Three-dimensional degradable porous scaffolds made by silk fibroin-chitosan composite / 中国组织工程研究

ABSTRACT

BACKGROUND: Simply used natural materials-prepared scaffolds such as collagen, gelatin and fibrin solve problems of biocompatibility, but its degradation is rapid, and cannot induce new tissues, but collapse is found as cell scaffolds.OBJECTIVE: To explore and determine the property of biological degradable three-dimensional porous scaffolds using silk fibroin-chitosan composite.DESIGN, TIME AND SETTING: The material observational study was performed at the Institute of Bioengineering, Zhejiang Academy of Medical Science from June 2008 to June 2009.MATERIALS: Spring silk cocoon was presented by a silkworm farmer from Huangdunmiao village, Maqiao town, Haining City,Zhejiang Province, China. Chitosan was produced by Shanghai Bo'ao Biological Technology.METHODS: 15 g/L silk fibroin solution was made by degumming, salvation and dialysis. Chitosan was dissolved in 2% acetic acid solution to prepare 25 g/L chitosan-acetic acid solution. Two solutions were mixed to prepare six silk fibroin/chitosan solutions, and mass ratio was 10: 0, 5: 5, 4: 6, 3: 7, 2: 8, 0:10. These solutions were separately sucked in a 24-well plate. Following exhausting gas vacuole at 4 ℃, precooling was performed at -20 ℃ for 12 hours, followed by cryodesiccate for 30 hours. Samples were then hydrated in ethanol, neutralized in NaOH-alcohol for 1 hour, washed and then frozen to dry.MAIN OUTCOME MEASURES: Optical microscope and scanning electron microscope were used to observe pore size and structure of various mass ratio-prepared scaffold. Modified liquid substitution method was utilized to measure porosity of various scaffolds. The degradable rate of various scaffolds was determined at 4 weeks in vitro.RESULTS: Silk fibroin/chitosan of 10: 0 mass ratio-prepared scaffold had rough fluffy pore, was brittle, with high dissolve-loss rates. On the contrary, chitosan-prepared scaffold was hard, without enough elasticity following freeze-dry. The composite scaffold of 5: 5, 4: 6, 3: 7 and 2: 8 following freeze-dry was loose and soft, similar to sponge. With increased chitosan concentration,scaffold hardness increased. There were evenly distributed, detailed eyelets on the scaffold. Under the optical microscope,various pores were irregular; each pore closely connected and linked together; pore size was even, 20-100 μm. With increased chitosan concentration, pore size was gradually reduced. Scaffold porosity determination results displayed that mass ratio of silk fibroin/chitosan 4: 6 group > 5: 5 group > 3: 7 group > 2: 8 group. Compared with 2: 8 group, the porosity was significantly increased in the 5: 5 and 4: 6 groups (P < 0.05). No significant difference was detected in volume expansibility in the silk fibroin/chitosan composite scaffold of various mass ratios (P > 0.05). The degradation was slowest in the 2: 8 group, and fastest in the 5: 5 group at 4 weeks.CONCLUSION: Regarding physical and chemical properties, composite scaffold made by silk fibroin/chitosan showed significant superiority compared with scaffold made by silk fibroin or chitosan alone. Of them, silk fibroin/chitosan mass ratio of 5: 5 and 4: 6 are accorded with the requirement of cartilage tissue engineering.