Fabrication of 3D-nanofibrous fibrinogen scaffolds using salt-induced self assembly

Fibrinogen has become highly attractive for tissue engineering scaffolds since it is a naturally occurring blood protein, which contains important binding sites to facilitate cell adhesion. Here, we introduce a novel biofabrication technique to prepare three-dimensional, nanofibrous fibrinogen scaffolds by salt-induced self assembly. For the first time, we were able to fabricate either free-standing or immobilized fibrinogen scaffolds on demand by tailoring the underlying substrate material and adding a fixation and washing procedure after the fiber assembly. Using scanning electron microscopy we observed that different buffers including phosphate buffered saline and sodium phosphate reproducibly yielded dense fiber networks on bare and silanized glass surfaces, gold as well as polystyrene upon drying. Fibrillogenesis could be induced with a fibrinogen concentration of at least 2 mg ml^-1 in a pH regime of 7-9. Fiber diameters ranged from 100 to 300 nm, thus resembling native fibrin and ECM protein fibers. By adjusting the salt concentration we could prepare fibrinogen scaffolds with overall dimensions in the centimeter range and a thickness of 3 to 5 μm. Using FTIR analysis we observed peak shifts of the amide bands for fibrinogen nanofibers in comparison to planar fibrinogen, which indicates changes in the secondary structure. Since fibrillogenesis was only induced upon drying when salt ions were present we assume that protein molecules were locally oriented in the respective buffers, which-in combination with the observed conformational changes-led to the assembly of individual molecules into fibers. In summary, our novel self assembly process offers a simple and well controllable method to prepare large scale 3D-scaffolds of fibrinogen nanofibers under physiological conditions. The unique possibility to chose between free-standing and immobilized scaffolds makes our novel biofabrication process highly attractive for the preparation of versatile tissue engineering scaffolds.