Influence of three-dimensional culture in a type II collagen sponge on primary cultured and dedifferentiated chondrocytes
Influence of three-dimensional culture in a type II collagen sponge on primary cultured and dedifferentiated chondrocytes
复制标题
DOI：
10.1007/s00776-005-0930-8
复制
发表时间：
2005-09-01
影响因子：
1.7
通讯作者：
Itoman, M
中科院分区：
文献类型：
--
作者：
Mukaida, T;Urabe, K;Itoman, M
关节软骨一旦被破坏，其内在修复能力较差。因此，各种技术已经发展到修复关节缺陷。多种支架已被用于培养软骨细胞移植。在这项研究中，我们开发了一种由II型胶原蛋白组成的海绵。研究三维培养对软骨细胞表型维持和去分化软骨细胞再分化的影响。从大鼠肋软骨中分离软骨细胞，在塑料培养皿中培养一周（PO）。然后用胰蛋白酶分离细胞，再传代培养2周（P1）。原代分离软骨细胞在II型胶原海绵中培养3周（S1）。我们通过逆转录聚合酶链反应（RT-PCR）比较了软骨形成标志物中S1基因的表达与PO和PI的表达。然后用胰蛋白酶分离细胞，再传代培养2周（P1），再传代培养6周（P3）。P1细胞随后在II型胶原海绵中培养4周（P1r）。在每个时间点，用rt - pcr检测成软骨标志物的基因表达。COL2A1、COL10A1和aggrecan基因在S1中的表达与PO相同。COL10A1和聚集蛋白在P1r中的表达高于P1和P3。COL1A1、COL2A1和SOX9基因在P1r中的表达低于P1和P3。检测P1r中ALP和骨钙素的基因表达。这些结果表明，在II型胶原海绵中培养可以维持软骨细胞表型；然而，去分化软骨细胞分化为肥大软骨细胞。这些发现表明，细胞和支架与原代细胞的复合物在实验室和临床应用中比与去分化软骨细胞的复合物更有用。
Background Once articular cartilage is destroyed, the intrinsic reparative ability is poor. Therefore, various techniques have been developed to repair articular defects. Many kinds of scaffolds have been used for cultured chondrocyte transplantation. In this study, we developed a sponge consisting of type II collagen. We investigated the influence of three-dimensional culture on the maintenance of the chondrocyte phenotype and on the redifferentiation of dedifferentiated chondrocytes.Methods. Chondrocytes were isolated from the rib cartilage of rats and were cultured in plastic dishes for a week (PO). The cells were then dissociated with trypsin and subcultured for another 2 weeks (P1). Primary isolated chondrocytes were cultured in the type II collagen sponges for 3 weeks (S1). We compared the gene expression of S1 for chondrogenic markers with the expression of PO and PI by reverse transcription-polymerase chain reaction (RT-PCR). The cells were then dissociated with trypsin and subcultured for another 2 weeks (P1) and then another 6 weeks (P3). Cells of P1 were subsequently cultured in type II collagen sponges for 4 weeks (P1r). At each time point, gene expression of chondrogenic markers was examined by RT-PCR.Results. Gene expression of COL2A1, COL10A1, and aggrecan in S1 was the same as in PO. Gene expression of COL10A1 and aggrecan in P1r was higher than in P1 and P3. Gene expression of COL1A1, COL2A1, and SOX9 in P1r was lower than in P1 and P3. Gene expression of ALP and osteocalcin in P1r was detected.Conclusions. These results show that culture in type II collagen sponges could maintain the chondrocyte phenotype; however, dedifferentiated chondrocytes differentiated to hypertrophic chondrocytes. These finding suggest that the complex of cells and scaffolds with primary cells was more useful than that with dedifferentiated chondrocytes in laboratory and clinical application.