Constrained Adherable Area of Nanotopographic Surfaces Promotes

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Biological and Medical Applications of Materials and Interfaces

Constrained Adherable Area of Nanotopographic Surfaces Promotes Cell Migration through the Regulation of Focal Adhesion via Focal Adhesion Kinase/Rac1 Activation Jiwon Lim, Andrew Choi, Hyung Woo Kim, Hyungjun Yoon, Sang Min Park, Dylan Tsai, Makoto Kaneko, and Dong Sung Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18954 • Publication Date (Web): 12 Apr 2018 Downloaded from http://pubs.acs.org on April 12, 2018

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ACS Applied Materials & Interfaces

Constrained Adherable Area of Nanotopographic Surfaces Promotes Cell Migration through the Regulation of Focal Adhesion via Focal Adhesion Kinase/Rac1 Activation

Jiwon Lim†, #, Andrew Choi†,#, Hyung Woo Kim†, #, Hyungjun Yoon†, Sang Min Park†, Dylan Tsai‡,§, Makoto Kaneko‡, and Dong Sung Kim*,† †

Department of Mechanical Engineering, Pohang University of Science and Technology

(POSTECH), 77 Cheongam-Ro, Nam-gu, Pohang, Gyeongbuk, 37673, Korea ‡

Department of Mechanical Engineering, Osaka University, 1-1 Yamadaoka, Suita, Osaka

565-0871, Japan *

Address correspondence to [email protected].

§

Present address: Department of Mechanical Engineering, National Chiao Tung University,

1001 University Toad, Hsinchu, Taiwan 300, ROC #

J Lim, A Choi and HW Kim contributed equally to this work as first authors

KEYWORDS: adherable area, polystyrene nanopore surface, fibroblast cell migration, focal adhesion, FAK, Rac1 1

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ABSTRACT Cell migration is crucial in physiological and pathological processes such as embryonic development and wound healing; such migration is strongly guided by the surrounding nanostructured extracellular matrix. Previous studies have extensively studied the cell migration on anisotropic nanotopographic surfaces, however, only a few studies have reported cell migration on isotropic nanotopographic surfaces. We herein, for the first time, propose a novel concept of adherable area on cell migration using isotropic nanopore surfaces with sufficient nanopore depth by adopting high-aspect-ratio. As the pore size of the nanopore surface was controlled to 200, 300, and 400 nm in a fixed center-to-center distance of 480 nm, it produced 86, 68, and 36 % of adherable area, respectively, on the fabricated surface. A meticulous investigation of the cell migration in response to changes in the constrained adherable area of the nanotopographic surface showed 1.4-fold, 1.5-fold, and 1-6 fold increase in migration speeds and a 1.4-fold, 2-fold, and 2.5-fold decrease in the number of focal adhesions as the adherable area was decreased to 86, 68, and 36 %, respectively. Furthermore, a strong activation of FAK/Rac1 signaling was observed to be involved in the promoted cell migration. These results suggest that the reduced adherable area promotes cell migration through decreasing the FA formation, which in turn upregulates FAK/Rac1 activation. The findings in this study can be utilized to control the cell migration behaviors, which is a powerful tool in the research fields involving cell migration such as promoting wound healing and tissue repair.

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1. INTRODUCTION Cell migration plays a pivotal role in many physiological and pathological processes, such as embryonic development, immune response, cancer metastasis, and wound healing.1-2 During wound healing, the human body immediately initiates the sequential migration of diverse cells upon the recognition of injury. In the early stages of this process, fibroblasts migrate to the injury site and stimulate migration and differentiation of neighboring cells by providing them with newly formed extracellular matrix (ECM).3-5 In other words, the cell migration behavior of fibroblasts predominantly determines the nature of wound healing through the regulation of the sequential migration of various cells. Therefore, research on wound healing has focused on promoting the migration of fibroblasts while continuing to understand the general mechanism of cell migration. The mechanism of cell migration is quite complicated and is influenced by interactions between the cell and its surrounding nanostructured ECM.6-7 To regulate cell migration and reveal the underlying mechanism of cell–ECM interactions, researchers have fabricated various kinds of nanotopographies, such as grooves, ridges, pillars, and pore structures. Anisotropic nanotopographic surfaces, including grooves and ridges, have been intensively studied and are known to promote cell migration along the aligned structure through the modulation of cell polarization or the apical contact guidance of cell protrusions.8-11 Recently, an isotropic nanopillar structures were used to investigate the correlation between the cell migration and the surface nanotopography or the underlying mechanisms. Slater et al. modulated the adhesion site through the nanopillar structures to show the correlation between the adhesion site and the cell migration speed.12 Liang et al. showed a similar correlation between the cell migration speed and the size of the nanopillars, and examined the assembly and disassembly of focal adhesion (FA) on the various nanopillar 3



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surfaces.13 In the case of isotropic nanopore structures, however, several contradictory results have highlighted the lack of comprehensive understanding.13-17 Nasrollahi et al. showed increased migration speed with increased pore size, while Pan et al. reported the opposite behaviors of the cell migration.14-15 Jeon et al. and Biggs et al. explained such antithetical observations by emphasizing the dimension of topographic depth.18-19 Because cell membranes cannot be deflected when they reach a certain threshold level, it is crucial to consider this threshold level during the fabrication of nanotopographic surfaces if they are being utilized for studying the cell migration mechanism. Until now, there has not been systematic approaches elucidating the relationship between the adherable area of isotropic nanopore surfaces and cell migration while ensuring sufficient topographic depth. In this study, the adherable area of the nanotopographic surface was controlled using a polystyrene (PS) surface by changing the wall thickness of the isotropic nanopore-arrayed surface to 280, 180, and 80 nm using anodic aluminum oxide (AAO) followed by thermal nanoimprinting, which resulted in 86%, 68%, and 36% of the adherable areas of the nanotopographic surface for wall thicknesses of 280, 180, and 80 nm, respectively. While controlling the adherable area of the nanotopographic surface, crowded and individual cell migration behaviors of fibroblasts were investigated. Moreover, FA formation and maturation were analyzed to speculate the relationship between cell migration behaviors and the nanotopographic surface adherable areas. Finally, focal adhesion kinase (FAK) signaling followed by Rac1 activation was investigated to reveal the underlying molecular mechanisms of enhanced cell migration on the nanotopographic surface. To the best of our knowledge, this is the first study to investigate correlations between cell adhesion and cell migration using the concept of an adherable area of a nanotopographic surface.

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2. MATERIALS AND METHODS 2.1. Fabrication of PS nanopore surfaces. To modulate the adherable area of the nanotopographic surface, a PS nanopore surface was fabricated using two-step aluminum anodization, nickel electroforming, and thermal nanoimprinting based on our previous works.20-22 An isotropic nanopore-arrayed AAO template with a high aspect ratio was fabricated with phosphoric acid by controlling anodization processing parameters; the applied voltage, anodizing temperature, and anodizing time were set to 187 V, −10°C, and 3 h, respectively. This resulted in a center-to-center distance of 480 nm and a nanopore depth of 500 nm. Then, the desired wall thicknesses of nanopore surfaces (280, 180, and 80 nm corresponding to pore diameters of 200, 300, and 400 nm, which were denoted as NPt280, NPt180, and NPt80, respectively) were achieved by adjusting the widening time (1.8, 4.2, and 6.5 h, respectively). Next, nickel mold inserts having a reverse shape of the nanopore-arrayed AAO surfaces were formed using nickel electroforming. After ensuring uniform conductivity on the AAO surface with a nickel seed layer of 20 nm, electroforming was carefully conducted until the electroformed nickel mold insert achieved the targeted thickness of 1 mm. Finally, NPts were fabricated using thermal nanoimprinting at an imprinting temperature of 110°C with an imprinting pressure of 5 MPa for 2.5 min. The fabricated PS FL and NPts were treated by oxygen plasma to enhance surface hydrophilicity. Apparent contact angle of sessile water droplet on FL and NPts before and after oxygen plasma treatment was measured (Smartdrop, Femtobiomed Inc.). The contact angles for oxygen plasma-treated FL and NPts were measured to be ~ 50° and 40°, respectively, with no statistical difference among all surfaces, which provided a suitable hydrophilic surface to allow the cells to adhere well to the surface.23-24 (Supplementary Fig. S1). The samples were sterilized by socking in 70% ethanol 5



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for 2 h and placing them under a UV lamp overnight for cell culture.

2.2. Cell culture. Murine fibroblasts (NIH3T3) were purchased from KCLB (Korean Cell Line Bank, Korea). The cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (HG-DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin/streptomycin (Hyclone) in a humidified incubator at 37°C with 5% CO2. The cells were subcultured with 0.25% trypsin/EDTA before reaching confluence, and the culture medium was changed every 2-3 days.

2.3. Scanning Electron Microscopy (SEM) and Focused Ion Beam (FIB). Fibroblasts were seeded on PS FL and NPts at a density of 1 × 104 cells/cm2 and cultured for 24 h to stabilize cell adhesions on the substrates. The next day, cells were fixed with pre-warm 2.5 % glutaraldehyde for at least 15 min, then, the samples were dehydrated through a series of ethanol (30 %, 50%, 70%, 90%, 100%, 100% and 100%) over 30 min at each steps. The dehydrated samples were dried in a freeze dryer overnight. And additionally to obtain a vertical section of the cell, the samples were cut through Dual BEAM focused ion beam (FIB) (Helios 650; FEI) using Ga+ ion beam with 30 kV acceleration voltage. A thin layer of Pt layer was sputtered on all prepared samples and the samples were observed by scanning electron microscopy (SEM) (SU6600; Hitachi) while 20 kV of acceleration voltage was applied to the samples.

2.4. Wound healing assay. A culture insert with two wells separated by a 500 µm gap (Ibidi GmbH) was attached to PS FL and NPts. Then, fibroblasts were seeded in the wells of the 6


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culture insert at a density of 3 × 104 cells/well. After 12 h of incubation, the culture insert was removed and washed twice with PBS; a cell-free wound gap of 500 µm remained on the surface of PS FL and NPts. A fresh complete growth medium (HG-DMEM supplemented with 10% FBS) was added for initiating cell migration with or without the indicated concentration of inhibitors. After 15.5 h, the cells were washed with PBS twice and fixed with 10% formalin for 10 min at room temperature. Then, they were washed with PBS again and stained with 0.1% crystal violet for 10 min. To remove the staining solution, the cells were washed with deionized water several times until the background stains completely disappeared. Stained cells were imaged using an inverted microscope (Eclipse TS100, Nikon).

2.5. Time-lapse imaging of cell movement. Fibroblasts were seeded on PS FL and NPts at a density of 1 × 104 cells/cm2. After 4 h of incubation, the cells were observed by inverted live imaging microscopy (JuLI; NanoEnTek) in a 5% CO2 atmosphere at 37°C. Images were captured using the time-lapse and phase-contrast modes at a time interval of 10 min for 72 h. For analyzing individual cell movement, time-lapse images were imported to ImageJ software (NIH), and the positions of individual cells were tracked in a Cartesian coordinate using a manual tracking plug-in. Then, the migration speeds of the cells were calculated by measuring the total distance traveled on the surface by each cell during the time interval.

2.6. Immunofluorescence staining and image analysis. The cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature and permeabilized with 0.3% Triton X-100 in PBS for 15 min at 4°C. To avoid nonspecific antibody binding, the cells were blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Then, the cells were incubated overnight with anti-vinculin primary antibody (Sigma-Aldrich) 7



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at 4°C. After that, the primary antibody solution was removed, and the cells were incubated with Alexa Fluor 488-conjugated secondary antibody (Molecular Probes) for 1 h at 4°C. Then, the cells were immunostained with TRITC-conjugated phalloidin (Sigma) for 30 min, and the nucleus was stained with DAPI (Invitrogen) for 3 min. Fluorescence images of FAs were obtained using a confocal microscope (LSM, Carl Zeiss). For the quantification of FAs, the images were analyzed using ImageJ. A “subtract background” with the sliding paraboloid option (rolling ball radius was 10 pixels) was applied for all captured images, and the images were then converted to grayscale (with threshold intensity values ranging from 0 to 255). ImageJ was then directed to trace/crop each randomly selected cell and the vinculin labels within it for a thorough analysis.

2.7. Western blot analysis. The fibroblasts were seeded on PS FL and NPt80 and incubated for 12 h. To reduce the basal level of FAK phosphorylation, serum starvation was performed with serum-free DMEM for 24 h. A fresh growth medium (with 10 % FBS) or a growth medium (with 10 % FBS) containing the FAK inhibitor (0.5 µM) was added for the initiation of movement and the activation of intracellular signals to the serum-starved cells. The cells were lysed with RIPA buffer (Biosesang) containing a protease and phosphatase inhibitor cocktail (Roche) for 30 min on ice followed by centrifugation at 14,000 rpm for 10 min. The total protein content of the cell lysate was determined using a BCA protein assay kit (Thermo Fisher Scientific). Twenty micrograms of total protein were separated using SDS-PAGE. The separated proteins were transferred to a nitrocellulose membrane, and the membrane was blocked with 3% BSA in PBS for 1 h at room temperature. Anti-p-FAK (Tyr 397) (Abcam) and anti-FAK (Santacruz) primary antibodies were incubated overnight at 4°C, and anti-βactin (Santacruz) primary antibody was incubated for 1 h at room temperature. After primary 8


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antibody incubation, HRP-conjugated anti-rabbit IgG secondary antibody (Thermo Fisher Scientific) for FAK/p-FAK and HRP-conjugated anti-mouse IgG secondary antibody (Thermo Fisher Scientific) for β-actin were incubated for 1 h at room temperature. Then, the signals were detected using a chemiluminescence imager, ImageQuant LAS 4000 (GE Healthcare).

2.8. Measurement of expression levels of activated Rac1. To measure Rac1 activation in fibroblasts on PS FL and NPt80, cells were seeded and serum starvation was performed to reduce the basal level of Rac1 activity as described in the above section. The cells were harvested, and the Rac1 activation assay was performed using the Rac1 Activation Assay Kit (Cytoskeleton, Inc.) according to the manufacturer’s instruction. This method is based on “pull-down” assay using a GST-PAK-PBD bead. The cells on PS FL and NPt80 were washed with ice-cold PBS and lysed using an ice-cold cell lysis buffer with a protease cocktail. The harvested cell lysate was snap-frozen in liquid nitrogen to maintain GTP-bound protein stability. Then, the protein concentration was determined using Precision Red Advanced Protein Assay. Pull-down assay was performed with 600 µg of total protein with PAK-PBD beads at 4°C for 1 h. After incubation, GTP-Rac1-bound beads were washed with a washing buffer provided in the kit. Washed samples were resuspended in 2× laemmli sample buffer then they were boiled for 2 min. The samples were analyzed by western blotting as described above.

2.9. Inhibitors. The FAK inhibitor (PF573228), Rac1 inhibitor (NSC23766), and Cdc42 inhibitor (ML141) were purchased from Tocris Bioscience. The Rho inhibitor (C3) was purchased from Cytoskeleton, Inc. Before starting the experiment, the ideal concentration of 9



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all chemicals used in this study was tested and determined (data not shown).

2.10. Statistical analysis. Experiments were conducted at least three times, and data are presented as mean ± SD. One-way analysis of variance (ANOVA) with Tukey’s HSD post hoc test was performed. A P-value of

Constrained Adherable Area of Nanotopographic Surfaces Promotes

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