Isolation, identification and chondrogenic differentiation of rat articular cartilage-derived progenitor cells

doi:10.3969/j.issn.2095-4344.1756

Abstract

Abstract:

BACKGROUND: Articular cartilage-derived progenitor cells have strong chondrogenic differentiation ability and limited osteogenic differentiation ability. However, it is unclear whether and how the cellular characteristics of articular cartilage-derived progenitor cells after amplification change.
OBJECTIVE: To observe the proliferation, osteogenesis and chondrogenesis ability of articular cartilage-derived progenitor cells in different generations as compared with bone marrow mesenchymal stem cells.
METHODS: Articular cartilage-derived progenitor cells were sorted from chondrocytes by using differential adhesion to fibronectin. Cell counting kit-8 and colony formation assay were used to observe the proliferative capacity of articular cartilage-derived progenitor cells. Osteogenic differentiation potential of articular cartilage-derived progenitor cells and bone marrow mesenchymal stem cells were measured by alizarin red staining and determination of osteogenic gene expression. Collagen II immunohistochemical staining and determination of chondrogenic-related gene expression were used to measure the change in the chondrogenic ability of articular cartilage-derived progenitor cells and bone marrow mesenchymal stem cells.
RESULTS AND CONCLUSION: Articular cartilage-derived progenitor cells sorted by fibronectin had strong proliferation ability, osteogenic and chondrogenic differentiation potential. The results of cell counting kit-8 and colony formation assay showed that articular cartilage-derived progenitor cells had a slight decrease in cell proliferation during cell expansion. Bone marrow mesenchymal stem cells presented with strong osteogenic potential throughout cell expansion, while the chondrogenic potential of the cells decreased gradually during cell expansion. Articular cartilage-derived progenitor cells had strong chondrogenic potential with no considerable change during cell expansion, while the osteogenic potential of the cells decreased gradually significantly during cell expansion. To conclude, the osteogenic potential of articular cartilage-derived progenitor cells was significantly lower than that of bone marrow mesenchymal stem cells, and it gradually decreased during cell expansion. The chondrogenic potential of articular cartilage-derived progenitor cells was higher than that of bone marrow mesenchymal stem cells, but there was no significant change during cell expansion. These findings indicate that articular cartilage-derived progenitor cells may be an ideal kind of seed cells for cartilage tissue engineering.

Key words: articular cartilage-derived progenitor cells, bone marrow mesenchymal stem cells, osteogenic potential, chondrogenic potential, cartilage tissue engineering, fibronectin sorting, cell proliferation, National Natural Science Foundation of China

CLC Number:

Shen Xu, Lu Yingjie, Lu Dongdong, Fang Yuepeng, Zhou Naihui, Zhu Xuesong, Zhu Yueqian. Isolation, identification and chondrogenic differentiation of rat articular cartilage-derived progenitor cells[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(21): 3364-3370.

Figures/Tables 2

2.1 ACPCs的细胞形态和细胞活性在倒置显微镜下观察，经过纤连蛋白分选后的ACPCs均为中间宽两边细长的短梭形，与间充质细胞的典型细胞形态类似。随着细胞代次的增加，在相同时间内ACPCs的细胞密度逐渐降低，但在细胞形态上未有明显改变，见图1A。 CCK-8结果显示，同一代次ACPCs的吸光度值随着培养时间延长持续升高；同一时间ACPCs的吸光度值在原代最高，其次是第1代，最后是第2代，培养1，3，5，7，9 d时原代与第1代，第1代与第2代差异均有显著性意义(P < 0.05)，见图1B。 2.2 ACPCs的流式细胞鉴定结果纤连蛋白分选后获得的大鼠ACPCs进行流式细胞鉴定，流式细胞仪计数结果显示：间充质细胞标志物CD29、CD73表达为强阳性，分别为96.8%和94.2%，CD90、CD105表达为阳性，分别为40.6%和67.0%，软骨来源祖细胞备选特异性标志物Notch-1表达为阳性，为33.7%；非干细胞标志物CD31、CD44、CD45表达弱阳性或阴性，分别为3.17%，5.15%和1.85%，见图1C。 2.3 ACPCs克隆形成实验结果在各个代次细胞培养皿底部均有多个ACPCs单细胞克隆形成，见图2A。随后对不同代次细胞形成的克隆数进行比较，可见随着细胞代次的增加，ACPCs克隆数明显下降。对直径大于2 mm的克隆进行计数，不同代次之间差异有显著性意义(P < 0.000 1)，见图2B。 2.4 成骨诱导结果在肉眼下观察，ACPCs成骨诱导组橙红色范围随着代次增加逐渐减少。原代橙红色范围为孔中心及周围，颜色较深；第1代仅孔周围有部分橙红色区域，中心为蓝色区域；第2代孔内基本为浅蓝色区域。BMSCs成骨诱导组在原代、第1代和第2代均可见孔内区域为橙红色，橙红色范围随着细胞代次增加未见明显改变，见图3A。在倒置显微镜下观察，ACPCs成骨诱导组在原代可见多个钙结节，第1代1个视野中仅可见1个钙结节，第2代无钙结节形成。BMSCs成骨诱导组在原代、第1代和第2代视野内基本均为橙红色钙结节，3个代次之间的差异不明显，见图3A。 ACPCs的RT-PCR结果显示，成骨诱导组的成骨相关基因AKP、osteocalcin均明显高于非诱导组，差异有显著性意义(P < 0.05)，成骨相关基因在不同代次之间是逐渐降低的，在原代与第1代之间的降低幅度尤为明显，AKP、osteocalcin的表达在原代与第1代之间差异无显著性意义(P > 0.05)，在第1代与第2代之间差异无显著性意义(P > 0.05)，但原代与第2代之间差异有显著性意义(P < 0.05)，见图3B。 BMSCs的RT-PCR结果显示，AKP、osteocalcin的表达在原代与第1代、第1代与第2代之间差异无显著性意义(P > 0.05)，原代与第2代之间差异也无显著性意义(P > 0.05)，见图3C。 2.5 成软骨诱导结果在成软骨诱导21 d后，对细胞团块进行组织学染色分析，ACPCs成软骨诱导组细胞团块中有软骨组织典型结构，即软骨陷窝产生；Ⅱ型胶原染色可见ACPCs构建的团块全部为棕色阳性染色，不同代次之间阳性染色没有明显差异。BMSCs成软骨诱导组细胞团块中仅有部分区域有软骨陷窝产生；Ⅱ型胶原染色可见BMSCs构建的团块大部分组织为棕色阳性染色，且阳性染色区域随着细胞代次的增加而减少，到第2代时Ⅱ型胶原染色明显降低，见图4A。 ACPCs的RT-PCR结果显示，成软骨诱导组成软骨相关基因Aggrecan、COLⅡ、SOX9均明显高于非诱导组，差异有显著性意义(P < 0.05)，Aggrecan、COL-Ⅱ、SOX9的表达在原代与第1代之间及第1代与第2代之间表达有减低，但基因表达差异无显著性意义(P > 0.05)，原代与第2代之间差异也无显著性意义(P > 0.05)，见图4B。 BMSCs的RT-PCR结果显示，成软骨诱导组成软骨相关基因Aggrecan、COL-Ⅱ、SOX9均明显高于非诱导组，差异有显著性意义(P < 0.05)，Aggrecan、COL-Ⅱ、SOX9的表达在原代与第1代及第1代与第2代之间表达有减低，基因表达差异均无显著性意义(P > 0.05)，但在原代与第2代之间差异有显著性意义(P < 0.05)，见图4C。"

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