Construction of tissue-engineered cartilage with cross-linked sodium hyaluronate as scaffold materials in vitro

doi:10.3969/j.issn.2095-4344.2014.08.008

Abstract

Abstract:

BACKGROUND: Tissue engineering provides new ideas and approaches for repair of cartilage defects.
OBJECTIVE: To develop a complete set of solutions for construction of tissue engineered cartilage in vitro, with chondrocytes as seed cells and cross-linked sodium hyaluronate as scaffold materials.
METHODS: New Zealand rabbit articular chondrocytes were isolated, counted, and then cultured and passaged to prepare cell suspension. Toluidine blue staining, RT-PCR and immunocytochemistry were exerted to evaluate the cultured cells. Chondrocytes were seeded and co-cultured with cross-linked sodium hyaluronate scaffold for 21 days. Then, RNA was isolated for RT-PCR, and frozen sections were prepared for morphological observation and immunohistochemistry study.
RESULTS AND CONCLUSION: The chondrocytes could adhere to the cross-linked sodium hyaluronate scaffold and aggregate, growing between fibers or adhering to the scaffold in a monolayer manner. The transcripts of cartilage specific aggrecan gene and collagen type II alpha 1 gene and cartilage specific protein collagen type II were expressed in cell-scaffold complexes to maintain the phenotype of chondrocytes. Cell-scaffold complexes co-cultured in vitro can form cartilage extracellular matrix, by which tissue engineered cartilage is expected to be obtained.

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

全文链接：

Key words: biocompatible materials, chondrocytes, hyaluronic acid, cross-linking reagents

CLC Number:

R318

Liu Shao-ying, Chen Jian-ying, Chen Qian-qian, Liu Fei, Liu Xia, Zhu Xi-qiang, Ling Pei-xue. Construction of tissue-engineered cartilage with cross-linked sodium hyaluronate as scaffold materials in vitro[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(8): 1191-1197.

Figures/Tables 4

2.2 软骨细胞的鉴定细胞形态观察：分离获得的软骨细胞体外培养48 h，细胞贴壁良好(图1A1)。体外传代3次后，细胞形态维持不变，胞体呈梭形或多角形，形状饱满，局部呈聚簇生长，胞外基质丰富(图1A2)。甲苯胺蓝染色：软骨细胞体外传代3次，甲苯胺蓝染色结果(图1B)，细胞整体染成蓝色，细胞核着色较深，细胞内外均有蓝紫色异染颗粒。 RT-PCR检测蛋白聚糖基因与Ⅱ型胶原α1基因的mRNA：同步提取体外培养软骨细胞与阴性对照Balb/3T3细胞的RNA，进行RT-PCR检测(图2)，Balb/3T3细胞中，出现内参基因gapdh的mRNA，而未出现软骨组织特异性基因蛋白聚糖基因与Ⅱ型胶原α1基因相应的mRNA；体外培养传代二三次的软骨细胞中，这两种软骨组织特异性基因的mRNA能够维持表达，且表达水平基本维持不变。免疫细胞化学检测Ⅱ型胶原蛋白的表达：空白对照组细胞表现免疫反应的DAB显色呈阴性结果，在苏木精染液的作用下，细胞核呈蓝紫色，细胞质与胞外基质着色较浅(图3A)。经DAB显色的实验组细胞大部分区域显示褐色 (图3B)，信号较强的区域呈鲜红色，在低倍显微镜下大部分区域呈现艳红色(图3C)，说明被检测细胞能够大量表达软骨组织特异性蛋白Ⅱ型胶原蛋白。 2.3 组织工程软骨组织的构建与鉴定大体观察：细胞-支架复合培养物体外培养21 d，支架材料的结构基本保持完整，局部区域形成乳白色、半透明的组织块，质地细腻，表面光滑，呈现出典型的透明软骨组织的表观特征(图4A)。冰冻切片观察：冰冻切片显微图像(图5)显示，种子细胞能够帖附在支架上生长，大量细胞聚集成团，在支架材料的纤维间隙中生长，或者呈单层细胞附着于支架材料纤维。"

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