*2.7. Evaluation of Cell Attachment and Proliferation on the Silk Hydrogels*

Hydrogels were prepared as mentioned above using autoclaved SF and UV-irradiated eADF4(C16) solutions, and the overall concentration of protein mixtures was fixed at 2% *w*/*v*. 100 μL of the protein mixtures were transferred to a tissue culture-treated 48-well pate and incubated at 37 ◦C under humidified atmosphere for at least 48 h to allow complete gelation. For pure SF solutions, gelation was accelerated using sonication at 40% amplitude for 30 s [19].

All hydrogels were hydrated with the complete media for 24 h prior to cell seeding. hASCs at a density of 5000 cell/cm2, L929 and SaOS-2 at a density of 10,000 cell/cm2 were seeded on the hydrogels. Seeding density was based on the proliferation profile of each cell, of which its logarithm growth phase should be achieved within 1 to 5 days. Cells cultured on the tissue culture-treated plate (TCP) were used as controls. The cell culture media, DMEM/F-12 + 10%FBS + 1% antibiotics and DMEM + 10%FBS + 1% antibiotics were used for culturing hASCs and L929 or SaOS-2, respectively, and the samples were stored in a CO2 incubator at 37 ◦C and 5% CO2 supplementation.

On day 1, 3, 5 and 7 after cell seeding, the cell proliferation was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Briefly, cells were washed with PBS, treated with 0.5 mg/mL MTT solution, and incubated at

37 ◦C in the dark for 30 min. After that, the MTT dye was removed and replaced with dimethyl sulfoxide (DMSO) to extract the precipitated formazan. The blue solution was then retrieved, and its absorbance at 570 nm was measured with a visible-light background correction at 650 nm.

Cell morphology was observed using a phase contrast imaging as well as fluorescent live-dead staining with calcein AM and propidium iodide (PI) dyes (Thermo Fisher Scientific, Waltham, MA, USA). At the designated time-points, cells were washed and stained with the fluorescent dyes. Bright-field and fluorescent images were obtained using a fluorescence microscope (Nikon Eclipse 80i, Nikon, Tokyo, Japan) with green and red filters to visualize calcein AM stained (lived) and PI stained (dead) cells, respectively.
