Electrospun Fibrous Mats on Lithographically Micropatterned

Nov 15, 2012 - Salima Nedjari , Sandy Eap , Anne Hébraud , Corinne R. Wittmer , Nadia Benkirane-Jessel , Guy Schlatter. Macromolecular Bioscience 201...
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Electrospun Fibrous Mats on Lithographically Micropatterned Collectors to Control Cellular Behaviors Yaowen Liu,† Lei Zhang,‡ Huinan Li,† Shili Yan,† Junsheng Yu,‡ Jie Weng,† and Xiaohong Li*,† †

Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, P.R. China ‡ State Key Laboratory of Electronic Thin Films and Integrated Devices, School of Optoelectronic Information, University of Electronic Science and Technology of China, Chengdu 610054, P.R. China ABSTRACT: Spatial arrangement of multiple cell types plays a critical role in maintaining the viability of cells and functionality of tissues. Micropatterning has been used to fabricate scaffolds to modulate cell distribution, growth, and functions for reconstructing the anisotropy in native tissues. In the current study, a glass substrate patterned with an electrically conductive circuit was prepared by lithography as a collector for electrospinning. Densely packed fibers were deposited on the top of silver strips and patterned fibrous mats were obtained with distinct ridge and groove areas. Orthogonal alignment was shown for fibers in the ridge and groove areas, and the pattern feature and fiber alignment were well maintained in the ridge during incubation of cells with patterned fibrous mats. Sequential confocal laser scanning from the top of cell-loaded fibrous mats indicated that a larger number of cells were spread in the ridge than that in the groove areas, and cells penetrated into the fibrous mats in the ridge. Microscopic observation and immunofluorescent staining indicated that cells and collagen deposition appeared to have distinct patterns on the fibrous scaffold and aligned along the directionality of fibers with an elongated morphology. It is concluded that lithography can provide the design flexibility of collectors with micrometer-scale precision patterning, and cells can be confined to precise locations, sizes, and shapes by the use of micropatterned fibrous scaffolds without any adverse effect on the cell viability and function. The results suggest the potential of patterned electrospun fibrous mats to construct complex tissues of well organized multiple cell types and with spatially distributed extracellular matrices. lining of the intrahepatic biliary apparatus.3 Therefore, the fabrication of functional tissue relies considerably on the spatial orientation and distribution of scaffolds to guide cell growth and proliferation, to support cell−cell interactions, as well as to direct cell alignment in mimicking the tissue structure. To reconstruct the anisotropy in native tissues, micropatterning has been widely used to fabricate scaffolds with spatial arrangement of topical and chemical signals to modulate cell attachment, growth, differentiation, distribution, and functions.4 Topographical cues have been made on culture substrates to guide cell alignment through a variety of methods,5 including photolithography,6 embossing,7 and microcontact printing.8 Zhang et al. modified glass and titanium by a hydrophilic chitosan layer and then protein patterns with varying shapes and sizes were deposited onto the surfaces

1. INTRODUCTION Despite the success in thin layer tissues such as bioengineered skin, regeneration of large and more complex organs is met with more significant challenges. One of the most important issues is to mimic the complex organization of multiple cell types and complex extracellular matrices. As evidenced in vivo, the well-defined architectures in complex tissues not only provide a physical microenvironment for cells but also give tissues with optimal functional capabilities. For example, the native artery vessel is a layered tubular structure, conferred by aligned endothelial monolayers along the blood fluid direction on the inner wall, surrounded by smooth muscle layers perpendicular to the blood flow in the outer portion of vessels.1 Native myocardial tissue is three-dimensionally (3D) arranged as the stacking sheetlike tissues of aligned cardiomyocytes, and the complex tissue systematically organizes mechanical and electrical anisotropy for producing unique electrical propagation in vivo.2 In native liver, the proliferating endodermal cells give rise to interlacing cords of liver cells and the epithelial © 2012 American Chemical Society

Received: August 29, 2012 Revised: November 12, 2012 Published: November 15, 2012 17134

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ment in an organic solvent.18 However, it was not easy to bring this approach into practice, and the patterned fibrous mats lost many fiber characteristics. Zhang and Chang applied metal weave19 and metal protrusions20 to form patterned collectors, and the resulting fibrous meshes had topological structures similar to that of the template collector. Xie et al. used an array of stainless steel beads as the collector for electrospinning, and PCL fibrous mats were fabricated with arrayed microwells and controllable structural cues on the surface.21 However, the template collectors, constructed by weaving or engraving, indicated significant shortcomings for micrometer-scale precision patterning and complex patterns. Herein we present a simple, versatile method to selective deposition of electrospun fibers on a micropatterned collector prepared by lithography. It was aimed to provide design flexibility that could be utilized to assess the structure and functions of patterned fibrous mats. The viability, distribution, and alignment of fibroblasts and the deposition of extracellular matrices on patterned electrospun mats were clarified.

through a microcontact-printing process. Osteoblast-like cells preferentially adhered and grew on protein-patterned areas, and the cell morphology and distribution direction were dependent on the width and spaces of the protein patterns.9 However, the lack of porous structure, the use of nondegradable materials, and the two-dimensional (2D) feature might be problematic upon its wide application for tissue regeneration. To overcome these problems and maintain phenotypic functions of cells, engineers have explored various methods to fabricate 3D patterned scaffolds for tissues engineering.10 Zhang et al. utilized a microfluidic bioreactor for culturing high-density arrays of hepatocytes to mimic the physiological mass transport and nutrients supply of liver tissues, and hepatocytes sustained viability for over 1 week.11 Alternatively, layer-by-layer deposition of patterned 2D substrate in the zdirection was applied to control 3D arrangement of scaffolds, biomolecules and cells in microscale. Chen et al. cast a polymer solution onto patterned poly(dimethylsiloxane) matrix, and formed a solid polymer layer containing microstructures. A 3D scaffold was constructed by lamination of the patterned polymer layers, and the cell shape was found to be governed by multiple focal adhesion-sized islands.12 Although the sequential layer-by-layer approach provided a convenient method to 3D patterning, it generally suffered from the loss of fidelity in the z-direction during the iterative procedures.13 Another strategy to create 3D scaffolds is to provide independent control over the porous structure and the macroscopically patterned feature though combination of lithographic and pore formation techniques. Sarkar et al. prepared porous micropatterned polycaprolactone (PCL) scaffolds using a technique that integrated soft lithography, melt molding and particulate leaching processes. Diffusion of media through the scaffolds was 6-fold greater than through nonporous scaffolds, but the cell alignment was similar to that on micropatterned nonporous scaffolds, indicating no loss in cellular organization on micropatterned particulate-leached scaffolds.14 Bryant et al. used a sphere template to produce a highly uniform porous structure, and utilized a photomask and light initiating to create a patterned and porous hydrogel scaffold. 3D architectures can be patterned into porous hydrogels in one step to create a wide range of tissue engineering scaffolds that may be tailored for cell elongation, spreading, and fibrillar formation.15 Electrospun fibers have offered several advantages in mimicking the size scale of fibrous extracellular matrix (ECM), and providing a very high fraction of surface available to interact with cells. The porous feature possesses microscale interconnected pores, which are essential to transport oxygen and nutrient supplies for cell growth, and are beneficial in the adhesion, viability, proliferation and maturation or differentiation of different cell types.16 Up to now, few attempts have been employed to obtain electrospun mats with various patterned architectures through fiber ablation, selective photocross-linking and woven metal substrates. Wu et al. devised femtosecond ablation of electrospun PCL mats to yield welldefined 3D patterns with holes of an average diameter of 436 μm and a center-to-center spacing of 1000 μm.17 However, most materials could not maintain well under that high temperature produced by a femtosecond laser. Carlberg et al. incorporated a photoinitiator into electrospun polyurethane matrix, which was selectively cross-linked by standard photolithographic methods, and electrospun fibrous mats with spatially defined microstructures were obtained after develop-

2. EXPERIMENTAL SECTION 2.1. Materials. Poly(ethylene glycol)−poly(DL-lactide) (PELA, Mw = 42.3 kDa, Mw/Mn = 1.23) was prepared by bulk ring-opening polymerization of lactide/PEG using stannous chloride as initiator.22 The molecular weight and its distribution were determined by gel permeation chromatography (GPC, Waters 2695 and 2414, Milford, MA). Toluidine blue and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich Inc. (St. Louis, MO). Rabbit anti-mouse antibody of collagen I and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit secondary antibody were purchased from Biosynthesis Biotechnology Co., Ltd. (Beijing, China). All other chemicals and solvents were of reagent grade or better, and purchased from Chengdu Kelong Reagent Co. (Chengdu, China) unless otherwise indicated. 2.2. Fabrication of Micropatterned Collector. Figure 1 shows the schematic illustration of fabrication process of a glass template collector patterned with an electrically conductive circuit. The designed template contained several squares, each containing an array of parallel strips, which were drawn by tanner L-edit software and printed with high resolution on a photomask by E-beam mask lithography system (Mark 40, CHA Industries, Fremont, CA). An insulating glass substrate with dimensions of 15 × 15 cm2 was deposited with a silver layer by DC sputtering (Sunicoat 594L, Sunic, Korea), coated with a layer of photoresist (MicroChem Inc., Newton, MA), then covered with the photomask, and exposed by a lithography machine (Suss mircotec MA6, Germany). The exposed regions became soluble, and the glass substrate was rinsed to remove the photoresist, followed by etching away silver in the exposed area. After etching and removing the rest of the photoresist, the glass substrate was obtained with micropatterned silver circuit as a collector for electrospinning process. 2.3. Preparation of Patterned Fibrous Mats. Electrospun patterned fibrous mats were obtained as described previously with some modifications.23 Briefly, PELA solution in acetone was added in a 2 mL syringe, attached with a clinic-shaped metal capillary. The patterned collector was located about 15 cm from the capillary tip. The flow rate was controlled within 0.6 mL/h by a precision pump (Zhejiang University Medical Instrument Company, Hangzhou, China) to maintain a steady flow from the capillary outlet. The applied voltage was controlled within the range of 20 kV using a high voltage statitron (Tianjing High Voltage Power Supply Company, Tianjing, China). The fiber collections were vacuum-dried at room temperature for 2 days to completely remove any solvent residue prior to further use. 2.4. Characterization of Patterned Fibrous Mats. The morphology of patterned fibrous mats was examined by using a scanning electron microscope (SEM, FEI Quanta 200, The Nether17135

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scaffold before the addition of 1.0 mL of culture medium into each well. The cell-seeded scaffolds were replenished with fresh media every 2 days. Nonpatterned fibrous mats were prepared through deposition on a grounded plate-type collector and included in the cell study as control. 2.6. Cell Growth on Patterned Fibrous Mats. Cell viability and attachment were assayed with CCK-8 cell counting kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Briefly, after removal of culture medium at predetermined time intervals, cells were rinsed twice with phosphate buffer saline (PBS), moved to another 48-well TCP, and immersed into 400 μL of fresh culture medium in each well. Then 40 μL CCK-8 reagent was added into each well and incubated for 2 h according to the reagent instruction. An aliquot (150 μL) of incubated medium was pipetted into a 96-well TCP and the absorbance at 450 nm was measured for each well using a μQuant microplate spectrophotometer (Elx-800, Bio-Tek Instrument Inc., Winooski, VT). The cell attachment was examined 4 h after cell seeding, and the cell viability test was repeated on days 1, 4, and 7 after incubation. All experiments were performed with n = 6. 2.7. Cell Distribution on Patterned Fibrous Mats. The number of cells in the ridge and groove areas of patterned fibrous mats was quantified after DAPI staining.25 Briefly, the fibrous samples were extracted from the culture plate after 7 days of culture, washed twice with PBS gently, and fixed with 4% glutaraldehyde for 24 h at 4 °C. After incubation into 1.0 g/mL DAPI solution for 5 min, the stained fibrous samples were observed under a confocal laser scanning microscope (CLSM, Olympus FV1000S, Tokyo, Japan). The images were taken by z-stack scanning with step size of 20 μm, and a 3D image reconstruction was used to measure the density of cells in the ridge and groove areas of fibrous scaffolds. The cell density was evaluated with the total cell number in each microfeature, divided by the periodic area, and then averaged through 10 random fields of view per sample.25 SEM was used to observe cell morphologies on fibrous mats. Briefly, cell-loaded fibrous mats were dehydrated through a series of graded ethanol solutions and then freeze-dried. Dry constructs were sputter-coated with gold and observed by SEM as above. 2.8. ECM Secretion of Cells on Patterned Fibrous Mats. Collagen I secretion on patterned fibrous mats was determined by immunofluorescent staining. Briefly, cell-loaded fibrous scaffolds were rinsed three times in PBS, fixed with 4% paraformaldehyde and permeabilized with 0.1% (v/v) Triton X-100 in PBS for 15 min at room temperature. The samples were then washed three times in PBS and then blocked with 10% normal goat serum for 20 min at 37 °C. After PBS rinsing, the fibrous samples were incubated with rabbit antimouse antibody of collagen I at a dilution of 1:200 at 4 °C overnight. After washing with PBS three times, the samples were incubated with FITC-labeled goat anti-rabbit secondary antibody at a dilution of 1:500 for 30 min at room temperature. Then the samples were washed with PBS, and stained with DAPI for 5 min. After rinsing with distilled water, fibrous samples were observed by CLSM, and fluorescence signals of DAPI and FITC were merged at each sample by ImageJ software. 2.9. Statistical Analysis. The values were expressed as means ± standard deviation (SD). Whenever appropriate, two-tailed Student’s t test was used to discern the statistical difference between groups. A probability value (p) of less than 0.05 was considered to be statistically significant.

Figure 1. (a) Digital image of the photomask printed by E-beam mask lithography system. (b) Schematic illustration of the fabrication process of the patterned collector with silver circuit. (c) Digital image of the patterned collector, and inset shows the amplified images of a squares containing silver strips. (d) Digital image of fibrous mats after deposition of fibers on the patterned collector through ordinary electrospinning process. Bars represent 20 mm. (e) Amplified image of patterned electrospun mats deposited on patterned collectors with the ridge width of 200 μm and the groove width of 400 μm. lands) equipped with a field-emission gun and Robinson detector after being vacuum-coated with a thin layer of gold to minimize the charging effect. The thickness of fibrous mats was measured from a vertical section image taken with an optical microscope (Nikon Eclipse TS100, Japan) and processed by ImageJ software. The diameter, pore size, and alignment of fibers were determined manually from SEM images using ImageJ, and the specific methodology was referred to http://rsbweb.nih.gov/ij/plugins/index.html. The analysis of fiber diameter was evaluated at three randomly selected SEM images with a magnification of 3000, and at least 50 different sites from each image were randomly chosen and measured to generate an average value. The alignment of fibers indicated the percentage of fibers distributed from −10 to 10° respect to the total fibers counted. The pore size of the scaffolds was evaluated on the basis of void fraction as seen in top view of SEM images. An ImageJ script was written to properly convert gray scale images into a black and white format, with fibers and voids associated with black and white pixels, respectively, and to compute the void fraction in an automated fashion.24 2.5. Cell Seeding on Patterned Fibrous Mats. Swiss mouse embryo fibroblasts NIH3T3 (American Type Culture Collection, Rockville, MD) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Rockville, MD) supplemented with 10% heat inactivated fetal bovine serum (FBS, Gibco BRL, Grand Island, NY). The patterned fibrous mats were punched into squares with a length of 6 mm, attached to coverslips to fit into 48-well tissue culture plates (TCP), and sterilized by electron-beam irradiation using linear accelerator (PreciseTM, Elekta, Crawley, U.K.) with a total dose of 80 cGy. Then 500 μL of cell suspension containing 5000 cells was seeded onto the patterned fibrous mats presoaked with culture medium. The cell-seeded mats were incubated at 37 °C in a humidified atmosphere for 4 h to make cells diffuse into and adhere onto the

3. RESULTS AND DISCUSSION 3.1. Characterization of Micropatterned Collector. Generally, electrospun fibers are deposited on a conductive collector as a randomly oriented mat, due to their chaotic whipping nature.26 Thus, there have been many efforts to control the fiber architecture, for example, by aligning the fibers and/or depositing them in a selective area.27 One of the most simple and effective methods is to use a patterned electrode as the collector. However, it is still a challenge to achieve structurally accurate, arbitrarily shaped micropatterns of 17136

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Figure 2. (a) SEM images of fibrous mats deposited on patterned collectors with the ridge width of 50, 100, 200, and 400 μm and the groove width of 200 and 400 μm. Bars represent 500 μm. SEM images of fibrous mats (b) between the ridge and groove, (c) in the ridge, and (d) groove areas. (e) Optical image of a vertical section of the fibrous mats deposited on the patterned collector with the ridge width of 400 μm and the groove width of 400 μm. Inset shows ImageJ analysis results of SEM images of the fibrous mat.

forces drove fibers to move toward the silver strips of the patterned collector. When electrospun fibers were close to collector, Coulombic interactions between the opposite charges led to a preferential deposition of fibers on the silver circuit,29 resulting in a higher density on the silver strips than those areas between the strips. 3.2. Characterization of Patterned Fibrous Mats. Figure 2a summarizes SEM morphologies of fibrous mats deposited on the patterned collector containing silver strips of different width. All the fibrous mats presented distinct ridge and groove morphologies, and only the fibrous mats obtained from narrow sliver strips (50 μm) and space areas (200 μm) indicated a poorly patterned feature. The width of ridge and groove areas was close to that of silver strips and spaces between them, respectively. Figure 2b−d shows the typical images of fibrous mats between the ridge and groove, in the ridge and groove areas of patterned scaffolds, indicating fiber alignment in both areas. During the electrospinning process Coulombic interactions between the opposite charges induced fibers to arrange along the strip, resulting in a parallel alignment of fibers, as it was the lowest energy configuration for this type of system with highly charged fibers.28 Fibers deposited in the space between silver strips followed an orthogonal alignment to the ridges and had a lower density (Figure 2b). Considering that Coulombic interactions were inversely proportional to the square of the separation between charges, the two ends of a fiber closest to the electrodes should generate the strongest

electrospun fibrous mats. To investigate the cellular behaviors on fibrous mats with patterned strips, a mask containing lines with different width was designed in the current study (Figure 1a). After sliver deposition on a glass substrate, a photolithography process was explored to remove silver layer in the exposed area (Figure 1b). Figure 1c shows the digital image of the patterned collector with a sliver circuit on the substrate, indicating patterning features similar to the photomask. The collector contained squares with size of 6.0 × 6.0 mm2, which fitted the well size of 48-well TCP. In each square the width of silver strips and gaps between strips was designed to vary from 50 to 400 μm. As shown in Figure 1d, fibrous mats with patterned fiber densities were obtained after deposition of fibers on the patterned collector through ordinary electrospinning process. In contrast to randomly assembled electrospun mats, electrospun fibers on the patterned collector can form well organized topological structures as evident in Figure 1e. The majority of the fibers were deposited onto the conductive areas of the collector, and electrospun mats showed a similar topological structure and dimensions to the collector configuration (Figure 1c). It was indicated that there were two kinds of electric forces in the electrospinning process that could influence the arrangement of electrospun fibers, namely, the electrostatic force resulting from the electric field and the Coulombic interactions between the positive charges on the nanofibers and the negative charges on the collector.28 Compared with a plate collector, the structure of the electric field changed, and the electrostatic 17137

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During the electrospinning process, fibers tended to be separated from each other because of repulsive interactions between the residual charges on their surfaces,26 resulting in a highly porous structure of electrospun fibrous mats. The SEM photographs of porous mats were processed into bmp form, and the border of each pore in top layer was defined according to a gradient method. The top layer images were obtained, and each pore area within statistical range were calculated from the number of pixels in the picture.31 Figure 3b shows the quantitative analysis of the pore size of fibrous mats. The fibrous mats deposited in the ridge area indicated an average pore size of around 5.9 μm, independent of the width of sliver strips and gaps between the strips. Significantly larger pore sizes of around 7.2 μm were found in the groove areas (p < 0.05), due to fewer fibers deposited and stronger electrostatic repulsion between fibers deposited. Figure 3c summarizes the fiber alignment within fibrous mats of different patterned feature, indicating significantly higher fiber alignment in the groove than that in the ridge areas (p < 0.05). Li et al. suggested that electrospun fibers could be uniaxially aligned by introducing insulating gaps into conductive collectors.32 Unlike fibers directly deposited on the silver strips where they can be immediately discharged, the fibers deposited across the gap between the strips should remain highly charged. The electrostatic repulsion between the deposited and the depositing fibers enhanced the parallel alignment, which represented the lowest energy configuration of highly charged fibers.29 As shown in Figure 3c, the increase in the width of silver strips led to a better alignment of fibers in the ridge area. Meanwhile, the alignment of fibers in the groove area of 400 μm width was better than that of 200 μm. Thus it was indicated that the width of the silver strips and the gaps between them could influence the fiber alignment. 3.3. Cell Attachment and Proliferation on Patterned Fibrous Mats. The cell attachment and proliferation behaviors on patterned fibrous mats were investigated after up to 7 days of incubation compared with TCP and flat fibrous mat. As shown in Figure 4, a significantly lower cell attachment was found on fibrous mats than that on TCP (p < 0.05). This may be attributed to the hydrophobic surface of electrospun PELA fibers.33 The cell adhesion on a surface is considered to be

electrostatic force, which would stretch the fiber across the gap to have it positioned perpendicularly to the edge of electrode.29 To reveal the topography of patterned fibrous mats, SEM images were analyzed by ImageJ software. As shown in the inset of Figure 2e, the ridge area was darker than the groove, indicating a significantly different thickness between the ridge and groove areas. Figure 2e shows the optical image of a vertical section of the patterned fibrous mat. The height of ridge and groove areas was around 300 and 80 μm, respectively. As a result of the conductive silver circuit, fibers formed a densely packed structure on the top of silver strips and ultimately dispersed few fibers between the strips. The diameter, pore size, and alignment of fibers were determined manually from SEM images using ImageJ software. As shown in Figure 3a, the fiber size of the patterned mats was

Figure 3. (a) Diameter, (b) pore size, and (c) alignment of fibers in the ridge and groove areas of fibrous mats determined from SEM images using ImageJ software. The left panel depicts the manual determination profile of a fibrous mat. The right panel summarizes the results of fibrous mats deposited on patterned collectors with the ridge width of 50, 100, 200, and 400 μm and the groove width of 200 and 400 μm.

around 1 μm. A slightly smaller average diameter was found in the nonconductive areas than those deposited on the silver strips. This may be due to that fibers suffered more electrostatic stretching between the two conductive silver strips. When polymer liquid droplet ejected, fibers straightly moved to metal areas. In the nonconductive gap, the polymer liquid droplet was cleaved easily by Coulombic interactions,30 resulting in a decrease in the fiber size. As shown in Figure 3a, no significant difference was found in the size of fibers obtained form the patterned collector with different width of silver strips and gaps between them (p > 0.05).

Figure 4. Cell adhesion and proliferation of NIH3T3 on patterned fibrous mats with the ridge width of 50, 100, 200, and 400 μm and the groove width of 200 and 400 μm (P50/200 represents fibrous mats with the ridge width of 50 μm and the groove width of 200 μm), compared with flat fibrous mats and TCP. The cell attachment was examined 4 h after cell seeding, and the cell viability test was repeated on days 1, 4, and 7 after incubation; n = 6. 17138

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Figure 5. (a) CLSM images of DAPI stained cell-loaded patterned fibrous mats with the ridge width of 200 μm and the groove width of 200 μm after 5 days of incubation. Images were taken by z-stack scanning with step size of 20 μm from the top of a fibrous mat. Bars represent 200 μm. (b) Images reconstructed by side view and (c) z-stacking of the fluorescent images. (d) Binary image converted from reconstructed fluorescent images with a global threshold. Bright areas show the existence of cells. (e) Cell densities on the ridge and groove areas of patterned fibrous mats with the ridge width of 50, 100, 200, and 400 μm and the groove width of 200 and 400 μm.

of individual fibrous mat. Fewer cells existed on the surface of the fibrous mat in the groove (the fifth layer of Figure 5a), while clustered cells penetrated into and were distributed throughout the fibrous mat in the ridge area (from the first to fourth layer of Figure 5a). Side views of the fluorescence images were reconstructed with a global threshold generated through image processing algorithms in Matlab to show the vertical distribution of cells in the patterned fibrous mats.39 As shown in Figure 5b, most of the cells penetrated into the fibrous mat in the ridge areas. Compared with the microscopic observation of the cross section of fibrous mats (Figure 2e), the thickness of ridge and groove areas collapsed after 7 days of incubation, and the shrinking rates were different between the ridge and groove areas. The height of ridge areas decreased from around 300 to 120 μm, while there was a large decrease from around 80 to 20 μm in the groove. In order to quantify the cell numbers in the groove and ridge areas, a binary image was created from 3D fluorescence images as above. Figure 5c shows a typical image reconstructed by the z-stack 3D scanning of the patterned fibrous mat with the ridge width of 200 μm and groove width of 200 μm, indicating most of the cells were distributed in the ridge areas. As shown in Figure 5c, the intersection of this central axis with the ridge of the pattern fibrous mats defined the origin of the local coordinate system, (x, y), where x was the width of sample and y the range of groove and ridge areas. The cell body covered a square region of 30 × 10 pixels (0.6 × 0.2 mm2) was obtained for each patterned fibrous mat. Figure 5d shows a typical cell density map extracted from the fluorescence images. The cell nuclei with size of more than 4 pixels were counted and the nuclei areas of all cells throughout the image were exported

strongly influenced by the balance of hydrophilicity and hydrophobicity. Many studies have demonstrated that cells adhere, spread, and grow more easily on moderately hydrophilic substrates than on hydrophobic or very hydrophilic ones.34 In addition, the cell density on flat fibrous mats was slightly higher than that of patterned mats after 4 h incubation (Figure 4). This may be due to the isotropic and random structure of flat fibrous mats, which could provide more direction guide for cell attachment.35 As shown in Figure 4, the cell density on patterned and flat fibrous mats showed no significant difference after up to 4 days of incubation (p > 0.05). This may be ascribed to the fiber alignment and ordered multilayer structure of patterned fibrous mats, resulting in higher proliferation rate of cells than that of flat fibrous mats.36 However, the enhancement of cell growth was balanced with the geometrical confinement effects of the patterns, which may cause cells to approach the stationary growth phase earlier and led to a lower proliferation rate on patterned fibrous mats.37 As shown in Figure 4, after 7 days of culture the numbers of cells on patterned fibrous mats were slightly larger than those on the flat mats. This may be due to the aid of excreted collagen and geometry together resulting in a high cell proliferation after several days. Lee et al. cultured human fibroblasts on aligned polyurethane nanofiber, indicating significantly higher collagen secretion than randomly oriented scaffold after incubation for 7 days.38 3.4. Cell Distribution on Patterned Fibrous Mats. To better understand the 3D distribution of cells, the cells densities in the ridge and groove areas of patterned fibrous mats were quantified after DAPI staining. Figure 5a depicts z-stack scanning CLSM images with step size of 20 μm from the top 17139

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through ImageJ software. Quantitative analyses of cells within the ridge and groove areas were performed on fibrous mats with different patterning features. Figure 5e summarizes the cell distribution on patterned fibrous mats, indicating significantly lower cell densities in groove than those in the ridge areas. Cells were sensitive to the topography of the supporting surface, but the exact reasons for this observation were unclear. Yang et al. interpreted that the ridgelike regions acted as a robust framework in nestlike-patterned scaffolds, so the existence of ridge regions played an important role in maintaining relatively smaller shrinkage, which was favorable for cell infiltration, growth, and proliferation in tissue-engineering applications.40 During the electrospinning process, a stream of polymer solution was subjected to a high electric field resulting in the formation of nanodimensional fibers, which induced inner stress, high degree of alignment, and orientation of polymer chains within electrospun fibers. With the increased surrounding temperature, the polymer chains could rapidly relax to random coil state that caused the dimensional shrinkage of fibrous mat. Due to the formation of fiber bundles in the ridge area (Figure 2c), fibers were likely to have a beneficial effect on the structural integrity by maintaining the size and shape of the original ridge, without observable macroscopic shrinkage. However, few and highly aligned fibers were deposited in the groove areas of fibrous mats, resulting in a high shrinking rate. Cell attachment was influenced directly by the shrinking of pattern fibrous sheets, causing more compliant behavior of cells.35 Another reason was the cellular adhesive domains, which were mainly located at the crossing points of fibers, due to the larger surface areas and higher stability than other areas. It was indicated from the cell distribution behaviors that the cross-points between fibers may be important in increasing the initial attachment and spreading of cells.41 It was assumed that higher fiber density in the ridge areas had more crossing points between fibers, and cells recognized these points and started to spread on the scaffolds. Blakeney et al. confirmed that cells seeded on the cotton ball-like fibrous scaffold had higher growth rate and stronger infiltration of cells than sheet-like fibrous scaffold over a 7 day period.42 The morphology of cells on patterned fibrous mats was evaluated by SEM. Figure 6a shows that cells appeared to align along the directionality of fibers in ridge and groove areas with an elongated morphology. It can be noted that cell growth in the ridge areas linked up into a single stretch compared to the parallel isolated growth of cells in the groove. The cell attachment was covering several fibers and entangled by them in the ridge areas (Figure 6b). Numerous lamellipodia and filopodia of cells were anchored on fibers, and cell bodies elongated in a bipolar manner on patterned electrospun mats, which could be explained by the cell−matrix contact guidance by the prevailing orientation of aligned fibers.43 Cells only attached and spread on the superficial fibers in the groove areas (Figure 6c), while cells penetrated into the ridge of fibrous mats (Figure 6b). This was due to the initial shrinkage of fibrous mats and significant decrease in the pore size in the groove areas after cell incubation. Compared with the fiber morphology before cell incubation (Figure 2), the fibrous mats indicated a significant shrinkage in the groove areas after incubation with cells, while the ridge of fibrous mats was intertwined by cells without deformation (Figure 6d). The fibrous mats in the ridge were stable and pulled fibers in the groove areas contracting toward the ridge.

Figure 6. (a) SEM image of cell−loaded patterned fibrous mats with the ridge width of 200 μm and the groove width of 400 μm after 5 daysof incubation. (b) Magnified images of cell-loaded fibrous mats in the ridge and (c) groove areas. Arrows indicate the fiber alignment directions. (d) SEM image of the patterned fibrous mats after 5 days of incubation.

3.5. ECM Secretion on Patterned Fibrous Mats. Collagen is the main component of ECM secreted by fibroblasts, so its secretion is another parameter important to the properties of the seeded graft. Collagen I secretion on patterned fibrous mats was determined by immunofluorescent staining. As shown in Figure 7, the secretion of collagen I was found in the ridge areas of patterned fibrous mats, indicating that cells spread in ridge successfully sustained the cellular functionality. Meanwhile, the groove could not promote a favorable biological response, due to the lack of cell guidance, and then affected the cellular proliferation. It was indicated that scaffolds with unique patterns may have specific biological performance in tissue engineering.44 As shown in inset of Figure 7c, the collagen deposition was not random, but aligned in the parallel direction of fibers. It was in agreement with the results of cell distribution on the fibrous mats observed by SEM (Figure 6), which provided anisotropy to collagen deposition compared with the random distribution in flat fibrous mats (Figure 7b). These data showed that cells can be confined to precise locations, sizes, and shapes through the use of patterned fibrous scaffolds without any adverse effect on the cell viability and ECM secretion, and that the ECM secretion was highly organized and aligned along the cell distribution on the fibrous mats. These findings indicate the potential to coculture different types of cells on the patterned fibrous mats for evaluation of cell−cell and cell−scaffold interactions, and ultimately to guide and orient multiple types of cells into organized structures that closely mimic native tissues.

4. CONCLUSIONS Patterned fibrous mats with distinct ridge and groove areas were obtained through ordinary electrospinning process after deposition of fibers on the patterned collector with a sliver circuit on the substrate. Orthogonal alignment was shown for 17140

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Figure 7. (a) Immunofluorescent staining of collagen I secretion, counterstained by DAPI, on patterned fibrous mats with the ridge width of 50, 100, 200, and 400 μm and the groove width of 200 and 400 μm, and (b) on flat fibrous mats after 5 days of incubation. Red lines show the border of ridge and groove areas of patterned fibrous mats. Bars represent 100 μm. (c) Magnified image of immunofluorescent staining of collagen I secretion by cells on patterned fibrous mats.

fibers in the ridge and groove areas, and the pattern feature and fiber alignment were well maintained in the ridge during incubation cells with patterned fibrous mats. There was no significant difference in the cell density between on patterned and on flat fibrous mats. A larger number of cells were spread in the ridge than those in the groove areas, and cells penetrated into the fibrous mats in the ridge area. Cells and the ECM secretion appeared to align along the directionality of fibers with an elongated morphology. It is suggested that patterned electrospun fibrous mats are capable to guide cell spatial distribution within patterned areas, penetration into the fibrous scaffolds, and alignment along the fiber orientation, which provide the potential to construct complex tissues of well organized multiple cell types and spatially distributed ECM.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +8628-87634068. Fax: +8628-87634649. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (51073130 and 21274117), and National Scientific and Technical Supporting Programs (2012BAI17B06).

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