Biological Engineering Program, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok, 10140, Thailand.
Department of Orthopaedic Surgery, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand.
BMC Biotechnol. 2018 Mar 14;18(1):14. doi: 10.1186/s12896-018-0426-1.
Dedifferentiation of chondrocytes during cell expansion is one of the barriers in tissue construction for cartilage repair. To understand chondrocyte behavior and improve cell expansion in monolayer culture, this study investigated the effects of morphological changes and cellular aggregation on the maintenance of chondrogenic capacity by observing the expression patterns of chondrogenic (collagen type II and aggrecan) and dedifferentiation (collagen type I) markers. Primary human chondrocytes were cultured on either a polystyrene surface (PS) or a polyamidoamine dendrimer surface with a fifth-generation (G5) dendron structure to create a one-step process of cell expansion and the maintenance of chondrogenic activities prior to the construction of cell sheets.
During the first two passages (P0 - P2), the relative mRNA level of collagen type II decreased in all cultures, while that of collagen type I increased. Remarkably, the level of collagen type II was higher and aggrecan was retained in the chondrocytes, forming cell aggregates and showing some round-shaped cells with less production of stress fibers on the G5 surface compared to fibroblast-like chondrocytes with abundant stress fibers on the PS surface. The numbers of P2 chondrocytes on the G5 and PS surfaces were nearly the same and sufficient for construction of chondrocyte sheets using a temperature-responsive plate. Without a supporting material during cell sheet manipulation, chondrocyte sheets spontaneously detached and exhibited a honeycomb-like structure of stress fibers. Unlike the chondrocyte sheets constructed from cells on the PS surface, the chondrocyte sheets from cells on the G5 surface had higher chondrogenic activities, as evidenced by the high expression of chondrogenic markers and the low expression of dedifferentiation markers.
The one-step process of cell expansion and maintenance of chondrogenic activity could be obtained using the G5 surface. Human chondrocyte sheets were successfully constructed with high chondrogenic activity. These findings may lead to an alternative cultivation technique for human chondrocytes that offers high clinical potential in autologous chondrocyte implantation.
在细胞扩增过程中软骨细胞去分化是软骨修复组织构建的障碍之一。为了了解软骨细胞的行为并改善单层培养中的细胞扩增，本研究通过观察软骨形成（胶原 II 型和聚集蛋白聚糖）和去分化（胶原 I 型）标志物的表达模式，研究了形态变化和细胞聚集对维持软骨形成能力的影响。原代人软骨细胞分别在聚苯乙烯（PS）或具有第五代（G5）树枝状结构的聚酰胺胺树状大分子（PAMAM）表面上培养，以创建一个在构建细胞片之前进行细胞扩增和维持软骨形成活性的一步法过程。
在最初的两个传代（P0-P2）中，所有培养物中的胶原 II 型相对 mRNA 水平降低，而胶原 I 型增加。值得注意的是，与 PS 表面上具有丰富的应激纤维的成纤维样软骨细胞相比，G5 表面上的软骨细胞中胶原 II 型的水平更高，聚集蛋白聚糖保留下来，形成细胞聚集，并显示出一些圆形细胞，产生的应激纤维较少。G5 和 PS 表面上的 P2 软骨细胞数量几乎相同，足以使用温度响应盘构建软骨细胞片。在没有支撑材料的情况下进行细胞片操作时，软骨细胞片会自发脱离并呈现出具有应激纤维的蜂窝状结构。与从 PS 表面上的细胞构建的软骨细胞片不同，从 G5 表面上的细胞构建的软骨细胞片具有更高的软骨形成活性，这表现为软骨形成标志物的高表达和去分化标志物的低表达。
可以使用 G5 表面获得细胞扩增和维持软骨形成活性的一步法过程。成功构建了具有高软骨形成活性的人软骨细胞片。这些发现可能为自体软骨细胞移植提供了一种替代的人软骨细胞培养技术，具有很高的临床潜力。
