THREE-DIMENSIONAL SCAFFOLDS MADE OF REGENERATED SILK FIBROIN PREPARED BY FREEZE DRYING

Regenerated silk fibroin (SF) produced by Bombyx mori exhibits mechanical properties and biocompatibility suitable to be used as starting material in tissue engineering. To guarantee satisfactory robustness of scaffolds, SF is required to assume the native β-sheet conformation (Silk II) which is generally induced by several treatments, such as heating, immersion in polar organic solvents, shearing and blending with natural or synthetic polymers. Three-dimensional SF scaffolds are prepare by freeze-drying. This work aimed to evaluate the impact of formulation and freezing conditions on molecular conformation of SF, mechanical stability and morphology of scaffolds. The effects of SF solution concentrations ranging from 1.3 to 4.0 % (w/w) on the mechanical properties of scaffolds imbibed in pH 7.4 PBS were also investigated. Small amounts of tert-butyl alcohol (TBA) and dimethyl sulfoxide (DMSO) were also added since these solvents are commonly used to speed-up the lyophilization process. Moreover, the impact of sterilization by water vapor under pressure on fibroin conformation and mechanical properties of scaffolds was also evaluated. Scaffolds were prepared from SF solutions frozen at -20 °C or -40 °C for a minimum of 8 hours to obtain suitable pore interconnectivity. Varying the process conditions, either SF fibers were highly oriented perpendicular to the horizontal surface, or randomly oriented within the scaffold structures, or a combination thereof. Removal of water through lyophilization had a negligible impact on the transition towards the β-sheet structure since the amide absorbance bands in the ATR-FTIR spectra were centered at the typical wavelengths of the random coil structure. The addition of 1% (w/w) TBA or DMSO permitted to raise the β-sheet fraction from 28.5% to 31% and 33%, respectively. Porosity was about 85 % and well-preserved after steam sterilization at 121 °C for 15 min. A significant increase in β-sheet content was induced by sterilization (34%). The scaffolds resistance to compression was dependent on the SF solution concentrations ranging from 0.1 N to 0.5 N. When SF solution concentration was higher than 1.3% (w/w), the values remained constant over a 2-week period. In conclusion, the freezing step resulted critical in determining the resistance to sterilization and the selected process conditions allowed preparing scaffolds made of SF having suitable properties for tissue engineering.