Detailed examination of cartilage formation and endochondral ossification using human mesenchymal stem cells
Detailed examination of cartilage formation and endochondral ossification using human mesenchymal stem cells
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DOI：
10.1111/j.1440-1681.2005.04231.x
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发表时间：
2005-07-01
影响因子：
2.9
通讯作者：
Tagami, M
中科院分区：
文献类型：
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作者：
Ichinose, S;Yamagata, K;Tagami, M
1.软骨形成是生物学中最复杂的过程之一。本研究的目的是建立一个简化的体外体系来解析其复杂性.将人间充质干细胞（hMSC）维持在具有含有10 ng/mL转化生长因子（TGF）-β 3.3的软骨形成诱导培养基的藻酸盐珠粒中。在培养的第0、2、4、8、12、16和19天，我们使用光学显微镜和透射电子显微镜检查细胞。我们还采用免疫冷冻超薄切片法对细胞进行了评价。本研究表明，hMSC产生了许多细胞外基质含有异常的胶原纤维后，他们暴露于软骨诱导培养基中的藻酸盐珠。此时，茜素红染色可见钙化，细胞质和细胞外均可见由羟基磷灰石组成的电子致密颗粒.此外，免疫冷冻超微切片显示，胶原蛋白II型，X型和蛋白多糖是突出的，骨钙素是可检测的第2天。培养8-16 d，X型胶原蛋白表达持续增强，骨钙素表达明显增加。相反，II型胶原和蛋白多糖的表达随着时间的推移而减少.这些发现表明hMSC快速分化为表达II型胶原和蛋白聚糖的软骨细胞。II型胶原和蛋白多糖的表达下降，X型胶原的活性与之前相同（4-8天）。结果，这些细胞发育成下一种细胞类型，即所谓的肥大软骨细胞。最后，骨钙素活性和细胞体和细胞外基质的钙化变得明显，表明软骨内骨化。因此，我们得出结论，hMSC迅速分化成软骨细胞，随后发育成肥大的软骨细胞。软骨内骨化是这种培养的最终形式。本研究的结果表明，我们的三维培养是一个方便的体外模型的软骨形成和软骨内骨化的调控机制的调查。
1. Cartilage formation is one of the most complex processes in biology. The aim of the present study was to produce a simplified in vitro system to resolve its complexities.2. Human mesenchymal stem cells (hMSC) were maintained in alginate beads with a chondrogenesis-induction medium containing 10 ng/mL transforming growth factor (TGF)-beta 3.3. At days 0, 2, 4, 8, 12, 16 and 19 of culture, we examined the cells using a light microscope and a transmission electron microscope. We also evaluated the cells using immunocryo-ultramicrotomy.4. The present study demonstrated that hMSC produced numerous extracellular matrices containing abnormal collagen fibres following their exposure to a chondrogenesis-induction medium in alginate beads. At this time, calcification was detected by alizarin red staining and electron-dense particles, composed of hydroxyapatite, appeared in both the cytoplasm and the extracellular spaces.5. In addition immunocryo-ultramicrotomy revealed that collagen type II, type X and proteoglycan were prominent and that osteocalcin was detectable at day 2. During 8-16 days of culture, collagen type X maintained its strong expression and the expression of osteocalcin increased markedly. In contrast, the expression of collagen type II and proteoglycan decreased with time.6. These findings demonstrate that hMSC rapidly differentiate into chondrocytes expressing collagen type II and proteoglycan.7. The expression of collagen type II and proteoglycan then dropped and the activity of collagen type X was the same as before (4-8 days). As a result, the cells developed into the next cell type, so-called hypertrophic chondrocytes. Finally, both osteocalcin activity and the calcification of cell bodies and extracellular matrices became evident, indicating endochondral ossification. Thus, we conclude that hMSC rapidly differentiate into chondrocytes, followed by the development of hypertrophic chondrocytes. Endochondral ossification is the final form in this culture.8. The findings of the present study indicate that our three-dimensional culture is a convenient in vitro model for the investigation of the regulatory mechanisms of cartilage formation and endochondral ossification.