## 4 Discussion
In contrast to skeletal maturation-related stiffness alterations, changes in AC biomechanics during the onset and progression of OA are still a matter of debate, with conflicting results in the literature. Applying AFM with a micro-scale flat (100 \(\upmu\)m) indenter on human articular cartilage specimens, a decreasing cartilage shear modulus was reported with increasing OA grade [18]. In human, an inverse correlation between stiffness and higher OA grades was demonstrated with IT-AFM by using pyramidal nano-scale tips (nominal radius = 30 nm) but not with micro-scale spherical tips (radius = 2.5 \(\upmu\)m) [17]. RegardingPT-OA, probing the surface with spherical AFM tips, a markedly lower stiffness of the mouse medial condyle was found from 1 to 8 weeks after DMM compared to Sham [15,36]. Interestingly, at 12 weeks post-surgery, which is considered as a late OA time point, the effective indentation modulus of the DMM AC had dramatically increased compared to the earlier stages and approached the stiffness level of the Sham [15]. This unexpected stiffness increase at late PT-OA could be due to erosion of the surface exposing deeper layers of the articular cartilage with higher compressive modulus. On the other hand, in the same study, the lateral condyle appeared to be significantly softer only at 4, 8 and 12 weeks post-DMM compared to Sham [15]. More recently, micromechanical properties of the middle/deep zone of the tibial plateau cartilage were assessed on cross-sections during the course of DMM-induced PT-OA between 3 days and 8 weeks [30]. AFM-based indentation using spherical tips (with a radius of 2.25 \(\upmu\)m) revealed reduced stiffness from 3 days in the PCM and 1 week in the ITM in DMM animals compared to Sham controls [30], suggesting that OA is potentially initiated in the pericellular matrix [40]. In contrast, our AFM indentation with a sharp, pyramidal tip (radius = 20 nm) in the ITM revealed higher stiffness of both the MTP and LTP in DMM animals compared to Sham 2 weeks after surgery. At this early stage of PT-OA, proteoglycan structural changes and GAG depletion may represent alterations that could affect cartilage biomechanics. Proteoglycan degradation through cathepsin D digestion in porcine articular cartilage samples resulted in stiffening of the matrix detected via nano-scale AFM indentation [21]. We previously demonstrated by nano-scale IT-AFM that reduced level of the major proteoglycan aggrecan in a mouse genetic model leads to stiffening of the AC zones of the tibial articular cartilage at 6 months of age, predisposing to OA-like cartilage degeneration at 12 months of age [24]. Furthermore, we showed that the loss of the small leucine-rich proteoglycan Decorin in mice increases proteoglycan nanostiffness and, to a lesser extent, collagen nanostiffness, in the articular cartilage [41]. Thus, the tissue stiffening that we observed in DMM mice 2 weeks post-surgery could be the biomechanical manifestation of the initial, subtle proteoglycan loss typical of early-OA. The contrasting results for AC biomechanics in OA studies may be related to the indenter geometry, where, as suggested previously, measurements using micro-scale spherical tip reflect integrated responses of cartilage ECM composition and structure, whereas nano-scale indentation using pyramidal tip represents the local nanomechanics of the ECM proteoglycan gel and collagen network [17,42,43]. Between 2 weeks and 8 weeks post-operatively, a marked stiffening of both MTP and LTP was observed in Sham animals, which can be explained by the maturation of AC tissue as described above. In DMM mice, however, there was only a modest increase in overall indentation modulus in the MTP and no change in stiffness in the LTP. The normal age-related increase in nanostiffness, especially in the MTP, was accompanied, by a stronger birefringence of the collagen network in Sham, while in DMM samples, polarized light signals were apparently reduced at 8 weeks compared to 2 weeks.