Directional cell elongation through filopodia-steered lamellipodial extension on patterned silk fibroin films Renchuan You, Xiufang Li, Zuwei Luo, Jing Qu, and Mingzhong Li
Citation: Biointerphases 10, 011005 (2015); doi: 10.1116/1.4914028 View online: http://dx.doi.org/10.1116/1.4914028 View Table of Contents: http://avs.scitation.org/toc/bip/10/1 Published by the American Vacuum Society
Directional cell elongation through filopodia-steered lamellipodial extension on patterned silk fibroin films Renchuan You, Xiufang Li, Zuwei Luo, Jing Qu, and Mingzhong Lia) National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, No. 199 Ren’ai Road, Industrial Park, Suzhou 215123, China
(Received 26 January 2015; accepted 19 February 2015; published 5 March 2015) Micropatterned biomaterials have been used to direct cell alignment for specific tissue engineering applications. However, the understanding of how cells respond to guidance cues remains limited. Plasticity in protrusion formation has been proposed to enable cells to adapt their motility mode to microenvironment. In this study, the authors investigated the key role of protrusion response in cell guidance on patterned silk fibroin films. The results revealed that the ability to transform between filopodia and small lamellipodia played important roles in directional cell guidance. Filopodia did not show directional extension on patterned substrates prior to spreading, but they transduced topographical cues to the cell to trigger the formation of small lamellipodia along the direction of a microgrooved or parallel nanofiber pattern. The polar lamellipodia formation provided not only a path with directionality, but a driving force for directional cell elongation. Moreover, aligned nanofibers coating provided better mechanical support for the traction of filopodia and lamellipodia, promoting cell attachment, spreading, and migration. This study provides new insight into how cells respond to guidance cues and how filopodia and lamellipodia control cell contact guidance on micropatterned biomaterial surfaces. C 2015 American Vacuum Society. [http://dx.doi.org/10.1116/1.4914028] V
I. INTRODUCTION Directional cell motility is critical for tissue morphogenesis and wound healing.1 Microscale topographical cues influence cell adhesion, morphology, migration, and differentiation in numerous cell types ranging from fibroblasts to mesenchymal stem cells.2,3 Contact guidance is characterized by the cellular response to the substrate topography and leads to morphological changes and functional alterations that can be directly observed as an aligned cytoskeleton, elongated morphology, and oriented cell migration.4–8 Aligned micropatterns, such as parallel grooves and nanofibers, have been used to direct cell migration through contact guidance for tissue regeneration applications, such as musculoskeletal and nervous tissue repair.9,10 Despite the long awareness of contact guidance, our understanding of how cells detect and respond to guidance cues remains limited. As vital “organelles” involved in cell motility, filopodia and lamellipodia play important roles in sensing the surrounding environment and guiding cell motility.11–17 Filopodia are thin fingerlike structures (0.1–0.3 lm) that are filled with tight parallel bundles of filamentous F-actin.11 By contrast, a lamellipodium is a highly dynamic broad membrane protrusion filled with a branched network of actin, which exhibits varying breadth from 1 to 5 lm depending on cell type and condition.11,16 On a substrate surface, cells sense their surrounding environment through filopodia that detect biophysical and biochemical stimuli,11–14 and the lamellipodia drive cell spreading and migration through actin polymerization and cytoskeletal rearrangement.15–17
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Filopodia exhibited varied responses to monolayers of beads with different sizes, indicating that filopodia play a crucial role in the recognition of topographical features.18 Moreover, the filopodia of fibroblasts and macrophages were able to pull surrounding targets toward the cell body, in a manner analogously to tentacles.19,20 On patterned substrates with grooves and ridges, lamellipodia periodically extended and retracted in the direction of the pattern to pull the cell into alignment.6 These results demonstrate the important functions of filopodia and lamellipodia as sensors and as a source of mechanical traction for cell alignment. In addition to the filopodial response to guidance cues, directional lamellipodia extension was observed on microscale adhesive islands, where cells preferentially extended lamellipodia from the corner regions of different geometric forms,1,21 however, the response mechanism of lamellipodia to surface topography to guide cell mobility has not been determined. Plasticity in protrusion formation has been showed to enable cells to adapt their migration mode to surrounding microenvironment. The mode of filopodial adhesion was shown to modulate lamellipodial protrusion in fibroblasts and neurons.22,23 In fibroblasts, filopodia nucleated and converted into lamellipodialike extensions starting at their tips in response to integrin occupancy by a matrix substrate and downstream Rac1 signaling.22 Neuronal growth cone filopodia have the capacity to nucleate the formation of lamellipodia along their length.23 These results indicate that filopodial activity influences the formation and advance of lamellipodia. Recently, it was shown that lamellipodial extension could be induced by filopodial traction on micropatterned substrates, implying that the synergetic cooperation between filopodia and lamellipodia plays critical roles in cell
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C 2015 American Vacuum Society V
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guidance.24 Therefore, understanding how filopodia direct the activities of lamellipodia is essential to understanding the response mechanism of cell contact guidance on a micropatterned surface. Silk fibroin (SF) is a promising biomaterial due to its abundance, mechanical robustness, biocompatibility, and tunable biodegradability.25,26 Regenerated SF can be processed in both aqueous and organic solvents, making it a versatile option for the simple and inexpensive fabrication of biomaterials with various microstructures.24,27 In the present study, we investigated the effect of physical guidance cues on cell orientation by dissecting the response of the cell to patterned SF films over time, and we clarified the key roles of filopodia and lamellipodia in contact guidance. II. EXPERIMENT A. Preparation of regenerated SF
The regenerated SF solution was obtained as previously described.24 Briefly, Bombyx mori raw silk fibers (Huzhou, China) were degummed three times in 0.05 wt. % Na2CO3 at 98–100 C for 30 min, and then, the dried fibroin extract was dissolved in a solution of CaCl2/EtOH/H2O (mole ratio, 1:2:8) at 72 6 2 C for 1 h. The SF solution was obtained after dialysis (MWCO 9–12 kDa) for 4 day, and it was stored at 4 C after filtration. For electrospinning, the SF solution was lyophilized to produce porous sheets and stored at 4 C. B. Preparation of SF substrates
Polydimethylsiloxane (PDMS; Sylgard 184, Dow Corning) was cast on diffraction grating (Edmund Optics, Inc.; 600 lines/mm and 2.5 2.5 cm) surfaces. The PDMS substrates were peeled from the grating after solidification for 4 h at 65 C and then rinsed with ethanol and deionized water. The SF solution was cast on the patterned PDMS surface to generate micropatterned films. Flat SF films were produced by pouring the solution into polystyrene dishes. The thickness of the films was determined by the ratio between the SF solution and the mold area. Porous SF sheets were dissolved in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP; Dupont Chemical, Co., USA) to generate a 10% (wt./v) solution. A constant flow rate of 1 ml/h was maintained using a syringe pump (KDS 100, KD Scientific), and the distance between the needle tip and the collector was 10 cm. The solution was drawn into fibers using a high voltage of 10 kV, and it was deposited on a collector made of two parallel steel strips to prepare the aligned fibers. The flat SF film was cut into disks with a 1.5-cm diameter and then the aligned fibers were carefully transferred onto the surface of the SF films to obtain a nanofiber-coated surface. All SF samples were immersed in 80% ethanol for 2 h to render the films waterinsoluble by inducing the formation of b-sheet secondary structure. The average diameter of the electrospun nanofibers was calculated by measuring the diameters of 100 different nanofibers in scanning electron microscopy (SEM) images. Biointerphases, Vol. 10, No. 1, March 2015
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C. Cell culture
The animal experiments were approved by Jiangsu Province according to the rules for the management of experimental animals [(2008) No. 26]. Bone marrow-derived mesenchymal stem cell (BMSC) were isolated as previously described.24 The cells were cultured in DMEM/F-12 (HyClone) supplemented with 10% fetal bovine serum (FBS, HyClone), 100 U/ml penicillin, and 100 lg/ml streptomycin (Invitrogen, Carlsbad, CA). SF films were cut to the size of the wells in a 24-well plate and sterilized for 15 min with 75% EtOH. The films were subsequently washed three times with sterile phosphate-buffered saline (PBS; 0.1 M, pH 7.4). A sterilized polystyrene tube with an outer diameter of 15.4 mm and an inner diameter 11.6 mm was placed into the wells to fix the film to the bottom of the culture plates. BMSCs were seeded on the SF films at 4 104 cells/cm2. D. SEM and atomic force microscopy observations
The substrate morphology was characterized by scanning electron microscopy (SEM; S-4800, Hitachi, Japan). The features of the microgrooved films were quantitatively evaluated using atomic force microscopy (AFM; Nanoscope V, Veeco, USA). The cell culture samples were rinsed three times in 0.1 M PBS and fixed with 2.5% glutaraldehyde at 4 C overnight, followed by three washes in PBS. The fixed samples were dehydrated in an ascending series of graded ethanol (50%, 70%, 90%, and 99.7%) for 5 min and then further dried using hexamethyldisiloxane (HMDS, SigmaAldrich) for 3 min. The dried samples were sputter coated with gold for 90 s prior to SEM observation. The polarization of small lamellipodia on patterned surfaces was analyzed by measuring the orientation angle of the lamellipodial rim, which has a minor included angle with pattern direction in SEM images, with 10 cells in each group, and the direction of grooves and nanofibers was defined as 0 . E. Cell staining and observation
After fixation in 4% paraformaldehyde-PBS for 15 min, the samples were permeabilized with 0.2% Triton X-100 for 5 min and blocked with 2% BSA-PBS for 30 min. The samples were incubated with 5 lg/ml FITC-phalloidin (Sigma-Aldrich) for 2 h at room temperature. After thoroughly rinsing, the nuclei were labeled with 5 lg/ml 40 ,6-diamidino-2-phenylindole (DAPI, Sigma) for 10 min, rinsed three times with PBS for 5 min each and observed using a confocal laser scanning microscope (CLSM; IX81/FV1000, Olympus, Japan). To investigate the elongation of nuclei, the nuclear shape index was calculated by measuring the aspect ratio of 30 different cells for each group (aspect ratio: major axis to minor axis of each nucleus). F. Cell viability assay
Cell proliferation was measured with a cell count kit-8 assay (CCK-8; Sigma-Aldrich). A 500-ll volume of fresh
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culture medium containing 50 ll of CCK-8 solution was added to each well containing the BMSC-seeded SF films, and the plates were kept in an incubator with 5% CO2 at 37 C for 3 h. The formazan derivative formed by cell metabolism is soluble in the culture medium and was detected by measuring the absorbance at 450 nm using a microplate reader (Bio-Tek Synergy HT, USA). The background absorbance measured from a sample containing the CCK-8 reagent in culture medium (without cells) was subtracted from all the samples. G. Statistical analysis
Statistical comparisons were performed using SPSS version 16.0 software (SPSS, Inc., Chicago, Illinois). The deductive statistics (t-test, analysis of variance) were conducted, and the data were presented as mean 6 standard deviation, and p < 0.05 was considered to be statistically significant. III. RESULTS A. Surface characteristic of SF substrates
As shown in Fig. 1, the unpatterned SF films presented a smooth surface [Fig. 1(a)], and the microgrooved pattern was successfully replicated from the PDMS molds [Fig. 1(b)]. The microgrooved pattern displayed a saw tooth shape with grooves of 1720 6 94 nm width and 142 6 15 nm depth [Fig. 1(c)]. The aligned SF nanofibers on the parallel electrode were easily transferred to the flat SF films. The nanofibers adhered to the film surface after aqueous alcohol treatment [Figs. 1(d) and 1(e)], and the average diameter of the nanofibers was 963 6 213 nm.
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B. Initial cell response to guidance cues
To characterize how the BMSCs interacted with the guidance cues of the substrates prior to spreading, the cell morphology was observed after 30 min of culture. As shown in Fig. 2, the cells anchored to the films and nanofibers through numerous protruding filopodia. On the flat surface, the cells randomly extended filopodia to anchor to the substrate [Figs. 2(a) and 2(d)]. Filopodia serve as “sensory organelles” that sense the mechanical and chemical environment of a cell.12,13 However, the cells also randomly protruded filopodia in multiple directions on the patterned film [Figs. 2(b) and 2(c)], indicating that the filopodia did not show directional extension prior to spreading. Although the cells predominantly exhibited a rounded shape, they protruded small lamellipodia along the direction of the microgrooved and nanofiber pattern [long arrows, Figs. 2(b) and 2(c)]. Filopodia can mature into different structures, such as become spines, dendrites, and lamellipodia,22–24,28–30 and the process of transformation from filopodia to lamellipodia has been observed in previous studies.22,24 Strikingly, the filopodia that extended along the direction of the patterns were switched into small lamellipodia [long arrows, Figs. 2(e) and 2(f)] and provided scaffolds for small lamellipodia formation [short arrows, Figs. 2(e) and 2(f)]. Although the extension direction of filopodia was random on the both flat and patterned SF films prior to spreading, the extension of small lamellipodia was oriented toward the direction of patterns. The extension direction of lamellipodia on flat surface was random, whereas the protruded small lamellipodia on both grooved and nanofiber-coated films exhibited oriented extension, which had the orientation angles
FIG. 1. SEM images of the surfaces of silk fibroin films with (a) flat, (b) microgrooved, and (d) and (e) nanofiber-coated topographies. (c) AFM image of the microgrooved film surface. Scale bars: (a) and (b) 50 lm, (d) 500 lm, and (e) 10 lm. Biointerphases, Vol. 10, No. 1, March 2015
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FIG. 2. SEM images showing the initial morphology of cells cultured on (a) flat, (b) microgrooved, and (c) nanofiber-coated films 30 min after plating. (d)–(f) Magnified images of the filopodia and lamellipodia shown in (a), (b), and (c), respectively. The short and long arrows indicate filopodia and lamellipodia, respectively. Scale bars: (a), (d), (e), and (f) 5 lm and (b) and (c) 10 lm.
of 5.3 6 4.3 and 15.4 6 6.4 , respectively. These results suggested that the filopodia transduced the substrate information to the cell nucleus and determined the direction of the lamellipodial protrusion. Filopodia cannot drive cell motility alone whereas lamellipodia can,30 and the directional advance of small lamellipodia can pull cell spreading in the direction of the pattern and thus trigger the initiation of contact guidance. C. Cellular response to microtopography over time
To visualize the course of cell spreading over time, the cell morphology was observed after culturing for 1, 2, and 4 h. After 1 h on an unpatterned film, small lamellipodia protruded in different directions from the sites of filopodial adhesion to form a stable anchor and to flat cell body [long arrows, Figs. 3(a) and 3(b)], indicating that the protrusion of lamellipodia was a prerequisite for BMSC spreading. The adhered filopodia served as the navigators and scaffolds for lamellipodial protrusion [short arrows, Fig. 3(b)]. On the patterned films, small lamellipodia matured into lamellipodia protrusion and directed cell elongation along the direction of the patterns [long arrows, Figs. 3(e) and 3(j)]. The front filopodia extended along the grooves and fibers to direct the advancement of the lamellipodia [short arrows, Figs. 3(f) and 3(k)]. Moreover, peripheral lamella formed around the cell body on the nanofiber-coated film, indicating that the cells had begun to undergo full spreading [Fig. 3(j)]. After 2 h, the peripheral lamella protruded from the cell body, resulting in a flattened spreading on both flat [Fig. 3(c)] and microgrooved films [Fig. 3(g)]. The cells that were cultured on the microgrooved film were elongated and aligned in the direction of the pattern [Fig. 3(g)], and the filopodia and lamellipodia at the leading edge extended in the direction of the groove [arrows, Biointerphases, Vol. 10, No. 1, March 2015
Fig. 3(g)]. By contrast, the cells cultured on the smooth film were mostly round, and the lamellipodial protrusions displayed random orientations [Fig. 3(c)]. Moreover, the cells cultured on the nanofiber-coated surface exhibited a distinct spreading and elongation compared to those cultured on the grooved surface [Fig. 3(l)], suggesting the SF nanofiber coating accelerated cell spreading on the SF film. After 4 h, the cells had fully spread [Figs. 3(d), 3(h), and 3(m)], and the lamellipodial protrusion extended along the bottom of the groove to drive cell migration in the direction of the pattern [Fig. 3(i)]. Furthermore, the cell morphology was observed by SEM and CLSM after 24 h. On the flat surfaces, the cells presented a polygonal shape and randomly protruded plasma membranes [Figs. 4(a) and 4(d)], whereas the cells cultured on patterned films presented a significantly elongated body shape and extended numerous membrane protrusions in the direction of the pattern [Figs. 4(b), 4(c), 4(e), and 4(f)]. Generally, the cell nuclei were spherical or ellipsoid, and it is possible that the nuclear shape is regulated by the substrate pattern.31 The nuclear elongation was quantitated by calculating the aspect ratio. The aspect ratio showed significant difference on various substrate surfaces (P < 0.05). The cell nuclei on flat film were found to be more spherical [Fig. 4(d)], which had lower aspect ratio (1.41 6 0.16). The cell nuclei became more elongated [Figs. 4(e) and 4(f)] and had higher aspect ratios on both micropatterned and nanofiber-coated surface (1.78 6 0.27 and 3.11 6 0.80, respectively). D. Cell proliferation
After 7 days of cell culture, the cells grown on SF substrates nearly covered the surface (Fig. 5). The cells on both the microgrooved surface and the aligned nanofibers were
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FIG. 3. Cell spreading after 1, 2, and 4 h of culture on (a)–(d) flat, (e)–(f) microgrooved, and (j)–(m) nanofiber-coated films. The images shown in (b), (f), (i), and (k) are magnified images of the regions indicated by boxes in (a), (e), (h), and (j), respectively. The short and long arrows indicate filopodia and lamellipodia, respectively. Scale bars: (a) and (k) 10 lm; (b), (c), (e), (g), and (i) 5 lm; (d), (h), and (m) 50 lm; (f) 3 lm; and (j) and (l) 20 lm.
FIG. 4. SEM images of BMSCs after 24 h of cultivation on (a) flat, (b) micropatterned, and (c) nanofiber-coated films. Fluorescent staining of BMSCs after 24 h cultivation on (d) flat, (e) micropatterned, and (f) nanofiber-coated films. Green indicates fluorescent actin staining, and blue indicates the nuclei stained with DAPI. Scale bars: (a) and (f) 100 lm and (b)–(e) 50 lm. Biointerphases, Vol. 10, No. 1, March 2015
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FIG. 5. SEM images of BMSCs after 7 days of cultivation on (a) flat, (b) microgrooved, and (c) nanofiber-coated films. Fluorescent staining of BMSCs after 7 days of cultivation on (d) flat, (e) micropatterned, and (f) nanofiber-coated films. Scale bars: 100 lm.
oriented along the direction of patterns [Figs. 5(e) and 5(f)]. By contrast, the cells cultured on the flat film grew in a circular pattern [Figs. 5(a) and 5(d)]. The results demonstrated that both the microgroove pattern and the nanofiber coating on SF films provided a suitable environment to control cell alignment. Furthermore, cell attachment and proliferation were measured after 7 days of culture on the SF substrates (Fig. 6). The optical density values obtained for cells cultured on the flat and microgrooved surfaces were not significantly different after 4 h or 7 days in culture, whereas the aligned nanofiber coating significantly promoted cell
FIG. 6. Viability of BMSCs on various silk fibroin films after 4 h and 7 days of cultivation (n ¼ 3, *p < 0.05, **p < 0.01, error bars indicate SD). Biointerphases, Vol. 10, No. 1, March 2015
attachment and proliferation (Fig. 6). This result was consistent with the SEM observations and suggested that nanofiber coating accelerated cell spreading (Fig. 3). IV. DISCUSSION The interaction between cells and the extracellular matrix (ECM) influences directional cell motility, which is critical for tissue development and wound repair.1 Therefore, cellmaterial interaction is a fundamental topic in the fields of tissue engineering and regenerative medicine. Understanding the mechanisms of topography sensing is also crucial for designing biomaterials that have the ability to promote healing and tissue regeneration. Conventionally, the nucleus is considered the site of integration of external physical and chemical signals.31,32 The shapes of cell nuclei have been shown to correlate with the pattern of the substrate,31 and nuclear shape and deformation in turn correlate with changes in gene expression.32 Filopodia act as cellular antennae that sense the surrounding microenvironment; they detect biophysical and biochemical stimuli and direct feedback to the nucleus to modulate cellular morphology and function.33 Dynamic lamellipodial protrusions provide traction to drive cell motility through actin polymerization and disassembly.16,17 Filopodia and lamellipodia generally coexist at the protruding front of migrating cells. Filopodia cannot drive cell motility without lamellipodia, but they do participate in cell guidance.30 Thus, filopodia and lamellipodia are intimate collaborators for cell motility. We cultured cells on a grooved pattern and aligned nanofibers to understand how they respond to topographical cues of SF-based biomaterials.
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Filopodia often form the first adhesive contact with the ECM, pathogens or adjacent cells, and they are believed to be involved in environment sensing and cell motility.12 However, filopodia protruded randomly on both flat and patterned SF films prior to spreading, suggesting that filopodia play a major role in the formation of initial attachments to anchor the cell onto the substrate surface. The dynamic activities of the lamellipodia drive cell spreading and migration on substrate surfaces,15–17 and the direction of lamellipodial protrusions is crucial to cell guidance.6 In cells cultured on a microgrooved surface, lamellipodia extended and retracted in the direction of the grooves to guide cell alignment.6 Actin-related protein 2/3 (Arp2/3) complex is a major actin nucleator of lamellipodia, cell guidance on microgrooved surface and endothelial cell layers could be disturbed after Arp2/3 complex inhibition.34,35 Furthermore, the direction of lamellipodial protrusions was impacted by geometric features of the adhesive islands,36 but the response mechanism of lamellipodia in sensing surface topography has not been determined. Filopodial adhesion plays important roles on lamellipodial adhesion sites during cell migration.17 Filopodia can provide the scaffold for lamellipodia nucleation, and they can mature into lamellipodia along their length.22,24 We found that after forming stable attachments, the filopodia served as scaffolds to guide small lamellipodial advance along their length in cells cultured on SF films [Figs. 3(a) and 3(b)], and the mature lamellipodia began to flatten the cell body on the substrate surface [Fig. 3(b)]. More importantly, small lamellipodia formed along the direction of the patterns [Figs. 2(b) and 2(c)], indicating that lamellipodial advance is controlled by the extension of exploratory filopodia. Exploratory filopodia guided lamellipodial polarization, and the directional lamellipodial extension dominated cell elongation in the direction of the grooves and nanofibers [Figs. 3(f) and 3(k)]. These results revealed the synergistic cooperation between filopodia and lamellipodia in cell guidance. Filopodia first served as sensors for path finding, and then they integrated and transduced the topographical cues to the nucleus to direct the cell response after stable anchoring. The lowest level of resistance is present along the direction of the grooves or fibers, the exploratory activity of the filopodia steered the directional advance of small lamellipodia along the direction of patterns to direct cell elongation. Therefore, filopodia-steered lamellipodial extension is an important mechanism in cell contact guidance. However, to further confirm the critical role filopodiaguided lamellipodia polarization on cell guidance, the mechanism of lamellipodia polarization should be further investigated in the future, including the change of actin filament structures during polar lamellipodia formation and the lamellipodial variation after filopodia inhibition. Surface patterning may be used to control material–cell interactions in SF-based implantable medical devices.37 Both the microgroove pattern and the aligned nanofibers were able to guide the cells. Nuclear shape was significantly impacted by microgrooves and nanofibers on SF films, and aligned nanofibers further increased the nuclear elongation Biointerphases, Vol. 10, No. 1, March 2015
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compared to groove pattern. Furthermore, the nanofiber coating significantly promoted cell spreading and proliferation. The ECM consists of complex fibrillar networks composed of fibrous proteins and glycosaminoglycans.38 ECM nanofibers are smaller than the cell itself and are an important part of the microenvironment that provides structural support to cells.39 On micropatterned islands, filopodia preferentially adhered to nanofibrillar island compared to flat surfaces, and the filopodia that initially extended quickly adhered to nanowires and then aligned with nanowires by pulling on them.19 On nanofiber-coated SF films, the protruding filopodia preferentially adhered to the nanofibers to form sites of stable attachment [Fig. 2(c)], and the small lamellipodia tightly wrapped around the nanofibers to drive cell elongation along the fiber axis [Fig. 2(f)]. The nanofibers provided better mechanical support than the microgrooves for the traction of filopodia and lamellipodia, and this may be a crucial factor for promoting cell attachment, spreading, and migration on a nanofiber-coated film. Moreover, the fabrication of aligned electrospun SF nanofibers is simpler and less expensive than the fabrication of a microgrooved pattern, thus offering an efficient approach for surface modification of biomaterials. V. CONCLUSIONS In this study, we investigated how guidance cues control cell elongation on patterned SF films. Filopodia supported and dominated lamellipodial extension, which is crucial for cell directionality. Exploratory filopodia sensed guidance cues and then guided lamellipodial polarization, and which ultimately resulted in the elongation of the cell along the direction of patterns. The results highlighted the important role of the diversity and plasticity of protrusion in directing cell contact guidance. Moreover, aligned nanofibers coating not only provided strong guidance cues, but also provided better mechanical support for the traction of filopodia and lamellipodia, promoting cell attachment, spreading, and migration, which is a useful approach for surface optimization of biomedical materials. These results reveal the critical roles of filopodia-steered lamellipodial extension in the control of cell motility and contact guidance in response to physical cues. ACKNOWLEDGMENTS This work was supported by the National Nature Science Foundation of China (31370968), Nature Science Foundation of Jiangsu Province (BK20131177), College Natural Science Research Project of Jiangsu Province (12KJA430003), Priority Academic Program Development of Jiangsu Higher Education Institutions and Jiangsu Province Ordinary Universities and Colleges Graduate Scientific and Innovation Plan (KYLX_1243). 1
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A. K. Mongiu, E. L. Weitzke, O. Y. Chaga, and G. G. Borisy, J. Cell Sci. 120, 1113 (2007). 24 R. You, X. Li, Y. Liu, G. Liu, S. Lu, and M. Li. J. Biomed. Mater. Res. Part A 102, 4206 (2014). 25 B. Kundu, R. Rajkhowa, S. C. Kundu, and X. Wang, Adv. Drug Delivery Rev. 65, 457 (2013). 26 R. You, Y. Xu, G. Liu, Y. Liu, X. Li, and M. Li, Polym. Degrad. Stab. 109, 226 (2014). 27 D. N. Rockwood, R. C. Preda, T. Y€ ucel, X. Wang, M. L. Lovett, and D. L. Kaplan, Nat. Protoc. 6, 1612 (2011). 28 M. G. Heiman and S. Shaham, Curr. Opin. Neurobiol. 20, 86 (2010). 29 E. Menna, G. Fossati, G. Scita, and M. Matteoli, Eur. J. Neurosci. 34, 1655 (2011). 30 M. Nemethova, S. Auinger, and J. V. Small, J. Cell Biol. 180, 1233 (2008). 31 Z. Pan, C. Yan, R. Peng, Y. Zhao, Y. He, and J. Ding, Biomaterials 33, 1730 (2012). 32 A. S. Nathan, B. M. Baker, N. L. Nerurkar, and R. L. Mauck, Acta Biomater. 7, 57 (2011). 33 C. Y. Tay, S. A. Irvine, F. Y. C. Boey, L. P. Tan, and S. Venkatraman, Small 7, 1361 (2011). 34 K. W. Kwon, H. Park, and J. Doh, PloS One 8, e73960(2013). 35 K. H. Song, K. W. Kwon, J.-C. Choi, J. Jung, Y. Park, K.-Y. Suh, and J. Doh, Integr. Biol. 6, 450(2014). 36 M. Kanada, A. Nagasaki, and T. Q. P. Uyeda, Mol. Biol. Cell 19, 8 (2008). 37 B. D. Lawrence, J. K. Marchant, M. A. Pindrus, F. G. Omenetto, and D. L. Kaplan, Biomaterials 30, 1299 (2009). 38 T. G. Kim, H. Shin, and D. W. Lim, Adv. Funct. Mater. 22, 2446 (2012). 39 C. P. Barnes, S. A. Sell, E. D. Boland, D. G. Simpson, and G. L. Bowlin, Adv. Drug Delivery Rev. 59, 1413 (2007).
Citation: Biointerphases 10, 011005 (2015); doi: 10.1116/1.4914028 View online: http://dx.doi.org/10.1116/1.4914028 View Table of Contents: http://avs.scitation.org/toc/bip/10/1 Published by the American Vacuum Society
Directional cell elongation through filopodia-steered lamellipodial extension on patterned silk fibroin films Renchuan You, Xiufang Li, Zuwei Luo, Jing Qu, and Mingzhong Lia) National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, No. 199 Ren’ai Road, Industrial Park, Suzhou 215123, China
(Received 26 January 2015; accepted 19 February 2015; published 5 March 2015) Micropatterned biomaterials have been used to direct cell alignment for specific tissue engineering applications. However, the understanding of how cells respond to guidance cues remains limited. Plasticity in protrusion formation has been proposed to enable cells to adapt their motility mode to microenvironment. In this study, the authors investigated the key role of protrusion response in cell guidance on patterned silk fibroin films. The results revealed that the ability to transform between filopodia and small lamellipodia played important roles in directional cell guidance. Filopodia did not show directional extension on patterned substrates prior to spreading, but they transduced topographical cues to the cell to trigger the formation of small lamellipodia along the direction of a microgrooved or parallel nanofiber pattern. The polar lamellipodia formation provided not only a path with directionality, but a driving force for directional cell elongation. Moreover, aligned nanofibers coating provided better mechanical support for the traction of filopodia and lamellipodia, promoting cell attachment, spreading, and migration. This study provides new insight into how cells respond to guidance cues and how filopodia and lamellipodia control cell contact guidance on micropatterned biomaterial surfaces. C 2015 American Vacuum Society. [http://dx.doi.org/10.1116/1.4914028] V
I. INTRODUCTION Directional cell motility is critical for tissue morphogenesis and wound healing.1 Microscale topographical cues influence cell adhesion, morphology, migration, and differentiation in numerous cell types ranging from fibroblasts to mesenchymal stem cells.2,3 Contact guidance is characterized by the cellular response to the substrate topography and leads to morphological changes and functional alterations that can be directly observed as an aligned cytoskeleton, elongated morphology, and oriented cell migration.4–8 Aligned micropatterns, such as parallel grooves and nanofibers, have been used to direct cell migration through contact guidance for tissue regeneration applications, such as musculoskeletal and nervous tissue repair.9,10 Despite the long awareness of contact guidance, our understanding of how cells detect and respond to guidance cues remains limited. As vital “organelles” involved in cell motility, filopodia and lamellipodia play important roles in sensing the surrounding environment and guiding cell motility.11–17 Filopodia are thin fingerlike structures (0.1–0.3 lm) that are filled with tight parallel bundles of filamentous F-actin.11 By contrast, a lamellipodium is a highly dynamic broad membrane protrusion filled with a branched network of actin, which exhibits varying breadth from 1 to 5 lm depending on cell type and condition.11,16 On a substrate surface, cells sense their surrounding environment through filopodia that detect biophysical and biochemical stimuli,11–14 and the lamellipodia drive cell spreading and migration through actin polymerization and cytoskeletal rearrangement.15–17
a)
Electronic mail: [email protected]
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Filopodia exhibited varied responses to monolayers of beads with different sizes, indicating that filopodia play a crucial role in the recognition of topographical features.18 Moreover, the filopodia of fibroblasts and macrophages were able to pull surrounding targets toward the cell body, in a manner analogously to tentacles.19,20 On patterned substrates with grooves and ridges, lamellipodia periodically extended and retracted in the direction of the pattern to pull the cell into alignment.6 These results demonstrate the important functions of filopodia and lamellipodia as sensors and as a source of mechanical traction for cell alignment. In addition to the filopodial response to guidance cues, directional lamellipodia extension was observed on microscale adhesive islands, where cells preferentially extended lamellipodia from the corner regions of different geometric forms,1,21 however, the response mechanism of lamellipodia to surface topography to guide cell mobility has not been determined. Plasticity in protrusion formation has been showed to enable cells to adapt their migration mode to surrounding microenvironment. The mode of filopodial adhesion was shown to modulate lamellipodial protrusion in fibroblasts and neurons.22,23 In fibroblasts, filopodia nucleated and converted into lamellipodialike extensions starting at their tips in response to integrin occupancy by a matrix substrate and downstream Rac1 signaling.22 Neuronal growth cone filopodia have the capacity to nucleate the formation of lamellipodia along their length.23 These results indicate that filopodial activity influences the formation and advance of lamellipodia. Recently, it was shown that lamellipodial extension could be induced by filopodial traction on micropatterned substrates, implying that the synergetic cooperation between filopodia and lamellipodia plays critical roles in cell
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C 2015 American Vacuum Society V
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guidance.24 Therefore, understanding how filopodia direct the activities of lamellipodia is essential to understanding the response mechanism of cell contact guidance on a micropatterned surface. Silk fibroin (SF) is a promising biomaterial due to its abundance, mechanical robustness, biocompatibility, and tunable biodegradability.25,26 Regenerated SF can be processed in both aqueous and organic solvents, making it a versatile option for the simple and inexpensive fabrication of biomaterials with various microstructures.24,27 In the present study, we investigated the effect of physical guidance cues on cell orientation by dissecting the response of the cell to patterned SF films over time, and we clarified the key roles of filopodia and lamellipodia in contact guidance. II. EXPERIMENT A. Preparation of regenerated SF
The regenerated SF solution was obtained as previously described.24 Briefly, Bombyx mori raw silk fibers (Huzhou, China) were degummed three times in 0.05 wt. % Na2CO3 at 98–100 C for 30 min, and then, the dried fibroin extract was dissolved in a solution of CaCl2/EtOH/H2O (mole ratio, 1:2:8) at 72 6 2 C for 1 h. The SF solution was obtained after dialysis (MWCO 9–12 kDa) for 4 day, and it was stored at 4 C after filtration. For electrospinning, the SF solution was lyophilized to produce porous sheets and stored at 4 C. B. Preparation of SF substrates
Polydimethylsiloxane (PDMS; Sylgard 184, Dow Corning) was cast on diffraction grating (Edmund Optics, Inc.; 600 lines/mm and 2.5 2.5 cm) surfaces. The PDMS substrates were peeled from the grating after solidification for 4 h at 65 C and then rinsed with ethanol and deionized water. The SF solution was cast on the patterned PDMS surface to generate micropatterned films. Flat SF films were produced by pouring the solution into polystyrene dishes. The thickness of the films was determined by the ratio between the SF solution and the mold area. Porous SF sheets were dissolved in 1,1,1,3,3,3-hexafluoroisopropanol (HFIP; Dupont Chemical, Co., USA) to generate a 10% (wt./v) solution. A constant flow rate of 1 ml/h was maintained using a syringe pump (KDS 100, KD Scientific), and the distance between the needle tip and the collector was 10 cm. The solution was drawn into fibers using a high voltage of 10 kV, and it was deposited on a collector made of two parallel steel strips to prepare the aligned fibers. The flat SF film was cut into disks with a 1.5-cm diameter and then the aligned fibers were carefully transferred onto the surface of the SF films to obtain a nanofiber-coated surface. All SF samples were immersed in 80% ethanol for 2 h to render the films waterinsoluble by inducing the formation of b-sheet secondary structure. The average diameter of the electrospun nanofibers was calculated by measuring the diameters of 100 different nanofibers in scanning electron microscopy (SEM) images. Biointerphases, Vol. 10, No. 1, March 2015
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C. Cell culture
The animal experiments were approved by Jiangsu Province according to the rules for the management of experimental animals [(2008) No. 26]. Bone marrow-derived mesenchymal stem cell (BMSC) were isolated as previously described.24 The cells were cultured in DMEM/F-12 (HyClone) supplemented with 10% fetal bovine serum (FBS, HyClone), 100 U/ml penicillin, and 100 lg/ml streptomycin (Invitrogen, Carlsbad, CA). SF films were cut to the size of the wells in a 24-well plate and sterilized for 15 min with 75% EtOH. The films were subsequently washed three times with sterile phosphate-buffered saline (PBS; 0.1 M, pH 7.4). A sterilized polystyrene tube with an outer diameter of 15.4 mm and an inner diameter 11.6 mm was placed into the wells to fix the film to the bottom of the culture plates. BMSCs were seeded on the SF films at 4 104 cells/cm2. D. SEM and atomic force microscopy observations
The substrate morphology was characterized by scanning electron microscopy (SEM; S-4800, Hitachi, Japan). The features of the microgrooved films were quantitatively evaluated using atomic force microscopy (AFM; Nanoscope V, Veeco, USA). The cell culture samples were rinsed three times in 0.1 M PBS and fixed with 2.5% glutaraldehyde at 4 C overnight, followed by three washes in PBS. The fixed samples were dehydrated in an ascending series of graded ethanol (50%, 70%, 90%, and 99.7%) for 5 min and then further dried using hexamethyldisiloxane (HMDS, SigmaAldrich) for 3 min. The dried samples were sputter coated with gold for 90 s prior to SEM observation. The polarization of small lamellipodia on patterned surfaces was analyzed by measuring the orientation angle of the lamellipodial rim, which has a minor included angle with pattern direction in SEM images, with 10 cells in each group, and the direction of grooves and nanofibers was defined as 0 . E. Cell staining and observation
After fixation in 4% paraformaldehyde-PBS for 15 min, the samples were permeabilized with 0.2% Triton X-100 for 5 min and blocked with 2% BSA-PBS for 30 min. The samples were incubated with 5 lg/ml FITC-phalloidin (Sigma-Aldrich) for 2 h at room temperature. After thoroughly rinsing, the nuclei were labeled with 5 lg/ml 40 ,6-diamidino-2-phenylindole (DAPI, Sigma) for 10 min, rinsed three times with PBS for 5 min each and observed using a confocal laser scanning microscope (CLSM; IX81/FV1000, Olympus, Japan). To investigate the elongation of nuclei, the nuclear shape index was calculated by measuring the aspect ratio of 30 different cells for each group (aspect ratio: major axis to minor axis of each nucleus). F. Cell viability assay
Cell proliferation was measured with a cell count kit-8 assay (CCK-8; Sigma-Aldrich). A 500-ll volume of fresh
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culture medium containing 50 ll of CCK-8 solution was added to each well containing the BMSC-seeded SF films, and the plates were kept in an incubator with 5% CO2 at 37 C for 3 h. The formazan derivative formed by cell metabolism is soluble in the culture medium and was detected by measuring the absorbance at 450 nm using a microplate reader (Bio-Tek Synergy HT, USA). The background absorbance measured from a sample containing the CCK-8 reagent in culture medium (without cells) was subtracted from all the samples. G. Statistical analysis
Statistical comparisons were performed using SPSS version 16.0 software (SPSS, Inc., Chicago, Illinois). The deductive statistics (t-test, analysis of variance) were conducted, and the data were presented as mean 6 standard deviation, and p < 0.05 was considered to be statistically significant. III. RESULTS A. Surface characteristic of SF substrates
As shown in Fig. 1, the unpatterned SF films presented a smooth surface [Fig. 1(a)], and the microgrooved pattern was successfully replicated from the PDMS molds [Fig. 1(b)]. The microgrooved pattern displayed a saw tooth shape with grooves of 1720 6 94 nm width and 142 6 15 nm depth [Fig. 1(c)]. The aligned SF nanofibers on the parallel electrode were easily transferred to the flat SF films. The nanofibers adhered to the film surface after aqueous alcohol treatment [Figs. 1(d) and 1(e)], and the average diameter of the nanofibers was 963 6 213 nm.
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B. Initial cell response to guidance cues
To characterize how the BMSCs interacted with the guidance cues of the substrates prior to spreading, the cell morphology was observed after 30 min of culture. As shown in Fig. 2, the cells anchored to the films and nanofibers through numerous protruding filopodia. On the flat surface, the cells randomly extended filopodia to anchor to the substrate [Figs. 2(a) and 2(d)]. Filopodia serve as “sensory organelles” that sense the mechanical and chemical environment of a cell.12,13 However, the cells also randomly protruded filopodia in multiple directions on the patterned film [Figs. 2(b) and 2(c)], indicating that the filopodia did not show directional extension prior to spreading. Although the cells predominantly exhibited a rounded shape, they protruded small lamellipodia along the direction of the microgrooved and nanofiber pattern [long arrows, Figs. 2(b) and 2(c)]. Filopodia can mature into different structures, such as become spines, dendrites, and lamellipodia,22–24,28–30 and the process of transformation from filopodia to lamellipodia has been observed in previous studies.22,24 Strikingly, the filopodia that extended along the direction of the patterns were switched into small lamellipodia [long arrows, Figs. 2(e) and 2(f)] and provided scaffolds for small lamellipodia formation [short arrows, Figs. 2(e) and 2(f)]. Although the extension direction of filopodia was random on the both flat and patterned SF films prior to spreading, the extension of small lamellipodia was oriented toward the direction of patterns. The extension direction of lamellipodia on flat surface was random, whereas the protruded small lamellipodia on both grooved and nanofiber-coated films exhibited oriented extension, which had the orientation angles
FIG. 1. SEM images of the surfaces of silk fibroin films with (a) flat, (b) microgrooved, and (d) and (e) nanofiber-coated topographies. (c) AFM image of the microgrooved film surface. Scale bars: (a) and (b) 50 lm, (d) 500 lm, and (e) 10 lm. Biointerphases, Vol. 10, No. 1, March 2015
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FIG. 2. SEM images showing the initial morphology of cells cultured on (a) flat, (b) microgrooved, and (c) nanofiber-coated films 30 min after plating. (d)–(f) Magnified images of the filopodia and lamellipodia shown in (a), (b), and (c), respectively. The short and long arrows indicate filopodia and lamellipodia, respectively. Scale bars: (a), (d), (e), and (f) 5 lm and (b) and (c) 10 lm.
of 5.3 6 4.3 and 15.4 6 6.4 , respectively. These results suggested that the filopodia transduced the substrate information to the cell nucleus and determined the direction of the lamellipodial protrusion. Filopodia cannot drive cell motility alone whereas lamellipodia can,30 and the directional advance of small lamellipodia can pull cell spreading in the direction of the pattern and thus trigger the initiation of contact guidance. C. Cellular response to microtopography over time
To visualize the course of cell spreading over time, the cell morphology was observed after culturing for 1, 2, and 4 h. After 1 h on an unpatterned film, small lamellipodia protruded in different directions from the sites of filopodial adhesion to form a stable anchor and to flat cell body [long arrows, Figs. 3(a) and 3(b)], indicating that the protrusion of lamellipodia was a prerequisite for BMSC spreading. The adhered filopodia served as the navigators and scaffolds for lamellipodial protrusion [short arrows, Fig. 3(b)]. On the patterned films, small lamellipodia matured into lamellipodia protrusion and directed cell elongation along the direction of the patterns [long arrows, Figs. 3(e) and 3(j)]. The front filopodia extended along the grooves and fibers to direct the advancement of the lamellipodia [short arrows, Figs. 3(f) and 3(k)]. Moreover, peripheral lamella formed around the cell body on the nanofiber-coated film, indicating that the cells had begun to undergo full spreading [Fig. 3(j)]. After 2 h, the peripheral lamella protruded from the cell body, resulting in a flattened spreading on both flat [Fig. 3(c)] and microgrooved films [Fig. 3(g)]. The cells that were cultured on the microgrooved film were elongated and aligned in the direction of the pattern [Fig. 3(g)], and the filopodia and lamellipodia at the leading edge extended in the direction of the groove [arrows, Biointerphases, Vol. 10, No. 1, March 2015
Fig. 3(g)]. By contrast, the cells cultured on the smooth film were mostly round, and the lamellipodial protrusions displayed random orientations [Fig. 3(c)]. Moreover, the cells cultured on the nanofiber-coated surface exhibited a distinct spreading and elongation compared to those cultured on the grooved surface [Fig. 3(l)], suggesting the SF nanofiber coating accelerated cell spreading on the SF film. After 4 h, the cells had fully spread [Figs. 3(d), 3(h), and 3(m)], and the lamellipodial protrusion extended along the bottom of the groove to drive cell migration in the direction of the pattern [Fig. 3(i)]. Furthermore, the cell morphology was observed by SEM and CLSM after 24 h. On the flat surfaces, the cells presented a polygonal shape and randomly protruded plasma membranes [Figs. 4(a) and 4(d)], whereas the cells cultured on patterned films presented a significantly elongated body shape and extended numerous membrane protrusions in the direction of the pattern [Figs. 4(b), 4(c), 4(e), and 4(f)]. Generally, the cell nuclei were spherical or ellipsoid, and it is possible that the nuclear shape is regulated by the substrate pattern.31 The nuclear elongation was quantitated by calculating the aspect ratio. The aspect ratio showed significant difference on various substrate surfaces (P < 0.05). The cell nuclei on flat film were found to be more spherical [Fig. 4(d)], which had lower aspect ratio (1.41 6 0.16). The cell nuclei became more elongated [Figs. 4(e) and 4(f)] and had higher aspect ratios on both micropatterned and nanofiber-coated surface (1.78 6 0.27 and 3.11 6 0.80, respectively). D. Cell proliferation
After 7 days of cell culture, the cells grown on SF substrates nearly covered the surface (Fig. 5). The cells on both the microgrooved surface and the aligned nanofibers were
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FIG. 3. Cell spreading after 1, 2, and 4 h of culture on (a)–(d) flat, (e)–(f) microgrooved, and (j)–(m) nanofiber-coated films. The images shown in (b), (f), (i), and (k) are magnified images of the regions indicated by boxes in (a), (e), (h), and (j), respectively. The short and long arrows indicate filopodia and lamellipodia, respectively. Scale bars: (a) and (k) 10 lm; (b), (c), (e), (g), and (i) 5 lm; (d), (h), and (m) 50 lm; (f) 3 lm; and (j) and (l) 20 lm.
FIG. 4. SEM images of BMSCs after 24 h of cultivation on (a) flat, (b) micropatterned, and (c) nanofiber-coated films. Fluorescent staining of BMSCs after 24 h cultivation on (d) flat, (e) micropatterned, and (f) nanofiber-coated films. Green indicates fluorescent actin staining, and blue indicates the nuclei stained with DAPI. Scale bars: (a) and (f) 100 lm and (b)–(e) 50 lm. Biointerphases, Vol. 10, No. 1, March 2015
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FIG. 5. SEM images of BMSCs after 7 days of cultivation on (a) flat, (b) microgrooved, and (c) nanofiber-coated films. Fluorescent staining of BMSCs after 7 days of cultivation on (d) flat, (e) micropatterned, and (f) nanofiber-coated films. Scale bars: 100 lm.
oriented along the direction of patterns [Figs. 5(e) and 5(f)]. By contrast, the cells cultured on the flat film grew in a circular pattern [Figs. 5(a) and 5(d)]. The results demonstrated that both the microgroove pattern and the nanofiber coating on SF films provided a suitable environment to control cell alignment. Furthermore, cell attachment and proliferation were measured after 7 days of culture on the SF substrates (Fig. 6). The optical density values obtained for cells cultured on the flat and microgrooved surfaces were not significantly different after 4 h or 7 days in culture, whereas the aligned nanofiber coating significantly promoted cell
FIG. 6. Viability of BMSCs on various silk fibroin films after 4 h and 7 days of cultivation (n ¼ 3, *p < 0.05, **p < 0.01, error bars indicate SD). Biointerphases, Vol. 10, No. 1, March 2015
attachment and proliferation (Fig. 6). This result was consistent with the SEM observations and suggested that nanofiber coating accelerated cell spreading (Fig. 3). IV. DISCUSSION The interaction between cells and the extracellular matrix (ECM) influences directional cell motility, which is critical for tissue development and wound repair.1 Therefore, cellmaterial interaction is a fundamental topic in the fields of tissue engineering and regenerative medicine. Understanding the mechanisms of topography sensing is also crucial for designing biomaterials that have the ability to promote healing and tissue regeneration. Conventionally, the nucleus is considered the site of integration of external physical and chemical signals.31,32 The shapes of cell nuclei have been shown to correlate with the pattern of the substrate,31 and nuclear shape and deformation in turn correlate with changes in gene expression.32 Filopodia act as cellular antennae that sense the surrounding microenvironment; they detect biophysical and biochemical stimuli and direct feedback to the nucleus to modulate cellular morphology and function.33 Dynamic lamellipodial protrusions provide traction to drive cell motility through actin polymerization and disassembly.16,17 Filopodia and lamellipodia generally coexist at the protruding front of migrating cells. Filopodia cannot drive cell motility without lamellipodia, but they do participate in cell guidance.30 Thus, filopodia and lamellipodia are intimate collaborators for cell motility. We cultured cells on a grooved pattern and aligned nanofibers to understand how they respond to topographical cues of SF-based biomaterials.
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Filopodia often form the first adhesive contact with the ECM, pathogens or adjacent cells, and they are believed to be involved in environment sensing and cell motility.12 However, filopodia protruded randomly on both flat and patterned SF films prior to spreading, suggesting that filopodia play a major role in the formation of initial attachments to anchor the cell onto the substrate surface. The dynamic activities of the lamellipodia drive cell spreading and migration on substrate surfaces,15–17 and the direction of lamellipodial protrusions is crucial to cell guidance.6 In cells cultured on a microgrooved surface, lamellipodia extended and retracted in the direction of the grooves to guide cell alignment.6 Actin-related protein 2/3 (Arp2/3) complex is a major actin nucleator of lamellipodia, cell guidance on microgrooved surface and endothelial cell layers could be disturbed after Arp2/3 complex inhibition.34,35 Furthermore, the direction of lamellipodial protrusions was impacted by geometric features of the adhesive islands,36 but the response mechanism of lamellipodia in sensing surface topography has not been determined. Filopodial adhesion plays important roles on lamellipodial adhesion sites during cell migration.17 Filopodia can provide the scaffold for lamellipodia nucleation, and they can mature into lamellipodia along their length.22,24 We found that after forming stable attachments, the filopodia served as scaffolds to guide small lamellipodial advance along their length in cells cultured on SF films [Figs. 3(a) and 3(b)], and the mature lamellipodia began to flatten the cell body on the substrate surface [Fig. 3(b)]. More importantly, small lamellipodia formed along the direction of the patterns [Figs. 2(b) and 2(c)], indicating that lamellipodial advance is controlled by the extension of exploratory filopodia. Exploratory filopodia guided lamellipodial polarization, and the directional lamellipodial extension dominated cell elongation in the direction of the grooves and nanofibers [Figs. 3(f) and 3(k)]. These results revealed the synergistic cooperation between filopodia and lamellipodia in cell guidance. Filopodia first served as sensors for path finding, and then they integrated and transduced the topographical cues to the nucleus to direct the cell response after stable anchoring. The lowest level of resistance is present along the direction of the grooves or fibers, the exploratory activity of the filopodia steered the directional advance of small lamellipodia along the direction of patterns to direct cell elongation. Therefore, filopodia-steered lamellipodial extension is an important mechanism in cell contact guidance. However, to further confirm the critical role filopodiaguided lamellipodia polarization on cell guidance, the mechanism of lamellipodia polarization should be further investigated in the future, including the change of actin filament structures during polar lamellipodia formation and the lamellipodial variation after filopodia inhibition. Surface patterning may be used to control material–cell interactions in SF-based implantable medical devices.37 Both the microgroove pattern and the aligned nanofibers were able to guide the cells. Nuclear shape was significantly impacted by microgrooves and nanofibers on SF films, and aligned nanofibers further increased the nuclear elongation Biointerphases, Vol. 10, No. 1, March 2015
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compared to groove pattern. Furthermore, the nanofiber coating significantly promoted cell spreading and proliferation. The ECM consists of complex fibrillar networks composed of fibrous proteins and glycosaminoglycans.38 ECM nanofibers are smaller than the cell itself and are an important part of the microenvironment that provides structural support to cells.39 On micropatterned islands, filopodia preferentially adhered to nanofibrillar island compared to flat surfaces, and the filopodia that initially extended quickly adhered to nanowires and then aligned with nanowires by pulling on them.19 On nanofiber-coated SF films, the protruding filopodia preferentially adhered to the nanofibers to form sites of stable attachment [Fig. 2(c)], and the small lamellipodia tightly wrapped around the nanofibers to drive cell elongation along the fiber axis [Fig. 2(f)]. The nanofibers provided better mechanical support than the microgrooves for the traction of filopodia and lamellipodia, and this may be a crucial factor for promoting cell attachment, spreading, and migration on a nanofiber-coated film. Moreover, the fabrication of aligned electrospun SF nanofibers is simpler and less expensive than the fabrication of a microgrooved pattern, thus offering an efficient approach for surface modification of biomaterials. V. CONCLUSIONS In this study, we investigated how guidance cues control cell elongation on patterned SF films. Filopodia supported and dominated lamellipodial extension, which is crucial for cell directionality. Exploratory filopodia sensed guidance cues and then guided lamellipodial polarization, and which ultimately resulted in the elongation of the cell along the direction of patterns. The results highlighted the important role of the diversity and plasticity of protrusion in directing cell contact guidance. Moreover, aligned nanofibers coating not only provided strong guidance cues, but also provided better mechanical support for the traction of filopodia and lamellipodia, promoting cell attachment, spreading, and migration, which is a useful approach for surface optimization of biomedical materials. These results reveal the critical roles of filopodia-steered lamellipodial extension in the control of cell motility and contact guidance in response to physical cues. ACKNOWLEDGMENTS This work was supported by the National Nature Science Foundation of China (31370968), Nature Science Foundation of Jiangsu Province (BK20131177), College Natural Science Research Project of Jiangsu Province (12KJA430003), Priority Academic Program Development of Jiangsu Higher Education Institutions and Jiangsu Province Ordinary Universities and Colleges Graduate Scientific and Innovation Plan (KYLX_1243). 1
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