Chondrogenesis of synovial mesenchymal stem cells co-cultured with chondrocytes on the three-dimensional scaffold

doi:10.3969/j.issn.2095-4344.2014.34.005

Abstract

Abstract:

BACKGROUND: Articular chondrocytes with the ability of autocrine and paracrine can provide the growth factors and microenvironment for synovial mesenchymal stem cells differentiating into the chondrocyte. The three-dimensional scaffold could provide space for cells adhesion, proliferation and differentiation.

OBJECTIVE: To study the ability of chondrogenesis by co-culturing synovial mesenchymal stem cells and chondrocytes under the three-dimensional condition.

METHODS: The synovial membrane and articular cartilage were harvested from rat knee joint. The synovial mesenchymal stem cells and chondrocytes were obtained through the method of enzyme digestion. The passage 3 synovial mesenchymal stem cells and passage 2 chondrocytes were co-cultured in the chitosan/I collagen composite scaffolds at the ratio of 1:2. Then, the cells/scaffold composite was harvested to be examined morphologically, histologically and immunohistochemically after being cultured 21 days. The confocal laser was also employed to detect the cells distribution in the scaffold.

RESULTS AND CONCLUSION: After being cultured 72 hours, it could be observed from the cells/scaffold composite examined through the scanning electron microscope that the cells adhered on the surface of the scaffold and extracellular matrix surrounding the cells was seen on the scaffold. After being cultured 21 days, it could be found through the confocal laser scanning that the cells were well-distributed on the scaffold, and cells decreased gradually. Type II collagen was positive in the extracellular matrix immunohistochamically. It suggested from this study that the synovial mesenchymal stem cells could be co-cultured with chondrocytes in the chitosan/I collagen composite scaffolds and have the ability of chondrogenesis differentiation.

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: synovial membrane, mesenchymal stem cells, chondrocytes, coculture techniques, chitosan, collagen type I

CLC Number:

R318

Ning Xiao-ting, Shao Bo, Gong Zhong-cheng, Liu Hui, Ling Bin, Keremu Abass, Lin Zhao-quan, . Chondrogenesis of synovial mesenchymal stem cells co-cultured with chondrocytes on the three-dimensional scaffold[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(34): 5434-5440.

Figures/Tables 2

2.1 细胞形态学观察倒置显微镜观察，原代滑膜间充质干细胞培养24 h后，可见细胞呈短梭形贴壁生长，胞核位于细胞中央，细胞周边可见少许异形细胞。48 h后可见细胞增殖明显且细胞呈长梭形，随着细胞的传代培养，细胞逐渐纯化，细胞边缘光滑，胞核位于中央，大致呈平行或漩涡状方向排列生长，具有成纤维细胞生长的特性(图1A)；原代软骨细胞培养24 h后可见细胞呈贴壁生长，个别细胞可见伸展，72 h后可见细胞形态呈多角形(图1B)，呈集落样生长，显微镜下观察其形态类似“铺路石”，传代后细胞迅速生长，部分区域可见复层生长(图1C)。 2.2 扫描电镜检查结果 2.2.1 空白支架扫描电镜观察扫描电镜下可见壳聚糖/Ⅰ胶原复合支架材料呈蜂窝状，各孔隙之间相互连接，支架表面光滑，孔径大小100 μm左右(图2A)。 2.2.2 混合细胞培养于支架后扫描电镜观察细胞混合培养于支架材料72 h后电镜下观察细胞与支架的生长情况，了解混合培养细胞的黏附情况及细胞表面结构。镜下可见细胞黏附于支架材料表面及各空隙之间(图2B)。滑膜间充质干细胞呈长梭形黏附于支架材料表面，细胞充分舒展，胞核处隆起，细胞周边可见有少量细胞基质附着。软骨细胞在镜下呈现圆球状黏附于支架表面，细胞表面附着有分泌的基质(图2C)。 2.3 激光共聚焦显微镜观察细胞在支架材料上的分布情况实验选取ADPI荧光染料标记支架材料中细胞的胞核，激光共聚焦显微镜下观察21 d后细胞在支架上的分布情况。结果显示由于支架材料对ADPI的吸附能力较强，激光共聚焦扫描后支架材料呈荧光蓝色，网孔状分布。诱导21 d后细胞均匀分布于支架材料表面，细胞核蓝染呈圆球状分布于支架材料当中(图3A)，逐层扫描支架后细胞逐渐变少(图3B)，细胞整体分布比较均匀(图3C)。 2.4 混合培养细胞支架复合物冰冻切片的苏木精-伊红染色苏木精-伊红染色后显微镜下观察细胞在支架材料中的生长情况，镜下可以观察到支架材料成红色，成网孔状，细胞沿支架材料网孔表面生长，细胞成多角形，胞核呈圆形染成深蓝色，胞浆成淡紫色(图4A)。 2.5 混合培养细胞支架复合物冰冻切片的甲苯胺蓝染色采用甲苯胺蓝染色检测软骨基质的形成，镜下可见诱导 21 d后的细胞支架复合物甲苯胺蓝染色阳性，并且可见细胞伸入支架生长，可以证明有软骨基质成分形成(图4B)。 2.6 混合培养细胞支架复合物冰冻切片的免疫组化染色实验用免疫组化检测细胞支架复合物Ⅱ型胶原的表达，Ⅱ型胶原是软骨特异性的标记物，也是软骨细胞外基质成分之一，检测结果显示混合培养21 d后，可以在细胞外基质中有棕褐色阳性的表达(图4C)。"

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