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

Abstract

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.

Successful Low-Cost Scaffold-Free Cartilage Tissue Engineering Using Human Cartilage Progenitor Cell Spheroids Formed by Micromolded Nonadhesive Hydrogel

“…The commonly scaffold-free 3D technologies employed for cartilage tissue engineering are based on pellet culture [ 20 ], hanging-drop [ 23 ], and 96-well plate [ 10 , 24 ], with the last two methodologies responsible for spheroid formation. Hanging-drop is a scalable technology; however, spheroids can be maintained in culture only for few days [ 23 ]. Recently, micromolded hydrogels have emerged as an alternative for spheroid culture due to the automation capacity and long-term culture for differentiation assays [ 25 ].…”

Chondrocyte secretome: a source of novel insights and exploratory biomarkers of osteoarthritis

“…Quantitative assessment of proteins secretome using these proteomic techniques remain hazardous, because the method promotes the The abundance of diverse models used in these proteomics studies also make them difficult to interpret. In monolayer, chondrocytes certainly have a different secretome than in their native 3D environment [31]. In explant culture, the majority of the newly synthesized proteins remains entrapped in the ECM and only degraded products are released into the supernatant [18].…”

“…In this part of the review, we focused on proteins identified either directly in the secretome of cartilage explants [12][13][14][15][16][17][18][19][20] or chondrocytes cultures [18,[21][22][23][24][25][26][27][28][29][30][31]), both of which are listed in Table 1. We further complete this list with proteins recently identified by proteomic analysis performed directly on fresh chondrocytes or cartilage tissue [32][33][34][35][36] (sometimes de-cellularized [37]) whereby different locations in the joint were compared, or healthy joint tissues were compared with 6 OA tissues.…”

Differential Secretome Profiling of Human Osteoarthritic Synoviocytes Treated with Biotechnological Unsulfated and Marine Sulfated Chondroitins

“…In recent years, global proteomic studies based on mass spectrometry approaches have been widely applied to investigate the pathophysiology of articular cartilage (extensively reviewed in [26]). To date, most studies have been focused on proteins directly identified in the secretome of chondrocyte cultures [20,22,24,[27][28][29][30][31][32][33][34][35]. In addition, proteomic analyses were also performed on cartilage tissues and cartilage explants [26].…”