The Study On The Genipin Crosslinked Bombyx Mori Silk Fibroin Materials

Bombyx mori silk fibroin materials have been investigated as biomaterials due to the unique chemical and mechanical properties and biocompatibility. But the fibroin materials without treatment would dissolve in the water easily, which limits the range of biomedical applications. Nowadays there are many reports about improving water resistance of fibroin materials, but improving the properties of silk fibroin materials have certain negative effects on biocompatibility. Genipin(GP) is manufactured from geniposide by usingβ-glucosidase. As a water-solube bi-functional crosslinking reagent, it was not cytotoxic for the animal and human cells so far examined. In this paper, Genipin crosslinked regenerated silk fibroin materials were prepared, at the same time,a systemic research on the structure, properties of the materials were carried on and the cytocompatibility of different crosslinked silk fibroin films was studied.The GP crosslinked fibrion films were prepared by casting GP/B.m mixture solution on a polyethylene plate and air-dried. Water solubility of the crosslinked films showed that the GP is able to improve water resistance effectively. At the same time the amino acid analysis, infrared spectroscopy results showed that lysine content of the silk fibroin films reduced after crosslinking, and the new covalent bonds emerged, noting effective crosslinking reaction was taken place between fibrion and GP. The condensed structure of the films was studied by XRD,FT-IR spectra and thermal Analysis and so on in this paper. The results indicated that the content of random coil and -helix structures in the films decreased as the content of GP increased, as well as more -sheet structure was formed in films after crosslinked; the GP crosslinked films was more thermal stable than uncrosslinked ones. The fixation index of crosslinked films increased as the content of GP increased. The mechanical properties of the GP crosslinked films did not change significantly. This paper also studied the influence of the reaction temperature and glycerol addition on the structure and properties of cross-linked films. The results showed that the reaction temperature and glycerol content had little effect on the water solubility; with the reaction temperature and glycerol conten increased, the crosslinked films structure tended to -sheet structure and the thermal stability improved; elevated temperature can improve the fixation index,but did not have significant effect on the mechanical properties of crosslinked films;glycerol additions can significantly improve the the elongation of crosslinked films, reducing its Young's modulus volume, improving the brittleness of the crosslinked film.The 30%GP crosslinked porous materials were prepared by freeze-drying with different fibroin concentrations and different pre-freezing temperatures. Water solubility of the porous materials reduced significantly. The X-ray diffraction and FT-IR spectroscopy results showed that the new covalent bonds appeared in the crosslinked porous materials; fibroin concentration and freezing temperature had lilltle affecton the silk fibroin structure; there were still mainly -helix structure and random coil in the crosslinked porous materials, while the structural regularity of silk fibroin increased slightly. Thermal analysis results showed that the the thermal stability of crosslinked porous materials increased slightly. When the concentration of fibroin increased and the freezing temperature droped, the pore size being smaller and the pore density being larger.At last, this paper studied the cytocompatibility between L929 cell and different crosslinked silk fibroin films. All types of silk fibroin films were able to support cell adhesion and proliferation, cells vitality on the 20% GP crosslinked films was same as fibroin films treatment by alcohol, better than the other crosslinked groups by means of microscopy, MTT and Bradford assay. Cell apoptosis and cell cycle of crosslinked fibroin fiims measured by Flow Cytometry(FCM). The results showed comparing pure fibroin films, the 20% GP crosslinked films was no significant difference, while was superior to the other crosslinked groups, having no cell genotoxicity. Overall, 20% GP crosslinked fibroin films had good cell compatibility.