DMM surgery affects more profoundly and ACLT + pMx more strongly the medial tibial compartment.
After the DMM and the ACLT + pMx surgeries, osteoarthritis develops mostly in the medial tibial plateau and this area was scored according to the histochemical-histological scoring system modified from (16). For the DMM model, animals were sacrificed at days 3, 5, 7, 14 and 28. Because ACLT with meniscectomy results in more severe OA compared to DMM (9), earlier time points were chosen for this model and animals were sacrificed at days 1, 3, 5, 7 and 14. After DMM surgery, loss of matrix staining in cartilage was apparent already after three days as illustrated on Fig. 1A and on the matrix staining sub-score (Figure S1) but cartilage defects appeared only at day 28 in most of the animals (Fig. 1E and sub-score cartilage structure in Figure S1). Most of the other sub-scores such as cellularity, alteration of the tidemark and thickening of the subchondral bone started to be evident at day 14. Small to medium-sized osteophytes were observed in a minority of animals 14 or 28 days after the surgery. As a result, the total histological score was significantly elevated at day 14 and 28 in comparison to the contralateral medial tibial plateau (Fig. 1) for this model. After the ACLT + pMx surgery, the total histological score (Fig. 1) was significantly elevated from the first day compared to the contralateral knee and this was mainly driven by the cartilage structure and the matrix staining sub-scores (Figure S2). The other sub-scores were barely affected, and no osteophyte was observed. The comparison of both models at days 3–14 shows that the total histological scores were higher (except at day 5) for the ACLT + pMx model, and this seems to be mostly driven by the higher sub-scores for cartilage structure and matrix staining. However, because more sub-scores were affected in the DMM compared to ACLT + pMx, it might indicate that in DMM induces more profound changes in the joint.
OxPTM type II collagen and CX signal in the deep zone are early markers of OA
ACLT + pMx and DMM knees were stained by anti-oxPTM-CII and antibody specific to CX (Fig. 2). For both the ACLT + pMx and DMM knees, a strong CX staining is observable in the deep zone located where matrix loss or cartilage damages were also visible. Otherwise, in the rest of the deep zone the staining was weak all along the tidemark. Similarly, most of the cartilage was negative for oxPTM-CII but a positive signal can be observed in the deep zone in the region where cartilage degradation occurred. Interestingly, both staining were already observable at early time points, before visible cartilage damages started to develop. Both staining were also particularly strong where hypertrophic chondrocytes in the deep zone were visible. In addition, in the DMM model at day 28 more severe damages could be observed. In this case, oxPTM-CII and CX staining extended beyond the deep zone at the damage site and the all depth of cartilage was positive for CX.
We also looked at the lateral tibial compartment of the operated knees (FigureS3) and the contralateral knees (Figure S4). For both the ACLT + pMx and DMM models at early timepoints (days 1 or 7 or days 3 or 14 respectively), the staining for CX and oxPTM-CII in the lateral tibial compartment were weak or not visible and this irrespectively of the histological score obtained on the medial side (Indicated in brackets Figure S3A). However, at day 14 in the ACLT + pMx model and day 28 in the DMM model signal for both collagens appeared. On the contrary to the medial tibial cartilage, the staining was not restricted to a specific location in the cartilage. These results illustrate that at later timpoints the disease progressed to the lateral side of the operated joint. The lateral tibial compartment was scored as well (Figure S3B). The histological scores were low in all groups and no difference could be observed to the contralateral lateral tibial compartment at all timepoint tested.
Similarly, the contralateral (Figure S4) knees showed only a weak CX and oxPTM-CII staining at day 1 or 3 in the ACLT + pMx or DMM models respectively but an increased staining intensity was visible on the medial side for both CX and oxPTM-CII after ACLT + pMx at day 14 and for oxPTM-CII after DMM at day 28. As for the ipsilateral knee, CX and oxPTM-CII were strictly localized in the deep zone around the large hypertrophic chondrocytes in the medial tibial compartment, and the staining was more diffuse in the lateral tibial compartment.
Finally, condylar cartilage was found to be mostly negative for oxPTM-CII (not shown) but started to be positive when large damages develop on the tibial side (see example on Fig. 3D-E). Condylar cartilage was also weakly positive for CX, which was strictly localized in the deep zone. A stronger CX staining appeared with the progression of the disease but at later time points compared to the tibial cartilage (data not shown).
OxPTM-CII and CX staining partially co-localize.
Higher magnifications of the medial tibial cartilage from DMM knees are shown Fig. 3 for cartilage with matrix staining loss but no defect (Fig. 3A and B) or for cartilage with a small (Fig. 3C) or a large defect (Fig. 3D). An example for condylar cartilage is also shown (Fig. 3E). Only the DMM knees are shown as the double-staining for CX and oxPTM-CII enable to better determine whether both markers co-localized. In the cartilage with no apparent defect, CX and oxPTM-CII staining co-localized in the deep zone mostly in the pericellular and territorial matrix of the hypertrophic chondrocytes. In panel A, chondrocytes that were positive for oxPTM-CII but negative for CX can also be observed (see arrows). In cartilage presenting a small defect (Panel C), the pattern was similar. However, in the case of larger defect (panel D), CX staining was found in the interterritorial matrix and extended to the middle zone while oxPTM-CII staining extended to the middle and superficial zone. Interterritorial staining of oxPTM-CII was also observed in the middle and superficial zones in the area of the damaged fibrillar cartilage (Panel D). In addition, in the middle and superficial zones, cells positive for oxPTM-CII but nor CX were observed (see arrows). These results indicate that oxPTM-CII and CX staining mainly co-localize (around the larger chondrocytes in the deep zone) in early OA but might show a slightly different pattern at later OA stages. Finally, condylar cartilage facing a large defect (here picture from panels D and E are from the same animal) was positive for both staining and the staining were observed in all zones of cartilage.
The growth plate and the meniscus are positive for oxPTM -CII
As expected, the growth plate and the calcified part of the meniscus were positive for CX (Fig. 4). These tissues were also found to be positive for oxPTM-CII. In these two tissues, both staining appear to co-localize.