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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

Analysis of cytokine expression in culture media. Human-specific cytokine antibody microarray membranes (RayBio Human Cytokine Array V) were probed with culture media from monolayers and spheroids. (A) Two representative array membranes corresponding to monolayers and spheroids from the same patient. (B) Comprehensive microarray maps of proteins secreted by chondrocytes in 2-D and 3-D after combining the results obtained from 3 different donors.
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fig4-1947603510383856: Analysis of cytokine expression in culture media. Human-specific cytokine antibody microarray membranes (RayBio Human Cytokine Array V) were probed with culture media from monolayers and spheroids. (A) Two representative array membranes corresponding to monolayers and spheroids from the same patient. (B) Comprehensive microarray maps of proteins secreted by chondrocytes in 2-D and 3-D after combining the results obtained from 3 different donors.

Mentions: A large number of dots were unambiguously identified after 1-minute exposure of membranes to x-ray films (Fig. 4A). Comprehensive location maps for the membranes were prepared by a gray-scale code following the different intensities of the dots obtained after the reaction (Fig. 4B). It should be noted that the relative intensities of spots in each array may not reflect necessarily the relative amounts of proteins revealed after each reaction because the affinity of proteins for their respective antibodies may differ substantially. However, this technique allows an accurate comparison between the different experimental groups. The protein microarray results showed similar patterns of proteins produced by chondrocytes established in MLs and 3-D spheroids. However, the intensity of some of the dots in the arrays was significantly different between the 2 experimental groups. Monolayer growth showed enhanced production of growth-related oncogene protein (GRO), monocyte chemoattractant protein-1 (MCP-1), IL- 8, angiogenin, and insulin-like growth factor binding protein-2 (IGFBP-2). After redifferentiation of chondrocytes in spheroids, the arrays showed overexpression of CKb 8-1, macrophage colony–stimulating factor (MCSF), and vascular endothelial growth factor (VEGF) when compared to chondrocytes in MLs.


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

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

Analysis of cytokine expression in culture media. Human-specific cytokine antibody microarray membranes (RayBio Human Cytokine Array V) were probed with culture media from monolayers and spheroids. (A) Two representative array membranes corresponding to monolayers and spheroids from the same patient. (B) Comprehensive microarray maps of proteins secreted by chondrocytes in 2-D and 3-D after combining the results obtained from 3 different donors.
© Copyright Policy
Related In: Results  -  Collection

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

fig4-1947603510383856: Analysis of cytokine expression in culture media. Human-specific cytokine antibody microarray membranes (RayBio Human Cytokine Array V) were probed with culture media from monolayers and spheroids. (A) Two representative array membranes corresponding to monolayers and spheroids from the same patient. (B) Comprehensive microarray maps of proteins secreted by chondrocytes in 2-D and 3-D after combining the results obtained from 3 different donors.
Mentions: A large number of dots were unambiguously identified after 1-minute exposure of membranes to x-ray films (Fig. 4A). Comprehensive location maps for the membranes were prepared by a gray-scale code following the different intensities of the dots obtained after the reaction (Fig. 4B). It should be noted that the relative intensities of spots in each array may not reflect necessarily the relative amounts of proteins revealed after each reaction because the affinity of proteins for their respective antibodies may differ substantially. However, this technique allows an accurate comparison between the different experimental groups. The protein microarray results showed similar patterns of proteins produced by chondrocytes established in MLs and 3-D spheroids. However, the intensity of some of the dots in the arrays was significantly different between the 2 experimental groups. Monolayer growth showed enhanced production of growth-related oncogene protein (GRO), monocyte chemoattractant protein-1 (MCP-1), IL- 8, angiogenin, and insulin-like growth factor binding protein-2 (IGFBP-2). After redifferentiation of chondrocytes in spheroids, the arrays showed overexpression of CKb 8-1, macrophage colony–stimulating factor (MCSF), and vascular endothelial growth factor (VEGF) when compared to chondrocytes in MLs.

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