FIGURE 3.

Effect of 4-MU on chondrocytes and cartilage explants induced by IL-1β, LPS, or HA oligosaccharides. To explore the effect of 4-MU on other reagents known to induce a pro-catabolic state in chondrocytes, high density monolayer cultures of bovine cells were treated without or with (−/+) 250 μg/ml HA oligosaccharides (A) or 10 ng/ml LPS (B) along with co-treatment with 0 or 1.0 mm 4-MU. After 24 h, cell lysates and serum-free media were isolated and processed for Western blotting for MMP13 protein. Shown are representative examples of three replicated, independent experiments. Aliquots of equal protein were loaded onto gels from the cell lysate fractions, and the medium samples represent aliquots of equal volume. All blots with cell lysates were stripped and re-probed for β-actin. To explore the effect of 4-MU on release of glycosaminoglycan, 6-mm cored disks of bovine cartilage (C, D, and G–L) or human OA cartilage (E and F) were treated without or with 1.0 ng/ml IL-1β or 250 μg/ml HA oligosaccharides as labeled (−/+) and including co-treatment with 1.0 nm 4-MU. In some experiments, serum-free medium was collected after 48 h and processed for glycosaminoglycan content using a dimethylmethylene blue colorimetric assay. Black bars depict values of control cultures. Values, shown in units of micrograms/ml, represent the average ± S.D. of data derived from five separate explant cultures of bovine cartilage for each condition (C and D) and seven separate explant cultures of human OA cartilage for each condition, performed on tissues derived from each of three patients (E and F). For statistical analysis, a two-way ANOVA followed by Tukey-Kramer test was used; *, p < 0.05; **, p < 0.01. F represents individual (non-averaged) data values that comprise the averaged data shown in E, wherein each individual IL-1β + 4-MU culture was compared with its control experiment (+IL-1β) set to a unit value of 100%. G–L depict 8-μm sections of fixed/paraffin-embedded bovine cartilage cores processed at the end of the experiment and stained with safranin O/fast green. G, H, J, and K show a representative experiment of explants cultured for 1 week in serum-free medium without (Ctrl) or with 1 ng/ml IL-1β and with 0 or 0.5 mm 4-MU as labeled. I and L represent bovine cartilage cultured without or with 0.5 mm 4-MU for 4 weeks. All images are of equivalent magnification and time exposure settings.