In vitro chondrogenesis in mouse limb mesenchymal cells; changes in ultrastructure and proteoglycan synthesis.

Mouse chondrocytes were allowed to differentiate from 11-day dissociated limb-bud mesenchymal cells cultured at high density. Over a period of 5 days in culture these cells differentiated from a culture containing a population of mesenchymal cells and myoblasts into one containing cartilage nodules. Chondrogenesis, as demonstrated by the sulfated proteoglycans (PGS) synthesized, was monitored quantitatively and qualitatively by molecular sieve chromatography on polyacrylamide and 1 % agarose columns, and by examination of cultures with phase contrast and transmission electron microscopy. Chromatographically, the PGS synthesized could be separated into three fractions (la, lb, and II). In the 24 h cultures a small amount of PGS was synthesized and was predominantly of fraction II. As time in culture elapsed there was, concomitant with chondrogenesis, an increase in PGS synthesis with a preferential increase in peaks la and lb. Peaks la and lb were shown to have an aggregate-subunit relationship. Fraction la could be dissociated to chromatograph at the lb position, but the disaggregation was not reversible. Digestion of the various chromatographic fractions extracted from the cell layer of the 5-day cultures by chondroitinase ABC and AC revealed that fraction I contained 80 % chondroitin-4-sulfate, 20 % chondroitin-6sulfate, and negligible amounts of dermatan sulfate, while chondroitinase ABC and AC digestion of fraction II showed 55 % chondroitin-4-sulfate, 0-3-7 % chondroitin-6-sulfate, and 20 % dermatan sulfate. Cytological studies revealed that as nodule development progressed there was a concurrent development of the endoplasmic reticulum and extracellular matrix, and a reduction in mitochondria.