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Approaches to Fabricating Multiple-Layered Vascular Scaffolds Using Hybrid Electrospinning and Thermally Induced Phase Separation Methods Hao-Yang Mi,†,‡,⊥ Xin Jing,† Jason McNulty,‡,⊥ Max R. Salick,‡,§ Xiang-Fang Peng,*,† and Lih-Sheng Turng*,‡,⊥ †

The Key Laboratory for Polymer Processing Engineering of Ministry of Education, South China University of Technology, Guangzhou, China ‡ Wisconsin Institute for Discovery, §Department of Engineering Physics, and ⊥Department of Mechanical Engineering, University of Wisconsin, Madison, Wisconsin, United States ABSTRACT: Fabrication of small-diameter vascular scaffolds has been a challenge in recent years, especially scaffolds with multiple layers. In this study, two approaches were proposed to fabricate triple-layered vascular scaffolds based on the electrospinning method and the thermally induced phase separation (TIPS) method. It was found that the electrospun fibers had a compact fibrous structure that provided good mechanical properties. The porous TIPS layer had high porosity and pore interconnectivity to facilitate cell penetration; however, this structure alone could not ensure sufficient mechanical properties for surgical applications. The triple-layered scaffolds, which consisted of electrospun TPU, TIPS TPU, and electrospun PPC layers, showed the highest mechanical properties and best structure and dimensions for vascular graft applications. Preliminary endothelial cell culture results showed that the cells could attach to and proliferate on the inside surface of the scaffolds with >95% viability.

1. INTRODUCTION The number of patients suffering from cardiovascular disease has increased in recent years. However, there are limited options for vascular grafts using autologous saphenous veins and mammary arteries for bypass surgery to treat vascular disease.1 Although many attempts have been made to prepare vascular grafts using decellularized tissue skeletons, biopolymers, and biodegradable synthetic polymers via various methods, several issues have not yet been resolved, such as the inability to mimic the layered structure of native blood vessels and improper mechanical properties.2,3 Developing vascular grafts for small-diameter vessel replacement (2−5 mm diameter) remains a challenge because of the risk of thrombosis and intimal hyperplasia, lack of suture retention, and mechanical failure.4−6 It is well-known that native blood vessels consist of three different layers: (i) intima, (ii) media, and (iii) adventitia. The intima, or inner layer, consists of a continuous monolayer of endothelial cells (ECs). The media, or middle layer, is composed of a dense population of smooth muscle cells (SMCs). The adventitia, or outer layer, mainly contains fibroblasts and perivascular nerve cells.3,7 Efforts have been made to produce vascular grafts that consist of multiple layers to mimic the structure of native blood vessels via different methods. A self-assembly method has been used to prepare vascular grafts that consist of three layers of cells by wrapping cell sheets to form a tubular structure.8 Decellularized bovine aortas have been combined with synthetic polymers to fabricate porous triple-layered vascular grafts as well.9 However, these methods are not practical due to their high cost and timeconsuming nature. Natural materials, such as silk protein, © XXXX American Chemical Society

collagen, and gelatin, have been used in the preparation of vascular grafts because of their high biocompatibility and cell affinity. Bilayered silk protein vascular grafts with a fibrous inner layer and a porous outer layer have been prepared using gel spinning and lyophilization methods.5 Silk fibroin, collagen, elastin, and polycaprolactone (PCL) have been electrospun to create a trilayered fibrous structure with mechanical properties that could be altered by varying the proportion of each component.10 Several kinds of synthetic polymers, such as poly(ε-caprolactone) (PCL),11 polyurethane (PU),12 and poly(L-lactide-co-glycolide) (PLGA),13 have been confirmed to be suitable for vascular grafts due to high endothelial cell adhesion and fast endothelialization. The biocompatibility of the grafts can usually be improved by combining them with natural materials, such as introducing gelatin to poly(ethylene glycol) (PEG)14 or adding fibrin to PCL.15 Electrospinning has been reported as a versatile technique to produce fibrous scaffolds with fiber diameters ranging from tens of nanometers to a few micrometers. This method has been widely used to prepare vascular grafts by electrospinning fibers directly onto a rotating mandrel.16,17 Hybrid or multiplelayered vascular grafts can be fabricated using dual-electrospinning or by electrospinning different materials.13,18 Electrospun vascular grafts could provide appropriate mechanical properties, such as suture retention and burst pressure, for surgical applications.19,20 However, the dense fibrous structure Received: September 16, 2015 Revised: December 16, 2015 Accepted: January 13, 2016

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DOI: 10.1021/acs.iecr.5b03462 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research

Figure 1. Schematic illustration of ele−TPU+TIPS−TPU+ele−PPC triple-layer scaffold fabrication: (a) electrospinning of TPU inner layer; (b) quenching TPU solution in ice to form the middle layer; (c) freeze-drying to remove solvent; (d) obtained double-layer scaffold; (e) electrospinning PPC outer layer; (f) final triple-layer scaffold.

vascular cells to proliferate over the scaffold and build up networks to maintain proper mechanical support.29,30 Conversely, the slow degradation of PU occupies space and restricts cell growth. Therefore, combining PU with a fast-degrading polymer would solve this problem. Poly(propylene carbonate) (PPC), an aliphatic polycarbonate composed of propylene epoxide and carbon dioxide (CO2), is a biocompatible and biodegradable polymer that has been used in tissue engineering in recent years.31,32 PPC scaffolds can be easily prepared via the electrospinning33 and TIPS methods.34 Moreover, PPC possesses higher mechanical performance than PU. The combination of PPC with TPU would enhance the scaffold stiffness as well. In this study, single-, double-, and triple-layered vascular scaffolds were prepared by combining the electrospinning and TIPS methods. Two approaches were proposed to prepare triple-layered vascular scaffolds. The microstructure and mechanical properties of the scaffolds, including the tensile strength, suture retention, and burst pressure, were investigated and compared. Preliminary endothelial cell cultures were performed by seeding with ECs for 2 weeks.

of the electrospun vascular grafts limits cell growth to the inner part of the grafts. The cells typically migrate to the surface of the substrate and can penetrate into the grafts only by degrading the material. For this reason, researchers have tried to combine different methods with electrospinning to prepare vascular grafts. For example, electrospinning has been combined with hydrogels,21 sponge coating,22 and fuse deposition23 to give vascular grafts diverse structures. Thermally induced phase separation (TIPS) is one of the more traditional techniques for fabricating porous tissue engineering scaffolds. High porosity and interconnectivity can be achieved by dissolving polymers in poor solvents or ternary solvents, followed by subsequent lyophilization.24 This method can easily be applied to vascular scaffold fabrication via an annular cylinder mold with a mandrel.25 Among all of the synthetic polymers, PU has been the most widely used material for vascular grafts because of its high flexibility and tear resistance. It has been found to be biocompatible with endothelial cells, and its cell affinity can be further improved through chemical modification with proteins.12,26 Thermoplastic polyurethane (TPU) is a class of PU that has a linear molecular structure and is easy to process. TPU has been used to fabricate vascular grafts via the TIPS method.27 The most significant defect of the TIPS method in the preparation of vascular grafts is the poor mechanical properties that are obtained due to the low mass-to-volume ratio. Therefore, the combination of electrospinning and TIPS could theoretically provide moderate mechanical properties and both a fibrous and porous microstructure simultaneously. Furthermore, triplelayered vascular scaffolds could be fabricated by repeating the electrospinning or TIPS process, yielding a triple-layered structure that would better resemble a native blood vessel. In addition, the degradation of the vascular scaffold needs to be coordinated with the speed of the tissue growth. Otherwise, issues such as blood leakage and mechanical failure could happen after implantation. PU typically has a slow biodegradation rate, taking >6 months to lose 30% of its mass in vivo.28 This slow degradation rate is usually favorable in vascular tissue engineering because it takes a long time for

2. MATERIALS AND METHODS 2.1. Materials. Medical grade TPU (Texin Rx85A) was supplied by Bayer Material Science, Inc. PPC (QPAC 40) was purchased from Empower Materials, Inc. N,N-Dimethylformamide (DMF), chloroform, 1,4-dioxane, acetic acid, and chitosan (media molecular weight 190 000−310 000 Da) were purchased from Sigma-Aldrich. All chemicals were used as received without further purification. The water used in this study was Milli-Q deionized water. 2.2. Solution Preparation. The multiple-layered scaffolds were fabricated from polymeric solutions via the electrospinning and TIPS methods. The TPU solution used for electrospinning was prepared by dissolving 1100 mg of TPU pellets into 10 mL of DMF in an oil bath with vigorous magnetic stirring at 70 °C for 8 h. The solution used to fabricate the TIPS TPU layer was prepared by dissolving 800 mg of TPU pellets into 10 mL of dioxane/water cosolvent B


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Figure 2. Schematic illustration of ele−TPU+TIPS−TPU+TIPS−PPC triple-layered scaffold fabrication: (a) electrospinning TPU inner layer; (b) quenching PPC solution in ice to form outer layer; (c) removing the barrier tube and freezing outer layer at −20 °C; (d) filling the gap with TPU solution and quickly freezing it in liquid nitrogen to form the middle layer; (e) freeze-drying to remove solvent; (f) final triple-layer scaffold.

freeze the solution. The middle layer was produced by lyophilization using a freeze-dryer (FreeZone 4.5 Freeze-Dry System, LABCONCO) for 5 days as illustrated in Figure 1c,d. The outer layer of the tubular scaffold was prepared by electrospinning PPC fibers out from the middle layer. The procedure was the same as electrospinning the TPU as illustrated in Figure 1e. The processing parameters used were as follows: 20 kV voltage, 0.5 mL/h flow rate, and 150 mm working distance. The prepared triple-layered scaffolds were named ele−TPU+TIPS−TPU+ele−PPC according to the methods and materials used in their fabrication. 2.3.2. Approach 2: Electrospinning + TIPS + TIPS. The second triple-layer tubular scaffold approach combined electrospinning TPU, TIPS TPU, and TIPS PPC. In this approach, the inner layer was still made by electrospinning TPU fibers coated with a chitosan layer. A mold with two sections was used to prepare the middle and outer layers. As illustrated in Figure 2b, the PPC TIPS solution was poured into the outer section of the mold, which had an inner diameter of 8 mm, and quenched in ice for 30 min. The middle barrier was removed after the PPC solution was frozen (Figure 2c). The TIPS TPU solution was then quickly injected into the gap between the inner and outer layer and frozen in liquid nitrogen to prevent the frozen PPC solution from being melted by the TPU solution (Figure 2d). The scaffolds were harvested after lyophilization for 5 days and named ele−TPU+TIPS−TPU+TIPS−PPC. 2.3.3. Fabrication of Single-Layered and Double-Layered Scaffolds. For comparison with the triple-layered scaffolds, single-layered and double-layered tubular scaffolds were prepared as well. The single-layered electrospun TPU scaffolds (ele−TPU) were prepared by electrospinning the TPU solution using the same working distance and flow rate. To increase the tube’s thickness, the electrospinning was performed for 6 h. The voltage in the first 3 h was 18 kV and was enhanced to 20 kV in the next 3 h. The single-layered TIPS TPU scaffolds (TIPS−TPU) were prepared by pouring the TIPS TPU solution into the cylindrical mold used in the first approach, followed by an ice quench, then frozen, and lyophilized. The same aluminum rod used to collect the electrospinning fibers was used as a mandrel to create the lumen of the scaffold. The

(dioxane/water = 8.5:1.5 vol). The pellets were dissolved in an oil bath with vigorous magnetic stirring at 70 °C for 6 h. PPC pellets (1600 mg) were dissolved in 10 mL of chloroform at room temperature for 6 h with vigorous magnetic stirring to prepare the solution for electrospinning. The TIPS PPC solution was prepared by dissolving 800 mg of PPC pellets into 10 mL of dioxane/water solvent (dioxane/water = 8.5:1.5 vol) at room temperature for 6 h with vigorous magnetic stirring. 2.3. Scaffold Fabrication Approaches. 2.3.1. Approach 1: Electrospinning + TIPS + Electrospinning. The first approach employed to fabricate triple-layered tubular scaffolds is shown in Figure 1. As shown in Figure 1a, the inner layer was produced by an electrospinning TPU solution on a rotating rod. Briefly, the prepared TPU electrospinning solution was loaded in a syringe that was connected to an 18 gauge blunt needle and mounted on a syringe pump with a constant solution flow rate of 0.5 mL/h. Next the fibers were created in a high-voltage field (18 kV) and were collected with a rotating aluminum rod (3.18 mm in diameter, purchased from McMaster Corp.) that was connected to a mini-motor rotating at a speed of about 1000 rpm. The needle-to-target distance (working distance) was 150 mm, and the electrospinning was performed for 2 h. The middle layer of the tubular scaffold was prepared using the TPU TIPS solution. To prevent the electrospinning TPU inner layer from being dissolved by the TPU dioxane/water solution and to improve the biocompatibility of the scaffold, a chitosan coating was added to the outside surface of the inner layer before the middle layer was produced. Briefly, the electrospun TPU tube was submerged in the chitosan solution (1% wt/vol chitosan in 3% vol acetic acid/water solvent) for 1 min, followed by overnight air drying in a fume hood. After the chitosan coating, the rod with the TPU inner layer was mounted in the center of a cylindrical aluminum mold having an inner diameter of 4.85 mm, the same as the mandrel of the tubular scaffold. The TPU TIPS solution was then poured into the gap between the mandrel and the mold, followed by ice quenching for 20 min to allow the occurrence of phase separation as shown in Figure 1b. Then the mold was placed in a −80 °C refrigerator (Thermo Scientific) for 3 h to completely C


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Figure 3. SEM images of single-layer vascular scaffolds prepared by (a−c) electrospinning TPU and (d−f) TIPS TPU at different magnifications.

double-layered TPU scaffolds (ele−TPU+TIPS−TPU) were prepared by the electrospinning and TIPS methods. The scaffolds were the middle product of the first approach and are shown in Figure 1d. 2.4. Human Umbilical Vein Endothelial Cell (HUVEC) Culture. HUVECs (Lonza) were maintained on T75 tissue culture-treated polystyrene flasks. Cells were fed every other day with an endothelial cell growth medium EGM-2-MV bullet kit (Lonza). Cells were passaged at a 1:6 ratio every 4−6 days with TrypLE Express (Life Technologies). Maintained cultures were regularly checked for mycoplasma. Before cell seeding, the tubular scaffolds were first sterilized with 70% ethanol for 30 min, followed by a series of phosphate buffer solution (PBS) washes, and then sterilized with ultraviolet (UV) light for another 30 min. The scaffolds were placed in 24-well TCPs and fixed with sterilized polyester double-sided adhesive tape (ARcare90106, Adhesive Research Inc., USA) to prevent the scaffolds from floating in the media. Cells were then seeded at a density of 5 × 104 cells/cm2. Spent medium was aspirated and replaced with 1 mL of fresh medium daily for screening samples. 2.5. Characterization. 2.5.1. Scanning Electron Microscopy (SEM). The structure of the prepared tubular scaffolds was observed via SEM. The scaffolds were quenched in liquid nitrogen for 30 min and fractured in cross section and then sputtered with a thin film of gold for 40 s. The whole view of the cross section was taken by a JEOL Neoscope SEM (Nikon) with an accelerating voltage of 10 kV. The high magnification images were taken by a fully digital LEO GEMINI 1530 SEM with a 3 kV accelerating voltage. Average measurements of the fiber diameter, pore size, and thickness of each layer were performed on the images using ImageJ software. 2.5.2. Tensile Tests. Tensile tests were performed on a mechanical testing machine (Instron 5967) in wet conditions at ambient temperature (23 °C). Electrospun membranes were soaked in PBS for 1 h and then stretched with a crosshead speed of 5 mm/min until the sample fractured. Statistical results were the average of five samples. Cyclical tensile tests were performed to investigate the sustainability of the mechanical properties of the scaffolds. The scaffolds were stretched to 50% strain at a crosshead speed of 5

mm/min and then fully released. The change of stress depended on the strain and was recorded for five cycles. 2.5.3. Circumferential Tensile Strength and Burst Pressure Measurements. The burst pressure strength of the electrospun tubes was estimated using the circumferential tensile strength according to a previously published method.13 A section of tube with a length (L) of 5 mm was cut and immersed in PBS for 1 h before being stretched in the circumferential direction by two custom-made L clamps at a constant rate of 5 mm/min using the same tensile testing machine. The ultimate circumferential tensile strength (UCTS) was defined as the sample fracture stress. The burst pressure of the tubular scaffolds was estimated from the UCTS values from an adaptation of Laplace’s law for intraluminal pressure.35,36 Five specimens of each group were tested. burst pressure (mmHg) = (UCTS) × t /r

t is the thickness of the tubular scaffold, and r is the intraluminal radius of the tubular scaffold at atmospheric pressure. 2.5.4. Suture Retention. The suture retention strength of the prepared tubular scaffolds was measured according to the standard ISO 7198. Scaffolds were cut into 2 cm long segments and immersed in PBS for 1 h prior to the test. One end of the scaffold was clamped by the fixed clamp, whereas the other end was pierced through at 2 mm from the edge using a tapered noncutting needle and connected to the movable clamp by a commercial suture (5−0 prolene suture, Ethicon Inc., USA). The crosshead speed to stretch the tube was 5 mm/min until the tube fractured. The maximum load was recorded as the suture retention strength. Five specimens of each group were tested. 2.5.5. Cell Viability and Proliferation. HUVEC viability was assessed 1 and 2 weeks after seeding via a Live/Dead Kit (Invitrogen). This kit allows simultaneous visualization of both live and dead cells. The assay was carried out following the manufacturer’s instructions. Briefly, cells were washed twice with PBS and then stained with 0.2% ethidium homodimer-1 (EthD-1) and 0.05% calcein-AM diluted in PBS for 45 min at room temperature. The stain utilized green fluorescent calceinAM to target esterase activity within the cytoplasm of living cells and red fluorescent EthD-1 to indicate cell death. After 45 D


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Figure 4. SEM images of double-layer vascular scaffolds prepared by electrospinning TPU+TIPS TPU at different magnifications. Scale bars: (a) 1 mm; (b) 500 μm; (c) 100 μm. The boundary of the layer is indicated by the red line.

Figure 5. SEM images of triple-layer vascular scaffolds prepared by (a−c) ele−TPU+TIPS− TPU+ele−PPC and (d−f) ele−TPU+TIPS−TPU +TIPS−PPC at different magnifications. The boundaries of the layers are indicated by the red lines.

specimens. The ele−TPU scaffolds were prepared via a layerby-layer deposition of electrospun nanofibers resulting in a 3D structure that consisted of numerous nanofibers. This fibrous structure has been reported to be similar to the structure of ECM and has shown a promising affinity in cell culture. However, low cell penetration is a drawback in real application. The cells could grow into the fibrous structure only by degrading the material. It has been reported that smooth surfaces with few but large pores were suitable for SMC penetration.37 Shalumon et al.38 and Yin et al.39 found that interconnected large pores with a multiscale structure or with the assistance of air pressure could promote SMC penetration into the inner part of the scaffolds. The TIPS method created an interconnected porous structure by phase separation. This structure was much easier for cells to migrate into compared with electrospun fibers. This scaffold, however, usually exhibited poor mechanical properties because of its high volume-to-mass ratio. The combination of these two methods might provide both good mechanical properties and cell affinity. The structure of the double-layered ele−TPU+TIPS− TPU scaffold is shown in Figure 4. The scaffold showed a compacted fibrous structure in the inner layer and a porous structure in the outer layer. The thickness of the inner layer was about 192.4 ± 11.6 μm and that of the outer layer about 424.5 ± 19.9 μm.

min, the stains were aspirated, and cells were washed with PBS twice. The stained cells were then imaged with a Nikon Ti-E confocal microscope. Advanced Research v.3.22 software was used for image processing. To quantitatively evaluate the proliferation and viability of the cells on the scaffolds, the following procedure was used. The stained cells were detached using TrypLE (Life Technologies) for 5 min at 37 °C and then collected and centrifuged at 200 rpm for 5 min. The supernatant was aspirated, and the cells were resuspended in 600 μL of PBS and filtered prior to analysis. The number and percentage of green fluorescent cells were acquired with an Accuri C6 flow cytometer (BD Biosciences).

3. RESULTS AND DISCUSSION 3.1. Morphology of the Multiple-Layered Scaffolds. 3.1.1. Cross-Section Structure of the Multiple-Layered Scaffolds. The cross section morphology of the single-layered scaffolds is shown in Figure 3. The ele−TPU scaffold (Figure 3a−c) presented a compact layered nanofibrous structure, whereas the TIPS−TPU scaffold (Figure 3d−f) showed a porous structure with pore sizes ranging from 20 to 60 μm. The inner diameters were the same for both scaffoldsm and the average thicknesses of the tubes for the ele−TPU specimens were 521.0 ± 24.2 and 534.4 ± 26.5 μm for the TIPS−TPU E


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Figure 6. SEM images of the inside and outside surfaces of vascular scaffolds prepared via different methods: (a) inside surface of electrospun TPU vascular scaffold; (b) outside surface of electrospun TPU vascular scaffold; (c) inside surface of TIPS TPU vascular scaffold; (d) outside surface of TIPS TPU vascular scaffold; (e) outside surface of electrospun PPC vascular scaffold; (f) outside surface of TIPS PPC vascular scaffold.

for the inner layer, 735.7 ± 37.1 μm for the middle layer, and 1063.3 ± 59.4 μm for the outer layer. It was noted that the pore size of the middle layer, which ranged from 4 to 20 μm, was obviously smaller than that of the outer layer, which ranged from 20 to 100 μm. This difference was caused by the fabrication procedure. Recall that the TIPS−PPC layer was quenched in ice, whereas the TIPS−TPU layer was directly frozen in liquid nitrogen to prevent the TIPS−PPC layer from being melted by the TPU solution. However, the solution could not experience sufficient phase separation in liquid nitrogen, which led to the formation of small solvent crystals when frozen and, therefore, small pore sizes after lyophilization. It is wellknown that the vessel diameter, wall thickness, and thickness of each layer are different for various vessels. The proposed approaches in this study are capable of preparing tubular scaffolds with various dimensions by controlling the conditions in the preparation of each layersuch as fiber collecting time, mold design, and solution concentrationto mimic the structure of the target vessel. 3.1.2. Inside and Outside Surfaces of the Multiple-Layered Scaffolds. The inside and outside surfaces of the multiplelayered scaffolds are the main areas of the vascular scaffolds that are in contact with the cells. Their structures are shown in

Native blood vessels consist of three layers of different cells as mentioned previously. The tubular scaffolds that have three layers are expected to better mimic the structure of native blood vessels. Two approaches were proposed to prepare triplelayered tubular scaffolds. PPC was introduced as the outer layer in the triple-layered scaffolds to improve the toughness of the scaffolds and modify the scaffold degradation rate. The structure of the triple-layered scaffolds is shown in Figure 5. The ele−TPU+TIPS−TPU+ele−PPC scaffolds are shown in Figure 5a−c; the thickness of the tube was 603.5 ± 48.3 μm. The thickness of the ele−TPU inner layer was 186.2 ± 15.6 μm; it was 331.0 ± 24.2 μm and 85.4 ± 19.4 μm for the middle TIPS−TPU layer and the outer ele−PPC layer, respectively. The inner layer and middle layer of the scaffolds were the same as the previously produced double-layered scaffold, whereas the ele−PPC layer showed a fibrous structure with much larger fibers than ele−TPU. The average thickness of the ele−TPU+TIPS−TPU+TIPS− PPC scaffold was 2204.2 ± 96.2 μm. The thickness of the middle layer and outer layer was determinate by the dimension of the cylinder mold. It was hard to fill the tubular mold with polymer solution if a smaller dimension cylinder mold was used. The average thickness of each layer was 302.4 ± 30.3 μm F


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Figure 7. Tensile test results of multiple-layered vascular scaffolds: (a) representative tensile test curves; (b) Young’s modulus; (c) tensile strength; (d) elongation at break.

ele−PPC and TIPS−PPC. Figure 6e shows the structure of ele−PPC. The average PPC fiber diameter was 3.1 μm, and few beads were observed from the image. The formation of beads was because the PPC fibers were electrospun onto the doublelayered tubular scaffold, which had low conductivity. The TIPS−PPC structure is shown in Figure 6f and presented a similar structure to that of the TIPS−TPU outside surface (Figure 6d). Its pore size ranged from 12 to 57 μm. 3.2. Mechanical Properties. Vascular grafts have strict mechanical property requirements to mimic native blood vessels and fulfill surgical needs. Fully mimicking the mechanical performance of native blood vessels has been a great challenge for artificial vascular grafts. Specifically, in our study, we prepared biodegradable vascular scaffolds that were eventually expected to be replaced by cells; in other words, they were not meant to be permanent grafts. Therefore, their mechanical strength needs to be coordinated with the speed of new tissue growth. To prevent mechanical failure during implantation, we chose scaffolds with mechanical performances superior to that of native blood vessels. Various tests were performed on the multiple-layered tubular scaffolds to evaluate their mechanical properties. Figure 7a shows the representative tensile test curves, from which it was found that the triple-

Figure 6. Panels a and b of Figure 6 show the inside and outside surfaces of ele−TPU, from which it was noted that the fibers in the inside surface were flatter than those on the outside surface. This might have been because some DMF residual remained as the inner fibers landed on the aluminum rod, which caused the fibers to soften. Moreover, aluminum has a high surface energy, which implies a low contact angle with liquids; therefore, the initial collected fibers got flattened as the DMF evaporated. However, the fibers in the other layers were in contact with TPU fibers that had a much higher surface roughness and lower surface energy than aluminum; hence, they were able to maintain their round structure. The average fiber diameter of the fibers in the outside layer was 327.8 ± 11.3 nm. Panels c and d of Figure 6 show the inside and outside surface structures of the TIPS−TPU scaffolds. The pore size on the inside surface ranged from 18 to 98 μm, whereas it was 16−60 μm for the outside surface. The smaller pore sizes on the outside surface were due to the faster cooling on the outside surfaces during the ice quench, which caused faster phase separation and smaller solvent crystals as compared to the inside surfaces. For the triple-layer tubular scaffolds prepared via the two approaches, the inside surfaces were the ele−TPU fibers, which is same as Figure 6a, whereas the outside surfaces were G


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Figure 8. Cyclical tensile tests of single- and multiple-layered vascular scaffolds.

Figure 9. (a) Suture retention strength and (b) burst pressure test results of single- and multiple-layered vascular scaffolds.

layered scaffolds showed different curve patterns than other specimens. Yield peaks were observed at about 50−60% strain on the triple-layered scaffolds, which was due to the failure of the rigid PPC layer. Figure 7b shows the Young’s modulus results, revealing that the TIPS−TPU scaffolds had the lowest Young’s modulus, whereas those scaffolds with a PPC outer layer showed relatively higher values. Overall, the Young’s modulus of all of the tubular scaffolds was higher than that of porcine coronary arteries, except for the TIPS−TPU scaffolds. The ele−TPU scaffolds showed the highest tensile strength, as shown in Figure 7c, due to their dense fibrous structure. The tensile strengths of the ele−TPU+TIPS−TPU and ele−TPU +TIPS−TPU+ ele−PPC scaffolds were close to porcine coronary artery values. The elongation-at-break of all scaffolds exceeded that of porcine coronary arteries, as shown in Figure 7d. Because the pulse is a repeated action that causes a repeated force on blood vessels, the cyclical mechanical properties of vascular scaffolds is also important. The cyclical tensile test results are shown in Figure 8. The stretch and release test was repeated five times, and the area enclosed in the stretch and

release curves at each time point represented the energy loss for each cycle. It was found that the TIPS−TPU and ele−TPU +TIPS−TPU scaffolds had low energy losses, whereas the ele− TPU+TIPS−TPU+TIPS−PPC showed the highest energy loss in the first cycle because of the porous PPC structure that could not regain its original shape after having been stretched. All of the scaffolds, however, showed low energy losses in the following cycles, which could be attributed to the flexibility provided by the TPU layers. The cyclical mechanical properties reflect the mechanical property sustainability of the scaffolds in the long term. The other two important mechanical variables are the suture retention strength (Figure 9a) and the burst pressure strength (Figure 9b). Values from human mammary arteries and human saphenous veins were used for comparison. The suture retention strengths of the TIPS−TPU scaffolds were lower than the referenced values, whereas all of the other vascular scaffolds showed higher suture retention strengths than the referenced values, which is sufficient for surgical applications. The burst pressure results showed that the TIPS−TPU scaffolds had lower values than the referenced human H


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Figure 10. Live/dead assay fluorescence images of HUVECs cultured on vascular scaffolds: (a) ele−TPU; (b) TIPS−TPU; (c) ele−TPU+TIPS− TPU; (d) ele−TPU+TIPS−TPU+ele−PPC; (e) ele−TPU+TIPS−TPU+TIPS−PPC after 2 weeks of cell culture. Green fluorescence indicates live cells, whereas red fluorescence represents dead cells. The substrate scaffold appears blue and purple due to the opening of the blue channel during imaging. The insets show the overview of the entire tubular scaffolds.

Figure 11. (a) Cell count and (b) cell viability statistical results for various tubular scaffolds after HUVEC seeding for 1 and 2 weeks.

results showed that the triple-layered scaffolds had 2−3 times higher mechanical performance, which should be sufficient for initial implantation. The gradual change of mechanical properties will be evaluated in future studies. 3.3. Scaffold Biocompatibility. Preliminary cell culture tests were performed to verify endothelial cell viability on prepared vascular scaffolds because the endothelial cells compose the intima of native blood vessels and have direct contact with the blood. A high coverage of endothelial cells on the vascular scaffold’s inside surface would be beneficial to prevent the occurrence of thrombi. After 2 weeks of cell culture, as shown in Figure 10, there were mostly live cells on the scaffolds, suggesting good biocompatibility. The substrate appeared blue in color because the blue channel was opened to enhance the image contrast during imaging. The quantitative cell proliferation and viability results are shown in Figure 11. The HUVECs were mostly observed on the inside surface of the elec−TPU scaffold (Figure 10a), which is ideal for vascular

mammary arteries and human saphenous veins, whereas the burst pressure of all of the other scaffolds were close to or higher than the referenced values. The difference in mechanical properties of the prepared multiple-layered tubular scaffolds was because of the difference in microstructure and materials. The poor mechanical performance of the TIPS−TPU scaffolds was due to its highly porous structure (Figure 3d−f). The ele− TPU scaffolds exhibited better mechanical performance due to their dense fibrous structure. The scaffolds made from the electrospinning and TIPS methods showed moderate mechanical properties. The involvement of a PPC third layer further improved scaffold mechanical properties because of the rigidity provided by the PPC outer layer. The triple-layered scaffolds with electrospun PPC as the outer layer showed a higher suture retention strength, burst pressure, and Young’s modulus than the ones with TIPS PPC as an outer layer, which proved that fibrous structures yield better mechanical properties for scaffolds than porous structures. The mechanical property I


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especially on the ele−TPU+TIPS−TPU+ele−PPC triplelayered scaffolds. Furthermore, PPC was found to have high endothelial cell affinity. The triple-layered scaffolds fabricated via the electrospinning and TIPS combined approach have great potential to be used as vascular grafts.

graft applications. Few cells were observed on the TIPS−TPU scaffold (Figure 10b), indicating that the HUVECs could not attach to the porous TPU structure. Similarly, on the doublelayered ele−TPU+TIPS−TPU scaffolds (Figure 10c), a layer of cells formed on the inside surface, whereas few cells dispersed on the outside layer. The number of cells grown on the triplelayered scaffold was higher than on the other scaffolds. The cell count trends were the same for 1 and 2 week time periods. The cell viability results showed that all of the scaffolds had >85% live cells after 1 and 2 weeks of culture except for the TIPS− TPU scaffold, which could not facilitate HUVEC attachment. The average cell viability improved about 5% during the second week of culture, and the number of cells increased significantly after 2 weeks of cell culture. These results suggest that endothelial cells can attach and grow on tubular scaffolds containing an electrospun fibrous TPU inner layer. Moreover, we noted that the triple-layered scaffolds with a PPC outer layer (Figure 10d,e) showed interesting results. HUVECs were observed not only on the internal surface but also on the outer PPC layer, regardless of their electrospun fibrous structure or porous structure. This finding is important because it may indicate that PPC might be able to interact actively with endothelial cells. PPC is a relatively new biocompatible material for tissue-engineering scaffolds. There are very few publications about its potential use in vascular grafts. Zhang et al. seeded bone marrow mesenchymal stem cells (MSCs) on PPC fibrous tubes to investigate the possibility of their being used for vascular grafts.40 Polyurethane, as a flexible biocompatible material, has been widely used in vascular recovery as previously mentioned. Therefore, the combination of these materials may provide better attributes when used in vascular grafts. Herein, the biological differences among scaffolds were mainly caused by the substrate material and the fibrous or porous structure differences. Moreover, it has been recently found that immobilization of specific peptides and genes on the inner surface of vascular scaffolds could further improve endothelialization and prevent thrombosis at the same time.41 Further optimization and functionalization of our multilayered vascular scaffolds will be conducted in future studies.

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AUTHOR INFORMATION

Corresponding Authors

*(L.-S.T.) E-mail: [email protected]. *(X.-F.P.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the support of the China Scholarship Council, the Wisconsin Institutes for Discovery (WID), the financial support of the National Nature Science Foundation of China (No. 51073061, No. 21174044), and the Fundamental Research Funds for the Central Universities (2015ZM093).

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REFERENCES

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4. CONCLUSION In this study, two approaches were proposed to fabricate triplelayered vascular scaffolds based on the electrospinning and TIPS methods to mimic the structure of native blood vessels. The inner layer of the scaffolds consisted of electrospun TPU nanofibers, the middle layer was composed of porous TIPS TPU, and the outer layer was made up of either electrospun PPC microfibers or porous TIPS PPC. Single- and doublelayered vascular scaffolds were also prepared to compare the structure and mechanical properties. It was found that the electrospun fibers had a compact layer-by-layer fibrous structure that yielded high values for Young’s modulus, suture retention, and burst pressure to the scaffold. The TIPS layer, which had high porosity and pore interconnectivity, facilitated cell penetration, whereas its mechanical properties were insufficient for surgical applications. The ele−TPU+TIPS− TPU+ele−PPC triple-layered scaffolds showed the highest mechanical properties and proper dimensions for vascular graft applications. The endothelial cell culture tests revealed that the cells could attach to the inner surface of the electrospun fibers, which is ideal to prevent potential thromboses, and that the cells proliferated on the vascular scaffolds with high viability, J


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