*3.4. Protein Adsorptivity of the Hydrogels*

Adsorption of soluble proteins in SF:eADF4(C16) hydrogels was determined using FBS as a model (Figure 3C). For all samples, FBS can be rapidly adsorbed onto the hydrogels in the first hour of immersion before maintaining a plateau. The adsorption of the proteins in the SF hydrogel (10:0) was slower than in the others, but the identical protein level could be achieved within 6 h.

#### *3.5. Proliferation of Cells Cultured on the Silk Hydrogels*

Due to the very long gelation time, gelation of pure SF was accelerated using ultrasonication, and the 7:3 SF:eADF4(C16) hydrogel was omitted from the cell culture experiment. Cell proliferation was determined from MTT assay (Figure 4). For primary cells, such as hASC, the results showed no significant difference among all samples. Cells on TCP presented the growth phase in the first five days before plateauing on day 7 (population

doubling time (PDT) = 61.3 h), while those on the silk hydrogels showed low proliferation rates after 3 days (Figure 4A). The proliferation of two cell lines, L929 and SaOS-2 was depended on the sample composition, as samples with higher SF content showed enhanced cellular activities. L929 cells (PDT on TCP = 21.0 h) cultured on hydrogels containing SF (10:0, 5:5, 3:7 SF:eADF4(C16)) showed a similar behavior, while the growth of cells on the eADF4(C16) (0:10) hydrogel was significantly lower (Figure 4B). Similar results were noticed for SaOS-2 (PDT on TCP = 25.9 h) (Figure 4C). The cells on 2% SF hydrogels presented the highest growth rate, which was not significantly different from those on TCP for all time-points. The absorbance values were lower depending on an increasing eADF4(C16) content, with the lowest cell activity for 2% eADF4(C16).

**Figure 4.** Cell proliferation determined using the MTT assay of (**A**) human adipose-derived stromal cells (hASC), (**B**) mouse L929 fibroblasts, and (**C**) human SaOS-2 osteosarcoma cells cultured on 2% SF:eADF4(C16) hydrogels for 7 days. Cells cultured on tissue culture. The asterisk (\*) indicates the statistical difference at *p*-value ≤ 0.05.
