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

Polacek M, Bruun JA, Johansen O, Martinez I - Cartilage (2011)

Bottom Line: The results from the proteomic analyses were validated by immunoblotting.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Institute of Clinical Medicine, University of Tromsø, Tromsø, Norway ; Department of Orthopaedic Surgery, University Hospital of North Norway, Tromsø, Norway.

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.

No MeSH data available.


Related in: MedlinePlus

Verification of the protein expression by Western blot. Immunodetection of 8 relevant proteins including some overexpressed in monolayers (OMD, GAS6, CTGF), some overexpressed in spheroids (aggrecan, CHI3L2, MMP3), and some equally expressed in both conditions (SPARC, TIMP1) performed on the same conditioned media used for mixed MS analyses. Molecular weight markers are shown at the left side of each gel. Bands appearing at the predicted molecular weight of each individual protein are shown.
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fig3-1947603510383856: Verification of the protein expression by Western blot. Immunodetection of 8 relevant proteins including some overexpressed in monolayers (OMD, GAS6, CTGF), some overexpressed in spheroids (aggrecan, CHI3L2, MMP3), and some equally expressed in both conditions (SPARC, TIMP1) performed on the same conditioned media used for mixed MS analyses. Molecular weight markers are shown at the left side of each gel. Bands appearing at the predicted molecular weight of each individual protein are shown.

Mentions: Validation of the MS analyses was done by specific immunodetection of 8 relevant proteins (Fig. 3). Two of the selected proteins for validation analyses were similarly expressed in both conditions corresponding to SPARC and tissue inhibitor of metalloproteinase 1 (TIMP1). Among the differently expressed proteins, osteomodulin (OMD), growth arrest–specific protein 6 (GAS6), and connective tissue growth factor (CTGF) were upregulated in ML and weakly expressed or not found in spheroid supernatants. On the opposite end, aggrecan, CHI3L2 (chitinase-3–like protein 2), and stromelysin-1 (MMP3) were upregulated in spheroids and less expressed in ML. The results obtained by Western blots with all proteins tested confirmed accurately the original findings obtained by quantitative SILAC analyses. The size of the bands revealed in the Western blots corresponded to the expected size of the full-length proteins. Additionally, the intensity of the bands after immunoreaction matched up with the Mascot scores in the proteomic analysis.


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

Polacek M, Bruun JA, Johansen O, Martinez I - Cartilage (2011)

Verification of the protein expression by Western blot. Immunodetection of 8 relevant proteins including some overexpressed in monolayers (OMD, GAS6, CTGF), some overexpressed in spheroids (aggrecan, CHI3L2, MMP3), and some equally expressed in both conditions (SPARC, TIMP1) performed on the same conditioned media used for mixed MS analyses. Molecular weight markers are shown at the left side of each gel. Bands appearing at the predicted molecular weight of each individual protein are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4300778&req=5

fig3-1947603510383856: Verification of the protein expression by Western blot. Immunodetection of 8 relevant proteins including some overexpressed in monolayers (OMD, GAS6, CTGF), some overexpressed in spheroids (aggrecan, CHI3L2, MMP3), and some equally expressed in both conditions (SPARC, TIMP1) performed on the same conditioned media used for mixed MS analyses. Molecular weight markers are shown at the left side of each gel. Bands appearing at the predicted molecular weight of each individual protein are shown.
Mentions: Validation of the MS analyses was done by specific immunodetection of 8 relevant proteins (Fig. 3). Two of the selected proteins for validation analyses were similarly expressed in both conditions corresponding to SPARC and tissue inhibitor of metalloproteinase 1 (TIMP1). Among the differently expressed proteins, osteomodulin (OMD), growth arrest–specific protein 6 (GAS6), and connective tissue growth factor (CTGF) were upregulated in ML and weakly expressed or not found in spheroid supernatants. On the opposite end, aggrecan, CHI3L2 (chitinase-3–like protein 2), and stromelysin-1 (MMP3) were upregulated in spheroids and less expressed in ML. The results obtained by Western blots with all proteins tested confirmed accurately the original findings obtained by quantitative SILAC analyses. The size of the bands revealed in the Western blots corresponded to the expected size of the full-length proteins. Additionally, the intensity of the bands after immunoreaction matched up with the Mascot scores in the proteomic analysis.

Bottom Line: The results from the proteomic analyses were validated by immunoblotting.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, Institute of Clinical Medicine, University of Tromsø, Tromsø, Norway ; Department of Orthopaedic Surgery, University Hospital of North Norway, Tromsø, Norway.

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.

No MeSH data available.


Related in: MedlinePlus