A Novel 3D Culture Model to Study TSP2 Proteolysis
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The extracellular matrix (ECM) gives bone its strength and integrity and provides discrete structural and biological contributions to bone form and function. The Thrombospondin protein family is comprised of five secreted matrix proteins that share significant structural homology. Specifically, thrombospondin-2 (TSP2) affects marrow derived multipotent mesenchymal stromal cell (MSC) proliferation and lineage commitment. Once osteoblast differentiation has commenced, TSP2 expression increases and the protein promotes both osteoblast maturation and mineralization. Two immunoreactive isoforms of TSP2 sized approximately 125 and 200 kDa have been observed from whole protein extracts from the tibias and femurs of developing but not skeletally mature mice, yet the function of these isoforms remains to be uncovered. In vitro observations point to the possibility that extracellular proteolysis cleaves TSP2, releasing the two isoforms. However, concrete evidence is necessary to pose further mechanistic questions about the functional importance of two TSP2 isoforms. In this study, we investigated whether a 3D culture and matrix environment is necessary for the activation of osteoblast-derived proteases and liberation of the 125 kDa TSP2 species in vitro by culturing cells in Type I collagen gels. This 3D Type I collagen environment did not consistently yield liberation of both TSP2 isoforms. Future studies may seek to isolate both TSP2 isoforms by manipulating or altering the proposed culture microenvironment.