Figure 5

shRNA construct against matrix metalloproteinase 2 (MMP-2) (shMMP2) inhibits localized gelatin degradation surrounding annulus fibrosus (AF) cells. Microscopy images of gelatin films seeded with (a) no cells, (b) noninfected AF cells, (c) cells transduced with shMMP2, and (d) cells transduced with shNon. Films were stained at 4 days in culture with Ponceau S and visualized immediately. Scale bars represent 200 μm. All images were taken at 100×. Films with no cells (a) stained evenly red, whereas films seeded with noninfected AF cells (b) showed focal degradation of gelatin in the areas immediately surrounding cells, which is shown by the lighter-stained regions. Similar results are seen with cells transduced with shNon (d). Cells transduced with shMMP2 (c) show little to no focal degradation of gelatin, implying that MMP-2 is directly involved in gelatin breakdown. (e) Quantification of gelatin degradation. Gelatin films seeded with noninfected control AF cells and shNon-transduced cells were similar in percentage degradation of gelatin, which corroborated visual results. Compared with the shMMP2-transduced cells, degradation was significantly higher in the noninfected AF control group (P = 0.001) and shNon-transduced group (0.002), as observed visually.