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Fig. 4. Treatment with DAPT did not affect the adhesion of surface and deep zone chondrocytes to fibronectin (A) but abolished the CFE of surface zone cells at 6 and 10 (B) days. Indeed, the CFE of DAPT-incubated cells was not different from that of deep zone cells at either time point. Transfection with NICD rescued this abolition of CFE (C). NICD transfection did not increase CFE in cells not treated with DAPT (P>0.05). Cartilage explants were removed from 7-day bovine articular cartilage and cultured in the presence (D) or absence (E) of 50 nM DAPT for 7 days as described in Materials and Methods. Note that in the presence of DAPT, an acellular weakly stained band is present beneath the surface zone (arrows). These images represent a selection from 3 separate experiments each containing 6 explants per treatment. Note that the image in D is the other half of the explant from that shown in E. Using a graduated grid, the number of cells 0-100 and 101-200 µm from the articular surface was counted and the region 101-200 µm from the articular surface was shown to contain fewer cells in treated samples relative to controls (F). Explants were treated with DAPT for 7 days with the addition of BrdU on days 4, 5 and 6. Localisation of BrdU in controls (G) reveals cell proliferation, whereas there was no BrdU localisation in DAPT-treated samples (H). ns, P>0.05 compared with surf control; *P<0.01 compared with DAPT treated; **P<0.05 compared with all other cohorts, DMSO; 0.1%, dimethyl sulfoxide, Mock; no plasmid, PBS; fibronectin only.
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