Analysis of type II and type X collagen synthesis in cultured growth plate chondrocytes by in situ hybridization: rapid induction of type X collagen in culture

• Published in: Journal of bone and mineral research Vol. 9; no. 11; p. 1713
• Main Authors: O'Keefe, R J, Puzas, J E, Loveys, L, Hicks, D G, Rosier, R N
• Format: Journal Article
• Language: English
• Published: United States 01.11.1994
• Subjects: Animals; Autoradiography; Blotting, Northern; Cell Fractionation; Cells, Cultured; Chickens; Collagen - biosynthesis; Collagen - genetics; DNA, Complementary - genetics; DNA, Complementary - metabolism; Electrophoresis, Polyacrylamide Gel; Growth Plate - cytology; Growth Plate - metabolism; In Situ Hybridization; Oligonucleotide Probes; RNA, Messenger - metabolism
• ISSN: 0884-0431
• DOI: 10.1002/jbmr.5650091107

Type X collagen is produced by hypertrophic chondrocytes and serves as a highly specific marker for chondrocyte maturation. This study was designed to compare the expression of type II and type X collagen in growth plate sections and in distinct populations of chondrocytes in culture by in situ hybridization. Growth plate sections were treated with type II and type X collagen cDNA probes. Type II collagen mRNA was present throughout the growth plate but greatest in the lower proliferating and upper hypertrophic regions. In contrast, type X collagen was expressed only in the hypertrophic region. Northern analysis confirmed the specificity of the probe for type X collagen mRNA. Chick growth plate chondrocytes were separated by countercurrent centrifugal elutriation into five distinct populations and plated in serum-containing medium. These cultures were examined at varying times after plating for the expression of type II and type X collagen mRNA. At 3 h, type II collagen was present in the majority of the cells in all fractions, and approximately 15-20% of the cells expressed type X collagen mRNA. The cells expressing type X were from the hypertrophic region. At 24 h, however, nearly all cells in culture expressed type X mRNA, and there was a decrease in expression of type II collagen mRNA. Similar results were obtained in cultures in the absence of serum, and SDS-PAGE analysis of collagen synthesis confirmed the expression of type X collagen in all populations of fractionated cells at 24 h at the protein level. Type X collagen is an important marker through which cellular matruation can be evaluated in culture.