## 4 Discussion  
The search for ON therapies capable of controlling the joint microenvironment is a unique challenge [1,2]. A holistic view of biological responses during ON can produce insights for generating efficient strategies [22]. Our pilot study showed different histopathological features in two groups of ON patients by opening up interesting biological perspectives. All specimens with NTFH and PTFH forms displayed basal integration with the underlying bone. However, PTFH samples showed noticeable fibrotic reactions and a reduced stem cell pool in the bone marrow. Interrupting blood supply likely contributed to mediating the death of bone and marrow cells. As the bone supports the exchange of nutrients with cartilage by facilitating joint force distribution, bone alterations might speed up cartilage degeneration [22,45]. The subchondral bone changes may lead to misalignment of the articulating surfaces with consequences on the mechanosensory cells in the bone [45]. Thus, first-line approaches for shifting the bone matrix turn-over are essential to avoid progressive degenerative changes in cartilage tissue. Bisphosphonate treatment could restore the balance between bone resorption and formation [26]. Biopsies from patients with post-traumatic aetiology reported several OA features, including impaired extracellular matrix and vascular infiltration in cartilage because of the mechanical stresses. We determined the relevance of matrix composition and tidemark presence using a semi-quantitative analysis with the ICRS score, by observing different histological scores in the PTFH and NTFH groups. This latter displayed similar results to the control group by reporting intact cartilage but several changes in the subchondral bone. These findings are in line with other studies, which showed intact articular cartilage in the NTFH group with corticosteroid treatments [22]. The two groups even exhibited distinct protein expression for mediators modulating fibrotic and catabolic responses. Protein assessment for collagen type I and autotaxin corroborated the fibrotic aspect in both cartilage and bone from the PTFH group. The autotaxin-lysophosphatidic acid (LPA) axis is emerging as a critical regulator in various biological responses [46]. Autotaxin exerts a fibrotic activity by catalysing LPA, which promotes fibrosis responses by regulating collagen type I biosynthesis [34,47]. Moreover, autotaxin controls the fusion and bone resorption capacity of osteoclasts [35]. Another peculiar feature observed in the cartilage and bone tissues from the two ON aetiologies was the protein expression for MMP-13. It is a molecule which stimulates collagen and proteoglycan degradation in cartilage and bone [48]. Along this path, [PERSON] et al. provided first evidence of MMPs in the ON setting. ON patients with PTFH showed increased gene expression levels of MMP-2 and a low amount of the tissue inhibitor of metalloproteinases (TIMPs) [49]. The increased protein levels of MMP-13 in the PTFH group may likely depend on the up-regulated activity of chondrocytes in producing MMPs after stress forces. These findings open preliminary biological insights into considering MMPs and fibrotic markers as attractive therapeutic targets by exploiting future technological advances like CRISPR-Cas9 [50]. Combining current ON strategies with small molecule inhibitors to block collagen type I, MMP-13 and ATX could improve the success of PTFH treatment [47,51]. Regardless of the ON aetiology, samples from both groups displayed a moderate positivity for caspase-3, showing higher values than the control. Beyond the classical signalling pathway, we considered the role of CB-2 involved in the endocannabinoid system. [PERSON] et al. reported that this system promotes specific signalling pathways in response to pathogenic events to launch repair processes [52]. In specimens from both groups, we showed positive immunostaining for CB-2, especially in cartilage, with higher values than healthy controls.