Articular cartilage contains a subpopulation of tissue-specific progenitors that are an ideal cell type for cell therapies and generating neocartilage for tissue engineering applications. However, it is unclear whether the standard chondrogenic medium using transforming growth factor beta (TGFβ) isoforms is optimal to differentiate these cells. We therefore used pellet culture to screen progenitors from immature bovine articular cartilage with a number of chondrogenic factors and discovered that bone morphogenetic protein-9 (BMP9) precociously induces their differentiation. This difference was apparent with toluidine blue staining and confirmed by biochemical and transcriptional analyses with BMP9-treated progenitors exhibiting 11-fold and 5-fold greater aggrecan and collagen type II (COL2A1) gene expression than TGFβ1-treated progenitors. Quantitative gene expression analysis over 14 days highlighted the rapid and phased nature of BMP9-induced chondrogenesis with sequential activation of aggrecan then collagen type II, and negligible collagen type X gene expression. The extracellular matrix of TGFβ1-treated progenitors analyzed using atomic force microscopy was fibrillar and stiff whist BMP9-induced matrix of cells more compliant and correspondingly less fibrillar. Polarized light microscopy revealed an annular pattern of collagen fibril deposition typified by TGFβ1-treated pellets, whereas BMP9-treated pellets displayed a birefringence pattern that was more anisotropic. Remarkably, differentiated immature chondrocytes incubated as high-density cultures in vitro with BMP9 generated a pronounced anisotropic organization of collagen fibrils indistinguishable from mature adult articular cartilage, with cells in deeper zones arranged in columnar manner. This contrasted with cells grown with TGFβ1, where a concentric pattern of collagen fibrils was visualized within tissue pellets. In summary, BMP9 is a potent chondrogenic factor for articular cartilage progenitors and is also capable of inducing morphogenesis of adult-like cartilage, a highly desirable attribute for in vitro tissue-engineered cartilage.

中文翻译：

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Bone Morphogenetic Protein-9 是关节软骨软骨祖细胞的有效软骨形成和形态形成因子。

关节软骨包含一个组织特异性祖细胞亚群，它们是细胞治疗的理想细胞类型，并为组织工程应用生成新软骨。然而，目前尚不清楚使用转化生长因子 β (TGFβ) 同种型的标准软骨形成培养基是否最适合区分这些细胞。因此，我们使用颗粒培养来筛选具有多种软骨形成因子的未成熟牛关节软骨的祖细胞，并发现骨形态发生蛋白 9 (BMP9) 早熟地诱导了它们的分化。这种差异在甲苯胺蓝染色中很明显，并通过 BMP9 处理的祖细胞的生化和转录分析证实，与 TGFβ1 处理的祖细胞相比，聚集蛋白聚糖和 II 型胶原 (COL2A1) 基因表达高 11 倍和 5 倍。超过 14 天的定量基因表达分析突出了 BMP9 诱导的软骨形成的快速和分阶段性质，聚集蛋白聚糖依次激活，然后是 II 型胶原蛋白，X 型胶原蛋白基因表达可忽略不计。使用原子力显微镜分析的 TGFβ1 处理的祖细胞的细胞外基质是纤维状和坚硬的，而 BMP9 诱导的细胞基质更顺从，相应地纤维状更少。偏光显微镜显示出以 TGFβ1 处理的颗粒为代表的环状胶原纤维沉积模式，而 BMP9 处理的颗粒显示出更具各向异性的双折射模式。值得注意的是，分化的未成熟软骨细胞与 BMP9 在体外作为高密度培养物孵育，产生明显的胶原纤维各向异性组织，与成熟的成人关节软骨无法区分，更深区域的细胞以柱状方式排列。这与用 TGFβ1 生长的细胞形成对比，其中在组织颗粒中可以看到胶原纤维的同心圆图案。总之，BMP9 是关节软骨祖细胞的有效软骨形成因子，并且还能够诱导成人样软骨的形态发生，这是体外组织工程软骨非常理想的属性。