Limits...
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

Schematic diagram showing the experimental strategy used in our study. (A) Details of the cell isolation and culturing procedures, the metabolic labeling of cells, and the generation of the conditioned media are given sequentially. (B) The comparative proteomic analyses are described. Labeled and nonlabeled supernatants are mixed in a 1:1 ratio, and the total protein content is separated by 1-D gel electrophoresis. Thereafter, bands are excised, trypsinized, and analyzed by LC/MS-MS (liquid chromatography, tandem mass spectrometry). Peaks of labeled and nonlabeled peptides are compared and quantified. For qualitative comparison, identified proteins are contrasted in databases (KEGG analyses) and classified after functionality by literature search. Finally, validation of relevant proteins is done by specific immunodetection.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4300778&req=5

fig1-1947603510383856: Schematic diagram showing the experimental strategy used in our study. (A) Details of the cell isolation and culturing procedures, the metabolic labeling of cells, and the generation of the conditioned media are given sequentially. (B) The comparative proteomic analyses are described. Labeled and nonlabeled supernatants are mixed in a 1:1 ratio, and the total protein content is separated by 1-D gel electrophoresis. Thereafter, bands are excised, trypsinized, and analyzed by LC/MS-MS (liquid chromatography, tandem mass spectrometry). Peaks of labeled and nonlabeled peptides are compared and quantified. For qualitative comparison, identified proteins are contrasted in databases (KEGG analyses) and classified after functionality by literature search. Finally, validation of relevant proteins is done by specific immunodetection.

Mentions: DMEM and amino acids were from the SILAC Protein Identification and Quantitation Kit purchased from Invitrogen (Cat. no. SM10002, Carlsbad, CA). Basal medium was supplemented with ascorbic acid, L-glutamine, dexamethasone, antibiotics, and ITS supplement (Cat. no. I3146-5ML, Sigma-Aldrich). Amino acids [U-13C6] L-arginine and [U-13C6] L-lysine were added to the medium as described in the protocol for the kit. After initial cell expansion, 2 × 106 chondrocytes were seeded in T-175 cell culture flasks. Culture medium was originally supplemented with 10% serum to promote cell adherence during the first 24 hours. After cell attachment, cultures were extensively washed with basal DMEM medium, and thereafter, cells were incubated in 12 mL of [U-13C6] L-arginine and [U-13C6] L-lysine culture medium. Supernatants were removed after 5 days and replaced by new labeled medium. The new supernatants were conditioned during an additional 5 days and collected for analyses (Fig. 1A). The cell number was checked both in ML and spheroids at the end of the experiment by trypsinization and collagenase digestion, respectively. Numbers always ranged between 1.5 and 2 × 106. Later in the text, labeled cultures refer to cells maintained in special serum-free medium containing heavy amino acids (13C6 isotopes), whereas non-labeled cultures refer to cells maintained in serum-free media with regular amino acids (12C6 isotopes). Labeled and non-labeled cultures had been run in parallel and handled in the same way (Fig. 1).


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

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

Schematic diagram showing the experimental strategy used in our study. (A) Details of the cell isolation and culturing procedures, the metabolic labeling of cells, and the generation of the conditioned media are given sequentially. (B) The comparative proteomic analyses are described. Labeled and nonlabeled supernatants are mixed in a 1:1 ratio, and the total protein content is separated by 1-D gel electrophoresis. Thereafter, bands are excised, trypsinized, and analyzed by LC/MS-MS (liquid chromatography, tandem mass spectrometry). Peaks of labeled and nonlabeled peptides are compared and quantified. For qualitative comparison, identified proteins are contrasted in databases (KEGG analyses) and classified after functionality by literature search. Finally, validation of relevant proteins is done by specific immunodetection.
© Copyright Policy
Related In: Results  -  Collection

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

fig1-1947603510383856: Schematic diagram showing the experimental strategy used in our study. (A) Details of the cell isolation and culturing procedures, the metabolic labeling of cells, and the generation of the conditioned media are given sequentially. (B) The comparative proteomic analyses are described. Labeled and nonlabeled supernatants are mixed in a 1:1 ratio, and the total protein content is separated by 1-D gel electrophoresis. Thereafter, bands are excised, trypsinized, and analyzed by LC/MS-MS (liquid chromatography, tandem mass spectrometry). Peaks of labeled and nonlabeled peptides are compared and quantified. For qualitative comparison, identified proteins are contrasted in databases (KEGG analyses) and classified after functionality by literature search. Finally, validation of relevant proteins is done by specific immunodetection.
Mentions: DMEM and amino acids were from the SILAC Protein Identification and Quantitation Kit purchased from Invitrogen (Cat. no. SM10002, Carlsbad, CA). Basal medium was supplemented with ascorbic acid, L-glutamine, dexamethasone, antibiotics, and ITS supplement (Cat. no. I3146-5ML, Sigma-Aldrich). Amino acids [U-13C6] L-arginine and [U-13C6] L-lysine were added to the medium as described in the protocol for the kit. After initial cell expansion, 2 × 106 chondrocytes were seeded in T-175 cell culture flasks. Culture medium was originally supplemented with 10% serum to promote cell adherence during the first 24 hours. After cell attachment, cultures were extensively washed with basal DMEM medium, and thereafter, cells were incubated in 12 mL of [U-13C6] L-arginine and [U-13C6] L-lysine culture medium. Supernatants were removed after 5 days and replaced by new labeled medium. The new supernatants were conditioned during an additional 5 days and collected for analyses (Fig. 1A). The cell number was checked both in ML and spheroids at the end of the experiment by trypsinization and collagenase digestion, respectively. Numbers always ranged between 1.5 and 2 × 106. Later in the text, labeled cultures refer to cells maintained in special serum-free medium containing heavy amino acids (13C6 isotopes), whereas non-labeled cultures refer to cells maintained in serum-free media with regular amino acids (12C6 isotopes). Labeled and non-labeled cultures had been run in parallel and handled in the same way (Fig. 1).

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