壳聚糖-丝素-磷酸三钙复合支架的细胞毒性研究

The cytotoxicity research of chitosan-silk fibroin-tricalcium phosphate composite scaffold

目的对壳聚糖-丝素-磷酸三钙复合支架体外进行细胞毒性实验,为该材料在牙周组织工程中的应用提供依据。方法制备壳聚糖-丝素-磷酸三钙复合支架,并制备该支架材料的浸提液,通过四甲基偶氮唑盐(MTT)法检测25%、50%、100%浓度的壳聚糖-丝素-磷酸三钙浸提液对小鼠皮肤成纤维细胞系L-929细胞的毒性作用,以第1、3、5天为检测时间点,观察细胞生长状况,计算细胞相对增殖率,并对材料的细胞毒性进行分级。以正常培养基为阴性对照组,含0.64%苯酚的培养液为阳性对照组。结果 L-929在不同浓度实验组及阴性对照组中生长均好,细胞呈长梭形等形态,细胞形态饱满有光泽,MTT法结果表明同一时间点各浓度实验组与阴性对照组间之间差异均无统计学意义(P>0.05),各时间点各浓度浸提液对L-929的相对增殖率均在75%以上,毒性分级为0级或1级。结论壳聚糖-丝素-磷酸三钙复合支架符合生物材料细胞毒性试验的安全标准,是一种有潜力的牙周组织工程支架。

Objective To performed cytotoxicity test on chitosan-silk fibroin-tricalcium phosphate（TCP） composite scaffold,and to provide the basis for the application of this material in periodontal tissue engineering.Methods Chitosan-silk fibroin-TCP composite scaffold was firstly established and their liquid immersion was then prepared.MMT method was used to detect the cytotoxic effects of chitosan-silk fibroin-TCP liquid immersion of 25%,50% and 100% concentrations on mouse skin fibroblasts（L-929）.The growth of cells was observed at 1,3 and 5 days.Thereafter,the proliferation rates of cells were calculated,and the cytotoxicity of the material was then graded.The normal culture media was assigned as negative control group,while the media with 0.64% phenol solution as positive control group.Results L-929 cells grew well in both the experimental groups of different concentrations and negative control group,with full and burnish fusiform morphology.MMT method showed no difference in morphology between experimental groups of different concentrations and negative control group at the same time spot（P〉0.05）.The relative proliferation rates of the L-929 cells were all above 75% in the experimental groups at every time spot,and the cytotoxicity was graded as 0 or 1 level.Conclusion Chitosan-silk fibroin-TCP composite scaffold conformed to the safety standard of cytotoxicity test,which might be a potential scaffold for periodontal tissue engineering.