Functional Modification of Electrospun Poly(Îµ-caprolactone) Vascular

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Functional Modification of Electrospun PCL Vascular Grafts with the Fusion Protein VEGF-HGFI Enhanced Vascular Regeneration Kai Wang, Qiuying Zhang, Liqiang Zhao, Yiwa Pan, Ting Wang, Dengke Zhi, Shaoyang Ma, Peixin Zhang, Tiechan Zhao, Siming Zhang, Wen Li, Meifeng Zhu, Yan Zhu, Jun Zhang, Mingqiang Qiao, and Deling Kong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b16713 • Publication Date (Web): 09 Mar 2017 Downloaded from http://pubs.acs.org on March 13, 2017

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Functional Modification of Electrospun PCL Vascular Grafts with the Fusion Protein VEGF-HGFI Enhanced Vascular Regeneration

Kai Wang,1# Qiuying Zhang,1# Liqiang Zhao,2 Yiwa Pan,1 Ting Wang,3 Dengke Zhi,1 Shaoyang Ma,1 Peixin Zhang,1 Tiechan Zhao,4 Siming Zhang,4 Wen Li,1 Meifeng Zhu,1 Yan Zhu,4 Jun Zhang,1,* Mingqiang Qiao,2,* Deling Kong1,*

1

Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China

2

Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China

3

Urban Transport Emission Control Research Centre, College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China

4

Center for Research and Development of Chinese Medicine, Tianjin State Key

Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China

#

Authors contributed equally to this work

Corresponding authors: Dr. Deling Kong: [email protected] Dr. Mingqiang Qiao: [email protected] Dr. Jun Zhang: [email protected]

Abstract 1

ACS Paragon Plus Environment

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Synthetic artificial vascular grafts have exhibited low patency rate and severe neointimal hyperplasia in replacing small-caliber arteries (< 6 mm) because of their failure to generate functional endothelium. In this study, small-caliber (2.0 mm) electrospun poly(ε-caprolactone) (PCL) vascular grafts were modified with a fusion protein VEGF-HGFI which consists of the class I hydrophobin (HGFI) and vascular endothelial growth factor (VEGF), via hydrophobic interactions. Immunofluorescence staining with the anti-VEGF antibody showed that VEGF-HGFI formed a protein layer on the surface of fibers in the grafts. Scanning electron microscopy (SEM) and mechanical measurements showed that VEGF-HGFI modification had no effect on the structure and mechanical properties of PCL grafts. Blood compatibility tests demonstrated lower level of fibrinogen (FGN) absorption, platelet activation and aggregation on the VEGF-HGFI modified PCL mats than that on the bare PCL mats. The haemolysis rate was comparable in the both modified and bare PCL mats. In vitro culture of human umbilical vein endothelial cells (HUVECs) demonstrated that VEGF-HGFI modification could remarkably enhance nitric oxide (NO) production, prostacyclin2 (PGI2) release and the uptake of acetylated low-density lipoprotein (Ac-LDL) by HUVECs. The healing characteristics of the modified grafts were examined in the replacement of rat abdominal aorta for up to 1 month. Immunofluorescence staining revealed that endothelialization, vascularization and smooth muscle cells (SMCs) regeneration was markedly improved in the VEGF-HGFI modified PCL grafts. These results suggest that modification with fusion protein VEGF-HGFI is an effective method to improve the regeneration capacity of synthetic vascular grafts.

Keywords: fusion protein VEGF-HGFI; PCL; vascular grafts; surface modification; vascular regeneration

1. Introduction Cardiovascular diseases are one of the most prevalent diseases worldwide that 2



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cause very high motality and massive social burden.1 Clinical treatment often requires bypass surgery.2 Autografts, particularly the saphenous vein and internal mammary artery, are regarded as the gold standards for the treatment of vascular occlusions.3 However, autografts have several limitations, such as mismatched size or length, prior surgery and morbidity at the donor site.4-5 At present, frequently used synthetic vascular grafts,

including polyethylene terephthalate (PET) and expanded

polytetrafluoroethylene (ePTFE), perform well for large-diameter blood vessels in clinical settings, but they are not suitable for the replacement of small-caliber (＜5 to 6 mm) blood vessels due to severe neointimal hyperplasia and thrombosis.6 A functional endothelial layer can effectively prevent thrombosis and intimal hyperplasia, and improve the patency of the graft.7 Therefore, the rapid endothelialization of implanted vascular grafts is of paramount importance. Preculturing endothelial cells (ECs) on the luminal side of the vascular prostheses before implantation is an effective method to achieve fast endothelialization.8 However, the isolation, culture and seeding of ECs is time-consuming, costly and usually causes donor-site morbidity.9 Moreover, the preseeded ECs are easily washed away by the blood flow upon implantation. As a solution to enhance the endothelialization and prevent the disadvantages associated with cell seeding, researchers have functionalized the surface of vascular grafts with bioactive molecules to improve in situ endothelialization.10 VEGF is a potentially effective molecule because it can regulate the proliferation, migration and survival pathways of ECs. The common methods used to immobilize or load proteins and growth factors onto scaffolds are chemical conjugation,11 coaxial12 or emulsion13 electrospinning, entrapment14 and adsorption.15 Although covalent conjugation has the advantage that the immobilized molecules are not easily removed by rinsing, surface degradation and polymeric chain incision during the preparation process may cause alterations in structural and mechanical properties.6 In addition, non-specific conjugation may yield heterogeneous products which might influence the function and orientation of individual protein molecule.16 Coaxial or emulsion electrospinning can directly encapsulate VEGF into core/shell fibers, which avoids the subsequent chemical 3


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reaction and help to maintain the physico-chemical properties of the grafts. However, the use of an organic solvent may cause the denaturation of VEGF during the electrospinning process. Entrapment can successfully achieve protein load and release, however, the multiple fabrication steps may reduce the bioactivity and receptor-binding ability of the protein.15 Adsorption of protein or growth factor on pre-fabricated scaffolds is a relatively easy-to-achieve and a straightforward method. The interactions between the adsorbed protein and the scaffold surface include ionic bonds, hydrogen bonds, van der Waals and hydrophobic forces.15 Recently, synthetic polymers used to fabricate vascular grafts are usually hydrophobic, such as ePTFE, PET, polylactide (PLA) and PCL. Thus, adsorption based on hydrophobic interactions is particularly suitable to modify these polymers with proteins. Among these hydrophobic polymers, PCL is a promising polymer for the construction of small-caliber vascular grafts due to its excellent biocompatibility and suitable mechanical strength.17 Walpoth’s group found that electrospun PCL grafts exhibited better endothelial coverage, extracellular matrix (ECM) formation and polymer biodegradation compared to ePTFE grafts (2 mm diameter) in the rat abdominal aorta.18 Besides, cellular infiltration was also improved in the electrospun PCL grafts.18 Cellularization of the vascular graft, also known as engraftment, is critical for tissue regeneration and implant integration with the host environment. The survival and growth of cells migrated into scaffolds, similar to the cells in most tissues and organs, rely on the nutrients and oxygen diffused from capillaries.19 However, the maximum distance between these capillaries is 200 µm, which means that any scaffold that exceeds 200 µm in any dimension must be vascularized.20 In order to meet the mechanical requirements of vascular implantation, the wall thickness of electrospun PCL vascular grafts prepared in previous studies was usually in the range of 500-850 µm.4, 17, 21 So sufficient vascularization within graft wall is important for vascular regeneration. Therefore, an ideal functional modification should enhance the endothelialization and capillary formation within the graft walls at the same time. Class I hydrophobin HGFI, isolated from the edible mushroom Grifolafrondosa, is 4



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a small amphipathic protein and possesses hydrophobic and hydrophilic moieties.22 Owing to its special structural characteristics, HGFI can form amphipathic membranes at hydrophilic-hydrophobic interfaces by hydrophobic interactions, which strongly improves the hydrophilicity of PCL scaffold and enhances cell attachment.22 In our previous report, we synthesised the fusion protein VEGF-HGFI by gene recombination technology.23 To avoid unfavourable protein folding and poor activity, a flexible peptide chain (GGGGS)3 was used as a linker to connect VEGF and HGFI. The fusion protein could form a VEGF-HGFI coating layer on the surface of PCL fibers via the hydrophobic interactions between the PCL fibers and the hydrophobic moiety of HGFI, which could significantly enhance the cellularization and vascularization of PCL scaffolds. In this report, we aimed to investigate the effect of VEGF-HGFI modification on endothelialization, smooth muscle regeneration, and vascularization of the electrospun PCL grafts.

2. Materials and Methods 2.1 Materials PCL pellets (average Mn=80,000) and Human FGN were purchased from Sigma Aldrich (Shanghai, China). Methanol, chloroform and alcohol were purchased from Tianjin Chemical Reagent Company (Tianjin, China). Triton X-100 was purchased from Alfa Aesar (London, England). 3-amino-4-aminomethyl-2’, 7’-difluorescein, diacetate (DAF-FM) was purchased from Beyotime Biotechnology Co., Ltd (Shanghai, China). Human platelet-rich plasma (PRP) was purchased from the Tianjin Blood Center. HUVECs were purchased from Science Cell (USA). The rat aortic SMCs line A10 was obtained from the American Type Culture Collection (Bethesda, MD). Human FGN enzyme-linked immunosorbent assay (ELISA) kit, P-selectin ELISA kit and human PGI2 ELISA kit were purchased from UNOCI Biotechnology Co., Ltd (Shanghai, China). Griess Reaction assay kit was purchased from Nanjing Jiancheng

Bioengineering

Institute

1,1’-dioctadecyl-3,3,3’,3’-tetramethylindo-carbocyanine

(Nanjing, perchlorate

China). labeled

acetylated low-density lipoprotein (DiI-ac-LDL) was purchased from Melone 5


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Pharmaceutical Co., Ltd (Dalian, China). CyQUANT Cell Proliferation Assay Kit was obtained from Invitrogen (Grand Island, NY). New Zealand white rabbits (male, weight 2–2.5 kg) and Sprague Dawley (SD) rats (male, weight 280-320g) were obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China). All animal experiments were approved by the Animal Experiments Ethical Committee of Nankai University

2.2 Electrospinning PCL mats and vascular grafts 25 wt% of PCL solution was prepared by dissolving PCL in a mixture of methanol and chloroform (1:5, volume ratio) at room temperature (RT) for 12 h. The PCL solution was loaded into a 10-mL glass syringe with a 21-gauge needle and injected using a syringe pump (Cole-Parmer, USA) at a flow rate of 8 mL/h. A grounded rotating mandrel (15 cm in diameter) was used as a collector, and the speed of mandrel rotation was 300 rpm. The distance between the needle tip and the collector was 17 cm, and a voltage of 11 kV was supplied by a high-voltage generator (DWP503-1AC, Dong-Wen High Voltage power supply factory, Tianjin, China). The electrospun PCL mats were fabricated by collecting fibers for 30 min on an aluminium foil wrapped around the rotating mandrel. To construct PCL vascular grafts, a grounded rotating steel rod (2 mm in diameter) was used as the collector. The rotation rate was set to approximately 150 rpm. The other electrospinning parameters were the same as those used for electrospun PCL mats. After electrospinning for 4 min, the steel rod was removed, yielding a PCL vascular graft. Finally, the electrospun PCL mats and vascular grafts were dried in a vacuum oven at RT for 2 days before further treatment.

2.3 Modification of PCL mats and vascular grafts with VEGF-HGFI The fusion protein VEGF-HGFI was constructed and purified according to our previous report.23 The PCL mats and grafts were sterilized by immersion in a 75% (v/v) ethanol solution for 1 h and air-dried in a sterile biological containment hood at RT. After that, the sterile PCL mats and grafts were immersed in a sterilized aqueous 6



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HGFI or VEGF-HGFI solution (200 µg/mL) at 4 °C for 12 h, followed by taking out the mats and grafts from the protein solution and washing thrice with phosphate-buffered saline (PBS). PCL and HGFI-PCL or (VEGF-HGFI)-PCL identification codes have been used to indicate the unmodified and protein modified PCL mats and grafts.

2.4 Characterization of PCL grafts with or without protein modification The

cross-sections

and

luminal

surfaces

of

PCL,

HGFI-PCL

and

(VEGF-HGFI)-PCL grafts were mounted on aluminium foil and sputter-coated with gold. SEM (HITACHI, X-650, Japan) at an accelerating voltage of 15 kV was used to observe the structure of the grafts. The outer and luminal perimeters of the grafts were manually measured from SEM images of the cross-section with Image-Pro Plus (IPP) software to determine the luminal diameter and wall thickness of the grafts (n=3). Based on the SEM micrographs of the luminal surface of grafts, the fiber diameter and pore diameter were analysed using IPP software. At least ten fibers per image, three images per sample and three samples per group were included to calculate the fibers diameter. The pore size was measured according to the previously described procedures.24 The surface area of the pores was calculated and normalized to a circular area. The circular diameter could be obtained from the area of the circle and was regarded as the pore diameter. At least six pores per image, five images per sample and three samples per group were used to calculate the average pore diameter. The porosity of the grafts was evaluated using a specific gravity bottle based on Archimedes' Principle following a previous report.25 In brief, the porosity of the grafts was calculated by the following equation: Vs=[W1-(W2-W3)]/ρe Vw=L×[(d1/2)2-(d2/2)2]×π Porosity(%) = (1-Vs/Vw)×100% where W1 is the weight of the gravity bottle filled with ethanol, W2 is the weight of gravity bottle containing ethanol and the graft immersed in ethanol completely, W3 is the weight of the graft, Vs is the volume of the graft skeleton, Vw is the volume of the 7


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whole graft, ρe is the density of ethanol, and L, d1 and d2 are the length, outer diameter and luminal diameter of the graft, respectively. To visualize the distribution of VEGF-HGFI modification, (VEGF-HGFI)-PCL grafts were incubated with a VEGF antibody (1:100, Abcam, USA) at 4 °C for 12 h. Samples were washed with 0.1M PBS 5 times, blocked with a 1% bovine serum albumin (BSA) solution for 0.5 h, and treated with an Alexa Fluor 488-conjugated goat anti-mouse IgG (1:200, Invitrogen) secondary antibody at RT for 2h. After washing the samples 5 times with 0.1M PBS, their cross-section was observed with a confocal laser scanning microscope (CLSM, Leica TCS SP5) to observe the distribution of VEGF-HGFI. The PCL and HGFI-PCL grafts as control groups were stained and observed using the same protocol. 2.5 Mechanical testing Longitudinal mechanical properties were determined using a tensile-testing machine with a load capacity of 100 N (Instron-3345, Norwood, MA) at RT. Grafts (length, 2.0 cm; gauge length, 1.0 cm) were pulled longitudinally at a strain rate of 10 mm/min until rupture. Tensile strength and ultimate elongation at break were measured. Young’s modulus was calculated from the initial linear region (up to 5% strain) of the stress-strain curve. As described in a previous report,26 burst pressure was measured by filling a graft of 3 cm in length with soft paraffin (Vaseline), clamping one end and hermetically sealing the other end to a vascular catheter. After the sample was incubated at 37 °C for 30 min, the Vaseline was filled at a rate of 0.1 mL/min. The filling pressure was recorded until the graft wall burst. These mechanical tests were performed in triplicate. 2.6 In vitro hemocompatibility evaluation To test clotting time, the PCL, HGFI-PCL and (VEGF-HGFI)-PCL mats were cut into circular samples (1 cm in diameter, n = 3). Then, 500 µL of human plasma was placed into a tapered tube, and a circular sample was added. A tapered tube with 8



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human plasma but no sample was used as the control. After incubation at 37°C for 1 h, the mats were removed and the full human plasma activated partial thromboplastin time (APTT), prothrombin time (PT) and thromboplastin time (TT) of the plasmas were measured with an automatic SYSMEX CA-5100 coagulation analyser. The average data from three measurements is reported. For the haemolysis assay, blood was obtained from the heart of the rabbit and mixed with 0.5 mL of 2 wt% solution of potassium oxalate in physiological saline (0.9 % NaCl solution). Anticoagulated blood solution was obtained by mixing abovementioned anticoagulated blood with 0.9 % NaCl solution at a 4:5 volume ratio. The samples (1×2 cm) were placed in 15 mL centrifuge tubes with 10 mL of 0.9% NaCl solution. Tubes containing only saline or distilled water were used as a negative and a positive control group, respectively. After static equilibration at 37 °C for 30 min, 0.2 mL of anticoagulated blood solution was added into the centrifuge tubes and incubated at 37°C with shaking for another 60 min. Subsequently, the tubes were centrifuged for 5 min at 2500 rpm and the absorbance of the supernatant was recorded at 545 nm with a UV–visible spectrophotometer (1900PC, Shanghai Spectrum Instruments Co. LTD, Shanghai, China). The haemolysis percentage was calculated according to the following equation: Haemolysis (%)= (A1–A2)/(A3–A2) × 100%, where A1, A2 and A3 are the absorbance of the sample, negative control and positive control, respectively. The data were averaged from measurements on five specimens. FGN adsorption to mats was determined by ELISA. Modified and unmodified PCL mats were placed into a 96-well plate and incubated with 100 µL of human FGN at 37 °C for 1 h to allow protein adsorption. After the sample was washed with PBS (5 min × 3 times), the human FGN ELISA Kit was used to measure FGN according to the literature. The absorbance of the substrate solution was measured at 450 nm using a microplate reader (iMark, Bio-Rad, USA). The data were averaged from measurements on five specimens. To test platelet adhesion and activation, PCL, HGFI-PCL and (VEGF-HGFI)-PCL mats of appropriate size were placed in 48-well cell culture plates (n=5). To each well, 300 µL human PRP was added and the samples were incubated at 37 °C for 1 h. The 9


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unattached platelets were removed by rinsing 3 times with PBS and the samples were characterized for platelet adhesion and activation. For the adhesion assay, the samples were fixed with 2.5% glutaraldehyde and dehydrated with gradient ethanol. Platelet adhesion to the sample surface was observed by SEM, and the quantitative analysis was performed from 4 randomly selected fields. The activation state of the platelets that had adhered to the mats was measured using a human P-selectin ELISA Kit according to the manufacturer’s instructions and the absorbance was measured at 492 nm using a microplate reader.

2.7 In vitro endothelial function assay To demonstrate the potential of VEGF-HGFI modification on endothelial NO production, HUVECs were seeded on bare glass-bottom dishes (15 mm in diameter), HGFI and VEGF-HGFI modified glass-bottom dishes at a cell density of 2.0×104 per well and cultured in endothelial basal media-2 (EBM-2, Lonza, Basel, Switzerland) without VEGF. After cultured for 24 h, real-time NO production in HUVECs was stained with the DAF-FM solution (5 µM, 1 mL) at 37 °C for 30 min. The cells were then washed PBS 5 times and fixed in 4% paraformaldehyde. The nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) containing mounting solution (DAPI Fluoromount G, Southern Biotech, England). The NO production was visualized with the fluorescent indicator using a CLSM with excitation and emission maxima at 495 and 515 nm, respectively. To evaluate the effect of VEGF-HGFI modification on endothelial Ac-LDL uptake, HUVECs were seeded and cultured by the same method as that described above. After cultured for 24 h, DiI-ac-LDL (10 µg/mL in EBM-2 medium without VEGF) was added to each well and incubated at 37 °C for 4 h. The cells were then washed 5 times with PBS and fixed in 4% paraformaldehyde. Afterward, the nuclei were counterstained with DAPI, and the samples were viewed using a CLSM. The DAF-FM and DiI-ac-LDL intensities were then analysed using IPP software by outlining individual cells and measuring the fluorescence intensity. At least 90 cells were measured in each group. HUVECs were cultured on bare glass-bottom dishes as a control, and the fluorescence intensity of the control group was set as 100%. 10



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The amount of NO and PGI2 released into the cell culture supernatants was also measured

to

evaluate

HUVECs

function.

The

PCL,

HGFI-PCL

and

(VEGF-HGFI)-PCL mats were placed in 6-well culture plates (n = 5). HUVECs were seeded on mats at 3.0×105 cells per well and cultured for 24 h in EBM-2 medium without VEGF. Then, the original medium was replaced with 700 µL fresh EBM-2 medium without VEGF. After 4 h of incubation, the cell culture supernatants were collected. The amount of NO released into the medium was quantified with the Griess Reaction assay kit. The absorbance was measured at 540 nm with a microplate reader. The concentration of nitrite was counted by comparison with the absorbance at 540 nm of standard solutions of 0-200 µM NaNO2 prepared in EBM-2 medium without VEGF. The amount of PGI2 released into the medium was analysed with a human PGI2 ELISA kit. The absorbance at 450 nm was measured with a microplate reader. The number of HUVECs per well was quantified using a CyQUANT Cell Proliferation Assay Kit. The amount of NO and PGI2 was finally normalized to cell number.

2.8 Co-culture of HUVECs and SMCs The co-culture experiments were performed in 6-well Transwell plate (BD Falcon) cell culture inserts incorporating PET membranes with 0.4 µm pores. The PCL, HGFI-PCL and (VEGF-HGFI)-PCL mats were placed in the lower well. HUVECs were seeded on the mats at 3.0×105 per well and cultured for 24 h in EBM-2 medium without VEGF. Then, the original medium was replaced with 1 mL fresh EBM-2 without VEGF. SMCs (A10) were harvested with trypsin and suspended in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 3 × l06 cells/mL. 1 mL of SMCs suspension was seeded on the upper cell culture inserts. After co-culturing the cells for 24 h, the total protein of SMCs was extracted by adding lysis buffer [0.1% sodium dodecyl sulfonate (SDS), 10 mM Tris (pH 7.4), 150 mM NaCl, 10 mM ethylene diamine tetraacetic Acid (EDTA), 1% Triton X-100, 1% sodium deoxycholate, and protease inhibitor cocktail] to the membrane while on ice. The cells were disrupted using a cell scraper and the lysates were centrifuged at 13000 rpm for 10 min at 4°C. The supernatants were collected, and protein concentrations was tested using the BCA assay (Beyotime, China). After being boiled in water with SDS-PAGE loading buffers, the protein samples were 11


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separated by electrophoresis using 8% SDS-polyacrylamide gels. Then the separated proteins were transferred onto polyvinylidene difluoride membranes (Roche, U.S.A.) and incubated with one of the following primary antibodies: mouse anti-alpha smooth muscle actin (a-SMA, 1:1000, Abcam, USA), mouse anti calponin (1:1000, Abcam, USA), or mouse anti β-actin antibody (1:1000, Cell Signaling Technology). The membranes were then incubated with an HRP-labelled goat anti-mouse IgG (H+L) antibody (1:1000, Beyotime) and washed with Tris-buffered saline with Tween. The intensity of all bands quantified using Quantity One v.4.62 software.

2.9 In vivo implantation in rats Fifteen SD rats were randomly divided into three experimental groups to receive PCL, HGFI-PCL and (VEGF-HGFI)-PCL grafts. As previously reported,3 after intraperitoneal injection of chloral hydrate (330 mg/kg) for anaesthesia and tail vein injection of heparin (100 units/kg) for anticoagulation, a midline laparotomy incision was performed, and the abdominal aorta was isolated and clamped at the proximal and distal sides. The vascular grafts (inner diameter, 2.0 mm; and length, 1.1 cm) were then anatomised in an end-to-end fashion using interrupted 9-0 monofilament nylon sutures (Jin Huan, Shanghai, China). After restoring the blood flow, the wound was washed with a 0.9% NaCl solution containing gentamicin (200 U/mL) and sutured using 3-0 knitted polyester sutures (Jin Huan, Shanghai, China). No anticoagulation drug was administered to the rats after surgery. Four weeks post-operation, the rats were sacrificed by injection of an overdose of chloral hydrate, and the grafts were explanted. After rinsed with 0.9% NaCl solution, the explanted grafts were cut into two parts from the middle. One part was snap-frozen in optimal cutting temperature (OCT) Compound (Tissue Tek) for frozen cross-sections. The other part was longitudinally cut into two pieces. One piece was observed under a stereomicroscope (LEICA S8AP0, Germany) and then snap-frozen in OCT for longitudinal sections. The other piece was prepared for SEM. 2.10 SEM The longitudinal samples were fixed in 2.5% (v/v) glutaraldehyde for 12 h, and dehydrated in ascending series of ethanol. Then, the samples were mounted onto aluminium stubs, sputter-coated with gold, and observed by SEM.

2.11 Histological analysis 12



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For histological analysis, cross-sections of explanted grafts (6 µm in thickness) were

stained

with

haematoxylin

&

eosin

(H&E),

Masson's

trichrome,

Verhoeffe-VanGieson (VVG), Safranin O and VonKossa. Slides were observed under an upright microscope (Leica DM3000, Germany), and images were acquired with a digital camera (Leica DFC450, Germany).

2.12 Immunofluorescence staining The frozen sections were fixed in acetone at -20 °C for 10 min, air-dried, and rinsed once with 0.01 mM PBS. To stain cell surface antigen, slides were blocked with 5% normal goat serum for 45 min at 4 °C and then incubated with mouse anti-CD31 (1:100, Abcam, USA) and mouse anti-CD68 (1:200, Abcam, USA) antibodies for 12 h at 4°C. To stain intracellular antigens, 0.1% Triton-PBS was used to permeate the membrane before incubation with 5% normal goat serum. Then the slides were incubated with mouse anti-α-SMA (1:100, Abcam, USA) and mouse anti-smooth muscle myosin heavy chain I (MYH, 1:500, Abcam, USA) antibodies for 12 h at 4°C. After finishing the incubation with primary antibodies, the slides were rinsed 5 times with PBS. Alexa Fluor 488-conjugated goat anti-mouse IgG (1:200, Invitrogen) was applied for 2 h at RT. The cell nuclei were stained with a DAPI containing mounting solution. Slides incubated with secondary antibody only served as negative controls. Slides were observed under a fluorescence microscope (Zeiss Axio Imager Z1, Germany) and images were acquired with a digital camera (AxioCam MRm, Germany). After cross-sections of grafts were stained with anti-CD31 antibody, all CD31+ capillaries within graft walls were counted to assess vascularization. The endothelialisation rate was quantified by adding the length of CD31-positive monolayer and dividing this sum by the length of the longitudinal section of the graft. The coverage rate by α-SMA+ SMCs and MYH+ SMCs was quantified by the same method as endothelialization. The longitudinal sections stained with anti-α-SMA and anti-MYH antibodies were used to measure the area of α-SMA+ SMCs and MYH+ SMCs, respectively, using IPP software. For the above analysis, one image per sample and five samples per group were included to obtain the statistical results.

13


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2.13 Statistical analysis GraphPad Prism Software Version 5.0 (San Diego, CA, USA) was used for statistical analysis. Single comparisons were carried out using a paired Student's t-test. Multiple comparisons were performed using a one-way ANOVA and Tukey’s post-hoc analysis. Significance level was accepted at a p-value below 0.05 and 0.001. Data are espressed as the mean ± standard error of the mean (SEM).

3. Results 3.1Characterization of VEGF-HGFI modified PCL graft As can be seen form the cross-sectional SEM images (Figure 1A), HGFI-PCL grafts and (VEGF-HGFI)-PCL grafts maintained good tubular and porous structure similar to the unmodified PCL grafts. The statistical analysis showed that the luminal diameters (1.81 ± 0.02 mm for PCL grafts, 1.81 ± 0.01 mm for HGFI-PCL grafts, 1.82 ± 0.01 mm for (VEGF-HGFI)-PCL grafts) and wall thickness (532.44 ± 12.65 µm for PCL grafts, 522.20 ± 13.77 µm for HGFI-PCL grafts and 528.05 ± 10.28 µm for (VEGF-HGFI)-PCL grafts) of the three types of grafts were almost the same (Table 1). The porosity of the PCL, HGFI-PCL and (VEGF-HGFI)-PCL grafts was 72.83 ± 1.86%, 72.05 ± 2.46% and 71.72 ± 0.89%, respectively (Table 1). The SEM images of the luminal surface showed that all fibers of the three types of grafts had smooth surface with well-defined fiber morphology (Figure 1B). The average fiber diameter and pore size of (VEGF-HGFI)-PCL grafts were 7.06 ± 0.76 µm and 29.99 ± 4.99 µm, respectively, which are almost similar to those of PCL and HGFI-PCL grafts (Table 1). Burst pressure, tensile strength, young’s modulus and elongation at break of the (VFGF-HGFI)-PCL grafts was 2125.92 ± 110.86 mmHg, 3.82 ± 0.72 MPa, 10.46 ± 1.03 MPa and 713.02 ± 29.82%, respectively, which are similar to those of HGFI-PCL and bare PCL grafts (Table 1). These results demonstrate that the HGFI and VEGF-HGFI modification did not alter the structure and mechanical properties of PCL grafts. To visualize the distribution of VEGF-HGFI modification within the PCL grafts, 14



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the (VEGF-HGFI)-PCL grafts were stained with a VEGF antibody and then cross-sections of the grafts were observed by CLSM. The (VEGF-HGFI)-PCL graft showed high fluorescence intensity compared to PCL and HGFI-PCL grafts (Figure 1C). A magnified view of CLSM image showed that the green fluorescence mainly surrounded the PCL fibers and could not be observed in the space between the PCL fibers. This indicates that the fusion protein VEGF-HGFI could form a VEGF coating layer on the surface of PCL fibers in the grafts by hydrophobic interactions, but it did not occupy the pores of PCL grafts.

Table 1. Structural characterization and mechanical properties of the grafts Luminal Diameter (mm) Wall Thickness (µm) Fibers Diameter (µm) Pore Size (µm) Porosity (%) Burst Pressure (mmHg) Maximum Stress (MPa) Young's Modulus (MPa) Strain at Rupture (%)

PCL

HGFI-PCL

(VEGF-HGFI)-PCL

1.81±0.02

1.81±0.01

1.82±0.01

532.44±12.65

522.20±13.77

528.05±10.28

7.03±0.55

6.93±0.68

7.06±0.77

30.27±5.66

31.07±4.24

29.99±4.99

72.83±1.86

72.05±2.46

71.72±0.89

2125.93±110.86

2250.18±77.08

2145.43±45.22

3.99±0.17

3.38±0.78

3.82±0.72

10.28±1.48

10.61±1.69

10.46±1.03

764.90±17.89

695.80±53.78

713.02±29.82

15


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Figure 1. Characterization of VEGF-HGFI-modified PCL vascular grafts. (A): SEM images of the cross-sections of the three types of grafts. (B): SEM images of the lumen surface of the three types of grafts. (C): After staining with a VEGF antibody, the distribution of VEGF-HGFI modification within the PCL grafts was observed by CLSM. The region outlined by the white square in the CLSM image was enlarged and shown in the inset. 3.2 Haemocompatibility One of the main factors hindering the clinical application of small-caliber vascular grafts is acute thrombosis. The blood clotting times of the HGFI-PCL and (VEGF-HGFI)-PCL grafts were similar to those of bare PCL grafts (Table S1), which indicates that HGFI and VEGF-HGFI modification did not induce acute thrombosis. The haemolysis rate of (VEGF-HGFI)-PCL (0.52 ± 0.04%) was lower than that of PCL (0.64 ± 0.10%) and HGFI-PCL (0.62 ± 0.05%) and was very close to that of commercial ePTFE vessels (0.48 ± 0.09%). In addition, HGFI and VEGF-HGFI modifications significantly reduced FGN adsorption (Figure 2B). Platelet adhesion on 16



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various PCL mats was also tested by SEM (Figure 2C). A quantitative comparison of platelet adhesion on various mats is given in Figure 2D. HGFI and VEGF-HGFI modification markedly reduced platelet adhesion. The expression of P-selectin on the adherent platelets was considered as an activation marker.27 The expression of P-selectin was significantly lower in HGFI-PCL and (VEGF-HGFI)-PCL compared with that of the PCL mat (Figure 2E), which indicates that HGFI and VEGF-HGFI modification decreased the activation of adherent platelets. Collectively, these results demonstrate

that

HGFI

and

VEGF-HGFI

modification

improved

the

haemocompatibility of PCL grafts.

Figure 2. Evaluation of the haemocompatibility of PCL mats with or without protein modification. (A): Haemolysis ratios of PCL mats with or without protein modification. Modification with HGFI or VEGF-HGFI had no influence on the haemolysis of PCL mats. (B): The FGN adsorbed to mats was tested using a Human FGN ELISA Kit. The FGN adsorbed to bare PCL mats was defined as 100%. Relative quantification showed that HGFI and VEGF-HGFI modification significantly decrease FGN absorption on PCL mats. (C): Platelet adhesion on various PCL mats was observed by SEM. (D): The number of platelet adhesion was calculated based on the SEM images. (E): Activation of platelets on various PCL mats was quantified by a P-selectin ELISA Kit. The activation of platelets on the bare PCL mats was defined as 100%.*p < 0.05, #p

Functional Modification of Electrospun Poly(Îµ-caprolactone) Vascular

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