In vitro cell-free synthesis of fibronectin fibrils: Their conformation and effects on platelet function

Abstract

In insoluble fibrillar form, fibronectin exerts its most significant biological activities. Inducing assembly of fibrillar FN on scaffold prior to cell seeding provides controllable early interactions between cells and the biomaterial. This study aimed to synthesize fibronectin fibrils under cell-free conditions, evaluate the conformation of induced fibrils and their effect on platelet adhesion. To induce fibrillogenesis, purified fibronectin were dialyzed against urea 2 M in 16 h followed by dialysis against PBS pH 7.3. Microscopic images revealed that urea induced heterogeneous formation of fibronectin fibrils with various morphologies ranging from aggregation form to fibrillar form. Fibrillar fibronectin with diameter in range of 30–130 µm tend to assembly into matrix, whereas fibronectin aggregates with size around 50 µm suspend in solution. Our data showed that among 4 tested concentrations, 1 mg/ml, as it yields the most aggregated fibronectin and fibrillar matrix, is the optimal condition for fibronectin polymerization. At lower concentrations, less fibronectin fibrils were formed and they did not link together to form a matrix. Platelet adhesion assay indicated a stronger platelet adhesion on surfaces coated with fibronectin fibrils than those with untreated fibronectin. Adherent platelets gathered into small groups around aggregated fibronectin and separate fibrils while on fibrillar matrix, they adhere into networks of larger clusters. Denaturation of plasma fibronectin by urea induces irreversible heterogeneous formation of fibronectin fibrils and improves platelet adhesion.