Osteogenic and angiogenic activity of hypertrophic chondrocytes and its clinical application

1.分离的间充质基质细胞向软骨细胞分化方法的建立我们从表达绿色荧光蛋白（GFP）的转基因小鼠细胞系中分离出在成骨细胞特异性I型胶原启动子控制下的间充质基质细胞。使用这些细胞作为检测器，我们筛选了各种软骨因子的组合，以获得最佳信号。因此，我们确定Sox5、Sox6和Sox9的组合是最有效的、与细胞类型无关的信号传导单元。联合处理诱导软骨标记基因mRNA表达，如II型胶原、IX型胶原、XI型胶原、聚集蛋白和软骨调节素-1。通过免疫组化检测II型胶原蛋白和甲苯胺蓝染色，该处理还诱导了软骨特异性基质的表达。诱导软骨细胞增生性分化方法的建立我们用多种信号分子处理软骨细胞分化的间充质间质细胞、未分化的间充质细胞系C3H10T1/2和初代肋骨软骨细胞，通过实时RT-PCRT检测X型胶原，发现典型的Wnt信号通路和Runx2信号通路促进增生性分化。相反，刺激G蛋白（一种假定的肥厚抑制因子）基因的缺失会导致体内软骨细胞加速肥厚。矿化增生性软骨细胞诱导血管生成和成骨我们从兔肋中分离软骨细胞，在成骨培养基中用典型的Wnt信号处理，诱导其增生性分化和钙化。我们将这些细胞制成微球，并将其皮下移植到兔子的背部。术后2周观察到颗粒内血管新生。术后4周，出现骨髓间隙成骨。

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.