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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

Qualitative analyses of proteins identified in culture media. (A) Microscopic photograph of 4-week-old human articular chondrocyte monolayer culture (bar = 100 µm). (B) Microscopic photograph of 3-D chondrocyte spheroids 7 days after initiation of the hanging drop culture (bar = 250 µm). (C and D) Percentage of metabolically labeled proteins found in the spent medium of chondrocytes in monolayers and spheroids, respectively, after 10 days of incubation in SILAC media. (E and F) Classification of identified proteins by previously described functionality.
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fig2-1947603510383856: Qualitative analyses of proteins identified in culture media. (A) Microscopic photograph of 4-week-old human articular chondrocyte monolayer culture (bar = 100 µm). (B) Microscopic photograph of 3-D chondrocyte spheroids 7 days after initiation of the hanging drop culture (bar = 250 µm). (C and D) Percentage of metabolically labeled proteins found in the spent medium of chondrocytes in monolayers and spheroids, respectively, after 10 days of incubation in SILAC media. (E and F) Classification of identified proteins by previously described functionality.

Mentions: Metabolic labeling of newly produced proteins was allowed for 10 days by cells cultured in either 2-D or 3-D environments. A total of 93 proteins were consistently identified in the supernatants of chondrocyte ML from the 3 individuals included in the study, whereas in spheroids, the number of identified proteins was reduced to 62. The cell cultures from all 3 patients were treated identically throughout all the experimental parts. Qualitatively, the data showed only minor variations among the different individuals. In supernatants from ML, over 95% of proteins incorporated 13C-arg and 13C-lys, whereas in spheroids, the proportion of nonlabeled or weakly labeled proteins was approximately 20% of all identified proteins (Fig. 2 C and D). Most of the nonlabeled proteins found in the media corresponded to plasma-related proteins stemming from the serum used in early stages of cell expansion. In 3-D conditions, some of the nonlabeled proteins corresponded to chondrocyte-derived components originating from the pre-SILAC period. All labeled (newly produced) proteins found in the supernatants were categorized by their functionality, and the 2 conditions were compared (Fig. 2 E and F). Qualitative comparison of the 2 groups show that a larger number of extracellular matrix components were released into the media during ML growth (50% v. 36%); however, chondrocytes in 3-D expressed a lower number of matrix catabolic agents (10% v. 9%), higher number of matrix anabolic agents (5% v. 11%), and higher number of anti-inflammatory and antioxidant proteins (13% v. 24%).


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

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

Qualitative analyses of proteins identified in culture media. (A) Microscopic photograph of 4-week-old human articular chondrocyte monolayer culture (bar = 100 µm). (B) Microscopic photograph of 3-D chondrocyte spheroids 7 days after initiation of the hanging drop culture (bar = 250 µm). (C and D) Percentage of metabolically labeled proteins found in the spent medium of chondrocytes in monolayers and spheroids, respectively, after 10 days of incubation in SILAC media. (E and F) Classification of identified proteins by previously described functionality.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4300778&req=5

fig2-1947603510383856: Qualitative analyses of proteins identified in culture media. (A) Microscopic photograph of 4-week-old human articular chondrocyte monolayer culture (bar = 100 µm). (B) Microscopic photograph of 3-D chondrocyte spheroids 7 days after initiation of the hanging drop culture (bar = 250 µm). (C and D) Percentage of metabolically labeled proteins found in the spent medium of chondrocytes in monolayers and spheroids, respectively, after 10 days of incubation in SILAC media. (E and F) Classification of identified proteins by previously described functionality.
Mentions: Metabolic labeling of newly produced proteins was allowed for 10 days by cells cultured in either 2-D or 3-D environments. A total of 93 proteins were consistently identified in the supernatants of chondrocyte ML from the 3 individuals included in the study, whereas in spheroids, the number of identified proteins was reduced to 62. The cell cultures from all 3 patients were treated identically throughout all the experimental parts. Qualitatively, the data showed only minor variations among the different individuals. In supernatants from ML, over 95% of proteins incorporated 13C-arg and 13C-lys, whereas in spheroids, the proportion of nonlabeled or weakly labeled proteins was approximately 20% of all identified proteins (Fig. 2 C and D). Most of the nonlabeled proteins found in the media corresponded to plasma-related proteins stemming from the serum used in early stages of cell expansion. In 3-D conditions, some of the nonlabeled proteins corresponded to chondrocyte-derived components originating from the pre-SILAC period. All labeled (newly produced) proteins found in the supernatants were categorized by their functionality, and the 2 conditions were compared (Fig. 2 E and F). Qualitative comparison of the 2 groups show that a larger number of extracellular matrix components were released into the media during ML growth (50% v. 36%); however, chondrocytes in 3-D expressed a lower number of matrix catabolic agents (10% v. 9%), higher number of matrix anabolic agents (5% v. 11%), and higher number of anti-inflammatory and antioxidant proteins (13% v. 24%).

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