生物玻璃/壳聚糖三维多孔支架的抗感染性能和生物相容性研究  

Antibacterial property and biocompatibility of three dimensional porous bioactive glass/chitosan scaffolds

作　　者：李扬 刘怡卓[2] 沈超[1] 朱俊峰[1] 彭建平[1] 肖飞[1] 崔一民[1] 郭亚平[2] 陈晓东[1] Li Yang;Liu Yizhuo;Shen Chao;Zhu Junfeng;Peng Jianping;Xiao Fei;Cui Yimin;Guo Yaping;Chen Xiaodong(Department of Orthopedic Surgery,Xin Hua Hospital Affiliated to Shanghai Jiaotong University,School of Medicine,Shanghai,200092;The Education Ministry Key Lab of Resource Chemistry and Shanghai Key Laboratory of Rare Earth Functional Materials,Shanghai Normal University,Shanghai,200234,China)

机构地区：[1]上海交通大学医学院附属新华医院骨科,上海200092 [2]上海师范大学生命与环境科学学院,上海200234

出　　处：《生物骨科材料与临床研究》2020年第2期49-54,共6页Orthopaedic Biomechanics Materials and Clinical Study

基　　金：国家自然科学基金(81702123);上海市青年科技英才扬帆计划(17YF1411800);上海交通大学“医工交叉研究基金”项目(YG2016QN67)。

摘　　要：目的研发制备生物玻璃/壳聚糖三维多孔支架(BG/CS),对其生物相容性及抗感染性能进行研究,阐明其抗感染作用的关键因素。方法通过模板法制备多孔生物玻璃(BG)支架,注入壳聚糖乙酸,合成生物玻璃/壳聚糖多孔支架,此后以BG作为对照组,通过场发射扫描电镜(FESEM)观察各组材料的表面形貌和孔结构;接种Balb/c小鼠胚胎成纤维细胞(Balb/c 3T3 cells)于不同材料表面培养,分析其对细胞黏附及增殖的影响;各组支架材料分别与标准菌株ATCC 35984(表皮葡萄球菌)和ATCC 25923(金黄色葡萄球菌)共培养,以复合染料染色,在激光共聚焦显微镜(CLSM)下观察各组支架材料表面活菌和死菌的荧光强度;采用结晶紫染色法,定量分析材料表面细菌生物膜形成情况;进行细菌涂板并计数菌落(CFUs/cm2),测算抑菌效率。结果 BG/CS支架材料表面被覆一层壳聚糖,支架呈多孔结构,孔径较均一(200~400 m);材料细胞共培养4 h,BG/CS和BG表面的黏附细胞数量比较差异无统计学意义;共培养1 d,两组材料表面细胞均可正常黏附和铺展;第1~7 d的增殖过程中,各组材料表面细胞的增殖趋势接近,组间细胞增殖率比较差异无统计学意义;经过8 h细菌与材料共培养,CLSM下观测BG支架表面存在大量的细菌黏附(绿色荧光),BG/CS表面则显示出代表死亡细菌的红色荧光;生物膜定量实验显示在2~5 h,5~8 h,与BG组相比,BG/CS支架则明显抑制了生物膜的形成(P<0.01);共培养12 h,与BG组相比,BG/CS组胰酶大豆琼脂平板(TSA)培养板表面细菌的单克隆菌落数明显较少,表现出较高的抑菌效率(P<0.01)。结论生物玻璃/壳聚糖三维多孔支架(BG/CS)具有较好的体外生物相容性和抗感染性能,有望作为一种应用于骨关节感染预防及治疗的骨修复填充材料。Objective To fabricate a porous bioactive glass/chitosan scaffolds(BG/CS) and to investigate its in vitro biocompatibility and antibacterial property. Methods BG/CS were prepared by template method. The morphology and microstructures of the samples were investigated by using FESEM. The proliferation of Balb/c 3 T3 cells on samples was examined with a CCK-8 assay. The cell morphology was investigated by using SEM. Samples were co-cultured with standard strains ATCC 35984(staphylococcus epidermidis) and ATCC 25923(staphylococcus aureus), respectively. The antibacterial ability and biofilm formation on the surface of samples were analyzed using the spread plate method and crystal violet stain method. The bacterial adherence was observed under CLSM. Results All the samples have ordered pores with uniform pore size of approximately 200-400 m. At time points of 4 h and 1 to 7 days, the initial numbers of cell adhesion, morphology observed under FESEM and proliferation rate show a similar trend on the surfaces of both groups of samples. The CLSM images indicated that BG/CS posses a better antibacterial ability than the BG, as confirmed by quantitative analysis using spread plate method and crystal violet stain. Conclusion The excellent in vitro biocompatibility and antibacterial property of the BG/CS suggest that it has great potential for bone-implantable applications to prevent and treat orthopedic infections.

关 键 词：多孔生物玻璃支架 壳聚糖 生物相容性 抑菌性能 

分 类 号：R318[医药卫生—生物医学工程]