Bone Morphogenetic Protein-9 Is a Potent Chondrogenic and Morphogenic Factor for Articular Cartilage Chondroprogenitors

Articular cartilage contains a subpopulation of tissue-specific progenitors that are an ideal cell type for cell therapies and generating neo-cartilage for tissue engineering applications. However, it is unclear whether the standard chondrogenic medium employing transforming growth factor-β (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 factor-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 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 TGFβ1treated progenitors analysed using atomic force microscopy was fibrillar and stiff whist BMP9-induced matrix of cells more compliant and correspondingly less fibrillar. Polarised 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 organisation of collagen fibrils indistinguishable from mature adult articular cartilage, with cells in deeper zones arranged in columnar fashion. This contrasted with cells grown with TGFβ1 where a concentric pattern of collagen fibrils was visualised 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