Complementary Density Gradient of Poly(hydroxyethyl methacrylate

May 19, 2014 - Complementary Density Gradient of Poly(hydroxyethyl methacrylate) and YIGSR Selectively Guides Migration of Endotheliocytes. Tanchen Re...
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Complementary Density Gradient of Poly(hydroxyethyl methacrylate) and YIGSR Selectively Guides Migration of Endotheliocytes Tanchen Ren,† Shan Yu,† Zhengwei Mao,*,† Sergio Enrique Moya,‡ Lulu Han,† and Changyou Gao*,† †

MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China ‡ CIC biomaGUNE, San Sebastian 20009, Gipuzkoa, Spain S Supporting Information *

ABSTRACT: Selective enhancement of directional migration of endotheliocytes (ECs) over vascular smooth muscle cells (SMCs) plays a significant role for the fast endothelialization of blood-contacting implants, in particular for the antirestenosis of vascular stents. Herein, a complementary density gradient of poly(2-hydroxyethyl methacrylate) (PHEMA) brushes and YIGSR peptide, a sequence specifically improving the mobility of ECs, was fabricated using a dynamically controlled reaction process. The gradients were visualized by fluorescent labeling and further quantified by X-ray photoelectron spectrometry (XPS) and quartz crystal microbalance with dissipation (QCM-d). The ECs exhibited preferential orientation and enhanced directional migration behavior on the gradient surface toward the region of lower PHEMA density and higher YIGSR density. The migration rate of the ECs was significantly enhanced to 5-fold, whereas the mobility of SMCs was not significantly influenced, leading to faster migration of ECs than SMCs. Therefore, the success of the complementary gradient relies on the appropriate interplay between the PHEMA brushes and the cell-specific ligands, enabling the selective guidance of EC migration.

1. INTRODUCTION Various important physiological processes are dependent on selective cell migration. Undesired cell migration will cause diseases or improper regeneration of tissues such as atherosclerosis, a chronic inflammatory disease of arterial wall.1 During atherosclerosis, endothelium, composed of endotheliocytes (ECs), is damaged, and subsequent migration of vascular smooth muscle cells (SMCs), which naturally move much faster than ECs to the impaired vessels, is stimulated by various inflammatory factors,2 leading to further damage of the vasculature. In-stent restenosis (ISR), a particular refractory form of neointimal hyperplasia,3 is another example. Stent implantation has become the main method to treat coronary artery diseases. However, the implantation may induce a series of pathological processes such as thrombosis and abnormal release of cytokines. These pathological events subsequently trigger the migration and proliferation of SMCs, and thereby induce ISR.4 Therefore, it is of great importance to develop a material which is able to specifically guide the migration of ECs rather than SMCs. Many physical and chemical signals have been implemented to materials to enhance the mobility of ECs, such as micropatterns,5−8 extracellular matrix proteins9−13 and their derived peptides,14−17 growth factors,18−20 and other bioactive molecules, such as heparin21 and polydopamine.22,23 However, the majority of the materials are not cell-selective. Only a few studies © 2014 American Chemical Society

have recently demonstrated that immobilization of an EC-specific ligand onto a cytocompatible matrix can specifically promote EC adhesion and rapid in situ endothelialization.14,21,24,25 Furthermore, some of the materials with EC-binding peptides can also release nitric oxide (NO), which decreases the adhesion and migration of SMCs.26 However, selective enhancement of EC migration by biomaterials has scarcely been achieved so far. The major difficulties in achieving a selective EC migration are (1) ECs naturally have much slower mobility compared to SMCs,27 and (2) the migration direction of ECs is hard to control. Fortunately, some important governing factors on the cell migration have been explored recently. For example, by adjusting the cell adhesion force on the substrates, the cell migration rate can be modulated by the grafting amount of poly(ethylene glycol) (PEG),28 the brush length of poly(hydroxyethyl methacrylate) (PHEMA),29 and the stiffness of salt-treated multilayers.30 Cells migrate fastest on a surface with a moderate cell adhesion force.31 The migration rate of cells can be further enhanced on a material with a gradient change of physiochemical properties.11,12,32 In addition, gradient signals can also guide cell migration in a preferential direction.33−36 However, Received: March 12, 2014 Revised: May 17, 2014 Published: May 19, 2014 2256

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diameter of 1.6 cm, into which the BCS toluene solution (0.05% v/v) was injected continuously by a microinfusion pump (WZS-50F2, Zhejiang University Medical Instrument Co., Ltd., China) at room temperature with a rate of 2 mL/h. A total of 30 min later, the slide was taken out and sequentially washed in toluene, acetone and ethanol under ultrasonication. The −Br groups were then substituted by azide groups as described in literature.46 In brief, the slide was immersed in a NaN3 dimethylformamide (DMF) solution (65 mg in 10 mL of DMF) at 80 °C for 24 h. After thoroughly washing in plenty of water 5×, the slide was dried under a nitrogen flow and immersed in a BES toluene solution (0.05% v/v) for 30 min at room temperature. The slide was then washed with toluene 5× and dried under a nitrogen flow. 2.3. Preparation of PHEMA/YIGSR Complementary Density Gradient. The PHEMA brushes were generated on the pretreated slide by surface-initiated atom transfer radial polymerization (SI-ATRP).29 In brief, the slide was first placed in a sealed Pyrex test tube purged with nitrogen, into which the oxygen-free ATRP reaction solution (the molar ratio of HEMA/CuCl2/ascorbic acid/PMDETA was 100:1:2:1, and the v/v ratio of HEMA/water was 1:50) was rapidly injected. After reaction at room temperature for a predetermined time period, the slide was thoroughly washed in plenty of ethanol and water under ultrasonication 3 times, respectively. To visualize the PHEMA thin film by fluorescence microscopy, fluorescein O-methacrylate (FMA, Sigma) was copolymerized with HEMA at a molar ratio of 1:20 at the same condition. It has been demonstrated previously that copolymerizing with FMA at this ratio does not change the property of the polymer chain.47 The peptide alkynyl modified YIGSR was “clicked” onto the gradient surface by referring to the work of Becker’s group.48,49 The PHEMA/ azide groups-functionalized slide was immersed in 10 mL of oxygen-free peptide aqueous solution containing 0.2 mmol sodium L-ascorbate, 0.04 mmol copper(II) sulfate pentahydrate, and 1 mg alkynyl YIGSR peptide at 37 °C under shaking. After 5 h, the slide was placed in 0.01 mM ethylene diamine tetraacetic acid disodium salt (EDTA) aqueous solution overnight at 4 °C to remove the bound copper ions. YIGSR peptide was labeled with rhodamine B isothiocyanate (RITC) in order to visualize its distribution on the gradient. 2.4. Characterization of the Gradients. The chemical compositions of the surfaces were characterized by X-ray photoelectron spectroscopy (XPS) using an Axis Ultra spectrometer (Kratos Analytical, U.K.) with a monochromatized Al Kα source at pass energy of 160 eV for survey scans and 80 eV for core level spectra. Data was analyzed by Kratos Vision Processing and XPS Peak software. The binding energy was corrected by setting the lowest binding energy of C 1s peak at 284.6 eV. Thicknesses of the PHEMA films and peptide layers were measured by a variable angle spectroscopic ellipsometer (model M2000D, J. A. Woollam Inc., Lincoln, NE, U.S.A.) at a 70° angle of incidence and a wavelength range of 300−1700 nm. A Cauchy model provided with the instrument for transparent layer (Cauchy layer/silicon substrate) was used to fit the data. Here we used Si substrates for the ellipsometry tests. The thickness of the silica layer on the Si wafer was measured first and subtracted to get the thickness of the organic layer. The mass growth of PHEMA brushes and grafted peptide on uniform surfaces was quantitatively monitored by quartz crystal microbalance with dissipation (QCM-d, Model Q-SENSE E4, Sweden) according to the change of resonance frequency using a Sauerbrey model embedded in the QTools software.50 Three independent measurements were carried out for all the samples. 2.5. Cell Culture. Human vein endotheliocytes (ECs) and human vein smooth muscle cells (SMCs) were obtained from the Cell Bank of Typical Culture Collection of Chinese Academy of Sciences (Shanghai, China). The ECs were maintained with Roswell Park Memorial Institute-1640 medium (RPMI-1640) and the SMCs were maintained with a regular growth medium consisting of high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, U.S.A.). Both mediums were supplemented with 10% fetal bovine serum (FBS, Sijiqing Inc., Hangzhou, China), 100 U/mL penicillin, and 100 μg/mL streptomycin and cultured at 37 °C in a 5% CO2 humidified environment.

several types of cells show a similar migration tendency on these gradient materials, suggesting they are not cell-selective. Only Fittkau et al. demonstrated that a PEG hydrogel modified with Thy−Ile−Gly−Ser−Arg (YIGSR) and Arg−Gly−Asp (RGD) peptides can selectively enhance the migration of ECs by 25% but do not promote that of SMCs.15 In vivo, the selective directional migration is driven by the synergistic effect of attractants secreted from the destination point and the repellant from the cells passing by.37 In this study, we mimic this phenomenon and integrate a complementary gradient of PHEMA brushes and EC-specific ligands on the same substrate (Scheme 1). PHEMA is a typical hydrophilic biocompatible polymer Scheme 1. Schematic Illustration to Show the Structure of a Complementary Density Gradient of PHEMA and YIGSR and Its Influence on the Mobility of ECs and SMCsa

a

The direction of increased YIGSR density and decreased PHEMA density is defined as “+X” direction.

that is commercially available in artificial corneas. The antifouling effect of PHEMA polymer brushes has been widely demonstrated.38−40 Decrease of the density of PHEMA brushes provides a gradually enhanced cell adhesion force regardless of the cell types, whereas the complementary increase of EC-specific ligand density is expected to selectively enhance the mobility of ECs. Due to the synergetic effects of PHEMA repelling and ligand attraction, ECs shall be guided in the direction of the ligand gradient. YIGSR is an active sequence derived from β1 chain of laminin, and can interact with the 67 kDa laminin binding protein (67LR), which is highly expressed on membrane surface of ECs.41 Therefore, the surfacetethered YIGSR can improve the attachment,42 spreading,43 and migration16 of ECs, rather than other types of cells such as SMCs.15,26,44 To the best of our knowledge, this is the first time to obtain such a complementary gradient of polymer brushes and cellbinding biomolecules, and it is also the first time to observe a selective directional migration of one type of cells over another type based on the complementary gradient.

2. EXPERIMENTAL SECTION 2.1. Materials. Hydroxyethyl methacrylate (HEMA) was purchased from J&K company (Guangzhou, China) and was purified by neutral aluminum oxide columns before use. Alkynylated peptide, HCC-GGGYIGSRGGGK, was synthesized by Sangon Biotech (Shanghai, China). 3-(2-Bromoisobutyramido)propyl(triethoxy)silane (BES) was synthesized according to literature.45 All other chemicals were of analytical grade and used as received. Milli-Q water and ultrapure nitrogen were used through all the experiments. 2.2. Preparation of Complementary Density Gradient of Azide Groups and ATRP Initiators. A microinjection method was used to create the gradient of (3-bromopropyl)trichlorosilane (BCS) density gradient by controlling the reaction time. A clean glass/silica slide was vertically placed in a cylindrical plastic container with a 2257

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2.6. Cell Migration. The samples were sterilized in 75% ethanol for 1 h, followed with six washes in phosphate buffered saline (PBS, pH 7.4). Cell migration behaviors were studied on various positions of the gradient surfaces and their corresponding uniform surfaces with same densities of PHEMA and YIGSR brushes. The cells were seeded at a relatively low density (5 × 103 cells/cm2) to minimize cell−cell interaction. The cell migration behaviors were monitored for 12 h in situ using a time-lapse phase-contrast microscope (DMI6000B, Leica) equipped with an incubation chamber (37 °C and 5% CO2 humidified atmosphere) 8 h post cell seeding. The manual tracking plugin in NIH ImageJ software was used to track each individual cell to yield a sequence of (x, y) position coordinates versus time. The original position of each cell was defined as the origin (0, 0) of the trajectory. The cell trajectories over the entire observation period were reconstructed from the center positions of individual cells. Cell migration distance and rate were then calculated by Chemotaxis Tool (Ibids, Germany) at 15 min time intervals over the observation period. All mitotic and spherical dead cells were excluded from the analyses. At least 50 cells were chosen randomly from each sample for analysis. Three independent replicate experiments were carried out. The data was analyzed by Rayleigh test and a significant level of p < 0.05 was set, suggesting a statistically significant asymmetric distribution of the end points. Rayleigh test embedded in ImageJ software was used to measure the significance of the mean direction of a circular distribution,51 which can be used to represent the directionality of cell migration.52 In cell sheet migration experiments, the glass slide was covered with a polydimethylsiloxane (PDMS) stamp with a slit (around 0.5 mm × 10 mm). The slit was placed vertically to the direction of gradient, exposing a desired stripe for initial cell adhesion (Scheme S2). The stamps were pretreated with plasma to increase surface hydrophilicity. Cells were seeded onto the desired positions of the gradients at a density of 5 × 105 cells/cm2 and were allowed to form a confluent monolayer. At 8 h post cell seeding, the PDMS films were gently removed to allow cell migration on the gradients. The cell sheet morphology and location were observed under the time-lapse microscope for 24 h. Three independent experiments were carried out for each sample. 2.7. Cell−Substrate Interaction. Fluorescent staining of 67LR and nucleus was carried out. Briefly, after 24 h culture in the medium containing 10% FBS, the cells were carefully washed with PBS 3× and then fixed with 4% paraformaldehyde at 37 °C for 30 min, followed by three washes in PBS. The cells were further treated in 0.5% (v/v) Triton X- 100/PBS at 4 °C for 10 min to increase the permeability of the cell membrane. After being rinsed 3× with PBS, they were incubated in 1% bovine serum albumin (BSA)/PBS at 37 °C for 30 min to block the nonspecific interactions. Then the cells were incubated with a mouse monoclonal antibody against human 67LR (Abcam) for 1 h. After being washed twice in 1% BSA/PBS, they were further stained with FITC-labeled goat antimouse IgG (Beyotime, China) and 4′,6-diamidino2-phenylindole (DAPI) (Sigma) at room temperature for 1 h, followed by three washes in PBS. The cells were observed under confocal laser scanning microscopy (CLSM, SP5, Leica) and the relative fluorescent intensity was analyzed by ImageJ software from at least 20 cells. The distribution of 67LR on ECs was further analyzed by dividing cells into three regions along the gradient direction according to the position of nucleus (as shown in Figure 5h): rear, middle, and anterior. The relative fluorescent intensity of each region was also analyzed by ImageJ software from at least 20 cells in each experiment. Three independent experiments were carried out. 2.8. Protein Adsorption. Uniform surfaces represent specific positions of gradients were used for protein adsorption experiments. To test total protein adsorption, the slides (8 mm × 8 mm) were incubated in cell culture medium containing 10% FBS at 37 °C for certain time, washed with copious PBS for 3× to remove free proteins and ions. Then the surface-adsorbed proteins were detached in 200 μL 1% sodium dodecyl sulfate (SDS) solution at 37 °C for 30 min. After that, the total amount of adsorbed proteins was determined by micro BCA protein assay kit (Thermo) by referring to a standard protein concentration curve. The activity of surface-adsorbed fibronectin (Fn) was detected by enzyme-linked immunosorbent assay (ELISA). After incubated with 10% FBS at 37 °C for 4 h and washed with PBS 3×, the substrates were

treated with Fn antibody (C6F10, Santa Cruz) in 1% BSA/PBS solution (1:200) for 1 h at 37 °C. After washed with 1% BSA/PBS solutions three times, the substrates were further treated with enzyme linked IgG and subsequently tetramethyl benzidine (TMB) solution. After the chromogenic reaction was stopped with 0.5 M H2SO4, the absorbance of each resulting solution at 450 nm was measured. The PHEMA/YIGSR complementary gradient was pretreated in 30 μg/mL Fn/PBS solution at 37 °C for 4 h. Cell migration on the prinstine and Fn preadsorbed gradients in serum free medium and complete medium containing 10% FBS was studied to evaluate the effect of proteins, respectively. 2.9. Statistical Tests. Statistical difference of the data was analyzed by one-way ANOVA with a Turkey post hoc method, and a significant level of p < 0.05 was chosen for all the tests, unless otherwise stated. The error bars represent standard deviation of the mean value of each type of experiments.

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of the Complementary Gradient. In this study, a linear density gradient of YIGSR is designed, which shall be backfilled with PHEMA. Therefore, the density range of YIGSR is preferred to change from 0 to a value of as high as possible. As illustrated in Scheme S1, the complementary density gradient of azide groups and ATRP initiators, BES, were first created on a glass/silica surface. In brief, into a glass vial having the vertically placed slide, the (3-bromopropyl)trichlorosilane (BCS) solution was injected continuously by a microinfusion pump to generate the gradient −Br end-capped surface (from top to bottom, the defined gradient direction; 5 mm long). The Br/C ratio, measured by XPS (Figure S1a), reflects that the relative content of −Br on the surface had a positive correlation with the gradient position, that is, the BCS reaction time (X-coordinate). On this surface, a water drop moved to the reverse direction of the gradient with more −OH groups, revealing asymmetrical nature (Figure S2).53,54 By nucleophilic substitution reaction with NaN3 in DMF solution, the −Br groups gradient was transformed into an azide gradient (Figure S1b), which was used to click the YIGSR peptide after HEMA polymerization. The remaining −OH groups on the substrate, then reacted with BES, yielding a complementary density gradient of ATRP initiators consisting of a −C(CH3)2Br group (Figure S1c). Namely, a higher density of −C(CH3)2Br groups is formed at a upper position where the BCS reaction time is shorter. The complementary density gradient of PHEMA brushes and YIGSR peptide was subsequently obtained because they are dependent on the densities of initiators and azide groups, respectively. One of the important advantages of this strategy is that the reaction processes are basically thermodynamically controlled except for the first BCS reaction, leading to the good control over the quality of samples in different batches. According to our preliminary experiments, the PHEMA thickness was controlled at ∼3 nm so that the polymer brushes would not bury the peptide ligands (the length of HCC-GGGYIGSRGGGK is estimated to be ∼5 nm in a complete extension state). As PHEMA brushes grew almost linearly at gradient surfaces (Figure S3), 3 min was chosen as the polymerization time. According to Figure S4, the PHEMA brushes were built first, and then the peptides were clicked onto the surface. Figure S4 shows also that the YIGSR density on the complementary surface is almost the same to that of the individual surface, suggesting the presence of the very thin PHEMA brushes did not jeopardize the peptide immobilization. This is reasonable since the substrate with the same density and distribution of BES and BCS is used to 2258

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Figure 1. (a) Fluorescent images showing the complementary gradient of PHEMA (upper) and YIGSR (lower) at 4 mm position, respectively. HEMA was copolymerized with FMA (green) and YIGSR was stained with RITC (red), respectively. (b) Fluorescent intensity as a function of position corresponding to the gradient shown in (a). (c) Mass densities of PHEMA and YIGSR on the complementary gradient as a function of gradient position and BCS reaction time, respectively. The data are linear fitted with a R2 = 0.86 and 0.88 for PHEMA and YIGSR, respectively.

prepare the individual (without YIGSR immobilization) and complementary gradients. Since it is difficult to directly quantify the grafting mass/ density of the polymers and peptides on the gradient surface, their grafting processes on uniform surfaces with different BCS reaction time (corresponding to the respective gradient positions) were monitored by ellipsometry and QCM-d (Figures S3,5). In this study, the increasing density of YIGSR (i.e., the decreasing density of PHEMA) is set as the x coordinate (Scheme 1). In order to observe the YIGSR and PHEMA gradients directly, on the gradient surface YIGSR peptide was labeled with RITC, and FMA was copolymerized with HEMA, respectively. Figure 1a,b show that along with the gradient direction the fluorescent intensities of PHEMA and YIGSR gradients were gradually reduced and enhanced, respectively. The density gradient of the YIGSR peptide was also confirmed by XPS measurement (Figure S6). Taking into account the PHEMA and YIGSR densities on the uniform surfaces (Figure S7), their densities on the complementary gradient surface were obtained at different positions (Figure 1c).55 The grafting masses of PHEMA brushes and YIGSR almost linearly decreased (−43 ng/cm2·mm) and increased (73 ng/cm2·mm) along with the complementary gradient, respectively. For example, at 1, 2.5, and 4 mm positions, the densities of PHEMA brushes reached 341, 273, and 191 ng/cm2, whereas the densities of YIGSR reached 55, 178, and 314 ng/cm2, respectively. The deviation of the gradient slope is very small in the repeating experiments, suggesting the good reproducibility of the complementary gradient. All the results confirm that the complementary gradient with a linear decrease of PHEMA and increase of YIGSR densities is successfully prepared. 3.2. Cell Migration on the Complementary Gradient. Endotheliocytes form the endothelium of blood vessel, and smooth muscle cells are the major cell types in the tunica media

vasorum. In atherosclerosis, the endothelium formed by ECs is damaged and migration of SMCs at the impaired vessels is stimulated by a cocktail of inflammatory factors,56 resulting in the predominant migration of SMCs over ECs and further deterioration of vasculatures. Correspondingly, in cardiovascular tissue engineering, a delicate balance in growth and migration of ECs and SMCs is desired to favor fast endothelialization on biomaterials, which is critical to lower the risk of coagulation, restenosis, and so on. So it is of paramount significance to create materials that promote the migration of ECs and inhibit that of SMCs. In this research, PHEMA with a moderate thickness is expected to improve the migration of cells regardless of cell types, and the PHEMA gradient shall guide the cell migration direction. Together with the biospecific role of YIGSR peptide to ECs, the complementary gradient is then expected to selectively improve the ECs migration behaviors in terms of rate and direction. The ECs and SMCs were cultured on the complementary gradient surface by using single gradients of PHEMA and YIGSR, and tissue culture polystyrene (TCPS) as controls (Figure 2). Although the slope of the gradient is consistent through the surface, the selective guidance of the migration of different types of cells became prominent only at the 4 mm position (the densities of PHEMA and YIGSR were 193 and 308 ng/cm2, respectively). At this place, the complementary gradient showed strongest haptotaxis to ECs over SMCs in terms of migration rate and directionality (Figures 2−4 and Video S1). Furthermore, ECs also oriented on the complementary gradient surfaces, that is, about 50% of the cells aligned in ±30° of the X direction (Figure S8). Figure 2a shows that 82% ECs moved to the X direction of the PHEMA/YIGSR complementary gradient, suggesting almost unidirectional migration of ECs. By contrast, SMCs did not 2259

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Figure 2. Migration traces of ECs (a−d) and SMCs (e−h) on (a, e) PHEMA/YIGSR complementary gradient, (b, f) YIGSR gradient, and (c, g) PHEMA gradient at 4 mm position, and on (d, h) TCPS, respectively. Cells in the range of 1.2 mm, that is, 4 ± 0.6 mm were observed. The migration traces of the cells to the X direction (indicated by the number in the left-bottom corner) and −X direction are drawn in red and black lines, respectively. The cells were continuously tracked every 15 min for 12 h. The total number of the trajectories was 50 for each position. The data of the end points were analyzed by Rayleigh test, suggesting a statistically significant asymmetric distribution with p < 0.001 in (a) and p < 0.05 (f).

Figure 3. Statistic results of (a) the percentage of cells moving to the X direction and (b) migration rate on different gradient surfaces. *Indicates a statistical significant difference at p < 0.05 level.

Figure 3b). Indeed, Figure 3b reveals that ECs migrated faster than SMCs only on this complementary surface. On all other types of surfaces (single YIGSR and PHEMA gradients, and TCPS), SMCs moved faster than ECs. In detail, on the TCPS surface (Figure 2d), the ECs moved randomly to very limited distances during the observation period, revealing their extremely slow mobility (3.5 μm/h). The SMCs (Figure 2h) moved randomly, too, but they migrated obviously longer distances, revealing a faster mobility (8.5 μm/h). On the YIGSR density gradient ECs moved faster (7.1 μm/h) than those on TCPS, while the mobility of SMCs was not significantly enhanced. Nonetheless, the migration of ECs is still significantly slower than that of SMCs. It is known that the capabilities of other biomolecules including collagen,9 fibronectin,10 and vascular endothelial growth factor (VEGF)57 are similar to that of YIGSR15 (2×) in terms of enhancing ECs migration, showing the strong effect of the complementary YIGSR and PHEMA

present obvious migration directionality (Figure 2e, only 54% toward the gradient direction), showing no significant difference with those cultured on TCPS (Figure 2h). Moreover, the single gradient of YIGSR and PHEMA showed rather weak haptotactic effect to ECs (Figure 2b,c), leading to 56 and 64% directionality toward X direction, respectively. However, they showed a relatively stronger guidance effect to the SMCs (Figure 2f,g), leading to respective 68 and 60% directionality which are larger than that of the complementary gradient (54%). The statistical data in Figure 3a show the same tendency. These results reveal that the complementary structure of the gradient with appropriate densities of PHEMA and YIGSR is very effective to selectively guide the directional migration of ECs. Figure 2a,e also shows that ECs traveled significant longer distances than SMCs on the complementary gradient. Consequently, their migration rate (18.2 μm/h, 5-fold of that on TCPS) was significantly faster than that of SMCs (9.7 μm/h; 2260

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Figure 4. (a) Optical photos of EC (upper panel) and SMC (lower panel) sheets cultured on PHEMA/YIGSR, PHEMA, and YIGSR density gradient surfaces for 24 h, respectively. The red lines represent the original boundaries of the cell sheets, which are endowed with green color by Photoshop software for better discrimination from the substrates. (b) The statistic result of the migration distance of cell sheet in X direction. *Indicates statistical significant difference at p < 0.05 level.

However, as shown in Figure S9, the results could not provide a convincing explanation of significantly enhanced cell mobility on the complementary PHEMA/YIGSR density gradient than the PHEMA gradient. Other factors must come into play. It has been reported that YIGSR peptide is able to bind to 67LR, a membrane-associated protein highly expressed on the plasma membrane of ECs, and subsequently interacts with cytoskeleton.59 This interaction will induce a series of cell activity such as migration.41 Therefore, the 67LR was immunofluorescently stained (Figure 5a−f), and its expression level was quantified from the images (Figure 5g). 67LR was highly expressed on the ECs (Figure 5a−c). By contrast, almost no expression of 67LR was found on the SMCs (Figure 5d−f). The results suggest that the ECs may have stronger interaction with YIGSR surfaces through specific recognition, enhancing the migration of ECs rather than SMCs. On the different surfaces, the expression levels of 67LR by ECs were different (Figure 5g), with the sequence of YIGSR gradient > PHEMA/YIGSR complementary gradient > PHEMA gradient. The distribution of 67LR on ECs was more asymmetric on the complementary gradient (Figure 5i). It was found that only on the PHEMA/YIGSR complementary gradient the 67LR was expressed with a significantly higher value at the anterior region (toward high density YIGSR) than at the rear region. The obvious asymmetric distribution of 67LR toward gradient direction should be solely induced by the complementary gradient, which subsequently guides the directional migration of ECs. By contrast, the distribution of 67LR on ECs was statistically uniform on the YIGSR gradient. This fact may explain the random migration of ECs on the pure YIGSR gradient. In summary, the selectively enhanced mobility and directional migration of ECs over their competitive SMCs are achieved by the synergistic effects of PHEMA and YIGSR complementary gradient on the same substrate. 3.4. Protein Adsorption. When biomaterials are put into cell culture environment, first water will reach the materials

gradient. Figure 2c,g reveals that, on the PHEMA density gradient, both ECs and SMCs moved about 3× longer distance than those on the TCPS surfaces, respectively, revealing that the mobility of ECs (12.5 μm/h) and SMCs (21.9 μm/h) are significantly enhanced. However, the effect of PHEMA density gradient is not cell-selective. Therefore, only the synergistic effect of the complementary gradient can achieve the optimal effects (Figure 2a). As a consequence of change of the cell migration ability, the wound healing process shall be greatly influenced. Therefore, the cell sheet assay was further performed (Figure 4). For the movement of cell sheets, the cell−cell interaction will come into play. In this case, the cells are not likely to move freely and thereby exhibit lower mobility. Therefore, the results in Figure 2 and Figure 4 are not directly comparable in terms of the absolute values. The ECs sheets only directionally migrated toward +X direction on the PHEMA/YIGSR complementary gradient with almost 100 μm in +X direction and only a few micrometers in −X direction (Figure 4b). Since the cells do not duplicate very prominently during 24 h period, the movement of the cell sheet is mainly attributed to the collective migration of cells, proving again the strong guidance of the PHEMA/YIGSR complementary gradient. By contrast, the ECs moved similar distances toward both directions (50 and 37 μm) on the YIGSR gradient and did not move on the PHEMA gradient, suggesting that the single gradient alone could not effectively guide the collective cell migration. Moreover, the SMC sheets migrated to very limited distances on all three types of gradients and did not show any preferential directionality (Figure 4). All the results confirm the success of the present complementary gradient, which can effectively promote the ECs mobility, selectively guide the directional migration of ECs, and retard the migration of SMCs. 3.3. Cell−Substrate Interactions. Previous studies reveal that cell adhesion force on substrate plays an important role on the cell migration behaviors.28,29,58 Cells usually have the highest mobility on the surfaces with a moderate adhesion force. 2261

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Figure 5. CLSM images showing the distribution of 67LR (green) of (a−c) ECs and (d−f) SMCs at 4 mm position of (a, d) PHEMA/YIGSR complementary density gradient, (b, e) YIGSR density gradient, and (c, f) PHEMA density gradient, respectively. Nucleus (blue) was stained by DAPI. (g) Relative expression of 67LR on different surfaces. Data are calculated from (a−f) by ImageJ software. (h) Schematic illustration of rear, middle, and anterior regions of polarized ECs. (i) Relative expression of 67LR from different regions of ECs on different surfaces. *Indicates statistical significant difference at p < 0.05 level.

When cells were cultured in a medium without FBS (Figure 6c), the migration rate of SMCs significantly increased to 20.0 μm/h while that of ECs decreased to 8.7 μm/h. Besides, only 58% of ECs moved toward +X direction, suggesting the reduced directional migration (Figure 6d). The results reveal that the FBS is necessary to achieve the selective guidance of ECs. On the surface preadsorbed of Fn, the migration rate of SMCs decreased significantly (8.4 μm/h) in the medium without FBS, which was similar to that of SMCs in the medium containing 10% FBS. The results indicate that Fn-adsorption is a key factor in reducing the mobility of SMCs on the complementary gradient. In contrast, the preadsorption of Fn did not significantly influence the migration rate of ECs (11.0 μm/h) and directionality (63%) with or without the presence of FBS (p > 0.05). The results suggest that the presence of FBS, rather than Fn preadsorption, is a key factor in enhancing the directionality and mobility of ECs under the guidance of the complementary gradient. This is very promising since in the in situ adsorption assay, many different types of proteins can be competitively adsorbed onto the surfaces. In fact, the majority of the proteins should be BSA, which is the most abundant protein in serum. Therefore, the impact of protein adsorption on cell migration can be much more complex in this case. This finding suggests the

surface, following by some small molecules and then proteins. Cells arrive last.60 So, protein adsorption on biomaterials can influence the following cell behaviors. Fibronectin (Fn) is a glycoprotein in extracellular matrix that can not only bind to membrane-spanning receptor proteins (integrin) but also bind to other ECM components such as fibrin, collagen, and heparin sulfate proteoglycans.61 It plays a major role in cell adhesion,62 proliferation,63 migration,10 and differentiation.64 As a result, the adsorption of proteins, especially Fn, on the complementary gradient might also influence the migration of cells. First, the total protein adsorption amount on different surfaces was measured in culture medium containing 10% FBS. As shown in Figure 6a, the protein amount increased along with incubation time, but the increasing rate slowed down after 4 h on all surfaces. The protein adsorption amount on the YIGSR and PHEMA/YIGSR gradients was about 2.5 μg/cm2, which was much higher than that of PHEMA surface (1.5 μg/cm2). The adsorption of functional Fn had a similar trend on these surfaces (Figure 6b). The adsorbed amount of active Fn, which was slightly smaller than on TCPS, was highest on the YIGSR gradient. This amount was significantly reduced on the PHEMA and PHEMA/YIGSR gradients. The impact of protein adsorption on cell migration was further studied on the PHEMA/YIGSR complementary gradient. 2262

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Figure 6. (a) Protein adsorption amount on different surfaces in culture medium containing 10% FBS as a function of incubation time. (b) Relative active Fn amount adsorbed on different substrates determined by ELISA assay. TCPS was used as a positive control. (c) Cell migration rate of ECs and SMCs on PHEMA/YIGSR gradient. (d) The percentage of cells moving toward to the X direction at different environments. *Indicates statistical significant difference at p < 0.05 level.

potential application of such gradients in the vascular therapies, in a complicate environment containing a lot of different types of protein molecules.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Schemes for materials preparation and cell sheet migration experiments are included as Schemes S1 and S2. Additional data are included in Figures S1−S9. Cell migration videos are included in Video S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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ACKNOWLEDGMENTS

This study is financially supported by the National Basic Research Program of China (2011CB606203), the Natural Science Foundation of China (21374097 and 51120135001), and Open Project of State Key Laboratory of Supramolecular Structure and Materials (sklssm201303).

4. CONCLUSIONS In conclusion, PHEMA/YIGSR complementary gradient was successfully fabricated. On this surface, ECs migrated directionally along the gradient direction with significantly enhanced mobility. By contrast, SMCs did not show preferred directional migration nor enhanced mobility on the gradient. The specific interaction between ECs and the substrate played a decisive role in the selective guidance of EC migration over SMCs. This study opens new strategies for desired tissue regeneration by using a complementary gradient with a synergic effect of two signals to dominate the selective cell migration, highlighting a new perspective on designing complex biomaterials.

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AUTHOR INFORMATION

Corresponding Authors

*Fax: (+)86-571-87951108. E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest. 2263

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