The Mechanical Properties of Dry, Electrospun Fibrinogen Fibers.

Due to their low immunogenicity, biodegradability and native cell-binding domains, fibrinogen fibers may be good candidates for tissue engineering scaffolds, drug delivery vehicles and other medical devices. We used a combined atomic force microscope (AFM)/optical microscope technique to study the mechanical properties of individual, electrospun fibrinogen fibers in dry, ambient conditions. The AFM was used to stretch individual fibers suspended over 13.5 µm wide grooves in a transparent substrate. The optical microscope, located below the sample, was used to monitor the stretching process. Electrospun fibrinogen fibers (diameter, 30-200 nm) can stretch to 74 % beyond their original length before rupturing at a stress of 2.1 GPa. They can stretch elastically up to 15 % beyond their original length. Using incremental stress-strain curves the viscoelastic behavior of these fibers was determined. The total stretch modulus was 4.2 GPa while the relaxed elastic modulus was 3.7 GPa. When held at constant strain, fibrinogen fibers display stress relaxation with a fast and slow relaxation time of 1.2 s and 11 s.In comparison to native and electrospun collagen fibers, dry electrospun fibrinogen fibers are significantly more extensible and elastic. In comparison to wet electrospun fibrinogen fibers, dry fibers are about 1000 times stiffer.

Denaturing of single electrospun fibrinogen fibers studied by deep ultraviolet fluorescence microscopy.

Deep ultraviolet (DUV) microscopy is a fluorescence microscopy technique to image unlabeled proteins via the native fluorescence of some of their amino acids. We constructed a DUV fluorescence microscope, capable of 280 nm wavelength excitation by modifying an inverted optical microscope. Moreover, we integrated a nanomanipulator-controlled micropipette into this instrument for precise delivery of picoliter amounts of fluid to selected regions of the sample. In proof-of-principle experiments, we used this instrument to study, in situ, the effect of a denaturing agent on the autofluorescence intensity of single, unlabeled, electrospun fibrinogen nanofibers. Autofluorescence emission from the nanofibers was excited at 280 nm and detected at ∼350 nm. A denaturant solution was discretely applied to small, select sections of the nanofibers and a clear local reduction in autofluorescence intensity was observed. This reduction is attributed to the dissolution of the fibers and the unfolding of proteins in the fibers.

The mechanical properties of individual, electrospun fibrinogen fibers.

We used a combined atomic force microscopic (AFM)/fluorescence microscopic technique to study the mechanical properties of individual, electrospun fibrinogen fibers in aqueous buffer. Fibers (average diameter 208 nm) were suspended over 12 microm-wide grooves in a striated, transparent substrate. The AFM, situated above the sample, was used to laterally stretch the fibers and to measure the applied force. The fluorescence microscope, situated below the sample, was used to visualize the stretching process. The fibers could be stretched to 2.3 times their original length before breaking; the breaking stress was 22 x 10 (6)Pa. We collected incremental stress-strain curves to determine the viscoelastic behavior of these fibers. The total stretch modulus was 17.5 x 10 (6)Pa and the relaxed elastic modulus was 7.2 x 10 (6)Pa. When held at constant strain, electrospun fibrinogen fibers showed a fast and slow stress relaxation time of 3 and 55 s. Our fibers were spun from the typically used 90% 1,1,1,3,3,3-hexafluoro-2-propanol (90-HFP) electrospinning solution and re-suspended in aqueous buffer. Circular dichroism spectra indicate that alpha-helical content of fibrinogen is approximately 70% higher in 90-HFP than in aqueous solution. These data are needed to understand the mechanical behavior of electrospun fibrinogen structures. Our technique is also applicable to study other nanoscopic fibers.