Induction of Re-Differentiation of Passaged Rat Chondrocytes Using a Naturally Obtained Extracellular Matrix Microenvironment
Induction of Re-Differentiation of Passaged Rat Chondrocytes Using a Naturally Obtained Extracellular Matrix Microenvironment
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DOI：
10.1089/ten.tea.2012.0358
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发表时间：
2013-04-01
影响因子：
4.1
通讯作者：
Park, Kwideok
中科院分区：
文献类型：
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作者：
Cha, Myung Hwa;Do, Sun Hee;Park, Kwideok
去分化的人软骨细胞严重限制了临床实践中透明软骨修复的成功。本研究的主要目的是评估自然获得的细胞衍生基质（CDM）作为软骨细胞再分化的物理微环境。一旦将不同的细胞类型培养6天并使用去污剂和酶进行脱细胞，就获得成纤维细胞衍生的基质（FDM）、前成骨细胞衍生的基质（PDM）和软骨细胞衍生的基质（CHDM）。从扫描电子显微镜观察，发现每个CDM类似于具有自组装原纤维的纤维网。与CHDM相比，FDM和PDM都显示出更紧凑的基质结构。对于组成分析，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳显示大量的基质蛋白，这是很大的不同，从每个CDM的数量和类型。特定的基质成分，如纤连蛋白，I型胶原蛋白（Col I），和层粘连蛋白，使用免疫荧光染色检测。此外，水接触角表明FDM比PDM或CHDM更亲水。大鼠原代软骨细胞生长在CDMs上的增殖优于生长在塑料盖玻片（对照）或明胶上的细胞。同时，在CDMs上培养的传代软骨细胞（P4）合成糖胺聚糖（GAG）的效率更高，与在对照或明胶上生长的细胞相比差异显著。至于软骨特异性标志物的基因表达，CDMs随着时间的推移表现出良好的软骨细胞再分化：去分化标志物Col I受到抑制，而Col II和Col I之间的比率，以及聚集蛋白聚糖和Col I之间的比率，作为再分化的指标，大大提高。此外，Col II的免疫荧光显示在CDM上培养2周的软骨细胞中的非常阳性的信号。在另一项研究中，当将由平板生长或基质生长的去分化软骨细胞（P5）制成的三维细胞团培养4周时，番红-O染色、Col II免疫组织化学和总GAG测定的结果表明，基质生长的细胞在诱导软骨细胞再分化方面明显优于平板上生长的细胞。这项工作表明，天然存在的基质，CDM，可以提供一个有利的表面纹理细胞附着，增殖，更重要的是，软骨诱导的微环境去分化软骨细胞的再分化。
Dedifferentiated human chondrocytes severely limit successful hyaline cartilage repair in clinical practice. The primary interest of this study is to evaluate the naturally obtained cell-derived matrix (CDM) as a physical microenvironment for chondrocyte re-differentiation. Once different cell types were cultured for 6 days and decellularized using detergents and enzymes, the fibroblast-derived matrix (FDM), preosteoblast-derived matrix (PDM), and chondrocyte-derived matrix (CHDM) were obtained. From scanning electron microscope observation, each CDM was found to resemble a fibrous mesh with self-assembled fibrils. Both the FDM and PDM showed a more compact matrix structure compared to the CHDM. For compositional analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis displayed numerous matrix proteins, which were quite different from each CDM in quantity and type. Specific matrix components, such as fibronectin, type I collagen (Col I), and laminin, were detected using immunofluorescent staining. In addition, the water contact angle suggests that the FDM is more hydrophilic than the PDM or CHDM. The proliferation of rat primary chondrocytes growing on CDMs was better than those growing on a plastic coverslip (control) or gelatin. Meanwhile, synthesis of glycosaminoglycan (GAG) was more effective for passaged chondrocytes (P4) cultivated on CDMs, and the difference was significant compared to cells grown on the control or on gelatin. As for the gene expression of cartilage-specific markers, CDMs exhibited good chondrocyte re-differentiation with time: the dedifferentiating marker, Col I was restrained, whereas the ratio between Col II and Col I, and between aggrecan and Col I, as an indicator of re-differentiation, was greatly improved. In addition, immunofluorescence of Col II showed a very positive signal in chondrocytes cultivated for 2 weeks on the CDMs. In an additional study, when three-dimensional cell pellets made from either plate-grown or matrix-grown dedifferentiated chondrocytes (P5) were cultured for 4 weeks, the results of Safranin-O staining, immunohistochemistry of Col II, and total GAG assay suggested that matrix-grown cells were significantly better in the induction of chondrocyte re-differentiation, than those grown on the plate. This work suggests that the naturally occurring matrix, CDM, can provide a favorable surface texture for cell attachment, proliferation, and more importantly, a chondroinductive microenvironment for the re-differentiation of dedifferentiated chondrocytes.