Kinner B, Spector M
Department of Orthopaedic Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
J Orthop Res. 2001 Mar;19(2):233-41. doi: 10.1016/S0736-0266(00)00081-4.
Recent studies have demonstrated that human articular chondrocytes can express the gene for a contractile muscle actin, alpha-smooth muscle actin (SMA), in situ. One objective of this work was to evaluate the SMA-content of isolated human articular chondrocytes using Western blot analysis and to correlate the amount of SMA in the cells with passage number and the number of days in culture. A second objective was to determine if articular cartilage-derived cells expressing the gene for SMA in vitro also continue to express type II collagen. A final aim of the current study was to determine if SMA-containing cartilage-derived cells were capable of contracting a collagen glycosaminoglycan analog of extracellular matrix in vitro. Articular chondrocytes were isolated from 13 patients undergoing total joint arthroplasty. Cells were serially passaged through passage 7. Samples were allocated for Western blot analysis of SMA. Cells in monolayer culture were also stained immunohistochemically for SMA and type II collagen. Cells from passage 3 and 7 were seeded into a porous type I collagen-glycosaminoglycan matrix and the diameter of the scaffolds measured every other day for 21 days. Immunohistochemistry of the articular cartilage samples revealed SMA in the articular chondrocytes in situ with a greater percentage of cells staining positive in the superficial half (60 +/- 1.2%; mean +/- SEM) of the cartilage than in the basal half (28 +/- 1.3%). There was an increasing amount of SMA in the cells in monolayer culture with passage number and a meaningful correlation of the SMA content with the days in culture (linear regression analysis; R2 = 0.72). Double staining for SMA and type II collagen showed that type II collagen-expressing cells in monolayer could also express SMA. SMA-containing cells were found to contract the collagen glycosaminoglycan matrix, with the cells containing more SMA (passage 7 cells) displaying more matrix contraction than those with a lesser amount of SMA (passage 3 cells). The results indicate that control of the expression of SMA may be important when employing articular chondrocytes, expanded in monolayer culture, for implantation alone or in a cell-seeded matrix for cartilage repair procedures.
近期研究表明，人类关节软骨细胞能够在原位表达一种收缩性肌动蛋白基因，即α-平滑肌肌动蛋白（SMA）。本研究的一个目的是使用蛋白质印迹分析评估分离出的人类关节软骨细胞中的SMA含量，并将细胞中的SMA量与传代次数及培养天数相关联。第二个目的是确定在体外表达SMA基因的关节软骨来源细胞是否也继续表达II型胶原蛋白。本研究的最终目标是确定含有SMA的软骨来源细胞在体外是否能够收缩细胞外基质的胶原糖胺聚糖类似物。从13例接受全关节置换术的患者中分离出关节软骨细胞。细胞连续传代至第7代。将样本分配用于SMA的蛋白质印迹分析。单层培养的细胞也进行SMA和II型胶原蛋白的免疫组织化学染色。将第3代和第7代的细胞接种到多孔I型胶原-糖胺聚糖基质中，每隔一天测量支架直径，共测量21天。关节软骨样本的免疫组织化学显示，原位关节软骨细胞中有SMA，软骨表层（60±1.2%；平均值±标准误）中染色阳性的细胞百分比高于基底层（28±1.3%）。单层培养的细胞中SMA量随传代次数增加，且SMA含量与培养天数存在显著相关性（线性回归分析；R2 = 0.72）。SMA和II型胶原蛋白的双重染色表明，单层中表达II型胶原蛋白的细胞也能表达SMA。发现含有SMA的细胞能够收缩胶原糖胺聚糖基质，含有更多SMA的细胞（第7代细胞）比含有较少SMA的细胞（第3代细胞）表现出更多的基质收缩。结果表明，在单独使用单层培养扩增的关节软骨细胞进行植入或用于软骨修复程序的细胞接种基质时，控制SMA的表达可能很重要。
