Häuselmann H J, Fernandes R J, Mok S S, Schmid T M, Block J A, Aydelotte M B, Kuettner K E, Thonar E J
Department of Biochemistry, Rush Medical College, Rush-Presbyterian-St. Luke's Medical Center, Chicago, IL 60612.
J Cell Sci. 1994 Jan;107 ( Pt 1):17-27. doi: 10.1242/jcs.107.1.17.
Articular chondrocytes embedded in alginate gel produce de novo a matrix rich in collagens and proteoglycans. A major advantage of this culture system is that the cells can be recovered by chelating the calcium, which otherwise maintains the alginate in its gel state. Chondrocytes thus released are surrounded by tightly bound cell-associated matrix, which seems to correspond to the pericellular and territorial matrices identified in cartilage by electron microscopy. The cells and their associated matrix can be easily separated by mild centrifugation from more soluble matrix components derived principally from the 'interterritorial' matrix. This new cell culture system thus makes it possible to study the assembly and turnover of molecules present in two distinct matrix pools. Importantly, a significant proportion of the aggrecan molecules in each of these two pools can be extracted using a non-denaturing solvent, thereby making possible studies of the metabolism and turnover of native proteoglycan aggregates. We show in this report that chondrocytes isolated from the full depth of adult bovine articular cartilage and maintained for 8 months in alginate gel are still metabolically active and continue to synthesize cartilage-specific type II collagen and aggrecan. The cells did not synthesize large amounts of type I collagen or of the small nonaggregating proteoglycans as usually occurs when chondrocytes lose their phenotypic stability. After this extended period of time in culture, the cells were present as two populations exhibiting differences in size, shape and amount of extracellular matrix surrounding them. The first population was found only near the surface of the bead: these cells were flattened and surrounded by a matrix sparse in proteoglycans and collagen fibrils. The second population was found throughout the remaining depth of the bead: the cells were more round and almost always surrounded by a basket-like meshwork consisting of densely packed fibrils running tangential to the surface.
包埋在藻酸盐凝胶中的关节软骨细胞可重新合成富含胶原蛋白和蛋白聚糖的基质。该培养系统的一个主要优点是，通过螯合钙可以回收细胞，否则钙会使藻酸盐保持凝胶状态。这样释放出的软骨细胞被紧密结合的细胞相关基质所包围，这似乎与通过电子显微镜在软骨中鉴定出的细胞周和区域基质相对应。通过轻度离心，细胞及其相关基质可以很容易地与主要来自“区域间”基质的更易溶的基质成分分离。因此，这种新的细胞培养系统使得研究存在于两个不同基质库中的分子的组装和周转成为可能。重要的是，这两个库中每一个库中的很大一部分聚集蛋白聚糖分子都可以用非变性溶剂提取，从而使得对天然蛋白聚糖聚集体的代谢和周转进行研究成为可能。我们在本报告中表明，从成年牛关节软骨全层分离并在藻酸盐凝胶中培养8个月的软骨细胞仍然具有代谢活性，并继续合成软骨特异性的II型胶原蛋白和聚集蛋白聚糖。这些细胞没有像软骨细胞失去其表型稳定性时通常发生的那样合成大量的I型胶原蛋白或小的非聚集蛋白聚糖。在培养了这段较长时间后，细胞呈现为两个群体，它们在大小、形状以及周围细胞外基质的量上存在差异。第一个群体仅在珠子表面附近被发现：这些细胞扁平，周围是蛋白聚糖和胶原纤维稀疏的基质。第二个群体在珠子的其余深度都有发现：细胞更圆，几乎总是被一个由与表面相切排列的密集纤维组成的篮状网络所包围。
