Cell directional migration and oriented division on three-dimensional laser-induced periodic surface structures on polystyrene

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Biomaterials 29 (2008) 2049e2059 www.elsevier.com/locate/biomaterials

Cell directional migration and oriented division on three-dimensional laser-induced periodic surface structures on polystyrene Xuefeng Wang a,b, Christian A. Ohlin c, Qinghua Lu a,*, Jun Hu b a

School of Chemistry and Chemical Technology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, PR China b School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, PR China c Department of Chemistry, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA Received 2 October 2007; accepted 9 December 2007 Available online 12 February 2008

Abstract The extracellular matrix in animal tissues usually provides a three-dimensional structural support to cells in addition to performing various other important functions. In the present study, wavy submicrometer laser-irradiated periodic surface structures (LIPSS) were produced on a smooth polystyrene film by polarized laser irradiation with a wavelength of 266 nm. Rat C6 glioma cells exhibited directional migration and oriented division on laser-irradiated polystyrene, which was parallel to the direction of LIPSS. However, rat C6 glioma cells on smooth polystyrene moved in a three-step invasion cycle, with faster migration speed than that on laser-irradiated polystyrene. In addition, focal adhesions examined by immunostaining focal adhesion kinase in human epithelial carcinoma HeLa cells were punctuated on smooth polystyrene, whereas dash-like on laser-irradiated polystyrene. We hypothesized that LIPSS on laser-irradiated polystyrene acted as an anisotropic and persistent mechanical stimulus to guide cell anisotropic spreading, migration and division through focal adhesions. Ó 2008 Elsevier Ltd. All rights reserved. Keywords: Laser irradiation; Polystyrene; Cell migration; Cell division

1. Introduction Cell migration and cell division are not only essential for embryogenesis, tissue repair, and cancer cell invasion and formation of metastasis in vivo, but also for developing biological substitutes in tissue engineering in vitro. Cell migration speed and directionality are usually regulated by chemotactic stimuli, and a series of signal proteins are involved in, such as, phosphatidylinositol 3-kinase, cofilin, Cdc42 and Rac [1e4]. The orientation of the cell division axis, which determines the future position and fate of the daughter cells, is dependent on the mother cell shape in interphase [5e7]. Geometrical constraints inherent to cell shape anisotropy in interphase can provide a default guiding cue that orientates the division axis in metaphase in the absence of cell polarity [5]. In

* Corresponding author. Tel./fax: þ86 21 54 747 535. E-mail address: [email protected] (Q. Lu). 0142-9612/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2007.12.047

addition, migration of some mammalian cells in culture are also affected by properties of substrates, such as, rigidity [8], hydrophilicity [9], and random and patterned topography in micro- to nanometer scale [10e12]. The surface properties of biomaterials are also factors to be considered besides their mechanical properties, porosity, degradation rate and mouldability in the field of tissue engineering [13]. There are two approaches to modify substrate surfaces. One is to change the chemical composition to improve biocompatibility by forming hydrophilic functional groups by ion-assisted reaction [14] and plasma treatment [15], and incorporating integrin receptor-binding peptides and mono- and oligosaccharides by coating [11], molecular self-assembly [16] and grafting [17,18]. The other is to create micro- and nanostructures on substrate surfaces using such methods as electron-beam lithography [19], soft lithography [12], photolithography [20], interference lithography [21], LangmuireBlodgett lithography [22], colloidal lithography [19], nanoimprinting lithography [12], casting [23], polymer

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Fig. 1. AFM images of PS (A) and TPS (B).

demixing [24], and electrospinning [25e27]. The results of these studies show that the modified substrate surfaces affect cell attachment, spreading, migration, alignment, proliferation, and even the level of cytokine production and gene expression. However, the influence of these micro- and nanostructures on substrate surfaces on cell division is often neglected, and the modification methods are, in fact, rather complicated, usually including more than one-step. Polystyrene is widely used as two-dimensional (2D) cell culture substrates in vitro. Recently, some methods have been applied to produce three-dimensional (3D) structures on polystyrene surface, such as, microgrooves with the width/ spacing/depth of 10/20/3 mm by casting [23], nanogrooves with a periodicity of 500 nm and different depths of 50 nm and 150 nm by LangmuireBlodgett lithography [22], and nanofibres with the mean diameter from 1 mm to 0.66 mm by

electrospinning [26]. However, polystyrene with a hydrophobic surface does not provide a suitable environment for adherent cells to attach and grow. Therefore, hydrophilic groups also should be induced on polystyrene surface by plasma treatment [22,23,26]. In the present study, an efficient and effective onestep technique e p-polarized laser irradiation with a wavelength of 266 nm, was used to modify a smooth polystyrene film, not only generating wavy laser-induced periodic surface structures (LIPSS) with a periodicity of 250 nm and a depth of 60 nm, but also yielding hydrophilic carbonyl-containing groups [28]. A rat C6 glioma cell (C6 cell) was chosen as an experimental model system because glioblastoma is the most aggressive primary intracranial tumor [29]. In a previous study, we have studied the influence of physicochemical properties of laser-irradiated polystyrene on bovine serum albumin adsorption and C6 cell attachment, spreading, alignment and

Fig. 2. Frame-to-frame trajectory (mm) for 5 min interval of C6 cells on PS (A, 804 points of 23 cells during migration for 3 h with an average migration speed of 0.74 mm/min) and TPS (B, 1110 points of 18 cells for 6 h with an average migration speed of 0.29 mm/min). The units of the axes are micrometers.

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proliferation [28]. The present study focuses on cell migration and cell division on laser-irradiated polystyrene by time-lapse microscopy, fluorescent microscopy and scanning electron microscopy. The expression of gamma-tububin and focal adhesion kinase (FAK) was also examined by an immunocytochemical assay. 2. Materials and methods 2.1. Preparation and AFM characterization of the substrates A simple but effective method to generate large area wavy nanostructures on polymer surfaces by laser irradiation was used [30,31], and briefly summarized here. A spin-coated thin polystyrene film on a clean glass coverslip was fixed on a computer-controlled moveable platform, with a speed of 5 mm/s in the x direction and a speed of 0.01 mm/s in the y direction. A sample was irradiated by p-polarized laser with a wavelength of 266 nm and an energy flux of 2.98 mJ/cm2, of a Nd:YAG laser with Q-factor modulation. Surface profiles of spin-coated (PS) and laser-irradiated polystyrene (TPS) were characterized using atomic force microscopy (Vecco AFM NanoScope III) in contact mode.

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2.2. Cell culture C6 cells, HeLa cells (human epithelial carcinoma cell line), and SPCA-1 cells (human lung adenocarcinoma cell line) were provided by Shanghai Cancer Institute. Cells were cultured in 25 cm2 flasks (Corning) with high glucose Dulbecco’s Modified Eagle’s medium (DMEM, Hyclone) supplemented with 10% foetal bovine serum (FBS, Hyclone) in a humidified incubator (MCO15 AC, Sanyo) at 37  C in which the CO2 level was kept constant at 5%.

2.3. Inverted phase contrast light microscopy After culturing for 24 h, C6 cells and HeLa cells on PS and TPS were fixed with 3.7% formaldehyde for 20 min. The cells were observed by an inverted phase contrast light microscope (Nikon TE2000-S) and pictures were taken by a digital camera (Nikon Coolpix 4500).

2.4. Fluorescence microscopy C6 cells were cultured on both substrates for 24 h. The cells were then washed by sterile phosphate buffered saline solution (PBS, 0.01 M, pH ¼ 7.4) and dyed with acridine orange (AO, 100 mg/mL in PBS) for

Fig. 3. C6 cell random migration on PS with an irregular change in circularity (A) and alignment angle (B), and cell directional migration on TPS with a decrease in circularity (C) and alignment angle (D), and with a decreaseeincreaseedecrease change in circularity (E) and a decrease in alignment angle (F).

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10 min. C6 cells were observed using a fluorescence microscope (Olympus X71) and micrographs were acquired by an EMCCD camera system (Cascade 650, Photometrics). Both C6 cells on TPS and HeLa cells on PS and TPS were cultured for 12 h, and then fixed with 3.7% formaldehyde in PBS for 20 min and permeated with 0.1% Triton X-100 for 10 min. After blocking with 3% FBS in PBS (v/v) for 30 min, C6 cells were incubated with a primary mouse IgG anti-gammatubulin antibody (1:100, clone GTU-88, Sigma) at room temperature for 2 h, and HeLa cells were incubated with a primary mouse IgG1 anti-FAK antibody (1:100, BD Biosciences) at room temperature for 2 h. The samples were washed with PBS three times for 5 min, and then incubated with rhodamine-conjugated anti-mouse IgG (1:100, Kangcheng Biotech, China) for 30 min. The samples were observed under a fluorescence microscope and micrographs were taken by an EMCCD.

a 20 apochromat objective lens every 5 min for 3 h in one field. Cells cultured on TPS were followed and recorded during migration with a 63 apochromat objective lens driven by a PIFOC P-721 objective nanopositioner (Piezo device, Physik Instruments) every 5 min for 6 h in six fields and cells on PS were also tracked for 3 h under the same conditions. The outline of the individual cell without cell-to-cell contact was manually traced using Adobe Photoshop CS 8.0.1 software. Three parameters were used to analyse cell migration by ImageJ software (NIH) e cell frame-to-frame trajectory, which was the displacement of the cell centroid, cell circularity, which was defined by Eq. (1) (a circularity value of 1.0 indicates a perfect circle and the value approaching 0.0 indicates an increasingly elongated polygon), and alignment angle, which was defined as the angle between the longest axis of the cell and the direction of LIPSS on TPS, whereas a reference direction was picked arbitrarily on PS.

2.5. Scanning electron microscopy

area Circularity ¼ 4p perimeter2

C6 cells on PS and TPS, HeLa cells on TPS, and SPCA-1 cells on TPS were cultured for 24 h, and then fixed with 2.5% glutaraldehyde at 4  C for 1.5 h, post-fixed in 1% OsO4 for 2 h, and dehydrated using a graded ethanol series. Critical point-dried samples were sputtered with gold. Surface images of cells were acquired by field emission gun scanning electron microscopy (Philips FEG-SEM Sirion 200).

ð1Þ

The average of migration speed (V, mm/min) was calculated by Eq. (2), where x and y are the coordinates of the cell centroid in one image and n is the total number of images. V¼

n 1X 5n i¼1

qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ðxiþ1  xi Þ2 þðyiþ1  yi Þ2

ð2Þ

2.6. Time-lapse microscopy

3. Results C6 cells were trypsinized and seeded onto PS and TPS at the bottom of a home-made chamber in which the CO2 level was kept constant at 5% and the temperature was kept at 37  C. A phase contrast microscope (Leica DM IRE2) with AS-MDW (Application Solution Multi-Dimensional Workstation) was used to observe cell migration and live cell images were captured by a charge-coupled device camera (Cool SNAP HQ, Princetown Instruments). Cells cultured on PS were followed and recorded during migration with

3.1. AFM characterization of the substrates AFM images indicated that PS surface was quite smooth, with Z ranges smaller than 10 nm, and three-dimensional submicrometer LIPSS were yielded on TPS surface (Fig. 1).

Fig. 4. One isolated C6 cell migration on PS for 150 min with a three-step moving paradigm cycle. Bar: 20 mm.

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3.2. Cell motility Range and speed of moving C6 cells on PS and TPS were analysed by the displacement of the cell centroids. Cells on PS moved randomly and widely with an average moving speed of 0.74 mm/min during migration for 3 h (Fig. 2A). Cell trajectories on TPS for 6 h were in one direction, which was parallel to the direction of LIPSS, and the average moving speed was 0.29 mm/min (Fig. 2B). The moving speed of C6 cells on PS was 2.5 times of that on TPS. Cell circularity and alignment angle were used to characterize cell shape change during migration. A cell shape on PS during migration for 3 h changed from a circle to an elongated polygon, then to a circle again, and the alignment angle changed irregularly in the first 1 h, and from large to small, then to large again in the next 2 h (Fig. 3A and B). For cells on TPS, there were two types of cell shape change during migration. One was that the cell shape changed from a circle to

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an elongated polygon and the alignment angle changed from large to small during migration for 6 h (Fig. 3C and D). The other was that the cell shape changed from a circle to an elongated polygon, then to a circle again, whereas the alignment angle change was from large to small, which indicated that although the cell shape on TPS changed in a circle-elongated polygonecircle model like that on PS, the anisotropic cell shape was still kept uniform during migration (Fig. 3E and F). The moving paradigm of C6 cells on PS and TPS was further studied by analyzing cell images during cell migration. Images of a C6 cell on PS during migration for 150 min showed that its moving paradigm could be divided into three steps (Fig. 4). The first step was the exploring one lasting for 10 min or so in the images of the cell at 40, 100 and 150 min, in which the spherical cell extended randomly to explore its environment. The next step was the spreading one lasting for 40 min or so in the images of the cell at 0, 10, 20, 60, 70, 80, 110, 120 and 130 min, in which the cell spread

Fig. 5. Two isolated (marker < and ;) and five interacted (marker :, =, /, Y and )) C6 cells migrating on TPS with adjusting themselves to align along LIPSS. The double-ended arrow pointed the direction of LIPSS. Bar: 20 mm.

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and elongated. The last step was the pulling up one lasting for less than 10 min in the images of the cell at 30, 90 and 140 min, in which the anchored end of the polarized cell stabilized itself when retracing its cell body. The moving paradigm cycle of the cell on PS was repeated and the mean time for one cycle was 50 min or so. For cells on TPS tracked for 490 min, their moving paradigm was different from that on PS. Some isolated cells spread slowly along LIPSS after seeding with the cell shape changing from a circle to an elongated polygon (Fig. 5, marker < and ;). For some cells with interaction, two of them spread slowly along LIPSS in the first 325 min and still kept their anisotropic shape after encountering (Fig. 5, marker : and =), one elongated cell could stride over other cell and spread along LIPSS after interaction (Fig. 5, marker /), one elongated cell along LIPSS still kept its polarity after interaction (Fig. 5, marker Y) and one floating cell landed on the other cell, fell down, attached and spread along LIPSS (Fig. 5, marker )). A series of SEM images of a C6 cell on TPS showed that cell migrated along LIPSS with a complicated tree-like structure at its rear (Fig. 6A). The longest branch was almost 20 mm. The ends of branches were along the ridges of LIPSS and at the nodes there was at least one branch spread along LIPSS (Fig. 6BeD).

fluorescence microscopy. After culturing for 24 h, C6 cells on PS extended randomly and their nucleus distributed irregularly (Fig. 7A and D), whereas for C6 cells on TPS, both the longest axes of the cells and those of their elliptic nucleus aligned along LIPSS (Fig. 7B). When a mother cell enters mitosis, one of its two centrosomes moves to the opposite pole of the cell, and then the mitotic spindle, which segregates the duplicated chromosomes, forms between the two centrosomes [32]. The centrosome consists of two barrel-shaped centrioles arranged perpendicular to one another, surrounded by the pericentriolar material (PCM). Gamma-tubulin ring complexes, which nucleate microtubules, are in the PCM [33]. The line connecting the two centrosomes in metaphase indicates the orientation of cell division axis. The distribution of the two centrosomes, marked by immunostaining gamma-tubulin in the C6 cell on TPS, indicated that the direction of the cell division was parallel with LIPSS (Fig. 7C). The longest line connecting the two new daughter cell nucleus dyed with AO on TPS, which also indicated the direction of the cell division, was parallel with LIPSS (Fig. 7E). In addition, SEM images showed that the direction of the C6 cell division on PS was random, while for such cells on TPS as C6, HeLa and SPCA-1, the direction of the cell division was also parallel to LIPSS (Fig. 8).

3.3. Cell alignment and division

3.4. Cell spreading after division

Cell shape and nuclear shape of C6 cells on PS and TPS were observed using inverted phase contrast microscopy and

The morphology of cells on both substrates after division was observed using SEM. C6 cells on PS extended lamellipodia to spread, while cells on TPS, including C6 cells, HeLa cells and SPCA-1 cells, explored their environment using filopodia (Fig. 9), which agreed with the result of C6 cell spreading on these both substrates after seeding for 2 h without FBS in the culture medium [28]. 3.5. FAK expression Focal adhesions (FAs) in HeLa cells on both substrates marked by the immunostaining of FAK were examined using a fluorescent microscope. The result showed that a majority of FAs were punctuated in HeLa cells on PS (Fig. 10A and B), whereas dash-like in HeLa cells on TPS (Fig. 10C and D). In addition, most dash-like FAs distributed at the two ends of the cells on TPS. 4. Discussion

Fig. 6. SEM images of a C6 cell on TPS during migration after culturing for 24 h: an image of an intact cell (A, 2750), an image with magnification of an area (*) in A (B, 14,000), of an area (:) in A (C, 8000) and of an area (;) in A (D, 14,000).

Laser irradiation of such solid surfaces as metals, semiconductors and polymers generating submicrometer LIPSS is a universal phenomenon [30,31]. The periodicity of LIPSS from nanometer to micrometer scale can be obtained by adjusting laser sources and substrates. Unfortunately, the application of laser irradiation into modifying biomaterial surface is still in its preliminary step. Preparation and characterization of a laser-irradiated polystyrene film used in this study have been done in our previous study [28]. Irradiating a smooth polystyrene film by p-polarized laser with a wavelength of

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Fig. 7. Inverted phase contrast microscopy images of C6 cells cultured on PS (A) and TPS (B) for 24 h. Fluorescence microscopy image of C6 cells cultured on TPS for 24 h and the cells immunostained gamma-tubulin (C). Fluorescent microscopy images of C6 cells cultured on PS (D) and TPS (E) for 24 h and the cells stained with acridine orange. The double-ended arrows pointed the direction of LIPSS.

266 nm generates three-dimensional anisotropic nanostructures e LIPSS with a periodicity of 250 nm and a depth of 60 nm, in addition to oxidizing the surface to yield hydrophilic carbonyl-containing groups, which increases the apparent surface energy of polystyrene film with an anisotropic wetting behaviour. Attachment onto a substrate with forming FAs is the first step for an adherent cell to grow. FAs are dynamic groups of structural and regulatory proteins that transduce external signals to the cell interior and can also relay intracellular signals to generate an activated integrin state at the cell surface [34]. More C6 cells attachment per unit area on TPS than those on PS [28], and more human tongue squamous carcinomas cells selective adhesion in the laser-irradiated area of a polystyrene Petri dish [35], have showed that the hydrophilic polystyrene accelerates cells to attach and adhere by inducing FA assembly and stabilization. Cell migration involves in FA assembly and disassembly and the fine regulation of the cytoskeleton system, especially, actin polymerization and depolymerization [36]. The result of one study has revealed that the migration speed of 3T3 fibroblasts on patterned Ti4Al6V is similar to that on smooth Ti4Al6V [37]. Therefore, C6 cells on TPS moving slower than those on PS might be mainly due to that the hydrophilic TPS accelerated dash-like FAs to be assembled

and stabilized. For C6 cells on hydrophobic PS, with weak cell-substrate adhesiveness and fast migration speed, their punctuated FAs might be less stable. Lamellipodia at the leading edge of C6 cells on PS has showed that the rate of actin polymerization and depolymerization is faster [38], which consumes a lot of energy (ATP). However, for cells on TPS, with strong cell-substrate adhesiveness and slow migration speed, there might remain enough ATP to synthesize proteins and duplicate DNA, which agreed with the results that faster motility for human glioma cells is associated with reduced transcription of proliferation genes [39], and that the rate of cell proliferation on TPS is faster than on PS [28]. Therefore, it was the increased hydrophilicity of TPS surface that accelerated C6 cell attachment and proliferation, whereas inhibited cell migration, and the dynamics of FAs played an essential role in adjusting these cell behaviours. The cellular tensegrity model was used to explain the phenomenon of anisotropic LIPSS on TPS guiding cell spreading, migration and division. This model proposes that the whole cell is a prestressed tensegrity structure, and that geodesic structures are found in the cell at smaller size scale with tensional forces borne by cytoskeletal microfilaments (MFs) and intermediate filaments (IFs) and balanced by microtubules (MTs) and FAs [40]. Mechanical signals outside the cell

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Fig. 8. SEM images of C6 cell division on PS (A) and TPS (B), and SPCA-1 cell (C) and HeLa cell (D) division on TPS. The double-ended arrows pointed the direction of LIPSS.

transmitted across FAs can be converted into chemical and electrical signals inside the cell as a result of their transmission across discrete cytoskeletal linkages and associated changes in the cytoskeletal force balance [41]. After trypsinization or division, the cell is spherical and its cytoskeleton reorganizes gradually during attachment and spreading. Compared with the cell on PS, the cell on TPS increased its tension to balance the larger apparent surface energy of the substrate as a result of carbonyl-containing groups and LIPSS [28]. In addition, the apparent surface energy of TPS is anisotropic with slightly higher in the direction parallel than perpendicular to the LIPSS. The results of other studies have indicated that filopodium, which is the stiffened bundles of crosslinked actin filaments bearing compression, forms when cell membrane tension increases, while for lamellipodium, which is a branched actin network bearing tension, its extension rate is inversely correlated with the apparent cell membrane tension [40,42]. Therefore, the cells on TPS spread using filopodia with longer and thicker parallel with LIPSS, while the cells on PS extended lamellipodia in all directions. C6 cells on two-dimensional PS moved in a three-step moving paradigm, similar to invading a three-dimensional collagen type I matrix [43]. However, C6 cell on TPS moved along

LIPSS, which agreed with cell directional migration on grooved substrates, such as, osteoblasts on nanogrooved polystyrene [22], 3T3 Swiss embryo fibroblasts on microgrooved Ti4Al6V [37], and human corneal epithelial cells on nanogrooved silicon [20]. C6 cell directional migration on TPS might be explained that the anisotropic LIPSS acted as a permanent mechanical stimulus to guide the dynamics of FAs and the cytoskeleton inside the cell during migration, and the cell adjusted itself to align along LIPSS step by step to reach a balance between the cellular tensegrity structure and LIPSS. When the C6 cell on TPS was in interphase, both its whole body and its nucleus aligned along LIPSS. One study has revealed that the longest axis of elliptic primary human fibroblast nucleus is parallel to grooves on microgrooved quartz with gene expression change [44]. According to the tensegrity theory, the cell nucleus is a smaller self-stabilizing tensegrity module inside the cell, which can respond to the mechanical stimuli outside the cell and achieve its structural balance [40]. Therefore, LIPSS acted as an anisotropic and persistent mechanical stimulus to induce cell nucleus shape anisotropy by cytoskeleton reorganization in interphase. When the C6 cell on TPS entered mitosis, the direction of the cell division was parallel to LIPSS. The result of one study

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Fig. 9. SEM images of C6 cell spreading after division on PS (A and B) and TPS (C and D), and SPCA-1 cell (E) and HeLa cell (F) spreading on TPS. The doubleended arrows pointed the direction of LIPSS.

has indicated that the orientation of the cell division axis is regulated by the spatial distribution of fibronectin on mercaptoepropyulrimethoxyesilane, and the patterned fibronections determine the spindle orientation during mitosis through inducing the anisotropic distribution of the actin cytoskeleton and such associated cortical proteins as ezrin and cortacin in interphase [6]. LIPSS inducing dash-like FA assembly at the two ends of the cell, cell spreading with filopodia, and migration and alignment along LIPSS, these signals of spatial

anisotropy, might be expressed as the cell division axis along LIPSS in mitosis. Therefore, the oriented cell division on TPS was the result of celleLIPSS interaction. 5. Conclusion The commercially available smooth polystyrene film irradiated by p-polarized laser with a wavelength of 266 nm not only formed periodic three-dimensional nanostructures e LIPSS,

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Fig. 10. Fluorescence microscopy images of C6 cells cultured on PS (A and B) and TPS (C and D) for 24 h, and the cells immunostained focal adhesion kinase.

but also became hydrophilic. We found that hydrophilic TPS accelerated C6 cells to attach and adhere, but inhibited cells to migrate, which led to the rate of cell proliferation increasing. At the same time, LIPSS on TPS acted as an anisotropic and persistent mechanical stimulus outside the cell to induce dash-like FA assembly at the two ends of the cell, cell directional migration, and cell oriented. The most obvious cell behaviour change was the moving paradigm from a three-step invasion paradigm on PS to a LIPSS dependent directional moving paradigm on TPS. Acknowledgements The authors are grateful to Mr Yijian Lai of the Instrumental Analysis Centre, Shanghai Jiao Tong University, for SEM measurements. Funding for this work was provided by the National Natural Science Foundation of China (grand No. 60577049), and the Shanghai Municipal Science and Technology Commission (grant No. 0652nm017). References [1] Andrew N, Insall RH. Chemotaxis in shallow gradients is mediated independently of PtdIns 3-kinase by biased choices between random protrusions. Nat Cell Biol 2007;9:193e200. [2] Weiner OD. Regulation of cell polarity during eukaryotic chemotaxis: the chemotactic compass. Curr Opin Cell Biol 2002;14:196e202.

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