## 4 Discussion
As the presence and intensity of birefringence is indicative of the organization and density of collagen fibrils, polarization light microscopy is a tool to assess the structural disorganization and degradation of the collagen network, associated with AC degeneration [44]. We previously showed that limited collagenase digestion of bovine articular cartilage explants leads to reduced birefringence and nanostiffness of the surface zone [45]. Likewise, [PERSON] et al. demonstrated with micro-scale IT-AFM that collagen depletion with prolonged elastase treatment reduces stiffness in the surface of pig AC [21]. During the course of DMM-induced PT-OA, increased degradation of collagen was reported [46]; therefore, it is tempting to speculate that the eventual structural deterioration of the collagen network in DMM mice at 8 weeks counteracts ageing-associated stiffening, resulting in only a modest change of the nano-scale indentation modulus from 2 weeks to 8 weeks post-surgery. Osteoarthritis is a whole joint disease in which both the lateral and medial cartilage compartments are compromised. A realistic total human knee finite element model showed that medial meniscus damage also affects the lateral AC [24], and in a DMM PT-OA model, the micro-scale stiffness of the lateral condyle cartilage was significantly reduced from 4 weeks after surgery [15]. In the present study, we found that at 2 weeks post-surgery, the Young's moduli of MTP and LTP in Sham were in a similar range, whereas the LTP after DMM showed markedly increased stiffness compared to MTP, despite the significantly higher Mankin score in the MTP. Further investigation is needed to wisely interpret why the lateral side shows higher stiffening and degrades slower compared to the medial side in the DMM model [10]. Lastly, this study has some limitations that could be addressed in follow-up experiments. As has been widely discussed, the size and geometry of the indenter is a critical issue when measuring biomechanical properties of biological tissues with IT-AFM. Therefore, it would be interesting to directly compare nano-scale and micro-scale stiffness data, obtained by sharp pyramidal or spherical tips, respectively, on the same sample. OA may start in the PCM than spread to other ECM compartments. In our study, we focused on the ITM; therefore, it would be informative to complement our data with the biomechanical changes in the PCM. Degradation and structural modification of ECM components underlie the biomechanical changes that occur during the onset and progression of OA. For a better understanding of how changes at the molecular level are responsible for altering tissue biomechanics, a detailed immunohistochemical investigation of typical pro-teases and degradation epitopes of the major AC proteins is desirable. Finally, in this study, we only examined the early and middle stages of PT-OA development (namely, 2 and 8 weeks post-surgery) to challenge the ability of IT-AFM to detect biomechanical changes. It would also be useful to extend our analysis to later post-operative time points, when AC degradation is more advanced and, presumably, biomechanical changes are more pronounced.