Preparation And Properties Of Silk Fibroin Microspheres For Cell Cultture

Preparation of polymer carriers has been the hot spot in tissue engineering and tissue regeneration field. Silk fibroin, a natural and fibrous protein, presents excellent physical and chemical properties, cell biocompatibility and tissue biocompatibility. Besides, silk fibroin is a useful polymer for preserving the activity of biological macromolecules. If the silk fibroin microspheres with controlled structure and release of activity components can be prepared by physical technologies that can reduce the pollution caused by chemical methods, it will provide a high-performance carrier for cell proliferation, growth factor delivery and tissue engineering. In this paper, we construct porous silk fibroin microspheres with controllable size by electrostatic spraying and freeze-drying method. The relationship between preparation conditions and microsphere structure was investigated. Additionally the characterion of silk fibroin microspheres used as cell carriers and growth factor delivery system was studied.The silk fibroin solution was divided into small droplets by electrostic field and frozen in the liquid nitrogen. Then the frozen microspheres were lyophilized for 1 day. The change of silk fibroin structure was promoted by ethanol treatment and adding glycerin into silk fibroin solution. The influences of solution concentration, voltage, flow rate and diameter of syringe needle on the morphology and size of the spheres were observed by SEM. The condensed structure of silk fibroin microspheres was studied by XRD and FT-IR spectra. The results indicated that the microsphere diameter increased with the increasing of silk fibroin solution concentration, flow rate and syringe needle diameter, while decreased as the voltage increased. The conformation of silk fibroin microspheres changed to silkⅡstructure after ethanol treatment, while there was mainly silkⅡstructure in the microspheres with a small amout of SilkⅠstructure after adding glycerin into silk fibroin solution. The adhesion, spreading, proliferation and morphology of L929 cells on silk fibroin microspheres with different diameters were observed by LSCM, SEM and MTT. The results revealed that L929 cells fully stretched on the microspheres, exhibiting a normal morphology, and the cell number increased with the prolongation of the culture time. Compared with the bigger microspheres, the cells showed better proliferation and viability when cultured on smaller microspheres due to the larger specific surface. In addition, the cells expansion rate on silk fibroin microspheres was significantly higher than on general tissue culture plate.Silk fibroin microspheres loading basic fibroblast growth factor (bFGF) were prepared by penetration-absorption, chemical-crosslinking and direct embedding methods. The loading, encapsulation efficiency and release rate of microspheres were analyzed by ELISA. The microspheres prepared by the three methods all showed 97% encapsulation efficiency. The release behavior of bFGF-microspheres prepared by absorption showed that most bFGF released within 4 h, while remaining 17% bFGF could released slowly in the period of 4-48 h. The microspheres prepared by chemical-crosslinking and direct embedding methods exhibited a relative slow release rate. Approximately 50% bFGF remained in microspheres after 13 days release. In vitro cell culture revealed that L929 cells could adhere and proliferate on bFGF-microspheres, cells on silk fibroin microspheres incorporating bFGF showed better proliferation, which indicated that silk fibroin microspheres were effective carriers for maintaining the bioactivities of bFGF.In this paper, we prepared porous silk fibroin microspheres by physical technologies, which could used as cell culture carriers and biological macromolecules delivery system. The silk fibroin microspheres with excellent properties have broad development and application prospects in biomedical field.