In Vitro Cell-Free Synthesis of Fibronectin Fibrils: Their Conformation and Effects on Platelet Function
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
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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. Keywords
Fibronectin
1
Fibronectin fibril
Introduction
Fibronectin (FN) is an adhesive glycoprotein which plays different critical roles in cellular adhesion, migration and growth in the processes of wound healing and tissue repair. Presence of FN in the fibrin matrix is critical for the initiation of proliferative phase of wound healing [1]. Previous
P. Le S.-N. Mai-Thu T.-H. Nguyen T. Vo Van K. Huynh (&) Biomedical Engineering Department, International University-National University HCMC, Quarter 6, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, Vietnam e-mail: [email protected]
Platelet adhesion
Tissue engineering
reviews conclude that FN, in the incorporation into fibrin matrix, contributes to the induction of platelet adhesion, migration and aggregation [1–3]. Studies shows cells failed to adhere, spread, and migrate into the FN-depleted blood clot [1, 4, 5]. Furthermore, it has been investigated that the reversal of this effect caused by FN depletion could only be achieved by adding of FN into plasma before clotting, but not afterwards [4]. These findings suggest that plasma FN, as it is synthesized and circulates in a soluble and compact form, is able to extend its conformation within the fibrin-FN clot in order that cryptic cell-binding domains become available for cell adhesion and repopulation [2, 5]. In late wound-healing process, both cellular and plasma FN are assembled into fibrillar matrix. The FN-matrix
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