Micropatterned coculture of vascular endothelial and smooth muscle cells on layered electrospun fibrous mats toward blood vessel engineering Huinan Li,* Yaowen Liu,* Jinfu Lu, Jiaojun Wei, Xiaohong Li Key Laboratory of Advanced Technologies of Materials, Ministry of Education, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, People’s Republic of China Received 21 July 2014; revised 31 August 2014; accepted 5 September 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35332 Abstract: A major challenge in vascular engineering is the establishment of proper microenvironment to guide the spatial organization, growth, and extracellular matrix (ECM) productions of cells found in blood vessels. In the current study, micropatterned fibrous mats with distinct ridges and grooves of different width were created to load smooth muscle cells (SMCs), which were assembled by stacking on vascular endothelial cell (EC)-loaded flat fibrous mats to mimic the in vivolike organized structure of blood vessels. SMCs were mainly distributed in the ridges, and aligned fibers in the patterned regions led to the formation of elongated cell bodies, intense actin filaments, and expressions of collagen I and a-smooth muscle actin in a parallel direction with fibers. ECs spread over the flat fibrous mats and expressed collagen IV and laminin with a cobblestone-like feature. A z-stack scanning of fluorescently stained fibrous mats indicated that SMCs effectively infil-

trated into fibrous scaffolds at the depth of around 200 lm. Compared with SMCs cultured alone, the coculture with ECs enhanced the proliferation, infiltration, and cytoskeleton elongation of SMCs on patterned fibrous mats. Although the coculture of SMCs made no significant difference in the EC growth, the coculture system on patterned fibrous scaffolds promoted ECM productions of both ECs and SMCs. Thus, this patterned fibrous configuration not only offers a promising technology in the design of tissue engineering scaffolds to construct blood vessels with durable mechanical properties, but also provides a platform for patterned coculture to investigate cell–matrix C 2014 and cell–cell interactions in highly organized tissues. V Wiley Periodicals, Inc. J Biomed Mater Res Part A: 00A:000–000, 2014.

Key Words: micropatterned fibrous mat, cell coculture, blood vessel, cell infiltration, cell patterning

How to cite this article: Li H, Liu Y, Lu J, Wei J, Li X. 2014. Micropatterned coculture of vascular endothelial and smooth muscle cells on layered electrospun fibrous mats toward blood vessel engineering. J Biomed Mater Res Part A 2014:00A:000–000.

INTRODUCTION

There are many kinds of diseases or traumas that often damage human blood vessels irreversibly, and a surgical replacement with autologous vessels, allografts, xenografts, or synthetic materials is the most effective treatment. Autologous blood vessels are the best choice for transplantation, but suffer from scarce sources and donor site morbidity. Allografts and xenografts are heavily limited by the risk of immune rejections, resulting in significantly reduced durability and longevity of the implants. Synthetic polymers, like polyethylene terephthalate and expanded polytetrafluoroethylene, are rapidly gaining attention for substituting large diameter vessels.1 Although many strategies have been figured out to modulate vascular strength and biochemistry of the synthetic grafts, long-term follow-up studies reveal the

occurrence of material-related failures such as stenosis, thromboembolization, and calcium deposition.2 In this condition, researchers began to focus on tissue engineering to develop vascular substitutes, in which vascular endothelial cells (ECs) and smooth muscle cells (SMCs) were seeded on biodegradable scaffolds to facilitate the cell growth and extracellular matrix (ECM) productions for generating blood vessels in vitro.3 Although tissue-engineered blood vessels have seen important advances during recent years, proper mechanical strength and vasoactivity remain unsolved problems to withstand cyclic mechanical stresses generated by a pulsatile flow of blood.4 The proper function of many tissues depends critically on the structural organization of cells and ECMs of which they are comprised. Anatomically, the inner layer of a blood

*These authors contributed equally to this work. Correspondence to: X. Li; e-mail: [email protected] Contract grant sponsor: National Natural Science Foundation of China; contract grant numbers: 51073130, 21274117, and 31470922 Contract grant sponsor: Specialized Research Fund for the Doctoral Program of Higher Education; contract grant number: 20120184110004 Contract grant sponsor: National Scientific and Technical Supporting Programs; contract grant number: 2012BAI17B06 Contract grant sponsor: Construction Program for Innovative Research Team of University in Sichuan Province; contract grant number: 14TD0050

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vessel is a monolayer made of ECs, which serves as a structural barrier between the circulation and surrounding tissues, and as an antithrombogenic surface to minimize friction between the vessel wall and blood cells.5 The key feature of arterial vessels is the alignment of SMCs with their long axis extending in the circumferential direction, which provides durable mechanical properties for high pressure existing in the circulation and contractile force for the constriction or dilation of blood vessels.6 Therefore, a major challenge in vascular engineering is the establishment of proper microenvironment to guide the spatial organization, growth, proliferation, and ECM productions of cells found in blood vessels. A variety of techniques have been developed to create tissue engineering scaffolds that provide chemical and physical cues to mediate cell–matrix and even cell–cell interactions and to improve functionality of engineered tissues. To enhance EC adhesion, spreading, and proliferation, scaffolds were modified with ECM proteins, such as fibronectin and collagen, or functionalized with sequences corresponding to cell adhesion domains in ECM proteins.7 Zieris et al.8 fabricated poly(ethylene glycol)/heparin hydrogels to release basic fibroblast growth factor and vascular endothelial growth factor, indicating beneficial effects on the morphology, proliferation, and survival of ECs. To obtain an oriented structure of ECM productions, micropatterned surfaces have been developed to determine the effects of topographical cues on SMC alignment and functions. Chang et al.9 constructed patterned substrates with defined parallel microgrooves of 3 lm wide and 5 lm deep, and SMCs showed a spindle-like morphology associated with cytoskeletal rearrangement in the direction of micropatterns. To achieve a three-dimensional (3D) distribution of cells, Sarkar et al.10 fabricated porous micropatterned polycaprolactone (PCL) scaffolds using soft lithography, melt molding, and particulate leaching. Cells conformed to the porous microarchitecture mimicking blood vessels in vivo, and SMCs aligned on micropatterned scaffolds.10 The communication between ECs and SMCs in vascular walls occurs through the synthesis and release of mediators into the surrounding media, and cocultures of ECs and SMCs have been developed to better mimic the structure of blood vessels and understand their interactions. Direct coculture of EC and SMC layers and coculture of ECs and SMCs on opposite sides of porous membranes, collagen gels, and Transwells have been investigated to regulate the normal functions of both cell types.11 Wu et al. developed a collagenous vascular graft, and ECs and SMCs were seeded on different sides of the bilayer porous membrane. After coculture for 7 days, vascular substitutes were harvested for subcutaneous implantation, resulting in the formation of continuous layers of ECs and SMCs as the vascular lumen and tunic media, respectively, but the interactions between these cells were not determined.12 To construct 3D scaffolds for the coculture of ECs and SMCs, Yuan et al.13 designed a stress-induced rolling membrane and obtained a multilayered tube structure with the inoculation of ECs and SMCs in layers from inside to outside like blood vessels in vivo. The

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orientation of SMCs was controlled by the inside of tubes because of the topographical contact guidance.13 Although distinct advantages of the coculture techniques are the mimicry of an in vivo-like structure and SMCs can be controlled by the geometry of those layers, the films are not suitable for cell growth because they are not porous and cannot facilitate the transport of nutrients and oxygen to cells.14 Electrospun fibers have a nano- to microscale fibrous structure similar to protein fibers within native ECM and are capable of supporting the attachment and proliferation of a variety of cell types.15 McClure et al. fabricated a multilayered electrospun conduit composed of PCL with the addition of elastin and collagen to demonstrate mechanical properties indicative of native arterial tissues. Both the modulus and compliance data of the scaffold displayed values within the range of a native artery, while remaining conducive to tissue regeneration.16 Jeong et al.17 deposited electrospun fibers on porous tubular collagen scaffolds for the loading of both SMCs and ECs. Compared with a random cell alignment under a static condition, a pulsatile perfusion system enhanced cell proliferation, induced cellular alignment, and upregulated ECM productions. Janairo et al.18 developed a bilayered synthetic vascular graft of 1-mm diameter that consisted of a microfibrous luminal layer and a nanofibrous outer layer, resulting in EC and SMC organizations in the explanted grafts after implantation for 1 month.18 However, currently no study has examined the interactions between ECs and SMCs on a highly organized fibrous mat. We have recently developed patterned fibrous mats with distinct ridges and grooves through designing a glass substrate patterned with an electrically conductive circuit as a collector for electrospinning.19 The cell growth and collagen deposition seemed to be confined to precise locations, sizes, and shapes of patterned scaffolds without any adverse effects on the cell proliferation and ECM productions.19 In this study, we assessed the possibility to assemble flat and patterned fibrous mats for loading ECs and SMCs, respectively, and to establish their coculture for potential interactions. ECs were supposed to attach and spread on randomly fibrous mats, whereas SMCs were arranged in patterned fibrous mats to form a highly aligned structure as in vivolike. The effect of the cell coculture and dimensions of the grooves and ridges of patterned scaffolds on the proliferation and ECM productions was evaluated on both ECs and SMCs. The skeleton organization, distribution, and penetration of cells into fibrous mats were also determined. MATERIALS AND METHODS

Materials Poly(ethylene glycol)-poly(DL-lactide) (PELA, Mw 5 42.3 kDa, Mw/Mn 5 1.23) was prepared by bulk ring-opening polymerization of lactide/poly(ethylene glycol) using stannous chloride as the initiator. All the electrophoresis reagents, bovine serum albumin (BSA), toluidine blue, rhodamine B, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC)-phalloidin, and goat serum were procured from

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FIGURE 1. (a) Digital image of a photomask printed by E-beam mask lithography system and (b) a patterned collector with strips of 300 lm wide and gaps between the strips of 100 lm wide. Bars represent 300 lm. (c) Digital images of fibrous mats with the ridge/groove width of 200/ 50, 200/100, 300/50, and 300/100 lm after deposition on the patterned collectors. Bars represent 300 lm. (d) SEM images of patterned fibrous mats with the ridge/groove width of 300/100 lm and (e) fibers in the ridges and grooves. (f) SEM images of fibers in the ridges of patterned fibrous mats and (g) flat fibrous mats. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Sigma-Aldrich Inc. (St. Louis, MO). Protein molecular weight marker and RIPA lysis buffer were from Beyotime Institute of Biotechnology (Shanghai, China). Rabbit antihuman antibodies of collagen I, laminin, a-smooth muscle actin (a-SMA), and b-actin, goat anti-rabbit IgG2FITC, goat anti-rabbit IgG–horseradish peroxidase (HRP), and 3,30 diaminobenzidine (DAB) developer were purchased from Biosynthesis Biotechnology Co. (Beijing, China). All other chemicals and solvents were of reagent grade or better and received from Chengdu Kelong Reagent Co. (Chengdu, China), unless otherwise indicated. Fabrication of micropatterned collector The patterned collector was constructed on a glass template patterned with an electrically conductive circuit as described previously.19 In brief, the designed template contained an array of parallel strips was designed by tanner L-edit software. Micropatterned photomasks [Fig. 1(a)] with strip width of 200 and 300 lm and distance between the strips of 50 and 100 lm were fabricated by E-beam mask lithography system (Mark 40, CHA Industries, Fremont, CA). An insulating glass substrate was deposited with a silver layer by DC sputtering (Sunicoat 594L, Sunic, Korea) and coated with a layer of photoresist (MicroChem, Newton, MA). After exposure under the micropatterned photomask by a lithography machine (Suss Mircotec MA6, Germany), the glass substrate was rinsed to remove the photoresist in the exposed regions, followed by etching away silver in the exposed area. After etching and removing the rest of photoresist, a micropatterned silver circuit [Fig. 1(b)] was obtained on the glass substrate as a collector for electrospinning process. Preparation of flat and micropatterned electrospun fibrous mats Electrospun patterned fibrous mats were obtained as described previously with some modifications.19 In brief,

PELA solution in acetone (16.6%, w/w) was added in a 2mL syringe, attached with a blunt needle. The flow rate was controlled at 0.6 mL/h by a syringe pump (Zhejiang University Medical Instrument Company, Hangzhou, China). The electrospinning apparatus was equipped with a high-voltage power supply (Tianjin High Voltage Power Supply Co., Tianjing, China), and the applied voltage was controlled within the range of 20 kV. The distance between the needle tip and patterned collector was about 15 cm, and the collected fibers were vacuum dried to remove any solvent residue before further use. Micropatterned fibrous mats were obtained with the ridge/groove width of 200/50, 200/100, 300/50, and 300/100 lm on patterned collectors made from photomasks of different strip width. Alternatively, a grounded plate-type collector was used to collect flat fibrous mats. Characterization of flat and micropatterned fibrous mats The patterned distribution of fibers was observed by an optical microscope (Nikon Eclipse TS100, Japan). The fibrous morphology of flat and micropatterned fibrous mats was examined by using a scanning electron microscope (SEM, FEI Quanta 200, The Netherlands) equipped with a field-emission gun (20 kV) and Robinson detector after 2 min of gold coating to minimize the charging effect. The fiber diameter and alignment and the pore size of fibrous mats were measured from SEM images using ImageJ as described previously.19 In brief, the fiber diameter was evaluated from three randomly selected SEM images, 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 210 to 10 respect to the total fibers counted. The pore size was evaluated on the basis of void fraction as seen in top view of SEM images.19

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FIGURE 2. (a) Schematic illustration of the coculture process of stacking an SMC-loaded patterned fibrous mat on an EC-loaded flat fibrous mat, followed by fixing through two concentric glass tubes. (b) Merged fluorescent image after stacking an FITC-labeled patterned fibrous mat on a rhodamine B-labeled flat mat. (c) Optical microscope images of toluidine blue stained EC-loaded fibrous mats and SMC-loaded patterned fibrous mats with the ridge/groove width of 300/100 lm after 7 days of incubation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Cell coculture on flat and micropatterned fibrous mats The fibrous mats were punched into squares with a length of 10 mm and sterilized by e-beam radiation using a linear accelerator (PreciseTM, Elekta, Crawley, UK) with a total dose of 80 cGy. Flat and micropatterned fibrous mats were incubated into 1 mg/mL of rhodamine B and FITC solution overnight, respectively, before being stacked. As illustrated in Figure 2(a), two concentric glass tubes were used to fix the stacked fibrous mats, which were placed between the tubes. After washing with distilled water, the upper and lower layers of the scaffold were observed by a confocal laser scanning microscope (CLSM, Olympus FV1000S, Tokyo, Japan) under the excitation and emission wavelength of 490 and 520 nm for FITC and those of 550 and 620 nm for rhodamine B, respectively. The fluorescence images were merged by ImageJ software to show the stacked fibrous mats. Human umbilical vein ECs and human aortic SMCs were from American Type Culture Collection (Rockville, MD) and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Rockville, MD) supplemented with 10% heatinactivated fetal bovine serum (FBS, Gibco BRL, Grand Island, NY). Sterilized fibrous mats were put on cover slips, and 400 mL of EC and SMC suspensions with a cell density of 5 3 104 cells/mL were seeded on flat and patterned fibrous mats, respectively. After incubation at 37 C for 1 day to make cells diffuse into and adhere to the scaffold, cell-loaded fibrous mats were assembled as shown in Figure 2(a) so that the ECs and SMCs were separated by the fibrous mats. The cell-loaded fibrous mats were further incubated in 24-well tissue culture plate (TCP) with DMEM containing 10% FBS, and the culture media was refreshed every 223 days. ECs were cocultured with SMCs on flat mats, patterns mats with the ridge/groove width of 200/50,

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200/100, 300/50, and 300/100 lm, which were defined as E-M, E-M200/50, E-M200/100, E-M300/50, and E-M300/ 100, respectively. ECs cultured alone on flat mats (E), and SMCs cultured alone on flat mats (M), and on patterns mats with the ridge/groove width of 200/50 (M200/50), 200/100 (M200/100), 300/50 (M300/50), and 300/100 lm (M300/100) were used as control. Cell growth profiles after coculture on fibrous mats After incubation for 7 days, cell-loaded fibrous scaffolds were washed with phosphate buffer saline (PBS) twice and fixed with 4% paraformaldehyde for 24 h at 4 C. The cell distributions were observed on fibrous mats after incubation with 0.5 mL of freshly prepared toluidine blue solution (0.04 wt %) for 30 min at ambient temperature. The stained mats were washed, dehydrated through a series of graded ethanol solutions, and observed with an inverted microscope (Nikon Eclipse TS100, Japan). Alternatively, the dehydrated fibrous mats were sputter coated with gold, and the cell morphologies were examined by SEM as above. The actin filaments (F-actin) in ECs and SMCs on fibrous mats were stained with phalloidin to assess their organizations. In brief, cell-loaded fibrous mats were incubated with TRITC-phalloidin of 10 lg/mL for 20 min at 37 C, followed by extensive wash with PBS. The samples were observed by CLSM, and the orientation index of F-actin staining was evaluated by analyzing the CLSM images using ImageJ software as described previously.20 In brief, a fast Fourier transformation (FFT) was applied on a CLSM image, rendering an image with pixel intensities. With the support of an oval profile plug-in, an FFT analysis was used to sum the pixel intensities along a circle for each one-degree sector, resulting in a graph of pixel intensities across 360 degrees. All the alignment data were normalized to a baseline value of 0

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and plotted in percentage of the arbitrary units ranging from 0 to 0.15 with respect to the total fibers counted.21 The infiltration of cells into fibrous mats was observed by CLSM after stained with 1.0 mg/mL DAPI solution for 5 min. The images were taken through z-stack scanning with a step size of 5 lm, and a 3D image reconstruction was used to measure the cell distribution within fibrous mats by ImageJ software as described previously.22 Cell proliferation after coculture on fibrous mats The cell proliferation on fibrous mats was assayed with CCK-8 cell counting kit (Dojindo Molecular Technologies, Kumamoto, Japan). In brief, culture media were removed after coculture for 1, 4, and 7 days, and cell-loaded fibrous scaffolds were rinsed three times with PBS. The flat and micropatterned fibrous mats were split for the determination of ECs and SMCs separately. Cell-loaded fibrous mats were moved to a 48-well TCP, and 400 lL of fresh culture media and 40 lL CCK-8 reagent were added into each well according to the reagent instruction. After incubation for 2 h, 150 lL of incubation media was pipetted into a 96-well TCP and the absorbance at 450 nm was measured for each well using a mQuant microplate spectrophotometer (Elx-800, Bio-Tek Instrument, Winooski, VT). ECM production of cells after coculture on fibrous mats The productions of collagen IV and laminin by ECs, and those of collagen I and a-SMA by SMCs were observed by immunofluorescent staining. In brief, after coculture for 7 days, the culture media were removed and cell-loaded fibrous mats were rinsed three times with PBS. The flat and micropatterned fibrous mats were split and then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 solution in PBS. After being blocked by incubation with 10% goat serum for 30 min at 37 C, EC-loaded fibrous mats were incubated with rabbit antihuman antibodies of collagen IV or laminin, whereas SMC-loaded fibrous mats were incubated with those of collagen I or a-SMA for 2 h at 37 C. After being washed three times with PBS, samples were incubated with goat anti-rabbit IgG–FITC for 1 h at 37 C. The fibrous mats were observed by CLSM as above after DAPI counterstaining. The ECM production of ECs and SMCs after coculture was quantified after Western blotting analysis as described previously.4 In brief, cell-loaded fibrous mats were homogenized in RIPA lysis buffer, and the total protein of the cell lysate was determined by BCA protein assay kit (Pierce, Rockford, IL). The cell lysate was mixed with the loading buffer (40 mM Tris-HCl, 1% sodium dodecylsulfate, 50 mM dithiothreitol, 7.5% glycerol, and 0.003% bromophenol blue) and then were subjected to electrophoresis on 10% polyacrylamide gel at 100 V, followed by electronically transferring to PVDF membrane (Millipore Corp., Bedford, MA). After being blocked with 5% BSA for 2 h at room temperature, the membrane was washed and incubated with rabbit antihuman antibodies of collagen IV, laminin, collagen I, or a-SMA overnight at 4 C. After washing with PBS containing 0.05% Tween 20, the membrane was incubated with

goat anti-rabbit IgG–HRP for 1 h. Antigen–antibody complexes were visualized by DAB developer, and expression of b-actin was used as protein loading control. The membranes were digitally scanned, and the levels of ECM proteins were assessed using Image pro software by measuring the integrated intensity of all the pixels in each band, excluding the local background.23 Results were obtained from at least three separate experiments, and the relative protein levels were normalized to the b-actin signals. Statistical analysis The values were expressed as means 6 standard deviation (SD). Whenever appropriate a 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. RESULTS

Characterization of flat and patterned fibrous mats Micropatterned fibrous mats with ridges and grooves of different width were created by combining lithography with electrospinning process. Figure 1(a) shows a typical photomask containing parallel strips of 300 lm wide and gaps between the strips of 100 lm. Silver was deposited on a glass substrate, and a photolithography process was used to remove the silver layer in the exposed areas under a photomask. As shown in Figure 1(b), patterned sliver strips of 300 lm wide were obtained on the substrate, indicating that patterning features of the photomask were well maintained. During the electrospinning process, fibers were preferentially deposited on the silver circuit, driven by Coulombic interactions between the positive charges on fibers and negative charges on the collector.19 Figure 1(c) summarizes optical images of patterned fibrous mats, indicating ridges of 200 and 300 mm wide and grooves of 50 and 100 mm wide. The micropatterned fibrous mats indicated topological structures and dimensions similar to the collector configuration, and few fibers were found within the gaps between strips. Figure 1(d,e) summarizes typical SEM morphologies of fibrous mats deposited on the patterned collector, presenting distinct ridge and groove regions. During the electrospinning process, Coulombic interactions between the opposite charges induced fibers to arrange along the strip,19 resulting in a parallel alignment of fibers in the patterned regions [Fig. 1(f)], although randomly distributed fibers were observed in flat fibrous mats [Fig. 1(g)]. The diameter of fibers in the patterned and flat fibrous mats was 0.85 6 0.17 and 0.96 6 0.18 mm, respectively. The pore size and fiber alignment in the patterned regions was 8.2 6 1.2 mm and 84.5 6 7.1%, respectively. Establishment of coculture systems of ECs and SMCs To mimic the vessel structures in vivo, ECs were seeded on flat fibrous mats, whereas SMC-loaded patterned fibrous mats were assembled with the EC layers to establish an ECSMC coculture system. To assist the cell coculture and define the patterns of fibrous mats, two concentric glass tubes were used to fix the stacked fibrous mats [Fig. 2(a)].

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Fluorescent labelling was used to visualize the scaffold morphology, and Figure 2(b) shows the merged fluorescence images after stacking patterned fibrous mats on flat ones. Toluidine blue is a dye with affinity for nucleic acids and primarily used for cell staining, and the distribution of ECs and SMCs on fibrous mats was observed after coculture for 7 days. As shown in Figure 2(c), ECs attached and spread on the surface of flat mats, although the SMC density in the ridges was significantly higher than that in the grooves, indicating uneven distribution according to the architecture of fibrous scaffolds. Compared with those in the grooves, the higher fiber density in the ridges created more crossing points as cellular adhesive domains, and the higher structural stability of fibrous morphologies was beneficial for cells to attach, spread, and infiltrate into the ridge regions.19 Cell proliferation after coculture on fibrous mats To determine the cell proliferation after coculture, the fibrous scaffolds were split and the behaviors of ECs and SMCs were determined separately. Figure 3 summarizes the cell growth after incubation for 1, 4, and 7 days, indicating a progressive proliferation after both coculture and monoculture of ECs and SMCs. At each time point, the EC growth after coculture showed no significant difference compared with that cultured alone (p > 0.05), indicating that the coculture with SMCs did not apparently affect the growth of ECs on fibrous mats. As shown in Figure 3(a), there was no significant difference in the viabilities of SMCs between cultured alone and cocultures for 1 day on flat fibrous mats or fibrous mats with different patterning profiles (p > 0.05). However, significantly higher SMC viabilities were detected on patterned fibrous mats after coculture for 4 days with ECs than those cultured alone (p < 0.05) [Fig. 3(b)], indicating that the coculture with EC enhanced SMC proliferation on patterned fibrous mats. This stimulatory effect progressively increased until day 7, for example, there was around 38 and 57% increase in the SMC proliferation on E-M300/ 100 after coculture with ECs for 4 and 7 days, respectively, compared with SMCs cultured alone [Fig. 3(c)]. Cellular morphologies after coculture on fibrous mats Figure 4(a) shows SEM morphologies of ECs on flat fibrous mats. ECs were tightly attached and spread over multiple fibers and stretched well in a flat profile. In contrast, SMCs seeded on patterned fibrous mats were elongated and organized within the ridges, which could be explained by the cell–matrix contact guidance.24 As shown in Figure 4(b), numerous lamellipodia and filopodia of SMCs were anchored on fibers, and cell bodies elongated in a bipolar manner on patterned fibrous mats, similar to those cells in vivo. The F-actin organization of cells on patterned fibrous mats was evaluated via staining with TRITC-phalloidin. As shown in Figure 4(c), ECs on flat fibrous mats were round and contained little or no organized filamentous actin. However, there was intense F-actin staining on SMCs, indicating that cell bodies elongated in a bipolar manner and linked up into a single stretch [Fig. 4(d,e)]. The orientation of

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FIGURE 3. Viabilities of ECs and SMCs after coculture on patterned fibrous mats with the ridge/groove width of 200/50, 200/100, 300/50, and 300/100 lm for (a) 1, (b) 4, and (c) 7 days, compared with ECs or SMCs cultured alone (n 5 6, *p < 0.05).

F-actin could be controlled by the alignment of fibers, and the orientation index was 78.6 6 8.4% for cocultured SMCs [Fig. 4(e)], which was slightly higher than that of 70.9 6 9.1% for SMCs cultured alone [Fig. 4(d)]. Cell distributions after coculture on fibrous mats To better understand the 3D distribution of cells in fibrous mats, z-stack scanning with a 5-lm step size was performed by CLSM after DAPI staining. Figure 5 shows CLSM images at different depth levels from the top of an individual fibrous mat. ECs distributed on the superficial layer of scaffolds, although few cells could be found in the depth of

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FIGURE 4. (a) SEM images of ECs cocultured on flat fibrous mats and (b) SMCs on patterned fibrous mats with the ridge/groove width of 300/100 lm. (c) CLSM images of TRITC-phalloidin stained F-actin of ECs cocultured on fibrous mats, (d) SMCs cultured alone and (e) SMCs cocultured on patterned fibrous mats. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

around 50 lm [Fig. 5(a)]. As shown in Figure 5(b), a patterned distribution feature of SMCs was observed on patterned fibrous mats, and few cells existed in the grooves. It should be noted that SMCs penetrated into and were distributed throughout the fibrous mat in the ridges, and a patterned distribution of SMCs was observed at the depth of around 150 lm. The coculture with ECs led to a deeper infiltration of SMC in the fibrous mats, and a patterned distribution of SMCs was detected at the depth of around 200 lm [Fig. 5(c)]. The side views of the fluorescence images were reconstructed to show the cell infiltration into fibrous mats. ECs mainly distributed in the upper region of scaffolds as seen by the illuminated blue [Fig. 5(d)]. As shown in Figure 5(e,f), SMCs existed not only in the top surface region but also in middle and bottom regions of fibrous mats, and the patterning distribution remained throughout the ridge regions of patterned fibrous mats. A denser blue level was observed for SMCs under coculture with ECs than that of SMCs cultured alone. ECM productions of cells after coculture on fibrous mats The ECM production is an important step for generating functional tissues. The substantial function of ECs is the vascular formation of basal lamina, which is composed of a number of macromolecules, including collagen, proteoglycans, and glycoproteins.25 SMCs play an important role in the maintenance of arterial integrity and functions. The growth of SMCs is characterized by expression of cytoskeletal and contractile proteins such as a-SMA, myosin, collagen I, and elastin.26 In the current study, the expressions of collagen IV and laminin by ECs and those of collagen I and a-SMA by SMCs were evaluated by immunofluorescent staining after coculture for 7 days. Figure 6(a) demonstrated strong expressions of collagen IV and laminin by ECs with a cobblestone-like feature [Fig. 6(c)]. As shown in Figure 6(b),

the productions of collagen I and a-SMA by SMCs were found in the ridges of patterned fibrous mats, indicating that cells in the ridges successfully sustained cellular functionality. In addition, the collagen deposition was not random and was aligned in the parallel direction [Fig. 6(d)]. The ECM expressions of ECs and SMCs after coculture were quantified through digitizing the Western blotting images and subsequently analyzing the gray values of the bands. Figure 7(a) summarizes the laminin and collagen IV expressions by ECs cultured alone and after coculture with SMCs, using b-actin as protein loading control. The band densities were assessed using Image pro software to reflect the levels of ECM proteins.27 As shown in Figure 7(b), the densities of laminin bands were significantly higher for cocultured ECs (p < 0.05), and there was more than twofold increase in the densities of laminin bands from cocultured ECs over those from ECs cultured alone. In addition, ECs after coculture with SMCs on patterned fibrous mats indicated significantly higher laminin expression than those after coculture with SMCs on flat mats (p < 0.05). Especially, the laminin expressions by ECs from E-M200/100 and EM300/100 were significantly higher than those after coculture with SMCs on other patterned fibrous mats. Figure 7(c) summarizes the relative collagen IV levels normalized to the b-actin signals, indicating a profile similar to laminin expressions. ECs cocultured on E-M300/100 produced significantly higher collagen IV levels compared with ECs cultured alone and after coculture on other patterned fibrous mats and flat ones (p < 0.05). Figure 8 shows Western blotting results of a-SMA and collagen I expressions by SMCs cultured alone on different patterned fibrous mats and after coculture with ECs. As shown in Figure 8(b), the a-SMA levels of SMCs on patterned fibrous mats after coculture with ECs were significantly higher than those cultured alone (p < 0.05), and indicated an almost twofold higher over SMCs cultured

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FIGURE 5. (a) CLSM images at different depth of DAPI-stained ECs after coculture on fibrous mats, (b) SMCs cultured alone, and (c) SMCs cocultured on patterned fibrous mats with the ridge/groove width of 300/100 lm after incubation for 7 days. Scale bar represents 200 lm. (d) Reconstructed images after postprocessing CLSM images taken by z-stack scanning of ECs cocultured on fibrous mats, (e) SMCs cultured alone, and (f) SMCs cocultured on patterned fibrous mats after incubation for 7 days. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

alone. SMCs on patterned fibrous mats produced significantly higher amount of a-SMA than those on flat fibrous mats either in coculture or monoculture system (p < 0.05). The patterned fibrous mats with ridges and grooves of different width made no significant difference in the a-SMA levels (p > 0.05), but slightly higher a-SMA expressions were obtained for SMCs from E-M200/100 and E-M300/ 100 compared with other patterned mats. Similarly, as shown in Figure 8(c), the collagen I production was significantly up-regulated by the coculture with SMCs compared with those cultured alone (p < 0.05), and SMCs from EM200/100 and E-M300/100 produced slightly higher levels of collagen I than those from other patterned mats.

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DISCUSSION

Distribution and penetration profiles of cocultured ECs and SMCs The function of engineered tissues is strongly affected anatomically by their 3D structure, so it is essential to control cell location and morphology on scaffolds. This is the case for engineering blood vessels, where the development of durable strength and elasticity remains elusive to deal with blood flow. However, to achieve anisotropic mechanical properties in functional vessels, it remains a great challenge to adequately mimic the structural organization of the vessel wall, which derives its properties from its complex organization of matrix proteins and cells.9 In the current study,

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FIGURE 6. (a) Immunofluorescent staining images of collagen IV and laminin productions by cocultured ECs, and (b) collagen I and a-SMA productions by SMCs after coculture on patterned fibrous mats with the ridge/groove width of 300/100 lm. (c) Magnified immunofluorescent images of laminin productions by cocultured ECs and (d) a-SMA by SMCs cocultured on patterned fibrous mats. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

SMCs were seeded on patterned fibrous mats with different ridge and groove width, which were assembled with ECloaded fibrous mats to develop an EC-SMC coculture model to investigate interactions between ECs and SMCs. In addition, cell-loaded fibrous mats can be wrapped to make a cylindrical tube, which would be meaningful to precisely mimic vessels in vivo. After loading on a flat fibrous mat, ECs spread over the mat [Fig. 4(a)], contained little or no

organized filamentous actin [Fig. 4(c)], and produced collagen IV and laminin with a cobblestone-like feature after incubation for 7 days [Fig. 6(a)]. It was indicated that the EC adhesion and proliferation on irregularly distributed fibers was important for achieving a continuous endothelium. A patterned fibrous mat was used to load SMCs, which were mainly distributed in the ridges observed from toluidine blue [Fig. 2(c)] and DAPI staining [Fig. 5]. The ridge

FIGURE 7. (a) Western blotting images of collagen IV and laminin productions by ECs after coculture with SMCs on flat fibrous mats, patterned fibrous mats with the ridge/groove width of 200/50, 200/100, 300/50, and 300/100 lm and ECs cultured alone. b-Actin was used as protein loading control. (b) Quantification analyses of laminin and (c) collagen IV expressions on the band densities of their Western blotting images, and the relative protein levels were normalized to the b-actin signals (n 5 3, *p < 0.05 compared with ECs cultured alone).

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FIGURE 8. (a) Western blotting images of collagen I and a-SMA productions by SMCs cocultured alone on flat fibrous mats and patterned fibrous mats with the ridge/groove width of 200/50, 200/100, 300/50, and 300/100 lm and after coculture with ECs. b-Actin was used as protein loading control. (b) Quantification analyses of a-SMA and (c) collagen I expressions on the band densities of their Western blotting images, and the relative protein levels were normalized to the b-actin signals (n 5 3, *p < 0.05).

regions contained a parallel alignment of fibers [Fig. 1(f)], leading to the formation of elongated cell bodies with numerous lamellipodia and filopodia because of the cell–matrix contact guidance [Fig. 4(b)]. The staining of actin filaments [Fig. 4(e)] and the productions of collagen I and a-SMA by SMCs [Fig. 6(b)] indicated a distinct alignment of SMCs, which could provide the contractile force and durable mechanical properties for a successful generation of blood vessels.28 It is known that traditionally electrospun mats allow cells to attach but always superficially and difficult to promote cellular infiltration because of tightly packed fibrous structure, which would likely prevent significant cell infiltration into the scaffolds.22 Numerous strategies have been tried to overcome this challenge, including the incorporation of nanoparticles, the use of larger microfibers, or the removal of embedded salt or water-soluble fibers to increase the porosity.29 In the current study, a z-stack CLSM scanning of cell-loaded fibrous mats after DAPI staining indicated that ECs were distributed into flat fibrous mats around 50 lm in depth [Fig. 5(a,d)]. SMCs effectively infiltrated into the fibrous mat of around 200 lm in depth, and a patterning distribution remained throughout the ridge regions [Fig. 5(c,f)]. This may be due to fewer crossing points between aligned fibers in the ridge regions of patterned fibrous mats compared with flat ones.19 In addition, the gradual degradation of fibrous scaffolds along with the incubation would create space for cell proliferation and ECM productions to achieve functional tissues. Compared with the micropatterned surface to induce the alignment of SMCs,4 the infiltration of SMCs into patterned fibrous mats

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should provide the formation of SMC layers with different thickness, which made it possible to construct vessels with comparable mechanical properties at different anatomical locations. Vasoactivity, the constriction or dilation of blood vessels, is controlled by the contractile force produced by circumferentially aligned SMCs. As indicated above, aligned fibers were effective to guide the elongation of SMCs along the fiber alignment and enhance the penetration of SMCs into fibrous scaffolds to form durable SMC layers. The cellloaded patterned fibrous mats can be further wrapped into a vessel tube to create a helical arrangement of SMCs by connecting adjacent ridges, which should provide additional advantages for the formation of functional vessels. It is known that SMCs and their ECM productions are arranged in a helical pattern around the circumference of a native vessel, and this helical arrangement should be effective to enhance the circumferential load-bearing properties, and more importantly, to provide the torsional stability of blood vessels.30 For example, compared with aligned fibers, the contraction of SMCs in the ridges of patterned fibrous mats could achieve a reduction of the vessel lumen diameter rather than its length because of the helical arrangement of SMCs and their ECMs. In addition, this torsional stability of SMC layers should also allow more efficient control of the vessel tone, which in turn dictated blood pressure and shear stress.9 Therefore, the layered fibrous scaffolds could provide cellular interactions of EC with the lumen, the infiltration of SMC into the outer layer, and the alignment and helical arrangement of SMCs and their ECMs, which could be successfully used for engineering blood vessels.

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Proliferation and ECM productions of cocultured ECs and SMCs Cocultures of ECs and SMCs have been established to investigate their interactions and influence in cellular growth, migration, differentiation, and function through the synthesis and release of growth factors.31 Wallace et al.32 examined the growth of human ECs on quiescent human aortic SMCs, indicating that ECs were attached firmly on the SMC layers and the cocultured SMCs showed a more contractile state than SMCs cultured alone. A further study from this research group indicated SMCs did not produce a true basement membrane, and SMCs easily migrated over ECs and inhibited EC proliferation.11 Therefore, it is necessary to establish a separate, but close, coculture of ECs and SMCs. The loading of ECs and SMCs on opposite sides of polymer membranes is an easy way to permit a separation of these cells and limit their overgrowth while bringing them within 100 lm of each other.11 In the current study, porous electrospun fibrous mats were used to coculture ECs and SMCs, and the effects of the coculture on the cell proliferation and ECM productions were initially clarified. Compared with ECs cultured alone for 7 days, ECs after coculture with SMCs on patterned or flat fibrous mats indicated no significant difference in the cell proliferation rate (Fig. 3), but produced significantly higher levels of laminin and collagen IV (Fig. 7). It was indicated that the coculture with SMCs promoted the ECM productions for ECs, and the coculture on patterned scaffolds made no difference in the promotion of ECM expressions. On the other hand, compared with SMCs cultured alone, the EC coculture led to significantly higher cell proliferation (Fig. 3) and higher productions of a-SMA and collagen I for SMCs on patterned fibrous mats (Fig. 8). There was no significant difference in the proliferation and ECM productions between SMCs cultured alone on micropatterned fibrous mats with different width of ridges and grooves and SMCs cocultured with ECs (p > 0.05), but EM300/100 and E-M200/100 coculture system indicated slightly higher cell proliferation and ECM productions for both ECs and SMCs than other patterning features (Figs. 3 and 8). Although there was no significant difference in the SMC proliferation on flat mats after coculture with ECs (Fig. 3), the coculture of SMCs on flat mats resulted in significantly higher ECM levels, compared with SMCs cultured alone (Fig. 8). The enhancement of SMC growth and functions after coculture with ECs was also reflected in cell infiltration into fibrous mats of around 200 lm (Fig. 5) and cytoskeleton distribution with a high orientation index in patterned fibrous scaffolds (Fig. 4). Therefore, the coculture system on fibrous scaffolds promoted their ECM productions of both ECs and SMCs and enhanced the proliferation, cell infiltration, and cytoskeleton elongation of SMCs on patterned fibrous mats. The ECM proteins of collagen I, collagen IV, a-SMA, and laminin represent the major composition in the vascular wall33 and could be used to promote rapid endothelialization and latter proliferation of SMCs, which promoted vascular tissue regeneration on the scaffold and provided mechanical properties for engineered vessels.34 The layered

fibrous scaffold would be meaningful to precisely mimic blood vessels in vivo and is of great importance to enhance EC2SMC interactions and promote vascular reconstruction process. Although the coculture of SMCs and ECs have shown influences on their protein expressions,35 the precise molecular mechanisms for regulating the migration, proliferation, and network formation of ECs and SMCs on patterned fibrous scaffolds should warrant further investigations. CONCLUSIONS

Micropatterned fibrous mats with distinct ridges and grooves were created to load SMCs, and layered fibrous scaffolds were constructed after stacking on EC-loaded flat fibrous mats. SMCs were mainly distributed in the ridges of patterned fibrous mats, and the fiber alignment in these regions led to the formation of elongated cell bodies and intense actin filaments with a high orientation index. ECs were spread over the flat fibrous mats, while SMCs effectively infiltrated into fibrous scaffolds and the patterning distribution remained throughout the ridge regions. The coculture of ECs with SMCs on fibrous mats enhanced the cell proliferation, cell infiltration, and cytoskeleton elongation of SMCs, and the coculture system on fibrous scaffolds promoted ECM productions of both ECs and SMCs. Therefore, this configuration not only offers a promising technology in the design of tissue engineering scaffolds to construct blood vessels with in vivolike organized structure and unique properties, but also provides a platform to investigate cell2matrix and cell2cell interactions in highly organized tissues. REFERENCES 1. Patterson J, Martino MM, Hubbell JA. Biomimetic materials in tissue engineering. Mater Today 2010;13:14–22. 2. Xu ZC, Zhang WJ, Li H, Cui L, Cen L, Zhou GD, Liu W, Cao Y. Engineering of an elastic large muscular vessel wall with pulsatile stimulation in bioreactor. Biomaterials 2008;29:1464–1472. 3. Naito Y, Shinoka T, Duncan D, Hibino N, Solomon D, Cleary M, Rathore A, Fein C, Church S, Breuer C. Vascular tissue engineering: Towards the next generation vascular grafts. Adv Drug Deliver Rev 2011;63:312–323. 4. Shen JY, Chan-Park MB, He B, Zhu AP, Zhu X, Beuerman RW, Yang EB, Chen W, Chan V. Three-dimensional microchannels in biodegradable polymeric films for control orientation and phenotype of vascular smooth muscle cells. Tissue Eng 2006;12:2229–2240. 5. Truskey GA. Endothelial cell vascular smooth muscle cell coculture assay for high throughput screening assays for discovery of anti-angiogenesis agents and other therapeutic molecules. Int J High T Scr 2010;1:171–181. 6. McClendon MT, Stupp SI. Tubular hydrogels of circumferentially aligned nanofibers to encapsulate and orient vascular cell. Biomaterials 2012;33:5713–5722. 7. Cai L, Heilshorn SC. Designing ECM-mimetic materials using protein engineering. Acta Biomater 2014;10:1751–1760. 8. Zieris A, Prokoph S, Levental KR, Welzel PB, Grimmer M, Freudenberg U, Werner C. FGF-2 and VEGF functionalization of star-PEG heparin hydrogels to modulate biomolecular and physical cues of angiogenesis. Biomaterials 2010;31:7985–7994. 9. Chang S, Song S, Lee J, Yoon J, Park J, Choi S, Park JK, Choi K, Choi C. Phenotypic modulation of primary vascular smooth muscle cells by short-term culture on micropatterned substrate. PLoS One 2014;9:88089. 10. Sarkar S, Lee GY, Wong JY, Desai TA. Biomaterials development and characterization of a porous micro-patterned scaffold for vascular tissue engineering applications. Biomaterials 2006;27: 4775–4782.

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