Measurement of Cell Migration on Surface-Bound Fibronectin

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Measurement of Cell Migration on Surface-Bound Fibronectin Gradients Jason T. Smith,† John K. Tomfohr,‡ Matthew C. Wells,§ Thomas P. Beebe, Jr.,§ Thomas B. Kepler,‡ and W. Monty Reichert*,† Department of Biomedical Engineering and Department of Biostatistics and Bioinformatics, Duke University, Durham, North Carolina 27708, and Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716 Received April 22, 2004. In Final Form: June 16, 2004 A novel technique for the quantitative observation of cell migration along linear gradient substrates functionalized with adhesive proteins is presented. Gradients of the cell adhesion molecule fibronectin are generated by the cross diffusion of functionalizable alkanethiols on gold and characterized by X-ray photoelectron spectroscopy and surface plasmon resonance. Two distinct migration assays are described that characterize the movement of either sparsely populated noncontacting cells or a confluent monolayer of cells into free space. The drift speed of bovine aortic endothelial cells is measured and shown to increase along a fibronectin gradient when compared to a uniform control substrate using both assays. The results of these experiments establish reproducible conditions for studies of cell migration on gradients of surfacebound ligands.

Introduction Cell movement in response to soluble and matrix-bound stimuli is essential to a wide variety of phenomena, such as the immune response, wound healing, and tumorgenesis.1 In addition to the large body of work that exists for leukocytes such as neutrophils2 and macrophages,3 significant effort has been focused on the migration of smooth muscle cells,4 endothelial cells (ECs),5-7 and fibroblasts8,9 that are involved in angiogenesis and wound healing. Cells respond to stimuli such as chemical gradients6 and substrate compliance.10 Attachment and migration also play a critical role in the patency of vascular graft materials with lumenal EC seeding.11 Several techniques have been developed to characterize the movement of both individual cells and whole populations. The wound assay,12 the Teflon fence assay,13 and * Corresponding author: tel., 1 919 660-5151; fax, 1 919 6844488; e-mail, [email protected]. † Department of Biomedical Engineering, Duke University. ‡ Department of Biostatistics and Bioinformatics, Duke University. § Department of Chemistry and Biochemistry, University of Delaware. (1) Singer, S. J.; Kupfer, A. Annu. Rev. Cell Biol. 1986, 2, 337-365. (2) Tranquillo, R. T.; Zigmond, S. H.; Lauffenburger, D. A. Cell Motil. Cytoskeleton 1988, 11 (1), 1-15. (3) Jones, G. E. J. Leukocyte Biol. 2000, 68 (5), 593-602. (4) Dimilla, P. A.; Stone, J. A.; Quinn, J. A.; Albelda, S. M.; Lauffenburger, D. A. J. Cell Biol. 1993, 122 (3), 729-737. (5) Soga, N.; Namba, N.; McAllister, S.; Cornelius, L.; Teitelbaum, S. L.; Dowdy, S. F.; Kawamura, J.; Hruska, K. A. Exp. Cell Res. 2001, 269 (1), 73-87. (6) Stokes, C. L.; Rupnick, M. A.; Williams, S. K.; Lauffenburger, D. A. Lab. Invest. 1990, 63 (5), 657-668. (7) Funasaka, T.; Haga, A.; Raz, A.; Nagase, H. Biochem. Biophys. Res. Commun. 2001, 284 (5), 1116-1125. (8) Maheshwari, G.; Wells, A.; Griffith, L. G.; Lauffenburger, D. A. Biophys. J. 1999, 76 (5), 2814-2823. (9) Bromberek, B. A.; Enever, P. A. J.; Shreiber, D. I.; Caldwell, M. D.; Tranquillo, R. T. Exp. Cell Res. 2002, 275 (2), 230-242. (10) Wong, J. Y.; Velasco, A.; Rajagopalan, P.; Pham, Q. Langmuir 2003, 19 (5), 1908-1913. (11) Hsu, P. P.; Li, S.; Li, Y. S.; Usami, S.; Ratcliffe, A.; Wang, X.; Chien, S. Biochem. Biophys. Res. Commun. 2001, 285 (3), 751-759. (12) Majack, R. A.; Clowes, A. W. J. Cell. Physiol. 1984, 118 (3), 253-256.

the phagokinetic track assay14 all measure cell movement on uniform substrates using information from only the beginning and end of the experiment. Cells migrate from known positions defined by the system geometry and are fixed for viewing at the end of the experiment to determine the net distance traveled during the experiment. Videomicroscopy techniques have provided the ability to continuously monitor cell behavior during migration and introduced the possibility of tracking discrete cell motion.15 To date, little work has been done to characterize cell motion in response to gradients of surface-bound molecules. Boyden et al. (1962) studied cell response to gradient stimuli; however, the lack of specific information about the gradient generated in the porous mesh and the inability to observe cell motion during experiments have limited the utility of the Boyden chamber.16 Carter et al. (1965) reported that mouse fibroblasts would move along gradients of increasing cell adhesiveness on acetate substrates evaporated with palladium. This first example of cell migration along gradient surfaces led to the coining of the term “haptotaxis” from the greek: haptein, to fasten; taxis, arrangement.17 Only recently have gradient surfaces seen a rebirth as a tool to study the behavior of anchoragedependent cells. Lee et al. reported cell seeding and protein adhesion on wettability gradients generated by radio frequency corona discharge treatment of polyethylene.18 Several researchers have generated functionalizable gradients using the cross-diffusion of alkanethiols on gold and silanes on glass.19 Bohn has demonstrated the generation of alkanethiol gradients using an electro(13) Pratt, B. M.; Harris, A. S.; Morrow, J. S.; Madri, J. A. Am. J. Pathol. 1984, 117 (3), 349-354. (14) Lewis, L.; Albrechtbuehler, G. Cell Motil. Cytoskeleton 1987, 7 (3), 282-290. (15) Dow, J. A. T.; Lackie, J. M.; Crocket, K. V. J. Cell. Sci. 1987, 87 171-182. (16) Boyden, W. V. J. Exp. Med. 1962, 115, 453. (17) Carter, S. B. Nature 1965, 208, 1183-1187. (18) Lee, J. H.; Lee, H. B. J. Biomed. Mater. Res. 1998, 41 (2), 304311. (19) Ruardy, T. G.; Schakenraad, J. M.; Vandermei, H. C.; Busscher, H. J. Surf. Sci. Rep. 1997, 29 (1), 3-30.

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chemical technique.20 Gradient techniques have significant potential for the study of cell migration in response to immobilized proteins; however, published work is limited to proteins adsorbed to gradients of wettability. Movement of anchorage-dependent cell types can be attributed to random motility, chemotaxis, haptotaxis, or the sum of their effects. Random motility is the persistent random movement of a cell that over a short time period, called the persistence time, tends to persist in one direction, but over a long period of time will result in no directional displacement. Random motility can be altered by both soluble factors and adhesion molecules on the surface21 but remains inherently nondirectional. Chemotaxis is a biased and persistent movement of cells in response to a gradient of soluble stimuli in conjunction with random motility, which can be either attractive or repulsive. Haptotaxis is essentially the same phenomenon as chemotaxis, except in response to a surface-bound gradient. The modeling of cell movement in response to chemotactic and haptotactic gradients is critical for differentiating random and directed components of cell motion. Dunn has demonstrated that modeling cell movement as a persistent random walk allows the determination of cell characteristics more properly than do other methods such as calculating the average distance migrated during a given time interval.22 The Langevin equation, which was originally used to describe the persistent random movement of pollen grains on the surface of water, is used in these experiments. Persistent random walk models are typically used to fit the mean squared displacement vs time curve to obtain parameters that define the speed (S), persistence time (P), and random motility coefficient (µ).23 Although these variables sufficiently characterize changes in cell movement in response to stimulus on uniform substrates, they provide little information about the directional nature of cell motility. The parameters used to describe cell motion in this work are random speed (Srandom) and drift speed (Sdrift) along the gradient, which are more descriptive of directed cell movement. However, µ values are also provided for comparison to previous studies. This paper presents a novel experimental technique for the quantitative observation of cell motion along linear gradients on substrates functionalized with adhesive proteins. Two distinct migration assays characterize the movement of either a monolayer of cells into free space or sparsely populated noncontacting cells. The initial demonstration of these assays measured the drift speed of bovine aortic endothelial cells (BAECs) on gradients of the cell adhesion molecule fibronectin (Fn). Surfaces with uniform fibronectin concentration served as negative controls. The results of these experiments established reproducible conditions for studies of cell migration on gradients of surface-bound ligands. Methods Materials. Absolute ethanol, methanol, hydrogen peroxide, ammonium hydroxide, hydrochloric acid, and acetic acid were purchased from VWR Scientific Products. Unless specifically mentioned, all other materials were purchased from Sigma and used without further purification. (20) Balss, K. M.; Coleman, B. D.; Lansford, C. H.; Haasch, R. T.; Bohn, P. W. J. Phys. Chem. B 2001, 105 (37), 8970-8978. (21) Maheshwari, G.; Lauffenburger, D. A. Microsc. Res. Tech. 1998, 43 (5), 358-368. (22) Dunn, G. A.; Brown, A. F. J. Cell Sci. 1987, 81-102. (23) Stokes, C. L.; Lauffenburger, D. A.; Williams, S. K. J. Cell Sci. 1991, 99, 419-430.

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Gradient Generation. All substrates were formed using self-assembled monolayers (SAMs) of alkanethiols on gold-coated glass slides. Glass slides were prepared for gold deposition by cleaning in 5% RBS detergent followed by cleaning in HCl/methanol 1:1 (vol/vol) and inductively coupled plasma treatment at 10 W for 5 min. A 4 nm Cr adhesion layer and a 20 nm Au layer were thermally evaporated on the inverted glass slides without breaking vacuum yielding optically transparent gold-coated slides for further experiments. Alkanethiol gradients were generated on optically transparent gold-coated slides by cross diffusion of the two alkanethiols using a previously described method.24,25 Briefly, gold-coated slides were placed in an 8.5 × 8.5 cm polystyrene Petri dish with 5 g of Sephadex gel (Amersham Pharmacia Biotech) and 16 g of ethanol causing swelling of the gel and forming a diffusion matrix on the slide surface. Evaporation was allowed to occur until the gel was firm yet moist with a final thickness of 5 mm and a final composition of 3.5-4.0 mL of ethanol g-1 Sephadex gel. Glass filters (Ace Glass) measuring 8 × 8 × 50 mm with pore sizes of 0.1-0.2 mm were gently placed into the gel at the ends of the glass slides. Solutions of either 16mercaptohexadecanoic acid (HDA) or 11-mercapto-1undecanol (MUD), 2 mM in an 85/10/5 (vol) solution of ethanol/H2O/acetic acid, were added to the filters on opposing ends of the glass substrates. The Petri dish was covered and sealed with Parafilm to avoid evaporation for 48 h. The slides were removed from the gel, initially rinsed with ethanol, and subsequently cleaned in an ultrasonic bath in ethanol (fresh solvent) for 1 min. Fibronectin Treatment. Fibronectin was covalently attached to the substrate utilizing the activity of the carboxy terminus of the HDA. Gradients containing HDA for covalent attachment were activated by treatment with N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) (0.05 and 0.2 M in PBS, respectively) for 20 min. All gradients were then incubated in a 10 µg/mL solution of fibronectin at 37 °C for over 3 h. Surface Plasmon Resonance (SPR) Measurements. The quantitative determination of surface concentration of protein using SPR has been described in detail previously.26 Experiments were carried out using a BIACORE X (Pharmacia Biosensor) microfluidic system. Protein solutions in phosphate buffer were brought into contact with treated substrates using a flow rate of 5 µL/ min for up to 20 min. After the loose protein was removed by washing with buffer, the absolute coupling angle change, given in response units (RU), was converted to weight of adsorbed protein using the relationship: 1000RU (0.1 degree change) corresponds to 1 ng/mm2 of surfacebound protein. X-ray Photoelectron Spectroscopy (XPS) Measurements. XPS data were obtained using a VG ESCALAB 220i-XL electron spectrometer (VG scientific Ltd., East Grinstead, U.K.). Monochromatic Al KR X-rays (1486.7 eV) were employed. Typical operation conditions for the X-ray source were a 400 µm nominal X-ray spot size (full width at half-maximum, fwhm) operating at 15 kV, 8.9 mA, and 124 W for both survey and high-resolution spectra. Survey spectra, from 0 to 1200 eV in binding energy were collected at a 100-eV pass energy with an energy resolution of ∼1.0 eV, a dwell time of 100 ms per (24) Liedberg, B.; Tengvall, P. Langmuir 1995, 11 (10), 3821-3827. (25) Smith, J. T.; Viglianti, B. L.; Reichert, W. M. Langmuir 2002, 18 (16), 6289-6293. (26) Stenberg, E.; Persson, B.; Roos, H.; Urbaniczky, C. J. Colloid Interface Sci. 1991, 143 (2), 513-526.

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point, and a total of two scans (averaged) in the respective binding energy ranges. The data acquisition and data processing were done using Eclipse data system software. The operating pressure of the spectrometer was typically in the 10-9 Torr range with a system base pressure of 2 × 10-10 Torr. Cell Culture. Bovine aortic endothelial cells (BAECs) were isolated from fetal calf aortas by mechanical scraping. Cells were maintained in an incubator at 37 °C with a humid atmosphere of 5% CO2 and 95% air. Dulbecco’s modified eagles medium (DMEM) supplemented with 10% fetal bovine serum 100 U/mL penicillin and 100 µg/mL streptomycin was used to nourish the cells. Cells were split 1:3 before a complete monolayer was formed. Cells in a 25-cm2 flask were subcultured by incubating in 2 mL of trypsin/EDTA (Clonetics) for 5 min at room temperature and resuspending in normal growth media. Cells were used in passages 4 through 9. Cell Attachment Assay. Gradients were exposed to a solution containing 10 000 BAECs/mL for 1 h and subsequently placed in fresh media for 23 h. After incubation, gradients were gently rinsed with PBS and images were captured along the length of the gradient for cell counting using a Zeiss Axiovert S100 inverted microscope equipped with a silicon-intensified target camera (VE1000SIT, Dage-MTI). Duplicate gradients were tested with at least three images taken at each fibronectin concentration. Migration Assays. Two distinct migration assays were used in these experiments to determine the effect of haptotactic gradients on BAEC motion. For all experiments, cell motion was tracked on the stage of a Zeiss Axiovert S100 inverted microscope equipped with temperature control and a silicon-intensified target camera (VE1000SIT, Dage-MTI) coupled to a computer operating an established time-lapse image capture macro in Scion Image (Scion Corp.). Images were taken every 20 min, processed using the standard “Sharpen” filter and placed sequentially into image stacks for further analysis. Cell locations were defined by center of mass and determined by manual tracking. The available static viewing field covered an area of 1 mm2. Cells were observed from 8 to 40 h, and duplicate experiments were conducted for all experimental conditions with a minimum of 20 cells tracked. All gradients used in these studies had a slope of 0.15 ng of Fn/mm. The Teflon Restraint Migration Assay. The previously described Teflon fence assay13 was modified to accommodate our gradient scheme. BAECs, 150 000, were plated on the low fibronectin concentration end of the gradient behind a Teflon restraint and allowed to reach confluence in growth media for at least 40 h. Figure 1A illustrates a glass slide with a confluent monolayer of cells behind the Teflon restraint. The barrier was positioned at 2030% of the length of the gradient transition region in order to allow sufficient Fn for cell attachment while positioning the cells in a linearly increasing Fn gradient. Cell positions for the Teflon restraint assay were recorded from t0 until cells moved out of the viewing field or until the cells moved away from the advancing front for more than 1 h and were considered “free” cells. The “Free” Cell Migration Assay. The free cell migration assay was used to determine the effect of only the haptotactic gradient on BAEC migration without the effects of cell-cell interaction. Cells were seeded on the surface of Fn gradients at a density of 3000-4000 cells/ cm2 yielding 30-40 cells/viewing field for observation. Positions of the cells in the free cell assay were tracked

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Figure 1. Schematic illustration of migration assays on haptotactic gradients: (A) the Teflon restraint assay on a linear Fn gradient prior to the removal of the restraint; (B) the Teflon restraint migration assay shortly after the removal of the restraint; (C) the “free” cell migration assay.

from t0 until the cells either moved out of the viewing field or made contact with a neighboring cell. Cell Motion Model. To determine the influence of different factors on the motion of endothelial cells, we look at what we will call the drift, Sdrift, and random, Srandom, speeds of cells under different experimental conditions. The drift speed is the average rate of motion along a specified direction (e.g., the direction of a chemical gradient), and the random speed is the root-mean-squared speed after removing the drift component. This section gives details about this analysis including precise definitions of the drift and random speeds and our method for estimating these quantities. Our analysis of the cell motion is based on a dynamical model for the motion of individual cells. Specifically, we assume the velocity b v ) vxxˆ + vyyˆ of an endothelial cell follows Langevin’s equation

dv b ) -βv b + σW B (t) dt

(1)

Here W B (t) is a random function (composed of x and y “white noise” components) that accounts for the effectively random impulses or tendencies of a cell to change the direction and rate of motion and σ is a parameter specifying the strength of these impulses. The term -βv b acts as a drag or resistance force. To include the possibility of a net drift along the direction of a chemical gradient, we also add a bias force to eq 1

dv b ) -βv b + σW B (t) + Rxˆ dt

(2)

Here, the gradient is assumed to be directed along the x-axis and the parameter R is a measure of the strength of the tendency of the cell to move up the gradient. Langevin’s equation was originally used as a model for Brownian motion but has since been applied in a variety of other areas including as a model for the motion of various cell types.22,23 It is the simplest dynamical equation that exhibits key aspects of endothelial cell motion including the basic element of randomness and also of persistence or inertiasthe tendency of a cell to continue moving in the same direction. (See Chandrasekhar27 and McQuarrie28 for general reviews of the Langevin equation (27) Chandrasekhar, S. Rev. Mod. Phys. 1943, 15 (1), 1. (28) McQuarrie, D. A. In Statistical Mechanics; University Science Books: Salusalito, CA, 2000; Chapter 20, p 452.

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Figure 2. XPS and SPR calibration curves used to determine fibronectin concentration across gradients: (A) directly measured XPS protein ratio as a function of distance along a typical gradient with a sigmoid fit and 95% confidence bands; (B) calibration curve relating fibronectin concentration measured by SPR on functionalized mixed SAMs to measured XPS protein ratio on the same samples. Y error bars represent standard error of the mean for SPR measured fibronectin concentration, and X error bars represent 5% error in XPS intensity.

in connection with Brownian motion and Stokes et al.23 for a detailed discussion of its use as a model for cell motion.) Equation 2 has three parameters: β, σ, and the bias R. In addition, we allow for the possibility of error in the measured cell positions. Specifically, the measurement errors in both the x and y positions are assumed to be Gaussian random variables with mean zero and variance σ. This amounts to four parameters that must be estimated for each cell trajectory. We take as the best parameter estimates the parameter values that maximize the probability of observing the given trajectory. The parameter values obtained in this way are called the maximum likelihood parameter estimates. For the Langevin equation, an analytic expression for the probability of a sequence of measured positions can be obtained and the maximum likelihood parameter estimates can be found using a function maximization algorithm. We used the “subplex” method of Rowan29 implemented in the Fortran subroutine of the same name available at netlib.org. However, any standard function maximization algorithm should suffice for this relatively small problem. For a particle moving according to eq 2, the drift speed (the expected rate of motion in the bias direction) is given by

Sdrift ≡ R/β

(3)

This is the speed at which the drag and bias forces balance. The drift speed is of more direct physical significance than the value of the bias R alone. In particular, higher bias (R) does not generally imply a higher rate of motion along the specified direction because it may be compensated by a higher drag (β). Only the ratio (Sdrift) gives the real quantity of interest: the rate of motion along the gradient. Next, the total mean squared speed can be shown to be

S2 ≡ 〈vx2 + vy2〉 ) σ2/β + (R/β)2

(4)

This has a term from the net drift and another σ2/β coming entirely from the random motion of the particle. This motivates defining a random speed as

Srandom ≡ σ/β1/2

(5)

In terms of Srandom and Sdrift, the root-mean-squared speed is

S ) (Sdrift2 + Srandom2)1/2

(6)

We note that studies of cell motion have often described the random motion by specifying the directional persistence time, which here is the same as β-1, and the random motility coefficient. The persistence time is a measure of how long a cell tends to continue moving along its initial direction. The motility, µ, is mainly relevant to the longterm behavior of the density F of a large number of cells; this density typically satisfies the diffusion equation dF/ dt ) µ∇2F (see, e.g., McQuarrie). The motility is related to the parameters in the Langevin equation by µ ) σ2/2β2. Results Gradient Characterization. Wettability gradients generated by the cross-diffusion of ω-functionalized alkanethiols have been characterized by water contact angle and capillary rise techniques.25 Gradients for the current study were generated by a similar cross-diffusion of HDA and MUD, yielding the characteristic sigmoidal profile for conjugation using standard NHS-ester chemistry. A series of X-ray photoelectron spectroscopy (XPS) atomic intensity signals were measured iteratively along the gradient to characterize the atomic profile with high resolution through the transition region. Atomic intensity information was used to determine the ratio of protein related peaks to substrate related peaks, defined as the “protein ratio” in eq 7. The change in protein ratio over the gradient transition region is demonstrated in Figure 2a.

protein ratio ≡

C 1s + N 1s + O 1s Au 4f + S 2p

(7)

Surface plasmon resonance (SPR) was used to measure the mass of protein bound to the surfaces. Unfortunately direct SPR analysis of the fibronectin gradients was not possible. Instead, XPS was used in conjunction with SPR to convert the protein ratio of atomic intensity to fibronectin concentration on the gradient. XPS and SPR experiments were conducted on substrates functionalized with eight different HAD/MUD solutions to produce a calibration curve for fibronectin concentration as a function of the XPS-generated protein ratio. The mass of fibronectin on the gradient increased linearly with increasing protein ratio to saturation at a concentration of 1.83 ng/mm2 as shown in Figure 2b. This relationship was used to determine the spatial distribution of fibronectin across the diffusion-generated gradients as shown in Figure 3. In general, fibronectin gradients can be characterized by their maximum and minimum surface coverage and the width of the transition region. For all substrates used in (29) Rowan, T. PhD University of Texas at Austin, 1990.

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Figure 3. Fibronectin concentration as a function of distance on the surface of a diffusion-generated gradient. Dotted lines represent 95% confidence interval for the sigmoid fit. Figure 5. Cell images captured during a Teflon restraint migration assay on a fibronectin gradient. Cells migrate from left to right with increasing time from immediately after the removal of the restraint (image 1) to 28 h after restraint removal (image 4).

Figure 4. BAEC density after 24 h seeding on a fibronectin gradient. Error bars represent the standard error of the mean for cell density. Dotted lines are 95% confidence bands for fibronectin concentration on the right axis. High concentrations of HDA on the left-hand side of the gradient showed increased coupling of fibronectin to the surface and enhanced cell adhesion. High concentrations of MUD on the right side of the gradient are not functionalized with fibronectin and exhibit limited cell adhesion. Intermediate cell adhesion is observed in the transition region of the gradient.

this study, gradients were from 1.83 to 0.36 ng of Fn/mm2 with a 10 mm wide transition region and a slope of 0.15 ng of Fn/mm. All control substrates had a uniform fibronectin concentration of 1.83 ng/mm2. The verification of cell adhesion along a fibronectin gradient was performed by seeding BAECs to a gradient for 24 h, followed by measuring cell density along the length of the gradient. Figure 4 illustrates the relationship between cell density and position along the gradient. As expected, the cell density increased with increasing concentration of fibronectin further verifying the presence of a gradient. Migration Assays. Two different migration assays were used to verify the directed migration of BAECs on the fibronectin gradients. The “free” cell migration assay was used to determine the effect of the gradient on individual nonconfluent cell migration by continuously tracking cells until they either moved out of the viewing field or came in contact with another cell. A Teflon restraint assay was used to measure the effects of a fibronectin gradient on the migration of a confluent monolayer of cells. The Teflon restraint assay provided information on the motion of a population of cells in response to a fibronectin gradient including both the effect of the gradient and the motion of a confluent monolayer of cells into free space. A series of typical images from the Teflon restraint assay is shown in Figure 5. Teflon Restraint Assay on Uniform Fibronectin Substrates: Serum/No Serum. Migration experiments were carried out using the Teflon restraint assay in serum-free media (no serum) and media containing 10% fetal bovine serum (serum). Both the serum and no serum experiments

Figure 6. Speed data for cells grown in serum (black) and serum-free (gray) conditions on a uniform substrate. Error bars represent standard error of the mean. Asterisk represents P < 0.01 of difference in the random component of cell speed and total rms speed between the two cell populations by the twosided Mann-Whitney test.

were conducted on a substrate with a uniform fibronectin concentration of 1.83 ng/mm2. The mean cell speeds are presented in Figure 6. No serum and serum populations have similar mean drift speeds of 4.39 ( 0.46 µm/h and 4.45 ( 0.56 µm/h, respectively, due to the population push into free space generated in the Teflon restraint assay. However there is a clear difference in observed random cell speed of 11.24 ( 0.93 µm/h for serum and 6.48 ( 0.65 µm/h for no serum due to the deterioration of health for the cells in no serum media. Movement of cells in the no serum media slowed with some cells becoming rounded and detaching from the surface during the course of the experiment. This experiment was used to determine the ability of the model to differentiate between changes in drift speed and random speed. Media containing serum was used for all subsequent migration experiments. Teflon Restraint Migration Assay on Fibronectin Gradient. BAEC migration was tracked using a Teflon restraint assay on fibronectin gradients with a transition region considered to be linear with a slope of 0.15 ng of Fn/mm. The restraint was positioned 2-3 mm beyond the beginning of the transition region to facilitate the attachment of cells. Figure 7 shows the mean cell speeds on gradient substrates compared to cells tested using the restraint assay on control surfaces with a uniform fibronectin concentration of 1.83 ng/mm2. Here the mean random cell speeds are very similar at 12.37 ( 0.85 µm/h for cells on the gradient and 11.24 ( 0.93 µm/h on the control. The difference in speed distributions between the two populations appears in the drift component. Cells on

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Figure 7. Speed data for cells grown on gradient (black) and uniform control (gray) substrates using the Teflon restraint assay. Error bars represent standard error of the mean. Asterisk represents P < 0.001 of difference in the drift component of speed and total rms speed between the two cell populations by the two-sided Mann-Whitney test. Figure 9. Average cell displacement vs time for cells measured in the free cell assay with a haptotactic fibronectin gradient and on uniform control substrates. Movement in the direction of the gradient is considered positive by convention. Error bars represent the standard error of the mean for displacement.

Figure 8. Net speed data for “free” cells grown on gradient (black) and uniform control (gray) substrates. Error bars represent standard error of the mean. Asterisk represents P < 0.01 of difference in the drift component of velocity between the two cell populations by the two-sided Mann-Whitney test.

the gradient have a mean positive drift of 7.82 ( 0.31 µm/h while the control cells drift at an average speed of 4.45 ( 0.56 µm/h. “Free Cell” Migration Assay on Fibronectin Gradient. A second method for observing cell migration on fibronectin gradients was tracking noncontacting cells uniformly seeded at low density. This technique facilitated the measurement of independent cell motion without the additional effects of cell-cell contact. Free cells were seeded on both gradient and uniform control substrates at a density of 3000-4000 cells/cm2 yielding 30-40 cells per viewing field for observation. Gradients used in these experiments displayed a transition region that can be considered linear with slope of 0.15 ng of Fn/mm. The viewing field selected for observation of cell motion was at the center of the transition region. A summary of the observed cell speeds on gradient substrates is compared to cells on uniform control in Figure 8. As demonstrated in the restraint assay there is no significant difference between the random speeds of the cells on gradient or control substrates. Measured values of average random speed are 9.92 ( 1.04 µm/h and 8.93 ( 0.51 µm/h, respectively. The effect of the fibronectin gradient on drift speed is again clearly shown. The average drift speed for free cells was 1.71 ( 0.49 µm/h and -0.15 ( 0.53 µm/h on the gradient and control substrates, respectively. Demonstration of Haptotaxis. Cell motility was examined on fibronectin gradient substrates using two distinct assays, both of which significantly affected the observed drift velocity compared to cells on a substrate with uniform fibronectin coverage. Directed migration of endothelial cells along a fibronectin gradient suggests that the cells respond haptotactically. A common relationship presented in studies of cell migration is the change in

mean squared displacement over time.23,30 For gradient substrates that have a distinct directionality, it is important to maintain the directional nature of the data. Thus, a presentation of mean displacement vs time is more revealing. Figure 9 compares average displacement of BAECs on a fibronectin gradient and on a surface with uniform coverage in the free cell assay. The slope is the average rate of motion along the direction of the gradient, and it is very close, as should be expected, to the calculated value for the drift speed Sdrift. A complete presentation of cell speed and motility coefficient information for all cell motility assays is given in Table 1. Discussion Previous studies of cell motility have examined the effects of the presence of soluble growth factors,6,8,31,32 compliant substrates,10 and shear flow11,33 on cell movement. The first measurements of cell migration on adhesion gradients employed mouse fibroblasts on acetate substrates coated with a shadow mask evaporated gradient of palladium.17 Recent work has focused on cell and protein interactions with more chemically complex gradients. Gradients of functionalizable alkanethiols and silanes generated by diffusion techniques were developed to study the effects of wettability on protein adsorption34,35 and cell adhesion.36 The most sophisticated gradient system developed to date is an electrochemical technique using SAMs conjugated with fibronectin and poly(ethylene glycol).20,37 However, none of these gradients has been applied to the study of cell migration. The current study presents the initial demonstration of an assay for systematic study of substrate parameters that affect cell migration. Specifically the migration of (30) Dickinson, R. B.; Tranquillo, R. T. AIChE J. 1993, 39 (12), 19952010. (31) Sirois, M. G.; Bernatchez, P. N. Circulation 1999, 100 (18), 40. (32) Yuan, J.; Munn, L. L.; Jain, R. K. FASEB J. 1999, 13 (4), A2. (33) Albuquerque, M. L. C.; Waters, C. M.; Savla, U.; Schnaper, H. W.; Flozak, A. S. Am. J. Physiol.: Heart Circ. Physiol. 2000, 279 (1), H293-H302. (34) Golander, C. G.; Lin, Y. S.; Hlady, V.; Andrade, J. D. Colloids Surf., B 1990, 49 (3-4), 289-302. (35) Welin-Klintstrom, S.; Lestelius, M.; Liedberg, B.; Tengvall, P. Colloids Surf., B 1999, 15 (1), 81-87. (36) Lee, J. H.; Khang, G.; Lee, J. W.; Lee, H. B. J. Colloid Interface Sci. 1998, 205 (2), 323-330. (37) Wang, Q.; Bohn, P. W. J. Phys. Chem. B 2003, 107 (46), 1257812584.

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Table 1. BAEC Motility Parameters for the Teflon Restraint and “Free” Cell Migration Assays migration assay

serum

gradient

drift speed (µm/h)

random speed (µm/h)

total rms speed (µm/h)

motility coeff (µm2/h)

Teflon restraint Teflon restraint Teflon restraint “free cell” “free cell” “free cell”

no yes yes no yes yes

no no yes no no yes

4.39 ( 0.46 4.45 ( 0.56 7.82 ( 0.31 -0.04 ( 0.27 -0.15 ( 0.54 1.71 ( 0.49

6.48 ( 0.65 11.24 ( 0.93 12.37 ( 0.85 4.98 ( 0.36 8.93 ( 0.51 9.92 ( 1.04

7.82 ( 0.80 12.09 ( 1.09 14.64 ( 0.91 4.99 ( 0.45 8.93 ( 0.74 10.07 ( 1.15

14.61 ( 2.14 25.32 ( 4.10 13.51 ( 1.71 11.43 ( 2.22 29.17 ( 5.24 17.86 ( 1.56

BAECs was observed by video microscopy on a linear onedimensional haptotactic gradient produced by coupling fibronectin to cross-diffused alkanethiols. The effect of fibronectin on cell adhesion is well characterized and served as a system for testing the capability of this migration assay.38-40 However this assay is not limited to studying fibronectin gradients. This assay is designed to accommodate a variety of chemistries, to give precise control of nutrient media presented to the cells, and to provide flexibility regarding the physical dimensions of the gradients. It is well-known that cell types have different migratory behavior and respond differently to stimuli. This migration assay can be used to study the motility of virtually any anchorage-dependent cell on ligand concentration gradients. The Teflon restraint assay and the free cell assay provide information relating to different aspects of cell motility. Initial observations indicated that the effects of the confluent monolayer and the fibronectin gradient were confounded in the Teflon restraint assay. This limitation was overcome by using the free cell assay to isolate the effect of the gradient on individual cell movement. However, the Teflon restraint assay could be used to isolate the effect of cell-cell contact on cell motility on uniform substrates. Comparing data from Table 1, cell-cell contact in the Teflon restraint assay increased the drift speed and total root mean squared (rms) speed of migrating cells regardless of the presence of a fibronectin gradient. Cell drift speeds in the Teflon restraint assay for both serum and no serum conditions were 4.45 and 4.39 µm/h, while control cells in the free cell assay had a drift value much closer to zero at -0.15 and -0.04 µm/h. The results seem to indicate that cell-cell contact has a stronger effect on drift speed than the chosen fibronectin gradient slope (4.45 µm/h vs 1.71 µm/h) and that the joint influence of the two effects could be complementary (7.82 µm/h). We hypothesize that this behavior was due to both free space limitations caused by the monolayer and an organized cellular response to cell-cell contact resulting in net cell motion away from neighboring cells. Further experiments taking advantage of the Teflon restraint assay and soluble molecules designed to mimic the binding of cell-cell interaction could be used to elucidate the mechanism of this behavior. Although providing no directional information about the effect of fibronectin gradients on cell motion, the motility coefficient (µ ) σ2/2β2) and total rms speed were useful for comparison of values generated in these experiments with previous studies. Separate experiments conducted by Stokes et al.23 in 1991 and Kouvroukoglou et al.41 in 2000 on microvessel and pulmonary artery endothelial cells reported total rms speeds from 25 to 60 (38) Garcia, A. J.; Ducheyne, P.; Boettiger, D. Tissue Eng. 1997, 3 (2), 197-206. (39) Yamada, K. M.; Akiyama, S. K.; Hayashi, M. J. Colloid Interface Sci. 1985, 104 (1), 2. (40) Truskey, G. A.; Iuliano, D. J.; Saavedra, S. S. FASEB J. 1991, 5 (4), A372. (41) Kouvroukoglou, S.; Dee, K. C.; Bizios, R.; McIntire, L. V.; Zygourakis, K. Biomaterials 2000, 21 (17), 1725-1733.

µm/h and motility coefficient values from 375 to 800 µm2/ h. The rms speed values were very close to the values reported in Table 1, and the calculated motility coefficients were within an order of magnitude. It is also of note that the motility coefficient measured in the absence of a fibronectin gradient was significantly higher in both the Teflon restraint assay and the free cell assay in this study. Since the motility coefficient is known to be dependent on the surface concentration of fibronectin,4,42 it is likely that this effect was due to changes in the absolute ligand concentration between the gradient samples and the uniform control substrates. Future experiments exploring endothelial cell motion over a range of gradient slopes and uniform fibronectin concentrations using this system are necessary to fully describe this effect. The slope of the adhesion gradients can be varied by changing the diffusion conditions such as the hydration of the matrix or the alkanethiol solution concentrations. All gradients used in this study had a fibronectin concentration that ranged from 0.36 to 1.83 ng/mm2 with a slope of 0.15 ng of Fn/mm. Given that the BAECs had cell lengths of 10-17 µm, this yielded an estimated gradient of 0.4-0.7% in fibronectin concentration across the length of a cell. Changing the gradient parameters will facilitate the study of the relationship between gradient slope and cellular drift speeds. This system can also be used to measure the effects of soluble or surfacebound growth factors on cell migration in conjunction with a variety of haptotactic gradients. Proteins such as acidic fibroblast growth factor (aFGF), vascular endothelial growth factor (VEGF), or epidermal growth factor (EGF) that have been shown to affect cell motility could be titrated into the assay media to determine both their relative and cooperative effects on migration in a haptotactic gradient. These growth factors also could be titrated into the NHS/EDC reaction solution for covalent attachment to the surface as a permanent signaling moiety that cannot be internalized by the cells. Finally, the assay could be used to measure the haptotactic effects of substrate ligand gradients on cells experiencing shear by applying laminar flow to the migration chamber. These fundamental experiments have also established reproducible control conditions for the gradient surface migration assay. Two negative controls are cells seeded on a uniform surface concentration of fibronectin in both the presence and absence of serum-containing media. In the Teflon restraint assay, 4.4 µm/h drift was observed in both controls due to the effects of cell-cell contact and directed motion into free space. Negligible drift was observed in the “free cell” assay negative control as would be expected for a uniform substrate with no directional stimulus. For future studies the directed migration of BAECs on a fibronectin gradient with a slope of 0.15 ng of Fn/mm can serve a positive control with 7.8 and 1.7 µm/h drift speeds expected in the Teflon restraint and “free cell” assays, respectively. (42) Palecek, S. P.; Loftus, J. C.; Ginsberg, M. H.; Lauffenburger, D. A.; Horwitz, A. F. Nature 1997, 385 (6616), 537-540.

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Conclusions This pilot study was designed to demonstrate the ability to generate haptotactic fibronectin gradients and to use two migration assays to characterize their effects on cell motility. Gradients have been quantitatively characterized using a joint XPS/SPR technique and used with migration assays in conjunction with a Langevin model to measure cell behavior in terms of random and drift speeds along the linear gradient. The gradient slope of 0.15 ng of Fn/ mm2 was observed to increase the drift speed of the BAECs from -0.15 and 4.45 µm/h to 1.71 and 7.82 µm/h in the “free” and “Teflon restraint” assays, respectively. The presented technique has been applied to characterizing bovine aortic endothelial cell migration on fibronectin gradients but can easily be applied to other cells and signaling environments. We are currently developing a

Smith et al.

series of fibronectin gradients to characterize the dependence of drift speed on gradient slope. The ultimate utility of this technique for the quantitative observation of cell migration will be its application to improving the patency and integration of a wide range of implantable therapies. Acknowledgment. The authors thank Benjamin Viglianti for assistance in gradient development. This work was funded by the National Institutes of Health (EB00463 (T.P.B.) and DK54932 (W.M.R.)), the Duke University Center for Translational Research, the Lord Coorporation of North Carolina, and the North Carolina Biotechnology Center. LA0489763