Directional cell migration through cell–cell interaction on polyelectrolyte multilayers with swelling gradients

Biomaterials 34 (2013) 975e984

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Directional cell migration through cellecell interaction on polyelectrolyte multilayers with swelling gradients Lulu Han, Zhengwei Mao*, Jindan Wu, Yang Guo, Tanchen Ren, Changyou Gao* MOE of Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 August 2012 Accepted 12 October 2012 Available online 3 November 2012

The directional cell migration plays a crucial role in a variety of physiological and pathological processes. It can be controlled by the gradient cues immobilized on the substrate. The poly(sodium 4-styrenesulfonate) (PSS)/poly(diallyldimethylammonium) chloride (PDADMAC) multilayers were post-treated in a gradient NaCl solution with a concentration ranging from 3 M to 5 M, yielding the gradient multilayers with a similar chemistry composition (PSS domination) but gradually changed swelling ratio. The gradient nature and physicochemical properties were characterized by X-ray photoelectron spectroscopy and ellipsometry. Compared to the random migration with a lower rate at a smaller cell-seeding density, the vascular smooth muscle cells migrated directionally to the low hydration side at an appropriate cell-seeding density (1.5
104/cm2) under the assistance of cellecell interactions. The cell migration rates on the gradient surface were significantly larger than those on the corresponding uniform surfaces etched by salt solutions of the same concentrations. Relative cell adherent strength and focal adhesion formation were studied to unveil the intrinsic mechanism of the gradient multilayers on the cell migration. It was found that both the gradient cues and cellecell contact have major influences on the directional cell migration. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Cell migration Polyelectrolyte multilayers Gradient surface Cellecell contact Directional cell migration

1. Introduction The cell migration plays a crucial role in a variety of physiological and pathological processes ranging from embryonic development, cancer metastasis, blood vessel formation and remolding, tissue regeneration, immune surveillance, and inflammation [1,2]. For example, the leukocytes migrate toward the sites of inflammation and infection, the neurons send projections to specific regions of the brain to find their synaptic partners, and the fibroblasts move into the wound space [3]. Thus, in vivo directional cell migration to the special sites is critical rather than random motility. The cell’s compass is governed by various directional cues, such as soluble chemoattractants (chemotaxis), surface-attached molecules (haptotaxis), and mechanical cues (durotaxis) [4,5]. It is believed that controlling the cell migration by these various directional cues is one of the paramount problems in regenerative medicine and tissue engineering. So far the surfaces with chemical and physical gradients have been prepared and their influences on the cell migration have

* Corresponding authors. Fax: þ86 571 87951108. E-mail addresses: [email protected] (Z. Mao), [email protected] (C. Gao). 0142-9612/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2012.10.041

attracted increasing attention [6]. Immobilization of various directional cues on the biomaterials is necessary to guide cell migration, especially for the haptotaxis and durotaxis. For example, cell can directionally migrate on a density gradient of immobilized biomolecules (haptotaxis), such as extracellular matrix proteins (fibronectin (FN) [7], laminin [8], and collagen [9,10]), growth factors (epidermal growth factor (EGF) [11], basic fibroblast growth factor (bFGF) [12] and vascular endothelial growth factor (VEGF) [13]), and small ligands (arginine-glycine-aspartic acid (RGD)) [14e16]. Although many gradients of biological molecules are proved to be effective in inducing directional cell alignment and migration, the complexity of natural macromolecules and their interactions with cells still challenge the design of biomaterials for controlling cell migration because a variety of factors will influence the cell fate. Also, these proteins and growth factors are expensive and easy to reduce bioactivity or denature, limiting their practical applications. On the other hand, the physical signal is also very influential in modulating the cell migration. For instance, the motility of fibroblasts can be governed purely by substrate rigidity (durotaxis), and can directionally move from the soft region towards the rigid side of the substrate [17,18]. Among the various surface engineering methods, the layerby-layer (LBL) assembly can diversely tailor the substrate properties and is particularly suitable to modify the biomaterials surface [19e21]. The physicochemical properties of the assembled

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polyelectrolyte multilayers (PEMs) can be easily modulated by the deposition or post-treatment parameters, such as ionic type and strength [22e24], pH [25e27] and temperature [28]. Therefore, gradual change of these parameters can fabricate gradient polyelectrolyte multilayers (GPEMs). For example, Nolte et al. [29] generated thickness gradients of poly(allylamine hydrochloride) (PAH)/poly(acrylic acid) (PAA) multilayers by a salt etching method. Kirchhof et al. established a pH gradient (5e9) by a microfluidic device and generated the heparin/chitosan multilayers with gradient properties [30]. The gradient multilayers pre-adsorbed with fibronectin are able to guide MG-63 osteoblast-like cells’ movement from regions deposited under low pH to those under high pH, due to gradual changes in mechanical properties and fibronectin density. Recently, we found that the structure and physicochemical properties of the poly(sodium 4-styrenesulfonate)/poly(diallyldime thylammonium) chloride (PSS/PDADMAC) multilayers can be changed continuously by the post-treatment in NaCl solutions of increasing concentration (1e5 M) with a transition point at 3 M [31]. For the Multilayers-1M and Multilayers-2M (the multilayers posttreated with 1 M and 2 M NaCl solutions, respectively), the original structure and properties of the multilayers are mostly retained, and their surface is dominated by the positively charged PDADMAC. By contrast, the surface of Multiayers-3M, 4M and 5M is dominated by the negatively charged PSS as a result of larger loss of PDADMAC. The swelling ratio increases along with the salt concentration within a range from 3 M to 5 M. More recently, it was found that smooth muscle cells (SMCs) are preferable to adhere and spread on the negatively charged PSS-dominated surface (the Multilayers-3M, 4M and 5M), compared with the positively charged one (Multilayers1.2M) which shows cytotoxicity to some extent [32]. Furthermore, the mobility of SMCs is slowest on the dehydrated surface (Multilayers-3M) but can be effectively promoted on the highly hydrated surface such as the Multilayers-5M. Although these uniform multilayers provide a versatile means to control the cell mobility, they cannot govern the directional cell movement due to the lack of gradient cues. Herein, the gradient PSS/PDADMAC PEMs with a gradually increasing swelling ratio are prepared, which are expected to guide the directional cell migration by providing the cell adhesion force in a gradient manner. For this purpose, the as-prepared multilayers are etched in a gradient salt solution with a linearly increasing concentration from 3 M to 5 M (Fig. 1). For the first time we find that the directional migration of SMCs are guided by both the gradient physical cue and the cell-seeding density.

2. Experimental section 2.1. Material Polyethyleneimine (PEI, Mw ¼ 25 kDa), poly(diallyldimethylammonium chloride) (PDADMAC, Mw ¼ 200e350 kDa) and poly(sodium 4-styrenesulfonate) (PSS, Mw ¼ 70 kDa) were obtained from SigmaeAldrich. Water used in this experiment was purified by a Milli-Q water system (Millipore, U.S.A.). All the polyelectrolytes were prepared to a final concentration of 1 mg/L aqueous solutions. PEI was dissolved in water, and PSS and PDADMAC were supplemented with 1 M NaCl. Quartz, glass, and silicon wafers were cleaned in piranha solution (7:3 v/v% H2SO4/H2O2). After rinsed thoroughly with water, they were dried under a smooth stream of N2.

2.2. Assembly of polyelectrolyte multilayers To ensure the successful adsorption, a precursory layer of PEI was deposited on the substrates. PSS and PDADMAC were then alternately assembled by auto dipping at 20  C. Between alternate exposures to the two kinds of polymer solutions for 20 min, there were 3 rinses with 0.1 M NaCl solutions for 3 min. In the last step, the films were immersed in water for at least 5 min to eliminate the adsorbed salt. Taking into account our previous results on the physicochemical properties after salt etching [31] and the cell responses on the uniform salt-etched multilayers [32], herein a total of 7 bilayers were assembled and the multilayers are expressed as (PSS/PDADMAC)7.

2.3. Preparation of gradient solution A linear density gradient of NaCl solution was obtained by using a home-made device which is consisted of two reservoirs, two channels and a gradient column (Fig. 1a). A and B reservoirs are loaded with the same amount of NaCl solutions of high and low concentrations (high and low densities), respectively. The solution in the reservoir A was pumped into the reservoir B and mixed by a magnetic stirrer, and consequently the concentration (density) of the solution in reservoir B was gradually increased. The flow velocity(s) was controlled by the peristaltic pump, and the rate ratio of sA to sB was 1:2. When the solution in reservoir B was pumped into the gradient column with the prolongation of time, the gradient column would be filled first with the salt solution of a lower concentration and gradually higher concentration. Due to a higher gravity of the higher concentration, the new concentrated flow could locate stably in the bottom of the gradient column and then the solutions of lower densities were pushed upwards gradually (the outlet of the tube was mounted at the bottom of the gradient column). After the solutions in A and B reservoirs were exhausted eventually (and simultaneously), the pump B was turned off to avoid the generation of air bubbles in the gradient salt column. Finally, the solution of gradually increasing concentration (density) was collected in the gradient column. By this fabrication strategy, the concentration (density) of the gradient salt solution is continuously increased from the top to the bottom in the range from the initial concentration of the solution in the reservoir B to that in the reservoir A. The concentration range and gradient length in the gradient column could be adjusted by the initial concentration and volume of the solutions in the reservoirs A and B. In this study, the concentration range of the gradient solutions was tuned from 3 M to 5 M and the length was adjusted to 2.5, 5 and 20 mm through the various initial volumes of the solutions in the reservoirs A and B, respectively.

Fig. 1. Schematic illustration showing the fabrication of gradient salt solution and gradient polyelectrolyte multilayers thereof. (a) Generation of a continuous density gradient of NaCl solution. The details are presented in the Experimental section. (b) Vertical incubation of the as-prepared (PSS/PDADMAC)7 multilayers into the gradient NaCl solution for 2 h. (c) The gradient multilayer film is obtained after rinsing with water. The upper level of the salt gradient solution (b) and the multilayers treated at this place is defined as the 0 position.

L. Han et al. / Biomaterials 34 (2013) 975e984 2.4. Post-treatment of the multilayers in the gradient solution To generate the gradient multilayers the substrate assembled with the (PSS/ PDADMAC)7 multilayers was vertically immersed into the 3e5 M NaCl gradient solution (Fig. 1b) for 2 h, and was then washed with water and dried under a smooth stream of N2 (Fig. 1c). Since previous studies show that the outer layers of the saltetched multilayers will dissociate initially in the salt solution, and the multilayers treated with 3e5 M NaCl solutions terminated by the PSS and PDADMAC have the same physicochemical and surface properties [31], in this study only PDADMACterminated multilayers were used to prepare the gradient materials. 2.5. Spectroscopic ellipsometry The thickness and refractive index of the multilayers were determined in air and water by a spectroscopic ellipsometer (model M2000D, J. A. Woollam Inc., Lincoln, NE) at an incident angle of 75 within a wavelength range of 300e1700 nm, and 300e1100 nm, respectively. The measurement of the samples in water was carried out in a liquid cell. The thickness was calculated from the ellipsometric parameters, D and j, using a Cauchy model. The swelling ratio was defined as the ratio of the multilayer thicknesses in wet and dry states, respectively. All the measurements were independently repeated for 3 times. 2.6. X-ray photoelectron spectroscopy (XPS) The compositions of the gradient multilayers at different positions after salt etching were detected by an Axis Ultra spectrometer (Kratos Analytical, UK) with a monochromated Al Ka source at pass energies of 160 eV for survey spectra and 80 eV for core level spectra. The binding energy was corrected for static charging of C1s peak at 284.6 eV. The XPS images were acquired at 400 mm
400 mm areas at a pass energy of 80 eV. The background-corrected image was obtained by subtraction of the background region image from the image at the peak of interest. The data processing was undertaken using Casa XPS. Three gradient multilayers were used for parallel measurements. 2.7. Cell culture Human vascular smooth muscle cells (SMCs) were obtained from the Cell Bank of Typical Culture Collection of Chinese Academy of Sciences (Shanghai, China). The cells were maintained in a regular growth medium consisting of high-glucose DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Sijiqin Inc., Hangzhou, China), 100 U/ml penicillin and 100 mg/ml streptomycin, and cultured at 37  C in a 5% CO2 humidified environment. 2.8. Cell migration Before cell culture, the salt-treated multilayers were sterilized under UV light for 1 h. The SMCs were plated on the salt-etched multilayers at low (5
103/cm2) and high (1.5
104/cm2) seeding densities, respectively. Approximately 12 h after the cell seeding, the cell migration was in situ recorded using a time-lapse phasecontrast microscope (IX81, Olympus) equipped with an incubation chamber (37  C and 5% CO2 humidified atmosphere). The trajectories of SMCs were manually reconstructed from the centre positions of cells over the whole observation time by Imagepro Plus software, and then the cell migration distance S was automatically calculated by the software according to the following equations at 15 min time intervals over the observation time of 12 h (t). S ¼

t qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi X ðxi  xi1 Þ2 þðyi  yi1 Þ2 i¼1

If xt > 0, Sx ¼ xt If xt < 0, Sx ¼ xt where xi (or yi) and xi1 (or yi1) represent x-axis (or y-axis) of cell centre positions at the i and i1 time, respectively. x0 and y0 values are set as 0. xt is the coordinate of cell centre positions after 12 h migration. The cell migration rate (n) and cell migration velocity in x or x axis (nx or nx) thus obtained by n ¼ S/t, nx ¼ Sx/t or nx ¼ Sx/t, respectively. At least 15 cells were calculated for each sample. 2.9. Cell adhesion The relative cellesubstrate interaction was measured according to the method suggested in Rey’s work [33]. After cell seeding for 24 h, the multilayers-coated glass substrate was gently washed with PBS to remove the floating cells. The cell number was counted under microscope. Then the multilayers-coated substrate with cells was placed vertically at the bottom of a centrifuge tube filled with PBS. The number of cells remained on the substrate was counted and the fraction of adhesion cells was calculated after being centrifuged at 2000 rpm for 5 min. The fraction of

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adherent cells was defined as the original cell number divided by the remained cell number. 2.10. Cell immunostaining Fluorescent staining of actin, vinculin and cell nucleus was carried out to study the cell migration. Briefly, after cultured in the medium containing 10% FBS for 4 h or 24 h, the SMCs were carefully washed with PBS and then fixed with 4% paraformaldehyde at 37  C for 30 min. The cells were further treated with 0.5% (v/v) Triton X-100/PBS at 4  C for 10 min to increase the permeability of the cell membrane. After rinsed 3 times with PBS, they were incubated in 1% bovine serum albumin (BSA)/PBS for 30 min and then in a mouse monoclonal antibody against human vinculin (Sigma) for 1 h. After washed twice in 1% BSA/PBS, they were further stained with FITC-labeled goat anti-mouse IgG (Beyotime, China), rhodamine phalloidin (Invitrogen) and DAPI (Sigma) for 1 h, followed by 3 washes in PBS. The cells were observed under a confocal laser scanning microscope (CLSM, Leica TCS SP5). 2.11. Statistical analysis The data are expressed as mean  standard deviation (SD). The statistical significance between groups is determined by one-way analysis of variance (ANOVA) in the Origin software. The Tukey Means Comparison method is performed and the statistical significance is set as p < 0.05.

3. Results and discussion 3.1. Preparation and characterization of gradient multilayers The (PSS/PDADMAC)7 gradient multilayers were prepared by post-treatment in a gradient salt solution as shown in Fig. 1. A concentration gradient of the NaCl solution from 3 M to 5 M along the gradient column from top to bottom was firstly generated (Fig. 1a). The salt concentration had a linear pattern and was stable for at least 2 h, which was proved theoretically and experimentally (Figure S1e3, and the detailed discussion in the sections 1e4 of Supporting information). The steepness of the gradient can be modulated by controlling the initial volume of the NaCl solutions in A and B reservoirs. After vertical immersion for 2 h in the asprepared NaCl gradient solution, the gradient polyelectrolyte multilayers (GPEMs) were obtained (Fig. 1b). The topside of the GPEMs is defined as the 0 position, where the multilayers were treated with 3 M NaCl solution. Along with the extension of the position the GPEMs were treated with more concentrated salt solution (Fig. 1c). The surface chemical compositions of the GPEMs were characterized by XPS (Fig. 2). In this study three different GPEMs with 2.5, 5 and 20 mm length were prepared, respectively. As shown in Fig. 2a, the relative content of nitrogen (representative for PDADMAC) to sulfur (representative for PSS) on the 20 mm GPEMs gradually decreased from 48/52 (2 mm, 3.2 M), 46/54 (10 mm, 4 M), to 39/61 (18 mm, 4.8 M) (the corresponding salt concentration was calculated from Eq. (4) in SI). These ratios are identical to those on the multilayers (45/55, 44/56, and 38/62) homogeneously treated with 3 M, 4 M, and 5 M NaCl solutions, respectively [31]. The identical chemical compositions with those uniform multilayers were further confirmed by the distribution of Na counter ions on the GPEMs (Figure S4). Therefore, the GPEMs are expected to show the similar physicochemical properties of the uniform counterparts treated with the salt solutions of the same concentrations. On the 2.5 and 5 mm GPEMs the gradual reduction of nitrogen element and increase of sulfur element were similarly confirmed along the gradient direction (Fig. 2a). In this way the gradient slope is easily tuned by controlling the length of the GPEMs. The distribution of nitrogen element at 1.25 mm position (corresponding to 4 M NaCl treatment) on the 2.5 mm GPEMs was further visualized by in situ XPS imaging (Fig. 2b), intuitively illustrating that the nitrogen content was reduced along with the gradient position. Because the entire surface of the GPEMs is dominated by the negatively charged

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Fig. 2. The chemical compositions of the gradient (PSS/PDADMAC)7 multilayers treated by 3e5 M NaCl density gradient solution. (a) The atomic contents of nitrogen (open square) and sulfur (filled circle) as a function of gradient position over 20 mm (solid line), 5 mm (dashed line) and 2.5 mm (dotted line) gradient multilayers measured by XPS. (b) XPS imaging showing the distribution of N element at 1.25 mm position over the 2.5 mm gradient multilayers.

PSS whose content is increased from w52% to w61% along the GPEMs, the influence of the surface chemistry of the GPEMs on the cell responses can be safely ruled out and will not be considered according to our previous results [32]. As a result of the salt treatment, the physical properties including thickness, swelling ratio and refractive index were also continuously altered as representatively shown in Fig. 3 for the 20 mm GPEMs. The dry thickness of the GPEMs was reduced gradually from 145 nm (region close to 0 position, 3M) to 56 nm (region close to 20 mm, 5M) along with the extension of position (Fig. 3a). This is caused by the gradual disassembly of the

multilayers as a result of electrostatic screening by the gradient NaCl solution [33]. The wet thickness of the GPEMs in water was measured as well and the swelling ratio was calculated (Fig. 3b). It increased from about 1.4 (0 position, 3M) to 6.4 (20 mm, 5M), and these two values are close to those of the uniform Multilayers-3M (1.3) and Multilayers-5M (7.7), respectively. By contrast, the refractive index, which is an average of polymer matrix (w1.52) and water (1.33) and thereby proportional to the polymer content, decreased from 1.48 to 1.37 along with the gradient position (Fig. 3c), implying the hydration level of the multilayers is gradually enhanced. In conclusion, the GPEMs with a similar surface

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L. Han et al. / Biomaterials 34 (2013) 975e984

SMCs with a density of 5
103/cm2 and 1.5
104/cm2 were seeded on the GPEMs for 12 h and then monitored online to obtain their migration trajectories, respectively. The mobility of the cells at the low density is only dominated by the cellesubstrate interactions because the distance between cells is large enough to avoid direct contact. In this case, the percentages of cells moved to the reverse direction of the gradient (x direction) were nearly 50% at different positions (Fig. 4b), suggesting that the cells migrate almost equally to both x and þx directions without priority. The rates of cell migration on the four continuous regions of 0e0.63 mm, 0.63e1.25 mm, 1.25e1.88 mm and 1.88e2.5 mm were 10  3, 16  5, 18  8 and 21  8 mm/h, respectively (Fig. 4c, right group). Compared with those on the corresponding uniform PEMs (6  2, 10  2, 12  4 and 15  4 mm/h on Multilayers-3.25, 3.75, 4.25 and 4.75M, respectively) (Fig. 4c, left group), the rates of cell migration on the regions of 0e0.63 mm and 0.63e1.25 mm of GPEMs were significantly improved (p < 0.01). In order to explore the influence of cellecell interactions and better mimic the cell migration behaviors in vivo, the cell-seeding density was increased to 1.5
104/cm2. The cell-seeding density would not be improved further since the cell mobility was found to be largely blocked at a still higher cell density (3
104/cm2, Video 1). Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2012.10.041. The cell migration trajectories (Fig. 5a) show that the percentages of cells moving to the x direction were 60, 75, 89 and 95% on the four continuous regions, respectively, implying that the cells preferentially migrate to the lower hydration side (x direction). The cell migration velocity in x axis was separated into the þx and x direction (calculated by the equations shown in Section 2.8) to analyze the cell mobility along the gradient, which is proportional to the final displacement of a single cell in þx or x axis. The results show that the cells traveled much faster towards the x direction

3.2. Cell migration on the gradient polyelectrolyte multilayers It is known that the hydration degree or stiffness of the substrate plays a primary role in cell adhesion and other behaviors [32,34,35]. Our previous studies show that in general the negatively charged surfaces (PSS dominated) are favorable for cell adhesion and spreading. However, cells display the best adhesion behavior on the 3M-treated multilayers (with less PSS content), suggesting that the content of PSS has less influence on the cell behaviors on all the negatively charged surfaces [32]. Additionally, cells adhere and proliferate better on the Multilayers-3M (water contact angle of 32 ) than that on the Multilayers-5M (water contact angle of w50 ), which is different from the well-cognized value (50e70 ) for the best cell adhesion. This result clearly indicates that the wettability of the multilayers is not a major governing factor in our system [32]. Therefore, in this study the gradient hydration degree of the gradient multilayers but not the chemical composition or the surface wettability is expected to provide a continuous signaling cue to bias the directional cell movement. In order to effectively direct the cell migration, the gradient with a sharper slope is preferred because the cell will sense a larger extent of difference between its head and tail in a definite distance, i.e. the cell length [36]. Therefore, the GPEMs with the shortest length (2.5 mm) and thereby the sharpest slope were selected. The cell migration was observed at four continuous regions on the GPEMs: 0e0.63 mm (3e3.5 M), 0.63e1.25 mm (3.5e4 M), 1.25e1.88 mm (4e4.5 M), and 1.88e2.5 mm (4.5e5 M). The cell movement was separated into x (gradient direction) and y (vertical to the gradient direction) directions (Fig. 4a).

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Fig. 4. (a) Schematic illustration of the x-y axis of cell migration on the gradient multilayers. (b) Cell migration trajectories at 0e0.63 and 1.88e2.5 mm gradient for 12 h after being cultured for 12 h at a low seeding density of 5
103/cm2. The arrows indicate the percentages of cells migrating to the direction with the gradually reduced hydration degree. (c) Cell migration rate on the uniform (left group) and gradient (right group) multilayers. Data were averaged from 15 cells. * Indicates significant difference at p < 0.05.

L. Han et al. / Biomaterials 34 (2013) 975e984

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Fig. 5. (a) Cell migration trajectories at 0e0.63, 0.63e1.25, 1.25e1.88 and 1.88e2.5 mm gradient. The cells were randomly tracked for 12 h after being cultured for 12 h at a seeding density of 1.5
104/cm2. The arrows indicate the percentages of cells migrating to the direction with the gradually reduced hydration degree. (b) Cell migration velocity in x direction on the gradient multilayers as a function of gradient position. (c) Cell migration rate on the uniform (left group) and gradient (right group) multilayers as a function of gradient position. Data were averaged from 15 cells. * Indicates significant difference at p < 0.05.

with a value of 5  3, 7  6, 5  5 and 15  13 mm/h on the four continuous regions, respectively, than to the þx direction (higher hydration side) with a value of about þ3 mm/h (Fig. 5b). Therefore, the SMCs show a much higher directional migration tendency at an appropriate cell-seeding density. It is worth mentioning that the cell migration rate on the uniform Multilayers-3.25, 3.75, 4.25 and 4.75M with a higher cellseeding density were significantly improved to 27  5, 30  6, 32  6 and 39  8 mm/h (Fig. 5c, left group) compared with those measured at a lower cell-seeding density (Fig. 4c, left group) (p < 0.01), suggesting that the interactions between cells can enhance the cell mobility. The cell migration rates on the four gradient regions of 0e0.63 mm, 0.63e1.25 mm, 1.25e1.88 mm and 1.88e2.5 mm were improved to 30  7, 37  8, 43  9 and 53  10 mm/h (Fig. 5c, right group), respectively, which were significantly larger than those on the corresponding uniform surface (p < 0.05) except at 0e0.63 mm (3e3.5M) region (p > 0.05), and those measured on the same positions with a lower cellseeding density (p < 0.01) (Fig. 4c, right group). To the best of our knowledge the influence of cell-seeding density on the cell mobility in terms of direction and rate on the gradient surface has not been observed previously. These phenomena demonstrate that the interactions between cells enable the cells to sense the surface (either uniform or gradient) in a longer distance, i.e. the larger difference of the signals, and the gradient surface is beneficial of the cell polarization and further enhances their mobility [32]. Therefore, the directional migration of SMCs with an enhanced migration rate has been successfully achieved on the GPEMs with an appropriate cell-seeding density. In the following experiments, this cell-seeding density was adopted to

elucidate the long term effect of cell directional migration and possible mechanism. 3.3. Migration of cell populations on GPEMs In order to examine the relative long term effect of directional cell migration, the cell numbers on different positions of the GPEMs were quantified after being cultured for different time (Fig. 6). After first 2 h culture, the cell populations (Fig. 6a,d) decreased progressively along the extension of the position (1.4  0.2
104/cm2, 1.0  0.1
104/cm2, 0.87  0.10
104/cm2 and 0.71  0.07
104/cm2 on the positions of 0e0.63 mm (3e3.5M), 0.63e1.25 mm (3.5e4M), 1.25e1.88 mm (4e4.5M) and 1.88e2.5 mm (4.5e5M), respectively). After 24 h culture, the cell populations were changed to 1.8  0.2
104/cm2, 0.85  0.1
104/cm2, 0.71  0.08
104/cm2 and 0.57  0.06
104/cm2 at the corresponding positions. Since the total population of the cells on the whole GPEMs were almost constant and the previous results show that the PSS-dominated surface does not induce the cell death [32], one can conclude that the increased cell number in 3e3.5M region and the reduced cell number on other regions can be mainly attributed to the continuously directional migration of the cells to the lower hydration side, resulting in an eventual cell accumulation in the 3e3.5M region. With the prolongation of culture time, the remaining isolated cells on the higher hydration side have less probability to contact with other cells. Therefore, the directional migration induced by the cell interactions will become less effective and the cells cannot completely travel to the lowest hydration side. The doubling time of our cells is about 40 h. Since 12 h was used to allow the cell adhesion, the cell migration was only tracked for 24 h. If the observation time

L. Han et al. / Biomaterials 34 (2013) 975e984

4M 1.25mm

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d

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Fig. 6. Bright field images of SMCs on the gradient multilayers recorded at (a) 2, (b) 12 and (c) 24 h post cell seeding with a density of 1.5
104/cm2, respectively. (d) The cell number on the gradient multilayers at different regions after 2 h and 24 h culture.

3.4. The cellesubstrate interactions and cellecell interactions The cellesubstrate and cellecell interactions are the two main influence factors on cell migration [37]. When the cellesubstrate interactions are strong enough, the cells can stably attach and spread on the substrate with a slow migration rate. By contrast, when the cellesubstrate interactions are weaker than the cellecell interactions, the cells would prefer to interact with each other, resulting in the cell aggregates or cell clusters [38]. Therefore, the cellesubstrate interaction, i.e. the cell adherent strength, was further detected by the adherent ratio of cells on the substrate after centrifugation at 2000 r/min for 5 min Fig. 7 shows that 97% of the cells remained on the region of 3e3.5M, indicating a strong interaction between the cells and the surface. The proportions of residual cells on 3.5e4, 4e4.5 and 4.5e5M regions were reduced to 85, 80 and 78%, respectively, indicating that the interaction between cells and substrate is gradually weakened with the continuously enhanced hydration level [39]. Consequently, the cells are preferably to directionally migrate to the region with higher adherent strength. A few studies have shown that construction of cell densitysensing signals in a gradient manner on surface can effectively guide a single cell biased migration. For example, Chan et al. reported that when the cells are seeded on a low density region of a ligand gradient, their organelles are reoriented towards the side of higher ligand density [40]. Smith et al. found that the cells move faster on a fibronectin gradient with a larger slope [41]. However, in

our case the cells seeded at a low density are hardly to recognize and migrate along with the direction of gradient cues. Since the cells only can sense the gradient in a length scale of their body, i.e. less than 100 mm for a single cells at a low cell-seeding density, the gradient perceived by a single cell in such a small range may not be sharp enough to induce the directional migration. However, in the higher cell-seeding density model, the cellecell interactions and/or communications shall come into play and may help the cells to acknowledge a larger scale of the gradient.

Fraction of adherent cells

was further extended, the cells would start to duplicate, interfering the cell migration behaviors.

1.00 0.95 0.90

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3.5-4M 4-4.5M 4.5-5M 0.625-1.25 1.25-1.875 1.875-2.5

Fig. 7. The fraction of adherent cells on the gradient multilayers at (a) 0e0.63 mm (3e3.5M), (b) 0.63e1.25 mm (3.5e4M), (c) 1.25e1.88 mm (4e4.5M), and (d) 1.88e 2.5 mm (4.5e5M) regions after being centrifuged under 2000 rpm for 5 min. Cellseeding density was 1.5
104/cm2.

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Fig. 8. Bright field images of SMCs migrating on the gradient multilayers at 3.5e4M. The images were captured between 15 and 17 h. The arrows and red dots indicate the migration direction and central positions of the cells, respectively. Cell-seeding density was 1.5
104/cm2. Scale bar 50 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Previous studies reported that the cellecell contact plays an important role in regulating numerous cell functions. For example, Tang et al. reported that differentiation and its degree of mesenchymal stem cells to osteogenic and adipogenic are promoted by cellecell contact [42]. Gray et al. found that one contacting neighbor cell increases cell proliferation, while two or more neighbors partially inhibit this increase [43]. Itano et al. reported that the cellecell contact can promote cell migration [44]. In our study, the cellecell contact was in situ observed (Video 2 and Fig. 8). In this process, the lamellipodia initially extended to sense the cell surroundings. When a cell physically coupled to others, they tended to move together, and in most cases the right cells (on the substrate with a higher hydration level) were attracted by the left ones

(on the substrate with a lower hydration level) and moved to the left eventually (Fig. 8, arrows indicate the migration direction). Besides the gradient surface, this phenomenon could also be observed at the sharp edge of two uniform surfaces with different swelling behaviors (Video 3). The cells migrated effectively from the Multilayers-5M surface (a higher hydration level) to the Multilayers-3M surface (a lower hydration level) with the assistance of cellecell interactions, which is more obvious than that in a dispersed cell model. These results show that the cell contacts/ cellecell interactions indeed play an important role in the directional cell migration at a proper cell density. As a result, the cells continuously migrate to one end of the gradient multilayers under the assistance of cellecell interactions.

Fig. 9. Organization of focal adhesions and cytoskeletons of the SMCs cultured for 24 h on the gradient multilayers at (a) 0e0.63 mm (3e3.5M), (b) 0.63e1.25 mm (3.5e4M), (c) 1.25e1.88 mm (4e4.5M), and (d) 1.88e2.5 mm (4.5e5M) regions, respectively. Panel 1, the merge fluorescence images of vinculin (green), actin (red), and nucleus (blue). Panel 2 and Panel 3 show the vinculin and actin of cells, respectively. The arrows in Panel 3 indicate the cell conjunction points, which serve as the anchor points of cellecell interactions and transmit the contact-dependent signals and force. Cell-seeding density was 1.5
104/cm2. Scale bar 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

L. Han et al. / Biomaterials 34 (2013) 975e984

Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2012.10.041. Therefore, when the cells are seeded with an appropriate density to ensure the cellecell contact, they can migrate together and move like a group. The sensing area and distance of the cell group on the gradient surface is enlarged. Consequently, the imbalance force imposed by the substrate to the cells can be recognized effectively, which is transmitted to the adjacent cells by actin cytoskeleton interaction (as shown below). Finally, the cells move to the region with a higher adhesion force and result in the directional cell migration. To the best of our knowledge the celle cell contact dependence of the directional cell migration has not been observed previously. 3.5. Organization of focal adhesions and cytoskeleton on gradient multilayers

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enhanced compared to their corresponding counterparts of multilayers uniformly treated with the same concentrations of NaCl solutions. This study provides a versatile means to prepare gradient surfaces and thus dominate directional cell migration, highlighting a new perspective on designing future complex biomaterials for tissue regeneration. Acknowledgments This study is financially supported by the Natural Science Foundation of China (20934003), and the National Basic Research Program of China (2011CB606203). Z.M. thanks for the financial support from “Qianjiang” outstanding researcher funding of Zhejiang Province (J20110541). Appendix A. Supplementary material

The focal adhesion is highly related to the cellesubstrate interactions [45,46]. It contains a high concentration of activated and engaged integrins which modulate the major physical links between the outside and inside of a cell. Therefore, organization of the focal adhesions after 24 h cell seeding was investigated by staining vinculin (associated with focal adhesion) and F-actin (the major membrane-cytoskeletal protein in focal adhesion) on the different positions of GPEMs (Fig. 9). It has been known that large focal adhesion plaques (>1 mm2 in area, stable focal adhesions) between cells and substrate illustrate that the cells are tightly adhered with a slow mobility. Compared to the large focal adhesion plaques on the uniform multilayers [32], for example the Multilayers-3M, only small focal adhesion complexes could be found on the gradient surface (Fig. 9aed), suggesting the reduced cell adhesion strength. Since the reduced binding affinity between cells and substrate is preferred for the cell migration, the cells on all the positions of the GPEMs have faster migration rates compared to their counterparts on uniformly treated multilayers of the same salt concentrations. On the GPEMs, the F-actins were polymerized around the cell edge, which served as new adhesion points/protrusions, and then became lamellipodia or filopodia after extension (Fig. 9 a3ed3). It is known that the cell protrusions/lamellipodia can be stabilized through focal adhesions or adjacent cells [47]. Fig. 9 shows that conjunction points were formed (arrows in Fig. 9 a3ed3) between the adjacent cells possibly via transmembrane receptors linked to the actin cytoskeleton, which serve as anchor points of cellecell interactions and can transmit the contact-dependent signals and force [48]. Furthermore, through these anchor points more stable cells can exert a pulling force to the adjacent cells with weak adhesion to activate their compass. Among all the positions, the clustered actin filaments throughout the entire cells can be observed only from the region of 0e0.63 mm of the GPEMs (Fig. 9 a3), which is consistent with the fact that the cells near the 3M side of the gradient display good cell spreading and have stable adhesion. 4. Conclusions The gradient PSS/PDADMAC multilayers with a similar chemistry composition (PSS domination) but gradually changed swelling property were fabricated in a concentration gradient of salt solution ranging from 3 M to 5 M. The directional migration of SMCs was achieved on the gradient multilayers when the cells were seeded at an appropriate density. The SMCs finally accumulated preferentially on these regions with higher cell adherent strength. The cellecell interactions were found to play a decisive role in assistance of the directional cell migration through F-actin contact. Furthermore, the cell migration rates on the gradient multilayers were significantly

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