In Vitro Cell-Free Synthesis of Fibroneetin Fibrils: Their Conformation and Effects on Platelet Function

摘要

In insoluble fibrillar form, fibroneetin 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 fibroneetin fibrils under cell-free conditions, evaluate the conformation of induced fibrils and their effect on platelet adhesion. To induce fibrillogenesis, purified fibroneetin 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 fibroneetin fibrils with various morphologies ranging from aggregation form to fibrillar form. Fibrillar fibroneetin with diameter in range of 30-130 um tend to assembly into matrix, whereas fibroneetin aggregates with size around 50 um suspend in solution. Our data showed that among 4 tested concentrations, 1 mg/ml, as it yields the most aggregated fibroneetin and fibrillar matrix, is the optimal condition for fibroneetin polymerization. At lower concentrations, less fibroneetin 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 fibroneetin fibrils than those with untreated fibroneetin. Adherent platelets gathered into small groups around aggregated fibroneetin and separate fibrils while on fibrillar matrix, they adhere into networks of larger clusters. Denaturation of plasma fibroneetin by urea induces irreversible heterogeneous formation of fibroneetin fibrils and improves platelet adhesion.