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 neocartilage for tissue engineering applications. However, it is unclear whether the standard chondrogenic medium using transforming growth factor beta (TGFß) 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 TGFß1-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 TGFß1-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 TGFß1-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 TGFß1, 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.

Platelet-rich plasma induces post-natal maturation of immature articular cartilage and correlates with LOXL1 activation.

Platelet-rich plasma (PRP) is used to stimulate the repair of acute and chronic cartilage damage even though there is no definitive evidence of how this is achieved. Chondrocytes in injured and diseased situations frequently re-express phenotypic biomarkers of immature cartilage so tissue maturation is a potential pathway for restoration of normal structure and function. We used an in vitro model of growth factor-induced maturation to perform a comparative study in order to determine whether PRP can also induce this specific form of remodeling that is characterised by increased cellular proliferation and tissue stiffness. Gene expression patterns specific for maturation were mimicked in PRP treated cartilage, with chondromodulin, collagen types II/X downregulated, deiodinase II and netrin-1 upregulated. PRP increased cartilage surface cell density 1.5-fold (P < 0.05), confirmed by bromodeoxyuridine incorporation and proportionate increases in proliferating cell nuclear antigen gene expression. Atomic force microscopy analysis of PRP and growth factor treated cartilage gave a 5-fold increase in stiffness correlating with a 10-fold upregulation of lysyl oxidase like-1 gene expression (P < 0.001). These data show PRP induces key aspects of post-natal maturation in immature cartilage and provides the basis to evaluate a new biological rationale for its activity when used clinically to initiate joint repair.