Fig. 1. Northern blot analysis of aggrecan, procollagen (α₁) II, HAS-2 mRNA, GAPDH and 28 S ribosomal RNA. (A) Aggrecan, procollagen (α₁) II and HAS-2 expression on bovine primary chondrocytes at the end of culture period. (B) Total RNA samples were collected at 0, 4, 8 and 24 h after addition of different forms of sulphated and non-sulphated hexosamines at 1 mM concentrations. Representative Northern blots are presented. Three donors were used for three separate experiments. Glucosamine: GlcN, galactosamine: GalN, mannosamine: ManN, glucosamine 3-sulphate: GlcN-3S, glucosamine 6-sulphate: GlcN-6S, galactosamine 6-sulphate: GalN-6S.

Fig. 2. Effects of glucosamine sulphate (GS) salt or glucose (Glc) on aggrecan mRNA expression levels. (A) Representative Northern blot from the cultures of bovine primary chondrocytes. Chondrocytes from 13 individual animals were treated for 24 h with 0, 100 μM and 1 mM of glucosamine sulphate salt or glucose. Total RNAs (20 μg) were separated on a 1.0% agarose/formaldehyde gel, and the probes for aggrecan, GAPDH, and 28 S ribosomal RNA were used for analysis. (B) The average of densitometric analysis of aggrecan mRNAs from 13 donors analyzed with Northern blot assay. The data are presented as a percent of control ± standard deviation, and the control is the untreated culture.

Fig. 3. Effects of glucosamine sulphate (GS) salt on HAS-2 mRNA expression levels. Chondrocytes were treated for 24 h with 0, 10, 50, 100, 500 μM and 1 mM of glucosamine sulphate salt. Total RNAs (20 μg) were separated on a 1.0% agarose/formaldehyde gel, and the probes for aggrecan, HAS-2, GAPDH, and 28 S were used for analysis. HAS-2 could be detected in three cell isolates out of thirteen. (A) HAS-2 mRNA level was elevated at 100 μM concentration of glucosamine sulphate in one donor. (B) The average of densitometric analysis of HAS-2 mRNAs from 3 donors analyzed with Northern blot assay.

Effects of sulphated and non-sulphated hexosamines on glycosaminoglycan synthesis in high (4.5 g/l) or low (1.0 g/l) glucose concentration evaluated by ³⁵S-sulphate incorporation analysis

Chondrocytes were cultured in the presence of 1 mM glucosamine (GlcN), galactosamine (GalN), mannosamine (ManN) or glucosamine 3-sulphate (GlcN-3S) in high or low glucose concentration. The differences between control (untreated groups) and treated groups were evaluated with non-parametric K-independent (Kruskal–Wallis test) and post hoc test.

Glycosaminoglycan (GAG) synthesis measured in the culture medium by ³⁵S-sulphate incorporation analysis

Bovine primary chondrocytes were cultured in 0, 100 μM and 1 mM of glucosamine sulphate (GS) salt or glucose (Glc) in DMEM with or without serum for 24 or 72 h. The differences between control (untreated groups) and treated groups were evaluated with non-parametric K-independent (Kruskal–Wallis test) and post hoc test (no significant changes were observed).