Chang C, Lauffenburger D A, Morales T I
Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02114, USA.
Osteoarthritis Cartilage. 2003 Aug;11(8):603-12. doi: 10.1016/s1063-4584(03)00087-6.
To determine whether differentiated chondrocytes are motile.
Calf articular chondrocytes isolated from six animals were cultured in spinner flasks and removed on days 3 and 7. Boyden chamber assays and time-lapse videomicroscopy were performed to monitor and quantify cell migration. A novel method for selectively harvesting and metabolically labeling the migrated cells was developed, based on cell movement to the underside of the Boyden chamber membranes. The 3H-collagen synthesized by these cells was purified and analyzed by SDS-PAGE and autoradiography either before or after cyanogen bromide cleavage.
In Boyden chambers, locomotion of day 3 chondrocytes on fibronectin-coated membranes was approximately 3-fold higher than on bovine serum albumin-coated controls (39+/-15 vs 12+/-8 cells/mm(2), respectively (P=0.005)). Insulin-like growth factor-I (IGF-I, 10 ng/ml) was chemotactic, increasing motility to 87+/-16 cells/mm(-) (difference from fibronectin alone: P=0.0003). A similar response was observed for day 7 cells, but IGF-I activation was not as pronounced (P=0.055). The collagen patterns produced by the migrated cells closely resembled those of standard collagen type II, without any evidence of collagen I production. In videotracking experiments, motile cells attached on fibronectin exhibited typical lamellipodia and filopodia, and approximately 30% of attached cells were motile (speed >1 micro m/h and directional persistence >1h). Typical cell path lengths were 30-50 micro m, substantially greater than a full cell length displacement.
A population of well-differentiated chondrocytes capable of matrix (COL II) synthesis are motile in vitro. This original finding opens new avenues to study the potential of motile cells for cartilage repair.
确定分化的软骨细胞是否具有运动能力。
从六只动物分离的小牛关节软骨细胞在旋转瓶中培养，并在第3天和第7天取出。进行博伊登室试验和延时视频显微镜检查以监测和量化细胞迁移。基于细胞向博伊登室膜下侧的移动，开发了一种选择性收获和代谢标记迁移细胞的新方法。这些细胞合成的3H-胶原在溴化氰裂解之前或之后通过SDS-PAGE和放射自显影进行纯化和分析。
在博伊登室中，第3天软骨细胞在纤连蛋白包被膜上的运动能力比在牛血清白蛋白包被的对照膜上高约3倍（分别为39±15个细胞/mm²和12±8个细胞/mm²，P = 0.005）。胰岛素样生长因子-I（IGF-I，10 ng/ml）具有趋化作用，将运动能力提高到87±16个细胞/mm²（与单独纤连蛋白相比差异：P = 0.0003）。第7天的细胞观察到类似反应，但IGF-I的激活不那么明显（P = 0.055）。迁移细胞产生的胶原模式与标准II型胶原非常相似，没有任何I型胶原产生的证据。在视频追踪实验中，附着在纤连蛋白上的运动细胞表现出典型的片状伪足和丝状伪足，约30%的附着细胞具有运动能力（速度>1μm/h且方向持续性>1小时）。典型的细胞路径长度为30 - 50μm，远大于整个细胞长度的位移。
一群能够合成基质（COL II）的高度分化软骨细胞在体外具有运动能力。这一原始发现为研究运动细胞在软骨修复中的潜力开辟了新途径。
