Interrelationship between cyclic AMP level and chondrogenesis in vitro.

A consistent chondrogenesis takes place in micro-mass cultures of stage 23-24 chicken limb bud mesenchymal cells. In these cultures a short, marked elevation of cAMP level was detected at the time of the onset of cartilage phenotype expression. On the other hand, exogeneous glycosaminoglycans which inhibited chondrogenesis caused a reduction in the cAMP level of the cells. These correlations between cAMP level and phenotypic characteristics suggest that, among other things required in chondrogenesis, cAMP level may be a prominent factor.

Exogenous glycosaminoglycans modulate chondrogenesis, cyclic AMP level and cell growth in limb bud mesenchyme cultures.

Effects of hyaluronate, heparin and chondroitin-6-sulfate were studied on micromass cultures of chick limb bud mesenchyme (Hamburger and Hamilton stages 23-24). Histochemical, electron microscopical, biochemical and radiochemical investigations of day 4 cultures revealed dose-dependent inhibitory effects of these glycosaminoglycans on chondrogenesis, cyclic AMP level and growth of cells. In addition, hyaluronate with 100 micrograms/ml dose caused a displacement of newly formed proteoglycan from cultures into the medium. It is supposed that exogenous glycosaminoglycans influence ionic equilibrium in the immediate vicinity of cells and disturb the organization of the prechondrogenic extracellular matrix resulting in alterations of cell membrane--cytoskeleton associations. These alterations may provoke a reduction in cyclic AMP level and DNA synthesis. It is suggested that a reduction in cyclic AMP level preceding the expression of cartilage phenotype results in the inhibition of chondrogenesis.

Proteoglycan biosynthesis is stimulated by D-penicillamine in chondrifying high density cell cultures.

Chondrifying high density cell cultures of stage 22-24 chick embryo limb bud mesenchyme were treated with 5, 10 and 15 mmol/l D-penicillamine (DPA) for 4 and 6 days. The cultures were analyzed with morphological and biochemical techniques to learn more about the effect of DPA on the metabolism of cartilage glycosaminoglycans (GAGs). Using light and electron microscopic histochemical reactions for GAG, a considerable increase in the intensity of staining of the cartilage matrix could be detected in cultures treated with DPA as compared to the untreated controls. The uronic acid content of the treated cultures was higher than that of the controls. Liquid scintillation measurements and autoradiography revealed that DPA treatment increased the 35S-sulfate into the cultures. These data suggest that DPA - besides its well known inhibitory effect on collagen crosslink formation - alters the metabolism of sulfated GAGs in differentiating cartilage. It is supposed that DPA stimulates the biosynthesis of these macromolecules.

In vitro cartilage differentiation: modification of division cycle and effect of some polyanions.

A modification of division cell cycle under conditions of in vitro cartilage differentiation could be demonstrated. The frequency distribution of histograms showed that the G1 cells represented 50% of total cells number at zero time (i.e. in suspension before inoculation), 69% on day 2 and about 90% in following period of days 4, 6 and 14. Dextran sulfate and hyaluronic acid produced a conspicuous inhibition and the chondroitin sulfate as well as heparin exerted a moderate inhibiting effect on the cell proliferation.

Studies on cartilage formation. XXII. Investigations of certain oxidative metabolic processes in regenerating articular cartilage.

The distal articular surface of the femur was surgically removed in 57 dogs. Succinate dehydrogenase and cytochrome oxidase activities were assayed on postoperative days 7, 20, 26, 33 and 70 in the regenerating, chondrifying articular surface and in the granulation tissue adhering to the capsule. In the 70-day samples, the cyanide-induced inhibition of oxygen consumption was determined and enzyme histochemical reactions (cytochrome oxidase, monoamine oxidase, xanthine oxidase, peroxidase and "catalase") were performed. The succinate dehydrogenase activity was the highest in the early postoperative stage in both tissues. This was followed by a definite decrease and a subsequent significant increase in activity when chondrification took place. Measurement of cytochrome oxidase activity could not reveal any convincing result, presumably because of the properties of the tissues studied. The oxygen consumption by the chondrifying articular surface at 70 days was inhibited to about 50% by cyanide, and about 90% inhibition was observed in the tissue adhering to the capsule. The cells of the regenerating articular surface possess cytochrome oxidase and a cyanide- (and sodium azide-) resistant oxidase activity. The enzyme activity of the cartilaginous islets exceeded that of their connective tissue environment. The cytochrome oxidase activity increased in the cells during cartilage differentiation. Presumably, some further cyanide-sensitive and cyanide-resistant oxidases are present in chondroblasts and young chondrocytes.