The development of a mature collagen network in cartilage from human bone marrow stem cells in Transwell culture

Damaged hyaline cartilage shows a limited capacity for innate repair. Potential sources of cells to augment the clinical repair of cartilage defects include autologous chondrocytes and mesenchymal stem cells. We have reported that culture of human bone marrow mesenchymal stem cells with specific growth and differentiation factors as shallow multilayers on Transwell permeable membranes provided ideal conditions for chondrogenesis. Rigid translucent cartilaginous disks formed and expressed cartilage-specific structural proteins aggrecan and type II collagen. We report here the analysis of the collagen network assembled in these cartilage constructs and identify key features of the network as it became mature during 28 days of culture. The type II collagen was co-polymerized with types XI and IX collagens in a fibrillar network stabilized by hydroxylysyl pyridinoline cross-links as in epiphyseal and hyaline cartilages. Tandem ion-trap mass-spectrometry identified 3-hydroxylation of Proline 986 and Proline 944 of the α1(II) chains, a post-translational feature of human epiphyseal cartilage type II collagen. The formation of a type II collagen based hydroxy-lysyl pyridinoline cross-linked network typical of cartilage in 28 days shows that the Transwell system not only produces, secretes and assembles cartilage collagens, but also provides all the extracellular mechanisms to modify and generate covalent cross-links that determine a robust collagen network. This organized assembly explains the stiff, flexible nature of the cartilage constructs developed from hMSCs in this culture system.