Self-assembled fibrinogen nanofibers: a multiscale scaffold platform for wound healing
As a key player in haemostasis and wound healing the blood plasma protein fibrinogen is an ideal building block for tissue engineering scaffolds. To mimic the native blood clot nanoarchitecture it can be assembled, extruded or electrospun into nanofibers. These methods often have a low nanofiber yield, which limits the production of 3D scaffolds, or they induce changes in protein conformation, often associated with pathogenic amyloid formation. Hence, we recently established a new process where salt in combination with controlled drying induces fibrinogen nanofiber assembly. This in vitro method yields either free-standing or immobilized scaffolds with overall dimensions in the centimeter range [1-2].
Nanofibrous fibrinogen was assembled in the presence of PBS solution, and planar fibrinogen films were prepared in NH4HCO3 buffer. Scanning electron microscopy analysis revealed dense networks of fibrinogen nanofibers in the presence of PBS with fiber diameters between 200 and 300 nm. By combining circular dichroism and Fourier-transform infrared spectroscopy we found changes in the secondary structure. During transition from planar to nanofibrous fibrinogen we observed partial transitions from a-helices to b-strands (see Fig. 1). Moreover, fluorescence staining with thioflavin T revealed that the observed conformational changes were not associated with any pathogenic amyloid formation. Toward novel scaffolds for wound healing, which are stable in aqueous environment, we introduced scaffold cross-linking in formaldehyde vapor. This treatment allowed us to maintain the nanofibrous morphology while the conformation of fibrinogen nanofibers was redeveloped toward a more native state after rehydration.
When we studied the mechanical characteristics of wet, free-standing fibrinogen scaffolds with tensile testing, nanofibrous scaffolds were found to exhibit a Young’s modulus in the range of native skin. In subsequent biocompatibility studies with 3T3 fibroblasts we observed very good proliferation on both, nanofibrous and planar, fibrinogen scaffolds. No significant differences in cell viability were found on the different fibrinogen scaffolds after 72 hours.
In summary, self-assembled fibrinogen scaffolds provide a multiscale biomaterial platform, which can be controlled from the molecular level via the nanomorphology to the macroscale. With these features, self-assembled fibrinogen nanofibers are highly attractive for future applications in wound healing.