Jakob M, Démarteau O, Schäfer D, Hintermann B, Dick W, Heberer M, Martin I
Department of Surgery, Research Division, University of Basel, Switzerland.
J Cell Biochem. 2001 Mar 26;81(2):368-77. doi: 10.1002/1097-4644(20010501)81:2<368::aid-jcb1051>3.0.co;2-j.
Adult human articular chondrocytes were expanded in a medium with 10% serum (CTR) or further supplemented with different mitogens (i.e., EGF, PDGFbb, FGF-2, TGF beta 1, or FGF-2/TGF beta 1). Cells were then induced to redifferentiate in 3D pellets using serum-supplemented medium (SSM), serum-free medium (SFM), or SFM supplemented with factors inducing differentiation of chondroprogenitor cells (i.e., TGF beta 1 and/or dexamethasone). All factors tested during expansion enhanced chondrocyte proliferation and dedifferentiation, as assessed by the mRNA ratios of collagen type II to type I (CII/CI) and aggrecan to versican (Agg/Ver), using real-time PCR. FGF-2/TGF beta 1-expanded chondrocytes displayed the lowest doubling times, CII/CI and Agg/Ver ratios, averaging, respectively, 50, 0.2 and 15% of CTR-expanded cells. Redifferentiation in pellets was more efficient in SFM than SSM only for EGF-, PDGFbb- or FGF-2-expanded chondrocytes. Upon supplementation of SFM with TGF beta and dexamethasone (SFM TD), CII/CI ratios decreased 4.4-fold for EGF- and PDGFbb-expanded chondrocytes, but increased 96-fold for FGF-2/TGF beta 1-expanded cells. Chondrocytes expanded with FGF-2/TGF beta 1 and redifferentiated in SFM TD expressed the largest mRNA amounts of CII and aggrecan and generated cartilaginous tissues with the highest accumulation of glycosaminoglycans and collagen type II. Our results provide evidence that growth factors during chondrocyte expansion not only influence cell proliferation and differentiation, but also the cell potential to redifferentiate and respond to regulatory molecules upon transfer into a 3D environment.
将成人关节软骨细胞在含10%血清的培养基（CTR）中培养，或进一步添加不同的促有丝分裂原（即表皮生长因子（EGF）、血小板源性生长因子BB（PDGFbb）、成纤维细胞生长因子-2（FGF-2）、转化生长因子β1（TGF beta 1）或FGF-2/TGF beta 1）进行扩增。然后使用含血清培养基（SSM）、无血清培养基（SFM）或添加诱导软骨祖细胞分化因子（即TGF beta 1和/或地塞米松）的SFM诱导细胞在三维微团中重新分化。通过实时聚合酶链反应（PCR）检测II型胶原与I型胶原的mRNA比值（CII/CI）以及聚集蛋白聚糖与多功能蛋白聚糖的mRNA比值（Agg/Ver），评估扩增过程中所测试的所有因子均增强了软骨细胞的增殖和去分化。FGF-2/TGF beta 1扩增的软骨细胞具有最短的倍增时间、最低的CII/CI和Agg/Ver比值，分别平均为CTR扩增细胞的50%、0.2和15%。仅对于EGF、PDGFbb或FGF-2扩增的软骨细胞，微团中的重新分化在SFM中比在SSM中更有效。在SFM中添加TGF beta和地塞米松（SFM TD）后，EGF和PDGFbb扩增的软骨细胞CII/CI比值降低了4.4倍，但FGF-2/TGF beta 1扩增的细胞该比值增加了96倍。用FGF-2/TGF beta 1扩增并在SFM TD中重新分化的软骨细胞表达的CII和聚集蛋白聚糖的mRNA量最大，并生成了糖胺聚糖和II型胶原积累量最高的软骨组织。我们的结果表明，软骨细胞扩增过程中的生长因子不仅影响细胞增殖和分化，还影响细胞在转移到三维环境后重新分化并对调节分子作出反应的潜能。
