## 4 Discussion  
The aim of this study was to analyze the biomechanical alterations in murine articular cartilage during the course of PT-OA induced by the DMM model. It is well accepted that during OA, changes in the biomechanical properties of the cartilage ECM precede histologically visible degradation [15; 17; 24; 26; 29; 30]. In this regard, we could clearly demonstrate pronounced changes in AC tissue stiffness at the onset and progression of PT-OA in DMM animals compared to Sham-operated groups, whereas histology and the application of a modified Mankin score could reveal only mild differences. For histopathological scoring, we used 20 \(\upmu\)m thick, native, non-decalcified sections, whereas the recommended guidelines for histology suggest 4-6 \(\upmu\)m thin, fixed, decalcified sections [31]. Nevertheless, the OA histological scores obtained showed a good comparison with our previous study [26]. After thawing and hydration, native frozen sections of 6-30 \(\upmu\)m are also routinely used to evaluate the biomechanical properties of cartilaginous tissues by IT-AFM [14; 24; 25; 26; 30]. Similarly to previous studies [13; 14; 24; 25; 26], we could identify the typical cartilaginous bimodal stiffness distribution, where the first peak represents the softer proteoglycan phase and the second peak the stiiffer collagenous network. The high comparability achieved by analyzing adjacent sections, and the fact that the same animal could be used for both histological and AFM investigations, further justifies our methodological approach to use thick, native sections. Articular cartilage ECM exhibits a decreasing stiffness gradient from the deep to the superficial zone, regardless of species [19; 24; 25; 32; 33]. This zone-specific biomechanical property, reflecting the composition, structure and function of AC regions, was confirmed in Sham mice by our nano-scale AFM indentation, which showed the highest Young's modulus in the deep zone and the lowest in the superficial zone for both the proteoglycan gel and the collagen fibrillary network. Furthermore, the time-dependent increase from weeks 2 to 8 in cartilage nanostiffness in Sham animals is indicative of tissue maturation, which is consistent with the previously reported stiffening associated with skeletal development and aging, and is most likely attributable to the maturation of the collagenous network [14; 17; 34; 35]. At 2 weeks after surgery, the nano-scale stiffness in all non-calcified AC zones of MTP and LTP of DMM mice was significantly elevated compared to Sham, which was associated with a significant increase in histological grade in MTP from normal AC (Sham, 1.33 \(\pm\) 0.47) to mild OA (DMM, 4.67 \(\pm\) 0.47), and with no significant histopathological change in LTP (Sham: 2.0 \(\pm\) 0.00; DMM: 2.33 \(\pm\) 0.47). Previous studies by surface AFM indentation showed significant changes in the stiffness of the intact medial condiple as early as 1 week post-operatively between DMM and Sham, whereas significant histopathological differences were observed only from 8 weeks post-DMM [15; 36]. In contrast, [PERSON] et al. reported significant differences in OARSI scores between DMM and Sham in MTP 2 weeks after surgery; however, concomitant changes in bone mineral density of the subchondral bone were significant only from 5 weeks [37]. Using native knee sections, micro- and nano-scale AFM indentation experiments revealed significant modulus changes from 3 days in the PCM and from 1-2 weeks in the ITM, preceding obvious histological differences at 4-8 weeks post-DMM [26; 30]. In the present and the aforementioned studies, DMM surgical procedures were performed in 12-week-old male mice, reducing experimental variations between studies that may be caused by the age or gender of the operated mice [38; 39]. Thus, our results, which show early changes in cartilage stiffness after DMM, confirm that biomechanical changes are among the first to occur during the onset of PT-OA [15; 29].