Composition and organization of the collagen network produced by fetal bovine chondrocytes cultured at high density.
Résumé
Fetal bovine chondrocytes isolated from the resting zone of epiphyseal cartilage were maintained in high-density culture for 4 weeks. From Day 2 in culture, the chondrocytes deposited an extracellular matrix composed of Types II, IX, and XI collagen. Types IX and XI collagen were restricted to the pericellular domain from Day 5. By 2 weeks the entire cell layer stained for antibodies to Type II and IX collagens. Type XI could be demonstrated throughout the cell layer by pepsinization of the sections. Results from both rotary shadowing and immunochemistry showed that the fibrils formed in culture were heterotypic, with Type IX collagen arranged along the surface and with Type XI collagen buried in Type II fibrils. Nonspecific Type VI collagen and the glycoproteins tenascin and fibrillin, previously described in cartilaginous tissue, were identified by their ultrastructural characteristics in the cell layer homogenate. Although the cells presented morphological characteristics of chondrocytes and still expressed cartilage-specific collagens, the appearance of Type I collagen in the culture cell layer after 4 weeks of culture demonstrates a partial dedifferentiation of the chondrocytes. The culture system described in this report provides an interesting tool for maintaining chondrocytes in a cartilage-like matrix to study the influence of different physical and chemical factors on the expression and differentiation of the cells.Fetal bovine chondrocytes isolated from the resting zone of epiphyseal cartilage were maintained in high-density culture for 4 weeks. From Day 2 in culture, the chondrocytes deposited an extracellular matrix composed of Types II, IX, and XI collagen. Types IX and XI collagen were restricted to the pericellular domain from Day 5. By 2 weeks the entire cell layer stained for antibodies to Type II and IX collagens. Type XI could be demonstrated throughout the cell layer by pepsinization of the sections. Results from both rotary shadowing and immunochemistry showed that the fibrils formed in culture were heterotypic, with Type IX collagen arranged along the surface and with Type XI collagen buried in Type II fibrils. Nonspecific Type VI collagen and the glycoproteins tenascin and fibrillin, previously described in cartilaginous tissue, were identified by their ultrastructural characteristics in the cell layer homogenate. Although the cells presented morphological characteristics of chondrocytes and still expressed cartilage-specific collagens, the appearance of Type I collagen in the culture cell layer after 4 weeks of culture demonstrates a partial dedifferentiation of the chondrocytes. The culture system described in this report provides an interesting tool for maintaining chondrocytes in a cartilage-like matrix to study the influence of different physical and chemical factors on the expression and differentiation of the cells.