We wished to determine whether NO modulates the pathogenesis of OA, inducing apoptosis by means of the inhibition of mitochondrial function Materials and methods Cartilage acquisition and cell isolation Normal human cartilage from femoral heads and knees was obtained from 11 adult donors without history http://www.selleckchem.com/products/AP24534.html of joint disease and who had macroscopically normal cartilage, human OA cartilage was obtained from the femoral heads of 12 patients. All patients and healthy donors have signed the informed consent and the project was approved by the Regional Ethical Committee from Galicia. Small cartilage fragments were digested as previously described. Primary culture of chondrocytes Chondrocytes were recovered and plated at high density in DMEM supple mented with 100 units ml penicillin, 100 ug ml strepto mycin, 1% glutamine, and 10% FCS.

Chondrocytes were incubated at 37 C in a humidified gas mixture Inhibitors,Modulators,Libraries containing 5% CO2 balanced with air. Chondrocytes were used at weeks 2 to 3 at confluence in primary culture. Cell viability was assessed by trypan blue dye exclusion, and stained cells were discarded to carry out experiments. Inhibitors,Modulators,Libraries General procedure and NO donor compounds employed NO donor compounds were added from 60 mM stock solution dissolved in 0. 1 M NaOH and 10 mM stock solution dissolved in medium. This NO donor compound was freshly prepared before each experiment but NOC 12 was stored as 60 mM stock solutions in 0. 1 mM NaOH at 20 C. Chondrocytes were first seeded in DMEM with 5% FCS inactivated for 24 hours, and then the NO donor compound was added directly to the culture medium and allowed to incubate for an additional 5, 12, 24 and 48 hour period, depending on each experiment.

Experiments without glucose Inhibitors,Modulators,Libraries were carried out in DMEM glucose free med ium supplemented in the same way as the standard. Quantification of nitrites The NO production of chondrocyte cells was measured by estimating nitrite Inhibitors,Modulators,Libraries accumulation using the Griess reagent ethylenediamine dihydrochloride in 5% H3PO4 as previously described. Chondro cytes were cultured in 96 well plates and sti mulated with different NO donors for 5, 24 and 48 hours. DNA labelling technique with propidium iodide for flow cytometry analysis Chondrocytes were incubated Inhibitors,Modulators,Libraries with different NO donors for 12, 24 and 48 hours. Then cells were fixed in 70% ethanol at 4 C for 60 min utes, washed and incubated with RNAse and propidium iodide for 15 minutes at room temperature in the dark and kept at 4 C.

PI fluor escence of nuclei was measured by flow cytometry on a FACScan using a 560 nm dichromatic mirror and a 600 nm band pass filter. Data are expressed as percent apoptotic nuclei. Morphological evidence find more of apoptosis For morphological studies, chondrocytes were cultured in 8 well slides and treated with 1 mM of different NO donors for 24 hours.