Osteogenic and angiogenic activity of hypertrophic chondrocytes and its clinical application

1.分离间充质干细胞向软骨细胞分化方法的建立我们从成骨细胞特异性I型胶原启动子控制下表达绿色荧光蛋白(GFP)的转基因小鼠系中分离出间充质基质细胞。使用这些细胞作为检测器，我们筛选出各种软骨形成因子的组合，以获得最佳的信号传导。因此，我们确定Sox5、Sox6和Sox9的组合是最有效和细胞类型独立的信号单位。联合治疗诱导软骨标志物基因的mRNA表达，如II型胶原、II型胶原、XI型胶原、聚集素和cbondromodin-1。2.软骨细胞高分化诱导方法的建立我们通过实时RT-PCRT检测到软骨细胞分化为间充质基质细胞、未分化间充质细胞系C3H10T1/2和含有不同信号分子的原代肋骨软骨细胞，发现规范的Wnt信号通路和Runx2信号通路促进了X型胶原的肥大分化。3.矿化肥大软骨细胞诱导血管生成和成骨我们从兔肋骨分离软骨细胞，在成骨介质中用规范的Wnt信号处理，诱导软骨细胞肥大分化和钙化。我们从这些细胞中形成了颗粒，并将其移植到兔子的背部。术后2周观察微球内血管生成情况。术后4周，骨髓腔内出现成骨现象。

1.Establishment of the method to differentiate isolated mesenchymal stromal cells into chondrocytesWe isolated mesenchymal stromal cells from a transgenic mouse line which expressed green fluorescent protein (GFP) under the control of the osteoblast-specific type I collagen promoter. Using these cells as a detector, we screened combinations of various chondrogenic factors for the optimal signaling. As a result, we identified the combination of Sox5, Sox6, and Sox9 was the most efficient and cell-type independent signaling unit. Treatment with the combination induced mRNA expression of cartilage marker genes, such as type II collagen, type IX collagen, and type XI collagen, aggrecan, and cbondromodulin-1. The treatment also induced expression of cartilage-specific matrix detected by immunohistuchemistry for type II Collagen and toluidine Blue stainin2.Establishment of the method to induce hypertcophic differentiation of chondrocytesWe treated chondrocyte-differentiated mesenchymal stromal cells, a undifferentiated mesenchymal cell line C3H10T1/2, and primary rib chondrocytes with various signaling molecules to find out that the canonical Wnt signaling pathway and Runx2 signaling pathway promoted hypertrophic differentiation detected by real-time RT-PCRT for type X collagen. In contrast, the loss of the gene endoding a stimulatory G protein, a putative inhibitory factor for hypertrophy, resulted in accelerated hypertrophy of chondrocytes in vivo.3.Induction of angiogenesis and osteogenesis by mineralized hypertrophic chondrocytesWe isolated chondrocytes from rabbit ribs and treated them with the canonical Wnt signal in osteogenic medium to induce hypertrophic differentiation and calcification. We formed pellets from these cells and transplanted subcutaneously into the back of rabbits. Angiogenesis into the pellets was observed at 2 weeks after the surgery. At 4 weeks after the surgery, osteogenesis occurred with bone marrow space.