*3.4. Stromal/Stem Cell and Chondropotency Markers and Genes Are Not Affected by Heparanase*

Flow cytometry demonstrated that the positivity of stromal/stem cell markers, CD73, CD90 and CD105 (Figure 6A), and the chondrogenic markers CD44, CD151, CD166, FGFR3 were unaffected by heparanase treatment (Figure 6B), although CD166 and FGFR3 showed

a high level of variability between donors. For the integrins, donor variability was also observed with CD49a, CD49b, CD49c and CD51/61, but not CD29, with no statistical difference between treated cells and controls for any of the integrins (Figure 6C). Chondrocytes from donor 17, the oldest donor, showed a marked heparanase-induced increase in CD166 and a noticeable decrease in CD49a, CD49b and CD49c compared to the cells from the other donors.

**Figure 6.** Flow cytometry analysis of the effects of heparanase treatment on surface markers of chondrocytes (*n* = 5). Results are shown as the percentage of positive cells for a particular marker on heparanase-treated chondrocytes and the matching control. Fluorochrome-conjugated antibodies were used to detect (**A**) stem cells markers, (**B**) integrins and (**C**) chondrogenic markers. No significant differences were observed. Matched samples are represented by the same colour dot; donor 16 (black), donor 17 (red), donor 18 (blue), donor 19 (green), and donor 20 (yellow).

Although the relative fold change in chondrogenic gene expression was not statistically significant between the heparinase-treated and untreated chondrocytes, there was a general decrease in SOX9 expression (median= −1.17), and increased expressions for ACAN (median = 1.1), COL2A1 (median =1.2), and FGFR3 (median = 1.1) following heparanase treatment (Figure 7). The relative fold change in expression of the hypertrophic genes COL10 (median = 1.6) and ALK-1 (median= 2.4) was also increased following heparanase treatment, but this was not statistically significant (Figure 7).

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**Figure 7.** Analysis of the effects of heparanase treatment on gene expression of chondrocytes in monolayer culture (*n* = 5). Results are presented as log-fold change in the expression of the chondrogenic genes SOX9, collagen type II, aggrecan, FGFR3 and hypertrophy genes collagen type X and ALK1 in chondrocytes that were treated with heparanase compared to the untreated controls. Matched samples are represented by the same colour dot; donor 16 (black), donor 17 (red), donor 18 (blue), donor 19 (green), and donor 20 (yellow). Error bars indicate medians and interquartile ranges.
