Direct Comparison of the Spread Area, Contractility, and Migration of balb/c 3T3 Fibroblasts Adhered to Fibronectin- and RGD-Modified Substrata Padmavathy Rajagopalan, William A. Marganski, Xin Q. Brown, and Joyce Y. Wong Department of Biomedical Engineering, Boston University, Boston, Massachusetts ABSTRACT Native proteins are often substituted by short peptide sequences. These peptides can recapitulate key, but not all biofunctional properties of the native proteins. Here, we quantify the similarities and differences in spread area, contractile activity, and migration speed for balb/c 3T3 fibroblasts adhered to fibronectin- (FN) and Arg-Gly-Asp (RGD)-modified substrata of varying surface density. In both cases spread area has a biphasic dependence on surface ligand density (s) with a maximum at s 200 molecules/mm2, whereas the total traction force increases and reaches a plateau as a function of s. In addition to these qualitative similarities, there are significant quantitative differences between fibroblasts adhered to FN and RGD. For example, fibroblasts on FN have a spread area that is on average greater by 200 mm2 over a 40-fold change in s. In addition, fibroblasts on FN exert 3���5 times more total force, which reaches a maximum at a value of s 5 times less than for cells adhered to RGD. The data also indicate that the differences in traction are not simply a function of the degree of spreading. In fact, fibroblasts on FN (s 2000 mm��2) and RGD (s 200 mm��2) have both similar spread area ( 600 mm2) and migration speed ( 11 mm/h), yet the total force production is five times higher on FN than RGD ( 0.05 dyn compared to 0.01 dyn). Thus, the specific interactions between fibroblasts and FN molecules must inherently allow for higher traction force generation in comparison to the interactions between fibroblasts and RGD. INTRODUCTION Specific interactions between cell surface receptors and extracellular matrix (ECM) molecules underlie a wide variety of processes such as cell adhesion, traction force generation, and migration (Akiyama et al., 1985 Zamir et al., 1999 Geiger et al., 2001). These interactions collectively give rise to even more complex physiological phenomena such as tumor metastasis, wound healing, and tissue morphogenesis (Trinkaus, 1969 Harris, 1986 Lauffenburger and Horwitz, 1996). Cells exert tractional forces through adhesive contacts (e.g., focal adhesions), and tractional forces in turn are required for cell migration. However, cell migration is not required for traction force generation. In fact, cells can exhibit low migration rates and yet exert substantial traction forces through contractile proteins. Moreover, both positive and negative correlations between traction and migration have been observed, depending on the cell type (Shreiber et al., 2003). The relationship between traction and migration also depends on the nature of the ECM, and numerous studies have investigated how cell-ECM interactions control adhesion and migration (Brandley and Schnaar, 1988 Massia and Hubbell, 1990 DiMilla et al., 1993 Garcia et al., 1999 Maheshwari et al., 2000 Hersel et al., 2003). In contrast, there have been relatively few studies that have directly probed the effect of specific cell-ECM interactions on traction force generation (Gaudet et al., 2003 Reinhart- King et al., 2003) or directly compared adhesion, traction, and migration (Gaudet et al., 2003). These types of studies are critical for engineering the cell-biomaterial interface to recapitulate phenomena such as wound healing and tissue morphogenesis. Here, we directly compare cellular response on the well-studied protein fibronectin (FN) and its cell adhesion peptide mimic Arg-Gly-Asp (RGD). Although there have been numerous qualitative studies comparing effects of RGD and FN, to our knowledge, the relationships between cell spreading, migration, and traction force gener- ation on RGD- versus FN-modified substrata have not yet been thoroughly explored. The cellular response to FN depends not only on the surface density of the molecule, but also its conformation. For example, recent in vitro studies have shown that the conformation of adsorbed FN is not only dependent on the properties of the substrate, but that these changes in FN conformation also lead to differences in cellular response (Garcia et al., 1999). Furthermore, the precise control of the surface conformation of ECM proteins such as FN requires site-specific immobilization and is rather challenging. In contrast, it is much easier to manipulate and preserve the conformation of short synthetic peptide sequences. There- fore, peptide sequences are frequently used in place of the Submitted November 11, 2003, and accepted for publication June 18, 2004. Padmavathy Rajagopalan and William A. Marganski contributed equally to this work. Address reprint requests to Joyce Y. Wong, Dept. of Biomedical Engineering, Boston University, 44 Cummington Street, Boston, MA 02215. Tel.: 617-353-2374 Fax: 617-353-6766 Email: jywong@bu.edu. Padmavathy Rajagopalan���s present address is Center for Engineering in Medicine, Harvard Medical School, Massachusetts General Hospital, Boston, MA. �� 2004 by the Biophysical Society 0006-3495/04/10/2818/10 $2.00 doi: 10.1529/biophysj.103.037218 2818 Biophysical Journal Volume 87 October 2004 2818���2827

native protein. The best example is RGD, which is found in the cell-binding domain of FN (Pierschbacher and Ruoslahti, 1984 Ruoslahti, 1996) and has been incorporated into a wide variety of otherwise nonadhesive substrata to promote cell adhesion, spreading, and focal contact formation (Massia and Hubbell, 1990, 1991). However, several studies have also shown that RGD-modified substrata do not completely recapitulate the same phenotype and behavior observed in cells adhered to FN-modified substrata. For instance, cells adhered to RGD-modified substrata exhibit significant dif- ferencesinmigrationspeed(Maheshwarietal.,2000),spread- ing and focal contact formation (Streeter and Rees, 1987) compared to cells adhered to FN-modified substrata. Although these studies clearly show differences in cell behavior on FN- versus RGD-modified substrata, they do not give any information regarding the traction stresses exerted by these cells. The lack of quantitative comparisons of cell traction on FN- and RGD-modified substrata can be at- tributed to the fact that methods to quantify cell traction have only recently been developed (Dembo and Wang, 1999 Balaban et al., 2001 Tan et al., 2003). Because focal contact formation has been linked to traction force generation (Balaban et al., 2001 Tan et al., 2003) and also because cells exert traction forces during migration (Lee et al., 1994 Oliver et al., 1994 Munevar et al., 2001b), we hypothesize that cellular traction force generation will differ on FN- and RGD-modified substrata. To quantify the differences and similarities between RGD and FN on cellular response, we directly compare the spread area, contractility, and migration of balb/c fibroblasts that are adhered to polyacrylamide substrata modified with varying surface densities of RGD and FN. Polyacrylamide substrata are utilized because they are nontoxic, elastic, and require the covalent attachment of a specific ligand to promote cell adhesion. Thus, we are able to follow the effects of both ligand type and surface density on three different aspects of cellular behavior. Spread area and contractility are measured using traction force microscopy (Dembo and Wang, 1999 Lo et al., 2000 Beningo et al., 2001 Munevar et al., 2001a,b Wang et al., 2001 Gaudet et al., 2003 Marganski et al., 2003a Doyle et al., 2004). Cell migration is quantified using a random walk model (Dunn, 1983 DiMilla et al., 1992b). This systematic approach provides a quantitative functional comparison of cellular behavior on RGD- and FN- modified substrata. MATERIALS AND METHODS Synthesis of modified polyacrylamide (PAAM) substrata PAAM substrata are synthesized by copolymerizing acrylamide (BioRad, Hercules, CA), bis-acrylamide (BioRad) and acrylic acid N-hydroxy succinimide (NHS) ester (Sigma-Aldrich, St. Louis, MO) (Brandley and Schnaar, 1988 Brandley et al., 1990). Specifically, the polymerization mixture contains 8% acrylamide, 0.04% bis-acrylamide, 10 mmol/ml acrylic acid NHS ester, 2 ml 0.25 M HEPES (4-(2-hydroxyethyl)-1-piperazinee- thanesulfonic acid Sigma-Aldrich), 200 ml 0.75 mm-diameter fluorescent marker beads (Fluoresbrite carboxylate microspheres Polysciences Inc., Warrington, PA), and 15 mL TEMED (N,N,N#,N#- tetramethylethylenedi- amine BioRad). The pH of the solution is adjusted to 6.0 by careful addition of 1 M HCl: it is critical to use a pH of 6.0, since a lower pH can inhibit polymerization whereas a higher pH can hydrolyze the NHS ester. Polymerization is initiated by adding 10% aqueous ammonium persulfate and the solution is cast onto glass cover slips that are activated with 3-aminopropyltrimethoxysilane and glutaraldehyde (Wang and Pelham, 1998). FN or the hexapeptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) is co- valently linked to the substratum through the NHS ester. Various plating concentrations (Table 1) of human plasma FN (Invitrogen, Carlsbad, CA) and the hexapeptide GRGDSP (Invitrogen) are prepared in cold, sterile PBS (phosphate-buffered saline Invitrogen) and HEPES (pH �� 8). The PAAM substrata are incubated in the peptide or protein solution at 4��C for 24 h. After the incubation period, ethanolamine (1 ml/ml in 50 mM HEPES and 10% ethanol) is added to react (15 min) with the remaining hydrolyzed ester groups to prevent nonspecific cell adhesion. Lastly, before cell culture, the substrata are rinsed in cold deionized water for 1 h and sterilized under UV radiation for 30 min. Quantification of FN and RGD covalently attached PAAM substrata The coupling efficiencies of FN and RGD to polyacrylamide substrata are quantified using radioactivity. Briefly, I-125 labeled FN (MP Biomedicals, Irvine, CA) and I-125 labeled YRGDS (Phoenix Pharmaceuticals, Belmont, CA) are added to unlabeled FN and RGD at a ratio of 0.8 mCi/mg FN and 10 mCi/mg GRGDSP. These mixtures are then covalently linked via the NHS ester to the polyacrylamide substrata. Finally, the substrata are rinsed in cold water three times, and the radioactivity of each gel is quantified with a gamma-counter (Cobra Auto-Gamma B5005 Gamma Counter, Packard Instruments, Meriden, CT). These radioactivity values are then converted into mass amounts using a standard curve that is generated by mea- suring the radioactivity of known amounts of I-125 labeled FN and RGD. TABLE 1 Relationship between plating and surface density for both fibronectin (FN) and GRGDSP (RGD) Concentration Number of cells Substrate Plating s ��A��, ��jFj��, ��jTj�� ��S�� (mg/ml) (Molecules/mm2) n n FN 0.5 10 - 9 FN 1.56 30 - 16 FN 3.13 60 8 - FN 6.25 120 14 - FN 12.5 240 15 27 FN 25 475 24 35 FN 50 950 21 37 FN 100 1900 15 40 RGD 25 50 13 23 RGD 50 100 12 25 RGD 100 200 10 25 RGD 250 500 17 27 RGD 500 1000 8 30 RGD 750 1500 6 - Polyacrylamide substrata were modified with indicated plating concen- trations of FN or RGD. The immobilized surface density (s) of FN or RGD on the substratum is given for each plating concentration. The total number of cells for each measurement is indicated by n. ��jTj��, ��jFj��, ��A��, and ��S�� are the population-averages of the traction magnitude, total absolute force, projected area, and migration speed, respectively. Fibroblast Behavior on Fibronectin- Versus RGD-Modified Substrata 2819 Biophysical Journal 87(4) 2818���2827