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Langmuir 2008, 24, 11734-11742
Probing Fibronectin-Surface Interactions: A Multitechnique Approach Elodie Velzenberger,*,† Isabelle Pezron,‡ Gilbert Legeay,§ Marie-Danielle Nagel,† and Karim El Kirat† UniVersite´ de Technologie de Compie`gne (UTC), BP 20529, 60205 Compie`gne Cedex, France, and Centre de Transfert de Technologie du Mans, 20 rue Thale`s de Milet, 72000 Le Mans, France ReceiVed June 4, 2008. ReVised Manuscript ReceiVed July 28, 2008 The development of adhesive as well as antiadhesive surfaces is essential in various biomaterial applications. In this study, we have used a multidisciplinary approach that combines biological and physicochemical methods to progress in our understanding of cell-surface interactions. Four model surfaces have been used to investigate fibronectin (Fn) adsorption and the subsequent morphology and adhesion of preosteoblasts. Such experimental conditions lead us to distinguish between anti- and proadhesive substrata. Our results indicate that Fn is not able to induce cell adhesion on antiadhesive materials. On adhesive substrata, Fn did not increase the number of adherent cells but favored their spreading. This work also examined Fn-surface interactions using ELISA immunoassays, fluorescent labeling of Fn, and force spectroscopy with Fn-modified tips. The results provided clear evidence of the advantages and limitations of each technique. All of the techniques confirmed the important adsorption of Fn on proadhesive surfaces for cells. By contrast, antiadhesive substrata for cells avoided Fn adsorption. Furthermore, ELISA experiments enabled us to verify the accessibility of cell binding sites to adsorbed Fn molecules.
Introduction Synthetic biomaterials are extensively used in therapeutics and diagnostics for cell-based assays,1 drug delivery,2-4 tissueengineering applications,3,5,6 and, more generally, for medical devices.3,7 The interactions of cells with materials modulate the cellular responses to implanted devices as well as cell culture supports.6,8 Controlling cell adhesion on polymeric materials is a key issue for biomaterials. Biochemical data highlight the necessity of extracellular matrix (ECM) proteins in promoting cell adhesion through specific interactions.9,10 Cells, via integrins (cell receptors), bind to specific amino acid sequences on ECM proteins, such as the arginine-glycine-aspartic acid (RGD) motif present in many ECM ligands including fibronectin (Fn), vitronectin, and thrombospondin.11,12 These proteins provide an attachment network for the adhesion and growth of specific cells in vivo. The adhesion molecules are also used to enhance cell attachment to various substrata in vitro. The difference in cell adherence behavior between various substrata may result from differences in the adsorption and the conformation of proteins on surfaces. The rapid adsorption of proteins effectively provides * Corresponding author. Phone: +33 (0)3 44 23 44 21. Fax: +33 (0)3 44 23 79 42. E-mail:
[email protected]. † Universite´ de Technologie de Compie`gne, CNRS UMR 6600, Biome´canique et Bioinge´nierie. ‡ Universite´ de Technologie de Compie`gne, EA Transformations Inte´gre´es de la Matie`re Renouvelable. § Centre de Transfert de Technologie du Mans.
(1) Thorsen, T.; Maerkl, S. J.; Quake, S. R. Science 2002, 298, 580–584. (2) Rothenfluh, D. A.; Bermudez, H.; O’Neil, C. P.; Hubbell, J. A. Nat. Mater. 2008, 7, 248–254. (3) Karp, J. M.; Langer, R. Curr. Opin. Biotechnol. 2007, 18, 454–459. (4) Langer, R. Science 2001, 293, 58–59. (5) Kohane, D. S.; Langer, R. Pediatr. Res. 2008, 63, 487–491. (6) Rosso, F.; Marino, G.; Giordano, A.; Barbarisi, M.; Parmeggiani, D.; Barbarisi, A. J. Cell Physiol. 2005, 203, 465–470. (7) Williams, D. Med. DeVice Technol. 2008, 19, 10–11. (8) Datta, N.; Holtorf, H. L.; Sikavitsas, V. I.; Jansen, J. A.; Mikos, A. G. Biomaterials 2005, 26, 971–977. (9) Tamada, Y.; Ikada, Y. Polymer 1993, 34, 2208–2212. (10) Akiyama, S. K.; LaFlamme, S. E. Colloids Surf., B 1994, 2, 241–250. (11) van der Flier, A.; Sonnenberg, A. Cell Tissue Res. 2001, 305, 285–298. (12) Ruoslahti, E.; Pierschbacher, M. D. Cell 1986, 44, 517–518.
the cell a biological interpretation of the structure and composition of the foreign surface. Several surface properties have been proposed to influence protein adsorption and cell behavior: charge,13 topography,13 surface energy,14 and surface chemistry.15 In this work, various techniques (antibody recognition, fluorescent labeling, and force spectroscopy) were used to investigate the capability of Fn to adsorb on four different materials and to induce the adhesion of preosteoblasts. Fn is one of the most studied proteins of the ECM: it plays a crucial role in the adhesion, spreading, proliferation, and differentiation of many cell types to ECM and artificial surfaces, including polystyrene supports for tissue culture.16 For this study, we have chosen materials for their biomaterial applications and/or for their use in cell culture: bacteriological-grade polystyrene (PS), tissue culture-grade polystyrene (tPS), poly(2-hydroxyethyl methacrylate) films (polyHEMA), and bilayered hydroxypropylmethylcellulose-carboxymethylcellulose-coated Petri dishes (CEL). PS and tPS are commonly used in cell culture, and several studies17,18 permitted the evaluation of their capacity to induce protein adsorption and cell adhesion. PolyHEMA, which is widely used for contact lenses,19 intraocular implants,20,21 and cell culture devices, prevents cell adhesion and spreading. Carboxymethylcellulose has been used as an antiadhesive coating for (13) Blanco, E. M.; Horton, M. A.; Mesquida, P. Langmuir 2008, 24, 2284– 2287. (14) Cha, P.; Krishnan, A.; Fiore, V. F.; Vogler, E. A. Langmuir 2008, 24, 2553–2563. (15) Fuse, Y.; Hirata, I.; Kurihara, H.; Okazaki, M. Dent. Mater. J. 2007, 26, 814–819. (16) Bentley, K. L.; Klebe, R. J. J. Biomed. Mater. Res. 1985, 19, 757–769. (17) Dewez, J.-L.; Doren, A.; Schneider, Y.-J.; Rouxhet, P. G. Biomaterials 1999, 20, 547–559. (18) Shen, M.; Garcia, I.; Maier, R. V.; Horbett, T. A. J. Biomed. Mater. Res. A 2004, 70, 533–541. (19) Lipson, M. J.; Musch, D. C. Optom. Vis. Sci. 2007, 84, 593–7. (20) Versura, P.; Torreggiani, A.; Cellini, M.; Caramazza, R. J. Cataract Refract. Surg. 1999, 25, 527–533. (21) Saika, S.; Miyamoto, T.; Ohnishi, Y. J. Cataract Refract. Surg. 2003, 29, 1198–1203.
10.1021/la801727p CCC: $40.75 2008 American Chemical Society Published on Web 09/25/2008
Probing Fibronectin-Surface Interactions
biomaterials, such as antiadhesive meshes,22-25 dressings,26-29 and drug delivery matrices.30-33 We have shown previously that CEL provides an antiadhesive substratum that is particularly suitable for studying the molecular mechanisms underlying cell-substratum and cell-cell interactions.34-36 Several authors have previously studied the mechanisms of interaction between Fn and surfaces. Sousa et al.37 assessed Fn adsorption on TiO2 by AFM (topography), ellipsometry, XPS, and radiolabeling of Fn with 125I. Their results provided evidence that Fn molecules spontaneously adsorb and form aggregate structures on TiO2 surfaces. Experiments with radiolabeled Fn also revealed that rapid adsorption occurred on TiO2 surfaces, but the authors also specified that radiolabeling modifies the structure and/or behavior of the protein significantly. Garcı´a et al.38 evaluated the impact of Fn conformational changes on cell proliferation and differenciation after protein adsorption on bacteriological-grade polystyrene, tissue culture polystyrene, and collagen layers with 125I radiolabeling and with antibody binding. The authors observed differences in Fn conformation that altered the quantity of bound cells and their proliferation. On each surface, they also established radiolabeled Fn adsorption isotherms after albumin saturation. 125I grafting can change the protein conformation; moreover, albumin may compete with radiolabeled Fn, which can change the initial amount of adsorbed Fn.39 Faucheux et al.40 examined the impact of different functional end groups (amines or carboxylic acids) of self-assembled monolayers on fluorescein-labeled Fn adsorption and subsequent human fibroblast spreading by fibrillar adhesion. Their observations indicated that the molecular composition of the substrata had a strong influence on Fn matrix formation by promoting or inhibiting the focal and fibrillar adhesions. However, the fluorescent labeling procedure had an impact on protein structure and on its biological activity.41 (22) Beck, D. E. Eur. J. Surg. Suppl. 1997, 49–55. (23) Buckenmaier, C. C., 3rd; Summers, M. A.; Hetz, S. P. Am. Surg. 2000, 66, 1041–1045. (24) Szabo, A.; Haj, M.; Waxsman, I.; Eitan, A. Eur. Surg. Res. 2000, 32, 125–128. (25) van ’t Riet, M.; de Vos van Steenwijk, P. J.; Bonthuis, F.; Marquet, R. L.; Steyerberg, E. W.; Jeekel, J.; Bonjer, H. J. Ann. Surg. 2003, 237, 123–128. (26) Caruso, D. M.; Foster, K. N.; Blome-Eberwein, S. A.; Twomey, J. A.; Herndon, D. N.; Luterman, A.; Silverstein, P.; Antimarino, J. R.; Bauer, G. J. J. Burn Care Res. 2006, 27, 298–309. (27) Guest, J. F.; Ruiz, F. J.; Mihai, A.; Lehman, A. Curr. Med. Res. Opin. 2005, 21, 81–92. (28) Guest, J. F.; Ruiz, F. J. Curr. Med. Res. Opin. 2005, 21, 281–290. (29) Ravenscroft, M. J.; Harker, J.; Buch, K. A. Ann. R. Coll. Surg. Engl. 2006, 88, 18–22. (30) Barbucci, R.; Leone, G.; Vecchiullo, A. J. Biomater. Sci. Polym. Ed. 2004, 15, 607–619. (31) Chiumiento, A.; Dominguez, A.; Lamponi, S.; Villalonga, R.; Barbucci, R. J. Mater. Sci. Mater. Med. 2006, 17, 427–435. (32) Liu, L.; Fishman, M.; Hicks, K. Cellulose 2007, 14, 15–24. (33) Wu, P. C.; Huang, Y. B.; Fang, J. Y.; Tsai, Y. H. Drug DeV. Ind. Pharm. 1998, 24, 179–182. (34) Hindie, M.; Legeay, G.; Vayssade, M.; Warocquier-Clerout, R.; Nagel, M. D. Biomol. Eng. 2005, 22, 205–208. (35) Hindie, M.; Vayssade, M.; Dufresne, M.; Queant, S.; Warocquier-Clerout, R.; Legeay, G.; Vigneron, P.; Olivier, V.; Duval, J. L.; Nagel, M. D. J. Cell Biochem. 2006, 99, 96–104. (36) Velzenberger, E.; Vayssade, M.; Legeay, G.; Nagel, M. D. Cellulose 2008, 347–357. (37) Sousa, S. R.; Bras, M. M.; Moradas-Ferreira, P.; Barbosa, M. A. Langmuir 2007, 23, 7046–7054. (38) Garcia, A. J.; Vega, M. D.; Boettiger, D. Mol. Biol. Cell 1999, 10, 785– 798. (39) Nonckreman, C. J.; Rouxhet, P. G.; Dupont-Gillain, C. C. J. Biomed. Mater. Res. A 2007, 81, 791–802. (40) Faucheux, N.; Tzoneva, R.; Nagel, M. D.; Groth, T. Biomaterials 2006, 27, 234–245. (41) Hoffmann, C.; Leroy-Dudal, J.; Patel, S.; Gallet, O.; Pauthe, E. Anal. Biochem. 2008, 372, 62–71.
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To date, the authors employed labeling methods (radio- or fluorescent labeling) for the characterization of Fn-surface interactions. According to the literature, these protocols can induce protein conformational changes with the grafting of FITC or 125I, respectively. Furthermore, the ELISA-like protocols, which are based on specific antibody recognition, need some rinsing steps that may accidentally desorb the proteins of interest. Therefore, we propose to evaluate the advantages and the limits of different approaches for the examination of instantaneous and long-term affinities of Fn for different surfaces. The present work examines the relationships between surface properties, Fn adsorption, and preosteoblast adhesion. Indeed, MC-3T3 preosteoblasts were widely studied for their Fn-dependent adhesion.42,43 Accordingly, this cell line constitutes a good model for evaluating cell responses to various biomaterials: adhesive-PS and tPS- and antiadhesive-polyHEMA and CEL, even if these two last materials are not employed for orthopedic applications. Substrata topography and wettability were also characterized by AFM and contact angle measurements, respectively. The Fn-surface interactions were explored by combining ELISA immunoassays, fluorescent labeling of Fn, and force spectroscopy with Fn-modified AFM tips. We believe that these combined methods and results will help us to understand better how Fn adsorption and cell behavior are influenced by the surface chemistry of well-known surfaces used in biomaterial and cell culture applications.
Materials and Methods Substrata. All studies were performed with the following materials: (1) Bacteriological-grade polystyrene (PS) standard culture dishes (Greiner Bio-One, Courtaboeuf, France). (2) Tissue culture surface-modified (corona treatment, manufacturer specified) polystyrene (tPS) culture dishes (Nunclon ∆ Surface, Issy-les-Moulineaux, France). (3) polyHEMA-coated (Sigma-Aldrich) PS plates. PolyHEMA films were prepared extemporaneously by coating PS plates with a polyHEMA solution (12 mg/mL in 95% ethanol) to obtain a final density of 0.8 mg/cm2. Plates were dried overnight at 37 °C. PolyHEMA-coated dishes were sterilized by incubation with penicillin (200 U/mL, Gibco Invitrogen, Cergy-Pontoise, France) and streptomycin (200 µg/mL, Gibco Invitrogen) for 1 h, followed by three washes with ultrapure water. (4) Cellulose derivatives E4 M hydroxypropylmethylcellulose (HPMC, lower layer) and 7LF carboxymethylcellulose (CMC, upper layer) were supplied by Colorcon (Dartford, U.K.) and the Benacel Company (Hercules International Ltd., Rijswijk, The Netherlands) as pharmaceutical-grade products. Bilayers with 0.2% (w/v) HPMC and 0.2% (w/v) CMC were prepared using PS activated by glow discharges (CTTM, Le Mans, France). The process is described in French patent no. 2,862,979 (03-06-2005). Cellulose-coated Petri dishes (CEL) were sterilized with ethylene oxide. Water Contact Angle Measurements. Three dishes of each material (PS, tPS, polyHEMA, and CEL) were cut into small squares. Contact angle measurements were performed at room temperature (21-24 °C) with a drop-shape analysis system (DSA-10, Kru¨ss GmbH, Palaiseau, France). Four droplets of ultrapure water were deposited on different areas of each square. Contact angles were determined by using image analysis software, taking into account the entire drop shape. Three independent experiments were performed in triplicate. The contact angles reported in this work result from the average of 36 measurements per material. (42) Stephansson, S. N.; Byers, B. A.; Garcia, A. J. Biomaterials 2002, 23, 2527–2534. (43) Sousa, S. R.; Lamghari, M.; Sampaio, P.; Moradas-Ferreira, P.; Barbosa, M. A. J. Biomed. Mater. Res. A 2008, 84, 281–290.
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Figure 1. (a) Representation of a monomer of human plasma Fn with the areas recognized by the different monoclonal Abs used in this study. (b) Schematic representation of the modified AFM tips. Fn tip: Fn molecules were covalently bound to a tip terminated with COOH groups using NHS/EDC chemistry. COOH tip: Control tip terminated with COOH alkanethiols. OH tip: Control tip terminated with OH alkanethiols. Table 1. Water Contact Angle Measurements and Terminal Treatment of the Various Substrata substratum
θwater (deg)a
surface treatment
PS tPS polyHEMA CEL
89.7 ( 0.9 70.8 ( 1.3 44.9 ( 0.8
