Cell culture in autologous fibrin scaffolds for applications in tissue engineering

In tissue engineering techniques, three-dimensional scaffolds are needed to adjust and guide cell growth and to allow tissue regeneration. The scaffold must be biocompatible, biodegradable and must benefit the interactions between cells and biomaterial. Some natural biomaterials such as fibrin provide a structure similar to the native extracellular matrix containing the cells. Fibrin was first used as a sealant based on pools of commercial fibrinogen. However, the high risk of viral transmission of these pools led to the development of techniques of viral inactivation and elimination and the use of autologous fibrins. In recent decades, fibrin has been used as a release system and three-dimensional scaffold for cell culture. Fibrin scaffolds have been widely used for the culture of different types of cells, and have found several applications in tissue engineering. The structure and development of scaffolds is a key point for cell culture because scaffolds of autologous fibrin offer an important alternative due to their low fibrinogen concentrations, which are more suitable for cell growth.     

Treatment of long tubular bone defect of rabbit using autologous cultured osteoblasts mixed with fibrin

The osteogenic potential of autologous cultured osteoblasts mixed with fibrin when transplanted to bone defects was evaluated. Radial shaft defects over 15 mm were made in 30 New Zealand white rabbits. A total of 15 rabbits in the control group underwent an iliac bone graft and 15 rabbits in the experimental group underwent an autologous cultured osteoblast injection mixed with fibrin. Both groups were compared radiologically and 5 rabbits in each group were sacrificed for histological evaluation using H-E and Masson’s trichrome stain at 3, 6, and 9 weeks. Osteogenesis in the control group progressed more rapidly than in the experimental group. However, at 9 weeks, bone formation in both groups were similar and showed no significant difference in terms of the amount of bone formation and the quality of bone union. Autologous cultured osteoblast transplantation mixed with fibrin in bone defects was found to produce bone efficiently.     

Applied tissue engineering in the closure of severe burns and chronic wounds using cultured human autologous keratinocytes in a natural fibrin matrix

Whereas in severe burns cultured human epithelial cells may well serve as a life saving method, the true value of tissue-engineered skin products in chronic wound care has yet to be clearly defined. Among other well-known clinical problems, the engraftment rate of commercially available multilayered "sheet grafts" has been shown to vary extremely. Adherence of transplanted cells to the wound bed--especially in the presence of potential wound contamination-- is one of the crucial aspects of this technique. Keratinocyte suspensions in a natural fibrin sealant matrix can potentially treat a variety of skin defects. In acute burn wounds, as well as in chronic wounds the clinical application of this type of tissue-engineered skin substitute demonstrates the capacity of cultured human autologous keratinocytes in a fibrin sealant matrix to adhere to wound beds, attach and spread over the wound resulting in reepithelialization of both acute and chronic wounds. In full thickness burns the combination of this new tool with allogenic dermis is a promising option to achieve complete dermal-epidermal reconstitution by means of tissue engineering and guided tissue repair. When transferring this technique into the treatment of chronic wounds we found an optimal preparation of such recipient wound beds to be crucial to the success. The additional application of continuous negative pressure (vacuum therapy) and preliminary chip skin grafting to optimally prepare the recipient site may be helpful tools to achieve such well-prepared and graftable surfaces. Prospective controlled comparative studies should be designed to further assess the clinical efficacy of this technique.     

Human endothelial cells produce a plasminogen activator inhibitor and a tissue-type plasminogen activator-inhibitor complex

Serum-free conditioned media and cell extracts from cultured human umbilical vein endothelial cells were analyzed for plasminogen activator by SDS-polyacrylamide gel electrophoresis and enzymography on fibrin-indicator gels. Active bands of free and complexed tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA) were identified by the incorporation of specific antibodies against, respectively, t-PA or u-PA in the indicator gel. The endothelial cells predominantly released a high-molecular-weight t-PA (95000–135000). This t-PA form was converted to M_r-72000 t-PA by 1.5 M NH₄OH/39 mM SDS. A component with high affinity for both t-PA and u-PA could be demonstrated in serum-free conditioned medium and endothelial cell extract. The complex between this component and M_r-72000 t-PA comigrated with high-molecular-weight t-PA. From the increase in M_r of t-PA or u-PA upon complex formation, the M_r of the endothelial cell component was estimated to be 50000–70000. The reaction between t-PA or u-PA and the plasminogen activator-binding component was blocked by 5 mM p-aminobenzamidine, while the complexes, once formed, could be cleaved by 1.5 M NH₄OH/39 mM SDS. These observations indicated that the active center of plasminogen activator was involed in the complex formation. It was further noted that serum-free conditioned medium of endothelial cell extract inhibited plasminogen activator activity when assayed by the fibrin-plate method. Evidence is provided that the plasminogen activator-binding component was different from a number of the known plasma serine proteinase inhibitors, the placenta inhibitor and the fibroblast surface protein, proteinase-nexin. We conclude that cultured endothelial cells produce a rapid inhibitor of u-PA and t-PA as well as a t-PA-inhibitor complex.     

Functional impact of oxidative posttranslational modifications on fibrinogen and fibrin clots

Fibrinogen is a circulating multifunctional plasma protein vital for hemostasis. Activation of the coagulation cascade converts soluble fibrinogen to insoluble polymerized fibrin, which, along with platelets, forms the hemostatic clot. However, inappropriate formation of fibrin clots may result in arterial and venous thrombotic disorders that may progress to life-threatening adverse events. Often thrombotic disorders are associated with inflammation and the production of oxidants. Fibrinogen represents a potential target for oxidants, and several oxidative posttranslational modifications that influence fibrinogen structure and function have been associated with disease pathogenesis. Here, we review various oxidative modifications of fibrinogen and the consequences of these modifications on protein structure and the ability to form fibrin and how the resulting alterations affect fibrin architecture and viscoelastic and biochemical properties that may contribute to disease.     

Influence of glyco-oxidation on complexes between fibrin(ogen) and insulin-like growth factor-binding protein-1 in patients with diabetes mellitus type 2

Fibrinogen and insulin-like growth factor-binding protein-1 (IGFBP-1) are tightly connected to metabolic changes and complications in patients with diabetes mellitus (DM), and since they mutually interact to form complexes in plasma, we investigated whether and to what extent IGFBP-1/fibrinogen complexes change due to glyco-oxidative processes in DM and whether they participate in fibrin clot formation. These complexes were determined by immunoblotting in plasma samples from healthy adults and patients with DM type 2 (DM2). The influence of glyco-oxidation in vitro on the complexes was also investigated. Amounts of IGFBP-1/fibrinogen complexes in plasma from patients with DM2 were slightly but not significantly lower than in healthy persons. Such complexes in patients’ samples participated in fibrin clot formation to a significantly decreased extent. In vitro experiments with glucose or methylglyoxal (MGO) as reactive agents demonstrated that the complexes underwent glyco-oxidative modification leading to reduced formation and/or stability. Extensively oxidized fibrinogen almost completely lost its ability to bind IGFBP-1. The reduced affinity of fibrinogen for IGFBP-1 accompanying diabetes may potentially shift the equilibrium to liberate more IGFBP-1 (and possibly insulin-like growth factor (IGF)-I) able to activate platelets during coagulation, so contributing to the hypercoagulation state together with other factors. This hypothesis, however, needs further examination.     

Regulating tension in three-dimensional culture environments

The processes of development, repair, and remodeling of virtually all tissues and organs, are dependent upon mechanical signals including external loading, cell-generated tension, and tissue stiffness. Over the past few decades, much has been learned about mechanotransduction pathways in specialized two-dimensional culture systems; however, it has also become clear that cells behave very differently in two- and three-dimensional (3D) environments. Three-dimensional in vitro models bring the ability to simulate the in vivo matrix environment and the complexity of cell–matrix interactions together. In this review, we describe the role of tension in regulating cell behavior in three-dimensional collagen and fibrin matrices with a focus on the effective use of global boundary conditions to modulate the tension generated by populations of cells acting in concert. The ability to control and measure the tension in these 3D culture systems has the potential to increase our understanding of mechanobiology and facilitate development of new ways to treat diseased tissues and to direct cell fate in regenerative medicine and tissue engineering applications.     

Aggregation of fibrinogen molecules by metal ions

The ability of metal ions to cause physical aggregation of neutral solutions of bovine fibrinogen has been studied. Three categories were found: (a) ions (such as Ca²⁺, Mg²⁺ and Mn²⁺) which did not cause aggregation even when present in 1–100 mm concentrations: (b) ions (such as Fe²⁺, Cu²⁺ and Ni²⁺) which caused aggregation in the 0–10 mm concentration range, (c) ions (such as Hg²⁺, Zn²⁺, Cr³⁺, La³⁺) which caused aggregation in the 0–1000 μm concentration range. Aggregation occurs immediately the metal ion is brought into contact with the fibrinogen, and product formation reaches a steady state within 5 min. With the exception of Zn²⁺, all the ions that caused aggregation exhibited a threshold concentration below which no observable aggregation took place. The threshold concentration for Hg²⁺, the most effective ion studied, was 6 μm. Addition of excess EDTA caused resolubilization of the aggregated fibrinogen due to removal of the metal ions. Aggregation is thus thought to be a physical process initiated by binding of metal ions to those carboxyl groups in fibrinogen responsible for keeping the monomers apart in solution. The aggregation does not involve prior proteolytic degradation of the fibrinogen.     

Characterization of Leukocyte-platelet Rich Fibrin,A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.     

Batroxobin Binds Fibrin with Higher Affinity and Promotes Clot Expansion to a Greater Extent than Thrombin

Batroxobin is a thrombin-like serine protease from the venom of Bothrops atrox moojeni that clots fibrinogen. In contrast to thrombin, which releases fibrinopeptide A and B from the NH₂-terminal domains of the Aα- and Bβ-chains of fibrinogen, respectively, batroxobin only releases fibrinopeptide A. Because the mechanism responsible for these differences is unknown, we compared the interactions of batroxobin and thrombin with the predominant γ_A/γ_A isoform of fibrin(ogen) and the γ_A/γ′ variant with an extended γ-chain. Thrombin binds to the γ′-chain and forms a higher affinity interaction with γ_A/γ′-fibrin(ogen) than γ_A/γ_A-fibrin(ogen). In contrast, batroxobin binds both fibrin(ogen) isoforms with similar high affinity (K_d values of about 0.5 μm) even though it does not interact with the γ′-chain. The batroxobin-binding sites on fibrin(ogen) only partially overlap with those of thrombin because thrombin attenuates, but does not abrogate, the interaction of γ_A/γ_A-fibrinogen with batroxobin. Furthermore, although both thrombin and batroxobin bind to the central E-region of fibrinogen with a K_d value of 2–5 μm, the α(17–51) and Bβ(1–42) regions bind thrombin but not batroxobin. Once bound to fibrin, the capacity of batroxobin to promote fibrin accretion is 18-fold greater than that of thrombin, a finding that may explain the microvascular thrombosis that complicates envenomation by B. atrox moojeni. Therefore, batroxobin binds fibrin(ogen) in a manner distinct from thrombin, which may contribute to its higher affinity interaction, selective fibrinopeptide A release, and prothrombotic properties.