Article pubs.acs.org/Biomac
Engineering Interaction between Bone Marrow Derived Endothelial Cells and Electrospun Surfaces for Artificial Vascular Graft Applications Furqan Ahmed,† Naba K. Dutta,† Andrew Zannettino,‡,§ Kate Vandyke,‡,§ and Namita Roy Choudhury*,† †
Ian Wark Research Institute, University of South Australia, Mawson Lakes Campus, South Australia, Australia Myeloma Research Laboratory, School of Medical Science, Faculty of Health Science, University of Adelaide, Adelaide, South Australia, Australia § Centre for Cancer Biology, SA Pathology, Adelaide, South Australia, Australia ‡
ABSTRACT: The aim of this investigation was to understand and engineer the interactions between endothelial cells and the electrospun (ES) polyvinylidene fluoride-co-hexafluoropropylene (PVDF-HFP) nanofiber surfaces and evaluate their potential for endothelialization. Elastomeric PVDF-HFP samples were electrospun to evaluate their potential use as small diameter artificial vascular graft scaffold (SDAVG) and compared with solvent cast (SC) PVDF-HFP films. We examined the consequences of fibrinogen adsorption onto the ES and SC samples for endothelialisation. Bone marrow derived endothelial cells (BMEC) of human origin were incubated with the test and control samples and their attachment, proliferation, and viability were examined. The nature of interaction of fibrinogen with SC and ES samples was investigated in detail using ELISA, XPS, and FTIR techniques. The pristine SC and ES PVDF-HFP samples displayed hydrophobic and ultrahydrophobic behavior and accordingly, exhibited minimal BMEC growth. Fibrinogen adsorbed SC samples did not significantly enhance endothelial cell binding or proliferation. In contrast, the fibrinogen adsorbed electrospun surfaces showed a clear ability to modulate endothelial cell behavior. This system also represents an ideal model system that enables us to understand the natural interaction between cells and their extracellular environment. The research reported shows potential of ES surfaces for artificial vascular graft applications.
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INTRODUCTION
years, one of the most important breakthroughs has been the development of fibrous scaffolds using electrospinning.7,8 The electrospinning technique has the advantage of being able to generate fibrous scaffolds of micro to nanoscale topography and high porosity that resemble the natural extracellular matrix (ECM) found in blood vessels.9,10 This technique provides the capacity to develop biomimetic materials that promote endothelial cell binding and proliferation and allow for transfer of the nutritional and oxygen to meet the demands of the engrafted cells. Besides the surface chemistry and topography of the original SDAVG, the post-treatment changes to the SDAVG, particularly the adsorption of serum proteins plays an important role in the success and rejection of the graft. Biomacromolecules such as proteins, polysaccharides, and proteoglycans can act as biological cues for adherent cells. Cell adhesion to ECM proteins, adsorbed from the environment or secreted by the cultured cells, is primarily mediated by
Formation of thrombus on the cardiovascular implants represents a significant limitation to their long-term success.1,2 In natural blood vessels, anticoagulants like prostacycline, nitric oxide and surface bound heparan sulfate to endothelial cells surface can prevent the formation of thrombus and atherosclerosis.3−5 A vascular graft without an adherent layer of endothelial cell lining can be rejected due to the nonspecific adsorption of biomolecules from the circulating blood, leading to thrombosis/occlusion. To limit thrombus formation, an uninterrupted endothelial cell lining on the implant surface is required in order to make the graft compatible with the blood and tissue environment.6 To date, the success of small diameter artificial vascular grafts (SDAVG) is in its formative stages due to the low patency rate, poor reliability and thrombosis issues. Careful selection of synthetic materials based on properties which improve blood and cytocompatibility has the potential to overcome many of these issues. As such, significant work has been focused on the material structure and composition, surface modifications, topographic changes, and surface and biochemical coatings to enhance endothelialization. In recent © 2014 American Chemical Society
Received: December 13, 2013 Revised: February 22, 2014 Published: February 24, 2014 1276
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environment for drying for 2 days at 65 °C. After solvent evaporation, the PVDF-HFP flat films were cut to make 1 cm discs. Nanofiber Fabrication. The fabrication of PVDF-HFP electrospun surfaces was performed using 10% solution, 13 kV voltage, 0.15 mL/h flow rate and 14 cm tip capillary distance as described in our previous study.22 Quantification of Fibrinogen Adsorption by Enzyme Linked Immunosorbent Assay (ELISA). The quantity of purified fibrinogen (Fg) on PVDF-HFP (SC and ES) samples was determined using a modified indirect ELISA method23 in 2-fold serial dilution of fibrinogen. A standard curve of fibrinogen adsorption on 96-well plates was established in two ways: first, surfaces were preincubated with Tris-buffered saline (TBS), followed by fibrinogen and antibodies; and second, surfaces were preincubated with 1% bovine serum albumin (BSA) blocking buffer, followed by incubation with fibrinogen and antibodies, as described below. The second standard curve step was established in order to account for the potential for fibrinogen to adsorb to the polystyrene wells at the time of ELISA assay for SC and ES samples. In brief, polystyrene wells were blocked with the addition of blocking buffer for 1 h, and then the PVDF-HFP (SC and ES) samples (after washing), which were cut to the size of the well were placed into the blocked well and subsequently exposed to purified bovine fibrinogen for 2 h at room temperature. These surfaces were rinsed with TBS to remove unattached proteins. The fibrinogen adsorbed PVDF-HFP samples were incubated with 1% BSA for 1 h to block surfaces; and subsequently exposed to an antifibrinogen antibody (0.25 μg mL−1) in TBS for 2 h. The samples were washed four times with TBS and then 100 μL of biotinylated goat anti-mouse IgG (0.25 μg mL−1 diluted in TBS) was added to all sample wells and incubated for 1 h. The samples were washed with TBS four times. The samples were exposed to 100 μL (at a 1:4000 dilution) of avidin-HRP for 30 min. The samples were again washed with TBS and the surfaces incubated for 5 min with 200 μL of ortho-phenylene diamine (OPD, Sigma; 1 tablet OPD reagent with 1 tablet buffer in 20 mL of water wrapped in foil for 20 min on a rocking platform). After this incubation, the reaction was stopped by adding 50 μL of 2.5 N sulphuric acid. Absorbance at 490 nm was measured on a 200 μL aliquot using an ELISA auto reader. The varying fibrinogen concentration assay was performed using TBS as control and, for each day, similar time intervals and concentrations were used with the same conditions. Characterization of ES and SC Samples. Water Contact Angle (WCA) Measurement. The static contact angle of water on pristine and fibrinogen preadsorbed samples (SC and ES) was measured at 25 °C using a proprietary contact angle goniometer by sessile drop method by placing a 10 μL drop of distilled water on surfaces of ES and SC samples of PVDF-HFP. The droplet shape was imaged with a video camera and contact angle calculated using proprietary software (Ian Wark Research Institute, Adelaide, Australia).24 Characterization by SEM. A Philips XL30 field emission gun Scanning Electron Microscopy (FEGSEM) with Oxford CT1500HF Cryo stage was used to characterize the morphology of cells on SC, polystyrene, and ES samples. To minimize the charging effect, platinum was deposited after cell fixation on each sample using sputtering and examined at an accelerating voltage of 10 KV. The analysis of the diameter and porosity of the ES samples was performed from the SEM image. Characterization by XPS. XPS analysis was performed with an AXIS HSi Spectrometer (Kratos Analytical Ltd., Manchester, U.K.), equipped with a monochromated Al Kα source at a power of 144 W (12 mA, 12 kV). The samples (∼6.5 mm) were mounted on a multisample holder stage. The charging of the samples during irradiation was compensated for by an electron flood gun in combination with a magnetic immersion lens. A reference binding energy of 285.0 eV for the aliphatic hydrocarbon C1s component was used to correct for any remaining offsets due to charge neutralization of specimens under irradiation.25 The pressure in the main vacuum chamber during analysis was typically 5 × 10−6 Pa. The spectra were recorded with the nominal photoelectron detection normal to the sample surface. The sampling depth was up to 10 nm depending on
integrins. ECM proteins, such as fibronectin, vitronectin, fibrinogen, and collagen, have the ability to support cell adhesion. Therefore, if biomaterial surfaces are modified with these bioactive macromolecules, the biocompatibility of the surfaces can be significantly improved. Fibrinogen is one of the most important serum proteins and displays both clotting and cell proliferation capabilities, with the concentration and conformation dictating the mobilization and recruitment of different cell; and, in turn, their adhesion and proliferation at the site of the implantation.11−13 The fibrinogen molecule has strong binding sites for platelets and endothelial cells, which defines its role in clotting and endothelial cell proliferation. Various materials have been used to study the role of fibrinogen on inhibition and proliferation of cells on synthetic materials like polyethylene terephthalate (PET, Dacron), expanded polytetrafluoroethylene (e-PTFE), polyurethane (PU), polycaprolactone (PCL), and copolylactic acid/ glycolic acid (PLGA),14−20 some of which are the subject of SDAVG development. However, most of these materials have shown very limited success either due to early thrombosis, antigen response, or loss of structural integrity due to poor mechanical (e.g., elasticity) performance. To overcome these deficiencies, we have selected poly(vinylidene-fluoride)-cohexafluoropropylene (PVDF-HFP) as it possesses robust, elastic, nonreactive, nontoxic, nonthrombogenic, resilient, and antibacterial properties.21 Moreover, PVDF-HFP can be easily processed using ES techniques, allowing for the generation of SDAVG with different diameter, morphology, porosity, and surface characteristics. Because of its biostability and biocompatibility, it also has the potential to minimize the issues of thrombosis and aneurysm formation. We have recently optimized the electrospinning process of PVDF-HFP and their physical/physicochemical characteristics.22 In this work, we used electrospinning to produce a morphologically relevant scaffold composed of PVDF-HFP fibres and examined in detail its capacity to support the attachment, proliferation, and viability of bone marrow-derived endothelial cells. We hypothesized that the physical and chemical characteristics of the biomimetic scaffold plays an important role in promoting adhesion of cells. To assess this hypothesis, we evaluated the ability of solvent cast (SC) and electrospun (ES) PVDF-HFP samples to support adhesion and proliferation of bone marrow derived endothelial cells (BMECs). Furthermore, we also assessed the effect of preadsorbing the surfaces with fibrinogen to BMEC adhesion and proliferation.
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MATERIALS AND METHODS
Reagents. PVDF-HFP, with an inherent viscosity of 2300−2700 Pa and Mw ∼ 400000, was procured from Sigma Aldrich, Australia. The polymer was dissolved in 70/30 ratio of N,N-dimethylacetamide (DMAc) and acetone (from Sigma Aldrich, Australia) at 10% (w/v) concentration and left for 24 h mixing with a magnetic stirrer at room temperature. Fibrinogen, from bovine plasma type 1-S, 65−85% protein, was purchased from Sigma Aldrich. To make 25 mg mL−1 aliquot, 450 mg (0.45 g) of the protein was diluted in PBS (the physiological concentration, i.e., 2 mg/mL of fibrinogen, was used for endothelial cell study). A monoclonal antifibrinogen antibody and a goat antimouse IgG, HRP conjugate were purchased from Sigma Aldrich and Millipore Australia, respectively. Preparation of PVDF-HFP SC Samples. Uniform flat films of the PVDF-HFP were prepared by casting of PVDF-HFP solution in a glass petri dish. The PVDF-HFP solution in petri dish was left in controlled 1277
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Figure 1. (A) Standard curve of fibrinogen adsorption on 96-well plates and scheme of ELISA assay on TCP (B) percentage of relative fibrinogen binding on SC and ES samples with scheme of ELISA assay (n = 3). resolution of 4 cm−1, 256 scans, and a mirror velocity of 0.158 cm s−1 was used. Three SC and ES samples of PVDF-HFP were analyzed for each characterization technique and the standard deviation of these samples was interpreted. Cell Culture. Human bone marrow derived endothelial cells (BMECs)26,27 were a gift from Professor Babette Weksler (Cornell University Medical College, New York, New York). The cells were plated in monolayer in T-75 tissue culture flasks and cultured to confluence in M199 culture medium containing 10% fetal bovine serum, growth factor, and heparin. The medium was replaced every 2 days and cultures were maintained in a tissue culture incubator at 37 °C with 5% CO2. Cell Seeding. For the cell culture studies, a preoptimized solution concentration (10% w/v) and electrospinning conditions (0.15 mL h−1 flow rate, 13 kV applied voltage and 14 cm tip capillary distance) for the production of ES samples were used. Control samples (∼1 cm)
the kinetic energy of the measured photoelectrons. The elemental composition of the samples was obtained from survey spectra (320 eV pass energy) using sensitivity factors supplied by the manufacturer. High resolution spectra of individual peaks were recorded at 40 eV pass energy. Characterization by PA-FTIR. The fibrinogen adsorbed surfaces were also investigated using Photoacoustic Fourier Transforms Infrared spectroscopy (PA-FTIR). PA-FTIR was performed using a Nicolet Magna Spectrometer (Model 750) equipped with a MTEC (model 300) photo acoustic cell. The sample was placed in a circular stainless steel cup, 3 mm deep and 10 mm in diameter, and sealed in a device with a potassium bromide salt window and a helium atmosphere to promote good heat transfer. A carbon black film was used as reference, as it absorbs all wavelengths of the infrared radiation and produces a spectrum that mirrors both the energy characteristics of the detector and optical performance of the instrument. The 1278
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Figure 2. X-ray photoelectron spectra of PVDF-HFP (A) pristine solvent cast, (B) pristine electrospun, (C) preadsorbed fibrinogen SC, and (D) preadsorbed fibrinogen ES, (E) C1s core level spectra of pristine SC, (F) C1s core level spectra of preadsorbed fibrinogen SC, (G) C1s core level spectra of pristine ES, and (H) C1s core level spectra of preadsorbed fibrinogen ES (n = 3). of TCP, SC, and optimized ES PVDF-HFP samples (after 2 h sterilization with ethanol) were placed at the bottom of a 24-well tissue culture plate and BMECs in M199 culture medium were seeded onto each surface at a density of 3 × 104, 6 × 104, and 12 × 104 cells cm−2.
After a seeding time of 5 h, each culture sample was analyzed under SEM and cell seeding efficiency was calculated with reference to TCP. Assessment of Cell Adhesion. The adhesion of BMECs to different samples was evaluated using cell counting. The SC and ES 1279
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samples were placed in a 24-well cell culture plate. All samples were sterilized in 70% ethanol for 2 h, followed by overnight drying. BMECs were detached with 0.05% trypsin-EDTA and their number was counted using a hemocytometer. Cell concentrations were adjusted in the culture medium to the corresponding plating densities. Cell Fixation. After 5 h of incubation, BMEC attached samples were washed with PBS to remove nonadherent cells and then fixed with 4% glutaraldehyde for 30 min at room temperature, rewashed with PBS for 5 min, and postfixed with 2% osmium tetraoxide for 30 min. The biomaterial/BMEC constructs were dehydrated with 70% ethanol for 10 min, with 90% ethanol for 10 min and 100% ethanol for 10 min followed by rinsing with hexamethyldisilazane (HMZ) diluted with equal amount of 100% ethanol for 10 min, then undiluted HMZ for 10 min, and left to dry overnight. After critical point drying, the biomaterial/BMEC constructs were sputter coated with platinum and observed under the SEM at an accelerating voltage of 10 kV. Cell count was determined by SEM image on 10 randomly selected locations using Image J software. Assessment of Cell Proliferation and Viability. For the proliferation studies, samples were sterilized as described above. BMECs were seeded separately on TCP and PVDF-HFP (SC and ES) samples, at a plating density of 6 x104 cells cm−2. The BMECs were allowed to proliferate for 1, 3, 7, and 9 days, after which proliferation and viability were determined using a WST-1 cell proliferation assay kit (Roche), as described below. Cell counting of SEM images was also performed in order to confirm our findings with the WST-1 assay. Experiments were performed in triplicate. The SEM images were taken from 10 randomly chosen locations for each sample condition. The cell densities were determined using Image J software. WST-1 Viability Assay. WST-1 reagent was diluted to 1:10 with phenol red free DMEM/10% FCS (prewarmed at 37 °C). BMEC/ biomaterial composites were transferred to adjacent wells to avoid counting cells which had adhered to the base and wall of the wells. A total of 400 μL of diluted WST-1 reagent was added to each well containing samples. The same amount of reagent was also added to a control well. The samples were incubated for 40 min at 37 °C/5% CO2 and triplicates of 100 μL from each well were transferred to a flat bottom 96-well plate for absorbance measurement on a ELISA plate reader with a wavelength of 490 nm. Cell Culture Study on Fibrinogen Adsorbed Samples. After proper sterilization with ethanol, the TCP, SC, and ES samples were soaked in 2 mg mL−1 fibrinogen solution for 2 h and rinsed with Milli Q water three times. All the samples (n = 3) were placed in a 24-well culture plate for adhesion and proliferation assays, as described above. Data Analysis. Statistical comparisons from different cell counts were performed in excel and data were presented as mean ± standard deviation (SD). The differences were considered statistically significant with
