Table 1.
Gene specific primers for qRT-PCR.
Fig 1.
STS inhibits murine joint chondrocytes mineralization.
(a) Alizarin red and Von Kossa staining of murine chondrocytes treated or not (Nt) with 25mM STS for 7 days in complete BGJb. Pictures represent one representative culture well of one experiment (five independent experiments were performed). Scale bar 100μm. The graph shows Alizarin red absorbance at 405nm, expressed in % over Nt cells at 1, 3 or 7 days of culture. Values represent means±SD of triplicates from one representative experiment of three independent experiments. (b) Percentage of cytotoxicity in murine chondrocytes treated or not with 25mM STS for 1, 3 or 7 days. Values represent means±SD of triplicates from one representative experiment of three independent experiments. (c) Alizarin red staining of murine chondrocytes treated or not (Nt) with different concentrations of STS for 7 days. Pictures represent triplicates from one experiment. Scale bar 100μm. The graph shows Alizarin red absorbance at 405nm, expressed in % over Nt cells at 7 days of culture. Values represent means±SD of triplicates from one representative experiment of three independent experiments. (d) qRT-PCR of the indicated genes in murine chondrocytes treated or not with 25 mM STS for 7 days in complete DMEM. Values represent means±SD of triplicate samples. *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. nd = not detectable
Fig 2.
STS inhibits chondrocyte IL-6 and MCP-1 secretion in a dose-dependent manner.
(a) IL-6 and MCP-1 secretion by primed murine joint chondrocytes treated or not with 25mM STS for 1, 3 or 7 days. Values represent means±SD of triplicates from one representative experiment of three independent experiments. (b) qRT-PCR of IL-6 gene in primed murine chondrocytes treated or not with 25 mM STS for 3 days. (c) IL-6 and MCP-1 secretion by primed murine joint chondrocytes treated or not with different concentrations of STS for 3 and 7 days. Values represent means±SD of triplicates from one experiment. *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Fig 3.
STS inhibits HA-induced cytokine and chemokine selectively in chondrocytes.
(a) IL-6 and MCP-1 secretion by primed murine joint chondrocytes and (b) IL-6, MCP-1, IL-1β and TNF-α secretion by primed murine BMDM, stimulated or not with HA crystals (500ug/ml) and treated or not with 25mM STS 25mM for 2, 6 and 24hrs. Values represent means±SD of triplicates from one representative experiment of three independent experiments. (c) IL-6 secretion by human cartilage explants was measured by ELISA. Explants were stimulated with 500μg/ml of HA crystals in presence or absence of STS 25Mm for 24 h. Matched-halves of cartilage explants are connected by a line (5 explants for patients 1, 3 for patient 2, 4 for patient 3). Values represent means±SD of triplicate samples. *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Fig 4.
STS inhibits HA-induced ROS production and enzymes involved in cartilage degradation.
ROS generation by (a) murine joint chondrocytes and (b) BMDM stimulated or not with HA crystals (500ug/ml) and treated or not with 25mM STS for 1h. Dihydroethidium (DHE) staining was used as an indicator of intracellular ROS generation. MitoSOX™ Red reagent was used as an indicator of mitochondrial ROS generation. Values represent means±SD of triplicates from one representative experiment of two independent experiments and are expressed as %of ROS in unstimulated cells. (c) qRT-PCR of the indicated genes in murine chondrocytes stimulated or not with HA crystals (500ug/ml) and treated or not with 25mM STS for 30 minutes in DMEM. Values represent means±SD of triplicate samples. *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Fig 5.
STS inhibits formation of new calcific deposits and cartilage degradation in experimental OA.
(a) Representative micro-CT scan images of menisectomized murine knee joints after vehicle (PBS) or STS treatment for two months after surgery. White arrows show periarticular deposits in menisectomized knees treated with vehicle and their reduction in STS treated mice. Graphs show CTAnalyzer quantitative analysis of new formation volume (mm3) and new formation crystal content (μg) in PBS- and STS-treated menisectomized mice. Data are expressed as the mean±SD (b) Representative histologies of PBS- and STS-treated menisectomized knees, stained with Safranin-O. Red arrows show degenerative OA changes in the articular cartilage of PBS-treated mice. Scale bars 150 μm. Graphs show tibial and femoral scoring of cartilage damage and Safranin-O loss, accordingly to OARSI method. Values represent means±SD. (c) Correlation graph between tibial cartilage damage score and new formation volume of pooled PBS- and STS-treated mice. (Mice number: PBS n = 20, STS n = 19).