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Langmuir 1997, 13, 3404-3413
Endothelial Cell Growth and Protein Adsorption on Terminally Functionalized, Self-Assembled Monolayers of Alkanethiolates on Gold Caren D. Tidwell, Sylvie I. Ertel, and Buddy D. Ratner* Center for Bioengineering and Department of Chemical Engineering, University of Washington, Seattle, Washington 98195
Barbara J. Tarasevich,† Sundar Atre, and David L. Allara* Departments of Materials Science and Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802 Received May 1, 1996X The effect of specific chemical functionalities on the growth of bovine aortic endothelial cells (BAEC) was investigated using a set of well-characterized, chemically functionalized surfaces prepared by selfassembly of alkanethiolate monolayers on gold surfaces using the molecules X(CH2)15SH with X ) -CH3, -CH2OH, -CO2CH3, and -CO2H. Cells seeded on a substrate in serum-containing culture medium interact with the adsorbed protein layer rather than the substrate. Therefore, the role of two serum proteins, albumin (Alb) (a nonadhesive or blocking protein) and fibronectin (Fn) (an adhesive protein), in cell growth was evaluated by measuring the amount of each protein bound and the tightness of binding (determined by resistance to SDS solubilization) on the self-assembled monolayers (SAMs). BAEC growth varied significantly with surface functionality. Cell growth increased in the following order: -CH2OH < -CO2CH3 < -CH3 , -CO2H, illustrating the effect of specific surface groups. Cell growth on all monolayer surfaces was lower than on tissue culture polystyrene suggesting that multiple chemical functionalities may be desirable for cell growth. Protein interaction studies demonstrated variations in protein adsorption and elutability in response to the SAM terminal functional group. Alb adsorption and Fn elutability did not differ significantly with terminal functional group. The best cell growth substrate (COOH SAM) demonstrated significantly higher Fn adsorption and Alb elutability than did the poor growth substrates (CO2CH3 and OH SAMs).
Introduction A high incidence of thrombotic occlusion in small diameter vascular grafts has been observed clinically.1 When endothelial cells, the natural antithrombotic surface contacting the blood in native vessels, are seeded onto synthetic grafts, improved graft healing is observed.2,3 Therefore, methods for promoting graft endothelialization such as cell seeding at implantation,4 cell culture prior to implantation,5 and graft surface coating with specific plasma proteins6,7 have been investigated to produce a thromboresistant surface. These methods all require a substrate that will promote endothelial cell attachment and growth. Anchorage-dependent cells, such as endothelial cells, require adhesion to a substrate in order for growth to occur. In addition, adhesion alone is not sufficient to ensure that cell growth will occur; cell spreading is a critical condition necessary for growth. The ability of polymer surfaces to support the attachment, spreading, and growth of anchorage-dependent cells varies significantly. The attachment and growth of endothelial cells † Currently at Pacific Northwest National Laboratory, Richland, WA 99352. X Abstract published in Advance ACS Abstracts, June 1, 1997.
(1) Chard, R. B.; Johnson, D. C.; Nunn, G. R.; Cartmill, T. B. J. Thorac. Cardiovasc. Surg. 1987, 94 (1), 132-134. (2) Zilla, P.; Fasol, R.; Deutsch, M.; Fischlein, T.; Minar, E.; Hammerle, A.; Krupicka, O.; Kadletz, M. J. Vasc. Surg. 1987, 6 (6), 535-541. (3) Ortenwall, P.; Wadenvick, H.; Kutti, J.; Risberg, B. J. Vasc. Surg. 1987, 6 (1), 17-25. (4) Graham, L. M.; Burkel, W. E.; Ford, J. W.; Vinter, D. W.; Kahn, R. H.; Stanley, J. C. Surgery 1982, 91 (5), 550-559. (5) Shindo, S.; Takagi, A.; Whittemore, A. D. J. Vasc. Surg. 1987, 6 (4), 325-332. (6) Joseph, J.; Sharma, C. P. J. Biomed. Mater. Res. 1986, 20, 677682. (7) Fischer, A. M.; Mauzac, M.; Tapon-Bretaudiere, J.; Jozefonvicz, J. Biomaterials 1985, 6, 198-202.
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onto synthetic surfaces have been associated with various surface properties including wettability,8 electronegativity,9 chemical composition,10,11 and adsorption of specific plasma proteins.12,13 Since plasma proteins rapidly adsorb to synthetic materials in a biological environment, material surface properties affect cellular interactions via the composition, structure, and conformation of the adsorbed protein layer.12,14-16 The composition of the adsorbed protein layer differs from the fluid phase composition, varies significantly with type of substrate, and has been demonstrated to undergo conformational and orientational changes with time.17-19 Therefore, protein adsorption onto a substrate is important in controlling cellular interactions with synthetic surfaces in vivo and also in vitro, where serum is added to culture media to sustain cell growth. Few studies, however, have addressed the effect of surface properties on cell behavior as mediated by the protein (8) van Wachem, P. B.; Beugeling, T.; Feijen, J.; Bantjes, A.; Detmers, J. P.; van Aken, W. G. Biomaterials 1985, 6, 403-408. (9) van Wachem, P. B.; Hogt, A. H.; Beugeling, T.; Feijen, J.; Bantjes, A.; Detmers, J. P.; van Aken, W. G. Biomaterials 1987, 8, 323-327. (10) Maroudas, N. G. J. Cell. Physiol. 1976, 90, 511-520. (11) Curtis, A. S. G.; Forrester, J. V.; McInnes, C.; Lawrie, F. J. Cell Biol. 1983, 97, 1500-1506. (12) van Wachem, P. B.; Vreriks, C. M.; Beugeling, T.; Feijen, J.; Bantjes, A.; Detmers, J. P.; van Aken, W. G. J. Biomed. Mater. Res. 1987, 21, 701-718. (13) Kaehler, J.; Zilla, P.; Fasol, R.; Deutsch, M.; Kadletz, M. J. Vasc. Surg. 1989, 9 (4), 535-541. (14) Horbett, T. A. In Biomaterials: Interfacial Phenomena and Applications; Cooper, S. L., Peppas, N. A., Eds.; ACS Symposium Series No. 199; American Chemical Society: Washington, DC, 1982; p 233. (15) Grinnell, F. In Biocompatible Polymers, Metals and Composites; Szycher, M., Ed.; Technomic Publishing Co.: Lancaster, 1983; p 674. (16) Lindon, J. N.; McManama, G.; Kushner, L.; Merrill, E. W.; Saltzman, E. W. Blood 1986, 68 (2), 355-362. (17) Horbett, T. A. J. Biomed. Mater. Res. 1981, 15, 673-695. (18) Uniyal, S.; Brash, J. L. Thromb. Haemostasis 1982, 47 (3), 285290. (19) Horbett, T. A. Colloids Surf. B: Biointerfaces 1994, 2, 225-240.
© 1997 American Chemical Society
BAEC Growth and Protein Adsorption on SAMs
layer adsorbed from complex solutions such as plasma and serum. The influence of polymer surface chemistry on cell attachment and growth is demonstrated by the finding that hydrophobic polymers such as polystyrene (PS) or Teflon do not support cell attachment unless precoated with an adhesive protein such as fibronectin (Fn) or vitronectin (Vn), while hydrophilic tissue culture polystyrene (TCPS) will support cell attachment without precoating with an adhesive serum protein.20,21 In general, hydrophobic surfaces, including currently available vascular graft materials such as expanded poly(tetrafluoroethylene) (PTFE), are poor supports for the growth of endothelial cells while hydrophilic surfaces are more supportive of cell attachment and growth. Very hydrophilic surfaces (i.e., hydrogels or agarose), however, are not supportive of cell attachment and growth, and maximal cell adhesion has been reported on surfaces of intermediate wettability.12 Surface wettability, therefore, is not solely predictive of the cell growth characteristics of a substrate. The improved cell growth characteristics of hydrophilic surfaces such as TCPS have been attributed to oxygencontaining functional groups. However, determination of the specific functionalities involved in cell growth is difficult, and contradictory results have been reported. Surface sulfonate, hydroxyl, and carboxyl groups have all been alternatively proposed to promote cell attachment and growth.10,11 For nitrogen-containing surfaces, amine and amide groups have been proposed to promote cell attachment and growth. Stenger et al. have demonstrated the promotion of neural cell adhesion and growth on aminoterminated alkylsilanes.22,23 Since protein adsorption from serum-containing culture medium occurs rapidly rendering direct recognition of surface functional groups by the cells virtually impossible, functional groups are believed to affect cell growth indirectly via the adsorbed protein layer. Therefore, differences observed in cell growth on different surfaces are most likely due to differences in the adsorption of proteins by the polymer surface.19,24 Polymer surface properties can affect the amounts and types of proteins bound as well as the conformation, orientation, or binding strength of the adsorbed protein. Additionally, for cells which secrete and assemble an extracellular matrix (ECM), such as endothelial cells, polymer surface properties may affect the ability of the cell to deposit its ECM by stabilizing the protein deposits or by affecting the orientation of cell-binding domains in the deposits.19,24 The role of surface chemistry, specifically chemical functional groups, and the effect of adsorbed proteins in promoting endothelial cell growth was investigated by Ertel et al.25-27 Bovine aortic endothelial cell (BAEC) growth on radio-frequency plasma-deposited films generally increased with surface oxygen content; however, it was proposed that cell growth was better correlated with specific oxygen-containing functionalities than with total (20) Steele, J. G.; Johnson, G.; Underwood, P. A. J. Biomed. Mater. Res. 1992, 26, 861-884. (21) Steele, J. G.; Dalton, B. A.; Johnson, G.; Underwood, P. A. J. Biomed. Mater. Res. 1993, 27, 927-940. (22) Stenger, D. A.; Georger, J. H.; Dulcey, C. S.; Hickman, J. J.; Rudolph, A. S.; Nielsen, T. B.; McCort, S. M.; Calvert, J. M. J. Am. Chem. Soc. 1992, 114 (22), 8435-8442. (23) Stenger, D. A.; Pike, C. J.; Hickman, J. J.; Cotman, C. W. Brain Res. 1993, 630, 136-147. (24) 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. (25) Ertel, S. I.; Ratner, B. D.; Horbett, T. A. J. Biomed. Mater. Res. 1990, 24, 1637-1659. (26) Ertel, S. I.; Chilkoti, A.; Horbett, T. A.; Ratner, B. D. J. Biomater. Sci., Polym. Edn. 1991, 3, 163-183. (27) Ertel, S. I.; Ratner, B. D.; Horbett, T. A. J. Colloid Interface Sci. 1991, 147, 433-442.
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oxygen content. BAEC growth on plasma-deposited films was subsequently correlated with surface carbonyl concentration but not with surface hydroxyl or carboxyl concentrations. Other groups have similarly investigated the role of chemical functionalities in cell growth; however, contradictory results have been reported.24 Comparison between studies investigating the role of chemical functionalities in cell attachment and growth is complicated by the use of different cell lines and different cell interaction protocols. Elucidating relationships between surface properties and cellular responses is often difficult since the polymer systems typically used in biointeraction studies do not allow for systematic, controlled variations in material surface properties. Cell culture surfaces (prepared by gas plasma (glow discharge) methods) are typically chemically complex and contain a broad spectrum of chemical functional groups rendering it difficult to determine the effect of any one chemical functionality on cell growth. Many polymer surfaces are dynamic and can undergo conformational rearrangements in response to environmental changes. When exposed to biological media, buried polar groups in a mobile polymer may reemerge at the surface and the adsorbing protein layer may adapt to the existing polymer surface which may differ compositionally from the composition of the polymer in air. To effectively determine the chemical functionalities and/or composition responsible for influencing protein adsorption and cell growth, a set of surfaces exhibiting systematic variations in surface chemistry which exhibit conformational stability in biological media is required. Molecular self-assembly techniques provide an effective means of fabricating organic surfaces with well-defined structure and chemistry. Surfaces which vary in surface chemistry can be prepared by the self-assembly of alkanethiols terminated with a variety of terminal functional groups onto gold substrates.28,29 Self-assembled monolayers (SAMs) of alkanethiols on gold are stable in a variety of organic and aqueous media, making them particularly suitable for studies of the present type. A few studies have demonstrated the utility of SAMs in investigating the effect of surface chemistry on protein and cellular interactions. Lewandowska et al. investigated cell growth on terminally functionalized SAMs of alkylsilanes on glass and found unique cell behavior that corresponded to the conformation of adsorbed Fn as mediated by the surface chemical functionality.30 Prime and Whitesides31,32 concluded that SAMs of alkanethiolates on gold were useful model systems for investigating mechanisms of protein adsorption in a study of monolayers containing mixtures of hydrophobic and hydrophilic alkanethiols. Lopez et al. recently described methods for controlling the attachment and spreading of mammalian cells on surfaces using patterned SAMs of alkanethiols on gold.33 SAMs terminated with an oligo(ethylene glycol) group uniformly prevented cell and protein attachment, while other functional groups including CH3 and CO2H promoted protein adsorption and cell attachment to varying degrees. In this study, we investigated the effect of specific chemical functionalities on the growth of BAEC using a (28) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558-569. (29) Dubois, L. H.; Nuzzo, R. G. Annu. Rev. Phys. Chem. 1992, 43, 437-463. (30) Lewandowska, K.; Pergament, E.; Sukenik, C. N.; Culp, L. A. J. Biomed. Mater. Res. 1992, 26, 1343-1363. (31) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167. (32) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714-10721. (33) Lopez, G. P.; Albers, M. W.; Schrieber, S. L.; Carroll, R.; Peralta, E.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 5877-5878.
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set of chemically functionalized surfaces prepared by the self-assembly of alkyl thiols terminally substituted with hydroxyl, methyl, methyl ester, and carboxyl groups. Since cells seeded on a substrate in serum-containing culture medium interact with the adsorbed protein layer rather than with the substrate itself, the effect of chemical functionality on the adsorbed protein layer was investigated in conjunction with cell growth. The role of two serum proteins, Alb (albumin, a nonadhesive or blocking protein) and Fn (an adhesive protein), in cell growth was evaluated by measuring the amount of each protein bound and the tightness of binding (determined by resistance to SDS solubilization) on the SAM substrates. We hypothesize that the amounts and types of proteins bound as well as the binding strength of the adsorbed proteins are affected by substrate chemistry. This is the first report in which protein-surface interactions (adsorption and binding strength) have been quantified and related to endothelial cell growth on wellcharacterized SAM surfaces. We determined that protein adsorption, protein elutability, and BAEC growth on the terminally functionalized surfaces differed in response to the presence of specific surface functional groups. Correlations between cell growth and adsorbed protein layer characteristics were observed. We found that BAEC growth on the SAM surfaces improved in the following sequence: hydroxyl < methyl ester < methyl , carboxyl. The significant differences in cell growth performance between different surfaces and the strong response to the carboxyl functionality are important observations. Materials and Methods Substrate Preparation. Si(100) wafers (Silicon Quest International, Santa Clara, CA; single-side polish, 3 in. diameter, ∼0.35 mm thick) were laser cut (EBTEC Radcliffe, Pasadena, CA) into 15 mm diameter circles (cell growth studies) or 10 × 10 mm squares (surface characterization studies). Double-side polished Si(100) wafers were laser cut into 10 × 10 mm squares (protein adsorption studies). Silicon wafers were cleaned by immersion in 1:4 H2O2/H2SO4 mixtures at 85 °C for 10 min (Warning: This solution reacts strongly with organic compounds and should not be stored in closed containers. It must be handled with extreme caution.) followed by rinses in deionized water and absolute ethanol. Substrates were coated by resistive evaporation of a 100 Å chromium adhesion layer followed by a 2000 Å gold layer (99.99%). Deposition of gold occurred at
