Fig 1.
H&E staining, Safranin O staining and immunohistochemical staining for Col2.
Osteochondral explants of control groups (top row) and OA model groups (bottom row) after Hematoxylin and Eosin (H&E) staining, Safranin O staining, and immunohistochemical staining for collagen type-2 (Col2) at week 3. For further analysis, the cartilage explants were divided into three zones (see top left micrograph): superficial zone (s), middle zone (m) and deep zone (d).
Fig 2.
Representative micrographs showing immunohistochemical staining of MMP13 in the middle zone of osteochondral explants over time.
The percentage of positive cells per zone was calculated for each time point, group and horse. Increase of MMP13 positive chondrocytes was detected in OA-model-1 compared to control-1 and in week 3.
Table 1.
Contrasts, lsmean (%positive cells), SE and significances of Immunohistochemistry for MMP1, MMP3, MMP13, ADAMTS5 and IL-6.
Values for statistical analysis were calculated from mean values of the three cartilage zones (superficial, middle, deep, detailed in S3 Table). Significant values are highlighted in bold font.
Fig 3.
Dot Plots showing the mean percentage of positive stained (IHC) cells of all three zones for a) MMP1, b) MMP3, c) MMP13 and d) ADAMTS5 for each individual horse for control groups (control 1, control 2) and OA-model groups (OA-model 1, OA-model 2) at different time points (week1 (W1), week 2 (W2), week 3 (W3)). Mean values of the three horses shown are displayed as a line.
Table 2.
Contrasts, lsmean, SE and significanes of mRNA expression relative to GAPDH (ct) for the 4 different groups (control-1, control-2, OA-model-1, OA-model-2) and 3 different time points (week 1, week 2, week 3) for Col1, Col2, Col10, Acan, MMP1, MMP3, MMP 13, ADAMTS5 and IL-6.
Significant values are highlighted in bold font.
Fig 4.
mRNA expression relative to GAPDH (Δct) for a)MMP1, b)MMP3, c)MMP13 and d)ADAMTS5 for control groups (control-1, control-2) and OA models (OA-model-1, OA-model-2) at week1, week2 and week3. * P<0,05 for differences between the groups; ** P< 0,05 for differences between time-points; the individual P-values are displayed in Table 2.
Table 3.
Contrasts, lsmean, SE and significanes of ELISA for the 4 different groups (control-1, control-2, OA-model-1, OA-model-2) and 3 different time points (week 1, week 2, week 3) for MMP1, MMP3, MMP 13, ADAMTS5 and IL-6.
Significant values are highlighted in bold font.
Table 4.
Statistically significant differences between the control and OA-model groups analysed by Immunohistochemistry (IHC), qPCR and ELISA.
Fig 5.
Mean NO/urea ratio of OA-model-2 and control-2 at week1 (W1), week2 (W2) and week3 (W3).
Control-2 showed a decrease in NO/urea ratio from week1 to week3, whereas OA-model-2 showed an increase from W1 to W2 and W3 indicating a shift of synovial macrophages toward the pro-inflammatory M1 phenotype.