Water-fast silk fibroin films were created from regenerated silk fibroin by drying at a controlled temperature and humidity and degeneration with ethanol.Silk fibroin membranes prepared under the conditions of 18% silk fibroin concentration and drying temperature controlled at 60°C exhibit a smooth surface and concavo-convex reticular formation as shown by scanning electron microscopy and many B-plate sheet structures as detected by Fourier infrared spectrum analysis.Mechanical testing showed that the tensile strength of these silk fibroin membranes was 25.4 MPa,the breaking strength was 1.09 cN/dt,and the elongation at break was 50.88%.The dissolubility in hot water was 18.6% and the swelling ratio was 38.67%.To evaluate the biosafety of these silk fibroin membranes,this study intraperitoneally injected Kunming mice with 100% silk fibroin membrane leaching liquor.After injection,no mice presented nose scratching,sneezing,piloerection,nonproductive cough,convulsion,dyspnea,urinary and fecal incontinence,shock,or death.After many injections of 100% silk fibroin membrane leaching liquor,no mice presented generalized anaphylaxis,such as asthma and astasia.All the mice that received injections of 100% silk fibroin membrane leaching liquor survived,exhibited normal body type,were lively,breathed normally,and had dense and glossy hair with no acute general toxic reactions.The body weight gradually increased within 72 hours.After in vitro cultured fibroblasts were treated with silk fibroin membrane leaching liquor,they proliferated appropriately at 96%,indicating the cytotoxicity of silk fibroin membrane leaching liquor is grade I.After injection of 100% silk fibroin membrane leaching liquor into the ear vein of Japanese big eared rabbits,the body temperature of rabbits increased by 0.37-0.51 °C,but did not exceed the upper limit of 0.6-1.4°C.At 24,48,and 72 hours after injection,neither the injection site nor surrounding region presented any stimulus reaction,eschar,erythema,or edema.The hemolysis test showed that the hemolytic rates of 100%,50%,25%,and 10% silk fibroin membrane leaching liquor were 3.88%,2.32%,1.29%,and 0.48%,respectively; all rates were lower than 50%.Taken together,the data indicate that regenerated silk fibroin films exhibit good mechanical properties and biosafety,proper water absorption,and good morphological stability in an aqueous phase medium.