A Facile Strategy to Generate Aligned Polymer Nanofibers: Effects on

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A Facile Strategy to Generate Aligned Polymer Nanofibers: Effects on Cell Adhesion Kui Wang, Liping Liu, Jun Xie, Lei Shen, Juan Tao, and Jintao Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b16057 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 28, 2017

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ACS Applied Materials & Interfaces

A Facile Strategy to Generate Aligned Polymer Nanofibers: Effects on Cell Adhesion Kui Wang†, Liping Liu† ,#, Jun Xie†, Lei Shen*,‡, Juan Tao§, Jintao Zhu*,†,# †

Key Laboratory of Materials Chemistry for Energy Conversion and Storage (HUST), Ministry

of Education, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology (HUST), Wuhan 430074, China #

‡

Shenzhen Research Institute of HUST, Shenzhen 51800, China School of Chemistry, Chemical Engineering and Life Science, Wuhan University of

Technology, Wuhan 430070, China §

Department of Dermatology, Affiliated Union Hospital, Tongji Medical College, HUST, Wuhan,

430022, China *Corresponding authors. E-mail: [email protected] (J. Z.); [email protected] (L. S.)

KEYWORDS: Dextran, Electrospinning, Nanofibers, Cell adhesion, Alignment

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ABSTRACT: Structure of polymer fiber membranes plays a vital role in controlling cell responses as applied to immobilize targets for specific cell interactions. Electrospinning is a simple and powerful method to prepare polymer fiber membranes with scales from nano- to micrometers. In this report, a facile yet versatile strategy has been developed for fabricating polymer nanofiber membranes with well-aligned structures by using a glass sheet between the needle and a static drum as the collector. Effects of solution concentration, polymer molecular weight, applied voltage and collection distance on the morphologies of the formed fibers were systematically studied. Adhesion of cells (e.g., mouse melanoma cells B16-F10 and fibroblast cells NIH-3T3) on the fiber membrane has been further investigated. Our results show that cell morphologies varied from elongated to spherical on the random fiber membrane when the pore area of membrane decreased. In contrast, on the membrane with aligned morphology, when decreasing the gap width of fiber membrane, cell is found to keep elongated state and spread along the alignment direction. This work provides a facile yet effective strategy to engineer surface structures of the fiber membranes for controlling cell adhesion.


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1. INTRODUCTION With highly porous and large surface, the fiber based materials find huge applications in tissue engineering and drug delivery system, etc.1-8 Various methods have been developed to fabricate nanofiber mats, such as electrospinning, self-assembly, phase separation, templating, etc.9 The self-assembly and phase separation methods could control the three dimensional (3D) pore arrangement. Yet, this procedure is complex and the fiber orientation is not well controlled. The templating method could control fiber orientation rather than dimension and arrangement, and also requires a sacrifice material. Electrospinning, as a simple and versatile method, is widely used to fabricate fibrous materials with nano- or micro- scaled dimensions from 1D to 3D, and the modification is easy to perform through chemical grafting of fibrous surface. Besides, the morphologies and diameters of fiber could be easily tuned by varying solution properties (e.g., polymer molecular weight and concentration) and electrospinning conditions.10-15 Therefore, electrospinning generated fiber materials are most widely used to simulate the biological structures and actions of natural extracellular matrix.16, 17 In general, chemical compositions, mechanical properties, patterns and pore areas of fiber membranes play dominant roles in these applications. Quality of membranes plays a crucial role in controlling cell responses. Structures and topologies of fiber membranes have been reported to strongly affect cells adhesion, spreading, differentiation and proliferation.18-22 Researchers have demonstated that fibroblasts could adhere and spread along with poly(methyl methacrylate) (PMMA) fiber axis with the fibrous diameter above 0.97 µm, and the fiber orientation could regulate cell behavior.23 Liu et al. found that osteoblasts have an improved phenotypic markers of differentiation on the patterned electrospun poly(lactic acid) fibers with diameter from 0.5 to 2.0 µm.24 It is obvious that the pattern and



diameter of electrospinning fibers could affect cell dynamics and bio-function, however, what is the role of pore area or gap width and the orientation of fiber membranes in cell adhesion and spreading still lack systematic researches. It has been demonstrated that electrospinning could control fiber arrangement and assembly. For example, some reports introduced an auxiliary electrode or an additional magnetic field between the needle and collector to control the fiber alignment.25-28 Besides, other works introduced wire drum, wheel edge or gap-like devices as collectors to prepare aligned fiber membranes by regulating external fields or a collector with complicated shape.29-34 However, despite these advances, there still exist many challenges for fabricating highly aligned electrospun fibers. Firstly, it is hard to harmonize degree of orientation and productivity of obtained fiber mats, and thickness for the highly aligned fibers collected through the above techniques is usually not enough. Secondly, up to now, aligned fibers are often collected between air gaps or on conductive surface, and it is still difficult to collect aligned fibers on substrates with varied properties (e.g., conductivity, hydrophilicity and transparency), limiting their further applications. Therefore, combination of existing methods and further development of fresh techniques are the two main directions for fabricating aligned fiber mats. In this paper, we demonstrated a facile yet effective strategy to control the polymer nanofibers orientation with the assistance of electrospinning technique. Electrospun fibers deposited on a glass sheet (2 cm × 2 cm) with well uniaxial aligned orientation are obtained, and thickness of the fiber mats could be tuned through regulating the collection time. Without any auxiliary electrodes, external magnetic field, or complicated modification of the conventional electcrospinning setup, we fixed a glass sheet between the needle and static drum as collector, which was demonstrated to successfully generate aligned nanofiber membranes. Furthermore, we


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investigated the influence of pore area or gap width and orientation of fiber membranes on cell adhesion and spreading. 2. EXPERIMENTAL SECTION 2.1. Materials: Dextran (Mw= 50 kDa (T50), Mw= 100 kDa (T100), Mw= 500 kDa (T500), Mw= 2000 kDa (T2000)) were purchased from Shanghai Ryon Biological Technology Co., Ltd., China. Magnesium chloride (MgCl2·6H2O, purity: ≥98%) were purchased from Sinopharm Chemical Reagent Co., Ltd., China. 50 wt% glutaraldehyde (reagent grade) were purchased from Adamas Reagent Co., Ltd., China. Roswell Park Memorial Institute-1640 medium (RPMI-1640), trypsin, fetal bovine serum (FBS), bovine serum albumin (BSA) and penicillin-streptomycin were purchased from Sigma-Aldrich. Phosphate buffer saline (PBS), 4% paraformaldehyde and 4, 6diamidino-2-phenylindole (DAPI) were purchased from Goodbio Technology Co., Ltd., China. Actin-Tracker Green was purchased from Beyotime Biotechnology Co., Ltd., China. Deionized water (Millipore Milli-Q grade) with resistivity of 18.0 MΩ was used in all the experiments. All the chemicals were used as received without further purification. 2.2. Preparation of Random, Aligned and Crossed Fibers: For randomly distributed fibers, a generally rotating cylinder drum was applied as collector, a stainless steel gauge-20 needle was chosen as the nozzle, and the flow rate was fixed at 0.15 mL/h. Influence of solution concentration (0.8-1.3 g/mL), polymer molecular weight (T100, T500, and T2000), applied voltage (11-16 kV) and collection distance (11.5-16.5 cm) were systematically investigated. All solutions were stirred for 24 h until a homogenous phase was obtained before electrospinning. For aligned and crossed fibers, with the help of a conventional electrospinning setup, we fixed a glass sheet (2 cm × 2 cm) between the needle and static drum as the collector. Schematic diagram for the experimental setup was shown in Figure 1a, the glass sheet was placed on the



edge of a nonconductive holder, and the cuboid holder (3 cm × 3 cm × 6 cm) was pasted on the drum surface with it’s long side perpendicular to the drum. T50 aqueous solution (1.1 g/mL) was selected for electrospinning with the applied voltage of 14 kV, and the collection distance was fixed at 13.5 cm. The crossed fibers could be easily obtained by rotating the received glass sheet with a designed angle (e.g., 45°, 90°) after having collected for a certain time. 2.3. Preparation of Crosslinked T50 Fiber Mats: All the polymer fiber mats were crosslinked using a previously reported method.35 Firstly, 0.03 g MgCl2·6H2O was mixed with the T50 aqueous solution through efficiently stirring at room temperature for 2 h. Then, 0.4 mL 50 wt% glutaraldehyde was added to the mixture. The mixed solution was stirred for 12 h until a homogenous phase was obtained. Secondly, electrospinning process was performed using this mixture, and fiber mats with various thickness were fabricated through changing the collection time. Finally, the electrospinning fiber mats were heated at 90 °C under vacuum for 24 h; then crosslinked fiber mats were obtained, which were found to maintain their original morphologies in water for at least 72 h. 2.4. Characterization of Fiber Morphology: Morphology of prepared dextran fibers were characterized through an inverted optical microscope (IX71, Olympus Optical Co., Ltd, Tokyo, Japan) and scanning electron microscope (SEM, Sirion 200) at a voltage at 10 kV. SEM samples were prepared though cutting an Al sheet with deposited fibers, and directly pasted it onto a SEM copper stage. Diameter distributions of the formed fibers were obtained from corresponding images by using IPWIN60 software, and more than 100 counts were randomly selected for each sample. 2.5. Mechanical Testing: Membrane specimens for mechanical testing were prepared by cutting an Al band covered with the electrospinning fiber mats. The film band was carefully removed


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from the Al sheet, and the final width, length and thickness scale of the rectangular film band was 1.0 cm, 5.0 cm and 50-100 µm, respectively. Thickness of the films was measured though a thickness gage, while conventional macro-tensile measurements were performed on an electromechanical tensile tester (HAAKE PolyDrive R600). All samples were fixed between two clamps with the distance of 2.0 cm. The tensile testing was conducted at a rate of 2 mm/min at room temperature. The entry point force was 0.05 N and the maximum force was set as 98 N, and the stress/strain curve and Young’s modulus were obtained after fitted by the HAAKE PolyDrive R600 software (SANS-Power Test). Meanwhile, a nano-indentor (Optics11 Piuma & Chiaro) was applied to test the mechanical property of an individual dextran fiber. With the help of an assistant microscope, this strategy could precisely control and move the indenter to the center of a single fiber prior to indentation testing, which ensured the testing is accurately and effectively. At last, the final Young’s modulus of the single fibers were obtained after being fitted by Hertz model. 2.6. Cell Adhesion: Cell adhesion and spreading experiments were performed in 24-well plate. The fiber coated glasses were transferred into the 24-well plate. Two types of adherent cells, mouse melanoma cells B16-F10 and fibroblast cells NIH-3T3 (Wuhan Boster Biological Engineering Co., Ltd) were applied. 1 × 105 cells were placed in each well under 0.5 mL cell medium (RPMI-1640 containing 10% fetal boving serum and 1% penicillin-streptomycin), which was cultured at 37 °C cell incubator with 5% CO2 for 24 h and 48 h. Optical microscope was used to characterize the cell morphology, and Adobe Photoshop CS4 was used to evaluate pore area or gap width of the films and cell areas. For fluorescence microscope characterization, cells on fiber mats-coated glass sheet were strained as follow. Firstly, the glass sheet was rinsed with PBS for three times to remove the non-



adherent cells. The adherent cells were immobilized with 100 µL of 4% paraformaldehyde for 5 min, followed by rinse with PBS. Then, the cells were soaked and permeabilized with PBS solution containing 0.1% Triton X-100 for 15 min at room temperature. Secondly, actin was stained with Actin-Tracker Green. The working solution was diluted for 100 folds by PBS solution containing 0.1% Triton X-100 and 3% BSA. The straining processes were carried out in a dark environment at room temperature for 30 min. After rinsing with PBS, the cells were strained again with 20 µL DAPI for 10 min, followed by rinsing with PBS. The stained cells were saved in dark environment at 4 °C for further fluorescence microscopy investigation. 3. RESULTS AND DISCUSSION 3.1. Preparation of Aligned and Crossed Fibers: Electrospinning is a facile yet effective method to prepare random polymer fibers. Generally, a grounded collector made from copper or aluminum was applied, and various shapes and configurations of fibers were obtained. However, there still exists a challenge in yielding aligned fibers with well-defined gaps or porosities. In this paper, we developed a facile yet effective route to prepare aligned fiber membranes without auxiliary electrodes or an additional magnetic field. Specifically, we set a small nonconductive glass sheet between the needle and a static drum as collector (Figure 1a) to collect the fibers before they arrived at the conductive drum. By this way, the obtained fibers demonstrated wellaligned structures as shown in Figure 1b. By rotating the glass sheet at a designed angle (e.g., 45° and 90°), the fiber membranes with crossed structure was obtained (Figure 1c and d). In addition, collection time demonstrates to affect the morphology and alignment of the formed fiber membrane during electrospinning experiments. As shown in Figure 2, the fibers exhibit excellent orientation, and all fibers displayed bright red signal under fluorescence microscopy investigation due to the staining of the polymer with 0.01 wt% rhdamine B. With the increase of


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collection time from 5 to 120 seconds, the gap width decreased from 134.06 ± 88.40 µm to 3.40 ± 2.24 µm, and relationship between gap width of the fibers and electrospinning time were shown in Figure S1 in the Supporting Information (SI). Generally, there are two main advantages of our method to generate aligned fibers. Firstly, the collector surface is parallel to the axial direction of fiber streams, triggering the full stretching and well alignment of the fibers on the collection surface. By this way, orientation of the deposition is precisely tuned. Secondly, nonlinear blending instability in the conventional electrospinning is the main reason for the formation of randomly distributed fibers.36 In our experiment, the collector is fixed between the drum and the jet, resulting in the decrease of the blending instability of fibers before its arrival of the target, triggering the collected fibers more smooth and stretchy. Therefore, this technique allows us to generate aligned and crossed fibers, and the gap width between fibers could be regulated through adjusting the collection time. 3.2. Influence of Experimental Parameters on Fiber Morphology: Characteristics of fiber membranes are highly dependent on the solution and the electrospinning conditions. Herein, we studied the influence of solution concentrations, polymer molecular weights, applied voltages and collection distances on electrospinning fibers. As shown in Figure 3a-f, the fibers are composed of spherical structure at low T50 concentrations of 0.8 g/mL and 0.9 g/mL. With the increasing of the polymer concentration, the number of polymer spheres decreased and the membrane contains no beads when the T50 concentration is higher than 1.1 g/mL. Diameter of fibers increased from 268.89 ± 56.94 nm to 1045.11 ± 166.89 nm with the increase of the T50 concentration (Figure 4a). Formation of smooth fibers and increase of the fiber diameters are due to the increase of mass throughput and solution viscosity,8,

35

which generates a larger

surface tension to decrease the division ability of electrospun droplets. Structure of fiber



membrane is also strongly affected by the polymer molecular weight. With other parameters as a constant, the fibers became much smoother and diameter of fiber increased by using higher molecular weight polymer (Figure S2). Statistics of the corresponding diameter were shown in Figure 4b. The reason for this can be ascribed to the increased solution viscosity and entanglements of dextran with higher molecular weight. In addition, fiber diameters also increased when increasing the applied voltage due to the increase of mass throughput by the increased applied voltage, as can be seen from the SEM images (Figure 3g-l) and the plots (Figure 4c). Besides, collection distance also affects the fiber diameter. For example, the fiber diameters increased from 756.72 ± 66.90 nm to 854.60 ± 59.68 nm by increasing the collection distances from 11.5 cm to 16.5 cm, as shown for the SEM images in Figure S3 and plots in Figure 4d. Clearly, effect of the collection distance on diameters was not as remarkable as the factors of concentration, molecular weight and applied voltage. 3.3. Mechanical Properties of Fiber Membranes: Mechanical properties of fibers were measured by a typical stress/strain testing and nano-indentation testing. Typical stress/strain testing results show that the modulus and elongation of original films was 61.16 ± 13.11 MPa and 9.43 ± 1.21% before crosslinking (Figure S4a). After crosslinking by glutaraldehyde, fibers could keep their morphology in water for at least 72 h, which ensures the successful performance of cell adhesion. Besides, after crosslinking, the modulus and elongation were changed to 78.34 ± 13.14 MPa and 6.62 ± 1.19% (Figure S4b), indicating that modulus improved with the sacrifice of toughness which agrees with the results in Ritchie’s report.37 To provide information about individual fiber, a nano-indentor was applied to test the mechanical property of individual crosslinked dextran fiber. Corresponding Young’s modulus fitting ranges from 5% to 80% by Hertz model, and Figure S4c displayed the force versus indentation depth curves. After fitting,


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the measured Young’s modulus ranged from 31.80 ± 30.56 MPa to 133.35 ± 41.90 MPa (Figure S4d). 3.4. Cell Adhesion and Spreading on Surface of T50 Fiber Mats: In cell experiment, crosslinking is believed to affect cell adhesion, and thus we just controlled the conditions unchanged in the crosslinking process and have no further repetition of the effect of this crosslinking on cell adhesion. We chose 1.1 g/mL T50 aqueous solution as the precursor to obtain fiber mats, and the crosslinking time was fixed at 24 h. The fiber membranes with different thickness and orientation were chosen as substrates to incubate two types of cells: mouse melanoma cell B16-F10 and fibroblast cell NIH-3T3. After culturing for 24 h and 48 h, the cells were immobilized on the substrates and stained with DAPI and actin-tracker green dyes. Figure 5a and Figure 5b displayed the fluorescence microscopy images of fibroblast cells NIH3T3 adhesion on fiber membrane with randomly patterned and aligned structures, respectively. Clearly, the fiber structures can significantly influence in the cell adhesion and spreading. For example, NIH-3T3 cell exhibits spherical morphology on the randomly patterned fiber membranes while spreading on aligned structures when the membranes was thick enough. The reason can be ascribed to the contact guidance between cell and the surface.38, 39 Random fiber membranes provided multiple adhesion sites while aligned fiber membranes guided cell adhesion and spreading along with the parallel direction. In general, stiffness of fibers have been demonstrated to affect cell behavior.40, 41 Due to the isotropic nature and softness of random T50 fiber mats, cells tended to less tension and change to round when the random fiber mats was thick enough. Thus, despite the influence of the stiffness, the arrangement of fiber mats played vital role in cell behaviors.



To look into the difference of cell behavior on random and aligned fiber membranes, we studied the cell adhesion and spreading on random mats with various pore areas and aligned mats with various gap widths systematically. Figure 6 showed the detailed statistics of random fiber membranes (e.g., pore area and contact angle) and adhesive cell area on corresponding surfaces. With the increase of electrospinning time from 0.5 min to 12.0 min, the average pore areas of fiber mats decreased from 30356 ± 39152 µm2 to 10 ± 5 µm2 (Figure 6a), and the water contact angle increased from 53 ± 4° to 77 ± 2° (Figure 6b). Interestingly, the adhesion cell areas decreased when decreasing the pore areas (Figure 6c and 6d). The cell area of B16-F10 decreased from 737 ± 315 µm2 (pore area: 30356 µm2) to 124 ± 36 µm2 (pore area: 10 µm2) after being cultured for 24 h and decreased from 741 ± 254 µm2 to 132 ± 37 µm2 after being cultured for 48 h (Figure 6c). Similarly, cell area of NIH-3T3 decreased from 463 ± 172 µm2 (pore area: 30356 µm2) to 121 ± 47 µm2 (pore area: 10 µm2) after being cultured for 24 h and decreased from 327 ± 130 µm2 to 105 ± 45 µm2 after being cultured for 48 h (Figure 6d). In short, on randomly patterned fibers, the cells exhibit spherical morphology on membranes with smaller pore areas and elongated structure on membranes with larger pore areas (Figure S5 and S6). In contrast, on the aligned fiber membranes, cells kept elongated along with the orientation of fibers at different gaps (Figure S7). Presumably, the reason can be ascribed to the different responses of glass and dextran surfaces to cell adhesion. It is well known that dextran displays a weak adhesion-cytoskeleton interaction while glass substrate is cell adhesive.42-44 On fiber membranes with larger pore area, the bare glass area between fibers provides a suitable environment for cell adhesion. Once the pore area decreases small enough, the cell could not penetrate into the membrane to contact the glass substrate, and the cell adhesion behavior mainly determined by the orientation of fibers and the contact guidance plays a key role in cell adhesion and spreading.


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Furthermore, we examined the influence of fiber morphology (with or without beads) and diameter (by increasing applied voltage) on cell behavior. In Figure 7, one can see that morphology and diameter slightly affect cell behavior comparing with orientation and pore area or gap width in our experiment. Spherical morphology of final adhered cell was mainly caused by the random orientation of fiber membranes. The structure of fiber membrane could effectively affect cell behavior and secretion of proinflammatory cytokines.45, 46 The cytokines are small molecule proteins that could regulate cell differentiation, immune function and physiological responses.47 Tumor necrosis factor alpha (TNF-a) could inhibit and kill tumor cells, and interleukin-6 (IL-6) correlated with the occurrence and development of tumor. Also, TNF-a and IL-6 could adjust cells function, and play a vital role in cells proliferation and differentiation. In this study, we investigated the influence of orientation and pore area or gap width of fiber membranes on the expression level of TNF-a and IL-6 by the enzyme-linked immuno sorbent assay (ELISA) method. Figure 8 showed the expression levels of both TNF-a and IL-6 after incubating fibroblast cells NIH-3T3 on the T50 fiber membranes for 24 h. Figure 8a and Figure 8b represented the expression level of TNF-a and IL-6 on the random fiber mats with various pore areas respectively, while Figure 8c and Figure 8d represented the expression levels of TNF-a and IL-6 on the aligned fiber mats with various gap widths respectively. Our results indicated that both pore area and gap width of fiber membrane could affect the expression level of TNF-a and IL-6, and there exist a maximum release value of both TNF-a and IL-6 on both random and aligned fiber membrane surface. In addition, there was no obvious difference of the expression levels between random and aligned fiber mats. In brief, the pore area or gap width of fiber membrane rather than fiber arrangement



could affect the secretion of proinflammatory cytokines, and it has an optimal pore area or gap width for the expression level of cytokines. 4. CONCLUSIONS In summary, we have developed a facile yet robust method for fabricating polymer fiber membranes with aligned structures. The effects of polymer solution properties (e.g., polymer molecular weight and concentration) and electrospinning conditions on the fiber diameters and structures have been systematically studied. Moreover, we compared the cell adhesion on fiber membranes with both aligned and randomly patterned structures. Our results show that the adhesive cells vary from elongated to spherical morphology on randomly patterned fibers and keep elongated on aligned fibers, which indicating that topological structure of fiber membrane significantly affected the cell behaviors. This work provides a new route to control the fiber orientation for electrospinning technique for tuning cell adhesion and spreading. ASSOCIATED CONTENT Supporting Information Available: The statistics of gap width between aligned fibers, fluorescence microscopy images of electrospinning fibers, characterization of mechanical properties of fiber membranes/single fiber, and optical microscopy images of cell adhesion and spreading on fiber mats surface. This material is available free of charge via the internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (J. Z.); [email protected] (L. S.). Notes


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The authors declare no competing financial interest. ACKNOWLEDGMENT This work was funded by the NSFC (51525302, 51473059 and 21404046), NSF of the Hubei Scientific Committee (2016CFA001) and Shenzhen Science and Technology Project (JCYJ20150630155150194). We also thank the HUST Analytical and Testing Center for allowing us to use its facilities.



Figures:

Figure 1. (a) Schematic diagram of the setup for electrospinning aligned fibers and (b-d) the fluorescence microscopy images of collected bilayer fibers by rotating the collector with 0°, 45°, and 90° after having collected for a period of time respectively. To obtain bright colored fibers and distinguish the individual fibers, the solutions were mixed with 0.01 wt% rhdamine B prior to electrospinning. The cartoons that inset in (b-d) show the aligned structures. The scale bar in (d) can be applied to (b) and (c).


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Figure 2. Fluorescence microscopy images of aligned fiber mats from various collection time: (a) 5 s, (b) 10 s, (c) 15 s, (d) 30 s, (e) 60 s and (f) 120 s. The fibers were labelled by 0.01 wt% rhdamine B to obtain bright color. The scale bar in (f) can be applied in the other images.



Figure 3. SEM images of electrospinning T50 fibers from different concentrations: (a) 0.8 g/mL, (b) 0.9 g/mL, (c) 1.0 g/mL, (d) 1.1 g/mL, (e) 1.2 g/mL and (f) 1.3 g/mL. SEM images of electrospinning T50 fibers from different applied voltages: (g) 11 kV, (h) 12 kV, (i) 13 kV, (j) 14 kV, (k) 15 kV and (l) 16 kV. The scale bars in the left image of each line can be applied to the other images in the same line.


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Figure 4. Average diameters of electrospinning dextran fibers: As the function of (a) solution concentration; (b) molecular weight; (c) applied electrostatic potential; (d) collection distance. T50 solution was used in (a, c, d) and T100, T500, T2000 solutions were used in (b).



Figure 5. Fluorescence microscopy images of fibroblast cells NIH-3T3 adhesion and spreading on (a) random and (b) uniaxially aligned fiber mats with enough thickness after being cultured for 24 h. Insets show the images with higher magnification.


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Figure 6. Average pore areas (a) and contact angles (b) of fiber mats obtained from different electrospinning time. Average cell areas of mouse melanoma cell B16-F10 (c) and fibroblast cell NIH-3T3 (d) after being cultured on fibrous mats with different pore areas. Black and red lines represented the culture time of 24 h and 48 h, respectively.



Figure 7. Optical microscopy (1st line) and fluorescence microscopy (2nd-5th lines) images of cell adhesion on fiber mats with different morphologies (some beads and no beads) and increased diameters (by increasing the applied voltage from 11 kV to 16 kV during electrospinning). T50 with the concentration of 0.8 g/mL was applied to prepare beaded fibers and 1.1 g/mL was applied to prepare the smooth fibers with different diameters. The scale bar in the last image can be applied to the other images.


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Figure 8. The expression levels of TNF-a and IL-6 after cultured fibroblast cells NIH-3T3 on the T50 fiber mats surface with different orientations and pore areas or gap widths: (a, b) represent the random fiber surfaces with various pore areas; (c, d) represent the aligned fiber surfaces with different space widths between fibers.



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A Facile Strategy to Generate Aligned Polymer Nanofibers: Effects on

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