## 3 Results
### Analysis of AC Biomechanics upon DMM  
Nano-scale IT-AFM measurements in the ITM were performed on the MTP and LTP to assess the biomechanical properties of the native articular cartilage 2 and 8 weeks after Sham or DMM surgery. Histograms of the Young's moduli demonstrated bimodal stiffness distribution in each cartilage zone (deep, middle and superficial), representing the proteoglycan (peak E1) and collagen (peak E2) phases of the tissue ECM (Figures 2 and 3). In all samples, the cumulative proteoglycan and collagen mean stiffness values (E) displayed a decreasing gradient from the deep towards the superficial zone (Figures 2-5), reflecting the composition and function of the AC. Two weeks after surgery, DMM mice showed an approximately 1.4-fold increase in the mean cumulative stiffness E in all three AC zones (E\({}_{\text{DZ}}\): 1.40-fold; E\({}_{\text{MZ}}\): 1.35-fold; E\({}_{\text{SZ}}\): 1.43-fold) of the MTP compared to the Sham group (Figures 2A and 4). The individual stiffness for the proteoglycan (E1) and collagen (E2) networks in the deep zone MTP of the DMM animals increased 2.04-fold and 1.71-fold, respectively, compared to Sham (Figures 2A and 5). In the middle zone, a 1.78-fold increase for E1 and a 1.51-fold increase for E2 were observed in DMM MTP compared to Sham MTP. The superficial zone of the MTP showed the highest DMM-versus-Sham shift with increasing E1 from 127 kPa to 287 kPa (2.25-fold) and E2 from 299 kPa to 567 kPa (1.99-fold). Interestingly, the LTP showed a higher cumulative stiffening (E\({}_{\text{DZ}}\): 2.02-fold; E\({}_{\text{MZ}}\): 1.98-fold; E\({}_{\text{SZ}}\): 2.28-fold) in DMM mice compared to Sham than the medial side (Figures 2B and 4). There was a general increase of LTP stiffness for both macromolecular networks, especially for the proteoglycans, in DMM animals compared to Sham in the deep zone (E1: 2.13-fold; E2: 1.98-fold), the middle zone (E1: 2.59-fold; E2: 2.17-fold) and the superficial zone (E1: 4.52-fold; E2: 2.76-fold). Taken together, nano-scale AFM indentation measurements demonstrated that in this very early phase of PT-OA, the articular cartilage zones in DMM mice were stiffer compared to Sham animals, the most pronounced biomechanical changes occurred in the superficial zone, the proteoglycan phase of cartilage ECM was more affected than the collagenous network, and the lateral DMM compartment was stiffer than the medial DMM compartment. On the other hand, cartilage stiffness in the corresponding articular cartilage zones of the lateral and medial cartilage compartments of Sham mice was comparable. At 8 weeks post-operatively, overall minor changes were observed compared to those detected in the 2 weeks group. When comparing DMM to Sham in MTP, we recorded a mild softening of the deep zone (E: 0.84-fold; E1: 0.70-fold; E2: 0.78-fold) and a slightly increased Young's modulus of the middle (E: 1.58; E1: 1.04-fold; E2: 1.35-fold) and superficial (E: 1.12; E1: 1.32-fold; E2: 1.22-fold) zones (Figures 3A, 4 and 5). However, in the LTP, a moderate stiffening in all non-calcified AC zones (E\({}_{\text{DZ}}\): 1.63-fold; E\({}_{\text{MZ}}\): 1.34-fold; E\({}_{\text{SZ}}\): 1.13-fold) was observed upon DMM surgery compared to Sham (Figures 3B, 4 and 5). The stiffness of the proteoglycan moiety and the collagen network in the deep zone of the DMM increased 1.42-fold and 1.50-fold, respectively, compared to the Sham. The mean individual stiffness values increased by 1.44-fold (E1) and 1.19-fold (E2) in the middle zone, and by 1.69-fold (E1) and 1.38-fold (E2) in the superficial zone relative to Sham. These data demonstrate that, in contrast to the 2 weeks, in the 8 weeks post-OP group, the stiffness of the proteoglycan and collagen networks changed more similarly; the stiffness difference between the LTP and MTP of the DMM mice was reduced (LTP/MTP 1.50-fold (E\({}_{\text{2w}}\)) and 1.15-fold (E\({}_{\text{8w}}\)), with E averaged over all three zones); and the MTP deep zone was softer in DMM compared to Sham.