Materials Science and Engineering C 49 (2015) 40–50

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Electrospinning thermoplastic polyurethane/graphene oxide scaffolds for small diameter vascular graft applications Xin Jing a,b,c, Hao-Yang Mi a, Max R. Salick c,d, Travis M. Cordie c,e, Xiang-Fang Peng a,⁎, Lih-Sheng Turng b,c,⁎⁎ a National Engineering Research Center of Novel Equipment for Polymer Processing, The Key Laboratory of Polymer Processing Engineering of Ministry of Education, South China University of Technology, Guangzhou, China b Department of Mechanical Engineering, University of Wisconsin–Madison, WI, USA c Wisconsin Institute for Discovery, University of Wisconsin–Madison, WI, USA d Department of Engineering Physics, University of Wisconsin–Madison, WI, USA e Department of Biomedical Engineering, University of Wisconsin–Madison, WI, USA

a r t i c l e

i n f o

Article history: Received 10 July 2014 Received in revised form 26 November 2014 Accepted 17 December 2014 Available online 18 December 2014 Keywords: Thermoplastic polyurethane (TPU) Graphene oxide (GO) Electrospinning Vascular grafts Human umbilical vein endothelial cells (HUVECs)

a b s t r a c t Fabrication of small diameter vascular grafts plays an important role in vascular tissue engineering. In this study, thermoplastic polyurethane (TPU)/graphene oxide (GO) scaffolds were fabricated via electrospinning at different GO contents as potential candidates for small diameter vascular grafts. In terms of mechanical and surface properties, the tensile strength, Young's modulus, and hydrophilicity of the scaffolds increased with an increase of GO content while plasma treatment dramatically improved the scaffold hydrophilicity. Mouse fibroblast (3T3) and human umbilical vein endothelial cells (HUVECs) were cultured on the scaffolds separately to study their biocompatibility and potential to be used as vascular grafts. It was found that cell viability for both types of cells, fibroblast proliferation, and HUVEC attachment were the highest at a 0.5 wt.% GO loading whereas oxygen plasma treatment also enhanced HUVEC viability and attachment significantly. In addition, the suture retention strength and burst pressure of tubular TPU/GO scaffolds containing 0.5 wt.% GO were found to meet the requirements of human blood vessels, and endothelial cells were able to attach to the inner surface of the tubular scaffolds. Platelet adhesion tests using mice blood indicated that vascular scaffolds containing 0.5% GO had low platelet adhesion and activation. Therefore, the electrospun TPU/GO tubular scaffolds have the potential to be used in vascular tissue engineering. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The number of patients suffering from cardiovascular disease has increased in recent years. Tissue engineering aimed at the regeneration of malfunctioning tissues provides a possible treatment for this disease [1,2]. Vascular tissue engineering attempts to fabricate functional vascular grafts that could be used in vivo to replace blood vessels and help blood vessel regeneration [3]. Although synthetic grafts such as woven poly(ethylene terephthalate) (Dacron) and extended polytetrafluoroethylene (ePTFE) have been successfully used as large diameter blood vessel substitutes [4], they are not suitable in small diameter vessel applications due to the risk of thrombosis and intimal hyperplasia [5,6]. Many researchers have been engaged in vascular scaffold fabrication to search for suitable material systems and processing methods for small

⁎ Corresponding author. ⁎⁎ Correspondence to: L.-S. Turng, Department of Mechanical Engineering, University of Wisconsin–Madison, WI, USA. E-mail addresses: [email protected] (X.-F. Peng), [email protected] (L.-S. Turng).

http://dx.doi.org/10.1016/j.msec.2014.12.060 0928-4931/© 2014 Elsevier B.V. All rights reserved.

diameter blood vessels. It has been found that electrospinning is a versatile technique which is capable of producing fibrous small diameter tubular scaffolds [7]. A grounded rotation mandrel is often used in electrospinning to collect fibers and form a tubular structure. Based on the electrospinning technique, many recent attempts have been made to fabricate capable small diameter vascular scaffolds. For example, bilayered tubes (3 mm in diameter) were prepared via electrospinning as vascular graft candidates [8], axially aligned 3D nanofibrous tubes were prepared for cardiovascular applications [9], vascular scaffolds containing heparin and chitosan were found to have anti-thrombogenic properties as well as endothelialization [10], and a melt electrospinning technique was employed to prepare vascular prostheses [11]. Synthetic polymeric materials such as polylactic acid (PLA) [12], polycaprolactone (PCL) [13], poly(propylene carbonate) (PPC) [14], polyhydroxybutyrate (PHB) [15], and polyurethane (PU) [16], as well as natural materials like silk fibroin [17] and gelatin [18], have been applied in vascular tissue engineering to fabricate vascular grafts in recent years. Among them, PU as a kind of flexible elastomer has properties similar to blood vessels. Thermoplastic polyurethane (TPU) as a class of PU has linear segmented molecular chains, good processability, high

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elongation, and excellent abrasion and tear resistance [19]. As its synthesis and purification technologies continue to improve, medical grade TPU has good biocompatibility and has been used in many medical devices such as catheters [20], cover for pacemaker leads [21], and skin replacements [22]. Therefore, its application to the field of tissue engineering scaffolds warrants further investigation. Polymer composites have shown promising superior attributes compared with polymers alone in many fields. The addition of biocompatible additives could improve the mechanical performance, hydrophilicity, and biocompatibility of scaffolds and stimulate cellular interactions between cells and scaffolds. Additives, such as hydroxyapatite (HA) [23], chitosan [24], and carbon nanotubes (CNT) [25], have been extensively studied in various scaffold applications. Graphene and its derivatives have attracted considerable attention in recent years as potential biomaterials because of their unique physico-chemical properties [26,27]. Graphene oxide (GO), one of the most important derivatives of graphene, has a large number of hydrophilic groups on its surface, which promote the possibility of GO being used in tissue engineering applications. Although GO has been used for drug delivery [28], gene delivery [29], and culturing different types of cells [30,31], the understanding of its cytotoxicity is still controversial. It has been reported that GO shows stronger affinity to human adipose-derived stem cells (hASCs) as compared to glass [32] and that GO could improve mouse fibroblast adhesion [33]. However, some specific studies have found that the cytotoxicity of graphene and GO depends on environmental exposure [34], applied dose, and time [35,36]. Typically, it is believed that a low content of GO is nontoxic and can even stimulate cell conduct, while high concentrations of GO can cause oxidative stress in cells and the loss of cell viability [37]. Although GO has been employed in many types of cells, it has not been used in vascular graft applications with TPU, especially for small diameter vascular graft applications. Therefore, it is highly valuable to do such research to explore the capability of this material combination in vascular tissue engineering. Considering the advantages of GO and the excellent property of TPU in preparation of vascular grafts, in this study, the structure, properties and cell response on the TPU/GO electrospun scaffolds were investigated in detail. Regarding to the inconsistencies on the GO biocompatibility, 3T3 fibroblasts were first used for verification. Human umbilical vein endothelial cells (HUVECs) were tested afterwards to further investigate the endothelial cell response on the TPU/GO electrospun nanofibers. In addition, cyclical tensile tests, suture retention tests, and burst pressure tests were performed on tubular TPU/GO scaffolds to verify their capability to be used in vascular scaffold applications. 2. Materials and methods 2.1. Materials Medical grade TPU (Texin® Rx85A) was supplied by Bayer Material Science, Inc. Other materials used in this study, such as N,Ndimethylformamide (DMF), graphite, sodium nitrate (NaNO3), concentrated sulfuric acid (H2SO4), potassium permanganate (KMnO4), hydrogen peroxide (H2O2), and hydrochloric acid (HCl) (37%), were purchased from Sigma-Aldrich. All chemicals were used as received without further purification. 2.2. Graphene oxide (GO) synthesis and purification GO was prepared using a modification of the Hummers' method [38, 39]. Briefly, 5 g of graphite, 5 g of NaNO3, and 230 ml of H2SO4 were stirred together in a round-bottom flask in an ice bath. Then, 30 g of KMnO4 was slowly added into the solution to prevent the temperature from exceeding 5 °C. Next, the suspension was transferred to an oil bath and maintained at 35 °C for 1 h. After forming a thick paste, 400 ml of water was slowly added into the mixture. The solution was then transferred to a 90 °C oil bath, where it stayed for 30 min. Finally, the

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Fig. 1. Raman spectra of GO, TPU, 0.5% GO, 1% GO, 2% GO scaffolds.

suspension was further diluted with 1000 ml of water, and then 30 ml H2O2 was added slowly, turning the color from dark brown to bright yellow. After being cooled, the product was filtered and then washed in succession with a 3% HCl aqueous solution and ethanol (2×). Multiple washes with deionized (DI-) water were then performed until the pH of the product was about 7. The solid paste was then dried for 5 days using a freeze drier (Freezone 4.5, Labconco, USA). 2.3. Scaffold fabrication 2.3.1. Electrospinning membrane scaffolds A certain amount of GO was dispersed into 5 ml of DMF with 1 h ultrasonication using a probe ultrasonic device (UP200H, Hielscher Ultrasoud Tech.). Two grams of pre-dried TPU pellets was dissolved in 15 ml of DMF for 8 h with 200 rpm magnetic stirring at 70 °C. The dispersed GO was then added into the TPU solution and stirred further at 500 rpm at 70 °C for 6 h. The TPU concentration in the solution was 10% w/v and the weight concentrations of GO were 0%, 0.5%, 1%, and 2% of TPU. The samples prepared from the different solutions were named TPU, 0.5% GO, 1% GO, and 2% GO according to their GO concentrations. The solutions were immediately used for electrospinning after preparation in order to prevent phase separation. The solution was loaded in a plastic syringe connected to an 18 gauge blunt-end needle and the syringe was mounted on a digital syringe pump (Harvard Bioscience Company). The electrospinning procedure was carried out using a voltage of 18 kV, a working distance of 150 mm, and a flow rate of 0.5 ml/h. The scaffold membranes were collected using a thin sheet of aluminum foil. The scaffolds for cell culture were directly electrospun on autoclaved sterilized stainless steel washers (McMaster-Carr). All scaffolds were collected after electrospinning for 1 h. 2.3.2. Electrospinning tubular scaffolds The tubular scaffolds were collected using a grounded aluminum tube (3.18 mm outer diameter, McMaster-Carr) which was connected to a mini motor whose rotating speed was about 1500 rpm. The applied voltage was 20 kV, the solution flow rate was 0.5 ml/h, and the distance between the needle tip and the collecting tube was 200 mm. The tubular scaffolds were removed from the aluminum tube after being electrospun for 3 h. 2.3.3. Plasma treatment of scaffolds The electrospun membranes were also treated with oxygen plasma using a plasma etcher (PE-200, Plasma Etch, Inc.) to activate the scaffold surface in order to improve the hydrophilicity of the scaffold. The

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Fig. 2. Microstructure of electrospun scaffolds (a) TPU, (b) 0.5% GO, (c) 1% GO, and (d) 2% GO at low (left) and high (right) magnifications. The presence of GO in the fibrous structure can be seen in the high magnification images of composites.

samples were placed in glass petri dishes and treated with oxygen plasma with an oxygen flow rate of 50 ml/min for 36 s at 100 W R.F. power. Samples were used for other tests (e.g., cell culture and water contact angle tests) immediately after plasma treatment. 2.4. 3T3 fibroblast cell culture 3T3 fibroblasts were cultured on the scaffolds to test the biocompatibility of the scaffolds. Cells were maintained on 6-well tissue culturetreated polystyrene plates (BD Falcon). Cells were fed every other day with a high-glucose 20% serum medium consisting of high-glucose DMEM (Gibco), 20% fetal bovine serum (WiCell), 2 mM L-glutamine (Invitrogen), and penicillin–streptomycin (Invitrogen). Cells were passaged at a 1:40 ratio every 6 days via 5-minute Trypsin-EDTA treatment (Invitrogen). Maintained cultures were regularly checked for mycoplasma. Test scaffolds were sterilized with UV light for 30 min on each side and then placed in 24-well tissue culture-treated polystyrene plates (TCPs) together with stainless washers to prevent scaffold flotation. 3T3 cells were treated with Trypsin-EDTA for 5 min and washed with phosphate-buffered saline (PBS) prior to seeding. Cells were then seeded at a density of 2.5 × 104 cells/cm2. Spent medium was aspirated and replaced with 1 ml of fresh medium daily. 2.5. HUVECs' cell culture Scaffolds with and without plasma treatment were used for HUVEC culture to investigate the scaffold affinity to blood vessel cells. Human umbilical vein endothelial cells (HUVEC, Lonza) were maintained on tissue cultured-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 1:6 ratio every 4 to 6 days with TrypLE Express (Life Technologies). Maintained cultures were regularly checked for mycoplasma. Test samples were first UV sterilized for 30 min on each side then placed in 24-well TCPs. The HUVECs were washed once with PBS then treated with TrypLE Express for 5 min. Cells were then seeded at a density of 2.5 × 104 cells/cm2. Spent medium was aspirated and replaced with 1 ml of fresh medium daily for screening samples. Table 1 Fiber diameter and porosity statistical results of electrospun TPU, 0.5% GO, 1% GO, 2% GO scaffolds. Data presented as average ± stand deviation. Sample

TPU

0.5% GO

1% GO

2% GO

Fiber diameter (nm) 295.3 ± 33.2 324.6 ± 49.4 330.4 ± 76.5 397.0 ± 121.8 Porosity (%) 84.3 ± 2.0 82.5 ± 3.3 79.2 ± 3.7 73.8 ± 5.5

2.6. Characterization 2.6.1. Raman spectra Raman spectra analysis of the scaffolds for material characterization and confirmation of GO in the TPU matrix was performed with a DXR Raman microspectrometer (Thermo Scientific). The membrane scaffolds were folded and fixed on glass slides for measurement. Raman spectra were recorded in the range of 100 to 3500 cm−1. 2.6.2. Scanning electron microscopy (SEM) SEM observations of the electrospun scaffolds and the scaffolds after cell culture were performed on a fully digital LEO GEMINI 1530 SEM with a 3 kV accelerating voltage after sputtering the samples with a thin film of gold for 40 s. The fiber diameter of the electrospun fibers was measured using Image Pro-Plus software. The average fiber diameter was the mean value of 50 individual fibers from three SEM images. The scaffolds with cells undergo a cell fixation process before gold sputtering. Briefly, samples were rinsed twice with Hanks' balanced salt solution (HBSS; Thermo Scientific). HyClone HyPure molecular biology grade water (Thermo Scientific) was mixed with paraformaldehyde to make a 4% solution. The rinsed samples were then immersed in the solution for 15 min. The samples were dehydrated using a series of ethanol washes (50%, 80%, 90%, and 100% for 30 min each), and dried in a vacuum desiccator for 2 to 3 h. 2.6.3. Porosity measurement The porosity of the prepared electrospun scaffolds was measured using apparent density method [40]. The scaffolds were trimmed into rectangular shape and their weight and dimensions were measured. Eq. (1) was used to calculate the porosity. The porosity value is the average of 5 samples.

Porosity ¼

V th ρ−W m  100% V th ρ

ð1Þ

where Wm is the measured weight, ρ is the density of TPU, which is 1.12 g/cm3 for the TPU used in this study, and Vth is the volume of the foamed sample. 2.6.4. Mechanical properties Tensile tests were performed on a mechanical testing machine (Instron 5967) in wet conditions at ambient temperature (23 °C). Electrospun membranes were cut into 8 mm × 30 mm rectangular shapes and soaked in PBS for 1 h then stretched at a crosshead speed

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Fig. 3. Tensile test results of TPU, 0.5% GO, 1% GO, 2% GO scaffolds. (a) Representative strain vs. stress curves, (b) statistical results of tensile modulus, tensile strength and strain at break.

of 5 mm/min until the sample fractured. Statistical results were the average of five samples. The burst pressure strength of the electrospun tubes was estimated using the circumferential tensile strength according to a previously published method [41]. 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 pressures of the tubular scaffolds were estimated from the UCTS values from the adaptation of Laplace's law for intraluminal pressure [42,43]. Five specimens from each group were tested. Burst pressure ðmm HgÞ ¼ ðUCTSÞ  t=r

ð2Þ

where, t was the thickness of the tubular scaffolds, and r was the intraluminal radius of the tubular scaffolds at atmospheric pressure. The suture retention strength of the electrospun tubes was measured according to the standard ISO 7198. Electrospun tubes were cut into 2 cm long segments and immersed in PBS for 1 h prior to the test. One end of the tube was clamped by the fixed clamp of the tensile test machine; the other end was connected to a movable clamp by a commercial suture (5–0 prolene suture, Ethicon Inc., USA). The suture was pierced through the test tube 2 mm from the edge using a tapered noncutting needle. 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 from each group were tested.

2.6.5. Water contact angle (WCA) test The wettability of the scaffolds and scaffolds with plasma treatment were evaluated via water contact angle tests. The tests were performed at room temperature in a Dataphysics OCA 15 optical contact angle measuring system using the sessile drop method. Four microliters of deionized water was dropped on the scaffolds and images at 0 and 5 s were taken and measured. The WCA of each scaffold was the average value of three different spots.

2.6.6. Fibroblast and HUVEC cell viability Cell viability was determined 3 days and 10 days after seeding. Viability was assessed via a Live/Dead Kit (Invitrogen). The stain utilized green fluorescent Calcein-AM to target esterase activity within the cytoplasm of living cells and red fluorescent ethidium homodimer-1 (EthD1) to indicate cell death. Stained cells were imaged with a Nikon Ti-E confocal microscope. Advanced Research v.3.22 software was used for image analysis, the number of live cells and dead cells were counted and the percentage of live cells was calculated as cell viability.

2.6.7. MTS assay for fibroblast cell culture Cell Titer 96 Aqueous One Solution Cell Proliferation assay (Promega) was used to determine the number of cells. Standard curves were established by performing the tests on cells seeded on the cell culture wells and confirmed by comparison to hemocytometer readings. Upon testing, cells were treated with an 83% media, 17% MTS solution and allowed to incubate for one hour. After incubation, 100 μl of spent media was removed and added to a clear 96-well plate. The absorbance of this plate at the 450 nm wavelength was then read with a GloMax-Multi + Multiplate Reader (Promega) and the subsequent number of cells was determined relative to a negative control. 2.6.8. HUVEC cell attachment The number of cells attached on the pristine scaffolds and plasma treated scaffolds were evaluated after culturing the cells for 2 h. The samples were rinsed with PBS to eliminate any unattached cells and the cell nucleus was stained with DAPI and imaged using the aforementioned confocal microscope. The number of cells attached was counted from multiple images using Image Pro-Plus software. 2.6.9. Platelet adhesion Based on the cell culture results, TPU scaffolds containing 0.5% GO were chosen to investigate the hemocompatibility and pure TPU scaffolds were used as controls according to the method proposed elsewhere [44]. Briefly, fresh animal platelet-rich plasma was obtained from mice. A quantity of 20 μl of platelet-rich plasma was carefully dropped on the electrospun TPU and TPU with 0.5% GO scaffolds. After incubation for 30 min at room temperature, the treated scaffolds were carefully rinsed twice by PBS. Adhered platelets were fixed with 2.5% glutaraldehyde for 30 min, followed by a dehydration procedure with a series of ethanol washes (30%, 50%, 70%, 90%, and 100% ethanol for 30 min each). Finally, the dehydrated scaffolds were dried in a vacuum desiccator for 2 h before gold sputtering for SEM. At the same time, TPU and TPU with 0.5% GO scaffolds experienced the same washing process without the platelet adhesion treatment. 2.6.10. Statistical analysis Statistical analysis was performed on biological experiments using a one-way analysis of variance (ANOVA) and the difference significance between groups was compared as well, with the level of significance set at p b 0.05. 3. Results and discussion 3.1. Raman spectra The Raman spectra results are shown in Fig. 1. On the TPU curve, the peaks at 2920, 2864, and 2801 cm− 1 corresponded to the stretching

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Fig. 4. Day 3 (a–h) and day 10 (i–p) fibroblast cell culture results of (a, e, i, m) TPU, (b, f, j, n) 0.5% GO, (c, g, k, o) 1% GO, (d, h, l, p) 2% GO scaffolds. (a–d and i–l) are fluorescence microscope images showing cell viability with cell viability on top right, (e–h and m–p) are SEM images showing cell morphologies. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

vibrations of the \CH2 groups. The strong peak at 1615 cm−1 was ascribed to the aromatic breathing mode of vibration. The peaks located at 1455 and 1479 cm−1 were due to the bending vibrations of \CH2

[45]. However, the intensity of these characteristic polymer bands decreased with increased GO in the composites. On the contrary, the D and G bands of GO could be observed in all spectra of the composites, with the peak intensity becoming stronger as the GO content increased [46]. This was due to the strong response of GO to the Raman spectra relative to the bonds in the polymer [39]. The results confirmed the presence of GO in the TPU matrix and the GO intensity increased with increasing content. 3.2. Electrospun scaffold morphologies and tensile properties

Fig. 5. 3T3 fibroblast MTS results on TPU, 0.5% GO, 1% GO, 2% GO scaffolds at day 3 and day 10.

Fig. 2 shows the microstructure of electrospun TPU and TPU/GO fibers at low and high magnifications. As can be seen, the collected fibers were bead-less and randomly dispersed, which represents a feasible imitation of the structure of extracellular matrix (ECM). The high magnification images show the presence of GO in the fibrous structure. GO plates that have larger sizes than the fiber diameters were embedded in TPU fibers in single layers or stacked in multiple layers. It has been reported that graphene has good miscibility and dispersion in TPU [47]. Some other studies found that GO formed small aggregates within a polyurethane matrix at high loading levels, and that dispersion could be enhanced via sufficient ultrasonication and surface modification

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Fig. 6. Water contact angle test results of (a) non-treated scaffolds and (b) plasma treated scaffolds at 0 s and 5 s. Inset images are the representative recorded images.

[48,49]. In the current study, stacked GO sheets were observed on the samples with 1% and 2% GO content as shown in Fig. 2(c) and (d). These observations indicated that the aggregation of GO may have happened at these GO loading levels. The statistical results of fiber diameter and porosity are listed in Table 1. It was found that the addition of GO increased the TPU fiber diameter slightly at low concentrations (b1%), while the fiber diameter increased dramatically when the GO loading was 2%. The fiber diameter deviation also increased as the GO loading level increased. This might have been caused by an increase of solution viscosity as the GO content increased. Similar changes in fiber diameter were also observed in electrospun polystyrene (PS)/carbon nanotubes (CNTs) [50]. The aggregation of GO might have induced a nonuniform fiber diameter in the high GO content scaffolds (e.g., 2% GO). The porosity of the scaffolds showed a decreasing trend as the GO content increased according to Table 1, which was due to the increased fiber diameter. Nevertheless, the porosities for TPU and 0.5% GO scaffolds still exceeded 80% which is high enough for many types of cells [51]. The representative stress versus strain curves and statistical resulting from the tensile tests of the electrospun scaffolds are shown in Fig. 3. It is obvious that the tensile modulus and tensile strength of the scaffolds increased as the GO content increased due to the high stiffness provided by GO and the increase of electrospun fiber diameter. Compared with neat TPU, the 2% GO scaffolds had a 5 times higher tensile modulus and a 3 times higher tensile strength. Similar improvements in mechanical properties of TPU were also reported in TPU/ graphite [52] and TPU/graphene [53] composites. The results for strain-at-break were not conclusive due to the relatively large deviation. During the tests, most samples either broke at the place where they had been clamped or slipped out of the clamp. Therefore, no solid conclusion could be drawn regarding the strain-at-break results.

spots) were observed from the fluorescent images. The cell viability for TPU and 0.5% GO scaffolds was still higher than 95%. While it was noticed that the 1% GO and 2% GO scaffolds showed more dead cells with viability of 92% and 87%, respectively. These results suggested that the cell viability of scaffolds has been reduced when the GO content exceed 1 wt.% The SEM images (Fig. 4(m–p)) show that the whole scaffold is covered by cells and it is hard to distinguish individual cells because the ECM produced by the cells obscures the cell boundary. The cracks on the SEM images were caused by dehydration during cell fixation for SEM. In order to further assess the cell proliferation behavior of scaffolds, MTS assays were performed at day 3 and day 10 time points. The results are shown in Fig. 5, from which it was found that, because of the large standard deviation of the data, the difference between neat TPU and TPU/GO composites was not statistically significant at day 3, but 0.5% GO scaffolds had significantly more cells than 2% GO scaffolds. At day 10, 0.5% GO scaffolds supported the largest cell population, while the cell number decreased as the GO content increased; in fact, the number of cells on 2% GO scaffolds was significantly lower than those on neat TPU scaffolds. These results suggest that a small amount of GO could slightly improve cell proliferation; while large GO doses will likely cause cell death and are not helpful for cell proliferation. Even though many publications have reported the biocompatibility of graphene and graphene derivatives [55,56], it is still widely agreed that GO has dose- and time-sensitive cytotoxicity [35,37], which means that while low concentrations of GO are nontoxic to most cells, and can even stimulate cell attachment and growth, high concentrations of GO usually cause dose-dependent oxidative stress in cells and induce loss of cell viability [37]. 3.4. Hydrophilicity and effects of GO and plasma treatment

3.3. Fibroblast cell culture 3T3 fibroblasts were seeded on the TPU and TPU/GO scaffolds for 3 days and 10 days to investigate the biocompatibility of the scaffolds by studying cell viability and proliferation. The fluorescent images and SEM images of day 3 cell culture are shown in Fig. 4(a–h). In Fig. 4(a– d), it was found that after 3 days of cell culture, there were already large amounts of cells on all scaffolds and a few dead cells were observed. The cell viability of all samples exceeded 95% at day 3, indicating good biocompatibility. The individual cell morphology can be observed on the SEM images (Fig. 4(e–h)), from which it was found that the fibroblasts attached to the scaffolds exhibited flat spindle shapes rather than round ball shapes, which demonstrated that the cells interacted and bonded well with the substrate fibers [54]. Fig. 4(i–p) shows day 10 3T3 fibroblast cell culture results. In consistent with the day 3 results, the majority of the cells on the scaffolds were live cells, as indicated by green spots, and only a few dead cells (red

Wettability has been recognized as an important characteristic for tissue engineering scaffolds since the hydrophilicity of scaffold surfaces affects cell attachment significantly [57]. Methods such as surface modification [58], surface coating [59], blending [60], and surface treatment [61] have been used to improve polymer scaffold surface hydrophilicity to stimulate cellular interactions. Among them, surface treatment with oxygen plasma is widely used and also the simplest method that could improve scaffold surface hydrophilicity [62,63]. Therefore, the effects of plasma treatment on TPU and TPU/GO composite scaffolds were investigated in this study via water contact angle (WCA) tests. The results are shown in Fig. 6. The WCAs of non-treated scaffolds decreased as GO content increased due to the hydrophilicity provided by the GO which has abundant hydroxyl and carboxyl groups on it. The WCA, however, was still more than 95° when the GO content reached 2%, with the WCA decreasing very slow over time. The scaffolds treated with

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addition of GO could improve the hydrophilicity of TPU slightly, while oxygen plasma treatment could dramatically increase the hydrophilicity of the scaffolds. 3.5. HUVEC culture

Fig. 7. Number of cells attached on non-treated and plasma treated TPU and TPU/GO composite scaffolds.

oxygen plasma, on the other hand, all showed much lower WCAs, and the difference between scaffolds with various GO contents was not significant. Moreover, the WCAs decreased very fast from 0 to 5 s and became zero eventually. These results indicate that the

HUVECs were cultured on the scaffolds to evaluate the feasibility of TPU and TPU/GO composite scaffolds to be used in vascular scaffold applications since endothelial cells are a crucial cell type that compose the interior surface of blood vessels and have direct contact with the blood [64]. They protect blood from forming thrombi. In the current study, the HUVEC interactions with the scaffolds were evaluated via cell attachment and cell viability tests. The number of cells attached on the non-treated and plasma-treated scaffolds is shown in Fig. 7, from which it was found that the number of cells attached on the plasma-treated scaffolds was much larger than that on non-treated scaffolds. Similar improvements were reported in other publications as well [65,66]. It was found that the scaffold with 0.5% GO showed the greatest cell attachment for both non-treated and plasma-treated groups. It has been reported that GO could greatly enhance the attachment and proliferation of mammalian cells [67]. However, as found in the fibroblast cell culture experiment and other references [35,37], large amounts of GO cause a decrease in cell viability.

Fig. 8. Day 3 HUVECs' cell culture results of (a, e) TPU, (b, f) 0.5% GO, (c, g) 1% GO, (d, h) 2% GO scaffolds and (i, m) TPU, (j, n) 0.5% GO, (k, o) 1% GO, and (l, p) 2% GO plasma treated scaffolds. Panels a–d and i–l are fluorescence microscope images showing cell viability with cell viability on top right, panels e–h and m–p are SEM images showing cell morphologies. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

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Fig. 9. Day 10 HUVECs' cell culture results of (a, e) TPU, (b, f) 0.5% GO, (c, g) 1% GO, (d, h) 2% GO scaffolds and (i, m) TPU, (j, n) 0.5% GO, (k, o) 1% GO, (l, p) 2% GO plasma treated scaffolds. Panels a–d and i–l are fluorescence microscope images showing cell viability with cell viability on top right, panels e–h and m–p are SEM images showing cell morphologies.

Fig. 10. Day 10 HUVECs' cell culture results on electrospinning 0.5% GO tubular scaffold. (a) Cross section fluorescent image, and (b) 3D fluorescent image.

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Fig. 11. (a) Photography of electrospinning 0.5% GO tubular scaffold, (b) cyclical tensile test curve of tubular scaffold, (c) suture retention results, and (d) burst pressure of tubular scaffold.

Thus, 0.5% GO might be the best concentration for HUVECs to attach on TPU/GO composite electrospun scaffolds. The viability of HUVECs on non-treated and plasma-treated scaffolds was tested via a Live/Dead Assay at day 3 and day 10 time points as shown in Figs. 8 and 9, respectively. It was found in Fig. 8 that, overall, the non-treated scaffolds had lower cell viability than their plasmatreated counterparts, by comparing Fig. 8(a–d) with Fig. 8(i–l), which proved that plasma treatment could enhance cell attachment as well as cell viability. By comparing within each group, it was found that 0.5% GO scaffolds had more live cells (green spots) than other scaffolds for both non-treated and plasma-treated groups, and the plasmatreated 0.5% GO scaffold had cell viability as high as 99%. SEM images show HUVEC morphologies. Unlike 3T3 fibroblasts, HUVECs spread out in various directions into flat sheet shapes, with cells on nontreated and plasma-treated scaffolds not showing any obvious differences in terms of cell morphology. The cell coverage area for the 0.5% GO scaffolds was found to be larger than other scaffolds for both nontreated and plasma-treated groups, which corresponds to the fluorescent images. Similar trends were found at day 10 time point as shown in Fig. 9. More obvious than day 3, the viability of cells on the scaffolds treated with oxygen plasma was higher than 95%; while for those without plasma treatment, it was only 65%. The 0.5% GO scaffolds still supported higher cell number and viability than other scaffolds. However, it was noticed that the cell viability for 2% GO scaffolds was the lowest in each group, which indicates that the high content of GO may lead to cell death as reported elsewhere [37]. The SEM images showed that larger areas of the scaffolds were covered by cells, indicating that cells migrated toward each other to form cell membranes by day 10. It is already hard to distinguish the morphology of individual cell from SEM

images. These results suggested that plasma treatment was favorable for HUVECs' attachment, which further leaded to quicker cell proliferation rate and higher cell viability as compared with scaffolds without plasma treatment. Hence, it was concluded that 0.5 wt.%. GO loading in TPU might be the best concentration for HUVECs; which is capable for vascular scaffold applications. HUVECs were cultured on 0.5% GO tubular scaffolds as well for 10 days to explore their growth behavior on tubular structures. As shown in Fig. 10, after 10 days of culture, the cells were mostly attached onto the inner surface of the scaffolds, forming a cell membrane layer. This cell conduct is favorable because endothelial cells make up the intima of blood vessels [68]. A high coverage of endothelial cells on the vascular scaffolds' inside the surface would be beneficial to prevent the occurrence of thrombi. Therefore, the results further indicated that the prepared TPU/GO electrospun tubular scaffolds with a low loading level of GO are suitable for vascular tissue engineering with regard to biocompatibility. 3.6. Mechanical performance of 0.5% GO tubular scaffold In order to further assess the capabilities of TPU/GO composite electrospun scaffolds in vascular graft applications, the mechanical performance of the 0.5% GO tubular scaffold was tested. The results are summarized in Fig. 11. Fig. 11(a) shows a photograph of the fabricated TPU/GO tubular scaffold with inner diameter and thickness 3.18 mm and 0.38 mm, respectively. Thus, the TPU/GO composite was able to be electrospun into a tubular shape with diameter smaller than 5 mm. Cyclical tensile tests were performed in the longitudinal direction; the results are shown in Fig. 11(b). It was found that the energy loss in the first cycle was the largest, and that the stress versus strain curve

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Fig. 12. Platelet adhesion results of (a) untreated TPU, (b) treated TPU, (c) untreated 0.5% GO, and (d) treated 0.5% GO scaffolds. (For interpretation of the references to color in this figure, the reader is referred to the web version of this article.)

followed a similar pattern in the following cycle, meaning that the scaffold had high flexibility which allowed it to recover from the cyclical force loading. This kind of behavior is required for vascular grafts. Suture retention strength (Fig. 11(c)) was found to be higher than human mammary arteries and human saphenous veins [43], demonstrating that the suture retention strength of the scaffolds met requirement for implantation (N2 N) [69]. The burst pressure (Fig. 11(d)) was also found to exceed that of human carotid arteries and human saphenous veins [70]. Those results suggested that the 0.5% GO tubular scaffold has the potential to be used in small diameter vascular graft applications.

3.7. Platelet adhesion on TPU and 0.5% GO scaffolds Generally, in the case of small-diameter tissue engineering vascular scaffolds, early thrombus formation often caused graft failure [71]. In this study, adhesion of blood platelets on the pure TPU and TPU with 0.5% GO scaffolds was investigated by SEM observation and the results are shown in Fig. 12. The TPU scaffolds after blood treatment (Fig. 12(b)) experienced some adhered platelets and spread into flat shapes on the fiber mat, showing an activated state of platelets (as indicated in the red ellipses). On the other hand, the scaffolds containing 0.5% GO (Fig. 12(d)) had very few platelets attached on the surface. Moreover, the observed round morphology of the platelets suggested that the adhered platelets were not activated. It has been reported that platelet spreading and aggregation are makers of platelet activation, which is linked to thrombotic events. Therefore, based on the preliminary platelet adhesion tests, the introduction of GO is unlikely to cause thrombosis on the scaffold, thereby making its use in vascular tissue engineering applications possible.

4. Conclusions Thermoplastic polyurethane (TPU) and TPU/graphene oxide (GO) composite membranes and tubular scaffolds (inner diameter of 3.18 mm) were fabricated via electrospinning at different GO loading (0.5%, 1% and 2%). It was found that the tensile strength, Young's modulus, and hydrophilicity of the scaffolds increased as GO increased. Plasma treatment improved scaffold hydrophilicity dramatically. The biocompatibility of the scaffolds was assessed by 3T3 fibroblast cell culture and the ability to be used as vascular grafts was also evaluated via human umbilical vein endothelial cell (HUVEC) culture. It was found that cell viability was the highest for both fibroblast and HUVECs on the scaffolds at a GO loading level of 0.5 wt.% and fibroblast proliferation and HUVEC attachment were the highest for this group of scaffolds as well. Oxygen plasma treatment was found to enhance HUVEC viability and attachment. The endothelial cells could form a cell layer on the inner surface of the tubular scaffolds which resembles the native blood vessel structure. Results of platelet adhesion tests indicated that a low loading of GO is unlikely to cause thrombosis on the scaffolds. Moreover, the suture retention strength and burst pressure of tubular TPU/GO scaffolds containing 0.5 wt.%. GO exceeded the mechanical requirements of human blood vessels, indicating that the TPU/GO electrospun scaffolds at low GO loading level have the potential to be used in small diameter vascular graft applications. Acknowledgments The authors would like to acknowledge the support of the Wisconsin Institute for Discovery (WID) at the University of Wisconsin–Madison, the China Scholarship Council, the financial support of the National Nature Science Foundation of China (No. 51073061, No. 21174044), the

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