Silk Scaffold Enzymatic Degradation

Silk scaffolds (n = 15) were exposed to protease XIV from Streptomyces griseus (Sigma–Aldrich, St. Louis, MO, USA) to monitor and compare enzymatic degradation profiles over the course of 21 days, as an in vitro model of scaffold degradation [47 (link),48 (link),49 (link)]. After recording the initial scaffold mass dry, scaffolds were plated in a 48-well plate and each submerged in 500 µL of 181 µg/mL (1 U/mL) protease XIV solution (prepared in sterile distilled water, with approximately 2% penicillin/streptomycin), with continuous incubation at 37 °C. The protease solution was changed daily for each scaffold. On three-day intervals, scaffolds were taken out of enzyme solution and rinsed 3 times in sterile distilled water before drying (8 h at 60 °C) to monitor changes in dry mass across measurement timepoints. Changes in dry mass for each scaffold were normalized to initial scaffold mass to compare percent reduction over the course of the study. Discarded protease solution was denatured by incubation for 20 min at 80 °C according to the manufacturer’s protocol.