Cartilage oligomeric matrix protein (COMP) and collagen IX are sensitive markers for the differentiation state of articular primary chondrocytes.
Primary chondrocytes dedifferentiate in serial monolayer with respect to their morphological and biosynthetic phenotype. They change from a round to a flattened fibroblast-like shape, and collagen I is secreted instead of the cartilage-specific collagen II. We analysed in detail the time course of dedifferentiation of mature bovine articular chondrocytes in monolayer for up to 32 weeks. Assessment of RNA expression by reverse transcription-PCR led to the identification of two novel phenotypical markers, the cartilage oligomeric matrix protein (COMP) and collagen IX, which are down-regulated faster than the widely accepted marker, collagen II. The different kinetics of COMP and collagen expression suggest differential regulation at the level of transcription. Immunostaining and metabolic labelling experiments confirmed the switch in the collagen expression pattern and the rapid down-regulation of de novo synthesis of COMP and collagen IX. Culture of chondrocytes in a three-dimensional matrix is known to stabilize the chondrocytic phenotype. We maintained cells for up to 28 weeks in an alginate bead system, which prevented dedifferentiation and led to a stabilization of collagen and COMP expression. Immunohistochemical analysis of the alginate beads revealed a similar distribution of matrix proteins to that found in vivo. Chondrocytes were transferred after a variable length of monolayer culture into the alginate matrix and the potential for redifferentiation was investigated. The re-expression of COMP and collagen IX was differentially regulated. The expression of COMP was re-induced within days after transfer into the three-dimensional matrix, while the expression of collagen IX was irreversibly down-regulated. In summary, these results demonstrate that the potential for redifferentiation decreases with increasing length of monolayer culture and show that the alginate bead system represents an attractive in vitro model to study the chondrocyte de- and re-differentiation processes, as well as extracellular matrix assembly.
Zaucke
F
F
Institute for Biochemistry II, Medical Faculty, University of Cologne, Joseph-Stelzmann-Strasse 52, D-50931 Cologne, Germany. frank.zaucke@uni-koeln.de
Dinser
R
R
Maurer
P
P
Paulsson
M
M
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis*; D002358:Cartilage, Articular / Q000166:cytology*; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism*; D003094:Collagen / Q000096:biosynthesis*; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D007150:Immunohistochemistry; D007700:Kinetics; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence; D010641:Phenotype; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic; D014166:Transferases
D001426:Bacterial Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D003094:Collagen; D014166:Transferases; C064995:comP protein, Bacillus subtilis