVCa was separated, reprecipitated, and then dried in UUCUO. The yield was 3.1 g (28%). Preparation of a Wettability Gradient Surface. A DMF solution of PVCa (2 wt %) was cast onto a glass plate and airdried at 70 "C. After drying a t room temperature in uucuo, the polymer-castglass plate was gradually immersed into 1.0 x M NaOH aqueous solution with a speed of 1.0 or 1.4 mm/min for 30 min using a custom-designed apparatus installed with a stepping motor and a control unit (Figure 2). The treated plate was rinsed three times with distilled water and dried in uucuo. Surface Analysis. Surface chemical composition of the outermost layers was analyzed by an X-ray photoelectron spectrometer (XPS,ESCA-750, Shimadzu Co., Kyoto, Japan). The X-ray source was MgKa radiation. The photoelectron takeoff angle was 90". The spectral data were collected using ESCAPAC 760 (Shimadzu Co.) and analyzed by a computer-
10 20 Hydrolysis time (min)
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Figure 3. Hydrolysis time dependence of the surface composition. (Abbreviation used is the same as in Figure 1; surface M NaOH aqueous solution; sample hydrolyzed in 1.0 x number n z 3.) aided curve-deconvolution method. All binding energies were referenced to the carbon 1s component set to 285 eV. Wettability of the films was evaluated by water contact angle measurement using a sessile drop technique with a contact angle goniometer (CA-D, Kyowa Kaimenkagaku Co., Ltd., Tokyo, Japan). QuantitativeAnalysis of CellularBehavior. BovineECs, harvested from bovine aorta, were cultured in Dulbecco's modified Eagle's medium (DMEM; purchased from Flow Laboratories, Irvine, Scotland, UK) supplemented with 15%fetal calf serum (purchased from Gibco Laboratories, Grand Island, NY)at 37 "C in 95%air and 5% C02. All experiments were performed between the 8th and 13thpassages. The hydrolyzedfilms, sterilized with 70% aqueous ethanol solution for 10 min, were preincubated in phosphate-buffered saline (PBS) before cell culture. ECs were seeded onto the film at a density of 2 x lo3-5 x lo4 cells/cm2. After incubation for 3 h, nonadherent cells were removed by a gentle wash with PBS, and morphology of the adhered cells was photographed with a phase-contrast microscope (DIAPHOT, Nikon, ,Tokyo, Japan) using Polaroid 667 films (Polaroid, Cambridge, MA) with a Nikon camera (HFX) connected to a microscope. The number of adherent ECs per unit area (0.25 mm2),average area (S)of adherent ECs, peripheral length (L), and circular coefficient(definedasL2/4nS)were determined from the phase-contrast microscopic images by a computerized imageanalyzing processor (Model LA-500, PIAS Co., Ltd., Osaka, Japan).ll Cell proliferation on the treated surface was expressed in terms of the number of cells attached per unit area. The migratory tracts of cells (2 x lo3-4 x lo3cells/cm2)cultured for 1day on the surface were recorded every 10 min for 2 h with a time-lapsed computerized video microscope. The migration rate was calculated from the average distance migrated every 10min. Detailed procedures for the quantitative analysis of cellular behavior were described in our previous paper. l2
Results The gradient surface, the wettability of which was gradually changed along the sample length direction, was prepared by the hydrolysis of PVCa film upon gradual immersion into a NaOH aqueous solution. The preparation of the gradient surface was verified by XPS and water contact angle measurements. The behaviors of adhered cells of the gradient surface were investigated with phasecontrast microscopy. Preparation and Characterization of the Wettability Gradient Surface. The hydrolyzed PVCa film was quantitatively analyzed by XPS,which enables us to determine the chemical composition up to several tenths of an angstrom in depth. Figure 3 shows the relationship between hydrolysis time and the mole fraction of PHM of (11)Takatsuka, M.; Kishida, A.; Matsuda, T. Trans.Am. SOC.Artif Intern. Organs 1992,38,M275. (12)Niu, S.; Matsuda, T. Cell Transplant. 1992,1 , 355. (13)Matsuda, T.;Kurumatani, H. Trans. Am. SOC.Artif Intern. Organs 1990,36,M565.
Langmuir, Vol. 11, No. 10, 1995 4137
Cellular Responses on a Wettability Gradient Surface
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the polymer surface. Upon immersion into 1.0 x low3M NaOH aqueous solution, the mole fraction of PHM, calculated from the relative ratio of C1, fraction assigned to the carbonate carbon (-OC(=O)O-) to that of hydroxymethyl carbon (>CH-OH), sharply increased within 10 min of hydrolysis, followed by a slow increase. A 24 h hydrolysis gave around 90%PHM on the surface (data not shown). Concomitantly,water contact angle decreased with hydrolysis time. PVCa films cast on glass plates were immersed into a NaOH aqueous solution at a constant speed (1.4 m d m i n ) using a custom-made apparatus. In Figure 4, an untreated virgin surface was relatively hydrophobic (advancing contact angle, 60"),while a 30 min treated region provided a quite hydrophilic surface (20"). The water contact angle-distance relationship of the treated surface showed that (1)both advancingand receding contact angles of the treated films gradually decreased with increasing distance from the untreated end of the films, (2) rapid decreases in both contact angles were noted a t surface regions with shorter periods of contact, and (3)receding contact angles were always smaller than advancing contact angles, and this difference was more significant for shorter periods of contact or shorter distance from the untreated end. This indicates that the hydrophilicity of the treated surface gradually increased with increasing distance from the untreated end of the film. This gradient surface thus prepared was subjected to cell culture as described below. Cell Adhesion and Spreading on the Gradient Surface. Cellular behavior, including adhesion, spreading, and growth, was examined on the gradient surface. ECs, a t 3 h of incubation after seeding on the gradient surface, adhered well on the untreated PVCa surface region. On the other hand, as shown in Figure 5, the number of adhered cells per unit square area (0.25 mm2) decreased with an increase in the distance from the untreated end of the film (note that the ending position calculated from the immersion speed and hydrolysis time was defined as the distance "zero"). A markedly reduced cell density was observed for surface regions with longer periods of contact. Apparently, little cell adhesion occurred on the well-treated or hydrophilic surface regions. Figure 6 shows the phase-contrast micrographs of ECs adhered on the gradient surface. Cells were observed to spread more on the hydrophobic surface than on the hydrophilic one.
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Figure 6. Phase-contrast micrographs of endothelial cells adhered on the wettability gradient surface after 3 h of incubation at 37 "C. (Number of seeded cells, 4.8 x 104/cm2. The distance and the value in parentheses represent the position of the film and advancing contact angle on the surface, respectively.)
Quantitative morphological analyses of cells adhered on the gradient surface a t 3 h of incubation after seeding were performed using computer-assisted morphological measurements,which included the average spreading area (S),peripheral length (L), and circular coefficient (defined asL2/4nS). These were plotted against the position of the film in Figure 7. All three morphological indices were found to have the highest values on the untreated PVCa surface and to rapidly decrease on the surface regions with shorter periods of hydrolysis, followed by a slower decrease with an increase in the distance from the untreated end or an increase in hydrophilicity. In principle, when the circular coefficient of a cell approaches
Ueda-Yukoshi and Matsuda
4138 Langmuir, Vol. 11, No. 10, 1995 1.5
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1, the shape of the cell approaches that of a circle; in contrast, a larger coefficient means a more elongated cell shape. 'The circular coefficient of adhered cells was 3.4 on the untreated surface but decreased in the more wettable surface regions: that of adhered cells on the surface for which distance was over 20 mm (advancing contact angle, 25")was found to be almost 1. Thus, these cells on the surface subjected to longer periods ofhydrolysis were almost completely circular. From these results, ECs adhered on the hydrophobic surface region were more elongated than those on the hydrophilic surface region of the gradient surface. Cell Migration, Growth, and Tissue Formation. After ECs seeded on the films with different degrees of hydrolysis were cultured for 1 day, phase-contrast microscopic images of adhered cells were recorded on a video recorder every 10 min. The migration rates were determined by computerized image analysis after digitizing the nucleus of adhered cells on every image. Figure 8 shows the cellular migration rate-wettability relationship thus obtained. The maximum migration rate appeared at around the surface with an advancing contact angle of 35". This value was almost 1.5 times higher than those
on less wettable surfaces with contact angles rangingfrom 45 to 60"and almost 1.7 times higher than that on a more wettable surface with a contact angle of 25". Cell culture on the gradient surface was continued up to 46 h after seeding, and a time-dependent cellular population change on the gradient surface is shown in Figure 9 (note that a more gentle washing was performed after 3 h of incubation, compared to that in Figure 5). Regardless of the culture time, the cells proliferated well on the surface regions with distances ranging from -30 to 5 mm. However, the proliferation of the cells on the surface with the distance exceeding 5 mm was markedly suppressed. This was more profound at about 2 days of incubation after seeding. Figure 10 shows the successively arranged phasecontrast micrographs of ECs that proliferated on the gradient surface after 3 days of incubation. ECs on the untreated PVCa surface formed a confluent monolayer that exhibited the characteristic cobblestone morphology. As shown in the middle portion of the successively arranged micrographs in Figure 10, a patchy region where adhered and nonadhered areas coexisted appeared, which was seen at a distance of around 5 mm (advancing contact angle, 35") in Figure 9. The continuity of monolayered adhered cells was disrupted in this zone, where shapes and sizes of the nonadhered areas were time-dependently changed: cells in this zone tended to aggregate and to detach from the surfaces, leaving a nonproliferative zone. On the more hydrophilic surface, a markedly reduced cell population was noted. The majority of the cells in this zone was circular.
Discussion Gradient surfaces whose surface chemical compositions continuously change in one direction have been recently fabricated for the diagnosis of biological responses in one
Cellular Responses on a Wettability Gradient Surface
Langmuir, Vol. 11, No. 10, 1995 4139
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Figure 10. Phase-contrast micrographs of endothelial cells grown on the wettability gradient surface after 3 days of incubation. The cell morphology around 5 mm (advancing contact angle, 35”) is shown in the successively arranged micrographs.
experiment on one surface. The pioneering work on the gradient surface by Elwing et al. led to the fabrication of a methylated silane-coupled glass surface in which hydroxyl groups were present at one end and methyl groups at the other.4 The ellipsometric technique5 and total internal reflectance fluorescence technique6 alllowed quantitative determination of the amount of adsorbed protein as a function of distance on the gradient surface. On the other hand, gradient polymeric surfaces were prepared by gradual exposure of radio-frequency plasma dischargeon poly(dimethylsi10xane)~ and corona discharge on polyethylene: the power of which gradually increased along the sample length direction. In these cases, oxygencontaining functional groups such as carboxyl, ketone, aldehyde, and alcohol may be produced on hydrophobic surfaces. Regardless of the methods utilized, surface chemical compositions were gradually changed from one end to the other end. Concomitantly, water wettability was gradually changed. In response to surface physicochemical properties, protein adsorption characteristics were drastically Cellular adhesion behavior was qualitatively ~ t u d i e d . ~ Many studies have shown that the cellularfundamental properties such as adhesion, spreading, migration, and proliferation are determined by surface physicochemical properties and surface composition (which includes type of functional groups and their density1.l For example, Curtis pointed out the cellular adhesivity dependence on surface functionalgroups almost 3 decades Recent molecular biological studies have shown that the cell adhesion and migration or movement on artificial substrates have been interpreted from cell-substrate interactions, in which adhesive proteins play an important role in cell behavior, and adherent machinery composed of a cell adhesion receptor which is a transmembrane protein assembly called integrin and cytoskeletal proteins such as actin and myosin.15 That is, adhesion is initiated by interaction between adhesive proteins (for example, fibronectin and vitronectin) adsorbed on a substrate and a cellular integrin receptor. Such information on adhesion is intracellularly transmitted to cytoskeletal proteins, which triggers polymerization of actin, producing actin filaments and its higher order aggregates, stress fibers. This induces cell spreading, which determines cell shape. However, depolymerization of actin filaments is a prerequisite for cell movement.15 Thus, the dynamic process of polymerization and depolymerization of actin takes place in the migration process.
There have been many studies on the surface wettability -cellular behavior relationship on individual samp1es.l In general, hydrophobic or polar surfaces tend to enhance cellular adhesion, spreading, and proliferation processes, all of which contribute to tissue formation. On the other hand, nonionic hydrophilic surfaces greatly suppress cellular adhesion and spreading. Therefore, tissue formation is hardly seen on these surfacs. Current understanding of this cellular response dependence on synthetic substrates is based on protein adsorptivity on a substrate. Thermodynamical considerations have shown that protein adsorption is driven by minimization of interfacial free energy between a substrate and water.l The general tendency obtained from accumulated experimental results is that a quite stable protein adsorption is observed for hydrophobic and polar surfaces, whereas a much less protein-adsorptive characteristic is found for nonionic hydrophilic nonswollen surfaces. A highly swollen surface-grafted surface minimizes protein adsorption especially under hydrodynamical shear stress. Hydrophobic and polar substrate surfaces exhibit high cell adhesion potentials, while the latter hydrophilic surfaces exhibit a low cell adhesion or nonadhesive potential due to a very weak protein-substrate interaction force, which is easily exceeded by an intracellularly generated mechanical force driven from highly elongated stress fibers. Since the adhesive interaction of a cell with the substrate or matrix influences the speed of movement, the local level of adhesiveness will influence the distribution of cells, by acting as a kinetic factor. The cells will only be found where the adhesive interaction with the surface permits the motile machinery to flatten the cell. An elegant demonstration of the influence of adhesion and movement comes from the experiment done by Carter 3 decades ago,16in which a gradient of adhesiveness was provided by shadowing palladium onto a cellulose acetate coated glass where cellulose is nonadhesive but the metal coated surface is suitable both for cellular adhesion and movement. Anchorage-dependent cells on such a hapatotactic gradient move toward the more adhesive region where they accumulate. The cells actually “perceive” a gradient and make a directional response (hapatotaxisis). In the present study, a surface hydroxylation reaction in solution was utilized to prepare gradient surfaces. In our previous paper, the kinetics of surface hydrolysis of a PVCa film was studied in detail by XPS analysis.1° The
(14) Curtis,A. S. G. Cell Adhesion. In The cell Surface;its molecular role in morpholgenesis; LondonLogodAcademic Press, 1967.
(15)Lackie, J. M. Cell Movement and Cell Behaviors; Allen & Unwin: London, 1986. (16) Carter, S. B. Nature 1967,213, 256.
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PVCa surface was hydrolyzed upon immersion into a NaOH aqueous solution at room temperature: upon hydrolysis, surface cyclic carbonate linkages were converted to hydroxyl groups. The degree of surface hydrolysis depended on the concentration of hydroxide ions and the contact time with an alkaline solution. As a continuation of a series of studies, the present study was undertaken in an attempt to prepare a wettability gradient surface by continuous immersion of a PVCa film into a NaOH aqueous solution. The surface molar fraction of PHM, determined by XPS measurements, depended on the hydrolysis time of PVCa at a fixed concentration of NaOH (Figure 3). The water contact angles of the treated surface decreased gradually with an increase in the distance from the untreated end of the film. A prolonged hydrolysis produced a more wettable surface. This result indicates that wettability of the treated surface changed continuously along one direction. As shown in Figures 4 and 5, the advancing contact angles on the surface with distances from -5 mm to 0 were lower than those on an untreated virgin PVCa surface. This may be due to the capillary action-driven elevation of the meniscus of the solution. Fundamental cellular properties were dependent on the location of the gradient surface. In the untreated hydrophobic surface region, a high degree of adherence and an elongated spreading state were attained, evident from the large adhesion area and large circular coefficient (Figures 5-7). On the other hand, in hydrophilic surface regions, a reduced adhesion potential as well as spreading potential was exhibited. In the intermediately wettable surface regions, those potentials gradually changed, depending on their wettability. On a wettability gradient surface, cell adhesion and spreading were gradually reduced as surface wettability increased, suggesting that adsorption of adhesive proteins from serum and/or stability
of the adsorbed protein layer are reduced with increasing wettability. The cellular migratory and proliferative potentials are also responsible for tissue formation on artificial materials. In the gradient surfaces prepared here, the spreading was very large but the migratory rate was relatively low on untreated surfaces. The migration rate on the slightly hydrophobic surface, for which the advancing contact angle was around 35", was the highest among those with different wettabilities studied (Figure 8). This is in good agreement with results obtained in our previous study13 that cellular migration of ECs on cellulose modified with n-propyl isocyanate appears to be significantly enhanced at the slightly modified surface, whereas adhesion, spreading, and proliferation potentials increase with an increase in the degree of surface derivatization. With increasing culture time, multicellular retraction occurred at this surface region, resulting in a thread-like or weblike structure. The cellular event occurring at the later stage of the tissue formation process may be due to increased cell-cell contact. That is, when a generated cell-cell interaction force which is strengthened with longer culture time exceeds the cell-substrate interaction force, an adherent cellular sheet will be delaminated from the surface to form cellular aggregates. As was observed in Figure 10, the patchy tissue with a web-like structure may be due to the above-mentioned mechanism. Our study indicates that each cellular potential of ECs is dependent on the wettability of the gradient surface. The gradient surface therefore serves as an excellent tool to investigate the cell-material interaction which is dependent on protein adsorption characteristics which reflect surface wettability, as was found in previous LA940971Q
