Structure, Properties and Degradation of Self-Assembled Fibrinogen Nanofiber Scaffolds

Self-assembled fibrinogen nanofibers are promising candidates for skin tissue engineering due to their biocompatibility and ability to mimic the native blood clot architecture. Here, we studied the structure–property relationship and degradation of rehydrated fibrinogen nanofibers prepared by salt-induced self-assembly, focusing on the effect of scaffold layering, cross-linking time and freeze-drying. Optimal fiber stability was achieved with cross-linking by formaldehyde (FA) vapor, while treatment with liquid aldehydes, genipin, EDC, and transglutaminase failed to preserve the nanofibrous architecture upon rehydration. Scaffold layering did not significantly influence the mechanical properties but changed the scaffold architecture, with bulk fiber scaffolds being more compact than layered scaffolds. Freeze-drying maintained the mechanical properties and interconnected pore network with average pore diameters around 20 μm, which will enhance the storage stability of self-assembled fibrinogen scaffolds. Varying cross-linking times altered the scaffold mechanics without affecting the swelling behavior, indicating that scaffold hydration can be controlled independently of the mechanical characteristics. Cross-linking times of 240 min increased scaffold stiffness and decreased elongation, while 30 min resulted in mechanical properties similar to native skin. Cross-linking for 120 min was found to reduce scaffold degradation by various enzymes in comparison to 60 min. Overall, after 35 days of incubation, plasmin and a combination of urokinase and plasminogen exhibited the strongest degradative effect, with nanofibers being more susceptible to enzymatic degradation than planar fibrinogen due to their higher specific surface area. Based on these results, self-assembled fibrinogen fiber scaffolds show great potential for future applications in soft tissue engineering that require controlled structure–function relationships and degradation characteristics.


S1: Preparation of nanofibrous fibrinogen scaffolds for mechanical testing
Table S1: Preparation of self-assembled fibrinogen (FG) nanofibers with varying amounts of layers and different protein amounts, yielding a final Fg:PBS ratio of 2:1 in all samples.To prepare fibrin (FN) scaffolds, different thrombin concentrations in PBS were added to fibrinogen.

S-3
Table S2: Thickness and cross-sectional area of wet fibrinogen (FG) and fibrin (FN) scaffolds with a dog-bone-shape prepared with varying cross-linking times and 15 mg protein were measured with a caliper after tensile testing and averaged for each sample type.All scaffolds had a threelayer design and an overall width of 8 mm.

Sample Type
Average

Figure S2: Molecular size of degradation products after short-term digestion of fibrinogen solution.
As a reference for long-term enzymatic fibrinogen scaffold degradation, the degradation of fibrinogen in solution was analyzed to evaluate the size of the degraded products.For this, 1 mg/ml fibrinogen in HEPES buffered saline was incubated at 37 °C overnight in the absence or presence of the respective enzymes and subsequently analyzed via SDS-PAGE.HEPES buffer control was additionally analyzed as a negative control (Buffer).
The molecular sizes of degraded products were compared against a protein standard.A prominent band between 460 kDa and 268 kDa, indicative of native fibrinogen protein having a molecular size of 340 kDa, was observed for control, plasminogen, urokinase, and thrombin treatments, whereas this band was slightly smaller (between 268 kDa and 171 kDa) in the presence of a combination of plasminogen and urokinase.The latter treatment also showed a smear of protein bands across the whole gel.The most effective digestion of soluble fibrinogen was observed after treatment with plasmin, where the native undigested fibrinogen band was replaced by a prominent band between molecular sizes of 117 kDa and 71 kDa as well as faint bands between 55 kDa and 41 kDa. S-5

Figure S1 :
Figure S1: Adjustable 3D-printed mobile phone holder.The holder was attached to the microscope eyepiece for thickness measurements of fibrinogen scaffolds.

Figure S3 :
Figure S3: SEM images of degraded planar fibrinogen scaffolds.Planar fibrinogen scaffolds were cross-linked with FA vapor for 120 min (A-G) or 60 min (H-N) and dried before incubation (untreated) or were incubated for 35 days in HEPES buffer (control) or HEPES buffer containing the respective enzymes, before being subjected to SEM imaging.The smooth topography of the scaffolds appeared to be unchanged even after 35 days of incubation in the absence or presence of different enzymes in comparison to untreated planar scaffolds without any prior treatment in an aqueous environment.

Table S3 :
Thickness measurements of rehydrated bulk scaffolds containing 15 mg fibrinogen that were cross-linked for 60 min or 120 min.Rectangular scaffolds were 16 mm x 32 mm in size and measured either with a caliper or under an optical microscope.

Table S4 :
Thickness and cross-sectional area of wet fibrinogen (FG) and fibrin (FN) scaffolds with dog-bone-shape prepared with varying layer number and protein amount were measured with a caliper after tensile testing and averaged for each sample type.All scaffolds were cross-linked for 120 min and had a width of 8 mm.