Fibroblast Cell Behavior on Bound and Adsorbed Fibronectin onto

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Articles Fibroblast Cell Behavior on Bound and Adsorbed Fibronectin onto Hyaluronan and Sulfated Hyaluronan Substrates R. Barbucci,* A. Magnani, A. Chiumiento, D. Pasqui, I. Cangioli, and S. Lamponi CRISMA and Department of Chemical and Biosystem Sciences and Technologies, University of Siena, Via A. Moro, 2 - Siena, Italy 53100 Received June 18, 2004; Revised Manuscript Received October 7, 2004

The effect of fibronectin protein (Fn) coating onto polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) on fibroblast cell adhesion was analyzed. The Hyal or HyalS were coated and grafted on the glass substrate by a photolithographic method. The Fn coating was achieved by two different routes: the immobilization of Fn by covalent bond to the polysaccharide layers and the simple adsorption of Fn onto Hyal and HyalS surfaces. AFM, SEM, and ATR-FTIR techniques were used for the chemical and topographical characterization of the surfaces. According to AFM and SEM data, the surface topography was dependent on the method used to cover the polysaccharide layers with the protein. ATR-FTIR analysis supplied information about the rearrangement of Fn after the interaction (adsorption or binding) with the Hyal and the HyalS. The conformational changes of the Fn were minimal when it was simply adsorbed on HyalS surfaces and larger once bound, whereas on the Hyal layer the protein underwent a bigger conformational change once adsorbed and covalently grafted. Then, the biological characterization was carried out by analyzing the human diploid skin fibroblasts adhesion on these surfaces. The morphology of fibroblasts was evaluated by SEM, whereas the dynamics of fibroblasts movement were recorded by a time-lapse system. Cell variations in area, perimeter, and length were analyzed at 2, 4, and 6 h. It was found that the addition of Fn (covalently bound or merely adsorbed) was fundamental in the promotion of fibroblasts adhesion and spreading. The greatest adhesion occurred onto HyalS layers covered by the adsorbed Fn. 1. Introduction Fibronectins, a family of structurally related soluble and matrix glycoproteins, have been implicated in numerous biological processes including opsonization, cell adhesion, wound healing, and organogenesis.1,2 Following the tissue injury, fibroblasts invade the fibrin clot in the wound space,3,4 after which they are activated.5 At least in part, this activation appears to be a switch from cell surface receptors binding type I collagen to those that bind provisional matrix proteins such as fibrin and fibronectin.6-8 The anchorage-dependent cell attachment is also mediated through the binding of integrin cell surface receptors to extracellular matrix (ECM) proteins, such as laminin, vitronectin, and fibronectin.9 They showed that they play an important role in cell adhesion to material surfaces.1,10,11 Cells producing a sufficient amount of fibronectin are able to spread on material surfaces in a serum-free medium, whereas cells that produce insufficient levels of fibronectin cannot spread in the absence of serum or added purified fibronectin.9 * To whom correspondence should be addressed. Tel/fax: + 39 0577 234382. E-mail: [email protected].

Fibronectin is composed of three general types of homologous repeating units, termed types I, II, and III,12 which are arranged into protease-resistant domains. The central cellbinding domain (CCBD) is composed of fibronectin type III repeating unit and includes an Arg-Gly-Asp (RGD) site in repeat III 10.13,14 Deletion of the RGD sequence eliminates cell attachment to fibronectin.15 The RGD sequence interacts with several cell surface integrins, the major one being R5β1.16,17 Many studies examined the ability of adsorbed ECM proteins to regulate cell behavior on biomaterial’s surfaces. The amount and conformation of adsorbed proteins vary significantly among different substrates.18,19 In addition, the composition of the adsorbed protein layers onto biomaterial surfaces vary after the exposure to protein-containing biological fluid such as serum.20 The vast number of publications on the role of proteins which interact with biomaterial underlines the great topical interest of this issue. Protein adsorption is the first step that occurs during biomedical materials applications: the nonspecific adsorption of proteins results in biological reactions to surfaces and may ultimately lead to the failure of the medical device. This protein film mediates the interactions

10.1021/bm049642v CCC: $30.25 © 2005 American Chemical Society Published on Web 01/27/2005

Fibroblast Cell Behavior on Fibronectin

with cells and surrounding tissues. Therefore, the characterization of the composition, conformation, and spatial distribution of adsorbed proteins is essential for understanding the interactions between the biomaterials surface and the biological environment.21 Currently, some different strategies are utilized for these investigations: the adsorption of a protein pool (i.e., bovine calf serum or human serum protein) on well-characterized materials,22 the step- by- step adsorption of 3-4 selected protein mixtures,23 and the chemical binding of only one protein on the material. In fact, a coating of an adsorbed or immobilized protein on a material influences all biological reactions at surfaces.24,25 Our approach to study the protein role with respect to cell adhesion in biomaterials is the immobilization of specific proteins on two different substrates. In the present study, we analyzed the dermal fibroblast behavior on polymeric surfaces, such as hyaluronic acid (Hyal) and its sulfated derivative (HyalS), containing bound or adsorbed fibronectin. Among the glycosamminoglycans, Hyal has the most simple chemical structure consisting of disaccharide units of N-acetil-glycosammine and D-glucuronic acid.26,27 The most part of glycosaminoglyans are synthesized by the cells in the Golgi complex, whereas Hyal is assembled in the internal art of cell membrane, subsequently extruded outside the membrane where specific protein complexes form. HyalS is chemically synthesized starting from the sodium salt of Hyal by the introduction of sulfate groups.28,29 In solution it shows anticoagulant properties thanks to the presence of sulfates which also render the polymer resistant to hyaluronan liaises enzymatic attachment.30 The protein was studied in terms of amount and conformation by AFM, SEM, and ATR FT-IR techniques. Fibroblasts behavior was analyzed in terms of adhesion and migration. 2. Materials and Methods 2.1. Materials. The sodium salt of hyaluronan (Hyal-Na, MW 240 000) was supplied by Biophyl S.p.A. (Italy). The Dowex 50W 8X resin, the sulfur trioxide complex, 4-azidoaniline hydrochloride, 1-ethyl-3-[3-(dimethyl-amino)propyl] carbodimide hydrochloride (EDC), and all of the other solvents used for the chemical reactions were purchased from Fluka-Sigma-Aldrich S.p.A (Germany), as well as Fibronectin from human plasma 0.1% solution in 0.5 M NaCl, 0.05 M Tris (pH 7.5), Dulbecco’s modified Eagle’s medium (DMEM), human serum (HS), trypsin solution, and all other reagents used for cell culture. 2.2. Methods 2.2.1. Preparation of Polysaccharide-Protein Surfaces. The preparation of polysaccharide-protein surfaces involved four successive steps: (1) Synthesis of the photoreactive polysaccharides (HyalN3 or HyalS-N3); (2) Grafting of the polysaccharide on the substrate; (3) Synthesis of the photoreactive fibronectin (Fn-N3); (4) Binding of Fn-N3 or adsorption of the native Fn above it.

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2.2.2. Synthesis of the PhotoreactiVe Polysaccharides. The conjugation of Hyal or HyalS, obtained as previously reported,29 with a photoreactive unit 4-azidoaniline31 was needed for the photoimmobilization process. Briefly, an established molar ratio (1:2:2) respectively of Hyal-Na or HyalS-Na repeat units, 4-azidoaniline hydrochloride, and EDC was dissolved in double distilled water. The solution was stirred at 4° C for 24 h in dark conditions. The solution was dialyzed against double distilled water in a dialysis tube with a 12 000 MW cut off and then lyophilised. Photoreactive polysaccharide was referred to as Hyal-N3 or HyalS-N3. 2.2.3. Polysaccharide Grafting on the Substrate. The photoreactive polysaccharide (Hyal-N3/HyalS-N3) was bound to glass cover slips (diameter 12 mm). To increase the hydrophilicity of these substrates, the glass disks were incubated in a 1% solution of 3-aminopropyl-3-aminoethyltrimethoxysilane in acetic ethanol (pH 5) for 20 min, followed by washing with ethanol and distilled water. Afterward, 100 µL of an aqueous solution (pH 7) of the photoreactive polysaccharide (1 mg/ml) was dropped on the pretreated glass substrates and dried at room temperature in dark conditions. Subsequently, the surfaces were irradiated by a UV source (Helios Italquarz GRE, power 400 W) at a distance of 40 cm for 60 s. The glass coverslip area was reduced at 4-5 mm2 by isolating a circle of surface with a silicon glue ring, which was positioned in the middle of the sample and left to dry under the hood. 2.2.4. Synthesis of the PhotoreactiVe Fibronectin. To bind Fn to Hyal/HyalS film using the photoimmobilization process, it was necessary also to conjugate the protein with the 4-azidoaniline molecule. The reaction was carried out in the same way as the Hyal or HyalS. A 1 mg/mL solution of fibronectin (Fn) (dissolved in NaCl 0.5 M plus Tris 0.05 M, pH 7.5), 4-azidoaniline hydrochloride, and 1-ethyl-3-[3(dimethyl-amino)propyl] carbodiimide hydrochloride (EDC) was dissolved in a NaCl 0.5 M, Tris 0.05 M solution using the established molar ratio of 1:2:2 respectively, under stirring at +4 °C for 24 h in dark conditions. The solution was dialyzed against a solution of NaCl 0.5 M, Tris 0.05M in a dialysis tube, with a cut off of 12 000 MW. At the end, the samples were frozen and then lyophilized. The photoreactive Fn was referred to as Fn-N3. The success of the reaction was checked by infrared spectroscopy, identifying the presence of the azide group. 2.2.5. Fibronectin Binding and Adsorption. A total of 100 µL of 0.33 mg/mL solution of Fn-N3 (same concentration of serum composition) was cast only onto the photoimmobilized polysaccharide spot and left to dry in dark conditions. Subsequently, the samples were irradiated by a UV lamp (Helios Italquarz 400 W) for 60 s at a distance of 40 cm from the source. These surfaces coated with Fn were employed for testing cell adhesion. Instead of binding the protein covalently, a NaCl 0.5 M, Tris 0.05 M solution of 0.33 mg/mL Fn was left to adsorb on the layers of Hyal and HyalS for 2 h at 37 °C. For each sample, 100 µL of the Fn containing buffer solution was dropped only onto the polysaccharide spots with the help of the silicon ring. All of the surfaces were washed by dipping in double distilled water for 20 min.

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Table 1. Main Wavenumbers Observed in the Protein Amide I Spectral Region Reflecting the Major Components of the Fn Secondary Structurea secondary structure components of amide I region R-helix native Fn Fn adsorbed on Hyal Fn adsorbed on HyalS Fn bound on Hyal Fn bound on HyalS a

β-sheet

β-turn

Random-coil

1610 s

1660 sh 1665 sh 1665 sh

1641 s 1635 s 1645 s 1648 s

1603 s 1617 sh

s ) strong, sh ) shoulder.

Four different samples were obtained: Hyal with bound fibronectin (Hyal-FnBnd), HyalS with bound fibronectin (HyalS-FnBnd), Hyal with adsorbed fibronectin (Hyal-FnAds) and HyalS with adsorbed fibronectin (HyalS-FnAds). 2.3. Physical-Chemical and Morphological Characterization 2.3.1. FTIR-ATR Analysis. Infrared analysis was performed by FT-IR-ATR spectrometer Bio-Rad FTS 6000 purged with nitrogen. The ATR spectra of dry samples were recorded with a horizontal (PIKE) ATR accessory equipped with a 45° Ge ATR crystal and a mercurium cadmium telluride (MCT) detector. Sixty-four scans at a resolution of 4 cm-1 were averaged for each spectrum. Using WIN-IR PRO version 2.6, recorded spectra were elaborated by baseline correction and smoothing (boxcar function; 9 N. of P.). First, the ATR-FT spectroscopy was used to verify that there were no changes in the conformation on the Fn-N3. Then, conformational changes of linked or adsorbed Fn were studied, taking into account the AMIDE I region (17001580 cm-1) of the spectrum. The polysaccharide (Hyal/ HyalS) spectrum was subtracted from the spectrum of the corresponding system containing Fn to obtain the difference spectrum of Fn, which reflected the secondary structure of the protein upon its interaction with the polymer. Protein characterization stemmed from the comparison of the difference spectrum of Fn and the native protein. Table 1 summarizes the main wavenumbers observed in the Amide I spectrum region. 2.3.2. Surface Morphological Analysis (AFM and SEM). The adsorbed and bound fibronectin layer morphology was evaluated by atomic force microscopy (AFM, Thermomicroscopes, Veeco) and scanning electron microscopy (XL20 Philips, The Netherlands). For the AFM analysis, dried samples were simply mounted on proper metallic stubs with double adhesive tape. Measurements were performed in different areas (scan size 50 × 50 µm2) operating in tapping mode in air using a silicon tip with a constant force of 13-100 N/m and a nominal resonance frequency between 240 and 420 kHz. The mean surface roughness was measured with the help of SPLMLab version 5.01 software. For SEM analysis the polysaccharide-protein coated surfaces were sputter-coated with gold using an automatic sputter coater (BAL-TEC SCD 050, Balzer, Germany). The observation was made using the SEM at 8 kV accelerating voltage.

2.3.3. Determination of Adsorbed Fn on Polysaccharide Layers. The amount of Fn adsorbed on Hyal and HyalS was assessed by removing the protein from the polysaccharide layers with the following eluants: PBS (8 mM sodium hydrogen phosphate, 2 mM potassium hydrogen phosphate, 3 mM potassium chloride in double distilled water, pH 7.4), sodium dodecyl sulfate (SDS) 0.1% in double distilled water and isoelectring focusing buffer (IEF) solution (urea, 3-[(3cholomidopropyl)dimethylamino]1-propansulfonate and dithioerythriol reagent). A total of 0.8 mL of each eluant buffer was added to the surfaces and kept in contact with them for 20 min at 37 °C. The amount of desorbed Fn was measured by Bradford assay on the eluants solution32 using the BioRad protein assay kit. The complete removal of Fn from the surfaces after the last elution step was checked by ATR FTIR analysis. 2.4. Biological Characterization 2.4.1. Cell Adhesion Analysis. The experiments were performed on six different substrates: Hyal, HyalS, HyalFnBnd, HyalS-FnBnd, Hyal-FnAds, and HyalS-FnAds. The experiments started at the cell deposition time, and cell adhesion was evaluated after 2, 4, 6, and 24 h. Human fibroblasts with diploid nature (as assumed from the source) from skin biopsies of normal individuals were used. Fibroblasts were propagated in DMEM containing glucose (4.5 g/L) and supplemented with 10% clotted human male whole blood serum (HS), at 37 °C in 5% CO2 humidified atmosphere. Following incubation, once at confluence, fibroblasts were washed with PBS, harvested with a 0.025% trypsin solution, and suspended in 10% HS DMEM solution. Fibroblasts were seeded only on the spots area in a number of 4 × 103 per 4-5 mm2 of the surface. The morphology of human fibroblasts was evaluated by phase contrast microscopy. The number of adhered cells at different times was determined by direct observation of samples at phase contrast microscopy. Furthermore, the possible dissolving effect of DMEM on Hyal-FnAds and HyalS-FnAds was taken into account. In fact, there was the possibility that an amount of adsorbed Fn would dissolve in the DMEM solution during the cell adhesion experiments. To evaluate whether the behavior of fibroblasts on Hyal-FnAds and HyalS-FnAds was only ascribable to the Fn adsorbed on Hyal and HyalS and not to the Fn dissolved in the medium solution, a cell adhesion experiment was performed by plating the cells in a complete DMEM solution containing also Fn (100 µL per mL of 0.3 mg/mL) on Hyal and HyalS surfaces. The cell response was compared to the one observed on Hyal-FnAds and HyalSFnAds surfaces. 2.4.2. Time-Lapse ObserVations and Analysis of Cell BehaVior. To measure the dynamics of cell movement and analyze cell shape at different times, time lapse video recordings of the cells were taken using an Olympus microscope and a CCD-Iris color video camera (Sony) at 200× magnification, PC powered by the Pinnacle DV500 DVD System with a frame rate of 1/min. The video analysis was performed on a PC computer using Premiere 6.0 program and cells were monitored for 6 h. The shape of the fibroblasts in terms of perimeter, area, and length was


Fibroblast Cell Behavior on Fibronectin Scheme 1. Schematic of the Synthesis of PhotoreactiveFibronectin (Fn-N3)

evaluated by using ImageJ 1.25s software (developed at the U.S. National Institute of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). Variations in cell area, perimeter and length were analyzed at 2, 4 and 6 h. 2.4.3. Data Analysis. All data were expressed as mean ( SE on three experiments. The significance was determined using the one-way ANOVA test and the level of significance was set at p < 0.05. For clarity purposes and biological relevance, we reported only the significant differences vs the controls (Hyal and HyalS in the cell adhesion experiments vs time zero in the analysis of time lapse registration) in figures and figure legends. Statistical analysis was performed using Microcal Origin (Microcal Software, Inc. Northampton, MA). 3. Results 3.1. Physical-Chemical and Morphological Characterization. Two different procedures were followed to coat Hyal and HyalS surfaces with Fn: one involves the simple adsorption of the protein onto a photoimmobilized polysaccharide layer, whereas the other one requires the photografting of the protein to the polysaccharide. The second procedure requires the synthesis of the photoreactive protein (Fn-N3) using the azidoaniline as reagent as reported in Scheme 1. 3.1.1. ATR FT-IR Analysis. ATR FT-IR analysis demonstrated that no structural changes occurred along the protein chains of Fn-N3 after the introduction of the 4-azidoaniline: the only difference between the IR spectrum of Fn-N3 and that of the native Fn was observed in the presence of the 1507 cm-1 band (Figure 1), distinctive of azide groups.

Figure 1. ATR-FTIR spectrum of Fn-N3 and native Fn.

The secondary structure of Fn adsorbed on Hyal and HyalS was investigated. As reported in Figure 2, FnAds showed a secondary structure different from the one belonging to the native Fn. The spectrum of the native Fn is characterized by a band centered at 1641 cm-1 related to a β-sheet structure; the spectrum of FnAds on the Hyal surface showed a shift of this amide I band to lower wavenumbers (1610 cm-1), more marked than on HyalS (1635 cm-1). The band shift can be ascribed to the different interaction occurring at the interface with Hyal or HyalS polymer. Moreover, a new band at 1665 cm-1, related to domains of undefined structure (random coil),33,34 was present in the spectrum of adsorbed Fn on both polysaccharides. The amide I spectral regions of FnBnd to Hyal and FnBnd to HyalS were very similar to each other (Figure 3). Fn bound to the polysaccharide showed a predominant component related to R-helix domains (bands at 1645 and 1648 cm-1, respectively) and another component (band at 1603 and 1617 cm-1, respectively) ascribable to the conformational changes induced by the interaction with the Hyal or the HyalS surfaces, respectively.

Figure 2. ATR-FTIR spectrum of FnAds on Hyal, FnAds on HyalS, and native Fn.

Figure 3. ATR-FTIR spectrum FnBnd on Hyal, FnBnd on HyalS, and native Fn.

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Table 2. Concentration of Adsorbed Fn on Hyal and HyalS Layers used eluants

concentration of Fn (µg/cm2) on Hyal

concentration of Fn (µg/cm2) on HyalS

PBS SDS 0,1% IEF sol. total

1.86 ( 0.09 1.32 ( 0.11 0.94 ( 0.07 4.12 ( 0.09

3.01 ( 0.24 1.46 ( 0.19 0.74 ( 0.15 5.21 ( 0.19

Figure 5. (a) SEM images showing FnAds on Hyal layer. The same surface Fn distribution has been observed on HyalS-FnAds. (b) SEM images showing FnBnd on Hyal layer. The same surface Fn distribution has been observed on HyalS-FnBnd.

Figure 4. AFM image of FnAds on a Hyal layer. (The same morphology is observed also on the other polysaccharide’s layer.); b. AFM image of FnBnd on a Hyal layer. (The same morphology is observed also on the other polysaccharide’s layer.)

Table 1 summarizes the main wavenumbers observed in the Amide I spectral region of the native, adsorbed and bound Fn together with their assignment in terms of protein secondary structure. 3.1.2. Fn Adsorption on Hyal and HyalS Layers. The amount of the adsorbed Fn on Hyal and HyalS layers was determined by the Bradford assay. The samples were first washed with double distilled water in order to remove the merely physically adsorbed Fn and then eluted by three different solutions: PBS, SDS 0.1%, and IEF solution. Only after the IEF treatment was the FT-IR spectrum of the sample surface the same as that of the photoimmobilized polysaccharide, meaning that no trace of Fn was left at least in the limit of FT-IR spectroscopy sensitivity. Table 2 shows the concentration of Fn on HyalS and on Hyal determined

after PBS, SDS, and IEF elution. After the PBS elution, the amount of Fn detected on HyalS was greater than the one found on Hyal, whereas the amount of FnAds eluted by the SDS and IEF solution on the two different polymeric layers is almost the same. 3.1.3. Surface Morphological Analysis (AFM and SEM). The Hyal and HyalS surfaces are not flat but characterized by several lamellas, as observed by optical microscope. A different morphology was observed when the protein is adsorbed or covalently bound to the polysaccharide layer despite its chemical composition (Hyal or HyalS). When the Fn was simply adsorbed on the polysaccharide layers, a homogeneous protein layer consisting of fibrous features in bundles with an irregular shape and different thickness was observed. These are visible in the AFM topographic images (Figure 4a). The surfaces were quite smooth with a mean roughness of Ra = 5 nm (SPLM-Lab version 5.01 software). On the contrary, Fn covalently bound to both polysaccharide layers showed randomly placed islands with different heights ranging from 700 nm up to 3000 nm and different shapes. The measured main diameter is ∼15µm (see Figure 4b). The islands formed during the photoimmobilization reaction are made of photocrosslinked Fn lying on a plain Fn layer as evidenced by ATR/FT-IR analysis. SEM analysis evidenced the same island morphology when the protein was bound to the polysaccharide layer, whereas when it was just adsorbed, the thick Hyal or HyalS lamellae randomly distributed were however visible (Figure 5a,b) 3.2. Biological Characterization 3.2.1. Cell Adhesion. The analysis of cell behavior showed that human fibroblasts preferentially adhered to the fibronec-


Figure 6. Number of adherent fibroblast cellson tested surfaces (oneway ANOVA test. *p < 0.05; **p < 0.01; ***p < 0.0001 vs Hyal and HyalS).

tin containing materials (Hyal-FnBnd, HyalS-FnBnd, HyalFnAds, and HyalS-FnAds) better than those without protein (Hyal and HyalS). In fact, the number of adhered cells on Hyal-FnBnd, HyalS-FnBnd, Hyal-FnAds, and HyalS-FnAds significantly increased with time, whereas on Hyal and HyalS the number remained almost the same (Figure 6). On the latter surfaces, fibroblasts showed a round shape (Figures 7a,d), showing a low ability of these substrates to stimulate cell adhesion. However, on Hyal-FnBnd, HyalS-FnBnd, HyalFnAds, and HyalS-FnAds, fibroblasts cells adhered and spread after 2 h of contact with the substrate assuming a completely flat shape (Figures 7b,c,e,f). On the Hyal-FnBnd and HyalS FnBnd surfaces, cells avoided the photocrosslinked Fn islands and spread only on the smooth areas (Figures 7c,f). The fibroblasts adhesion on HyalS-FnAds showed the highest number of adhered cells from the beginning of the experiment, which significantly increased in time. The number of


adhered cells followed this order: HyalS-FnAds > Hyal-FnAds > HyalS-FnBnd > Hyal-FnBnd. Furthermore, the possible presence of Fn in solution did not modify the behavior of cells; in fact plain Hyal or HyalS surfaces with added Fn in solution did not promote an improvement in cell adhesion compared to the one observed on Hyal and HyalS surfaces themselves. 3.2.2. Time-Lapse ObserVations. The time-lapse video recording demonstrated motility of fibroblasts on all of the different surfaces. The higher motility was observed on substrates containing lamellar or fibrous features; that is, the human fibroblasts preferentially moved toward these irregularities as evidenced by SEM analysis. There exists an important difference in fibroblasts behavior between substrates with Fn and with Hyal or HyalS alone. In fact on the latter substrates (Hyal and HyalS), fibroblasts moved to lamellae, but they did not adhere on them and showed a round shape, whereas on both polysaccharide substrates containing adsorbed Fn, fibroblasts moved and adhered to them showing a completely flat shape. Furthermore, during the first 6 h, the fibroblast shape was markedly different on Hyal or HyalS compared to that adhered on samples with Fn (Figures 8 and 9). Fibroblasts growth on plain Hyal and HyalS surfaces showed no significant differences in shape and area within the first 2h. Subsequently, HyalS surfaces supported cell adhesion better than Hyal ones inducing an increase of area spreading and perimeter length. However, cell adhesion on samples containing Fn led to a significant increase in area and a change in shape resulting in a rise in the cell length (Figures 8b,c and 9b,c). These results, in accordance with those of cell adhesion, led us to consider the presence of Fn as an important stimulating factor on cell growth. Among the different

Figure 7. Phase contrast microscope images (20×) of fibroblasts adhesion on Hyal (a); Hyal-FnAds (b); Hyal-FnBnd (c); HyalS (d); HyalS-FnAds (e); HyalS-FnBnd (f) after 2 h of culture.

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Figure 8. Morphological analysis, performed interms of area, perimeter and length measure of adherent fibroblast cells on (a) Hyal, (b) Hyal-FnBnd, and (c) Hyal-FnAds layers; (*p < 0.05; **p < 0.01; ***p < 0.001; #p < 0.0001).

surfaces, the HyalS with adsorbed Fn is the one which promote cell spreading more. 4. Conclusion The data reported in this paper emphasize the important role of fibronectin in mediating cell-surface interaction. In particular Hyal and HyalS layers which demonstrated to induce a low (in the case of HyalS) or no cell adhesion (in the case of Hyal) show a very low protein adsorption once put in contact with serum solution.22 The importance of Fn in the promotion of fibroblasts attachment and spreading as well as in development of focal adhesions was already described by Couchmann et al.35

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Figure 9. Morphological analysis in terms of area perimeter and length measure of adherent fibroblast cells on (a) HyalS, (b) HyalSFnBnd, and (c) HyalS-FnAds layers; (*p < 0.05; **p < 0.01; ***p < 0.001; #p < 0.0001).

However, in this study, another aspect has been pointed out: the importance of protein conformation. Once a protein is laying on a material surface, its conformation undergoes a rearrangement; the more its conformation is similar to the native one the more the protein biological activity is conserved. In this case, the covalent Fn grafting on Hyal layer induces a stronger shift of the amide I band with a consequent rearrangement of the protein secondary structure with respect to the both native protein structure and to the one adsorbed on HyalS layer. The cell number and spreading area were


greater on Fn containing HyalS surfaces. Moreover, both Hyal and HyalS surfaces show Fn containing islands of few micrometers, which might be due to the protein auto crosslinking during the photoirradiation step. The photocrosslinked Fn contained in the islands may undergoes to a stronger conformational change than the Fn on the surrounding surface areas. The fibroblasts are not able to emit pseudopodia and adhere on these microscaled islands, as cells usually do when the substrate show topographical cues.36,37 The fibronectin secondary structure does not change when it is just adsorbed on the sulfated hyaluronan layer maintaining a nativelike conformation. The higher surface wettability and the larger amount of negative charges, in comparison to the Hyal surfaces, induce an electrostatic interaction which affect less the protein secondary structure. In this case, many fibroblasts adhered with a large spreading area from the first 2 h of culture and their number increased fast in time. On the contrary, the Hyal coatings can give place to a bigger hydrophobic protein interaction promoting a larger Fn secondary structure rearrangement as seen by ATR/FtIR analysis which implies a less cell adhesion and spreading. From these data, it appears that the protein conformation plays a key role in the explication of fibronectin biological activity, less the conformation is affected by the interaction with the surface more the cell adhesion and proliferation are favored. Acknowledgment. Financial support from FIRB (Fondo per gli investimenti per la ricerca di base - MIUR) and PRIN 2003 (No. 2003037472) are gratefully acknowledged. References and Notes (1) Mosher, D. F. Prog. Hemostasis Thromb. 1980, 5, 111-51. (2) Hynes, R. O.; Yamada, K. M. J. Cell. Biol. 1982, 95, 369-77. (3) McCarthy J. B.; Chelberg, M. K., Mickelson, D. J.; Furcht, L. T. Biochemistry 1988, 27, 1380-88. (4) Iida, J., Skubitz, A. P., Furcht, L. T., Wayner, E. A.; McCarthy, J. B. J. Cell Biol. 1992, 431-44. (5) McClain, S. A.; Simon, M.; Jones, E.; et al. Am. J. Pathol. 1996, 1257-70. (6) Gailit, J.; Xu, J.; Bueller, H.; Clark, A. F. J. Cell Physiol. 1996, 169 (2), 281-89. (7) Xu, J.; Clark, A. F. J. Cell Biol. 1996, 132, 239-49. (8) Greiling, D.; Clark, A. F. J. Cell Sci. 1997, 861-70.

Biomacromolecules, Vol. 6, No. 2, 2005 645 (9) Hynes, R. O. Cell 1992, 69, 11-25. (10) Yamada, K. M.; Olden, K. Fibronectins-adhesive glycoproteins of cell surface and blood. Nature 1978, 275, 179-84. (11) Grinnel, F.; Minter, D. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 440812. (12) Yamada, K. N., Ed.; Hay ED. Plenum Press: New York, 1991; pp 111-45. (13) Ruoslahti, E. Annu. ReV. Biochem. 1998, 57, 375-423. (14) Hautanen, A.; Gailit, J.; Mann, D. M.; Ruoslahti, E. J. Biol. Chem. 1989, 264, 1437-42. (15) Obara, M.; Knag, M. S.; Yamada, K. M. Cell 1988, 53, 649-57. (16) Pytela, R., Pierschbacher, M. D.; Ruoslahti, E. Cell 1985, 40, 19198. (17) Ruoslahti, E.; Pierschbacher, M. D. Science 1987, 238, 291-97. (18) Juliano, D. J.; Saavedra, S. S.; Truskey, G. A. J. Biomed. Mater. Res. 1993, 1103-13. (19) Jonsson, U.; Ivarsson, B.; Lundstrom, I.; Berghem, L. J. Colloid Interface Sci. 1982, 90, 148-63. (20) Andrade, J. D.; Hlady, V. AdV. Polym. Sci. 1986, 79, 1-3. (21) Lhorest, J. B.; Wagner, M. S.; Tidwell, C. D.; Castner, D. G. J. Biomed. Mater. Res. 2001, 57, 432-40. (22) Magnani, A.; Barbucci, R.; Lamponi, S.; Trabalzini, L.; Martelli, P.; Santucci, A. Electrophoresis 2004, 25(14), 2413-2424. (23) Wagnaer, M. S.; Shen, M.; Horbett, T. A.; Castner, D. G. J. Biomed. Mater. Res. 2002, 64A, 1-11. (24) Zhang, Z.; Yoo, R.; Wells, M.; Beebe, T. P., Jr.; Biran, R.; Tresco, P.; Biomaterials 2004, in press. (25) Wagner M. S.; Castner, D. G. Appl. Surf. Sci. 2004, in press. (26) Mason, M.; Vercruysse, K.; Frish, R.; Marecak, D.; Prestwich, G.; Pitt, W. Biomaterials 2000, 31, 31-36. (27) Guss, J. M.; Hukins, D. W. L.; Smith, P. J. C.; Winter, W. T.; Arnott, S. R. J. Mol. Biol. 1975, 95, 359-384. (28) Chen, G.; Ito, Y.; Imanishi, Y.; Magnani, A.; Lamponi, S.; Barbucci, R. Bioconjugate Chem. 1997, 8, 730-734. (29) Magnani A.; Lamponi, S.; Rappuoli, R.; Barbucci, R. Polym. Int. 1998, 46, 225-240. (30) Girotto, D.; Urbani, S.; Brun, P.; Renier, D.; Barbucci, R.; Abatangelo, G. Biomaterials 2003, 24 (19), 265-75. (31) Barbucci, R.; Lamponi, S.; Magnani, A.; Pasqui, D. Biomol. Eng. 2002, 19, 161-70. (32) Bradford, M. Anal. Biochem. 1976, 72, 248-254. (33) Chittur, K. K. Biomaterials 1998, 19, 357-69. (34) Gendreau, R. M. Biomedical Fourier transform infrared spectroscopy: application to proteins. In Spectroscopy in the Biomedical Sciences; Gendreau, R. M., Ed.; CRC Press: Boca Raton, FL, 1986; pp 21-52. (35) Couchman, J. R.; Rees, D. A.; Green, M. R.; Smith, C. G. Cell Biol. 1982, 93, 402-410. (36) Dalby, M. J.; Riehle, M. O.; Johnstone, H.; Affrossman, S.; Curtis, A. S. G. Biomaterials 2002, 23, 2945-2954. (37) Dalby, M. J.; Childs, S.; Riehle, M. O.; Johnstone, H. J.; Affrossman, S.; Curtis, A. S. G. Biomaterials 2003, 24, 927-935.

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Fibroblast Cell Behavior on Bound and Adsorbed Fibronectin onto

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