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.

Effects of hyaluronate, heparin and chondroitin-6-sulfate were studied on micro-mass 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 μg/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.

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.

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.

In micro-mass cultures of stage 23-24 chicken embryos the expression of cartilage phenotype was density dependent and it always took place practically to the same extent at the density of 2 X 10(5) cells per culture. The cells formed aggregates on day 1 and cartilage nodules appeared on day 2. By day 3, 17% of the culture surface consisted of areas showing cartilage differentiation and these regions represented more than 50% on day 6. By day 14, the centre of th cultures contained a mass of cartilage. The progression of cartilage differentiation was indicated by the increasing amount of toluidine blue fixed by the cultures. By day 14 the DNA and protein contents increased 8.7 fold and 16.7 fold, respectively. Uronic acid and OH-proline were detected from the 2nd day. From day 3 to 14, the microgram uronic acid and microgram OH-proline per microgram DNA gradually increased 25 fold and 14 fold, respectively. A decreased density of the inoculum reduced the probability of cartilage formation and it failed to occur at the density of 1.25 X 10(4) cells per culture. Stage 19-20 limb cultures with identical density had the same morphological characteristics. Aggregates appeared but nodules failed to do so in cell cultures of stage 28 limb bud soft tissue region. It is supposed that an oxygen gradient may be present in the cell aggregates.

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.

Studies and comparison of the hexosamine and hydroxyprolin production by fibroblasts cultivated in vitro at different oxygen concentrations. The results show that the highest hexosamine production occurs at a low oxygen concentration (5%). The highest hydroxyprolin production occurs at 20% O2; this oxygen concentration is considered as an hyperoxic one for fibroblasts cultivated in vitro. Besides it has been ascertained that the highest sulphated MPS production occurs between 5 and 20% oxygen without any significant variation.