THE EXTRACELLULAR-MATRIX IN CARTILAGE ORGANOID CULTURE - BIOCHEMICAL, IMMUNOMORPHOLOGICAL AND ELECTRON-MICROSCOPIC STUDIES

Limb bud mesenchymal cells obtained from day-12 mouse embryos were grown at high density on a membrane filter (pore size 0.2-mu-m) at the medium/air interphase. Chondrogenesis in this so-called cartilage organoid culture was monitored quantitatively by immunological estimation of type I and type II collagen and qualitatively by indirect immunofluorescence and electron microscopy in the course of a 3 6 days culture period. Three stages of cartilage development could be substantiated: 1. Formation of cartilage between days 2 and 7; 2. maturation of cartilage between days 9 and 13; 3. degeneration of cartilage beginning at day 20. Differentiation in cell aggregates and a loose mesenchymal tissue occurred during the first two days of the culture period. Type II collagen synthesis started in cell aggregates two days after plating and after 6 days in culture distinct cartilage nodules had developed which were embedded in loose connective tissue that contained type I collagen. During this period the type II collagen content increased progressively from 2.3-mu-g (day 3) to nearly 40-mu-g (day 7) per mg dry weight, whereas the type I collagen level increased more linearly from 2.7 to 21.3-mu-g/mg dry weight. The second period was characterized by enlargement and fusion of cartilage nodules and a diminished increase in type II collagen content from 45 to 60-mu-g/mg dryweight. Enlargement and fusion occurred by matrix production as well as by transformation of perichondrial cells into chondroblasts. Type I collagen synthesis enhanced from 29 to 54-mu-g/mg. Hypertrophic chondrocytes could be demonstrated ultrastructurally. At the third stage a nearly continuous layer of cartilage on the membrane filter covered by noncartilagenous tissue had developed. To some extent chondrocytes lost their matrix capsule and changed into fibroblast-like cells accompanied by a switch of collagen synthesis from type II to type I collagen. Quantitative studies yielded a constant level of about 60-mu-g/mg type II collagen and a further increase in type I collagen from 77 to 116-mu-g/mg dry weight. This study reveals an in vitro model of a prolonged, but almost identical image of chondrogenesis in vivo prior to endochondral mineralization which may be useful for investigations on cartilage differentiation, maturation and degeneration.