Figure 4  .

No effects of MTX on cartilage proteoglycan metabolism in culture. (A) No inhibition by MTX of chondrocyte proteoglycan synthesis in vitro. Bovine articular cartilage was incubated for two days in the presence of MTX (0 to 100 µM). The samples were subsequently labelled with [³⁵S]-sulphate for four hours, hydrolysed with papain, and incorporation of the tracer into cetyl-pyridiumchloride precipitable macromolecules was determined. Bars represent means and SEM of eight cartilage explants per group. Identical results were obtained, when cartilage was cultured for 4, 8, or 16 days in the presence of MTX (not shown). (B) and (C) No alteration by MTX of the hydrodynamic size of newly synthesised (solid circles) or total (open circles) proteoglycan monomers in cultured articular cartilage. Bovine cartilage explants were incubated for 16 days in the absence (B) or presence (100 µM) of MTX (C). Media were replaced every two days, keeping the respective MTX concentrations (that is, 0 or 100 µM) constant throughout the entire incubation period. The samples were then labelled with [³⁵S]-sulphate for four hours by addition of the tracer. The PGs were extracted from the cartilage using 4 M guanidine-HCl, and subjected to size exclusion chromatography on a Sephacryl-S-200-HR column under dissociative conditions. In the respective fractions, the newly synthesised PGs (as [³⁵S]; solid circles) were determined by liquid scintillation counting, and the total PGs (as chondroitin sulphate (CS); open circles) measured using a dimethyl-methyleneblue dye binding assay. The smooth lines are based on a peak analysis performed with the SIGMA-Plot software package for microcomputers (Jandel Scientific, Erkrath, Germany). The arrows indicate v₀ of the column, v_t corresponds to fraction no 77. (D) No MTX effect on the spontaneous PG release from articular cartilage in culture. Bovine articular cartilage explants were labelled with [³⁵S]-sulphate for four hours. Free sulphate was subsequently removed by repeatedly washing the samples in PBS (verified by size exclusion chromatography). Incubation was then continued for 10 days in the presence of various MTX concentrations. Media were harvested every two days and replaced with fresh media containing MTX in concentrations corresponding to the respective treatment group. Radiolabelled PGs contained in the harvested media, and PGs remaining in the cartilage samples, respectively, were determined by liquid scintillation counting as described in methods. The rate of PG release was then calculated.