Fabrication of polycaprolactone electrospun fibers with different hierarchical structures mimicking collagen fibrils for tissue engineering scaffolds

Accepted Manuscript Title: Fabrication of Polycaprolactone Electrospun Fibers with Different Hierarchical Structures Mimicking Collagen Fibrils for Tissue Engineering Scaffolds Authors: Lin Jiang, Liwei Wang, Nathan Wang, Shaoqin Gong, Lixia Wang, Qian Li, Changyu Shen, Lih-Sheng Turng PII: DOI: Reference:

S0169-4332(17)32318-8 http://dx.doi.org/doi:10.1016/j.apsusc.2017.08.005 APSUSC 36842

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Received date: Revised date: Accepted date:

14-4-2017 25-7-2017 1-8-2017

Please cite this article as: Lin Jiang, Liwei Wang, Nathan Wang, Shaoqin Gong, Lixia Wang, Qian Li, Changyu Shen, Lih-Sheng Turng, Fabrication of Polycaprolactone Electrospun Fibers with Different Hierarchical Structures Mimicking Collagen Fibrils for Tissue Engineering Scaffolds, Applied Surface Sciencehttp://dx.doi.org/10.1016/j.apsusc.2017.08.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Fabrication of Polycaprolactone Electrospun Fibers with Different Hierarchical Structures Mimicking Collagen Fibrils for Tissue Engineering Scaffolds Lin Jiang a,b,c,d, Liwei Wang d, Nathan Wang d, Shaoqin Gong d, Lixia Wang a, Qian Li a*, Changyu Shena, and Lih-Sheng Turng c,d* a National Center for International Research of Micro-Nano Molding Technology, Zhengzhou University, Zhengzhou, China b School of Material Science and Engineering, Zhengzhou University, Zhengzhou, China c Department of Mechanical Engineering, University of Wisconsin–Madison, WI, USA d Wisconsin Institute for Discovery, University of Wisconsin–Madison, WI, USA

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* Corresponding author. Department of Mechanical Engineering, University of Wisconsin–Madison, WI 53706, USA. ** Corresponding author. E-mail addresses: [email protected] (Qian Li), [email protected] (Lih-Sheng Turng).

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Highlights:  Electrospun PCL nanofibers with a nanoporous surface or shish–kebab structure were prepared to mimic native collagen fibrils.  Two different methods were used to prepare PCL nanofibers with a shish–kebab structure and their effectiveness was compared.  The influence of three different electrospun fiber surface topographies on cell behavior was compared.

Abstract The ability to topographically mimic the surface features of collagen fibrils is an important step in the preparation of tissue engineering scaffolds. It is important to know which kinds of surface topographies of electrospun fibers are more favorable for cell growth. In this study, fibers with three kinds of hierarchical-structured surfaces were fabricated by electrospinning to mimic collagen fibrils. By combining thermally induced phase separation (TIPS) and non-solvent induced phase separation (NIPS), polycaprolactone (PCL) fibers with a porous surface were electrospun from PCL in a chloroform (CF)/dimethyl sulfoxide (DMSO) mixed solution. In addition, two additional types of fibrous membranes, with PCL fibers being the shish and decorated by PCL kebabs on the surface, were created by two different controlled homoepitaxic crystallization methods––the solution incubation method and the solvent evaporation method. It was found that the solvent evaporation method was more effective in forming kebabs and the primary optimal processing parameters were

identified. The presence of pores on the fiber surfaces contributed to a much larger surface area and a higher total volume of pores. To investigate the cellular response on such scaffolds, 3T3 fibroblast cell and human umbilical vein endothelial cell (HUVEC) assays were conducted and the results indicated that both of the nanotopographies on the surfaces of the scaffolds improved cell viability and proliferation. Furthermore, the porous surface was more beneficial for enhancing cellular responses, which suggests better biocompatibility and greater potential to mimic collagen fibrils for tissue engineering application, and especially as scaffolds for endothelial layers in blood vessels. 1. INTRODUCTION Collagen molecules are the main construction material of the extracellular matrix (ECM) [1-3]. They provide mechanical stability, elasticity, and strength to tissues. They also control cellular and biological processes [4-5]. Collagen molecules self-assemble into collagen fibrils with a characteristic staggered structure [3]. In addition, the staggered arrangement results in periodically alternating gaps and overlapping zones, also known as the D period [3]. In vivo, micro-/nano- scaled fibers are the basic structural building blocks for most connective tissues (e.g., blood vessels, tendons, nerves, etc.) [6-7]. In order to better mimic the properties of collagen fibrils, effective tissue engineering scaffolds should have the following characteristics: (1) appropriate mechanical properties to support the surrounding native tissue [6, 8]; (2) a fibrous structure with suitable sizes and porosities for allowing cell infiltration [9]; and (3) biomimetic surfaces with hierarchical nano-topographical features [10-11]. In light of these requirements, electrospinning as a fiber fabrication technique is one of the most promising methods for fabricating ECM- mimicking fibrous scaffolds [12-14]. Great progress has been made toward the fabrication of scaffolds by electrospinning that emulate the structures and properties of collagen fibrils. Hutmacher et al. created complex porous structures by melt electrospinning, which showed encouraging cell biocompatibility and migration [15-16]. Biomimicking and mechanically nonlinear articular cartilage scaffolds were also fabricated by combining a highly ordered melt electrospun network with hydrogels [6]. Xia et al. also successfully generated crimped features in electrospun nanofibers by ethanol treatment to mimic the morphology of collagen fibrils [17]. However, there are few systematic research studies focusing on emulating the hierarchical and nano-topographical features of collagen fibrils, which also play a vital role on cell behavior but have often been neglected during the fabrication process [18-20]. Surface nanotopography of electrospun fibers (i.e., the secondary nano-structures ‘inscribed’ onto individual fiber surface) can be obtained through the manipulation of electrospinning processing parameters or other post-processing steps [21-25]. Based on the previous studies, two main hierarchical structures on the surface of electrospun fibers can be obtained; namely, a nanoporous surface structure [26-32] and a nano-protruded structure [33-36]. Both of these hierarchical structures can be utilized as non-biological cues to mimic the surface topography of native collagen fibrils in

the extracellular matrix (ECM) due to their similarity to collagen fibrils. Zhang et al. found that the introduction of surface nanotopography in the form of elliptical nanopores onto the electrospun fibers allowed for enhancing the biomimetic properties of the fibrous substrate [31]. Feng et al. demonstrated that the combined fibrous and porous nanotopography of poly(L-lactic acid) fibers had a superior ability to promote primary hepatocyte culture as compared to smooth fibers [37]. Our group also introduced another nano-protruding shish–kebab structure onto electrospun poly(ε-caprolactone) fibers via controlled self-induced crystallization [35-36]. Specifically, the electrospun fibers acted as shishes, while the kebabs had lamellae crystals growing homoepitaxally on the shishes to yield the shish–kebab structure. The resultant fibrous scaffolds also showed enhanced cell biocompatibility. It is of great interest to compare the influence of these different surface topographies of electrospun fibers on cell behavior, which is important to better mimic the hierarchical nano-topographical features on collagen fibrils. However, to the best of our knowledge, no systematically comparative works have been reported. In this study, polycaprolactone (PCL), due to its superior mechanical properties and biocompatibility [38-39], was chosen to fabricate the different hierarchically structured fibers using the electrospinning technique. Nanoporous, shish– kebab-structured PCL fibers were prepared successfully. Surface properties of all fibrous scaffolds were characterized by scanning electron microscopy (SEM), water contact angle (WCA) tests, atomic force microscope (AFM), and Brunauer–Emmett– Teller (BET) surface area tests. For comparative studies on the cellular response of different hierarchical surface structured fibers, 3T3 mouse fibroblast cells were first used for preliminary cytocompatibility tests. Human umbilical vein endothelial cells (HUVECs) were then cultured to further investigate the endothelial cell response on smooth-surface PCL fibers, nanoporous PCL fibers, and nano-protruding shish– kebab-structured fibers. 2. MATERIALS AND METHODS 2.1 Materials Poly(ε-caprolactone) (PCL) (Mn = 80,000), chloroform (CF) (ACS reagent), N,N-dimethylformamide (DMF) (ACS reagent), acetic acid, and dimethyl sulfoxide (DMSO) were all purchased from Sigma–Aldrich (Milwaukee, WI, USA). The materials were used as received without further purification. 2.2 Electrospinning of Smooth-Surface PCL Fibers In preparation, a proper amount of PCL pellets were dissolved in a mixture of chloroform (CF) and N, N-dimethylformamide (DMF) (v/v=6:4) to achieve a 15% (W/V) solution. The prepared solution was then injected via a 10 ml syringe connected to a Teflon tube and an 18-gauge blunt needle. The flow rate was 0.5 ml/h controlled by a syringe pump, the applied voltage was 18 kV, the temperature was around 25 oC, and the humidity was around 30%. Sheets of grounded aluminum foil were used as the collectors for this experiment. The distance between the needle tip and the aluminum foil was 20 cm. The scaffolds were made to a thickness of 60–80

μm as measured by a caliper. 2.3 Electrospinning of Nanoporous PCL Fibers PCL pellets were dissolved in a binary solvent system comprised of chloroform with dimethyl sulfoxide (DMSO) at different ratios. The solvent system ratios were 9:1, 8:2, and 7:3 v/v. The solution concentration were kept at 12% (W/V) for different solvent system. After finishing preparing the solutions, all of the solutions were stirred at room temperature overnight to dissolve the PCL pellets. Then they were put separately into 10 ml standard syringes with 18- gauge blunt needles and used for the electrospinning process. The experimental conditions were kept the same for all solvent systems. The applied voltage was 18 kV, the flow rate was kept at 0.5 ml/h, and the distance from the needle to collector was 20 cm. The temperature and humidity at room temperature were around 25 oC and 30%, respectively. In order to obtain the best possible morphology, the concentration of the electrospinning solution was tuned according to the scanning electron microscopy (SEM) results. The thickness of the fibrous scaffolds was maintained at 60–80 μm as measured by a caliper. 2.4 Fabrication of Shish–Kebab-Structured Fibers by Self-Induced Crystallization 2.4.1 Homoepitaxy Crystallization by Solution Incubation A solution of 77% acetic acid/deionized (DI) water was used to prepare the PCL solution at 60 ºC for 2 hours. The concentrations of prepared PCL solutions were 0.5 w/v, 1% w/v, 1.5% w/v, and 2% w/v. The solution then rested at room temperature for 1 hour without stirring. PCL fiber meshes were incubated in the PCL/acetic acid/DI water solution at different concentrations over a range of incubation times. After incubation, the PCL fibrous scaffolds were washed in a 77% acetic acid/DI solution for various lengths of time at room temperature and then dried in a vacuum oven for 24 hours. 2.4.2 Homoepitaxy Crystallization by Solvent Evaporation Similar to the solution incubation method, 77% acetic acid/DI water was used to prepare a dilute PCL solution at 60 ºC and was rested at room temperature before it was used. The concentrations of prepared PCL solutions were also 0.5 w/v, 1% w/v, 1.5% w/v, and 2% w/v. The PCL nanofibers were collected by stainless steel washers. One drop (approximately 30 µl) of PCL acetic acid/DI water solution with different concentrations was slowly dropped on PCL fibrous scaffolds. Lastly, the fibers were dried at room temperature and then put in a vacuum oven to evaporate all remaining solvent for 24 hours to obtain the shish–kebab-structured PCL scaffolds. 2.5 Characterization Methods 2.5.1 Scanning Electron Microscopy (SEM) The morphologies of obtained fibrous scaffolds were analyzed by scanning electron microscopy (SEM; LEO GEMINI 1530) with an accelerating voltage of 10 kV. Before investigating, samples were coated with gold for 40 s. The fiber diameters

for all of the fibrous mats, the geometric information for the shish–kebab-structured fibers, and the pores sizes on the porous PCL fibers were all measured by Image-Pro Plus software from the SEM images. At least fifty fibers were measured from three SEM images to calculate the average geometric information. 2.5.2 Atomic Force Microscope (AFM) The surface topographies and morphologies of the kinds of fibers present were detected in tapping mode at room temperature by atomic force microscopy (AFM) (Keysight 7500, USA). Before AFM analysis, the samples were dried overnight in a vacuum oven. 2.5.3 Water Contact Angle (WCA) Tests Before water contact angle tests, the prepared scaffolds were placed in a vacuum oven at 30 oC for 3 days to ensure that all solvent was evaporated. Surface water contact angles (WCAs) of the fibrous scaffolds were measured using the sessile drop method at ambient temperature with a contact angle goniometer (OCA 15/20, Future Digital Scientific Corp., USA). The droplet size was fixed at 4 μL, and the surface contact angle was determined 10 s after the water droplet was deposited on the surface of the mats. Five measurements for each group were performed at different scaffold surface locations, and the average value was reported with standard deviation (±SD). 2.5.4 Brunauer–Emmett–Teller (BET) Surface Area Tests The specific surface area and the total pore volume of the fibrous scaffolds were measured at 77 K by nitrogen adsorption–desorption isotherms on a Micromeritics ASAP 2460 apparatus (USA). The specific surface area was derived from the adsorption data curve in relative pressure ranging from 0.07 to 0.25 according to the Brunauer–Emmett–Teller (BET) method. The total pore volume was obtained from the desorption branch of the data following the Barrett–Joyner–Halenda (BJH) method. 2.6 Biocompatibility Evaluations 2.6.1 3T3 Fibroblast Cell Culture and Seeding Swiss mouse NIH 3T3 fibroblast cells were used for cell culture studies on the prepared scaffolds. The cells were cultured in high-glucose DMEM (Invitrogen) supplemented with 10% fetal bovine serum (WiCell), 1% penicillin (Invitrogen), and 1% streptomycin (Invitrogen), and incubated at 5% CO2 and 37 oC. Cells were routinely subcultured at a 1:20 ratio before reaching ~80–90% confluence, and media was replaced every two days. Three groups of fibers were collected on sterilized stainless steel washers and placed on the bottom of a sterile standard 24-well plate. Before cell seeding, scaffolds were sterilized by ultraviolet (UV) sterilization for 30 minutes on each side. 3T3 cultures were dissociated for 5 min in trypsin-EDTA and then washed with 3T3 media. Cells were counted and seeded at a density of 20,000 cells/well onto membranes with washers resting in 24-well tissue culture-treated polystyrene plates (TCPs). The

culture plates with electrospun membranes were then placed in an incubator at 5% CO2 and 37 C. Media was replaced every two days and assays were performed at days 3, 5, and 10 to characterize cell viability and proliferation. All tests were done with at least three replicates. 2.6.2 Human Umbilical Vein Endothelial Cell (HUVEC) Culture Human umbilical vein endothelial cells (HUVEC, Lonza) were maintained in culture in RPMI-1640 (BioInd) supplemented with 10% fetal bovine serum (BioInd). Cells were routinely passaged with trypsin-EDTA at a 1:20 ratio every 4 to 6 days before reaching ~80–90% confluence, and media was replaced every two days. Prior to cell seeding, three groups of fibrous scaffolds were sterilized by UV sterilization for 30 min on each side and then placed in 24-well TCPs. The HUVECs were washed gently with phosphate-buffered saline (PBS) then treated with trypsin-EDTA for 5 min. Cells were then seeded at a density of 10,000 cells/well. Spent medium was aspirated and replaced with 1 ml of fresh medium daily for screening samples. 2.6.3 Cell Viability (Live/Dead Assay) The cell viability of the scaffolds was characterized using a Live/Dead Viability/Cytotoxicity Kit (Invitrogen) to determine how many cells were living and dead on days 3, 5, and 10 for 3T3 fibroblast cells and on day 3 and day 7 for HUVECs. This kit contained green fluorescent Calcein-AM to image the cytoplasm of living cells, and red fluorescent ethidium homodimer-1 (EthD-1) to image cell death by penetrating broken cellular membranes. The staining solution was prepared following the manufacturer’s instructions. Briefly, the medium was first removed from the scaffold and cells. Then PBS was used to gently wash the scaffold and cells. Following this, an appropriate amount of the staining solution was added directly to the scaffold with cells and incubated 30 min at room temperature. The cells were observed using a laser confocal microscope (LSCM, A1RsiTi-E, Nikon) and an inverted fluorescence microscope (Leica DMI3000 B). 2.6.4 Cell Proliferation (MTT Assay and CCK-8 assay) The number of cells living on days 3, 5, and 10 for 3T3 fibroblast cells was determined using an MTT assay (Promega). Five hundred µL of 500 µg/mL MTT dissolved in media were put onto five samples of each type. After incubating for 4 hours at room temperature, the media containing MTT was aspirated off and 200 µL of DMSO were added. The number of cells was calculated by measuring the absorbance at 560 nm in triplicate for each sample with a reference wavelength of 750 nm. The proliferation activity of HUVECs on the sample surfaces was investigated with a Cell Counting Kit-8 (CCK-8, DOJINDO) after 3 and 7 days of culture. The supernatant was removed and the samples were rinsed twice with PBS. Then, 200 μL of fresh media, which was consistent with the HUVEC’s seeding medium but contained 10% CCK-8 reagent, were added to the samples. After 2 hours of culture at 37 °C under 5% CO2, the absorbance at 450 nm was measured by a microplate reader

(Thermo Scientific). Quintuplicate samples were measured and averaged for each recorded result. 2.6.5 Cell Morphology/Adhesion (Cytoskeleton Assay) Cell morphology on days 3, 5, and 10 for 3T3 fibroblast cells was determined by Acti-stain™ 488 phalloidin (green F-actin) staining. Cells were gently rinsed with PBS three times and then fixed by incubating scaffolds in 4% paraformaldehyde in PBS for 15 minutes at room temperature. Following this, they were washed three times, again with PBS, and then permeabilized by incubating the scaffolds in 0.5% Triton-X in PBS for 10 minutes at room temperature. The cells were then washed with PBS and incubated in 0.3 µM Acti-stain™ 488 phalloidin with 4’, 6-diamidino-2phenylindole (DAPI) for 1 hour at room temperature. The cells were washed again with PBS and imaged using the same microscope used for the live/dead assay. The shape and cytoskeleton organization of the HUVECs were analyzed after 3 and 7 days of culture. For these assays, samples were fixed on ice with a 4% paraformaldehyde solution in PBS for 15 minutes. Cells were then gently rinsed with PBS three times and permeabilized with 0.5% Triton X-100 in PBS at room temperature for 10 minutes. Samples were rinsed three additional times with PBS and stained with biotium phalloidin CF™568 for the actin filament and 4’, 6-diamidino-2-phenylindole (DAPI) for the cell nuclei for 1 h at room temperature. Samples were then rinsed with PBS and imaged under a fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan). 2.6.6 Cell–Scaffold Interaction The interaction between HUVECs and the scaffold was determined by SEM at days 3 and 7. The samples were fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature and dehydrated by a series of ethanol dehydrations (30%, 50%, 60%, 70%, 90%, and 100% for 30 min each), then subsequently dried in a vacuum desiccator overnight before gold sputtering for SEM. 2.7 Statistical Analysis Data were expressed as the mean ± standard deviation (SD). Statistical analyses were performed using single-factor analysis of variance (ANOVA). All data were analyzed with SPSS software. 3. RESULTS AND DISCUSSION 3.1 Morphology of Electrospun PCL Fibers with a Smooth Surface PCL fibers were obtained by electrospinning a 15% (W/V) PCL/CF/DMF solution as discussed in the Materials and Methods section. Figure 1 (a) shows an SEM image of a random electrospun PCL fibrous scaffold that was smooth and beadless. Scaffolds were further characterized by measuring their fiber diameters. The average diameter of PCL fibers was 392 nm and the diameter of the fibers ranged from 190 to 866 nm, as illustrated in Figure 1 (b). The broad fiber diameter distribution may have been due to the low electrical conductivity of the solution.

3.2 Morphology of Electrospun PCL Fibers with a Porous Surface A plethora of methods has been adopted to prepare porous nanofibers. First, porous nanofibers can be made by selectively removing one component when electrospinning the composite or blended material. Many studies have been done using this mechanism [40]. Another method involves collecting porous polymer fibers directly by altering the humidity, which is generally believed to be in accordance with the “breath figure” or vapor-induced phase separation (VIPS) mechanism [41-42]. Non-solvent phase separation methods, which have been widely used to fabricate porous membranes [43-44], can also be used to prepare highly porous fibers. For example, Xia et al. reported that by immersing the collector in a bath of liquid nitrogen, porous polymer fibers could be obtained through electrospinning, followed by the removal of the solvent in a vacuum [43]. These methods require two steps to be effective, such as using water extraction or thermal annealing or a special additional device such as a humidifier or liquid nitrogen bath. Besides these methods, porous structured fibers can also be made using a highly volatile solvent that can remove heat during electrospinning, after which thermally induced phase separation (TIPS) can occur [37, 45-46]. However, not all of the polymers can achieve a porous surface using only a volatile solvent in electrospinning. A different approach combining a highly volatile solvent and a high boiling point non-solvent may be more effective in developing porous micro/nanofibers. Therefore, based on previous reports, thermally induced phase separation (TIPS) and non-solvent phase separation (NIPS) methods were combined in this study to electrospin PCL to obtain a porous surface. A solution of 12%w/v PCL was dissolved into a solvent made from three different ratios (9:1, 8:2, and 7:3) of CF and DMSO. The ratio of these two solvents was the only factor that varied during this experiment. The surface morphology of these scaffolds was analyzed using a scanning electron microscope (SEM). SEM micrographs showed that the solvent ratio of CF to DMSO greatly affected the fiber surface morphology and its size distribution. By changing the CF: DMSO ratio, the surface morphology could display a porous, eroded, or rough surface. At a ratio of 9:1 CF:DMSO, the fibers had small elliptical pores and large irregular pores on their surface, as shown in Figure 2 (a). By changing the blend ratio to 8:2 CF:DMSO, the surface roughness and microtexture could also be observed (Figure 2 (b)), but the structures were shallower and no distinct pores could be seen on the surface compared to those with fibers made from the 9:1 CF:DMSO solvent solution (Figure 2 (c)). These surface features disappeared when the fibers were spun from the 7:3 CF:DMSO solution, thereby leaving a smooth surface. Similar to others’ findings [26-29], the solvent vapor pressure could be changed by substituting CF with DMSO. Furthermore, the porous structure decreased as the volatility of the mixed solvent systems decreased [42]. The fiber diameters of all of the combinations were calculated and were in the range of 1599 ± 505 nm, 941 ± 361 nm, and 557 ± 181 nm for CF:DMSO ratios of 9:1, 8:2, and 7:3, respectively. The decrease in fiber diameter from 1599 nm to 557 nm accompanied the increase in the DMSO content in the electrospinning solution

from 10% to 30% v/v. This can be attributed to the increase in conductivity of the solution; a tendency also seen in others’ work [29]. Based on our experimental results, beadless fibers can be obtained at any solvent ratio, while an increased ratio between good and poor solvents, up to a certain limit, can facilitate pore formation. However, this is different from Qi’s findings [64]. In their research, they used a ternary system of non-solvent/solvent/poly(L-lactic acid) to fabricate porous fibers. They found high porosity electrospun fibers could be achieved by decreasing the ratio of non-solvent to solvent. This contrary tendency may be attributable to the high solution concentration we used (12% compared to 8%), since a high concentration normally induces a high viscosity which hinders polymer motion and pore formation. This observation is also in agreement with Georgiadou’s work [29]. Figure 2 (d) shows a schematic mechanism of porous structure formation on PCL fibers via electrospinning [28, 45-46]. When the solution just comes out of the spinning tip, the solution is homogenous and the polymer chains distribute in the fibers uniformly. During the slow acceleration process, the good solvent CF—which is a highly volatile solvent—evaporates quickly and causes a lower temperature on the fiber [27, 47]. On the one hand, this phenomenon induces phase separation. On the other hand, the water vapor begins to condense on the surface of the fibers [41-42, 48-49]. Meanwhile, while decreasing good solvent (i.e., CF) in the fibers, DMSO—which is a less volatile and a bad solvent for PCL—also starts to induce phase separation [50-51]. Due to the phase separations, there are concurrent polymer-rich phases and polymer-lean phases on the fibers. The fibers undergo a further elongation and the diameter decreases in the rapid acceleration process. After drying and solidification, there are two kinds of pores shown on the fiber’s surface (Figure 2 (a)); namely, elliptical pores and large irregular pores. Cell assays will be conducted on this interesting porous structure of the decorated scaffolds to test biocompatibility. 3.3 Shish–Kebab Structure of Electrospun PCL Fibers Induced by Different Methods The solvents selected to induce the shish–kebab structures on the PCL fibrous scaffolds should dissolve the deposited PCL material but not influence the existing PCL fibers. Based on our preliminary study [35], a binary solvent system of 77% acetic acid/DI water was able to form a kebab structure because it was a non-solvent for PCL at room temperature but could still dissolve PCL at 50 oC. Before preparing the shish–kebab structure, the dilute PCL/acetic acid/DI water solution should be rested for more than 1 hour to make sure it is cool enough. 3.3.1 Solution Incubation Method Mainly two methods of fabricating kebab structures on PCL fibers have been used. The first method, the solution incubation method, involves immersing PCL fiber meshes into a cooled dilute PCL solution and then using the solvent to wash the sample several times. The length of the wash times, concentration of the dilute PCL

solution, and immersion times are the main parameters for forming kebabs on PCL fibers. In this study, the immersion times were kept at five minutes for all experiments while the wash times and concentrations of the dilute PCL solution varied. The wash process and concentration of the incubation solution are important parameters for forming the kebab structure on PCL fibers based on our experiments. Different lengths of wash times with concentrations of 0.5% w/v and 1.0% w/v were conducted and the resulting morphologies are shown in Figure 3. Fibers melted together (Figures 3 (a) and (d)) if the samples were not washed. This was because the fibers remained covered by the solution after being took out of the dilute solution. After drying, the excess polymer deposited on the fibers, causing them to appear melted together. However, the morphologies (Figures 3 (c) and (f)) also did not look good when the samples were washed for a longer period of time (10 minutes). This may have been because the polymer chains did not assemble perfectly during the five minute immersion time (the incubation period) and the kebab structures were destroyed by the excessive wash time. Herein, five minutes of wash time seemed appropriate for leaching out the excess polymer adhered to the fibers while not destroying the kebabs, as can be seen in Figures 3 (b) and (e). In all of the following experiments, the wash time was kept the same (5 minutes). In addition to the wash time, the concentration of the dilute PCL solution was also a main parameter for forming kebabs on the PCL fibers. When the concentration was 0.5%, the kebab structure was hardly recognizable (Figure 4 (a) and Figures 3 (a)–(c)). This was because very few PCL chains surrounded the PCL fibers when the concentration was too low; hence, there was little chance for attachment to the fibers. In Figure 4 (b), tiny, isolated white spots (the kebab size was around 46.8 nm) could be seen on the surface of the PCL fibers. When the concentration was increased to 1.5%, the structure became larger and the average kebab size was 95.8 nm, which can be seen in Figure 4 (c). The size/degree of the kebabs looked similar when the concentration was increased to 2% (the average kebab size was 98.6 nm), but the fibers began melting together as can be seen in the circles in Figure 4 (d). This may have been due to the high concentration used and the PCL chains started aggregating together in some areas. 3.3.2 Solvent Evaporation Method The solvent evaporation method is another method that induces the kebab structure by solvent evaporation, which involves drop casting the cooled PCL solution on the fibers and then drying the sample completely. In this study, the volume of the drop cast PCL solution was kept the same at all concentrations. Similar to the previous solution incubation method, 0.5%, 1%, 1.5%, and 2% of PCL concentrations were tested to form the “shish–kebab” structure. When the concentration was too low, the kebab structure did not form, or only small kebabs showed on the surface of the fibers, as can be seen in Figures 5 (a) and (b). Increasing the concentration to 1.5% led to the formation of uniform kebabs surrounding the PCL fibers (Figure 5 (c)) and the average kebab size was larger (156.7 nm). Fibers gathered together when the concentration rose to 2% (Figure 5 (d)); this was likely because the concentration was

too high and the excess PCL chains aggregated together. Comparing Figure 4 (c) with Figure 5 (c), the morphologies produced using the solvent evaporation method were much better than those formed by the solution incubation method. This was because the fibers had to be washed with an acetic acid/DI water solvent quickly in the solution incubation method. However, with the solvent evaporation method, the fibers did not have to be washed and there was sufficient time for the polymer chains to grow on the fibers after drop casting the solution on the fibers. Figure 6 shows the schematic mechanism of the kebab structure formation on the PCL fibers that acted as shish [34-35]. First, the typical smooth PCL fibers were spun by electrospinning. Then the fibers were immersed in the dilute PCL acetic acid/DI water solution or the solution was drop casted onto the fibers. The PCL chains were homogeneous and existed around the fibers at the very beginning. Then the PCL chains began to adhere on the fibers due to hydrophobic–lipophilic interactions. As time went by, PCL chains started to self-assemble by homoepitaxic crystallization [33-35, 52-53]. Finally, the polymer chains aggregated large enough and the kebab decorated fibers could be obtained successfully. 3.4 Surface Properties and BET Surface Area Analysis of Prepared Scaffolds Based on the experimental results, three different structured fibrous scaffolds were fabricated via electrospinning; namely, normal electrospun fibers with smooth surfaces (labeled as PCL), nanoporous fibers electrospun from a PCL/CF:DMSO (9:1) solution (labeled as PCL-PO), and shish–kebab-structured fibers made using the solvent evaporation method (the concentration of the drop cast PCL solution was 1.5% w/v) ( labeled as PCL-SK). AFM analysis was performed to examine the surface topography of the three different hierarchical structured surfaces. Figure 7 shows the representative AFM topographic images corresponding to three samples prepared with different surface nanotopographies and morphologies. Similar to the SEM images, the surfaces of the electrospun PCL fibers were smooth (Figure 7 (a)) and there were no hierarchical structures on them. It can be seen from Figure 7 (b) that there were pores on the surface of the resultant PCL fibers. The pores were shallow and no internal porous structure existed on the PCL fibers. In addition, there were obvious protrusions on the PCL-SK fibers (Figure 7 (c)). This further suggests that the PCL-PO and PCL-SK fibers can be regarded as structurally hierarchical. The hydrophilicity of a polymer scaffold plays a major role in the protein absorption and interaction between the cells and the polymer matrix [11, 31, 54]. To evaluate the influence of hierarchical structures on the surface wettability of the electrospun scaffolds, a water contact angle (WCA) test was performed. As shown in Figure 7 (d), all three kinds of structured scaffolds were highly hydrophobic. The water contact angle for smooth PCL fibers was about 132.9o. Porous structured fibers did not show much change in the water contact angle (132.7o). However, this was not consistent with the findings of Venugopal et al. [26]. In their research, they used six different kinds of binary solvents to electrospin PCL with porous surfaces and all of

them were highly hydrophobic, except for the scaffold prepared from CF/DMSO (the same binary solvent used in this study), which showed high hydrophilicity. This discrepancy can mainly be attributed to the shallower pores formed on our fiber surfaces which can be seen from the AFM images (Figure 7 (b)). Finally, the water contact angles of the kebab-structured scaffolds were slightly higher (around 137.1o) than the other two scaffolds, due to the obviously increased roughness of the shish– kebab structures. Since all of our scaffolds were hydrophobic, a more meaningful comparison would be to study the influence of the surface topography alone on the cellular response. The nitrogen adsorption–desorption isotherms of the PCL fibrous scaffolds prepared with different surface nanotopographies were carried out to further investigate their surface structure, as shown in Figure 7 (e). It can be seen that all of the scaffolds have isotherms of type II from the Brunauer–Deming–Deming–Teller (BDDT) classification [55]. The Brunauer–Emmett–Teller (BET) specific surface area and Barrett–Joyner–Halenda (BJH) adsorption cumulative total pore volume of the three kinds of scaffolds are shown in Figure 7 (f). It can be observed that the PCL-PO mats showed the highest specific surface area of 6.07 m2/g with a total pore volume of 0.0213 cm3/g, which was much higher than the other two types of fibrous membranes. In this sense, PCL-PO fibers could be very attractive in tissue scaffolding application settings where the effective interfacial area of a supportive substrate is crucial [31]. 3.5 In Vitro Cell Culture Evaluation The biocompatibility of scaffolds plays an important role in inducing tissue regeneration. To explore the interactions between cells and fibrous scaffolds with different surface hierarchical structures, three different structured fibrous scaffolds were fabricated via electrospinning. One of them was comprised of normal electrospun fibers with smooth surfaces. The second one was composed of nanoporous fibers electrospun from a PCL/CF:DMSO (9:1) solution. The last one consisted of shish–kebab-structured fibers made using the solvent evaporation method (the concentration of the drop cast PCL solution was 1.5% w/v). Here, Swiss mouse NIH 3T3 fibroblast cells and HUVECs were chosen as an in vitro model for studying the effects of these different hierarchical structures on cell viability, morphology, and proliferation over the culture period. 3.5.1 Culture of 3T3 Fibroblast Cells on the Scaffolds Live/dead assays were carried out on scaffolds after 3, 5, and 10 days of in vitro cell culture. The results can be seen in Figure 8. After 3 days of cell culture, cells survived very well on all three scaffolds (green fluorescence indicates live cells) and few dead cells were seen on the scaffolds (red fluorescence indicates dead cells), especially on the fibrous scaffolds with a porous surface. More cells flourished after 5 days of cell culture than after 3 days of cell culture. In addition, cells spread out on the scaffolds, which demonstrated that they proliferated and differentiated quickly after 5 days of culture. After 10 days of in vitro cell culture, cells on the scaffolds showed a flourishing living state and single cells could not be differentiated from the

pictures, which indicated good biocompatibility for all of the scaffolds. The quantitative cell viability and proliferation results are shown in Figure 9 (a) and (b). The cell viability on day 3 was 84% for normal PCL fibers, 92% for porous PCL fibers, and 88% for shish–kebab fibers. A slight increase in cell viability of 92%, 97%, and 93% was seen on day 5 for PCL, PCL-PO, and PCL-SK fibers, respectively. Due to very high cell confluence, we were unable to see single cells, thus we were unable to analyze quantitative cell viability on day 10. MTT assays were used to determine the number of living cells on days 3, 5, and 10, as shown in Figure 9 (b). It can be seen that the largest number of cells were on porous-structured fibrous scaffolds on all three days cultured; with the shish–kebab-structured fiber scaffolds coming in second. These data indicated that PCL fibers with hierarchical structures had higher cell viability and cell proliferation, no matter if they had pores or a shish– kebab structure, as compared to normal fibrous scaffolds. Cell morphology and the organization of actin structures on PCL fibers with different surfaces were compared by actin staining, as shown in Figure 8. After 3 days of culture, cells grown on smooth PCL scaffolds displayed a spherical shape, which is a non-spreading and non-biocompatible morphology for fibroblasts. The desirable “spreading” fibroblastic morphologies were observed on both the porous- and shish– kebab-structured fibrous scaffolds. Compared to shish–kebab- structured fibrous scaffolds, cells were more spread out when grown on porous-structured fibrous scaffolds. After 5 days of culture, cells grew well on all three kinds of scaffolds, thus indicating good biocompatibility for the PCL fibers. All of the cells on the porous-structured fibrous scaffolds showed a highly stretched morphology. Similar to the live/dead assay, cells might have reached a saturated living state on the scaffolds after 10 days of culture. Cytoskeleton studies further verified that the scaffolds with hierarchical structures, especially those with a porous surface, enhanced cell adhesion. 3.5.2 Culture of HUVECs on the Scaffolds Endothelial cells are a crucial cell type for vascular grafts because they compose the interior surface of blood vessels [56]. Therefore HUVECs were also cultured on the fibrous scaffolds to evaluate the feasibility of scaffolds with different hierarchical structures to be potentially used in vascular scaffold applications. The viability of HUVECs on all three kinds of fibrous scaffolds was tested via a Live/Dead assay at day 3 and day 7 as shown in Figure 10. It was found that there were already many cells on all of the fibrous scaffolds and few dead cells were observed. The cell viability of all samples exceeded 85% at day 3 and exceeded 90% at day 7 (Figure 11 (a)), which demonstrated good biocompatibility for all scaffolds. The cell viability for PCL-PO scaffolds showed higher cell viability than the other two kinds of scaffolds, a result that is similar to the 3T3 fibroblast cell culture results. In order to further assess the cell proliferation behavior of scaffolds, CCK-8 assays were performed at day 3 and day 7 time points. Figure 11 (b) shows the proliferation of HUVECs on the PCL, PCL-PO, and PCL-SK scaffolds, as well as TCPS as a negative control, at different time points. It was found that, as the culture time

increased, there was an obvious increase in the optical density (OD). Particularly, the OD values of the PCL-PO scaffold were similar to the negative control at days 3 and 7, which indicated that the PCL-PO fibrous scaffold was non-toxic to the HUVECs. The cytoskeleton organization of HUVECs growing on the scaffolds was characterized by immunofluorescent microscopy with actin labeled with red and nuclei labeled with blue (Figure 10). It can be seen that HUVECs showed a healthy morphology on all scaffolds and did not show any obvious differences from the actin staining results. Cell spreading for the hierarchical structured scaffolds, especially on PCL-PO scaffolds at day 3 and day 7, was found to be larger than other scaffolds, which corresponds to the live/dead fluorescent images. In order to further investigated HUVECs morphologies and cell–scaffold interactions, SEM tests at day 3 and day 7 time points were also performed. It was found that HUVECs spread out in various directions into flat sheet shapes on all of the scaffolds (Figure 10). However, another phenomenon, that should be noted particularly, is that cells on the PCL-PO scaffolds interacted with each other very well. Obvious intercellular fibrils can be seen on PCL-PO scaffolds (Figure 10), however no similar phenomenon can be found on the other scaffolds (Figure 10). As culture time increased to day 7, cells on all of the scaffolds had migrated toward each other to form cell membranes and it was hard to distinguish the morphology of individual cells from the SEM images (Figure 10). Overall, these data suggest that electrospun PCL fibers with hierarchical structures had higher cell viability, cell proliferation, and better cell–cell interactions as compared to normal fibrous scaffolds. This is because cell filopodia, which play an instrumental role in the contact-guidance response, can ‘feel’ and interact with the surface features of a scaffold at a scale as small as 10 nm [31]. Furthermore, cells on porous fibrous scaffolds had the most flourishing living state. This can be attributed to the high surface area-to-volume ratio on the hierarchically structured scaffolds as compared to other scaffolds, which provided more binding sites for cell adhesion. Additionally, a relatively lower water contact angle on the porous-structured fibrous scaffolds, as compared to the shish–kebab-structured fibrous scaffolds, also promoted cell adhesion [57]. Together, these results indicate that the PCL-PO fibrous scaffold has great potential to support cell growth and utility as blood vessel endothelial layer scaffolds. 3.6 Limitations and Future Work There are several limitations to the work presented. The main challenge is obtaining sufficient porosity, appropriate pore sizes, and suitable height with the electrospun scaffolds. Tight fiber packing is another common problem for most electrospun membranes and is known to limit cell infiltration [10, 58]. A previous study reported that scaffolds with a pore size of at least 20 μm allowed for cellular infiltration into the scaffolds [59]. Another challenge is reproducing the nonlinear stiffening behavior of vascular grafts, which is mainly derived from the wavy/curly architecture of the collagen fibrils [7]. Therefore, incorporating large pores in the scaffolds and crimp structures on the fibers will be the focus of our future work. One method could be to combine hierarchical structured fibers as a blood vessel

endothelial layer with highly ordered melt electrospun microfibers as a tunica media layer to fabricate hybrid vascular grafts, while incorporating large pores in the scaffolds and mimicking the nonlinear stiffening behavior of vascular grafts [6, 16]. 4. CONCLUSIONS In summary, three kinds of hierarchically structured surfaces of PCL fibers were fabricated by electrospinning to mimic collagen fibrils. PCL fibers with a porous surface were obtained using a 9:1 CF:DMSO mixed solvent. The presence of pores on the surface suggested that the pores were formed by evaporative cooling of the fiber surface (TIPS), followed by induced moisture condensation on the fiber surface, which finally left an imprint on the surface. Furthermore, adding DMSO—which is a non-solvent for PCL—intensified the phase separation. Hierarchically shish– kebab-structured surfaces on electrospun PCL fibers were obtained using two types of controlled homoepitaxic crystallization methods. Compared with the solution incubation method, the solvent evaporation method was more effective and produced the best morphology when the concentration of the solution was 1.5%w/v. The formation of a kebab structure was ascribed to hydrophobic–lipophilic interactions at the beginning and homoepitaxic crystallization as time went by. AFM images also confirmed that the PCL-PO and PCL-SK fibers could be regarded as structurally hierarchical. Due to the obviously increased roughness of the shish–kebab structures, the water contact angle of the kebab-structured scaffolds was higher than that of the smooth and porous surface PCL fiber meshes. The specific surface area and the total pore volume of the PCL-PO fibers were much higher than the other two types of fibrous membranes. The 3T3 fibroblast cell and HUVEC cell assays on the electrospun PCL fibrous scaffolds with different hierarchical structures showed that cell viability and proliferation improved when the fibers were decorated by hierarchical structures. The scaffolds with porous surfaces provided a more favorable environment for cell growth based on live/dead assays and cell morphology analyses as compared to the shish–kebab-structured fibrous scaffolds. Collectively, these results suggested the potential utility of porous surface structured electrospun fibers to promote cell growth and serve as blood vessel endothelial layer scaffolds for tissue regeneration and biomaterial implants. Acknowledgments The authors would like to acknowledge the support of the Wisconsin Institute for Discovery (WID), National Center for International Joint Research of Micro-Nano Molding Technology and the China Scholarship Council (CSC), the financial support of the National Science Fund (Grant No. 11372286, 11372287, 51603192), the International Technological Cooperation Project (Grant No. 2015DFA30550), the Key Project of Science and Technology of the Education Department of Henan Province (15A430047) and the Key Project of International Cooperation of Henan Province (152102410013) for this project.

References

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Figure 1. (a) SEM images of an electrospun PCL fibrous scaffold with a smooth surface, and (b) histogram of the diameter distribution calculated from 60 random fiber measurements.

Figure 2. SEM images of the electrospun PCL fibrous scaffolds produced by CF/DMSO solutions.

The polymer concentration in the feed solution was 12%w/v in all cases. The ratio of poor solvent (DMSO) in the CF/DMSO mixture was (a) 10% v/v, the inset shows the fibers morphology at a higher magnification with arrows pointing to the elliptical pores and the circle encompassing a large irregular pore, (c) 20% v/v, and (e) 30% v/v. Scale bars are 2 m except for the inset, which is 1 m. (d) Schematic mechanism of porous structure formation on PCL fibers.

Figure 3. PCL fibers incubated in a PCL/acetic acid/DI water solution with different concentrations and various lengths of wash time: (a) 0.5% w/v and 0 min(the insert is the schematic of shish-kebab structure), (b) 0.5% w/v and 5 min, (c) 0.5% w/v and 10 min, (d) 1.0%

w/v and 0 min, (e) 1.0% w/v and 5 min, and (f) 1.0% w/v and 10 min.

Figure 4. PCL fibers incubated in a PCL/acetic acid/DI water solution with different concentrations: (a) 0.5% w/v, (b) 1% w/v, (c) 1.5% w/v, and (d) 2% w/v.

Figure 5. PCL fibers after drop casting 30 𝜇𝑙 of a PCL/acetic acid/DI water solution with different concentrations: (a) 0.5 w/v, (b) 1% w/v, (c) 1.5% w/v, and (d) 2% w/v.

Figure 6. Schematic mechanism of the kebab structure formation on PCL fibers.

Figure 7. AFM images of PCL fibers with different hierarchical structures: (a) PCL, (b) PCL-PO, and (c) PCL-SK. (d) Water contact angles of various structured PCL fibers. (e) Nitrogen adsorption–desorption isotherms of the different hierarchical structured PCL fibrous scaffolds with PCL fibers. (f) Specific surface area and total pore volume of PCL fibrous mats with different hierarchical structures derived from (e).

Figure 8. Live/dead viability results and cytoskeleton study of 3T3 fibroblast cells grown on different hierarchical structured fibers at various culture time points.

Figure 9. (a) Cell viability based on live/dead assay after culturing for 3 and 5 days. (b) Cell proliferation on the scaffolds after culturing for 3 days, 5 days, and 10 days as determined by MTT assay.

Figure 10. Live/dead viability results, cytoskeleton study, and SEM images of HUVECs grown on different hierarchical structured fibers at various culture time point.

Figure 11. (a) Cell viability based on live/dead assay after culturing for 3 and 7 days. (b) Cell proliferation on the scaffolds after culturing for 3 days and 7 days as determined by CCK-8 assay.