丝素蛋白溶液诱导成骨分化及其性能评价  

Osteoinductivity and performance of silk fibroin solution

作　　者：易兵成 张会兰[1] 余哲泡 袁卉华[1] 王先流 沈炎冰[1] 包佳煜 娄向新[1] 张彦中[1] 

机构地区：[1]东华大学化学化工与生物工程学院,上海市201620

出　　处：《中国组织工程研究》2016年第52期7788-7795,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(51073032;31570969);上海市科委基础研究重点项目(14JC1490100)~~

摘　　要：背景:丝素蛋白作为一种高性能生物材料常用于构建骨组织工程支架,但丝素蛋白自身是否具有成骨诱导性能尚未见报道。目的:体外观察不同浓度丝素蛋白溶液对大鼠骨髓间充质干细胞增殖和分化的影响。方法:分别从蚕茧和大鼠胫骨中提取丝素蛋白和骨髓间充质干细胞,并进行鉴定。然后用0.01%、0.05%和0.1%的丝素蛋白溶液对骨髓间充质干细胞进行培养,在不同时间点测定骨髓间充质干细胞增殖和碱性磷酸酶的表达情况。结果与结论:(1)FTIR结果证实了蚕茧中提取样品具有丝素蛋白的酰胺Ⅰ、酰胺Ⅱ和酰胺Ⅲ基团,为丝素蛋白;(2)细胞形貌观察、流式细胞术和三系分化鉴定提取的细胞具有骨髓间充质干细胞长梭形或星芒状形貌特征,能表达间充质干细胞表面抗原标志物,并具有三系分化潜能,证实了该细胞为骨髓间充质干细胞;(3)与不含丝素蛋白溶液培养的对照组相比,0.05%的丝素蛋白能够显著促进骨髓间充质干细胞黏附、铺展和增殖以及碱性磷酸酶表达,差异有非常显著性意义(P<0.01);(4)随着丝素蛋白浓度的增加,促进效果有所降低甚至产生抑制现象。这些结果说明丝素蛋白自身具有诱导成骨作用,可为发展高性能丝素蛋白基骨组织工程支架提供科学依据。BACKGROUND：Silk fibroin, as a kind of high-performance biomaterial, has been widely used to construct scaffolds in bone tissue engineering. However, whether silk fibroin itself holds osteoinductive ability has not been reported yet. OBJECTIVE：To investigate the impact of different concentrations of silk fibroin solution on the proliferation and differentiation of rat bone marrow mesenchymal stem cel s （BMSCs） in vitro. METHODS：Silk fibroin and BMSCs were respectively isolated from silkworm cocoon and rat tibia, and were identified. Then, BMSCs were cultured in different concentrations of silk fibroin solution （0.01%, 0.05%and 0.1%）, and the cell proliferation and the alkaline phosphatase activity were detected at different time points. RESULTS AND CONCLUSION：FTIR spectra of the sample extracted from silkworm cocoon showed distinct absorption peaks at 1 653 （amide I）, 1 530.5 （amide II） and 1 212.3 cm-1 （amide III）, which could be confirmed to be silk fibroin. Thus generated BMSCs showed long fusiform or astral morphology, positive for representative markers （CD29, CD44 and CD90） relating to mesenchymal stem cells, and could differentiate into osteocytes, chondrocytes and adipocytes under specific induction conditions, which further confirmed the extracted cells were BMSCs. Compared with the control group （without silk fibroin）, 0.05% silk fibroin not only significantly promoted the cell adhesion, migration and proliferation, but also enhanced the alkaline phosphatase activity （P〈0.01）. With the increasing of the silk fibroin concentrations, the osteodifferentiation capacity of the BMSCs was progressively improved within the range of 0-0.05%and then declined at 0.01%of silk fibroin solutions. These results suggest that silk fibroin can promote osteogenesis, thus providing scientific evidence for developing silk fibroin-based tissue-engineered scaffolds.

关 键 词：骨髓 间质干细胞 丝素蛋白 生物相容性材料 组织工程 生物材料 骨生物材料 骨髓间充质干细胞 细胞相容性 增殖 成骨分化 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]