The Mechanical Stress–Strain Properties of Single Electrospun Collagen Type I Nanofibers

Knowledge of the mechanical properties of electrospun fibers is important for their successful application in tissue engineering, material composites, filtration and drug delivery. In particular, electrospun collagen has great potential for biomedical applications due to its biocompatibility and promotion of cell growth and adhesion. Using a combined atomic force microscopy (AFM)/optical microscopy technique, the single fiber mechanical properties of dry, electrospun collagen type I were determined. The fibers were electrospun from a 80 mg ml−1 collagen solution in 1,1,1,3,3,3-hexafluro-2-propanol and collected on a striated surface suitable for lateral force manipulation by AFM. The small strain modulus, calculated from three-point bending analysis, was 2.82 GPa. The modulus showed significant softening as the strain increased. The average extensibility of the fibers was 33% of their initial length, and the average maximum stress (rupture stress) was 25 MPa. The fibers displayed significant energy loss and permanent deformations above 2% strai

Electrostatic Alignment of Electrospun PEO Fibers by the Gap Method Increases Individual Fiber Modulus in Comparison to Non-Aligned Fibers of Similar Diameter

Studies on the alignment, physical and mechanical properties of individual electrospun fibers provide insight to their formation, production and optimization. Here we measure the alignment, diameter and modulus of individual fibers formed using the electrostatic gap method. We find electrostatic alignment produces fibers with a smaller diameter than their nonaligned counterparts have. Therefore, due to the dependence of fiber modulus on diameter aligned fibers have a higher modulus. Furthermore, we show that aligned and nonaligned fibers of the similar diameter have different moduli. Aligned fibers have a modulus 1.5 to 2 times larger than nonaligned fibers of the similar diameter

Hemoglobin-Mediated Nitric Oxide Signaling

The rate that hemoglobin reacts with nitric oxide (NO) is limited by how fast NO can diffuse into the heme pocket. The reaction is as fast as any ligand/protein reaction can be and the result, when hemoglobin is in its oxygenated form, is formation of nitrate in what is known as the dioxygenation reaction. As nitrate, at the concentrations made through the deoxygenation reaction, is biologically inert, the only role hemoglobin was once thought to play in NO signaling was to inhibit it. However, there are now several mechanisms that have been discovered by which hemoglobin may preserve, control, and even create NO activity. These mechanisms involve compartmentalization of reacting species and conversion of NO from or into other species such as nitros othiols or nitrite which could transport NO activity. Despite the tremendous amount of work devoted to this field, major questions concerning precise mechanisms of NO activity preservation as well as if and how Hb creates NO activity remain unanswered

Recent Insights Into Nitrite Signaling Processes in Blood

Nitrite was once thought to be inert in human physiology. However, research over the past few decades has established a link between nitrite and the production of nitric oxide (NO) that is potentiated under hypoxic and acidic conditions. Under this new role nitrite acts as a storage pool for bioavailable NO. The NO so produced is likely to play important roles in decreasing platelet activation, contributing to hypoxic vasodilation and minimizing blood-cell adhesion to endothelial cells. Researchers have proposed multiple mechanisms for nitrite reduction in the blood. However, NO production in blood must somehow overcome rapid scavenging by hemoglobin in order to be effective. Here we review the role of red blood cell hemoglobin in the reduction of nitrite and present recent research into mechanisms that may allow nitric oxide and other reactive nitrogen signaling species to escape the red blood cell

Strength and Failure of Fibrin Fiber Branch Points

Blood clots form rapidly in the event of vascular injury, to prevent blood loss. They may also form in undesired places, causing heart attacks, strokes, and other diseases. Blood clots can rupture, and fragments of the clotmay lodge in distal blood vessels, causing, for example, ischemic strokes or embolisms. Thus, there has been great interest in understanding the mechanical behavior and failure mechanisms of blood clots and their constituents. To develop a mechanically realistic model of a blood clot, knowledge of the mechanical properties of its constituents is required. The major structural component providing mechanical strength to the clot is a mesh of fibrin fibers. Principally, three pieces of information are needed to develop realistic (fibrin fiber) network models: (i) the architecture of the network; (ii) the properties of the single fibers; and (iii) the properties of the fiber branchpoints

The Mechanical Properties of Single Fibrin Fibers

Background: Blood clots perform the mechanical task of stemming the flow of blood. Objectives: To advance understanding and realistic modeling of blood clot behavior we determined the mechanical properties of the major structural component of blood clots, fibrin fibers. Methods: We used a combined atomic force microscopy (AFM)/fluorescence microscopy technique to determine key mechanical properties of single crosslinked and uncrosslinked fibrin fibers. Results and conclusions: Overall, full crosslinking renders fibers less extensible, stiffer, and less elastic than their uncrosslinked counterparts. All fibers showed stress relaxation behavior (time-dependent weakening) with a fast and a slow relaxation time, 2 and 52 s. In detail, crosslinked and uncrosslinked fibrin fibers can be stretched to 2.5 and 3.3 times their original length before rupturing. Crosslinking increased the stiffness of fibers by a factor of 2, as the total elastic modulus, E0, increased from 3.9 to 8.0 MPa and the relaxed, elastic modulus, E∞, increased from 1.9 to 4.0 MPa upon crosslinking. Moreover, fibers stiffened with increasing strain (strain hardening), as E0 increased by a factor of 1.9 (crosslinked) and 3.0 (uncrosslinked) at strains ε \u3e 110%. At low strains, the portion of dissipated energy per stretch cycle was small (\u3c 10%) for uncrosslinked fibers, but significant (approximately 40%) for crosslinked fibers. At strains \u3e 100%, all fiber types dissipated about 70% of the input energy. We propose a molecular model to explain our data. Our single fiber data can now also be used to construct a realistic, mechanical model of a fibrin network

Erythrocytes and Vascular Function: Oxygen and Nitric Oxide

Erythrocytes regulate vascular function through the modulation of oxygen delivery and the scavenging and generation of nitric oxide (NO). First, hemoglobin inside the red blood cell binds oxygen in the lungs and delivers it to tissues throughout the body in an allosterically regulated process, modulated by oxygen, carbon dioxide and proton concentrations. The vasculature responds to low oxygen tensions through vasodilation, further recruiting blood flow and oxygen carrying erythrocytes. Research has shown multiple mechanisms are at play in this classical hypoxic vasodilatory response, with a potential role of red cell derived vasodilatory molecules, such as nitrite derived nitric oxide and red blood cell ATP, considered in the last 20 years. According to these hypotheses, red blood cells release vasodilatory molecules under low oxygen pressures. Candidate molecules released by erythrocytes and responsible for hypoxic vasodilation are nitric oxide, adenosine triphosphate and S-nitrosothiols. Our research group has characterized the biochemistry and physiological effects of the electron and proton transfer reactions from hemoglobin and other ferrous heme globins with nitrite to form NO. In addition to NO generation from nitrite during deoxygenation, hemoglobin has a high affinity for NO. Scavenging of NO by hemoglobin can cause vasoconstriction, which is greatly enhanced by cell free hemoglobin outside of the red cell. Therefore, compartmentalization of hemoglobin inside red blood cells and localization of red blood cells in the blood stream are important for healthy vascular function. Conditions where erythrocyte lysis leads to cell free hemoglobin or where erythrocytes adhere to the endothelium can result in hypertension and vaso constriction. These studies support a model where hemoglobin serves as an oxido-reductase, inhibiting NO and promoting higher vessel tone when oxygenated and reducing nitrite to form NO and vasodilate when deoxygenated. How erythrocytes modulate vascular tone has been widely studied over the last two decades. The vasodilation of the vasculature under hypoxic conditions has inspired much research ranging from the effect of oxygen partial pressure on smooth muscle cell contractility and endothelial nitric oxide synthase (eNOS) activity to nitrite reduction by hemoglobin (Hb) inside erythrocytes and subsequent production of nitric oxide. Here we review how red blood cells (RBCs) and hemoglobin regulate vascular function and blood flow

Exposure of fibrinogen and thrombin to nitric oxide donor ProliNONOate affects fibrin clot properties

Fibrin fibers form the structural backbone of blood clots. The structural properties of fibrin clots are highly dependent on formation kinetics. Environmental factors such as protein concentration, pH, salt, and protein modification, to name a few, can affect fiber kinetics through altered fibrinopeptide release, monomer association, and/or lateral aggregation. The objective of our study was to determine the effect of thrombin and fibrinogen exposed to nitric oxide on fibrin clot properties. ProliNONOate (5 [mu]mol/l) was added to fibrinogen and thrombin before clot initiation and immediately following the addition of thrombin to the fibrinogen solution. Resulting fibrin fibers were probed with an atomic force microscope to determine their diameter and extensibility and fibrin clots were analyzed for clot density using confocal microscopy. Fiber diameters were also determined by confocal microscopy and the rate of clot formation was recorded using UV-vis spectrophotometry. Protein oxidation and S-nitrosation was determined by UV-vis, ELISA, and chemiluminescence. The addition of ProliNONOate to fibrinogen or thrombin resulted in a change in clot structure. ProliNONOate exposure produced clots with lower fiber density, thicker fibers, and increased time to maximum turbidity. The effect of the exposure of nitric oxide to thrombin and fibrinogen were measured independently and indicated that each plays a role in altering clot properties. We detected thrombin S-nitrosation and protein carbonyl formation after nitric oxide exposure. Our study reveals a regulation of fibrin clot properties by nitric oxide exposure and suggests a role of peroxynitrite in oxidative modifications of the proteins. These results relate nitric oxide bioavailability and oxidative stress to altered clot properties

α−α Cross-Links Increase Fibrin Fiber Elasticity and Stiffness

Fibrin fibers, which are ∼100 nm in diameter, are the major structural component of a blood clot. The mechanical properties of single fibrin fibers determine the behavior of a blood clot and, thus, have a critical influence on heart attacks, strokes, and embolisms. Cross-linking is thought to fortify blood clots; though, the role of α–α cross-links in fibrin fiber assembly and their effect on the mechanical properties of single fibrin fibers are poorly understood. To address this knowledge gap, we used a combined fluorescence and atomic force microscope technique to determine the stiffness (modulus), extensibility, and elasticity of individual, uncross-linked, exclusively α–α cross-linked (γQ398N/Q399N/K406R fibrinogen variant), and completely cross-linked fibrin fibers. Exclusive α–α cross-linking results in 2.5× stiffer and 1.5× more elastic fibers, whereas full cross-linking results in 3.75× stiffer, 1.2× more elastic, but 1.2× less extensible fibers, as compared to uncross-linked fibers. On the basis of these results and data from the literature, we propose a model in which the α-C region plays a significant role in inter- and intralinking of fibrin molecules and protofibrils, endowing fibrin fibers with increased stiffness and elasticity

The Mechanical Properties of Individual, Electrospun Fibrinogen Fibers

We used a combined atomic force microscope (AFM)/fluorescence microscope technique to study the mechanical properties of individual, electrospun fibrinogen fibers in aqueous buffer. Fibers (average diameter 208 nm) were suspended over 12 μm-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 22·106 Pa. We collected incremental stress-strain curves to determine the viscoelastic behavior of these fibers. The total stretch modulus was 16·106 Pa and the relaxed, elastic modulus was 6.7·106 Pa. When held at constant strain, electrospun fibrinogen fibers showed a fast and slow stress relaxation time of 3 and 56 seconds. Our fibers were spun from the typically used 90% 1,1,1,3,3,3-hexafluoro-2-propanol (90-HFP) electrospinning solution and resuspended in aqueous buffer. Circular dichroism spectra indicate that alpha-helical content of fibrinogen is ~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