软骨细胞复合人脐带Wharton胶取向支架的体外评估  

IN VITRO EVALUATION OF CHONDROCYTES COMBINED WITH Wharton's JELLY OF HUMAN UMBILICAL CORD ORIENTED SCAFFOLD

作　　者：吕涵宁 徐国娇[1] 盖昱辛 陈礼[1] 刘舒云[2] 赵鹏[1] 卢世璧[2] 张莉[2] 郭全义[2] 杨建华[1] 

机构地区：[1]佳木斯大学附属第一医院骨外一科,黑龙江佳木斯154002 [2]解放军总医院骨科研究所

出　　处：《中国修复重建外科杂志》2014年第8期1017-1022,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：黑龙江省大学生创新创业训练资助项目(201310222006);国家级大学生创新创业训练资助项目(1110150221);国家高技术研究发展计划(863)资助项目(2102AA020502)~~

摘　　要：目的将软骨细胞复合于人脐带Wharton胶取向支架,体外评估该支架对软骨细胞的影响和作用。方法取成年新西兰大白兔肩关节软骨组织分离培养软骨细胞。取正常新生儿脐带组织,通过湿润粉碎结合化学脱细胞技术脱细胞,利用冷冻干燥及交联等技术制备人脐带Wharton胶取向支架。取第2代软骨细胞与人脐带Wharton胶取向支架复合培养,倒置显微镜、扫描电镜观察细胞在支架上分布及黏附情况;甲苯胺蓝、番红O染色观察细胞细胞外基质分泌情况;二乙酰荧光素-碘化丙啶双色荧光检测法染色及Hoechst33258染色评估细胞在支架上的活力及增殖情况;将体外PKH26荧光标记的软骨细胞与支架复合培养,荧光显微镜观察。结果倒置显微镜观察示,培养3 d细胞在支架孔隙内分布较均匀,呈圆形或椭圆形;扫描电镜观察示,培养7 d大量细胞黏附于支架孔隙间,呈圆形或椭圆形,且细胞沿取向孔道伸展排列,相互聚集,增殖旺盛;组织学观察示,培养7 d,细胞在支架中分布均匀且增殖旺盛,细胞分泌大量细胞外基质,取向支架能引导细胞迁徙和增殖,并能有效保留和促进细胞外基质分泌;细胞活力评估结果示,培养3 d见支架上黏附的大部分细胞为活细胞,细胞成活率高达90%以上。荧光显微镜观察示,荧光标记细胞与支架复合培养1 d,细胞在支架内部均匀分布,增殖旺盛。结论人脐带Wharton胶取向支架具有良好的亲和性和细胞相容性,有利于软骨细胞黏附和增殖,细胞存活率高,是一种比较理想的软骨组织工程支架材料。Objective To assess the role and effect of Wharton’s jelly of human umbilical cord oriented scaffold on chondrocytes co-cultured in vitro. Methods Chondrocytes from shoulder cartilage of adult New Zealand rabbits were isolated, cultured, amplified, and labelled using fluorescent dye PKH26. Cells were extracted from human umbilical cord tissue using wet-grinding chemical technology to prepare the Wharton’s jelly of human umbilical cord oriented scaffold by freeze-drying and cross-linking technology. Second generation of chondrocytes were cultured with Wharton’s jelly of human umbilical cord oriented scaffold. Inverted microscope and scanning electron microscope （SEM） were used to observe the cell distribution and adhesion on the scaffold; extracellular matrix secretion of the chondrocytes were observed by toluidine blue and safranin O staining. Cells distribution and proliferation on the scaffold were assessed by fluorescein diacetate-propidium iodide （FDA-PI） and Hoechst33258 staining. The viability of the in vitro cultured and PKH26 fluorescence labelled chondrocytes on the scaffold were assessed via fluorescence microscope. Results Inverted microscope showed that the cells cultured on the scaffold for 3 days were round or oval shaped and evenly distributed into space of the scaffold. SEM observation showed that large number of cultured cells adhered to the pores between the scaffolds and were round or oval shape, which aggregated, proliferated, and arranged vertically on longitudinally oriented scaffold at 7 days after culture. Histological observation showed that cells distributed and proliferated on the scaffold, and secreted large amount of extracellular matrix at 7 days. Scaffold could guide cell migration and proliferation, and could effectively preserve and promote the secretion of extracellular matrix. Cell viability assessments at 3 days after culture showed most of the adhered cells were living and the viability was more than 90%. PKH26 labelled chondrocytes were seen, which distributed u

关 键 词：软骨组织工程 人脐带Wharton胶 取向支架 软骨细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]