Fig 1.
Structural evaluation of the SF hydrogels after incubation in PBS at 37°C for 1, 3, 7, 10 and 14 days.
(a) Schematic illustration of the β-sheet structural transitions in the SF hydrogels. (b) Macroscopic images of the SF hydrogels (scale bar, 5 mm). (c) TEM micrographs of the SF hydrogels (scale bar, 500 nm for low magnification images; scale bar, 200 nm for high magnification image). (d) SF hydrogels labeled with thioflavin T (scale bar, 500 μm for low magnification images; scale bar, 100 μm for high magnification image) analyzed under the fluorescence microscope (scale bar, 200 μm). The black arrows indicate nanofibrils and the red arrows indicate aggregates. (e) OPT reconstructions of the SF hydrogels (scale bar, 500 μm). Movies from OPT reconstructions of the SF hydrogels are available in S1–S5 Movies.
Fig 2.
Physicochemical characterization of the SF hydrogel after incubation in PBS at 37°C for 3, 7 and 14 days.
(a) XRD patterns of the SF hydrogels. (b) ATR-FTIR spectra of the SF hydrogels. (c) Schematic illustration of nanoscale IR spectroscopy using AFM-IR: IR pulses are emitted in the sample increasing the local absorption on SF nanofibrils acquired by the AFM cantilever tip, corresponding to the absorption spectroscopic peaks. (d) Tapping-mode AFM nano-images (10 μm x 10 μm), and (e) IR nano-spectra of the SF hydrogels obtained by measuring the samples at different points selected by the AFM tip, corresponding to the absorption spectroscopic signatures and indicated as green, blue and red points.
Fig 3.
Rheological properties of the SF/HRP/H2O2 mixture before gelation, after SF hydrogels formation and incubation in PBS at 37°C for 1, 3, 7 and 14 days.
(a) Oscillatory experiments or frequency sweep curves: (i) storage modulus as a function of frequency, and (ii) loss modulus as a function of frequency for the SF hydrogels. (b) Dynamic moduli as function of: (i) temperature, and (ii) time for the SF/HRP/H2O2 mixture. (c) Rotational experiments: (i) shear viscosity, and (ii) shear stress as a function of shear rate for the SF/HRP/H2O2 mixture. (d) Macroscopic images of SF hydrogels analyzed at day 1 and day 7 (scale bar, 4 mm).
Table 1.
Damping factor of SF hydrogels after incubation in PBS at 37°C for 1, 3, 7, 10 and 14 days.
Fig 4.
U251 cell-laden SF hydrogels cultured for 1, 7, 10 and 14 days.
Cell viability and proliferation analyzed by (a) ATP assay and (b) DNA quantification, respectively. (c) Macroscopic images of the U251 cell-laden SF hydrogels (scale bar, 5 mm). (d) Live/Dead staining and fluorescence TUNEL assay of the U251 cell-laden SF hydrogels (scale bar, 200 μm). All the raw numerical data were provided in S1 and S2 Tables. **p < 0.01, ***p < 0.001.
Fig 5.
U251 cell-laden SF hydrogels cultured for 1, 7, 10 and 14 days.
(a) OPT projections (scale bar, 400 μm), OPT reconstructions (scale bar scale bar, 500 μm) and SPIM reconstructions (scale bar, 200 μm) of the U251 cell-laden SF hydrogels. Movies from OPT projections, OPT reconstructions and SPIM reconstructions of the cell-laden SF hydrogels are available in S6–S17 Movies. (b) Fluorescence TUNEL assay of the sections from the U251 cell-laden SF hydrogels for 1, 7, 10 and 14 days (scale bar, 50 μm). (c) Schematic illustration of the morphological changes of U251 cells during apoptosis induced by the conformational transitions of the cell-laden SF hydrogels.