In vitro evaluation of vascular endothelial cell behaviors on biomimetic vascular basement membranes

Colloids and Surfaces B: Biointerfaces 182 (2019) 110381

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In vitro evaluation of vascular endothelial cell behaviors on biomimetic vascular basement membranes ⁎

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Chenglong Yu, Meiyi Xing, Shibo Sun, Guoping Guan , Lu Wang

Engineering Research Center of Technical Textile, Ministry of Education, Key Laboratory of Textile Science and Technology of Ministry of Education, Key Laboratory of Textile Industry for Biomedical Textile materials and Technology, College of Textiles, Donghua University, 2999 North Renmin Road, Shanghai, 201620, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Biomimetic basement membrane Vascular endothelial cell Collagen type IV Elastic modulus Adhesion force

Vascular basement membrane (VBM) is a thin layer of fibrous extracellular matrix linking endothelium, and collagen type IV (COL IV) is its main composition. VBM plays a crucial role in anchoring down the endothelium to its loose connective tissue underneath. For vascular grafts, constructing biomimetic VBMs on the luminal surface is thus an effective approach to improve endothelialization in situ. In the present work, three types of polycaprolactone (PCL) membranes were produced and characterized through cell counting kit-8 (CCK-8) assay, adhesion force and elastic modulus test to examine the influence of fiber diameter and membrane composition on vascular endothelial cell (EC) behaviors. The PCL membranes with finer fibers of 54.77 nm (PCL-54) could biomimic the nanotopography of VBMs more efficiently than 544.64 nm (PCL-544), and they were more suitable for Pig iliac endothelium cells (PIECs) adhesion and proliferation, meanwhile, inducing higher elastic modulus and adhesion force of PIECs. On this foundation, we further immobilized COL IV onto PCL-54 (PCL-COL IV) to biomimic VBMs compositionally. Results showed that PIECs on PCL-COL IV exhibited the highest viability and proliferation. Besides, quantitative data indicated that the elastic modulus of the PIECs on PCL-COL IV (4441.00 Pa) was as two times higher than that on PCL-54 (2312.26 Pa), and the adhesion force grew to 1120.99 pN from 673.58 pN of PIECs on PCL-54. In summary, the PCL-COL IV membranes show high similarity with the native VBMs in terms of structure and composition, suggesting a promising potential for surface modification to vascular grafts for improved endothelialization.

1. Introduction Deaths caused by cardiovascular diseases have been increasing over the globe. Vascular transplantation is the most effective means of treating such diseases. However, the availability of healthy and mechanically robust tissue sources is limited, which has increased the necessity of artificial vascular graft development [1]. Currently, many commercial synthetic materials such as Dacron and extended-polytetrafluoroethylene (e-PTFE) have been successfully used for largediameter (inner diameter > 6 mm) artificial blood vessels [2,3]. While none of these materials has been proven suitable for the fabrication of small-diameter (inner diameter < 6 mm) vascular grafts due to thrombus formation and intimal hyperplasia [4–7]. Endothelium consisting of a continuous monolayer of endothelial cells (ECs) is the innermost layer of native blood vessels and involved in multifaceted blood vessel activities, such as inflammation, fibrinolysis, hemostasis, and extracellular matrix (ECM) production owing to direct contact with blood [2]. More importantly, the endothelium can be an antithrombotic

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barrier from avoiding thrombus formation by inhibiting platelet adhesion [8,9]. Therefore, the rapid formation of a functional endothelium on the luminal side of a vascular graft is a widely accepted means to prevent thrombosis [1,10]. Unfortunately, vascular grafts cannot occur spontaneously in situ due to poor ECs adhesion, spreading, and proliferation [11]. Therefore, various techniques have been developed to enhance the endothelialization of the vascular grafts, such as capturing ligands modification [12–14], oligopeptides like RGD [15] and REDV [16], vascular endothelial growth factors (VEGFs) [17], and fibroblast growth factors (FGFs) [11]. Besides, topography [18–22], stiffness [23,24] and hydrophilicity [25–27] of vascular grafts have also been considered to regulate the endothelialization process. However, the efficacy of these modifications has not been satisfactory in vivo. Actually, vascular ECs connect with tunica media of blood vessels in vivo through attaching vascular basement membranes (VBMs). Core compositions of native VBMs include collagen type IV (COL IV), laminins, nidogens, elastin, and other functional compositions. VBMs provide crucial support for vascular endothelium and offer some particular

Corresponding authors. E-mail addresses: [email protected] (G. Guan), [email protected] (L. Wang).

https://doi.org/10.1016/j.colsurfb.2019.110381 Received 23 April 2019; Received in revised form 15 July 2019; Accepted 18 July 2019 Available online 18 July 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. Schematic diagram illustrating the immobilization process of COL IV onto the PCL electrospun membranes.

endothelium cells (PIECs) were purchased from Cell Bank, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Roswell Park Memorial Institute (RPMI-1640), fetal bovine serum, penicillin–streptomycin solution and sodium pyruvate were obtained from Thermo Fisher Scientific. CCK-8, Triton X-100, Rhodamine phalloidin and 40,6-diamidino-2-phenylindole (DAPI) were purchased from Yeasen Biotechnology Co., Ltd. The water used in this study was purified by a Milli-Q system (Millipore Corp). All the chemicals were of analytical grade and used without further treatment if not specially mentioned.

signals to cells and tissues [28]. The BMs are a complex meshwork consisting of submicron (100–1000 nm) pores and nanoscale (1–100 nm) fibers and COL IV is the main constituent of the fibrillar network [29–31]. Recent work reported that tripeptide Cys-Ala-Gly (CAG) in COL IV could enhance the adhesion of ECs but inhibit smooth muscle cells adhesion [32]. The research found that bovine aortic ECs were more prone to attach onto the surfaces modified with COL IV than collagen type I, collagen type V and laminin [33]. Besides, various researches focused on the effect of ultrastructure on ECs behaviors [29,34–39]. Li et al. [39] evaluated the human umbilical vein endothelial cell behaviors on PCL electrospun substrates with varying diameters and orientations, and they found that nano-size fibers could enhance EC adhesion and proliferation remarkably. These results were in agreement with Ko’s studies [40]. It is hypothesized that electrospun membranes with micro- or nanofiber grafted by COL IV which could biomimic natural VBMs structurally and compositionally, would benefit for the adhesion and proliferation of vascular ECs on them. Polycaprolactone (PCL) has been catching increasing attention in the field of vascular grafts due to its easy processibility, robust mechanical property and excellent biocompatibility [41–43]. In the present work, two types of PCL electrospun membranes were designed and fabricated to mimic the ultrastructure of VBMs, followed by COL IV immobilization onto the surface of the membranes with ultrafine fibers through EDC/NHS coupling chemistry. A series of characterization tests had been carried out to determine the effects of the ultrastructure and the COL IV coating on the vascular ECs adhesion, spreading and proliferation. Moreover, the affinity of the VBMs to vascular ECs was evaluated quantitatively by elastic modulus and adhesion force through atomic force microscopy (AFM) and centrifuge technique. The present work might provide insights into the creation of a mesoscopic functional layer to modify the luminal surface of vascular grafts to obtain long-term patency in vivo through in situ endothelialization.

2.2. Preparation of PCL electrospun membranes with varying diameters PCL solution was made by dissolving in a mixture of formic acid and acetic acid (FA/AA, 7:3 v/v) at 12% (w/v) and then stirred for 72 h at room temperature. During electrospinning, the flow rate was controlled at 0.5 ml/h. The distance between the needle tip and the collector was 12.5 cm, and the voltage was 20 kV. Trichloromethane and N, N-dimethylformamide (CHL/DMF, 7:3 v/v) solvent system was applied to prepare another PCL electrospun membranes. The mass fraction of PCL was 12% (w/v) and stirred for 24 h at room temperature before electrospinning. The flow rate, spinning distance, and high voltage were 1 ml/h, 15 cm and 20KV, respectively. Besides, PCL flat films were prepared as the control group with trichloromethane and N, N-dimethylformamide (CHL/DMF, 7:3 v/v) solvent system, using a solvent casting method. All the samples were dried under vacuum overnight (> 12 h) and stored at room temperature. The membranes were sterilized by 75% ethanol for 30 min, washed three times with PBS prior to the following tests. 2.3. Immobilization of COL IV onto the PCL membranes The selected PCL electrospun membranes were wetted with 70% (v/ v) ethanol and deionized water firstly, then soaked in 0.4 M NaOH solution for 15 min at 40 ℃. After incubation, the activated membranes were reacted with EDC/NHS in 0.1 M MES buffer (EDC: 5 mg/mL; NHS: 5 mg/mL) for 6 h, then immersed in COL IV solution (20 μg/mL in 0.05 M HCl) for 24 h at room temperature (Fig. 1), followed by sufficient washing to remove the unreacted COL IV with PBS.

2. Materials and methods 2.1. Materials Polycaprolactone (PCL, average Mn 80000) and collagen type IV (COL IV, human placenta) were purchased from Sigma-Aldrich. N, Ndimethylformamide (DMF), Trichloromethane (CHL) and acetic acid (AA) were provided by Lingfeng Chemical Reagent Co., Ltd. Formic acid (FA), hydrochloric acid (HCl), alcohol and Sodium hydroxide were supplied by Sinopharm Chemical Reagent Co., Ltd. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and 2-(N-Morpholino) ethanesulfonic acid (MES) were obtained from Aladdin Chemical Reagent Co., Ltd. Pig iliac

2.4. Surface characterization of the PCL membranes The morphologies of the membranes were observed by the field emission scanning electron microscope (FESEM, SU8010, Hitachi, Japan), and the acceleration voltage was 15 kV. The diameters of fibers were measured using Image J software. Average pore diameters of PCL membranes were measured by a pore size analyzer (CFP-1100AI, 2

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Keysight, USA) were employed. Prior to the measurement, a silicon chip was used as the stiff surface to measure the deflection sensitivity of the cantilever. At 24 h after cell seeding, the probe was placed over the center of the target cells and the interested regions (90 μm × 90 μm) were scanned using contact mode. The force-distance curves were recorded by a constant approach velocity of 1 μm/s, and a force of 2 nN was applied to the cell by the AFM cantilever. Fifteen to twenty cells were selected, and measured for each sample and three curves for each cell were obtained. The cantilevers with cone tip were utilized here, so we employed the following Hertz-Sneddon model (1) to calculate the elastic modulus of the PIECs:

Porous Materials Inc., USA). Furthermore, the surface roughness was characterized by an atomic force microscope (AFM, 5500 AFM-SPM, Keysight, USA) with contact mode. Arithmetic mean roughness (Ra) was calculated with analyzing software (Pico Image Elements 7.3) in the present study, representing the arithmetic average of the absolute values of the profile height deviations from the mean line within the evaluation length. Wettability of the membranes was measured by a contact angle instrument (OCA15EC, Dataphysics, Germany), using the sessile drop method. A phosphate buffer saline (PBS) droplet (5 μL) was lightly injected onto the surfaces of membranes, and the images of the droplet were used to measure the contact angle. A Fourier transform infrared spectrophotometer (FTIR, Spectrum Two, PerkinElmer, UK) was applied to identify the functional groups on these membranes. FTIR was carried out in the range of 3800-600 cm−1 at a resolution of 4 cm−1. The X-ray photoelectron spectroscopy (XPS) analyses were performed on the ESCALAB 250Xi spectrometer (Thermo Fisher Scientific, USA) with monochromated Al Kα radiation (hv =1486.6 eV) to examine the surface element composition. XPS peak-fitting software (XPS PEAK41) was applied to compute the spectra.

F=

2 E tanαδ 2 π 1 − ν2

(1)

Where F is the applied loading force, E is Young's modulus of the cell, ν is the Poisson ratio of the sample (the cells were assumed to be nearly incompressible, so it was assumed that ν = 0.45). α is the half-opening angle of the sharp tip which is 17.5° here, and δ means the indentation depth. According to Schillers’s study [45], the elastic modulus of cells could be calculated from the slopes of the force1/2-deformation curves.

2.5. Cell culture and assays Prior to cell culture, the membranes were cut into round pieces with 15 mm in diameter and put into 24-well plates, then sterilized by 75% ethanol. After that, the samples were washed by PBS three times to remove ethanol. PIECs were seeded on the samples with standard culture medium (including 88% RPMI, 10% fetal bovine serum, 1% sodium pyruvate, and 1% penicillin-streptomycin solution) at a density of 4 × 104 cells per well for cell adhesion analysis and 1 × 104 cells per well for cell proliferation analysis, then incubated at 37 ℃ in a 5% (v/v) CO2 incubator. The culture medium was exchanged every two days. The CCK-8 assay was performed at 6 h after PIEC seeding to evaluate the cell initial metabolic activity on different samples, and at day 1, day 3, day 5 and day 7 to assess cell viability and proliferation. Firstly, the culture medium was removed, and the cell-adhered membranes were gently washed three times with PBS. Then, 450 μL RPMI and 50 μL CCK-8 solution were added into each well, followed by incubation for 2 h (37 ℃, 5% CO2). 100 μL supernatant solution was aspirated three copies per well, and transferred into wells of 96-well plates. The optical density (absorbance values) of the solutions were determined at 450 nm with a microplate reader (MULTISKAN FC, Thermo Fisher Scientific, USA). Fluorescent staining was applied to image F-actin organization and nucleus morphology of PIECs. After 12 h culture, PIECs on the samples were fixed with 4% paraformaldehyde at 37 ℃ for 30 min, then washed with PBS three times. Next, the cells were permeabilized using 0.5% Triton X-100 in PBS for 10 min, followed by washed with PBS three times. Afterwards, the cells were stained by Rhodamine phalloidin, protecting from light for 30 min at room temperature, washed with PBS three times. The cells were then stained by 40, 6-diamidino-2-phenylindole (DAPI) at room temperature for 2 min, avoiding light, followed by three washes with PBS. The cells were observed under a fluorescence microscope (Ti-S, Nikon, Japan), and the spreading areas of the cells were calculated using Image J software.

2.7. In situ adhesion force measurement of PIECs on the membranes by centrifuge technique Based on Reyes’s work [46], the relative PIECs adhesion force on the membranes was measured using a centrifuge technique. PIECs were seeded on the samples with standard culture medium at a density of 4 × 104 cells per well. At 24 h after cell seeding, the membranes were gently washed three times with PBS to remove floating cells, then placed vertically at the bottom of centrifuge tubes full of PBS. After centrifuging for 5 min at 900 rpm and 1500 rpm respectively, the cells still attached on the membranes were examined using CCK-8 assay. The adhesion forces of the PIECs on the membranes were calculated following Wu’s work [47].

f = (ρcell − ρPBS ) × Vcell × ω2 × r

(2)

Here, f represents the force exterting on each cell (the contrifugation force subtracts the buoyancy force), the densities of the PIECs (ρcell) and PBS (ρPBS) were assumed as 1.07 g/cm3 and 1.01 g/cm3 respectively, and the volume of the PIECs (Vcell) was determined as 1000 μm3. ω is the centrifugation angular velocity, and r corresponds to the distance between sample and rotation center. f is supposed to obey Normal distribution:

f ˜ N (μ, σ 2)

(3)

Here, μ is the mean value of the cell adhesion force and σ is the standard deviation. Regularizing Eq. (3) to standard normal distribution: 2

F=

f−μ ˜N (0,1) σ

(4)

Here, two forces (f) could be calculated through centrifugation process of 900 rpm and 1500 rpm, representing different cell detachment probablity (p). F was obtained from the standard normal distribution table with p value, and μ could be further figured out.

2.6. In situ elasticity measurement of PIECs on the membranes by AFM According to Kimberly’s study [44], there was no statistical difference between Young’s moduli of a single cell measured at room temperature (23 ℃) and in culture conditions (37 ℃ with 5% CO2 and 55% humidity) using an AFM (Student’s t-test), thus the elasticity measurement of the PIECs in the present work was performed at room temperature (23 ℃). The membrane samples were cut into round pieces with 20 mm in diameter and put into culture dishes. PIECs were seeded with standard culture medium at a density of 4 × 104 cells per dish. AFM and silicon nitride probes (9301-6471, spring constant 0.08,

2.8. Statistical analysis Data were presented as means ± standard deviation (SD). One-way analysis of variance (ANOVA) and Student-Newman-Keuls test were employed to statistically analyze the significant differences between groups of data with SPSS software (version 16.0). A difference was considered significant when P < 0.05(*), P < 0.01(**), and P < 0.001(***). 3

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Fig. 2. (A) FESEM photographs of PCL membranes. (B) Distribution of fiber diameters. (C) Average pore diameter distribution of PCL membranes.

3. Results and discussion

hydrophilic surfaces yet [48,49]. PCL-54 and PCL-544 were PCL electrospun membranes, so the roughness values of them were significantly higher than pure PCL cast film (Fig. 3B). Meanwhile, the roughness of PCL-544 was larger than PCL-54, so PCL-54 was more hydrophilic (Fig. 4). In the present study, PBS solution as the simulated body fluid was applied to measure the contact angles of PCL membranes rather than water. As shown in Fig. 4, contact angles of PCL-54 and PCL-544 were 131.80 ± 1.64° and 139.25 ± 0.50° respectively, much higher than that of PCL-FF (78.40 ± 3.84°) (P < 0.001). PCL is considered as a hydrophobic material, though PCL films are hydrophilic sometimes [50]. After immobilization of COL IV, the contact angle of PCL-COL IV decreased sharply to 61.80 ± 4.02°. COL IV is hydrophilic, which would contribute to the decrease of contact angles. Meanwhile, NaOH was used to hydrolyze the hydrophobic O] C–O- groups of PCL, leading to the increase of hydrophilic groups on the surface of PCL, such as −COOH.

3.1. Surface characteristics of the PCL membranes in vivo, VBMs exhibit a complex submicron-nano scale meshwork and COL IV is the main constituent of the fibrillar network. In the present study, PCL electrospun membranes with varying fiber diameters had been produced to evaluate the effects of surface ultrastructure on EC activities, and COL IV was further immobilized onto specific membranes to biomimic native VBMs structurally and compositionally. The high-resolution morphologies of different PCL membranes were observed by FESEM (Fig. 2A). Fig. 2A showed the flat surface of PCL cast films (PCL-FF) as the control group. As shown in Fig. 2B, the diameter of the PCL fibers prepared by CHL/DMF solvent system was 544.64 ± 131.47 nm (PCL-544) which was much thicker than the other two. The mean diameter of the PCL fibers prepared with FA/AA solvent system was 54.77 ± 12.92 nm (PCL-54). After COL IV immobilization, the diameter increased by 23.57% to 67.68 ± 14.89 nm, and these samples were named PCL-COL IV. Moreover, a significant difference between the fiber diameters of PCL-54 and PCL-COL IV groups was found from the results of Student-Newman-Keuls test, but the mean diameter values were both within 100 nm. Also, the fibrillar network of the PCL-COL IV exhibited crinkly morphology, which was more similar to the nanotopography of native VBMs than the PCL-54 (Fig. 2A). The average pore diameter results shown in Fig. 2C suggested that the PCL-544 got the highest value of 1.1532 ± 0.8008 μm, and there were two peaks in the PCL-544 curve which were between 0.8 μm to 1.0 μm, and 1.6 μm to 1.7 μm respectively. The thickest fiber diameter of the PCL-544 contributed to the largest pore size among all the PCL membranes, meanwhile the presentence of two peaks was due to the large deviation of fiber diameter. The average pore diameter of the PCL-54 was 0.2830 ± 0.2624 μm, smaller than that of the PCL-544 because of the finer fiber diameter of the PCL-54. After COL IV immobilization, the average pore diameter of the PCL-54 rose to 0.3260 ± 0.3285 μm of the PCL-COL IV. In Fig. 3, PCL-FF showed the smallest arithmetic mean roughness (Ra) of 65.23 nm, followed by PCL-54 of 146.09 nm, PCL-COL IV of 162.49 nm and PCL-544 of 589.67 nm. Among these three fiber-based samples, there was a direct correlation between the fiber diameter and the roughness, the larger the diameter matching the higher the roughness. Meanwhile, the roughness of the PCL-COL IV seemed close to that of a natural VBM [30]. It’s widely accepted that the surface roughness is a momentous factor which could influence the hydrophilicity of biomaterials. With the increase of surface roughness, the contact angle would rise for hydrophobic surfaces, decrease for

3.2. Chemical characterization of the PCL membranes In the present work, COL IV was introduced onto the surface PCL-54 to biomimic native VBMs compositionally (PCL-COL IV). The surface chemistry properties of PCL-54 and PCL-COL IV were analyzed by FTIR and XPS. In the FTIR spectra (Fig. 5A), the major carboxyl peak was observed in PCL-54 spectrum at 1730 cm−1 which was ascribed to the carbonyl group. In addition, -C–OeCe vibrations at 1186 and 1241 cm−1, as well as −CH2- bending vibrations at 1366, 1418 and 1471 cm−1 were the characteristic peaks of PCL. While comparing the PCL-54 and PCLCOL IV spectra, there were three distinct peaks could be identified. In the PCL-COL IV spectrum, the broader peak observed at 30003600 cm−1 could be related to the stretching of −OH, or –NH- groups of COL IV. The amide I and amide II peaks appeared respectively at 1636 cm−1 and 1540 cm−1 because of the successful immobilization of COL IV. Comparing the two spectra, the vibrations of the carboxyl peak shifted to lower intensity after COL IV immobilization, implying newer hydrogen bonds formation between the carboxyl groups and amine groups of COL IV [51]. Fig. 5B showed the XPS spectra which could provide information on the surface elements. Two peaks could be observed at binding energies of 285 eV and 532 eV on the PCL-54 spectrum, corresponding to carbon and oxygen respectively. After COL IV immobilization, besides carbon and oxygen, an additional peak at the binding energy of 399 eV which corresponded to nitrogen could be detected on PCL-COL IV spectrum, implying the successful immobilization of COL IV. Fig. 5C showed the high-resolution spectra of C1s. Three carbon 4

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Fig. 3. (A) AFM photographs of PCL membranes. (a) PCL-FF, (b) PCL-544, (c) PCL-54, (d) PCL-COL IV. (B) Ra values (arithmetic mean roughness) of PCL membranes (*** P < 0.001).

decreased to 11.28% from 14.94% of PCL-54 spectrum, because the aqueous solution of NaOH was used to hydrolyze the O] C–O, leading to the increase of O]C and C–O. The amide groups were introduced to the membranes after COL IV immobilization, resulting in the peak corresponding to C–O and C–N increasing to 24.40% from 22.39% (PCL-54 spectrum). Meanwhile, a new carbon peak at 287.6 eV which represented to O]C–N (5.00%) was seen in PCL-COL IV spectrum, because the amine groups of COL IV cross-linked with carboxyl groups. The formation of O]C–N demonstrated the successful covalent conjugation of collagen IV onto the surface of PCL-COL IV. Moreover, comparing the two spectra, C–H peak was slightly decreased from 62.67% of PCL-54 spectrum to 59.32% of PCL-COL IV spectrum. FTIR and XPS analyses confirmed the successful immobilization of COL IV.

3.3. Spreading and adhesion of the PIECs Fig. 4. PBS contact angles of PCL membranes (*** P < 0.001).

A growing body of evidence demonstrates that the pore size and porosity of scaffolds have important influence on cell spreading, proliferation, migration and infiltration [29,34,36,38,39]. As shown in Fig. 6A, PIECs cultured on PCL-54, PCL-544 and PCL-FF exhibited restricted-spread round shape, while widespread shape on the PCL-COL

peaks were presented in both spectra, O] C–O at 288.7 eV, C–O (C–N) at 286.0 eV and C–H at 284.8 eV, which were the major chemical groups of PCL. For PCL-COL IV spectrum, the peak of O] C–O

Fig. 5. Surface chemical characterization of PCL membranes. (A) FTIR spectra, (B) XPS survey spectra of and (C) High-resolution spectra of the carbon peak (C1s). 5

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Fig. 6. (A) Morphologies of PIECs on PCL membranes after 12 h culture. (a) PCL-FF, (b) PCL-544, (c) PCL-54, (d) PCL-COL IV. Nuclei were stained blue with DAPI, Factin was stained red with TRITC. (B) The coverage area of PIECs (*** P < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

[53,54]. Additionally, wettability, topography and chemical composition of surfaces are critical factors in regulating protein adsorption [55–59]. The contact angle of PCL-FF was 78.40 ± 3.84°, more hydrophilic than those of the PCL-54 (131.80 ± 1.64°) and PCL-544 (139.25 ± 0.50°). It might be evident that hydrophilic PCL-FF could facilitate less protein adsorption, but protein adsorption on material surfaces is a complex and dynamic process influenced by many factors, especially nanoscale topography of the surface because of its similar nano-dimensions with proteins and high surface area [56]. Even though the large surface area and hydrophobicity of materials could lead to the increase in protein adsorption, the conformation of proteins might change. Lord [60] and Miller [61] confirmed that the conformation of fibronectin adsorbed on the nano-rough surfaces had changed, leading to reduced binding sites for cell adhesion. The larger surface area and hydrophobicity of PCL-544 could both promote more proteins adsorption, while PCL-FF exhibited better PIEC adhesion. We speculated that the density of plasma proteins with normal conformation adsorbed on PCL-FF was higher than that on PCL544. Although PCL-544 might attract more proteins adsorption, the conformation of proteins had changed, thus leading to the limited PIEC adhesion. PCL-54 membranes attracted more PIECs attached than PCLFF, which meant that the surface area and hydrophobicity of PCL-54 could counteract the adverse effect of conformational change of proteins, resulting in a higher density of normal conformation proteins adsorbed.

IV. The results suggested that the mean spreading areas of PIECs on the PCL-544 and the PCL-54 were 584.23 μm2 and 565.04 μm2 respectively, significant larger than on the PCL-FF of 446.84 μm2. The reason may be that electrospun membranes mimic the morphological features of natural basement membrane architecture, which is beneficial to cell spreading and other behaviors. There was no significant difference on PIEC spreading area and PIEC morphology between the PCL-544 and the PCL-54 groups, even though these two membranes differed greatly in average pore diameter. For the PCL-COL IV, PIEC spreading area reached 716.37 μm2. The topographies of PCL-54 and PCL COL IV were similar, so we confirmed that COL IV could enhance the spreading of PIECs significantly [52]. The CCK-8 assay was carried out to evaluate the PIEC viability and proliferation. All the PCL membranes could support the adhesion and the proliferation of PIECs. As shown in Fig. 7A, the PIECs on all PCL membranes exhibited a similar pattern of time-dependent increase of the metabolic activity during the culture period. The absorbances of the PIECs on the PCL-FF, PCL-544, PCL-54 and PCL-COL IV were 0.1440 ± 0.0900, 0.1321 ± 0.0101, 0.1643 ± 0.0032 and 0.2379 ± 0.0131, respectively. PIEC adhered on PCL-FF exhibited higher activity than on PCL-544, lower than on PCL-54 yet. Meanwhile, the metabolic activity of the PIECs on the PCL-COL IV was the highest. It is believed that protein adsorption onto the material surfaces is the first event when a biomaterial implanted into a biological environment, followed by cellular interaction with the adsorbed protein layers, so the adsorbed proteins would further influence cell attachment

Fig. 7. CCK-8 assay for measuring PIEC viability after 6 h culture (A) and PIEC proliferation after 1 d, 3 d, 5 d and 7 d culture (B) on PCL membranes (* P < 0.05, ** P < 0.01, *** P < 0.001). 6

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FF, the PCL-544 and the PCL-54 were 633.23 Pa, 1273.51 Pa and 2312.26 Pa respectively, and that on the PCL-COL IV was the highest 4441.00 Pa. These data were in agreement with the observation of Takai who stated that the elastic modulus of cells (osteoblasts) adhered on extracellular matrix proteins was higher than on glass or poly-Llysine film [71]. The intracellular cytoskeleton of cells connects to material surfaces by integrins [66,72,73]. Researches suggested that when integrins bound to extracellular matrix ligands, like COL IV and some plasma proteins adhered on the membranes in this experiment, some cytoskeletal proteins, such as vinculin would bind and stabilize associated cytoskeletal proteins, also promote the accumulation of actin filaments of the cytoskeleton [63,64,69,74,75]. Thus, cell adhesion would be enhanced because of the stabilization of the cytoskeletal organization induced by vinculin. The elastic modulus of PIECs on PCL-54 was higher than on PCL-544 and PCL-FF, and PCL-FF as the control group presented the lowest value. According to Takai’s report [71], osteoblasts cultured on fibronectin were well spread with much fibrous structure, while demonstrating a more round morphology with no apparent fibrous structure formation on the glass. They assumed that the fibrous structure was much like F-actin bundles or stress fibers, and the better cell spreading might be a reason for a more extensive microtubule network, leading to a higher cell elastic modulus. However, the elastic modulus of cells was affected by various extraneous factors, so we can not establish links simplistically between the spreading area and the elastic modulus of cells. In the present study, PIECs cultured on PCL-54 showed significantly higher elastic modulus than on PCL-544, while there was no difference on the spreading area of PIECs between these two groups. Moreover, the stiffness of surfaces would influence EC behaviors significantly. High substrate stiffness could promote the adhesion and proliferation of ECs [23,24,76], also enhance cytoskeleton organization through generating new stress fibers and enriching actin structure which could bring about a higher cell elastic modulus [45,65,66]. Based on the study of Yim [73], the changes in mechanical properties of the cells (hMSCs) induced by nanopatterned topography varied with the stiffness of substrates, though nanotopography might be more dominant in determining the organization of the cytoskeleton and focal adhesions. Here, we hypothesized that different nanotopographies of PCL membranes in the present experiment influenced cytoskeletal organization differently, the differences on stiffness and surface composition (COL IV) led to the different elastic modulus of PIECs as well. In addition, temperature, CO2 concentration and other experimental factors might also affect the mechanical properties of PIECs [68,69,77]. As shown in Fig. 8, the results showed that the adhesion force and elastic modulus of PIEC on PCL-COL IV were the highest among all groups, in agreement with the viewpoints of Takai [71] and Simon [72] who held the point that an increasing apparent modulus of cells on ECM proteins implied a strong integrin-mediated binding, because the formation of actin stress fibers and focal adhesions was associated. While integrins binding to extracellular proteins, some cytoskeletal proteins like vinculin would induce stabilization of cytoskeleton and lead to strong cell adhesion [74,75]. ECs adhered onto the inner surface of implants are always exposed to physiological flow conditions. If the adhesion strength of ECs is not strong enough, the cells may be sloughed off from the surface [78,79]. In the present study, the mean adhesion force of PIECs cultured on the PCL-FF, the PCL-544, the PCL-54 and the PCL-COL IV were 121.83 pN, 436.55 pN, 673.58 pN and 1120.99 pN, respectively (Fig. 8B). PIECs interacted with the adsorbed protein layer on PCL membranes through integrins, besides fibronectin, vitronectin and other plasma proteins adsorbed, COL IV would also provide a mass of binding sites for integrins [32,33,53,63,69]. Therefore, more integrin-protein bonds brought about significantly higher adhesion force of PIECs on PCL-COL IV than on other membranes. On the PCL-FF, the PCL-544 and the PCL54, higher elastic modulus values of PIECs corresponded to higher adhesion forces, supporting the views of Takai [71] and Simon [72].

PIEC adhesion on PCL-COL IV was the best, besides the influence of topography that mimicked the ultrastructure of native VBMs, COL IV also played an important role. Cells adhere onto the surface of biomaterials via integrin proteins. Integrin proteins are cell transmembrane receptor proteins, serving as linker proteins, could bind to ECM proteins, such as collagens, fibronectins, and laminins [62,63]. Moreover, the contact angle of PCL-COL IV was 61.80 ± 4.02°, and it’s suitable for cell attachment [59]. Therefore, preferable contact angle and COL IV both contributed to the best EC adhesion of PCL-COL IV. 3.4. Proliferation of the PIECs As shown in Fig. 7B, the PIECs on all PCL membranes exhibited a similar pattern of time-dependent increase of the cell number during the culture period. PCL-FF as the control group always showed the lowest cell proliferation among all samples, implying the essential role of nanotopography on cell proliferation. The absorbance values of cells on the PCL-54 were higher than that on the PCL-544 with significant differences at 1d, 5d, and 7d, while lower at 3d with no significant difference, suggesting that the topography of PCL-54 was more suitable for PIEC proliferation. With the extending of incubation time, the difference in absorbance values among different groups became larger. In addition, PIEC proliferation on the PCL-COL IV was more favorable than other samples, exhibiting the best capacity in promoting PIEC proliferation. The mean absorbance of the PCL-COL IV was up to 3.3823 at 7d, while the values of PCL-54, PCL-544 and PCL-FF were 2.9113, 2.5676 and 1.2421 respectively. McKee [37] fabricated highly porous polyelectrolyte multilayer films (PEMs) to mimic the topography of VBMs, but ECs on the films showed a decreased proliferation rate compared to flat surfaces. Same results had also been corroborated by Lord [61], who confirmed that the protein adsorbed (fibronectin) onto the surfaces was changed conformationally, so the availability of the cell-binding domain of proteins was reduced. The proliferation of PIECs on PCL-54 was better than on PCL-544 or PCL-FF (Fig. 7B). We had speculated that the density of proteins with normal conformation adsorbed on PCL-54 was higher than on PCL-544 or PCL-FF under the influences of surface topography and wettability together, leading to more PIECs adhered. Thus, PCL-54 provided much more cell-binding sites, and promoted PIECs proliferation further. After immobilization of COL IV, besides the cell-binding domains of plasma proteins adsorbed (fibronectin, vitronectin, etc.), COL IV could also provide massive active sites to bind the integrin proteins of PIECs [32,33], contributing to the best adhesion and proliferation of PIECs on PCL-COL IV among all the groups. Heo [1] and Han [28] reported that COL IV could enhance EC adhesion and proliferation. Excellent PIEC spreading, adhesion and proliferation on the PCL-COL IV might improve cell-cell interaction for the maturation of PIEC layer [1] and further promote rapid endothelialization. 3.5. Elastic modulus and adhesion force of PIECs on the PCL membranes The elastic modulus of cells is closely related to the structure and functions of cells [45,64–68], and AFM is always applied to evaluate cell mechanical properties because of its wide range of applicable forces in cell studies (5pN–100nN) [45,66,69]. Sato proposed that mechanical properties of ECs should be considered while designing the inner surface of artificial blood vessels where ECs attached [68]. The cytoskeleton components, like actin fiber, microtubules, and intermediate filaments support the shape and structure of cells [70,71], and the changes of cell cytoskeleton would result in altered elastic properties of cells [66,72,73]. The modulus of ECs measured with AFM ranges from several hundred Pascal to 106 Pascal which is influenced by types of ECs, temperature, CO2 concentration, cell states, types of probes, and so on [45,64–68]. As shown in Fig. 8A, the mean elastic modulus of PIECs on the PCL7

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Fig. 8. The elastic modulus ((A); Red points represented the mean values) and adhesion force (B) of PIECs on PCL membranes (*** P < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

4. Conclusions

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A biomimetic VBM with ultrafine fibers was designed and fabricated by electrospinning method in the present work. Compared to PCL-544 and PCL-FF, PCL-54 showed more favorable cell adhesion and proliferation performance, as well as higher cell spreading area. According to the results of the elastic modulus and the adhesion force, PIECs attached more tightly on the PCL-54 with the ability to resist the detachment force. Furthermore, COL IV which was the main component of native VBMs was immobilized onto the PCL-54 to biomimic natural BMs structurally and compositionally. COL IV played a momentous role in modulating PIEC activities, such as spreading, adhesion, and proliferation. Moreover, the elastic modulus and the adhesion force were significantly improved. Therefore, PCL-COL IV has the promising potential in biomimicking VBMs to enhance endothelialization, making it a great candidate for modifying the luminal side of vascular grafts. Acknowledgments This work was supported by the Fundamental Research Funds of Central Universities (Grant No. 2232019G-06), the National Key Research and Development Program of China (2018YFC1106200, 2018YFC1106201), Innovation Program of Shanghai Municipal Education Commission (No. ZX201503000017, 15ZZ032) and “111 project” (B07024). References [1] Y. Heo, Y.M. Shin, Y. Bin Lee, Y.M. Lim, H. Shin, Effect of immobilized collagen type IV on biological properties of endothelial cells for the enhanced endothelialization of synthetic vascular graft materials, Colloid Surf. B 134 (2015) 196–203. [2] R. Chiesa, E.M. Marone, Y. Tshomba, D. Logaldo, R. Castellano, G. Melissano, Aortobifemoral bypass grafting using expanded polytetrafluoroethylene stretch grafts in patients with occlusive atherosclerotic disease, Ann. Vasc. Surg. 23 (2009) 764–769. [3] H. Kurobe, M.W. Maxfield, C.K. Breuer, T. Shinoka, Concise review: tissue-engineered vascular grafts for cardiac surgery: past, present, and future, Stem Cells Transl. Med. 1 (2012) 566–571. [4] X.W. Wang, P. Lin, Q.H. Yao, C.Y. Chen, Development of small-diameter vascular grafts, World J. Surg. 31 (2007) 682–689. [5] R.M. Nerem, D. Seliktar, Vascular tissue engineering, Annu. Rev. Biomed. Eng. 3 (2001) 225–243. [6] M.S. El-Kurdi, Y. Hong, J.J. Stankus, L. Soletti, W.R. Wagner, D.A. Vorp, Transient elastic support for vein grafts using a constricting microfibrillar polymer wrap, Biomaterials 29 (2008) 3213–3220. [7] G. Konig, T.N. McAllister, N. Dusserre, S.A. Garrido, C. Iyican, A. Marini, A. Fiorillo, H. Avila, W. Wystrychowski, K. Zagalski, M. Maruszewski, A.L. Jones, L. Cierpka, L.M. de la Fuente, N. L’Heureux, Mechanical properties of completely autologous human tissue engineered blood vessels compared to human saphenous vein and mammary artery, Biomaterials 30 (2009) 1542–1550. [8] D. Mehta, A.B. Malik, Signaling mechanisms regulating endothelial permeability, Physiol. Rev. 86 (2006) 279–367. [9] J.S. Pober, W.C. Sessa, Evolving functions of endothelial cells in inflammation, Nat. Rev. Immunol. 7 (2007) 803–815. [10] J.J.D. Henry, J. Yu, A.J. Wang, R. Lee, J. Fang, S. Li, Engineering the mechanical and biological properties of nanofibrous vascular grafts for in situ vascular tissue

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