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.

Changes in cyclic AMP and cyclic GMP levels during in vitro chondrogenesis.

cAMP and cGMP levels were measured in micro high-density cultures of chick limb bud mesenchyme cells stages 22-24 after 1, 2, 4 and 6 days of culturing. In these cultures, a consistent cartilage differentiation proceeds parallel to the progressive accumulation of cells in the G0 phase. The cAMP level increased by 45% by the time of the onset of cartilage phenotype expression, and significantly decreased thereafter. The cGMP level gradually diminished by a total of 39% during the period examined. It is suggested that the decrease in cell proliferation may be the consequence of the reduction of the cGMP level, and that a short-term marked elevation of cAMP level induces cartilage differentiation in the limb mesenchymal cells which are able to select only from a limited number of differentiation programmes.

Cartilage differentiation in micro-mass cultures of chicken limb buds.

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.

Effect of loading and prednisolone treatment on the glycosaminoglycan content of articular cartilage in dogs.

The effect of prolonged prednisolone administration on glycosaminoglycan components was studied by means of determinations of galactosamine, glucosamine, uronic acid, sulphate and sialic acid content in normally loaded and spared articular cartilages of the knee-joints in dogs. After 40 days, all glycosaminoglycan components decreased by 58% in the spared cartilage, as compated with the controls. Prolonged administration of 0.5 mg prednisolone/kg body weight (corresponding to a low therapeutic dose), elicited a further decrease in galactosamine, whereas the other components remained practically unchanged. In the normally loaded articular cartilage of prednisolone-treated dogs the glycosaminoglycan components decreased by about 11-31% compared with untreated controls. The quantitative data indicate that in the changes occurring the glycosaminoglycan composition of the articular cartilage due to relief of function and to prednisolone treatment, chondroitin suplphate is mainly involved. The changes due to prednisolone treatment are dependent on the functional demand of the joint.

Effect of prednisolone on the glycosaminoglycan components of the regenerating articular cartilage.

Investigations were performed on the effect of prednisolone (0.5 mg/kg) on the regenerating femoral articular cartilage of the knee joint in dogs that had been subjected to semiarthroplasty. After 70 days of prednisolone treatment the dogs were killed and the regenerating articular cartilage was removed, minced, and dried with acetone. The acetone-dried material was used for the determination of galactosamine, glucosamine, uronic acid, sulphate, sialic acid and hydroxyproline. Prednisolone treatment elicited a quantitative increase in galactosamine (30.2%), uronic acid (76.2%), and sulphate (9.1%), while no difference was observed in sialic acid content between the treated and untreated groups. From the molar ratio of the measured components it appears that prednisolone produced an increase in chondroitin sulphate and hyaluronic acid, and a decrease in the keratosulphate content of cartilage. By comparing the values measured in the regenerating articular cartilage of control and prednisolone-treated dogs with the values obtained in the mature articular cartilage, we may conclude that prednisolone--at least as regards the glycosaminoglycans of the ground substance--exerts an accelerating effect on cartilage regeneration.

Functional adaptation of the articular cartilage.

The effect of prolonged sparing and prolonged loading of the knee-joint of dogs on the glucosamine, sialic acid, sulphate and hydroxyproline contents of the articular cartilage was investigated. (a) In the articular cartilage of the spared leg the amount of sulphate decreased by 24.7%, while the sialic acid content remained unchanged. Hydroxyproline showed a slight decrease. (b) On increased loading, glucosamine augmented by 53% and sialic acid by 32.5%. No appreciable changes occurred in sulphate and hydroxyproline. It is concluded that an increased loading brings about accumulation of glycoproteins while the amount of sulphated glycosaminoglycans does not change appreciably; the glycoprotein content of the spared articular cartilage remains unchanged, whereas the chondroitin sulphate content decreases considerably.