Different Conformations of Fibronectin Fibrils Formed Under Cell-Free Condition in Vitro display Distinct Effects on Cell Adhesion and Spreading

Introduction: Insoluble fibrils are the active form of fibronectin (FN) with exposed functional domains capable of interacting with a variety of biomolecules and cells to support many cellular processes. Recent studies show that the formation of FN fibrils can occur in cell-free systems. Currently, the characteristic of FN fibrils and their potential in wound healing application are not fully understood. Our project aims at (i) using urea to induce the formation of FN fibrils with different conformations and (ii) to characterize their conformation-dependent effect on cellular adhesion and spreading.

Method: To induce FN fibrillogenesis, native plasma FN at various initial concentrations (0.25, 0.5, 0.75 and 1 mg/ml) was treated by dialysis in urea at concentrations of 1, 2, and 4 M for 16h at room temperature followed by dialysis in PBS buffer (to remove urea). Morphological analysis of formed FN fibrils was conducted bylight microscopy and scanning electron microscopy (SEM). To assess biological effects of FN fibrils on cells in relationto their morphologies, platelet and fibroblast cell adhesion assays were performed. Specifically, platelet rich plasma (5x10⁷ platelets/ml) or fibroblasts (L929, 10⁴ cells/ml) were incubated on surfaces pre-coated with 0.25 and 0.75 mg/ml native plasma FN or FN fibrils for 30 min at 37⁰C.

Results: Our cell-free FN fibrillogenesis experiments in vitro indicated that the optimal concentrations of FN and urea were 1 mg/ml and 2 M, respectively, to induce formation of FN fibrils with various conformations. FN only formed minuscule fibrils at 1 M urea while there was no fibril formation at 4 M urea. In contrast, FN fibrils with various conformations ranging from fibrillar FN to non-fibrillar FN (i.e., nodular FN aggregates) were formed when using 2 M urea. Fibril formation was shown to be dependent on the FN concentration. FN at low concentrations (0.25 or 0.5 mg/ml) formed a few insoluble minuscule fibrils or nodular FN aggregates with variation in size ranging from 10 to 50 µm. FN at higher concentrations (0.75 or 1 mg/ml) formed much more nodular FN and fibrillar FN with diameters mostly ranging from 10 to 30 µm that tended to interlace into the fibrillar matrix. SEM analysis documented morphological variations of fibrils formed under different conditions. For example, at low FN concentration (0.25 mg/ml) small fibrils with a smooth surface were formed. By contrast, larger fibrils with attached nodular FN and a resulting rough surface were formed at high FN concentration (≥ 0.75 mg/ml). Adhesion assays indicated that platelets adhered approximately 6 times more firmly on FN fibrils than on native plasma FN (n=3, p < 0.05).On immobilized native FN, adherent platelets were evenly distributed or formed small clusters, while fibrillar FN induced platelet accumulation on the matrix. Fibroblasts adhered both on non-fibrillar FN and matrix of small fibrils (diameter in range of 10-30 µm) but did not interact to larger fibrillar bundles (diameter > 292 µm). Investigations of FN-cell interaction by SEM revealed a structure-dependent effect of FN on fibroblast morphology after adhesion. Specifically, cells remained in spherical shape and separately adhered on nodular FN while extending their shape in multiple directions on the rough surface of FN fibrils.

Conclusion: Treatment of native plasma FN by urea at a concentration of 2 M induces irreversible formation of heterogeneous FN fibrils, displaying a rough surface due to attachment of FN nodules. The conformational and compositional changes of the FN matrix can lead to firm adhesion of platelets and fibroblasts and may thus provide a future tool to improve wound healing upon local application.