The molds were
sealed and kept at a controlled temperature in a thermostatic bath at 37 or 50 C until hydrogel formation. Gelation time was determined when the sample showed an opaque white color and did not flow when the mold was inverted for 30 s [28,29]. Part of these hydrogels was frozen at 20 C for 24 h to evaluate differences in the properties of non-frozen and frozen hydrogels. These hydrogels were identified with the letter F. Moreover, since fibroin is a natural polymer, the gelation time was evaluated from SF solutions prepared in different days to evaluate the reproducibility of the materials and to find a highly reproducible method for the formation of hydrogels. The data reported for gelation time represent the average of
ten replicates for each temperature.