*3.3. Heparanase Increases the Proliferation of Chondrocytes*

No discernible difference in morphology was observed in chondrocytes cultured in monolayer which had been treated with 200 ng/mL of heparanase compared to untreated controls after 48 h (Figure 5A). Separate and combined growth plots are shown for the individual donor cell populations tested in Figure 5B. Whilst there is variation between donors, a combined assessment of the cell populations showed that, for the first 20 h, the heparanase-treated and control chondrocytes showed similar growth rates, but diverged from 24 h onwards with treated cells showing significantly higher proliferation rates than untreated control cells between 32 and 48 h (Figure 5B, bottom right plot).

**Figure 5.** Morphology and growth kinetics of chondrocytes after treatment with heparanase (*n* = 5). (**A**) Phase contrast images were acquired for control and heparanase-treated chondrocytes every 10 min at precise locations for 48 h. The representative images shown are of chondrocytes from donor 20 at t = 0 h and t = 48 h. (**B**) Growth kinetics of chondrocytes were established using a live cell imaging platform and analysis software during the 48 h heparanase treatment period. Individual plots are shown for donors 16 to 20 with mean and SD of cell counts from three FOV from three separate wells. The combined data for all five donors at every time point are also shown (bottom right). FOV = field of view. \*\* *p* < 0.05, \*\*\* *p* < 0.01.
