Mechanical Properties Of Fibrin Research Articles

Physical, Mechanical, and Biological Properties of Fibrin Scaffolds for Cartilage Repair.

Articular cartilage is a highly organized tissue that provides remarkable load-bearing and low friction properties, allowing for smooth movement of diarthrodial joints; however, due to the avascular, aneural, and non-lymphatic characteristics of cartilage, joint cartilage has self-regeneration and repair limitations. Cartilage tissue engineering is a promising alternative for chondral defect repair. It proposes models that mimic natural tissue structure through the use of cells, scaffolds, and signaling factors to repair, replace, maintain, or improve the specific function of the tissue. In chondral tissue engineering, fibrin is a biocompatible biomaterial suitable for cell growth and differentiation with adequate properties to regenerate damaged cartilage. Additionally, its mechanical, biological, and physical properties can be enhanced by combining it with other materials or biological components. This review addresses the biological, physical, and mechanical properties of fibrin as a biomaterial for cartilage tissue engineering and as an element to enhance the regeneration or repair of chondral lesions.

Advances in Fibrin-Based Materials in Wound Repair: A Review.

The first bioprocess that occurs in response to wounding is the deterrence of local hemorrhage. This is accomplished by platelet aggregation and initiation of the hemostasis cascade. The resulting blood clot immediately enables the cessation of bleeding and then functions as a provisional matrix for wound healing, which begins a few days after injury. Here, fibrinogen and fibrin fibers are the key players, because they literally serve as scaffolds for tissue regeneration and promote the migration of cells, as well as the ingrowth of tissues. Fibrin is also an important modulator of healing and a host defense system against microbes that effectively maintains incoming leukocytes and acts as reservoir for growth factors. This review presents recent advances in the understanding and applications of fibrin and fibrin-fiber-incorporated biomedical materials applied to wound healing and subsequent tissue repair. It also discusses how fibrin-based materials function through several wound healing stages including physical barrier formation, the entrapment of bacteria, drug and cell delivery, and eventual degradation. Pure fibrin is not mechanically strong and stable enough to act as a singular wound repair material. To alleviate this problem, this paper will demonstrate recent advances in the modification of fibrin with next-generation materials exhibiting enhanced stability and medical efficacy, along with a detailed look at the mechanical properties of fibrin and fibrin-laden materials. Specifically, fibrin-based nanocomposites and their role in wound repair, sustained drug release, cell delivery to wound sites, skin reconstruction, and biomedical applications of drug-loaded fibrin-based materials will be demonstrated and discussed.

Influence of different concentrations of fibrinogen on the properties of a fibrin matrix for vascular tissue engineering

Aim. To evaluate the potential utility of fibrin matrices containing 10, 20, and 25 mg/ml of fibrinogen (fibrin-10, fibrin-20, and fibrin-30, respectively) in vascular tissue engineering (VTE).
 Materials and Methods. Fibrinogen was isolated using the method of ethanol cryoprecipitation and polymerized using a solution of thrombin and CaCl2. The fibrin structure was studied in a scanning electron microscope, and the physical and mechanical properties of the material were tested on a Zwick/Roell test machine. The metabolic activity of endothelial cells (EC) on the fibrin surface was evaluated by the MTT assay, and the viability of fibroblasts in the thickness of fibrin and possibility for migration by in fluorescent and light microscopy. Percent of fibrin shrinkage was determined from the difference in the sample volumes before and after removal of moisture.
 Results. The fiber diameter did not differ among all fibrin samples, but the pore diameter in fibrin-30 was smaller than those in fibrin-10 and fibrin-20. A possibility for migration of fibroblasts into the depth of the fibrin matrix and preservation of 97-100% viability of cells at a depth 5 mm was confirmed. The metabolic activity of EC on the surface of fibrin-20 and fibrin-30 exceeded that on collagen, fibronectin, and fibrin-10. All fibrin samples shrank in volume to 95.5-99.5%, and the highest shrinkage was seen in fibrin-10. The physical and mechanical properties of fibrin were inferior to those of human A. mammaria by a factor of 10.
 Conclusion. Fibrin with fibrinogen concentrations of 20 and 30 mg/ml maintains a high metabolic and proliferative activity of EC on the surface and also a high viability of fibroblasts in the matrix. Its availability, ease of preparation, and a number of other favorable properties make fibrin a promising material for VTE. However, the problem of insufficient strength requires further investigations.

Anomalous mechanics of Zn2+-modified fibrin networks

Fibrin is the main component of blood clots. The mechanical properties of fibrin are therefore of critical importance in successful hemostasis. One of the divalent cations released by platelets during hemostasis is Zn2+; however, its effect on the network structure of fibrin gels and on the resultant mechanical properties remains poorly understood. Here, by combining mechanical measurements with three-dimensional confocal microscopy imaging, we show that Zn2+ can tune the fibrin network structure and alter its mechanical properties. In the presence of Zn2+, fibrin protofibrils form large bundles that cause a coarsening of the fibrin network due to an increase in fiber diameter and reduction of the total fiber length. We further show that the protofibrils in these bundles are loosely coupled to one another, which results in a decrease of the elastic modulus with increasing Zn2+ concentrations. We explore the elastic properties of these networks at both low and high stress: At low stress, the elasticity originates from pulling the thermal slack out of the network, and this is consistent with the thermal bending of the fibers. By contrast, at high stress, the elasticity exhibits a common master curve consistent with the stretching of individual protofibrils. These results show that the mechanics of a fibrin network are closely correlated with its microscopic structure and inform our understanding of the structure and physical mechanisms leading to defective or excessive clot stiffness.

Lytic Susceptibility, Structure, and Mechanical Properties of Fibrin in Systemic Lupus Erythematosus.

Among complications of systemic lupus erythematosus (SLE), thrombotic events are relatively common and contribute significantly to the morbidity and mortality rates. An increased risk of thrombosis in various diseases has been shown to be associated with the lytic stability and mechanical stiffness of the fibrin clot determined by its structure. Here we studied alterations of the fibrin clot properties in relation to disease severity in SLE patients. Plasma clots from 28 SLE patients were characterized by the kinetics of formation and fibrinolytic dissolution (using dynamic turbidimetry), the network and fiber ultrastructure (scanning electron microscopy), viscoelasticity (shear rheometry), and the rate and degree of crosslinking (Western blotting) correlated with the disease activity, blood composition, and compared to clotting of pooled normal human plasma. Clots made from plasma of SLE patients were lysed faster with exogenous t-PA than control clots from normal plasma without a significant difference between those from active (SLEDAI>4) and inactive (SLEDAI<4) SLE patients. Clots from the blood of patients with active SLE were characterized by significantly slower onset, but faster rate of fibrin polymerization and a higher optical density due to thicker fibers compared to those from inactive SLE and control pooled normal plasma. The rheological parameters of the clots (storage and loss moduli) were significantly increased in the active SLE patients along with enhanced fibrin crosslinking and hyperfibrinogenemia. The structural and rheological alterations displayed a strong positive correlation with high fibrinogen levels and other laboratory markers of immune inflammation. In conclusion, changes in the blood composition associated with active systemic inflammation in SLE cause significant alterations in the lytic resistance of fibrin clots associated with changes in polymerization kinetics, viscoelastic properties, and structure. The formation of more rigid prothrombotic fibrin clots in the plasma of SLE patients is likely due to the inflammatory hyperfibrinogenemia and greater extent of crosslinking. However, the higher susceptibility of the SLE clots to fibrinolysis may be a protective and/or compensatory mechanism that reduces the risk of thrombotic complications and improves patient outcomes.

Structural Molecular Origins of Fibrin Mechanics

The mechanical properties of fibrin are essential to stop bleeding but also contribute to obstructive thrombi that cause heart attack and stroke. The deformation of fibrin polymer occurs at different spatial scales, including molecular unfolding, which is relatively unexplored. Here, the AFM-induced unfolding of fibrinogen monomers and oligomers was correlated with force-extension curves obtained using MD simulations. The unraveling of fibrin(ogen) comprised reversible extension-contraction of the α-helical coiled-coils and unfolding of the γ-nodules, which occurred at a ∼90-pN force and ∼25-nm peak-to-peak distance. All-atom MD simulations showed a transition from α-helix to β-sheet at higher extensions, confirmed experimentally by FTIR-spectroscopy of deformed fibrin clots. The α-to-β conversions correlated directly with the strain or pressure and were partially reversible at the conditions applied. The spectra characteristics of the nascent inter-chain β-sheets were consistent with protein aggregation and fiber bundling. Further MD studies revealed that the coiled-coils undergo ∼5-50 nm extension and 360-degree unwinding with three distinct regimes. In the linear regime, the coiled-coils unwind but not unfold. In the plastic regime, the triple α-helical segments rewind and re-unwind while undergoing a non-cooperative phase transition to form parallel β-sheets. We conclude that, under extension and/or compression, α-to-β conversion of the coiled-coils occurs in fibrin as a part of forced protein unfolding. These regimes of forced deformation of fibrin provide important qualitative and quantitative characteristics of the molecular mechanisms underlying fibrin mechanical properties at the microscopic and macroscopic scales. Furthermore, these structural characteristics of the dynamic mechanical behavior of fibrin at the nanometer scale determine whether or not clots have the ability to stanch bleeding and if thrombi become obstructive or embolize. Finally, this knowledge of the functional significance of different domains of fibrin(ogen) suggests new approaches for modulation of these properties as potential therapeutic interventions.

Molecular Basis of Biomechanics of Hemostasis and Thrombosis: Structural Molecular Transitions Underlying Deformation of Fibrin Clots and Thrombi.

Abstract 2217A new field of biomedical research, biomechanics of hemostasis and thrombosis, has been quickly developing over the past few years. The mechanical properties of fibrin are essential in vivo for the ability of clots to stop bleeding in flowing blood but also determine the likelihood of obstructive thrombi that cause heart attack and stroke. Despite such critical importance, the structural basis of clot mechanics is not well understood. The structural changes underlying deformation of fibrin polymer occur at different spatial scales from macroscopic to submolecular, including molecular unfolding, about which relatively little is known. In this work, fibrin mechanics was studied with respect to molecular structural changes during fibrin deformation. The results of atomic force microscopy-induced unfolding of fibrinogen monomers and oligomers were correlated with force-extension curves obtained using Molecular Dynamics simulations. The mechanical unraveling of fibrin(ogen) was shown to be determined by molecular transitions that couple reversible extension-contraction of the α-helical coiled-coil regions with unfolding of the terminal γ-nodules. The coiled-coils act as molecular springs to buffer external mechanical perturbations, transmitting and distributing force as the γ-nodules unfold. Unfolding of the γ-nodules, stabilized by strong inter-domain interactions with the neighboring β-nodules, was characterized by an average force of ∼90 pN and peak-to-peak distance of ∼25 nm. All-atom Molecular Dynamics simulations further showed a transition from α-helix to β-sheet at higher extensions. To reveal the force-induced α-helix to β-sheet transition in fibrin experimentally, we used Fourier Transform infrared spectroscopy of hydrated fibrin clots made from human blood plasma. When extended or compressed, fibrin showed a shift of absorbance intensity mainly in the amide I band but also in the amide II and III bands, demonstrating an increase of the β-sheets and a corresponding reduction of the α-helices. These structural conversions correlated directly with the strain or pressure and were partially reversible at the conditions applied. The spectra characteristic of the nascent inter-chain β-sheets were consistent with protein aggregation and fiber bundling during clot deformation observed using scanning electron microscopy. Additional information on the mechanically induced α-helix to β-sheet transition in fibrin was obtained from computational studies of the forced elongation of the entire fibrin molecule and its α-helical coiled-coil portions. We found that upon force application, the coiled-coils undergo ∼5–50 nm extension and 360-degree unwinding. The force-extension curves for the coiled-coils showed three distinct regimes: the linear elastic regime, the constant-force plastic regime, and the non-linear regime. In the linear regime, the coiled-coils unwind but not unfold. In the plastic regime, the triple α-helical segments rewind and re-unwind while undergoing a non-cooperative phase transition to form parallel β-sheets. We conclude that under extension and/or compression an α-helix to β-sheet conversion of the coiled-coils occurs in the fibrin clot as a part of forced protein unfolding. These regimes of forced elongation of fibrin provide important qualitative and quantitative characteristics of the molecular mechanisms underlying fibrin mechanical properties at the microscopic and macroscopic scales. Furthermore, these structural characteristics of the dynamic mechanical behavior of fibrin at the nanometer scale determine whether or not clots have the strength to stanch bleeding and if thrombi become obstructive or embolize. Finally, this knowledge of the functional significance of different domains of fibrin(ogen) suggests new approaches for modulation of these properties as potential therapeutic interventions. Disclosures:No relevant conflicts of interest to declare.

Fibrin gels and their clinical and bioengineering applications

Fibrin gels, prepared from fibrinogen and thrombin, the key proteins involved in blood clotting, were among the first biomaterials used to prevent bleeding and promote wound healing. The unique polymerization mechanism of fibrin, which allows control of gelation times and network architecture by variation in reaction conditions, allows formation of a wide array of soft substrates under physiological conditions. Fibrin gels have been extensively studied rheologically in part because their nonlinear elasticity, characterized by soft compliance at small strains and impressive stiffening to resist larger deformations, appears essential for their function as haemostatic plugs and as matrices for cell migration and wound healing. The filaments forming a fibrin network are among the softest in nature, allowing them to deform to large extents and stiffen but not break. The biochemical and mechanical properties of fibrin have recently been exploited in numerous studies that suggest its potential for applications in medicine and bioengineering.

The formation of tubular structures by endothelial cells is under the control of fibrinolysis and mechanical factors.

This study highlights the importance of several factors involved in the formation of capillary-like structure formation (CLS) using Human Umbilical Vein Endothelial Cells (HUVEC) and Bovine Retinal Endothelial Cells (BREC) cultured on fibrin gels. The fibrin concentration inducing (CLS) was 0.5 mg/ml for HUVEC and 8 mg/ml for BREC. The high fibrin concentration required for the latter cells appeared necessary to counterbalance the extensive fibrinolysis of the gel by the BREC. Fibrin degradation products measured in the culture media showed that fibrin degradation was mandatory but not sufficient for CLS formation. Fibrin degradation acted in concert with the mechanical, concentration dependent properties of the gels to induce CLS. For example, HUVEC did not form CLS on a rigid fibrin of 8 mg/ml in spite of fibrinolysis. As cell reorganisation occurred, the fibrin was disrupted (HUVEC) or pleated (BREC) giving indirect proof of the development of mechanical forces. During CLS formation, an increasing amount of latent TGFbeta1 was measured in the medium (1000-1700 pg/ml). The active form of TGFbeta1 was not, however, detected and the addition of anti-TGF-beta1 antibody to the medium did not influence the formation of the CLS network. Yet, added activated TGF-beta1 led to the formation of less organised structures, that were completely abolished by the concomitant addition of the same anti-TGF-beta1 antibody. Thus, it is likely that TGF-beta1 secreted by the endothelial cells remained in its latent form. In conclusion, a balance between the mechanical properties of fibrin and the fibrinolytic activity of each cell type may regulate CLS formation in our models. We think that the high fibrinolitic activity of the BREC may represent a defense mechanism to protect the retina against thrombosis-induced damage in vivo.