Detailed Study during Cartilage Formation by Human Mesenchymal Stern Cells In a Three Dimentional Culture and Implantation.

人间充质干细胞（hMSCs）具有分化为间充质组织谱系的潜力。本研究的目的是阐明hMSCs分化的详细过程。我们用三维培养和移植的方法研究了人间充质干细胞软骨的形成。三维培养-海藻酸珠培养-在海藻酸珠中维持hMSCs。采用光镜、透射电镜、免疫冷冻-超微切片检查。在海藻酸珠培养的情况下，hMSCs在第4天产生II型胶原、X型胶原、蛋白多糖和骨钙素。之后，出现了由羟基磷灰石组成的电子致密粒子。三维培养-颗粒培养-在颗粒培养的情况下，hMSCs在第1天与连接复合物连接。第7天，hMSCs产生I型、II型、X型胶原蛋白和蛋白多糖。第14-21天，II型胶原蛋白的表达下降。mso源性软骨植入兔软骨缺损。我们将MSCs作为微团在软骨形成培养基中培养14天，并在体外将MSCs分化为软骨。然后，将msc来源的软骨压入兔软骨缺损。植入8周后观察到软骨缺损完全修复。

Human mesenchymal stem cells (hMSCs) have the potential to differentiate into the lineages of mesenchymal tissues. The aim of this study Is to clarify the detailed process of differentiation In hMSCs. We examined cartilage formation of human mesenchymal stem cells using three dimentional culture and implantation.Three dimentional culture -alginate bead culture-: hMSCs were maintained in alginate beads. We examined them by light microscope, transmission electron microscope and immunocryo-ultramicrotomy. hMSCs produced collagen type II, X, proteoglycan and osteocalcin at day 4 in the case of alginate bead culture. Thereafter electron-dense particles, composed of hydroxyapatite, appeared.Three dimentional culture -pellet culture-: In the case of pellet culture, hMSCs were connected with junctional complex at day 1. At day 7, hMSCs produced collagen type I, II, X and proteoglycan. During day 14-21, the expression of collagen type II decreased.Implantation of MSO-derived cartilages into the rabbit cartilage defect.: We cultured MSCs as a micromass in chondrogenesis medium for 14 days and differentiated MSCs into cartilage in vitro. Thereafter, MSC-derived cartilages were press fitted into the rabbit cartilage defects. Eight weeks after implantation, we observed the cartilage defects were repaired.