All reagents obtained from Sigma Chemical Co., (St. Louis, MO, USA) unless noted. Nitrocellulose (Biorad Laboratories, Hercules, CA) was suspended and agitated for 24 hr at varying concentrations in acetone. Charged nylon (Nylon 66 derivatized with quaternary ammonium manufactured by Ambion of Austin, TX and sold as Brightstar-Plus Nylon membrane) was suspended and agitated for 24 hr in 1,1,1,3,3,3-hexaxafluoro-2-propanol (HFIP). A Brookfield RVDV-III Ultra programmable rheometer was used to measure solution viscosity.
Nitrocellulose was dissolved at various concentrations (60, 80, 100, 110, 120 and 140 mg/ml) in acetone under agitation for 24 hr. Electrospinning suspensions were loaded into a 20 ml Becton Davis syringe capped with an 18 gauge blunt tipped needle. The negative lead of a high voltage supply (Spellman CZE1000R; Spellman High Voltage Electronics Corporation) was attached by an alligator clip to the blunt tipped needle. This polarity was found to reduce drying of the acetone/nitrocellulose solution at the tip of the syringe and functioned to stabilize the Taylor cone. A 22 kV accelerating voltage was used in the electrospinning process. A Harvard perfusion pump was used to meter the delivery of the nitrocellulose solution to the electric field, the rate of delivery was set to the maximal rate that did not induce dripping from the tip of the syringe or the introduction of solvent defects in the resulting membrane (5–10 ml/hr, depending upon starting concentration).
Charged nylon was dissolved at various concentrations 60, 80, 100, 120 mg/ml in HFIP for 24 hr. Suspensions were electrospun from a 20 ml Becton Davis syringe capped with an 18 gauge blunt tipped needle. The negative lead was attached to the syringe and charged to 25 kV. The air gap distance between the source suspension and the grounded mandrel was set to 20 cm. A Harvard perfusion was used as described, the rate of solvent/polymer delivery was set at the maximal rate that did not induce dripping from the tip of the syringe (10–14 ml/hr). For all solutions, a stainless steel cylindrical mandrel (10 cm × 4.0 cm) was used as a grounded target. Each finished sheet had approximate dimensions of 15 cm × 4.0 cm.
Scanning electron microscopy (SEM)
Membranes were sputter coated and imaged with a Zeiss EVO 50 XVP scanning electron microscope equipped with digital image acquisition. Average fiber diameter was determined from representative samples using NIH ImageTool (UTHSCSA version 3). All measurements were taken perpendicular to the long axis of electrospun fibers. Measurements were calibrated from size bars incorporated into the SEM images at the time of capture. NIH Image J software was used to conduct fiber measurements [8,9].
Fiber diameter data sets were screened by One-way ANOVA to determine the effects of starting conditions on fiber diameter. A Tukey test was used in the post hoc analysis of these data sets. Statistical significance was determined at P
Slot blot experiments were conducted to characterize the performance of electrospun materials. Sheets of commercially available nitrocellulose and nitrocellulose electrospun from this parent material were wet in transfer solution and mounted into a slot blot apparatus (Hoefer, San Francisco CA). Each well was rinsed 3× in transfer buffer. A series of serial dilutions of human fibronectin (Fn) was prepared in PBS and added sequentially to the blotting wells (80, 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.1563, 0.078 μg total protein per well). After 5 minutes, the Fn solution was drawn through the blot apparatus as per slot blot manufacture's directions. The wells were each rinsed and blocked 3× with 250 μl of PBS supplemented with 1% BSA plus 0.1% Tween 20 (subsequently referred to as blocking buffer). Control lanes were incubated with blocking buffer for 5 minutes and rinsed as described.
Primary antibody (Sigma F3648) against human Fn was diluted 1:1000 in blocking buffer and applied to each lane and allowed to incubate for 5 minutes. At the conclusion of this incubation lanes were rinsed in blocking buffer 3×. Secondary antibodies (Vector Laboratories, Inc., Burlingame CA) tagged with HRP were prepared at a 1:10,000 dilution in blocking buffer and allowed to incubate for 5 minutes, the wells were re-rinsed 5× with 250 μl of blocking buffer. Samples were removed from the blot apparatus, rinsed in PBS and processed for chemiluminescence detection as per the manufacture's instructions (ECL Plus Western Blotting Detection System; Amersham, GE Health Care). Images were captured on Kodak Blue XB-1 film (Kodak). Gel images were captured with a BioRad Gel Doc 2000 system, relative optical density calculated from digital images with BioRad Gel Doc 2000 software.
Western blot analysis
Serial dilutions of human Fn (10.0, 5.0, 2.5, 1.0, 0.50, 0.25, 0.16 0.08, 0.02 μg Fn/lane) were prepared in Laemmli buffer and separated by SDS gel electrophoresis (10% acrylamide gels, run @150 V, BioRad). Separated samples were transferred overnight (12 hr, 125 mA @4°C) onto blotting membranes. Benchmark MW Standards were used in these experiments (Invitrogen, USA).
Membranes were blocked in 5.0% non-fat milk prepared in PBS plus 0.1% Tween 20 for 30 minutes at room temperature (this formulation referred to as Western blocking buffer). Primary antibody (Sigma F3648) against human Fn was diluted 1:1000 in Western blocking buffer and applied to the membranes for 1 hr at room temperature. Membranes were rinsed 30 minutes in Western Blocking buffer under agitation using 4–5× complete changes of buffer. Secondary antibodies (Vector Laboratories, Inc., Burlingame CA) were prepared at a 1:10,000 dilution in Western blocking buffer and allowed to incubate for 1 hr at room temperature. Membranes were once again rinsed 4–5× in fresh Western blocking buffer. Samples were rinsed in PBS and then processed for chemiluminescence as described for slot blotting. Images were captured on Kodak Blue XB-1 film (Kodak).