Influence of the polymerization method on the properties of fibrin matrices

Abstract

Highlights. We have proposed and patented a method for obtaining fully autologous fibrin without the use of exogenous thrombin, which can be implemented within the framework of a personalized approach in tissue engineering. Such fibrin, in addition to lower risk of infection and inflammation, possesses greater strength and resistance to degradation, as well as better hemocompatibility compared to fibrin polymerized with exogenous thrombin, which is an undoubted advantage when used as a coating for small-diameter vascular grafts.Background. Autologous fibrin can be used as a coating to impart biomimetic properties to various polymers used in vascular tissue engineering. Traditionally, fibrinogen polymerization is performed with the addition of exogenous thrombin and calcium chloride. Our patented method of obtaining fibrin without the use of exogenous thrombin allows us to obtain a completely autologous material that does not have the risk of infection. The differences in methods polymerization can change the fibrin properties.Aim. To compare the most important properties of vascular tissue  engineering  of fibrin obtained by various methods: using endogenous thrombin and exogenous thrombin in vitro.Methods. The fibrinogen precipitate was obtained with ethanol  precipitation  method  using low concentration of ethanol. The content of fibrinogen in the precipitate was normalized to a final concentration in fibrin of 30 mg/mL. Polymerization  of fibrin polymerized with exogenous thrombin was performed by adding 50 U/ mL and 0.2% calcium chloride to the thrombin precipitate, fibrin polymerized  by activation of endogenous thrombin only by adding 0.2% calcium chloride. The strength properties of the samples were tested on a Z tensile tester (Zwick/ Roell). The structure of fibrin was studied using scanning electron microscopy,   a quantitative analysis of the size of pores and fibers, the density of branching points was carried out. The quantitative content of FXIII in plasma and the resulting precipitate was determined by ELISA. FXIII activity was studied by the formation of γ-γ crosslinks by SDS-PAGE, the resistance of samples to proteolytic and fibrinolytic degradation. The ability of fibrin polymerized by activation of endogenous thrombin and fibrin polymerized with exogenous thrombin to activate contact coagulation and platelet aggregation was evaluated.Results. Thinner fibers predominated in the structure of the fibrin polymerized by activation of endogenous thrombin samples, while the samples were distinguished by greater strength and stiffness. The described features may be associated with  a more efficient activation of FXIII, which is confirmed by the formation of a larger number of γ-γ dimers in fibrin polymerized by activation of endogenous thrombin samples, as well as resistance to proteolytic degradation compared to fibrin polymerized with exogenous thrombin. Moreover, fibrin polymerized by activation of endogenous thrombin samples in vitro activated platelets less than fibrin polymerized with exogenous thrombin.Conclusion. The method for obtaining and polymerizing fibrin using endogenous thrombin makes it possible to obtain a completely autologous material that has better physical and mechanical properties, resistance to proteolytic degradation and lower thrombogenicity compared to traditional fibrin polymerization.