Article pubs.acs.org/Biomac
Near Superhydrophobic Fibrous Scaffold for Endothelialization: Fabrication, Characterization and Cellular Activities Furqan Ahmed,† Namita Roy Choudhury,*,† Naba K. Dutta,† Andrew Zannettino,‡ and Robert Knott§ †
Ian Wark Research Institute, University of South Australia, Mawson Lakes Campus, South Australia, Australia Bone and Cancer Research Laboratories, Haematology, Centre for Cancer Biology, SA Pathology and School of Medical Science, University of Adelaide, Adelaide, South Australia, Australia § Bragg Institute, ANSTO, Lucas Height, Sydney, Australia ‡
S Supporting Information *
ABSTRACT: In this work, we have applied an electrospinning method to control wettability and further hydrophobic modification of a hydrophobic polymer mat of poly(vinylidene fluoride-co-hexafluoropropylene). A correlation between the processing parameters, rheological properties of polymer solutions, and electrospinning ability was made using the polymer’s critical entanglement concentration, the boundary between the semidilute unentangled regime and the semidilute entangled regime. The wetting behavior, structural and thermal characteristics of electrospun (ES) mats were evaluated and compared with solvent cast sample using advancing and receding contact angle analyses, differential scanning calorimetry, and small-angle X-ray scattering. To demonstrate the feasibility, the best optimized ES samples were examined for their potential and ability to support bone marrow derived endothelial cell seeding efficiency, adhesion and proliferation. Our studies show that, while different processing techniques can effectively modulate physical and morphological changes such as porosity and hydrophobicity, the cellular adhesion and proliferation are highly time-dependent and controlled by chemical factors. As such, these results suggest that it is the interplay of both physical and chemical factors that determine the endothelialization of porous near superhydrophobic scaffolds. The developed electrospun samples demonstrate their feasibility for endothelialization.
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INTRODUCTION A material’s surface wettability directly influences the adsorption of protein/biomolecules that govern cell adhesion.1 The control of a material’s surface wetting properties is also central to their application as antifouling paints,2 protein suppression on membranes, blood contact medical devices, and microfluidics.3 Several fabrication techniques have been reported that can alter the surface wettability by combining material with low surface energy with optimum surface roughness and topography; such as laser ablation, phase separation, spin coating, and electrospinning.4 Compared with mechanical fiber-spinning technologies, electrospinning has emerged as a facile, inexpensive, and promising fabrication technique to produce ultrahydrophobic/superhydrophobic surfaces. The electrospinning technique utilizes electrical force to generate nanofibers of both synthetic and natural polymers with remarkable properties; including unique threedimensional characteristics, large surface area, tunable pore size, and fiber diameter, and novel physical properties.5 Recently, significant potential of electrospinning has also been realized to fabricate biomimetic nanofiber scaffold, similar to the natural extracellular matrix for tissue regeneration, treatment/rehabilitation, and improvement of quality of life.6 While a number of conventional methods and strategies have been developed to © 2013 American Chemical Society
generate a three-dimensional porous structure for tissue engineering scaffolds including rapid prototyping (RP), slurry-dipping, nanofiber self-assembly, particulate leaching, gas foaming, emulsification/freeze-drying, and thermally induced phase separation;7 many of these methods generate scaffolds that lack desirable topography and porosity and lack the characteristics of the extracellular matrix of native tissues. In tissue engineering, the combined use of living cells, biomolecules, and synthetic materials could provide a symbiotic relationship that mimics native tissues in terms of their function and growth.7 Significant research efforts8 have been expended to explore advanced electrospinning techniques to fabricate controllable porous scaffolds with desirable property, topology, morphology, and surface characteristics, which are necessary for the proper nutrition, growth, and oxygen supply to the cells in a native extracellular matrix. To this end, Badami et al.9 compared the adsorption of osteoblastic cells on spin-coated and electrospun (ES) poly(lactic acid) substrates and demonstrated superior cell infiltration on ES samples relative to smooth spin coated samples. They also observed greater Received: June 27, 2013 Revised: September 18, 2013 Published: September 18, 2013 3850
dx.doi.org/10.1021/bm400938n | Biomacromolecules 2013, 14, 3850−3860
Biomacromolecules
Article
adhesion of esophageal-derived epithelial cells on poly(D,Llactide) ES scaffolds compared with smooth films. High porosity, extensive pore interconnectivity, and large surface area to volume ratio of ES scaffolds make them desirable to achieve a 3-D environment that is highly conducive to cellular adhesion and growth. Ma et al. reported that artificial vascular grafts generated by electrospinning of polyethylene terephthalate (PET) nanofibers exhibited excellent cell adhesion/ growth due to a large surface area and porosity.10 The surface wettability of 3-D fibrous scaffolds plays a critical role in the adsorption of proteins/biomolecules that govern cell adhesion. Fluoropolymers have been used in a wide variety of bloodcontacting medical device applications such as arterial prostheses, drug-eluting leads of cardioverter-defibrillators, vascular sutures, and guide wire coatings.11 They are also known to possess inherent properties that contribute to thrombo-resistance, reduced inflammation, and increased reendothelialization in blood-contact applications in human and animal models. However, the importance of the physical parameters, such as, overall porosity, pore directionality, difference between 2D and 3D scaffold structures, are also highly critical. As an important thermoplastic material, poly(vinylidene fluoride) with its copolymer-co-hexafluoropropylene (PVDF-HFP) has been widely used as a polymer membrane12 for batteries, solar and fuel cells,13 medical devices,14 and tissue engineering. However, PVDF-HFP-based nanofibers have not been used for endothelialization. Compared with other biomaterials used as vascular scaffolds, PVDF-HFP-based nanofibers may be used to effectively mimic the native tissue, as PVDF-HFP is hydrophobic, elastic, resistant to infection, durable, resilient, and being a fluoropolymer, nonthrombogenic in nature.11 While ultrafine PVDF-HFP fibers can be produced easily by electrospinning,15 for the first time we have examined their endothelialization capacity; a feature which limits thrombosis. In addition to thromobogenicity, many grafts also fail due to the mechanical mismatch between the native tissue and the implant. In the present study, we investigate the effect of electrospinning parameters to generate PVDF-HFP scaffolds with optimal wettability/topography to support endothelial cell adhesion and proliferation. For this purpose, a series of PVDF-HFP electrospun (ES) mats with a variety of different surface morphologies were produced by tuning viscosity, surface tension, solution conductivity, and processing parameters. The formed nanofibers of different morphological features were characterized using scanning electron microscopy (SEM). The effect of different processing parameters on wettability were also examined using contact angle measurement, and their structural and morphological analyses were done by small-angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC). To demonstrate the feasibility, the best optimized nanofibrous scaffold was examined for the endothelial cell seeding efficiency; adhesion/proliferation in conjunction with solvent cast (SC) samples and the effect of wettability/ topography/crystallinity on cellularization was monitored.
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Scheme 1. Schematic Representation of the Chemical Structure of PVDF-HFP
temperature. The solutions of different concentrations, that is, 8, 10, 12, 14, and 16 wt % PVDF-HFP were prepared and electrospinning was carried out using them at room temperature. Solution Viscosity Measurement. Solutions viscosities were measured using controlled stress rheometer Rheolyst 1000 N (TA Instruments Delaware, U.S.A.). The measurement was carried out (at 25 °C) using 40 cm cone and plate geometry. The sample was placed between the fixed Peltier plate and a rotating cone attached to the driving motor spindle. The changes in shear stress with change in shear rate were measured and viscosity of solution was calculated. The viscosities of the solution are reported in Pa·s. Surface Tension Measurement. The surface tension of the solution was measured by drop shape method at 25 °C using a proprietary contact angle goniometer (Ian Wark Research Institute, Adelaide, Australia) Sessile Drop instrument. Hamilton 50 μL syringe was used to measure 10 μL pendant drops by SCA20 Version2 software. Solution Conductivity. The solution conductivity was measured by Corning digital conductivity meter with a range 0.00−199900 μS/ cm−1 at 25 °C with auto calibration points. KCl and distilled water were used as standards. Preparations of Electrospun (ES) Fibrous Samples. In a typical electrospinning process, polymer solution was placed in a 20 mL syringe with an 18 gauge blunt end needle that was mounted on a syringe pump (KD Scientific). In this study, a high voltage DC power supply was used to generate potential differences between 13 to 17 kV (nominal field strength E = V/H, where V = applied voltage and H, the tip-collector distance, is varied from 14 and 18 to 22 cm, field strength varies from 0.59 to 1.21 V/cm). High voltage was applied to the bluntend metal needle, which formed one electrode. Randomly oriented nanofibers were electrospun on the collector plate, when the high voltage was applied to the solution. During electrospinning process, charge repulsive forces oppose the surface tension of the solution, and a fluid jet elongates out of the capillary wall and forms a conical shape, which is called a Taylor cone. At a certain critical point the electric field overcomes the surface tension of the solution and a charged jet of solution is ejected from the Taylor cone.16 The fibers were obtained as a mat using an earthed collection system (electrode configuration point-plate), which consisted of an aluminum foil collector measuring 10 × 10 cm. Preparation of PVDF-HFP Solvent Cast (SC) Samples. Uniform thin films of the PVDF-HFP were prepared by casting of PVDF-HFP solution in glass Petri dish. The SC samples in Petri dish were left in controlled environment for drying for 2 days at 65 °C. After solvent evaporation, the PVDF-HFP thin films were cut to make 1 cm discs. Characterization of ES and SC Samples. Characterization by SEM. Different morphologies of ES samples and cell attached SC and ES samples were characterized by Philips XL30 FEGSEM with Oxford CT1500HF Cryo stage. To minimize charging effects, platinum was deposited after cell fixation on each sample by sputtering and examined at an accelerating voltage of 10 kV. Characterization by AFM. Prior to performing the cell culture study, best optimized ES and SC samples topography was assessed by atomic force microscopy (AFM). AFM provided topographical images using an NT-MDT NTEGRA SPM in noncontact mode. Silicon nitride noncontact tips coated with gold on the reflective side (NTMDT, NSG03) were used and had resonance frequencies between 65 and 100 kHz. The amplitude of oscillation was 10 nm, and the scan rate for 10 × 10 μm2 images was 0.5 Hz. The scanner used had a range of 100 μm and was calibrated using 1.5 μm standard grids.
EXPERIMENTAL SECTION
Polymer Solution Preparation and Properties. PVDF-HFP (Scheme 1) with an inherent viscosity of 2300−2700 Pa (avg molecular weight 400000) was purchased from Sigma Aldrich, Australia. The polymer was dissolved in 70/30 ratio of N,Ndimethylacetamide (DMAc) and acetone (Sigma Aldrich, Australia) and left overnight for mixing with a magnetic stirrer at room 3851
dx.doi.org/10.1021/bm400938n | Biomacromolecules 2013, 14, 3850−3860
Biomacromolecules
Article
Bulk Porosity Measurement. The average porosity of the samples was measured at room temperature using the liquid intrusion method. Briefly, the ES PVDF-HFP samples were weighed, after which 20 to 30 μL of fluorocarbon derivative of tetrahydrofuran (FC- 75) was spread on 1 cm diameter surface, and the samples weighed again (within 20 s to avoid the evaporation of solvent). The samples were subsequently immersed in water to allow it to penetrate into the sample voids. Pore volume was measured as below:
pore volume =
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, the cellular constructs were harvested, washed with PBS to remove nonadherent cells, and then fixed with primary fixative 4% glutaraldehyde for 0.5 h at room temperature, washed with PBS for 5 min, and post-fixed with 2% osmium tetraoxide for 30 min, then dehydrated with 70% ethanol for 10 min, with 90% ethanol for 10 min and 100% ethanol for 10 min. Finally, the constructs were rinsed with hexamethyldisilazine (HMZ) diluted with equal amount of 100% ethanol for 10 min then samples were exposed to undiluted HMZ for 10 min and left to dry for overnight. After critical point dry cellular constructs were sputter coated with platinum and observed under the SEM at an accelerating voltage of 10 kV. Cell count was determined by direct counting on SEM image on 10 randomly selected locations on triplicate samples 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, SC, and optimized PVDFHFP ES samples at a plating density of 6 × 104 cells/cm2. The cells were allowed to proliferate for 1, 5, and 9 days after which proliferation and viability was determined by direct cell counting and using the WST-1 cell proliferation and viability assay kit (from Roche). All experiments were performed in triplicate. SEM images were again taken from 10 randomly chosen locations for each sample condition. Cell densities were determined using Image J software as before. Data Analysis. Statistical comparisons for different cell counts were done and data were presented as mean ± standard deviation (SD). Differences were considered statistically significant with 120°, we use the term ultrahydrophobicity and when the contact angle is increased to >150° and the contact-angle hysteresis is
