Abstract: BACKGROUND: The pore-forming technology of silk fibroin scaffolds is mostly prepared by freezing or freeze-drying methods, but different concentrations and temperatures have an impact on the physical and biological properties of the scaffold. There is no clear study on which freezing method or concentration is used for the preparation of the bone tissue scaffold.  
OBJECTIVE: To study the effects of concentration, freezing condition and 10 g/L polyethylene glycol on the properties of silk fibroin scaffolds under the condition of pore-induced water, and to select the suitable production conditions for the fabrication of bone tissue engineering scaffolds.
METHODS: The silk fibroin protein solutions of 30, 60, 90 g/L were prepared. After pre-frozen at -20 ℃, the silk fibroin solution of each mass concentration was divided into four groups to prepare porous scaffolds: -20 ℃ freezing treatment group, -60 ℃ freezing treatment group, -20 ℃ lyophilization treatment group, and -60 ℃ lyophilization treatment group. The pore size, hydrophilicity, porosity and compression strength of the scaffolds and cell compatibility were determined in each group. The surface morphology of the material was observed by scanning electron microscope. Based on the above results, the optimal freezing conditions and the mass concentration of silk fibroin solution for preparing porous scaffolds were obtained. Polyethylene glycol and 60 g/L silk fibroin solution were mixed uniformly, and lyophilized method at -60 ℃ was used to prepare silk fibroin porous scaffolds. The compressive strength, tensile strength and cell compatibility of the composite scaffold were tested.  
RESULTS AND CONCLUSION: (1) The pore size, porosity, hydrophilicity, compressive strength and cell compatibility results showed that 60 g/L silk fibroin solution was pre-frozen at -20℃ and then lyophilized at -60 ℃ to obtain the best porous scaffold. The scaffold had a pore size of (213.07±37.89) µm and a porosity rate of 85%, which could promote the proliferation of bone marrow mesenchymal stem cells. (2) The addition of polyethylene glycol could increase the compressive and tensile strength of the silk fibroin scaffold (P < 0.05). (3) Bone marrow mesenchymal stem cells were seeded on a polyethylene glycol-silk fibroin composite scaffold. Scanning electron microscopy for 7 days of co-cultivation showed that the cells adhered to the scaffold sufficiently and the synapses on the cell surface were fully stretched. Hematoxylin-eosin staining at 2 weeks of co-cultivation showed that the cells adhered to the stent wall and the nuclei were deeply stained. At 4 weeks, the cells adhered tightly in the channel of the stent hole, filled the entire channel, and proliferated faster. Immunohistochemical staining for 2 weeks of co-culture showed that type I collagen, RUNX2, and osteocalcin were negatively expressed. RT-PCR detection of co-culture for 3 weeks showed that the composite scaffold had no effect on the differentiation of bone marrow mesenchymal stem cells. (4) It is concluded that at -60℃, 60 g/L silk fibroin freeze-dried porous scaffold is suitable for bone tissue engineering. 10 g/L polyethylene glycol can increase the tensile and compressive strength of the material. Silk fibroin scaffold has no induction effect on cell differentiation.

Key words: silk fibroin, polyethylene glycol, tissue engineering, porous scaffold, stem cells, biocompatibility