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

Abstract

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. The rapid induction of type X collagen in culture and its evaluation through in situ hybridization permit the investigation of factors that enhance or delay the development of cell hypertrophy and maturation. This may have important implications for the understanding and study of endochondral ossification and fracture repair.