A density gradient of basic fibroblast growth factor guides directional migration of vascular smooth muscle cells

Colloids and Surfaces B: Biointerfaces 117 (2014) 290–295

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A density gradient of basic fibroblast growth factor guides directional migration of vascular smooth muscle cells Jindan Wu a,b , Zhengwei Mao a , Lulu Han a , Yizhi Zhao a , Jiabin Xi a , Changyou Gao a,∗ a MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China b MOE Key Laboratory of Advanced Textile Materials & Manufacturing Technology, College of Materials and Textile, Zhejiang Sci-Tech University, Hangzhou 310018, China

a r t i c l e

i n f o

Article history: Received 17 December 2013 Received in revised form 8 February 2014 Accepted 17 February 2014 Available online 2 March 2014 Keywords: Gradient Basic fibroblast growth factor Cell migration Smooth muscle cells Biomaterials

a b s t r a c t The migration of vascular smooth muscle cells (VSMCs) is an important process in many physiological events. It is of paramount importance to control the migration rate and direction of VSMCs by biomaterials. In this paper, a density gradient of basic fibroblast growth factor (bFGF) was fabricated using an injection method and the bio-conjugation between heparin and bFGF. The density of bFGF gradually increased with a slope of 17 ng/cm2 /mm. Adhesion and migration of VSMCs were studied on the bFGF gradient. The VSMCs exhibited preferential orientation and an enhanced directional migration behavior on the gradient surface. Up to 70% cells migrated towards the region with a higher density of bFGF on the gradient. However, the bFGF gradient had no effect on the cell migration rate. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The cell migration involves in many significant biological processes such as embryo morphogenesis, angiogenesis, wound healing, immune response, and tumor metastasis etc. [1,2]. In vivo it is mediated by the so-called chemical and/or physical gradient signals [3–7]. Inspired by this fact, biomaterials with gradient chemical and/or physical cues have been constructed to spatiotemporally guide cell migration in vitro [8]. For example, the gradient materials with varying stiffness show a strong effect on guiding the directional migration of smooth muscle cells (SMCs) [9,10]. The density gradients of synthetic polymers, especially hydrophilic ones, have been generated to provide an adhesion force gradient to guide the directional cell migration [11,12]. The density gradients of extracellular matrix (ECM) proteins such as fibronectin, laminin and collagen as well as their derivative peptides have also been widely used to modify materials [13–17]. They are effective to regulate cell distribution, and induce cell alignment and even directional migration as well [18]. The growth factors are very effective in regulating cell behaviors and responses [19,20]. For example, basic fibroblast growth factor

∗ Corresponding author. Tel.: +86 571 87951108; fax: +86 571 87951108. E-mail addresses: [email protected], [email protected] (C. Gao). http://dx.doi.org/10.1016/j.colsurfb.2014.02.043 0927-7765/© 2014 Elsevier B.V. All rights reserved.

(bFGF) is a paradigm of a group of nine closely related, multifunctional proteins known as fibroblast growth factor family (FGFs). The FGFs induce proliferation, mitosis, migration, differentiation and other biological responses in most mesoderm and neuroectodermderived cells [21,22]. As a result, a variety of growth factors have been applied in tissue regeneration. However, much less attention is paid to the fundamental study to elucidate their influence on cell migration, especially after immobilization on substrates, leading to deficiency of mechanistic understanding of the structure–function correlation of growth factors-activated biomaterials. Recently, a few studies have proved the effectiveness of gradients of growth factors on cell migration. Stefonek–Puccinelli prepared a density gradient of epidermal growth factor (EGF), and showed that keratinocytes exhibited almost 10-fold directional migration in the presence of EGF gradient [19]. Liu et al. found that the directional migration of endothelial cells increased 2-fold on a vascular endothelial growth factor (VEGF) density gradient compared to a fibronectin gradient [20]. However, little work has been done on directing cell migration by bFGF gradient. We found that the surface-tethered bFGF molecules were able to control the mobility of VSMCs in a density-dependent manner in the range of 0 ∼ 300 ng/cm2 [23]. This effect is selectively effective to VSMCs but not to endothelial cells and mesenchymal stem cells. Since the migration of VSMCs is a key event in the development of arterial lesions, vessel generation and myocardium repairing [9,24,25], it is

J. Wu et al. / Colloids and Surfaces B: Biointerfaces 117 (2014) 290–295

Fig. 1. Schematic illustration to show the structure of bFGF gradient and cell migration on the surface.

of practical importance to develop a gradient of bFGF instead of the uniform counterpart, so that the migration direction of VSMCs can be effectively controlled. In this paper, a bFGF gradient is generated on a heparin density gradient via bio-conjugation between these two types of molecules. The gradient bFGF maintains its bioactivity, and implements the preferential orientation and directional migration of VSMCs (Fig. 1). 2. Experimental 2.1. Materials 3-(Trimethoxysilyl)propyl methacrylate (TMSPMA) and 3triethoxysilylpropylamine (APS) were purchased from J&K company. bFGF (Mn ∼ 18.5 kDa) was obtained from the Center of Biotechnology Research and Development, Jinan University, Guangdong, China. Heparin sodium (Mn ∼ 26 kDa) was purchased from Sinopharm Chemical Reagent Co., Ltd. N-EthylN -(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Aladdin Reagent Co., Ltd. Bovine serum albumin (BSA) was purchased from Sigma. All the chemicals were of analytical grade and used without further purification. The water was purified via a Milli-Q® Gradient System equipped with a quantumTM cartridge, and had a resistivity of 18.2 m/cm. Human VSMCs were purchased from the Cell Bank of Typical Culture Collection of Chinese Academy of Sciences (Shanghai, China). They were maintained in high-glucose DMEM culture medium (Gibco, USA), supplemented with 10% fetal bovine serum (FBS, Sijiqin Inc., Hangzhou, China), 100 U/mL penicillin and 100 ␮g/mL streptomycin, and cultured at 37 ◦ C in a 5% CO2 humidified environment. 2.2. Surface preparation The glass or silicon slide was cut into 0.5 × 1.5 cm pieces, which were cleaned in toluene, acetone and alcohol by ultrasound, respectively. They were further treated with “piranha” solution (a mixture of 30% of hydrogen peroxide and 70% of sulfuric acid (v/v)), followed by thorough wash with water, and dried under a nitrogen flow. A density gradient of amino groups was prepared on the slide. In brief, the glass slide was fixed vertically in a glass vessel. A continuous injection of TMSPMA toluene solution (25 ng/mL) with a microinfusion pump enabled the gradual elevation of the solution level. Therefore, the unreacted silane-hydroxyl groups formed a gradient along with the increase of slide position from bottom (0 mm) to top (10 mm) [26]. The injection rate was set at 40 mL/h, at which a 10 mm gradient was generated within 5 min. After washed with toluene five times, the slide was incubated in an APS solution (25 ng/mL in toluene) at 25 ◦ C for another 5 min, transforming the Si–OH gradient into the –NH2 gradient [27]. The slide was subsequently washed in toluene, acetone and alcohol under ultrasonication, respectively, before it was dried under a nitrogen flow.

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Finally, the slide was baked at 50 ◦ C for 4 h to stabilize the ultrathin silane layer. Heparin molecules were conjugated onto the amino groups by EDC/NHS chemistry. The slide with the amino group density was immersed into 1 mg/mL heparin solution containing 5 mg/mL EDC and 5 mg/mL NHS, and allowed to react for 4 h at 37 ◦ C. The slide was thoroughly washed against water for 4 h on a shaker to remove physically adsorbed heparin molecules. The unreacted NHS activated carboxyl groups of heparin, if any, were blocked with lysine. The slide with the heparin gradient was sterilized in 75% (v/v) ethanol for 1 h, followed with six washes by phosphate buffered saline (PBS, pH 7.4), and then incubated in 5 ␮g/mL bFGF/PBS solution containing 0.1% (w/v) BSA under aseptic condition at 4 ◦ C for 4 h. Finally, the slide with the bFGF gradient was gently washed with PBS five times, and used immediately.

2.3. Surface characterization Immunochemistry was adopted to quantitatively characterize the bioactive bFGF. The slide with bFGF was firstly blocked in 1% BSA/PBS solution for 1 h, and then was treated with a rabbit monoclonal antibody against human recombinant bFGF (Beyotime, China) at 37 ◦ C for 1 h. After washed three times with PBS, the slide was incubated with fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (Beyotime, China) at room temperature for 1 h, followed by five washes in PBS. Fluorescence images of various positions on the slide were recorded by a fluorescence microscope (IX81, Olympus) at the exactly same conditions, and their relative fluorescence intensity was analyzed by ImageJ software. The bFGF density at different positions of the gradient was obtained by an equation correlating the fluorescence intensity and bFGF density on homogenous bFGF surfaces [23]. The grafting mass of bFGF was quantitatively measured by quartz crystal microbalance with dissipation (QCM-D, Q-sense E4). The fluorescence intensity of homogenous bFGF surface increases with the bFGF density and the equation describing their relationship is y = 7.244 + 0.083x. The linear correlation coefficient is 0.9964. As a result, the bFGF density of the gradient surface at a given position was calculated by the fluorescence intensity at the corresponding position. The static contact angles of water and diiodomethane were measured by a sessile-drop method on a DSA 100 contact angle measuring system (Krüss, Germany). The volume of each solvent droplet was 2 ␮L. The results were averaged from five independent measurements. Surface energy was calculated based on these data using an Owens–Wendt–Rabel–Kaelble method.

2.4. Cell adhesion, orientation and migration The cells were seeded onto different surfaces at a density of 5 × 103 cells/cm2 in order to minimize the influence of cell–cell interactions. Approximately 8 h post cell plating in 0.4% FBS DMEM, the images of cells were recorded and the cell numbers were counted. The cell orientation was analyzed using the Image Pro Plus software to calculate the orientation angle of each cell. The cell migration behaviors were in situ recorded using a time-lapse phase-contrast microscope (IX81, Olympus) equipped with an incubation chamber (37 ◦ C and 5% CO2 humidified atmosphere) over a period of 12 h. Here the low concentration of FBS was used to rule out the potential influences of serum. The cell trajectories were reconstructed from the center positions of individual cells over the whole observation time. The cell migration distance S was calculated by an Image Pro Plus

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software according to the following equation at 0.5 h intervals over the observation time of 12 h (t = 12). S=

t  

(xi − xi−1 )2 + (yi − yi−1 )2

(1)

i=1

At least 40 cells were calculated for each sample. The cell migration rates () were thus obtained by  = S/t. The directionality (D) and haptotactic index (HI) was calculated according the method reported previously [28]. D=

Net cell displacement Total cell trajectory

(2)

CI =

Net displacement in gradient direction Total cell trajectory

(3)

According to the bFGF density which was measured by QCM-D and the correlation between fluorescence intensity and bFGF density [23], the bFGF density at different positions of the gradient (Fig. 2a) was obtained. It increased along with the gradient from the bottom (0 mm, ∼130 ng/cm2 ) to the top (10 mm, ∼300 ng/cm2 ). The slope of the gradient is 17 ng/cm2 /mm. Consequently, the difference of bFGF in the range of a single smooth muscle cell (∼100 ␮m) is ∼1.7 ng/cm2 . Fig. 2c shows that the water contact angle of the surface increased slightly from 56◦ to 65◦ , whereas the surface energy decreased correspondingly along the gradient. This slight variation is attributed to the increase of the density of relatively hydrophobic bFGF molecules. Since the alternation of surface hydrophilicity and surface energy is within a small range, it should not significantly influence on the cell adhesion (Fig. S1).

2.5. Cell focal adhesion complex formation and actin organization

3.2. Cell adhesion and orientation on bFGF gradient

Fluorescent staining of F-actin, vinculin and cell nucleus was carried out for the study of cell morphology and skeleton organization. Briefly, after the VSMCs were cultured for 24 h, they were carefully washed with PBS three times, and then were 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 enhance the permeability of cell membrane. After rinsed three times with PBS, they were incubated in 1% BSA/PBS at 37 ◦ C for 30 min to block the non-specific interactions. Then the cells were incubated with 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 antimouse IgG (Beyotime, China), rhodamine phalloidin (Invitrogen) and 4 ,6-diamidino-2-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, SP 5, Leica).

Cell migration is a complex process involving steps of cell adhesion, polarization, contraction, and forward movement [32]. Fig. S1 shows that the VSMCs adhered evenly on different positions of the bFGF gradient 8 h post cell seeding, suggesting the tested bFGF density did not directly influence on cell adhesion [23]. It is known that the assembly and disassembly of F-action is responsible for spreading and extrusion of cells [33]. Therefore, fluorescent staining of VSMCs was further performed to visualize the cell morphology, including cytoskeleton organization and focal adhesion (Fig. 3). A part of the VSMCs exhibited aligned focal adhesion plaques and actin filaments in parallel with the gradient direction on all three positions of the bFGF density gradient. Some cells also showed polarized morphology (Fig. 3, arrow headed), suggesting possible migration. Cell orientation was further quantified by measuring the angular separation between the major axis of the cell and the gradient direction 24 h post cell seeding. About 60% of the elongated cells orientated in ±30◦ to the X-direction (Fig. 4a) on the whole bFGF gradient, revealing that the VSMCs can sense the gradient surface and respond correspondingly. In contrast, the VSMCs always showed random orientation on the uniform bFGF surfaces and heparin gradient, and only 35–40% of them aligned in ±30◦ to the X-direction (Fig. 4b, c).

2.6. Statistic analysis Results are reported as mean ± standard deviation, and are analyzed using one-way ANOVA analysis for difference. The individual difference is further studied using an S–N–K method in the post-hoc test. The significant level is set at p < 0.05. 3. Results and discussion 3.1. Preparation and characterization bFGF gradient The density gradient of bFGF molecules was prepared by conjugation with immobilized heparin in a gradient manner (Fig. 1). Following the method reported previously [29,30], an amino density was created on a glass or silicone side by the successive reaction of TMSPMA and APS. Heparin molecules were covalently coupled onto the slide via EDC chemistry, forming a continuous increase of heparin density gradient from the bottom (0 mm) to the top (10 mm). Heparin is able to bind with bFGF with a high degree of specificity and keeps its bioactivity [31]. The processes allow good control over both the quality of samples in different batches and the maintenance of natural activity of the growth factor. The bFGF density gradient was characterized via immunochemistry (Fig. 2a). In our experiments, no obvious auto-fluoresce of the substrates was recorded at the excitation wavelength of 488 nm. The fluorescence intensity increased along with the gradient position, indicating the successful preparation of progressive increase of active bFGF molecules, since only those bFGF molecules maintaining their natural structure can be recognized by their antibody and thus emit fluorescence.

3.3. Cell migration The cell migration trajectories on various surfaces were recorded by a time-lapse microscope (Fig. S2). For the ease of discussion, the direction of the gradient with increasing bFGF density (from bottom to top) was set as the X coordinate. The same X coordinate was assigned to the uniform bFGF surfaces as the gradient substrate during cell culture. The VSMCs moved randomly without a preferential direction on the uniform bFGF surfaces regardless of its density (Fig. S2d–f). By contrast, about 70% of the VSMCs moved toward +X direction on the bFGF gradient (Fig. S2a–c), confirming the haptotactic effect of the bFGF gradient. However, the bFGF gradient did not have significant influence on the cell migration rate on all three positions compared to the corresponding uniform surfaces (Fig. 5c). The directionality and haptotactic index were calculated to further demonstrate the influence of the gradient surface on cell migration (Fig. 5a). The directionality was significantly enhanced on the gradient surface at 1 and 4 mm positions than the corresponding uniform surfaces (p < 0.05), indicating that the VSMCs are apt to move straightly rather than crooking their ways. As a result, the VSMCs on the gradient surface traveled longer distances than those on the uniform surfaces. The positive HI value which reflects the distance cells moving toward +X direction increased significantly on these positions of the gradient surface as well (p < 0.05)

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a

1mm

3mm

5mm

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300 250 200 150

Contact angle Surface energy

65

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Fig. 2. (a) Fluorescence images of bFGF gradient at different position after immunochemical staining. (b) Calculated bFGF density as a function of gradient position based on the correlation between fluorescence intensity and bFGF density obtained on uniform surfaces. (c) Contact angle and surface energy as a function of gradient position on bFGF gradient.

(Fig. 5b). Surprisingly, the directionality and HI was not obviously enhanced on the gradient surface at 7 mm position with a bFGF density of 262 ng/cm2 . This might be attributed to the already very high bFGF density and thus relatively small impact of bFGF density difference (∼1.7 ng/cm2 ) in the range of a cell-length. The importance of cell mobility for regeneration of tissues and organs in the right positions and time is recognized more recently. So far several strategies have been reported to control the cell

directional migration by chemical and/or physical signals. In our previous work, a PEG density gradient together with stripe patterns in parallel was fabricated. About 67% VSMCs unidirectionally traveled towards the gradient end with a reduced mPEG density, which is significantly larger than those on the unpatterned gradient (52%) and homogenous mPEG surface (46%) [11]. Bioactive molecules, such as extracellular matrix proteins and growth factors have a strong impact on cell functions. Therefore, it is highly possible to

Fig. 3. CLSM images of VSMCs on bFGF gradient surface 24 h post cell seeding: (a) 1 mm position, (b) 4 mm position, and (c) 7 mm position, respectively. The nucleus was stained by DAPI (blue, Column 1 and 2), vinculin was stained by its monoclonal antibody (green, Column 3), and F-actin was stained by phalloidine (red, Column 4). Column 1 and 2 were merged images of low magnification (scale bar 100 ␮m) and high magnification of nucleus, F-actin and vinculin, respectively. The scale bar in Column 2–4 is 50 ␮m. Arrows in Colum 3 indicate the direction of focal adhesion.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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Percentage of cells %

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Fig. 4. (a) The percentage of cells aligned in the direction within 30◦ as a function of position on () bFGF density gradient, (•) heparin density gradient, and () uniform surfaces with various bFGF densities (arrows headed). Distribution of the VSMCs angles to the gradient direction on (b) bFGF density gradient at 4 mm, (c) heparin density gradient, and (d) corresponding uniform bFGF surface (∼189 ng/cm2 ). The gradients were located parallel to the cells with the angle of 0◦ . At least 100 cells were analyzed for each data point. * Indicates significant difference at p < 0.05.

b

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bFGF gradients bFGF uniform surface

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Fig. 5. Histogram of (a) directionality, and (b) haptotactic index of VSMCs on various surfaces. (c) Migration rate of VSMCs on various surfaces. * Indicates significant difference at p < 0.05.

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achieve better heptotaxis effect by using these bioactive molecules. However, previous efforts do not always lead to satisfactory results, likely due to the direct immobilization of the bioactive molecules. For example, Gunawan et al. generated a series of laminin gradients with different slopes between 5 and 25 pg/dm2 ␮m. The corresponding directness probabilities (the probability of cells migrating ±60◦ in the direction of gradient) varied from 62% to 65% [34]. DeLong et al. prepared a bFGF density gradient on PEG hydrogels, and found that the percentages of aligned cells (align within ±30◦ ) were enhanced from about 40% to 55% [35]. The ratio of cells directional migrating up the bFGF gradient is increased by a maximum of 15%. In this work, the bFGF molecules were immobilized onto substrates via heparin conjugation and in a gradient manner. In this way, the bioactivity of bFGF molecules was maintained, and consequently, about 70% of the VSMCs selectively moved toward the direction of gradient with an increasing bFGF density, suggesting the success of this bFGF gradient on guiding directional migration of VSMCs. 4. Conclusion The bFGF density gradient was successfully fabricated using an injection method and the bio-conjugation between heparin and bFGF. The density of bFGF gradually increased from about 130 to 300 ng/cm2 with a slope of 17 ng/cm2 /mm. VSMCs were able to sense the bFGF density gradient and showed preferential orientation and directional migration. Up to 70% cells migrated towards the region with a higher density of bFGF on the gradient. However, the bFGF gradient had no effect on the cell migration rate. This versatile strategy can be effectively used to control the cell directional migration by the growth factor density gradient, providing a new perspective on designing complex biomaterials with advanced functions for tissue regeneration. Acknowledgments This study is financially supported by the Natural Science Foundation of China (21374097, 20934003 and 51003094), the Major State Basic Research Program of China (2011CB606203), Young Teacher Programs Foundation of Ministry of Education of China (20100101120034), and “Qianjiang” outstanding researcher funding of Zhejiang Province (J20110541). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2014. 02.043. References [1] P. Martin, Wound healing-aiming for perfect skin regeneration, Science 276 (1997) 75–81. [2] L.R. Bernstein, L.A. Liotta, Molecular mediators of interactions with extracellular matrix components in metastasis and angiogenesis, Curr. Opin. Oncol. 6 (1994) 106–113. [3] S. Zhou, Z. Cui, J.P. Urban, Nutrient gradients in engineered cartilage: metabolic kinetics measurement and mass transfer modeling, Biotechnol. Bioeng. 101 (2008) 408–421. [4] M.A. Swartz, M.E. Fleury, Interstitial flow and its effects in soft tissues, Annu. Rev. Biomed. Eng. 9 (2007) 229–256. [5] T. Lühmann, H. Hall, Cell guidance by 3D-gradients in hydrogel matrices: importance for biomedical applications, Materials 2 (2009) 1058–1083. [6] C. Ruhrberg, H. Gerhardt, M. Golding, R. Watson, S. Ioannidou, H. Fujisawa, C. Betsholtz, D.T. Shima, Spatially restricted patterning cues provided by heparinbinding VEGF-A control blood vessel branching morphogenesis, Genes Dev. 16 (2002) 2684–2698.

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