Chondrogenic activity of transforming growth factor-$\beta$1 and bone morphogenetic protein-2 on multipotential mesenchymal cells
The differentiation of multipotential mesenchymal cells into chondrocytes and osteoblasts is a fundamental process that occurs during skeletal development, growth and repair. These factors, many of which are members of the transforming growth factor-$\beta$ (TGF-$\beta$) superfamily, are found at high levels in bone matrix and can induce de novo cartilage and bone formation at ectopic sites in vivo, as well as enhance the chondrogenic phenotype in vitro, through processes similar to both embryonic development and bone repair. Repair of bone after serious injury such as fracture involves the recruitment of multipotential mesenchymal cells to the fracture site, and induction of chondrogenesis. While fracture repair is remarkably successful, articular cartilage repair is poor. Recently, the application of multipotential cells and/or these cartilage and bone promoting growth factors to sites of cartilage injury has been examined. However, the cellular events induced by these factors during chondrogenesis is unknown. The purpose of this investigation was to develop an in vitro system for induction of chondrogenesis in multipotential mesenchymal cells, and to examine mechanisms leading to cellular differentiation.^ Murine mesenchymal C3H10T1/2 cells have been shown to have the potential to differentiate into a number of connective tissue phenotypes, although chondrogenesis has been observed only rarely. The first part of this thesis aimed to test the hypothesis that the chondrogenic differentiation pathway can be selectively enhanced through culture conditions favorable to chondrogenesis, namely high density micromass culture and treatment with TGF-$\beta$1 or bone morphogenetic protein-2 (BMP-2). TGF-$\beta$1 treatment caused cells in the micromass culture to aggregate into a solid sphere, referred to as a spheroid. This spheroid displayed properties of cartilage matrix upon analysis using histochemical, biochemical and metabolic methods. In contrast, BMP-2 treatment did not result in spheroid formation, but instead caused the appearance of cells that morphologically, histologically and biochemically resemble chondrocytes in culture. Both effects were dependent on the dose of the factor as well as the density of the micromass culture.^ The second part of this thesis examined the mechanism(s) for the TGF-$\beta$1 and BMP-2 effects, and demonstrated that the two factors act by stimulating different cell adhesion events. TGF-$\beta$1 was found to enhance cell-matrix and cell-cell interactions, while BMP-2 acted mostly through cell-cell interactions. Specifically, TGF-$\beta$1 upregulated synthesis of the extracellular matrix protein fibronectin and the cell-cell adhesion protein N-CAM (neural cell adhesion molecule). BMP-2 cultures showed increased N-CAM and N-cadherin cell-cell adhesion, but did not change fibronectin levels. The N-cadherin effect was shown to be functional since a reduction of chondrogenesis was seen when cultures were treated with blocking peptides to N-cadherin mediated cell-cell adhesion. These results suggest that BMP-2 acted by enhancing cell-cell adhesion, whereas TGF-$\beta$1 caused the cells to become more adherent to other cells (N-CAM) and extracellular matrix (fibronectin) within the culture, than to the culture substrate, which resulted in spheroid formation and subsequent chondrogenesis. (Abstract shortened by UMI.) ^
Biology, Molecular|Biology, Cell|Health Sciences, Human Development
Denker, Andrew Evan, "Chondrogenic activity of transforming growth factor-$\beta$1 and bone morphogenetic protein-2 on multipotential mesenchymal cells" (1997). ETD Collection for Thomas Jefferson University. AAI9730317.