Comparative Analyses of the Secretome from Dedifferentiated and Redifferentiated Adult Articular Chondrocytes

OBJECTIVE: The main goal of this study was to compare the secretion products derived from human articular chondrocytes established in either long-term monolayer cultures or in scaffold-free 3-dimensional (3-D) cultures. METHODS: Stable isotope labeling of amino acids in cell culture (SILAC) was applied to investigate quantitatively the differences between proteins secreted from dedifferentiated and redifferentiated chondrocytes. Proteins in cell supernatants were resolved by 1-D gel electrophoresis and analyzed by mass spectrometry. The results from the proteomic analyses were validated by immunoblotting. Additionally, antibody arrays were used to screen culture supernatants for 79 different morphogens. RESULTS: Quantitative SILAC showed that some relevant growth factors such as CTGF or GAS6 were elevated in monolayers, along with proteins characteristic of a dedifferentiated phenotype such as collagen type I and tenascin. In spheroids, data showed overexpression of some cartilage-specific proteins such as aggrecan, together with important matrix regulators such as chitinase-3–like protein and stromelysin-1. Antibody arrays revealed that chondrocytes in monolayer secrete higher levels of leukocyte-activating agents such as MCP-1 and GRO, whereas the spheroid configuration favors the production of cell morphogens such as MCSF and VEGF. CONCLUSION: Our results show that some classic dedifferentiation and redifferentiation markers are differentially expressed in 2-D or 3-D culture configurations. Other cell/matrix regulatory molecules are also found to be differentially expressed by chondrocytes in 2-D and 3-D conditions by SILAC and antibody arrays. Our data bring new information for understanding the biology of chondrocytes in general and the process of cartilage tissue reconstruction in particular.