壳聚糖膜覆盖3D打印双相磷酸钙骨组织工程支架的制备和性能

目的：将壳聚糖（CS）膜覆于3D打印制备的双相磷酸钙（BCP）支架上制备成复合支架，探讨其在骨组织工程中的应用潜力。方法：采用3D打印制备BCP支架，采用CS膜覆盖制备BCP/CS支架。采用原子力显微镜和扫描电子显微镜分别观察2组支架的表面粗糙度和超微结构，通过平均称重法测定2组支架吸水率。培养小鼠前成骨细胞（MC3T3-E1），采用CCK-8法分别测定2种支架浸提液与细胞共培养不同时间（1、3和5 d）的毒性等级，同时设空白组；将2组支架分别与细胞共培养，采用CCK-8法检测不同时间（1、3、5和7 d）细胞的增殖活性。结果：原子力显微镜下观察，BCP/CS支架表面粗糙度低于BCP支架。扫描电子显微镜下观察，BCP支架平均孔径为350~450 μm，BCP/CS支架平均孔径为250~300 μm。BCP支架吸水率为（32.00±2.43）%，BCP/CS支架吸水率为（37.75±2.21）%，2组间吸水率比较差异有统计学意义（P<0.05）。按照国家标准（GB/T16886.5-2003）对2组支架浸提液毒性进行评级，在1、3和5d时，2组支架毒性等级均为0级、1级和1级。MC3T3-E1细胞可在BCP/CS和BCP支架上黏附并增殖，2组细胞数量均随着时间的延长均呈单纯性升高，培养至第7天时BCP/CS支架组细胞增殖活性明显高于BCP支架组和空白组（P<0.05）。结论：BCP/CS复合支架具有符合骨组织工程支架材料的表征，无细胞毒性，并且可以提高细胞的增殖能力，具有作为骨组织工程支架的潜力。

Preparation and properties of chitosan membrane covering 3D printingbiphasic calcium phosphate bone tissue engineering scaffold

Objective:To cover the chitosan(CS) membrane on the biphasic calcium phosphate(BCP) scaffold prepared with 3D printing to prepare the composite scaffold,and to explore its application potentiality in bone tissue engineering. Methods:The BCP scaffold was prepared by 3D printing,and BCP/CS composite scaffold was prepared by covering CS membrane on it.The experiment was divided into control group (BCP scaffold group) and experimental group(BCP/CS scaffold group).The surface roughness and ultrastructures of the scaffolds in two groups were observed by atomic force microscope(AFM) and scanning electron microscope (SEM).The water absorption rates of scaffolds in two groups were measured by mean weighting method.The mouse anterior osteoblasts (MC3T3-E1) were cultured,at the same time,blank group was set up; the toxicity degrees of two kinds of stent extracts after co-cultured with the cells for different time (1,3,and 5d) were determined by CCK-8 method.The proliferation activities of cells at different time (1,3,5,and 7d) were detected by CCK-8 method after the cells were co-cultured with the scaffolds in two groups. Results:The AFM results showed that the surface roughness of the scaffolds in BCP/CS group was lower than that in BCP group.The SEM results showed that the average aperture of the scaffolds in BCP/CS group was 250-300 μm,and the average aperture in BCP group was 350-450 μm.The water absorption rates of scaffolds were(37.75±2.21)% in BCP/CS group and (32.00±2.43)% in BCP group;there was significant difference between two groups(P<0.05).According to the National standard (GB/T16886.5-2003),the toxicities of stent extracts in two groups in 1,3, and 5 d were 0,1, and 1 degree.The MC3T3-E1 cells could adhere and proliferate on the BCP/CS and BCP scaffolds,and the number of the cells in two groups was simply increased with the prolongation of time.The proliferation activity of the cells in BCP/CS group was significantly higher than those in BCP group and blank group (P<0.05) on the 7th day. Conclusion:BCP/CS composite scaffold has the characteristics of bone tissue engineering scaffold material without cytotoxicity and can improve the cell proliferation ability.It has the potential as scaffold of bone tissue engineering.