Lü Hanning, Xu Guojiao, Gai Yuxin, Chen Li, Liu Shuyun, Zhao Peng, Lu Shibi, Zhang Li, Quanyi Guo, Yang Jianhua
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2014 Aug;28(8):1017-22.
To assess the role and effect of Wharton's jelly of human umbilical cord oriented scaffold on chondrocytes co-cultured in vitro.
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.
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 uniformly along the pore of oriented scaffold, and exuberantly proliferated.
Wharton's jelly of human umbilical cord oriented scaffold favors adhesion, proliferation, and survival of chondrocytes. It possesses a favorable affinity and cell compatibility. Thus, it is an ideal scaffold for cartilage tissue engineering.
评估人脐带华通氏胶定向支架在体外共培养软骨细胞中的作用及效果。
分离、培养、扩增成年新西兰兔肩软骨的软骨细胞，并用荧光染料PKH26进行标记。采用湿磨化学技术从人脐带组织中提取细胞，通过冷冻干燥和交联技术制备人脐带华通氏胶定向支架。将第二代软骨细胞与人脐带华通氏胶定向支架共培养。使用倒置显微镜和扫描电子显微镜（SEM）观察细胞在支架上的分布和黏附情况；通过甲苯胺蓝和番红O染色观察软骨细胞的细胞外基质分泌情况。采用二乙酸荧光素 - 碘化丙啶（FDA - PI）和Hoechst33258染色评估细胞在支架上的分布和增殖情况。通过荧光显微镜评估体外培养并经PKH26荧光标记的软骨细胞在支架上的活力。
倒置显微镜显示，在支架上培养3天的细胞呈圆形或椭圆形，均匀分布于支架空间。SEM观察显示，大量培养的细胞黏附于支架之间的孔隙，呈圆形或椭圆形，培养7天后在纵向定向支架上聚集、增殖并垂直排列。组织学观察显示，培养7天时细胞在支架上分布并增殖，分泌大量细胞外基质。支架可引导细胞迁移和增殖，并能有效保存和促进细胞外基质的分泌。培养3天后的细胞活力评估显示，大多数黏附细胞存活，活力超过90%。可见PKH26标记的软骨细胞，沿定向支架孔隙均匀分布且增殖旺盛。
人脐带华通氏胶定向支架有利于软骨细胞的黏附、增殖和存活。它具有良好的亲和力和细胞相容性。因此，它是软骨组织工程的理想支架。
