Three-dimensional scaffolds made of regenerated silk fibroin prepared by freeze drying: effects of process parameters

Regenerated silk fibroin (SF) produced by Bombyx mori finds application in tissue engineering as potential starting material for the preparation of scaffolds when mechanically robust and long-term degradable biocompatible polymer is required. In order to guarantee satisfactory mechanical properties, it is necessary that SF assumes the native β-sheet structure 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 relatively simple to prepare by freeze-drying. However, there are few reports focused on the effects of process parameters in molecular conformation of SF and features of scaffolds. The impact of freezing parameters, namely temperature and duration, on molecular conformation of SF, mechanical stability and morphology of scaffolds was studied. The effect of addition of small amount of tBuOH and DMSO, was also studied since these solvents are commonly used to speed-up the lyophilization process. Moreover, low molecular-weight poly(ethylene glycol) (PEG600) was also blended in the ratio 5/95 % w/w to investigate the possible transition to Silk II structure. Furthermore, since the scaffold has to be sterile, the impact of water vapor under pressure treatment on fibroin conformation and mechanical properties was also evaluated. Scaffolds prepared from 3.2% (w/w) SF solutions frozen at -20 °C or -40 °C appeared white cylinders. 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 tBuOH or DMSO permitted to raise the β-sheet fraction from 28.5% to 31% and 33%, respectively. To avoid collapsing of construct after sterilization, the freezing step was carried out for a minimum of 8 hours at -20 °C and the suitable pore interconnectivity of the obtain scaffolds was demonstrated by SEM. Porosity was about 85 % and well-preserved after steam sterilization at 121 °C for 15 min. After sterilization, a significant increase in β-sheet content was observed. By adding 5% PEG600, the resultant scaffolds presented a random coil conformation and the β-sheet content inducted by sterilization was slightly higher than that of the pure SF scaffold (36.5% vs 34%). The resistance to compression of SF/PEG600 scaffolds imbibed in pH 7.4 PBS resulted three time lower than that of pure SF scaffolds and remained constant over a three-week period. Moreover, the presence of PEG600 reduced the bust-effect release of a model protein, namely ovalbumin. In conclusion, the freezing step resulted critical in determination the resistance to sterilization and the selected process conditions allowed preparing scaffolds made of SF having suitable properties for tissue engineering. The presence of small amount of low molecular-weight poly(ethylene glycol) 600 tuned up the features of SF matrices in terms of mechanical strength and drug release.