<scp>I</scp>mpact of Sterilization on the Enzymatic Degradation and Mechanical Properties of Silk Biomaterials

The effect of some sterilization methods (autoclaving and ethanol treatments) on the degradation rate and mechanical properties of two types of porous silk scaffolds (aqueous- and hexafluoroisopropanol-derived) is evaluated. Changes in secondary structure, crystal size, and supramolecular features of silk fibroin, resulting from sterilization, are tracked to elucidate molecular level effects on protease XIV enzymatic degradation and compressive mechanical properties. The structural features and pore sizes of the silk scaffolds remain intact after both sterilization processes. Autoclave sterilization dramatically reduce the degradation rate of the silk scaffolds in response to protease XIV and significantly increase mechanical properties, in contrast to scaffolds sterilized with 70% ethanol. Higher β-sheet content and larger crystal size are observed after autoclaving, unlike in response to 70% ethanol sterilization, based on examination of Fourier transform (FT) IR spectroscopy and wide-angle X-ray scattering (WAXS). In addition, thermal analysis finds supramolecular features within silk fibroin amorphous regions, including the glass transition temperature (Tg ), heat capacity of glass transition (ΔCp-Tg ), and thermal gravimetric degradability. Such supramolecular level changes are related to the shift in enzymatic degradation and mechanical properties due to autoclaving versus treatment with 70% EtOH. The changes in supramolecular organization in amorphous regions can retard enzyme diffusion through the glassy regions of the silk matrix or/and hinder binding of enzymes, while also stiffening these matrices.