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Biomimetic modification of polyurethane-based nanofibrous vascular grafts: A promising approach towards stable endothelial lining
Accepted Manuscript Biomimetic modification of polyurethane-based nanofibrous vascular grafts: A promising approach towards stable endothelial lining
Parivash Davoudi, Shiva Assadpour, Mohammad Ali Derakhshan, Jafar Ai, Atefeh Solouk, Hossein Ghanbari PII: DOI: Reference:
S0928-4931(17)30153-4 doi: 10.1016/j.msec.2017.05.140 MSC 8122
To appear in:
Materials Science & Engineering C
Received date: Revised date: Accepted date:
12 January 2017 24 May 2017 28 May 2017
Please cite this article as: Parivash Davoudi, Shiva Assadpour, Mohammad Ali Derakhshan, Jafar Ai, Atefeh Solouk, Hossein Ghanbari , Biomimetic modification of polyurethane-based nanofibrous vascular grafts: A promising approach towards stable endothelial lining, Materials Science & Engineering C (2017), doi: 10.1016/ j.msec.2017.05.140
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ACCEPTED MANUSCRIPT Biomimetic Modification of Polyurethane-Based Nanofibrous Vascular Grafts: A Promising Approach towards Stable Endothelial Lining
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Parivash Davoudi1, Shiva Assadpour1, Mohammad Ali Derakhshan2, Jafar Ai1, Atefeh Solouk3, HosseinGhanbari2,4,*
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Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran 2
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Regenerative Nanomedicine Research Group, Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran 3
Biomedical Engineering Faculty, Amirkabir University of Technology, Tehran, Iran
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Research Center for Advanced Cardiovascular Techniques, Tehran Heart Center, Tehran University of Medical Sciences, Tehran, Iran
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*Corresponding author: Dr. Hossein Ghanbari, MD, PhD Associate Professor of Regenerative Nanomedicine Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Italia Street, 14177-55469, Tehran, Iran Tel: +98-21-4305 2200 Fax: +98-21-8899 1117 Email: [email protected]
ACCEPTED MANUSCRIPT Abstract:
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The emerging demand for small caliber vascular grafts to replace damaged vessels has attracted research attention. However, there is no perfect replacement in clinical use yet, mainly due to low patency rate of synthetic small caliber grafts. The main pathology behind low patency rate include thrombosis and graft/vessel hemodynamic mismatch, leading to intimal hyperplasia. Rapid in-situ endothelialization of vascular grafts is considered as one of the best strategies to overcome these complications. In the present study, Heparin and VEGF were immobilized via self-polymerization and deposition of polydopamine (PDA) on polyurethane (PU) nanofibrous scaffolds to improve endothelialization. Polyurethane nanofibrous scaffold (PUNF) that mimics vascular extracellular matrix (ECM) was chosen owing to its biocompatibility, biodegradability. Scanning electron microscopy (SEM), water contact angle (CA) measurement and Raman spectroscopy were used to characterize the surface, and tensile test was used to analyze mechanical properties before and after surface modification of the scaffolds. It was found that tensile strength and young´s modulus were significantly increased after PDA coating on PUNF membranes. The hemocompatibility tests revealed that surface heparinization significantly inhibited the adhesion of platelet on the scaffolds. Immobilization of VEGF on the scaffolds significantly enhanced the proliferation of human umbilical vein endothelial cells (HUVECs) through enhanced cells adhesion and improved cell-scaffold interactions. The results suggest that dual-factor immobilization resulted in not only confluent monolayer of endothelial cells but also conferred excellent antithrombotic properties to the surface. This method of surface modification (immobilization of Heparin, VEGF by PDA layer) is suggested as a promising modification technique to increase hemocompatibility of small-diameter vascular grafts.
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Keywords: Vascular grafts; Endothelialization; Polyurethane, Electrospun Nanofiber, Heparin; Poly dopamine.
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1. Introduction
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Many prosthetic vascular grafts made from biocompatible polymers such as polyurethane (PU) [1], expanded poly (tetrafluoroethylene) (ePTFE) [2], poly(ethylene terephthalate) (PET) [3], and polycaprolactone (PCL) [4] have been developed. However, they frequently suffer from low patency rate when applied as small diameter (<6mm) bypass grafts [5].
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Efforts to improve a small caliber vascular graft have been challenging over the past decade. A fully functional endothelial layer is essential for a small diameter vascular graft to be patent in long-term[6], owing to its role in the prohibition of excessive tissue ingrowth (intimal hyperplasia) and thrombogenesis that are two main reasons of graft failure. Therefore, both seeding of endothelial cells onto the luminal surface of the graft or in situ endothelialization improves long-term permanence of cardiovascular implants [7] and small-diameter prostheses. Since endothelial cells attach on the surface of an extracellular matrix (ECM) in nature, production of biomaterials equivalent to ECM can help the attachment, proliferation, and phenotypic maintenance of endothelial cells on vascular scaffolds [8].
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Recently, electrospinning technology has been extremely investigated as a method to make ECM-mimicking structures which can be noted as artificial ECM or scaffolds [9-11]. Electrospinning is a potent technique, which can generate fibrous structures from diverse materials such as polymers, ceramics , and composites [12]. Electrospun fibers are similar to the natural ECM components with many advantages such as the simplicity of the production process and scale-up, and also, capability to produce fibers with various diameters ranging from nanometer to micrometer sizes [13].
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Therewith, the high surface area to volume ratio and interconnected pores of these fibrous meshes result in desirable cell attachment and oxygen/nutrient transport, respectively. Also, multiple polymers entailing blood compatible ones (i.e. PTFE, PET, PCL and PU) can be electrospun into ultrafine fibers. All the mentioned benefits make electrospinning an ideal method for production of nanofibrous mats [14]. In comparison, PU-based materials have been vastly investigated for preparation of bio- and blood-compatible products. Recently, Adipurnama et al. have reviewed a variety of different surface modifications of PU and their effects on endothelialization for vascular graft applications [15].
Several surface modification methods have been applied to improve blood-compatibility of vascular grafts especially polyurethane based ones [16]. Functional groups or bioactive macromolecules can modify the surface of nanofibrous vascular grafts in order to accelerate
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endothelialization of electrospun mats [17]. For immobilization of biomolecules on polymeric biomaterials, various physical and chemical techniques are attainable. An appropriate functionalization method must be selected because most vascular grafts are produced from inert hydrophobic polymers lacking functional moieties for chemical conjugation. In our earlier attempts, collagen type I was grafted onto plasma-activated and acrylic acid coated PU nanocomposite [17]. In irradiation techniques such as plasma treatment high energy sources are used to introduce reactive groups for subsequent immobilization of desired biomolecules [18]. For versatile solid materials from metal to synthetic polymers, another facile surface modification method is simple dip-coating with dopamine solution [19]. Under slightly basic conditions, a stable layer that is adherent to the surface of materials, is created by oxidative polymerization of dopamine. The reaction environment in post-modification with polydopamine layer is simple and clean compared to other chemical immobilization methods and allows functionalization with bioactive molecules containing thiols or primary amines via imine formation and/or Micheal addition [18].
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It is known that rapid endothelialization is a prerequisite for artificial vascular grafts, and having non-thrombogenic surface before composition of a fully endothelial layer can make it ideal blood contacting surface. Vascular endothelial growth factor (VEGF) promotes proliferation and migration of endothelial cells (EC), results in angiogenic vascular growth, and induces differentiation of pluripotent stem cells to blood progenitor cells [20]. Heparin, one of the most commonly used clinical anticoagulant reagents, has positive effects on ECs growth and proliferation by binding and stabilizing cell growth factors(GFs) [21]. Binding of heparin and GFs principally happens via electrostatic interactions between N- and O-sulfated groups with negative charge of heparin and the basic lysine and arginine residues of FGF-2 or VEGF [22]. Diffusion of GFs is decelerated by binding to heparin [23], and interaction of growth factors with heparin is observed to be necessary for storage, release, and protection from pH, heat, and enzymatic degradation [24]. Recently, heparin modified biomaterials demonstrated excellent exploits in ECs growth and proliferation [25]. In the present investigation, nanofibrous electrospun vascular scaffolds based on elastic biodegradable PU substrates were fabricated to mimic the native vascular ECM. For surface modification, a poly dopamine-mediated immobilization platform together with a cell adhesive growth factor, VEGF, and an anti thrombogenic factor, heparin was developed. The effects of PDA-coating and VEGF and heparin immobilization on the morphology, hydrophilicity, hemocompatibility and mechanical properties of nanofibrous PU membranes were evaluated in details. Finally, the effect of PDA, VEGF, heparin and dual factors on regulation of HUVEC attachment and proliferation was evaluated. 2. Materials and Methods
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2.2.Preparation of Polyurethane Nanofibers (PUNF)
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Thermoplastic Polyurethane (PU,Tecoflex,SG-80A)was purchased from Lubrizol, USA. 3hydroxytyraminium chloride was purchased from Merck Co., Schuchardt OHG. Tris (hydroxymethyl) amino methane was obtained from Merck KGaA. Chloroform and Methanol were purchased from Merck co, Germany. Heparin sodium salt and VEGF were obtained from Sigma-Aldrich (St. Louis, MO, USA). HUVEC cells were obtained from the National Cell Bank of Iran (Pasteur Institute of Iran). Other unspecified chemicals were purchased from Sigma. All the chemicals were of analytical grade and used without further purification. Electrospinning machine was provided by Fanavarn Nano-meghyas Co. Ltd, Tehran, Iran.
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Nanofibrous scaffolds were produced utilizing a standard electrospinning setup. PU was dissolved in a mixture of chloroform and methanol with a ratio of 1:1 (v/v) as solvent system to prepare electro-spinning solution at 3/5% concentration (w/v), according to our previous study [26]. Solutions were pumped at a flow rate of 1 ml/h using a syringe pump while a potential of 20 kV was applied between the spinneret and a grounded collector located 10 cm apart from the spinneret. 2.3. Fabrication of Nanofibrous Vascular grafts
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A one-step electrospinning method was used in order to fabricate nanofibrous vascular graft. For this, a cylindrical mandrel with diameter of 4mm was fabricated using stainless steel alloy. Then it was used in the electrospinnig machine as a rotatory collector, in order to fabricate the tubal scaffolds in one-step process. Previously, optimized set up was used to fabricate nanofibrous scaffolds in the shape of small diameter vessel. 2.4. Surface modification of PUNF
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2.4.1. Poly dopamine coating(D-PUNF) For polydopamine (PDA) coating, the PUNF mats were cut into a circular shape (area: 1.99 cm2) which was submerged in 3,4-dihydroxyphenylamine (2 mg/ml) dissolved in Tris-HCl buffer (10mM,pH 8.5) and shaken on a rocker for 1 h. After this process, the samples were washed three times in deionized water to remove unbound PDA. The resultant membranes were used for characterization and further surface modifications. Then, polydopamine-coated PUNF (DPUNF) were modified with either heparin and VEGF or both of them. 2.4.2. Heparin immobilization(H-D-PUNF)
ACCEPTED MANUSCRIPT The prepared D-PUNF was immersed into the heparin solution (PBS buffer solution as solvent, pH 7.4) at 4◦C overnight. The heparin concentration(C) in solution was 2.0g/L. Then the membrane was taken out and rinsed with deionized water entirely in order to remove physically adsorbed heparin. Thereafter, the resultant modified membranes (H-D-PUNF) were used for characterization. 2.4.3.VEGF immobilization(V-D-PUNF)
2.4.4.VEGF and Heparin immobilization(VH-D-PUNF)
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For immobilization of VEGF, the PDA deposited mats were immersed in 1000ng/ml VEGF (dissolved in 10mMTris-HCl buffer, pH 8.5) overnight at4°C. The volume of VEGF solution was fixed to 400μL for the reaction.
2.5.1. Scanning electron microscopy (SEM)
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2.5.Characterization of the membrane surface
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For concurrent immobilization of VEGF and heparin, equal volumes of heparin and VEGF solutions (200μl) were added on the PDA-deposited mats (D-PUNF) for overnight at4°C.
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2.5.2. Raman spectroscopy:
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To investigate the surface morphology of the membranes and diameter of the obtained nanofibers, SEM (XL 30, Philips, USA) was used at an accelerating voltage of 25.0 kV. The samples were sputtered with gold prior to observation. Then, micrographs were analyzed utilizing ImageJ software to calculate the average diameter of nanofibers.
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Raman spectroscopy (Dispersive Raman Microscope, SENTERRA, 2009, BRUKER, Germany) in the spectral range of 200-3500cm−1 with resolution < 3 cm−1was used to display the structural changes in various functional groups on the scaffolds before and after surface modifications.
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2.5.3. Surface wettability
Surface wettability of membranes was characterized by static water contact angle measurements utilizing an optical video contact angle system (OCA-15-plus, Data physics, Germany). The measurement was performed at 20◦C and 70% relative humidity and in triplicate. 2.6. Mechanical evaluation Tensile test was performed for PUNF, D-PUNF, VH-D-PUNF samples using a uniaxial load testing machine (Instron 5565). The samples were cut into rectangular-shaped pieces (shaft length
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50mm, width 10mm, n=3). Thickness was measured using a digital electronic outside micrometer. The load cell capacity was 500 N and test speed uniaxial tension was applied to either ends of the scaffolds until failure was 10mm/min at room temperature. Stress (MPa) was calculated by dividing the force generated during stretching by the initial cross-sectional area. Strain was calculated as the ratio of the change in length in reference to the original sample length (%). The maximum tensile strength (maximum stress), elongation at break (maximum strain) and Young´s modulus for graft samples (n=3) were obtained.
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2.7.Platelet adhesion
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All four kinds of membrane samples (i.e. PUNF, D-PUNF, H-D-PUNF, and VH-D-PUNF) were placed in the wells of plate (24-well). 250µl of fresh platelet(obtained from Blood Transfusion Center) was dropped on each sample, and contact was maintained for 50min. Then the samples were carefully rinsed in PBS (PH 7.2) to remove non-firmly adsorbed platelet. After fixation with 4.0 wt% paraformaldehyde solution for 30min, the samples were washed twice with PBS. The samples were put in a freezer (-80◦C) overnight. Then, in the next day the samples were put in freeze dryer. Thereafter, the resultant samples were studied with SEM. 2.8.Cell culture on scaffolds
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HUVECs were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco, USA) supplemented with 10 % FBS, 50 U/ml penicillin and 50 U/ml streptomycin at 37 °C in a 5% CO2incubator. The culture medium was replaced every 2days. Before cell seeding, the unmodified scaffold (PUNF) and other modified scaffolds were cut into circular samples to fit a 24-well plate. Scaffolds were sterilized with ultraviolet light for 1 h. HUVECs were then seeded onto the top of the scaffold with a final density of 1×10 5 cells/scaffold in 24-well plates and incubated in DMEM/F12 for 1h allowing cells to attach onto the surface of the scaffold. For further incubation fresh medium was added. 2.9. Cell viability assay Metabolic activity of HUVECs cultured on the scaffolds was measured for 24, 48, and72h after cell seeding by applying the MTT assay. The activity of a mitochondrial dehydrogenase is assayed by transformation of light yellow MTT into purple formazan. For this purpose, 100μl of 5mg/ml MTT solution was added to each well. After 4h incubation at 37°C, the culture medium was removed and 300µL of dimethyl sulfoxide (DMSO) was added into each well. The solutions were transferred into a 96-well plate, and the absorbance was measured at 570 nm using an ELISA reader (Expert 96, Asys Hitch, Ec Austria). The experiments were repeated three times and the result of each experiment was reported as mean ± (SD).
ACCEPTED MANUSCRIPT 2.10.Cell adhesion and morphology study The morphologies of HUVECs on the surfaces of the scaffolds were observed by SEM. After 24h of culture, cell-containing scaffolds were fixed with 4% paraformaldehyde (pH 7.2) for 30 min, followed by washing with PBS and freezing in a freezer (-80◦C )for overnight, then the next day were put in freeze dryer. Finally, the resultant samples were observed with SEM.
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2.11. Statistical analysis
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All data are expressed as mean±SD. Statistical analysis was carried out by means of one-way ANOVA (p < 0.05).
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3. Results and discussion
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3.1. Fabrication of Nanofibrous Vascular graft
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3.2. Membrane Surface morphology
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Figure.1 shows the scaffolds fabricated using a one-step method. As shown in the figure a nanofibrous small diameter vessel with a diameter of 4mm has been successfully fabricated. The advantage of one-step process was uniform structure of the scaffold and feasibility of the technique.
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Electro spinning of polyurethane was led to the formation of bead-free nanofibers. The SEM images in Fig.2 display the effect of PDA coating and subsequent immobilization of biomolecules on the scaffolds. It can be noted that surface modification resulted in cross-linking between the fibers which was mainly the effect of PDA modification. The cross-linking effect of PDA has already been reported in previous study [27]. By the use of imageJ, the average fiber diameter was obtained 405±116, 410±119, 408±123, 418±98, 416± 100 nm for PUNF, D-PUNF, H-D-PUNF, V-D-PUNF, VH-D-PUNF, respectively. All the samples revealed uniformity and there was no significant difference in the average fiber diameter between unmodified and modified scaffolds. Since the coating layer was very thin, it did not change the average diameter of the nanofibers which was in accordance with previously published reports [26]. The average pore diameter of the nanofibrous scaffold was 4.57±2.5, 2.15±1.2, 2.2±1.67, 2.3±1.26, 2.28±1.04 m for PUNF, D-PUNF, H-D-PUNF, V-D-PUNF, VH-D-PUNF respectively. The porosity percentage was approximately 69%, 40%,45%,49.8%, and 47% for PUNF, D-PUNF, H-D-PUNF, V-D-PUNF, VH-DPUNF, respectively. The porosity of PUNF was significantly decreased after surface modification with polydopamine (p<0.05). This can be attributed to the cross-linking effect of polydopamine which decreased the free space between the fibers. It could also be related to the secondary immobilization of biomolecules on smaller pores and formation of tight junctions between the fibers. However, the difference between the modified samples was not statistically significant.
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3.3. Analysis of Raman spectroscopy: To further confirm the PDA coating and other surface modifications such as heparin and VEGF immobilization, Raman spectroscopy was utilized. 3.3.1. PDA coating
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As shown in Fig.3A the Raman spectra of PUNF and D-PUNF scaffolds revealed five distinct trends of peaks centered between the 1250and 1490 cm-1 wavenumbers. The intensity at 1266cm-1,1297cm-1,1438cm-1(CH3) in PUNF scaffold was greater than D-PUNF scaffold. Unlike the intensity of peaks 1356cm-1 (C-CH3) [28,29] and 1486cm -1(aromatic ring) was greater in DPUNF substrates. Raman new peaks at 1370/1600 cm-1 indicated the formation of PDA coating on PUNF scaffold [30]. In the Raman shift of D-PUNF, 1370cm-1 peak is related to stretching of catechol and the 1570cm-1peak is related to the deformation of catechol, which is a major component of the dopamine structure [31]. Hence, the peaks centered between the 1550 and 1600 cm-1wavenumbers(characteristic of stretching and deformation of aromatic ring)clearly shows PDA coating on PUNF scaffold [32].
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These results showed the existence of dopamine onto D-PUNF membrane surface.
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3.3.2. Heparin immobilization (H-D-PUNF)
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Prominent Raman peaks of heparin is appeared in the range of 900-1150 cm−1[33]. As shown in Fig.3B, Raman spectrum of H-D-PUNF scaffold revealed multiple peaks that some of them were new in comparison with Raman spectrum of D-PUNF, including 914cm-1 ,950cm-1 (Carboxylic acid dimer ), 1065 cm−1 (2-O-SO3 vibration), and 1088cm-1.The other peaks were also present in Raman spectrum of D-PUNF scaffold, but revealed higher intensity such as 1035cm-1 ((N-SO3 vibration) [34,35]. These results demonstrate that heparin is successfully immobilized onto the D-PUNF surface. 3.3.3. VEGF immobilization (V-D-PUNF) As shown in Fig.3C comparison between Raman spectrum of V-D-PUNF scaffold and D-PUNF scaffold revealed the novel peaks at 702,638 cm-1(C-S aliphatic ) and 914cm-1(C-O-C) [36]. Furthermore, another new peak with low density at 1065cm-1(aromatic rings) was seen on V-DPUNF samples. Some typical peaks attributing to D-PUNF scaffold but with lower intensity at837, 950cm-1(C-O-C) and with higher intensity at 729 cm-1 (C-S aliphatic) were also revealed. These results suggested that VEGF was successfully immobilized onto the D-PUNF membrane surface.
ACCEPTED MANUSCRIPT 3.3.4. VH immobilization (VH-D-PUNF) As shown in Fig.3D, Raman spectrum of VH-D-PUNF scaffold revealed characteristic peaks of V-D-PUNF (638,706,914 cm-1) together with similar relative intensity peaks at 1005 cm-1,and between 1035-1088 cm-1 with H-D-PUNF scaffold. All these emphasized the existence of VEGF and heparin on VH-D-PUNF scaffold. .
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3.4. Surface wettability
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Water contact angle (CA) measurement was used to characterize the surface relative hydrophilicity and wetting characteristics. Fig.4 shows water contact angle of unmodified and modified polyurethane. The water CA of unmodified PUNF was 125.17±3.89º which was significantly decreased to75.4±3.48º via dopamine modification(D-PUNF), and was further decreased after heparin immobilization to 67.5±9.87º(H-D-PUNF). This result indicated that the introduction of abundant hydrophilic groups such as –OH, –COOH and–NH2in the layer of polydopamine brought hydrophilicity to the hydrophobic PUNF scaffolds. Besides, high concentrations of –OH and ether linkages in heparin also enhanced surface hydrophilicity after heparinization[37]. In addition, immobilization of VEGF on D-PUNF subsequently further decreased the water CA to 40±8.6º(V-D-PUNF). The robust reduction of contact angle might be attributed to the combined hydrophilicity effect of PDA and VEGF[5]. The water CA of VH-DPUNF scaffolds was 54.03±0.89º, indicating the effect of simultaneous immobilization of VEGF and heparin on D-PUNF scaffolds. In vitro studies indicated that ECs display poor adhesion and proliferation behavior on hydrophobic materials[38], whereas EC behavior was enhanced on moderately wettable polymers[39]. A polymeric structure with water CA of 55◦ resulted in maximal cellular adhesion, enhanced proliferation rates, and improved cellular growth regardless of cell type[16],[40]. Therefore, taking only hydrophilicity into account, it was expected that surface modified scaffolds, in particular VH-D-PUNF scaffold with water CA 54.03±0.89º, could provide better interactions with the cells in order to create a confluent layer of endothelium. 3.5. Mechanical evaluations Mechanical properties of scaffolds are shown on Table.1.After PDA coating and biomolecules immobilization (i.e. D-PUNF and VH-D-PUNF), improved mechanical properties was observed compared to PUNF. The maximum tensile strength of PUNF samples was about 1.8±0.40MPa which was significantly increased to 3.69±0.28 in D-PUNF and 3.07±0.23 in VH-D-PUNF samples after surface modifications. The ultimate strain was also increased from 0.84± 0.05 for PUNF samples to 1.455±0.12 and 1.076±0.08 in D-PUNF and VH-D-PUNF, respectively. The reason behind this improvement in mechanical properties can be the presence of PDA layer within the void space between fibers that causes them to adhere to each other. In the other word, using
ACCEPTED MANUSCRIPT PDA, nonwoven PUNF scaffolds converted to cross-linked ones with improved mechanical properties [27].
3.6.Platelet adhesion
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As known, platelet adhesion and aggregation on the scaffold activates coagulation pathway that is one of the crucial factors in hemocompatibility evaluation. Therefore, platelet adhesion for anti-thrombogenic properties of the scaffolds was investigated. SEM images (Fig.5) represents that the quantity of platelets aggregation was markedly different from each other on the samples. The hydrophobic PUNF surface demonstrated the highest number of adherent platelets (Fig.5A). For interaction of platelets with a material surface, the nature of the surface and the adsorbed proteins are important [6]. Various plasma proteins, such as thrombin and fibrinogen, can be absorbed onto material surfaces and stimulate activation and aggregation of platelets on the scaffold. Each fibrinogen molecule by carboxylic terminus of chain interacts
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with integrin receptor on the platelet membrane. Resting platelets interact with immobilized fibrinogen because soluble fibrinogen in plasma is folded and ɤ chain is hidden. Therefore, fibrinogen must be unfolded or conformationally changed [41] to bind to the platelets. The conformational alteration of fibrinogen after absorption onto hydrophobic surfaces is higher than on hydrophilic surfaces as previously reported [42]. The data indicated that the adhesion and activation of platelets were clearly inhibited by PDA coating and heparin immobilization (Fig.5 B,C). Indeed, these hydrophilic surfaces preferred adsorption of water molecules to plasma proteins. Hydrophilic modification of PUNF surface resulted in enhanced hemocompatibility of the scaffolds. According to the Fig5, the quantity of adhered platelets on the samples declined in the order of: PUNF>D-PUNF>VH-D-PUNF>H-D-PUNF. Comparing two samples with heparin immobilized, i.e. H-D-PUNF and VH-D-PUNF, the results of platelet adhesion studies revealed that samples modified with heparin were more resistant to platelet adherence and consequent activation than VH-D-PUNF. This could be attributed to the platelet repellent effect of heparin conferred to the surface. When the surface simultaneously modified with heparin and VEGF, the presence of VEGF could partially compromise the effect of heparin, suggesting the importance of optimization of VEGF/Heparin ratio in surface modification method. 3.7. Cell viability assay MTT assay was used to evaluate viability of HUVECs on unmodified and modified PUNF scaffolds(i.e. PUNF, D-PUNF, H-D-PU, VH-D-PUNF and V-D-PUNF groups). As shown in Fig.6, all the surfaces showed acceptable cell viability which is increasing as time rises. The proliferation of HUVECs on surface modified PUNF was better than unmodified one in all the defined times.
ACCEPTED MANUSCRIPT Comparison between modified samples showed that VH-D-PUNF is the best substrate to support endothelial cell proliferation. Having both anti-thrombogenic property and endothelialisation capability is crucial for small caliber vascular grafts. These ensure the hemocompatibility and long term patency of the grafts which is desirable.
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3.8. Cell adhesion and spreading study
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Cell shape and spreading is relevant to surface wettability [43], that is one of the important modulators of cellular function. Fig.7 shows adhesion, spreading and morphologic features of HUVECs onto unmodified and modified PUNF scaffolds. Unmodified PUNF is highly hydrophobic polymer, and HUVECs grown on it displayed smooth border line without cellular processes, a typical non-adherent non-spreading morphology, demonstrating limited interaction and adhesion to the material (Fig.7.A). Cells appeared to be physically adsorbed instead of biologically attached. In contrast, cells seeded on D-PUNF scaffold exhibited limited spreading with formation of cellular processes, indicating that PDA deposition gained cell adhesion, which is attributed to either the adsorption /immobilization of serum proteins on PDA ad-layer or direct electrostatic interactions with cells or by both of them [44](Fig.7.B). The serum proteins connected to PDA layer act as cell adhesion sites [45]. With immobilization of heparin on DPUNF, flattened cells demonstrated robust cellular adhesion and their plasma membrane spread on the substratum, moreover indicated higher proliferation and interactions between them(Fig.7.C). The shape of a cell is closely dependent on cell function. Usually the shape of an EC in a vein wall is spindle or oval shape, demonstrating a biological function, proliferation only happens for cells that are spread out. Several studies pointed that heparin can advance endothelial cell proliferation [46]. As mentioned above, PDA-coating decreased water CA of PUNF scaffold from125º to 75º and with heparin immobilization more decreased to 67º, therefore, ECs better adhered and expanded on heparinized scaffolds. VEGF is known to stimulate cellular responses by coupling to and subsequent phosphorylation of tyrosine kinase receptors in cell membrane [5]. The cells seeded on VEGF-immobilized scaffolds (Fig.7.D,E) showed excellent spreading and adhesion compared to the others(PUNF,D-PUNF,H-D-PUNF). Planed area per cell calculations increased in the order of PUNF [224±25], D-PUNF [232±35], HD-PUNF [238±30], V-D-PUNF [245±42] µm2. However, the differences were not statistically significant. VEGF immobilization eventuated mitogenesis and migration of HUVECs along with cell adhesion affection of PDA coating composed a confluent mono-cellular layer of HUVECs which is important in endothelialization of vascular grafts with small diameter (Fig.7. D,E).The growth of HUVECs was intensified when dual factors(Heparin and VEGF) were simultaneously exist onto D-PUNF scaffold. As mentioned by Solouk et al [12], the best water CA for growing of the cells on scaffold is 55º, and water CA of VH-D-PUNF scaffold is 54±0.89º. Meanwhile, before
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4.Conclusion
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In vascular tissue engineering, efficient endothelialization of graft surfaces through adhesion, spreading and proliferation of ECs is essential. Synthetic polymers do not provide optimum interaction with cells to promote endothelialization. In this study a biomimetic coating was used as an efficient surface modification technique. For this purpose, electrospun nanofibrous scaffolds of PU were modified with a layer of PDA which were consequently, coated with Heparin and VEGF. These modifications not only improved the mechanical and surface properties of the scaffolds, but also enhanced cell adhesion, spreading, and proliferation on the scaffolds. The functional scaffolds demonstrated appropriate blood compatibility. These data suggest that modification of PU fibers with PDA layer and secondary immobilization with Heparin and VEGF is a potential improvement for small caliber vascular grafts and cardiovascular tissue engineering products. 5. Acknowledgements:
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REFERENCES
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This work was financially supported by Tehran University of Medical Sciences (grant number: 93-01-87-24919).
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Figure Captions
Fig.1: A nanofibrous small diameter vascular scaffold fabricated using one-step electrospinning method.
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Fig.2: Scanning electron microscopy of polyurethane nanofibers (PUNF) before and after modification: (a)PUNF, (b) D-PUNF, (c) H-D-PUNF, (d) V-D-PUNF, (e) VH-D-PUNF (scale bar5µm, inset10µm). The effect of surface modification on the pore size of nanofibrous scaffolds can be seen on modified samples.
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Fig.3: Comparison between Raman spectra of unmodified and modified scaffolds : A) PUNF(pu) and DPUNF(d), B)D-PUNF(d) and H-D-PUNF(h), C)D-PUNF(d) with V-D-PUNF(v), D)VH-D-PUNF(vh) with V-DPUNF(v) and H-D-PUNF(h).
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Fig.4: Surface wettability and water contact angle measurement and representative images of water droplet on the scaffolds. The differences between unmodified PUNF scaffolds with modified scaffolds were significant. (one way ANOVA, p<0.001). Fig.5: The morphology of platelets on the surface of unmodified and modified PUNF : (A)PUNF ,(B)DPUNF ,(C) H-D-PUNF , (D)VH-D-PUNF(T=50min). The hydrophobic PUNF surface had the highest number of adherent platelets (A) which was decreased in modified samples in the order of: PUNF > D-PUNF > VH-D-PUNF > H-D-PUNF. Fig.6: The results of MTT assay of HUVECs cultured on nanofibrous scaffolds. As it can be seen, modified samples revealed significantly higher level of cell viability compared to unmodified PUNF scaffold (one way ANOVA, *: p<0.05, **: p<0.01).
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Tables
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Table.1: Mechanical characterization of scaffolds
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Fig.1. A nanofibrous small diameter vascular scaffold fabricated using one-step electrospinning method.
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Fig.2. Scanning electron microscopy of polyurethane nanofibers (PUNF) before and after modification: (a)PUNF, (b) D-PUNF, (c) H-D-PUNF, (d) V-D-PUNF, (e) VH-D-PUNF (scale bar5µm, inset10µm). The effect of surface modification on the pore size of nanofibrous scaffolds can be seen on modified samples.
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Fig.3. Comparison between Raman spectra of unmodified and modified scaffolds: A) PUNF (pu) and DPUNF (d), B) D-PUNF (d) and H-D-PUNF (h), C) D-PUNF (d) with V-D-PUNF (v), D) VH-D-PUNF (vh) with V-D-PUNF (v) and H-D-PUNF (h).
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Fig.4. Surface wettability and water contact angle measurement and representative images of water droplet on the scaffolds. The differences between unmodified PUNF scaffolds with modified scaffolds were significant. (p<0.001).
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Fig.5. The morphology of platelets on the surface of unmodified and modified PUNF : (A)PUNF ,(B)D-PUNF ,(C) H-D-PUNF , (D)VH-D-PUNF(T=50min). The hydrophobic PUNF surface had the highest number of adherent platelets (A) which was decreased in modified samples in the order of: PUNF > D-PUNF > VH-D-PUNF > H-DPUNF.
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Fig.6. The results of MTT assay of HUVECs cultured on nanofibrous scaffolds. As it can be seen, modified samples revealed significantly higher level of cell viability compared to unmodified PUNF scaffold (one way ANOVA, *: p<0.05, **: p<0.01).
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Fig.7. SEM images of HUVEC adhesion and spreading on unmodified and modified PUNF scaffolds : (A) PUNF, (B) D-PUNF, (C) H-D-PUNF, (D) V-D-PUNF, (E) VH-D-PUNF, [Magnification in each of them from left to right : 500×/ scale bare=50µm, 2500×/ scale bare=10µm ].
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Table. 1 Mechanical characterization of scaffolds
1.8±0.40 3.69±0.28 3.07±0.23
Young, s modulus (E, MPa)
Toughness(UT,J/m3)
2.14±0.15 2.53±0.23 2.85±0.3
0.756±0.3 2.68±0.25 1.65±0.2
0.84± 0.05 1.455±0.12 1.076±0.08
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PUNF D-PUNF VH-D-PUNF
Ultimate Strain(εf)
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Max tensile strenght (σf, MPa)
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Sample name
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Highlights: This is an original article reporting our latest findings on developing polyurethane based
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nanofibrous scaffolds for vascular tissue engineering applications. In the present study,
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we immobilized Heparin and VEGF via self-polymerization and deposition of
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polydopamine (PDA) on polyurethane (PU) nanofibrous vascular grafts to improve endothelialization. Polyurethane nanofibrous scaffold (PUNF) that mimics vascular
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extracellular matrix (ECM) was chosen owing to its biocompatibility, biodegradability
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and enhanced mechanical properties. The results suggest that dual factors immobilization resulted in not only confluent monolayer of endothelial cells but also can
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confer excellent antithrombotic properties to the surface. This method of surface
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modification (immobilization of Heparin, VEGF by PDA layer) is suggested as a promising modification technique to increase hemcompatibility of small-diameter
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vascular grafts.
Parivash Davoudi, Shiva Assadpour, Mohammad Ali Derakhshan, Jafar Ai, Atefeh Solouk, Hossein Ghanbari PII: DOI: Reference:
S0928-4931(17)30153-4 doi: 10.1016/j.msec.2017.05.140 MSC 8122
To appear in:
Materials Science & Engineering C
Received date: Revised date: Accepted date:
12 January 2017 24 May 2017 28 May 2017
Please cite this article as: Parivash Davoudi, Shiva Assadpour, Mohammad Ali Derakhshan, Jafar Ai, Atefeh Solouk, Hossein Ghanbari , Biomimetic modification of polyurethane-based nanofibrous vascular grafts: A promising approach towards stable endothelial lining, Materials Science & Engineering C (2017), doi: 10.1016/ j.msec.2017.05.140
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Biomimetic Modification of Polyurethane-Based Nanofibrous Vascular Grafts: A Promising Approach towards Stable Endothelial Lining
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Parivash Davoudi1, Shiva Assadpour1, Mohammad Ali Derakhshan2, Jafar Ai1, Atefeh Solouk3, HosseinGhanbari2,4,*
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Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran 2
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Regenerative Nanomedicine Research Group, Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran 3
Biomedical Engineering Faculty, Amirkabir University of Technology, Tehran, Iran
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Research Center for Advanced Cardiovascular Techniques, Tehran Heart Center, Tehran University of Medical Sciences, Tehran, Iran
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*Corresponding author: Dr. Hossein Ghanbari, MD, PhD Associate Professor of Regenerative Nanomedicine Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Italia Street, 14177-55469, Tehran, Iran Tel: +98-21-4305 2200 Fax: +98-21-8899 1117 Email: [email protected]
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The emerging demand for small caliber vascular grafts to replace damaged vessels has attracted research attention. However, there is no perfect replacement in clinical use yet, mainly due to low patency rate of synthetic small caliber grafts. The main pathology behind low patency rate include thrombosis and graft/vessel hemodynamic mismatch, leading to intimal hyperplasia. Rapid in-situ endothelialization of vascular grafts is considered as one of the best strategies to overcome these complications. In the present study, Heparin and VEGF were immobilized via self-polymerization and deposition of polydopamine (PDA) on polyurethane (PU) nanofibrous scaffolds to improve endothelialization. Polyurethane nanofibrous scaffold (PUNF) that mimics vascular extracellular matrix (ECM) was chosen owing to its biocompatibility, biodegradability. Scanning electron microscopy (SEM), water contact angle (CA) measurement and Raman spectroscopy were used to characterize the surface, and tensile test was used to analyze mechanical properties before and after surface modification of the scaffolds. It was found that tensile strength and young´s modulus were significantly increased after PDA coating on PUNF membranes. The hemocompatibility tests revealed that surface heparinization significantly inhibited the adhesion of platelet on the scaffolds. Immobilization of VEGF on the scaffolds significantly enhanced the proliferation of human umbilical vein endothelial cells (HUVECs) through enhanced cells adhesion and improved cell-scaffold interactions. The results suggest that dual-factor immobilization resulted in not only confluent monolayer of endothelial cells but also conferred excellent antithrombotic properties to the surface. This method of surface modification (immobilization of Heparin, VEGF by PDA layer) is suggested as a promising modification technique to increase hemocompatibility of small-diameter vascular grafts.
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Keywords: Vascular grafts; Endothelialization; Polyurethane, Electrospun Nanofiber, Heparin; Poly dopamine.
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1. Introduction
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Many prosthetic vascular grafts made from biocompatible polymers such as polyurethane (PU) [1], expanded poly (tetrafluoroethylene) (ePTFE) [2], poly(ethylene terephthalate) (PET) [3], and polycaprolactone (PCL) [4] have been developed. However, they frequently suffer from low patency rate when applied as small diameter (<6mm) bypass grafts [5].
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Efforts to improve a small caliber vascular graft have been challenging over the past decade. A fully functional endothelial layer is essential for a small diameter vascular graft to be patent in long-term[6], owing to its role in the prohibition of excessive tissue ingrowth (intimal hyperplasia) and thrombogenesis that are two main reasons of graft failure. Therefore, both seeding of endothelial cells onto the luminal surface of the graft or in situ endothelialization improves long-term permanence of cardiovascular implants [7] and small-diameter prostheses. Since endothelial cells attach on the surface of an extracellular matrix (ECM) in nature, production of biomaterials equivalent to ECM can help the attachment, proliferation, and phenotypic maintenance of endothelial cells on vascular scaffolds [8].
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Recently, electrospinning technology has been extremely investigated as a method to make ECM-mimicking structures which can be noted as artificial ECM or scaffolds [9-11]. Electrospinning is a potent technique, which can generate fibrous structures from diverse materials such as polymers, ceramics , and composites [12]. Electrospun fibers are similar to the natural ECM components with many advantages such as the simplicity of the production process and scale-up, and also, capability to produce fibers with various diameters ranging from nanometer to micrometer sizes [13].
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Therewith, the high surface area to volume ratio and interconnected pores of these fibrous meshes result in desirable cell attachment and oxygen/nutrient transport, respectively. Also, multiple polymers entailing blood compatible ones (i.e. PTFE, PET, PCL and PU) can be electrospun into ultrafine fibers. All the mentioned benefits make electrospinning an ideal method for production of nanofibrous mats [14]. In comparison, PU-based materials have been vastly investigated for preparation of bio- and blood-compatible products. Recently, Adipurnama et al. have reviewed a variety of different surface modifications of PU and their effects on endothelialization for vascular graft applications [15].
Several surface modification methods have been applied to improve blood-compatibility of vascular grafts especially polyurethane based ones [16]. Functional groups or bioactive macromolecules can modify the surface of nanofibrous vascular grafts in order to accelerate
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endothelialization of electrospun mats [17]. For immobilization of biomolecules on polymeric biomaterials, various physical and chemical techniques are attainable. An appropriate functionalization method must be selected because most vascular grafts are produced from inert hydrophobic polymers lacking functional moieties for chemical conjugation. In our earlier attempts, collagen type I was grafted onto plasma-activated and acrylic acid coated PU nanocomposite [17]. In irradiation techniques such as plasma treatment high energy sources are used to introduce reactive groups for subsequent immobilization of desired biomolecules [18]. For versatile solid materials from metal to synthetic polymers, another facile surface modification method is simple dip-coating with dopamine solution [19]. Under slightly basic conditions, a stable layer that is adherent to the surface of materials, is created by oxidative polymerization of dopamine. The reaction environment in post-modification with polydopamine layer is simple and clean compared to other chemical immobilization methods and allows functionalization with bioactive molecules containing thiols or primary amines via imine formation and/or Micheal addition [18].
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It is known that rapid endothelialization is a prerequisite for artificial vascular grafts, and having non-thrombogenic surface before composition of a fully endothelial layer can make it ideal blood contacting surface. Vascular endothelial growth factor (VEGF) promotes proliferation and migration of endothelial cells (EC), results in angiogenic vascular growth, and induces differentiation of pluripotent stem cells to blood progenitor cells [20]. Heparin, one of the most commonly used clinical anticoagulant reagents, has positive effects on ECs growth and proliferation by binding and stabilizing cell growth factors(GFs) [21]. Binding of heparin and GFs principally happens via electrostatic interactions between N- and O-sulfated groups with negative charge of heparin and the basic lysine and arginine residues of FGF-2 or VEGF [22]. Diffusion of GFs is decelerated by binding to heparin [23], and interaction of growth factors with heparin is observed to be necessary for storage, release, and protection from pH, heat, and enzymatic degradation [24]. Recently, heparin modified biomaterials demonstrated excellent exploits in ECs growth and proliferation [25]. In the present investigation, nanofibrous electrospun vascular scaffolds based on elastic biodegradable PU substrates were fabricated to mimic the native vascular ECM. For surface modification, a poly dopamine-mediated immobilization platform together with a cell adhesive growth factor, VEGF, and an anti thrombogenic factor, heparin was developed. The effects of PDA-coating and VEGF and heparin immobilization on the morphology, hydrophilicity, hemocompatibility and mechanical properties of nanofibrous PU membranes were evaluated in details. Finally, the effect of PDA, VEGF, heparin and dual factors on regulation of HUVEC attachment and proliferation was evaluated. 2. Materials and Methods
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2.2.Preparation of Polyurethane Nanofibers (PUNF)
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Thermoplastic Polyurethane (PU,Tecoflex,SG-80A)was purchased from Lubrizol, USA. 3hydroxytyraminium chloride was purchased from Merck Co., Schuchardt OHG. Tris (hydroxymethyl) amino methane was obtained from Merck KGaA. Chloroform and Methanol were purchased from Merck co, Germany. Heparin sodium salt and VEGF were obtained from Sigma-Aldrich (St. Louis, MO, USA). HUVEC cells were obtained from the National Cell Bank of Iran (Pasteur Institute of Iran). Other unspecified chemicals were purchased from Sigma. All the chemicals were of analytical grade and used without further purification. Electrospinning machine was provided by Fanavarn Nano-meghyas Co. Ltd, Tehran, Iran.
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Nanofibrous scaffolds were produced utilizing a standard electrospinning setup. PU was dissolved in a mixture of chloroform and methanol with a ratio of 1:1 (v/v) as solvent system to prepare electro-spinning solution at 3/5% concentration (w/v), according to our previous study [26]. Solutions were pumped at a flow rate of 1 ml/h using a syringe pump while a potential of 20 kV was applied between the spinneret and a grounded collector located 10 cm apart from the spinneret. 2.3. Fabrication of Nanofibrous Vascular grafts
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A one-step electrospinning method was used in order to fabricate nanofibrous vascular graft. For this, a cylindrical mandrel with diameter of 4mm was fabricated using stainless steel alloy. Then it was used in the electrospinnig machine as a rotatory collector, in order to fabricate the tubal scaffolds in one-step process. Previously, optimized set up was used to fabricate nanofibrous scaffolds in the shape of small diameter vessel. 2.4. Surface modification of PUNF
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2.4.1. Poly dopamine coating(D-PUNF) For polydopamine (PDA) coating, the PUNF mats were cut into a circular shape (area: 1.99 cm2) which was submerged in 3,4-dihydroxyphenylamine (2 mg/ml) dissolved in Tris-HCl buffer (10mM,pH 8.5) and shaken on a rocker for 1 h. After this process, the samples were washed three times in deionized water to remove unbound PDA. The resultant membranes were used for characterization and further surface modifications. Then, polydopamine-coated PUNF (DPUNF) were modified with either heparin and VEGF or both of them. 2.4.2. Heparin immobilization(H-D-PUNF)
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2.4.4.VEGF and Heparin immobilization(VH-D-PUNF)
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For immobilization of VEGF, the PDA deposited mats were immersed in 1000ng/ml VEGF (dissolved in 10mMTris-HCl buffer, pH 8.5) overnight at4°C. The volume of VEGF solution was fixed to 400μL for the reaction.
2.5.1. Scanning electron microscopy (SEM)
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2.5.Characterization of the membrane surface
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For concurrent immobilization of VEGF and heparin, equal volumes of heparin and VEGF solutions (200μl) were added on the PDA-deposited mats (D-PUNF) for overnight at4°C.
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2.5.2. Raman spectroscopy:
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To investigate the surface morphology of the membranes and diameter of the obtained nanofibers, SEM (XL 30, Philips, USA) was used at an accelerating voltage of 25.0 kV. The samples were sputtered with gold prior to observation. Then, micrographs were analyzed utilizing ImageJ software to calculate the average diameter of nanofibers.
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Raman spectroscopy (Dispersive Raman Microscope, SENTERRA, 2009, BRUKER, Germany) in the spectral range of 200-3500cm−1 with resolution < 3 cm−1was used to display the structural changes in various functional groups on the scaffolds before and after surface modifications.
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2.5.3. Surface wettability
Surface wettability of membranes was characterized by static water contact angle measurements utilizing an optical video contact angle system (OCA-15-plus, Data physics, Germany). The measurement was performed at 20◦C and 70% relative humidity and in triplicate. 2.6. Mechanical evaluation Tensile test was performed for PUNF, D-PUNF, VH-D-PUNF samples using a uniaxial load testing machine (Instron 5565). The samples were cut into rectangular-shaped pieces (shaft length
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50mm, width 10mm, n=3). Thickness was measured using a digital electronic outside micrometer. The load cell capacity was 500 N and test speed uniaxial tension was applied to either ends of the scaffolds until failure was 10mm/min at room temperature. Stress (MPa) was calculated by dividing the force generated during stretching by the initial cross-sectional area. Strain was calculated as the ratio of the change in length in reference to the original sample length (%). The maximum tensile strength (maximum stress), elongation at break (maximum strain) and Young´s modulus for graft samples (n=3) were obtained.
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2.7.Platelet adhesion
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All four kinds of membrane samples (i.e. PUNF, D-PUNF, H-D-PUNF, and VH-D-PUNF) were placed in the wells of plate (24-well). 250µl of fresh platelet(obtained from Blood Transfusion Center) was dropped on each sample, and contact was maintained for 50min. Then the samples were carefully rinsed in PBS (PH 7.2) to remove non-firmly adsorbed platelet. After fixation with 4.0 wt% paraformaldehyde solution for 30min, the samples were washed twice with PBS. The samples were put in a freezer (-80◦C) overnight. Then, in the next day the samples were put in freeze dryer. Thereafter, the resultant samples were studied with SEM. 2.8.Cell culture on scaffolds
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HUVECs were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco, USA) supplemented with 10 % FBS, 50 U/ml penicillin and 50 U/ml streptomycin at 37 °C in a 5% CO2incubator. The culture medium was replaced every 2days. Before cell seeding, the unmodified scaffold (PUNF) and other modified scaffolds were cut into circular samples to fit a 24-well plate. Scaffolds were sterilized with ultraviolet light for 1 h. HUVECs were then seeded onto the top of the scaffold with a final density of 1×10 5 cells/scaffold in 24-well plates and incubated in DMEM/F12 for 1h allowing cells to attach onto the surface of the scaffold. For further incubation fresh medium was added. 2.9. Cell viability assay Metabolic activity of HUVECs cultured on the scaffolds was measured for 24, 48, and72h after cell seeding by applying the MTT assay. The activity of a mitochondrial dehydrogenase is assayed by transformation of light yellow MTT into purple formazan. For this purpose, 100μl of 5mg/ml MTT solution was added to each well. After 4h incubation at 37°C, the culture medium was removed and 300µL of dimethyl sulfoxide (DMSO) was added into each well. The solutions were transferred into a 96-well plate, and the absorbance was measured at 570 nm using an ELISA reader (Expert 96, Asys Hitch, Ec Austria). The experiments were repeated three times and the result of each experiment was reported as mean ± (SD).
ACCEPTED MANUSCRIPT 2.10.Cell adhesion and morphology study The morphologies of HUVECs on the surfaces of the scaffolds were observed by SEM. After 24h of culture, cell-containing scaffolds were fixed with 4% paraformaldehyde (pH 7.2) for 30 min, followed by washing with PBS and freezing in a freezer (-80◦C )for overnight, then the next day were put in freeze dryer. Finally, the resultant samples were observed with SEM.
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2.11. Statistical analysis
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All data are expressed as mean±SD. Statistical analysis was carried out by means of one-way ANOVA (p < 0.05).
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3. Results and discussion
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3.1. Fabrication of Nanofibrous Vascular graft
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3.2. Membrane Surface morphology
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Figure.1 shows the scaffolds fabricated using a one-step method. As shown in the figure a nanofibrous small diameter vessel with a diameter of 4mm has been successfully fabricated. The advantage of one-step process was uniform structure of the scaffold and feasibility of the technique.
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Electro spinning of polyurethane was led to the formation of bead-free nanofibers. The SEM images in Fig.2 display the effect of PDA coating and subsequent immobilization of biomolecules on the scaffolds. It can be noted that surface modification resulted in cross-linking between the fibers which was mainly the effect of PDA modification. The cross-linking effect of PDA has already been reported in previous study [27]. By the use of imageJ, the average fiber diameter was obtained 405±116, 410±119, 408±123, 418±98, 416± 100 nm for PUNF, D-PUNF, H-D-PUNF, V-D-PUNF, VH-D-PUNF, respectively. All the samples revealed uniformity and there was no significant difference in the average fiber diameter between unmodified and modified scaffolds. Since the coating layer was very thin, it did not change the average diameter of the nanofibers which was in accordance with previously published reports [26]. The average pore diameter of the nanofibrous scaffold was 4.57±2.5, 2.15±1.2, 2.2±1.67, 2.3±1.26, 2.28±1.04 m for PUNF, D-PUNF, H-D-PUNF, V-D-PUNF, VH-D-PUNF respectively. The porosity percentage was approximately 69%, 40%,45%,49.8%, and 47% for PUNF, D-PUNF, H-D-PUNF, V-D-PUNF, VH-DPUNF, respectively. The porosity of PUNF was significantly decreased after surface modification with polydopamine (p<0.05). This can be attributed to the cross-linking effect of polydopamine which decreased the free space between the fibers. It could also be related to the secondary immobilization of biomolecules on smaller pores and formation of tight junctions between the fibers. However, the difference between the modified samples was not statistically significant.
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3.3. Analysis of Raman spectroscopy: To further confirm the PDA coating and other surface modifications such as heparin and VEGF immobilization, Raman spectroscopy was utilized. 3.3.1. PDA coating
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As shown in Fig.3A the Raman spectra of PUNF and D-PUNF scaffolds revealed five distinct trends of peaks centered between the 1250and 1490 cm-1 wavenumbers. The intensity at 1266cm-1,1297cm-1,1438cm-1(CH3) in PUNF scaffold was greater than D-PUNF scaffold. Unlike the intensity of peaks 1356cm-1 (C-CH3) [28,29] and 1486cm -1(aromatic ring) was greater in DPUNF substrates. Raman new peaks at 1370/1600 cm-1 indicated the formation of PDA coating on PUNF scaffold [30]. In the Raman shift of D-PUNF, 1370cm-1 peak is related to stretching of catechol and the 1570cm-1peak is related to the deformation of catechol, which is a major component of the dopamine structure [31]. Hence, the peaks centered between the 1550 and 1600 cm-1wavenumbers(characteristic of stretching and deformation of aromatic ring)clearly shows PDA coating on PUNF scaffold [32].
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3.3.2. Heparin immobilization (H-D-PUNF)
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Prominent Raman peaks of heparin is appeared in the range of 900-1150 cm−1[33]. As shown in Fig.3B, Raman spectrum of H-D-PUNF scaffold revealed multiple peaks that some of them were new in comparison with Raman spectrum of D-PUNF, including 914cm-1 ,950cm-1 (Carboxylic acid dimer ), 1065 cm−1 (2-O-SO3 vibration), and 1088cm-1.The other peaks were also present in Raman spectrum of D-PUNF scaffold, but revealed higher intensity such as 1035cm-1 ((N-SO3 vibration) [34,35]. These results demonstrate that heparin is successfully immobilized onto the D-PUNF surface. 3.3.3. VEGF immobilization (V-D-PUNF) As shown in Fig.3C comparison between Raman spectrum of V-D-PUNF scaffold and D-PUNF scaffold revealed the novel peaks at 702,638 cm-1(C-S aliphatic ) and 914cm-1(C-O-C) [36]. Furthermore, another new peak with low density at 1065cm-1(aromatic rings) was seen on V-DPUNF samples. Some typical peaks attributing to D-PUNF scaffold but with lower intensity at837, 950cm-1(C-O-C) and with higher intensity at 729 cm-1 (C-S aliphatic) were also revealed. These results suggested that VEGF was successfully immobilized onto the D-PUNF membrane surface.
ACCEPTED MANUSCRIPT 3.3.4. VH immobilization (VH-D-PUNF) As shown in Fig.3D, Raman spectrum of VH-D-PUNF scaffold revealed characteristic peaks of V-D-PUNF (638,706,914 cm-1) together with similar relative intensity peaks at 1005 cm-1,and between 1035-1088 cm-1 with H-D-PUNF scaffold. All these emphasized the existence of VEGF and heparin on VH-D-PUNF scaffold. .
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3.4. Surface wettability
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Water contact angle (CA) measurement was used to characterize the surface relative hydrophilicity and wetting characteristics. Fig.4 shows water contact angle of unmodified and modified polyurethane. The water CA of unmodified PUNF was 125.17±3.89º which was significantly decreased to75.4±3.48º via dopamine modification(D-PUNF), and was further decreased after heparin immobilization to 67.5±9.87º(H-D-PUNF). This result indicated that the introduction of abundant hydrophilic groups such as –OH, –COOH and–NH2in the layer of polydopamine brought hydrophilicity to the hydrophobic PUNF scaffolds. Besides, high concentrations of –OH and ether linkages in heparin also enhanced surface hydrophilicity after heparinization[37]. In addition, immobilization of VEGF on D-PUNF subsequently further decreased the water CA to 40±8.6º(V-D-PUNF). The robust reduction of contact angle might be attributed to the combined hydrophilicity effect of PDA and VEGF[5]. The water CA of VH-DPUNF scaffolds was 54.03±0.89º, indicating the effect of simultaneous immobilization of VEGF and heparin on D-PUNF scaffolds. In vitro studies indicated that ECs display poor adhesion and proliferation behavior on hydrophobic materials[38], whereas EC behavior was enhanced on moderately wettable polymers[39]. A polymeric structure with water CA of 55◦ resulted in maximal cellular adhesion, enhanced proliferation rates, and improved cellular growth regardless of cell type[16],[40]. Therefore, taking only hydrophilicity into account, it was expected that surface modified scaffolds, in particular VH-D-PUNF scaffold with water CA 54.03±0.89º, could provide better interactions with the cells in order to create a confluent layer of endothelium. 3.5. Mechanical evaluations Mechanical properties of scaffolds are shown on Table.1.After PDA coating and biomolecules immobilization (i.e. D-PUNF and VH-D-PUNF), improved mechanical properties was observed compared to PUNF. The maximum tensile strength of PUNF samples was about 1.8±0.40MPa which was significantly increased to 3.69±0.28 in D-PUNF and 3.07±0.23 in VH-D-PUNF samples after surface modifications. The ultimate strain was also increased from 0.84± 0.05 for PUNF samples to 1.455±0.12 and 1.076±0.08 in D-PUNF and VH-D-PUNF, respectively. The reason behind this improvement in mechanical properties can be the presence of PDA layer within the void space between fibers that causes them to adhere to each other. In the other word, using
ACCEPTED MANUSCRIPT PDA, nonwoven PUNF scaffolds converted to cross-linked ones with improved mechanical properties [27].
3.6.Platelet adhesion
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As known, platelet adhesion and aggregation on the scaffold activates coagulation pathway that is one of the crucial factors in hemocompatibility evaluation. Therefore, platelet adhesion for anti-thrombogenic properties of the scaffolds was investigated. SEM images (Fig.5) represents that the quantity of platelets aggregation was markedly different from each other on the samples. The hydrophobic PUNF surface demonstrated the highest number of adherent platelets (Fig.5A). For interaction of platelets with a material surface, the nature of the surface and the adsorbed proteins are important [6]. Various plasma proteins, such as thrombin and fibrinogen, can be absorbed onto material surfaces and stimulate activation and aggregation of platelets on the scaffold. Each fibrinogen molecule by carboxylic terminus of chain interacts
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with integrin receptor on the platelet membrane. Resting platelets interact with immobilized fibrinogen because soluble fibrinogen in plasma is folded and ɤ chain is hidden. Therefore, fibrinogen must be unfolded or conformationally changed [41] to bind to the platelets. The conformational alteration of fibrinogen after absorption onto hydrophobic surfaces is higher than on hydrophilic surfaces as previously reported [42]. The data indicated that the adhesion and activation of platelets were clearly inhibited by PDA coating and heparin immobilization (Fig.5 B,C). Indeed, these hydrophilic surfaces preferred adsorption of water molecules to plasma proteins. Hydrophilic modification of PUNF surface resulted in enhanced hemocompatibility of the scaffolds. According to the Fig5, the quantity of adhered platelets on the samples declined in the order of: PUNF>D-PUNF>VH-D-PUNF>H-D-PUNF. Comparing two samples with heparin immobilized, i.e. H-D-PUNF and VH-D-PUNF, the results of platelet adhesion studies revealed that samples modified with heparin were more resistant to platelet adherence and consequent activation than VH-D-PUNF. This could be attributed to the platelet repellent effect of heparin conferred to the surface. When the surface simultaneously modified with heparin and VEGF, the presence of VEGF could partially compromise the effect of heparin, suggesting the importance of optimization of VEGF/Heparin ratio in surface modification method. 3.7. Cell viability assay MTT assay was used to evaluate viability of HUVECs on unmodified and modified PUNF scaffolds(i.e. PUNF, D-PUNF, H-D-PU, VH-D-PUNF and V-D-PUNF groups). As shown in Fig.6, all the surfaces showed acceptable cell viability which is increasing as time rises. The proliferation of HUVECs on surface modified PUNF was better than unmodified one in all the defined times.
ACCEPTED MANUSCRIPT Comparison between modified samples showed that VH-D-PUNF is the best substrate to support endothelial cell proliferation. Having both anti-thrombogenic property and endothelialisation capability is crucial for small caliber vascular grafts. These ensure the hemocompatibility and long term patency of the grafts which is desirable.
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3.8. Cell adhesion and spreading study
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Cell shape and spreading is relevant to surface wettability [43], that is one of the important modulators of cellular function. Fig.7 shows adhesion, spreading and morphologic features of HUVECs onto unmodified and modified PUNF scaffolds. Unmodified PUNF is highly hydrophobic polymer, and HUVECs grown on it displayed smooth border line without cellular processes, a typical non-adherent non-spreading morphology, demonstrating limited interaction and adhesion to the material (Fig.7.A). Cells appeared to be physically adsorbed instead of biologically attached. In contrast, cells seeded on D-PUNF scaffold exhibited limited spreading with formation of cellular processes, indicating that PDA deposition gained cell adhesion, which is attributed to either the adsorption /immobilization of serum proteins on PDA ad-layer or direct electrostatic interactions with cells or by both of them [44](Fig.7.B). The serum proteins connected to PDA layer act as cell adhesion sites [45]. With immobilization of heparin on DPUNF, flattened cells demonstrated robust cellular adhesion and their plasma membrane spread on the substratum, moreover indicated higher proliferation and interactions between them(Fig.7.C). The shape of a cell is closely dependent on cell function. Usually the shape of an EC in a vein wall is spindle or oval shape, demonstrating a biological function, proliferation only happens for cells that are spread out. Several studies pointed that heparin can advance endothelial cell proliferation [46]. As mentioned above, PDA-coating decreased water CA of PUNF scaffold from125º to 75º and with heparin immobilization more decreased to 67º, therefore, ECs better adhered and expanded on heparinized scaffolds. VEGF is known to stimulate cellular responses by coupling to and subsequent phosphorylation of tyrosine kinase receptors in cell membrane [5]. The cells seeded on VEGF-immobilized scaffolds (Fig.7.D,E) showed excellent spreading and adhesion compared to the others(PUNF,D-PUNF,H-D-PUNF). Planed area per cell calculations increased in the order of PUNF [224±25], D-PUNF [232±35], HD-PUNF [238±30], V-D-PUNF [245±42] µm2. However, the differences were not statistically significant. VEGF immobilization eventuated mitogenesis and migration of HUVECs along with cell adhesion affection of PDA coating composed a confluent mono-cellular layer of HUVECs which is important in endothelialization of vascular grafts with small diameter (Fig.7. D,E).The growth of HUVECs was intensified when dual factors(Heparin and VEGF) were simultaneously exist onto D-PUNF scaffold. As mentioned by Solouk et al [12], the best water CA for growing of the cells on scaffold is 55º, and water CA of VH-D-PUNF scaffold is 54±0.89º. Meanwhile, before
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4.Conclusion
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In vascular tissue engineering, efficient endothelialization of graft surfaces through adhesion, spreading and proliferation of ECs is essential. Synthetic polymers do not provide optimum interaction with cells to promote endothelialization. In this study a biomimetic coating was used as an efficient surface modification technique. For this purpose, electrospun nanofibrous scaffolds of PU were modified with a layer of PDA which were consequently, coated with Heparin and VEGF. These modifications not only improved the mechanical and surface properties of the scaffolds, but also enhanced cell adhesion, spreading, and proliferation on the scaffolds. The functional scaffolds demonstrated appropriate blood compatibility. These data suggest that modification of PU fibers with PDA layer and secondary immobilization with Heparin and VEGF is a potential improvement for small caliber vascular grafts and cardiovascular tissue engineering products. 5. Acknowledgements:
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REFERENCES
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This work was financially supported by Tehran University of Medical Sciences (grant number: 93-01-87-24919).
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Figure Captions
Fig.1: A nanofibrous small diameter vascular scaffold fabricated using one-step electrospinning method.
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Fig.2: Scanning electron microscopy of polyurethane nanofibers (PUNF) before and after modification: (a)PUNF, (b) D-PUNF, (c) H-D-PUNF, (d) V-D-PUNF, (e) VH-D-PUNF (scale bar5µm, inset10µm). The effect of surface modification on the pore size of nanofibrous scaffolds can be seen on modified samples.
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Fig.3: Comparison between Raman spectra of unmodified and modified scaffolds : A) PUNF(pu) and DPUNF(d), B)D-PUNF(d) and H-D-PUNF(h), C)D-PUNF(d) with V-D-PUNF(v), D)VH-D-PUNF(vh) with V-DPUNF(v) and H-D-PUNF(h).
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Fig.4: Surface wettability and water contact angle measurement and representative images of water droplet on the scaffolds. The differences between unmodified PUNF scaffolds with modified scaffolds were significant. (one way ANOVA, p<0.001). Fig.5: The morphology of platelets on the surface of unmodified and modified PUNF : (A)PUNF ,(B)DPUNF ,(C) H-D-PUNF , (D)VH-D-PUNF(T=50min). The hydrophobic PUNF surface had the highest number of adherent platelets (A) which was decreased in modified samples in the order of: PUNF > D-PUNF > VH-D-PUNF > H-D-PUNF. Fig.6: The results of MTT assay of HUVECs cultured on nanofibrous scaffolds. As it can be seen, modified samples revealed significantly higher level of cell viability compared to unmodified PUNF scaffold (one way ANOVA, *: p<0.05, **: p<0.01).
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Tables
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Table.1: Mechanical characterization of scaffolds
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Fig.1. A nanofibrous small diameter vascular scaffold fabricated using one-step electrospinning method.
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Fig.2. Scanning electron microscopy of polyurethane nanofibers (PUNF) before and after modification: (a)PUNF, (b) D-PUNF, (c) H-D-PUNF, (d) V-D-PUNF, (e) VH-D-PUNF (scale bar5µm, inset10µm). The effect of surface modification on the pore size of nanofibrous scaffolds can be seen on modified samples.
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Fig.3. Comparison between Raman spectra of unmodified and modified scaffolds: A) PUNF (pu) and DPUNF (d), B) D-PUNF (d) and H-D-PUNF (h), C) D-PUNF (d) with V-D-PUNF (v), D) VH-D-PUNF (vh) with V-D-PUNF (v) and H-D-PUNF (h).
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Fig.4. Surface wettability and water contact angle measurement and representative images of water droplet on the scaffolds. The differences between unmodified PUNF scaffolds with modified scaffolds were significant. (p<0.001).
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Fig.5. The morphology of platelets on the surface of unmodified and modified PUNF : (A)PUNF ,(B)D-PUNF ,(C) H-D-PUNF , (D)VH-D-PUNF(T=50min). The hydrophobic PUNF surface had the highest number of adherent platelets (A) which was decreased in modified samples in the order of: PUNF > D-PUNF > VH-D-PUNF > H-DPUNF.
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Fig.6. The results of MTT assay of HUVECs cultured on nanofibrous scaffolds. As it can be seen, modified samples revealed significantly higher level of cell viability compared to unmodified PUNF scaffold (one way ANOVA, *: p<0.05, **: p<0.01).
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Fig.7. SEM images of HUVEC adhesion and spreading on unmodified and modified PUNF scaffolds : (A) PUNF, (B) D-PUNF, (C) H-D-PUNF, (D) V-D-PUNF, (E) VH-D-PUNF, [Magnification in each of them from left to right : 500×/ scale bare=50µm, 2500×/ scale bare=10µm ].
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Table. 1 Mechanical characterization of scaffolds
1.8±0.40 3.69±0.28 3.07±0.23
Young, s modulus (E, MPa)
Toughness(UT,J/m3)
2.14±0.15 2.53±0.23 2.85±0.3
0.756±0.3 2.68±0.25 1.65±0.2
0.84± 0.05 1.455±0.12 1.076±0.08
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PUNF D-PUNF VH-D-PUNF
Ultimate Strain(εf)
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Highlights: This is an original article reporting our latest findings on developing polyurethane based
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nanofibrous scaffolds for vascular tissue engineering applications. In the present study,
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we immobilized Heparin and VEGF via self-polymerization and deposition of
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polydopamine (PDA) on polyurethane (PU) nanofibrous vascular grafts to improve endothelialization. Polyurethane nanofibrous scaffold (PUNF) that mimics vascular
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extracellular matrix (ECM) was chosen owing to its biocompatibility, biodegradability
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and enhanced mechanical properties. The results suggest that dual factors immobilization resulted in not only confluent monolayer of endothelial cells but also can
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confer excellent antithrombotic properties to the surface. This method of surface
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modification (immobilization of Heparin, VEGF by PDA layer) is suggested as a promising modification technique to increase hemcompatibility of small-diameter
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vascular grafts.