*3.2. Micromorphology of the Freeze-Dried Hydrogels*

The microstructures of freeze-dried hydrogels were visualized using FESEM (Figure 2). The microstructures of all samples presented a high porosity with a ridge- or wall-like structure, and the fracture surfaces displayed an accumulation of nanofibers.

#### *3.3. Secondary Structures of the Hydrogels*

Figure 3A,B display the FTIR spectra and the secondary structure content of the freeze-dried SF:eADF4(C16) hydrogels, respectively. Comparing in sol and gel state, the peak shift from approximately 1650 cm−<sup>1</sup> of the sol groups toward a lower wavenumber (1625 cm−1) of the gel group can be noticed, indicating a transition of the predominated random coil structure in the sol state to a beta sheet structure after gelation. The results were in accordance with the amount of secondary structures quantified using FSD and curve-fitting. A reduction in random coil and an increase of beta sheet structure could be clearly observed, especially in samples containing SF. The samples with higher eADF4(C16) content possessed a higher beta sheet and lower random coil content in the sol state, which slightly changed after gelation.

**Figure 1.** Gelation time of (**A**) 2% and (**B**) 3% *w*/*v* of SF:eADF4(C16) solutions with different ratios as indicated and upon addition of Dulbecco s Modified Eagle s Medium (DMEM), phosphate buffer saline (PBS) and normal saline solution (NSS) at 37 ◦C. "Plain" refers to SF:eADF4(C16) blends in the absence of salts. The gelation time was interpreted from the time-point at which the absorbance values reached the half-maxima. The experimental time was limited to 40 h. The ratio indicates the volume ratio of the respective 2% and 3% protein solutions.

**Figure 2.** FESEM images of freeze-dried hydrogels. The number on the left indicates the SF:eADF4(C16) volume ratio. The scale bars of 500× and 15 kx magnification are 10 and 1 μm, respectively.

**Figure 3.** Physico-chemical properties of the SF:eADF4(C16). (**A**) FTIR spectra of freeze-dried hydrogels, (**B**) The content of protein conformation, quantified from the amide I region of FTIR spectra, and (**C**) Adsorption of proteins in the hydrogels after immersion in 10% FBS. NS: nonsignificant difference.
