Study On Water-insoluble Silk Scaffolds With Lower Crystallization And Their Biocompatibility In Vitro

Silk is a promising biomaterial for tissue engineering because of its biocompatibility, unique mechanical properties, ease of chemical modification and biodegradability. However, following their increasing applications, there remains a demand to further improve the bioactivity of silk scaffolds. Achieving the designed structure and performance of scaffolds suitable for various tissues is becoming one of the most challenges.Freeze-drying is a common way in preparing silk scaffolds, but it often results in separate lamellar structure. In this study, silk nanofibers were firstly regulated in aqueous solution via a slowly increasing concentration process to facilitate porous structure formation of silk in lyophilization process. It was found that improved porous structures were achieved from the nanofiber solution. Glycerol was added to the silk nanofiber solution at different silk/glycerol weight ratios to obtain water-insoluble scaffolds directly. The insoluble silk scaffolds with reduced silk II content and different mechanical properties were prepared since glycerol could tune the self-assembly of silk in freeze-dried process.Then different ways were used to further tune silk self-assembly in an all-aqueous process for water-insoluble scaffold fabrication. Through culturing silk solution at higher temperature or freezing-thawing the solution repeatedly, we achieved insoluble scaffolds with lower silk II structure. The secondary structures, degradation behaviors as well as mechanical properties could be changed through adjusting the conditions.Finally, the rat bone marrow mesenchymal stem cells(r BMSC) were cultured on these scaffolds to reveal the influence of the different scaffolds on cell growth and differentiation. It was found that r BMSCs grew well on all the scaffolds. The r BMSCs had various differentiation capability on the scaffolds with different stiffness. The stem cells differentiated into muscle cells on the silk scaffolds with stiffness between 8k Pa and 17 k Pa, but into endothelial cells on the scaffolds with stiffness between 1k Pa and 7k Pa.Therefore, the present study provided an effective process to prepare silk scaffolds with tunable mechanical properties and secondary structures. These scaffolds with tuned properties would be promising platforms for studying the interaction between stem cell and biomaterials, and offer valuable matrices for different soft tissue regeneration.