*3.5. Hyt@tgel Protects against H2O2-Mediated Chondrocyte ECM Degradation*

Once damaged, the cartilage is enabled to repair itself due to its special physiological structure. In the early stages of OA, the production of inflammatory mediators including cytokines and prostaglandins by the cartilage and synovial cells lead to activation of matrix metalloproteinases (MMPs) [70]. Among them, matrix metalloproteinases MMP-3 and MMP-13 can further promote cartilage inflammation, chondrocyte apoptosis, and ROS production, via a positive feedback loop [71,72]. Emerging evidence has shown that MMP13 is considered a significant biomarker to assess OA therapeutic effects and OA progression [73,74]. In this context, bioactive molecules able to suppress these inflammatory mediators or block the involved signaling pathway may help to reduce the OA pathological process [75]. To further corroborate the Hyt@tgel anti-inflammatory action, the expression of catabolic genes such as those coding for MMP-3 and MMP-13 were evaluated (Figure 5).

**Figure 5.** Hyt released by Hyt@tgel10 prevents the expression of OA-related genes in chondrocytes treated with H2O2. (**A**) C20A4 chondrocytes were pre-treated with Hyt@tgel10 for 24 h, then stimulated with H2O2 for 4 h (±SD, *n* = 3, \*\* *p* < 0.01 vs. H2O2 group). (**B**) Beta-galactosidase senescence assay. The graph shows the mean percentage value of senescent cells in every experimental condition (±SD, *n* = 3, \*\*\* *p* < 0.005).

Compared with the untreated group, the mRNA expression of MMP-3 and MMP-13 increased significantly (*p* < 0.01) in H2O2-treated cells, while Hyt@tgel10 pre-treatment was able to reduce about 40% of the increases provoked by H2O2. These results, in line with Facchini et al. [25] corroborated the capacity of Hyt to antagonize the activation of pro-inflammatory pathways like NF-κB even in chondrocytes.

Activation of catabolic enzymes degrades proteoglycan and collagen in the articular cartilage. Moreover, inflammatory states lead to de-differentiation of chondrocytes accompanied by decreased expression of chondrocyte-specific proteins [76]. As reported in Figure 5A, the expression of SOX9 [77] (an early marker of the formation of a cartilage-like tissue matrix), COL2A1, ACAN [78], and COMP (markers of the final stage of chondrogenic differentiation) was significantly rescued by incubation with Hyt@tgel10. These data indicate that Hyt released by Hyt@tgel influenced the ECM balance and gene expression in the chondrocytes under pathological state maintaining their metabolic activity and proliferation in their differentiated phenotype.

Although various cell types are involved in OA pathology, chondrocytes play a major role in OA induction by cellular senescence [79]. It has been shown that chondrocytes have telomere shortening with age. For this reason, chondrocyte senescence, caused by chronic stress in the cells or caused by post-traumatic inflammation, is believed to be closely related to OA [80]. Therefore, the regulation of hypertrophic or senescent chondrocytes using natural phytochemicals known to have a powerful anti-inflammatory and antioxidant activity could be a potential therapeutic target to slow or stop the progression of OA [76]. Data demonstrated that senescence was noticeably reduced in cells treated with Hyt@tgel as detected in an in-situ beta-galactosidase assay (Figure 5B). In particular, Hyt@tgel10 treatment reduced more than two times the percentage of senescent cells compared to untreated chondrocytes.

Two different mechanisms of senescence are suggested in chondrocytes: replicative senescence and stress-induced premature senescence [81,82]. Upregulation of inflammatory cytokines expression induces senescence directly, while downregulation of chondrocyte phenotypic maintenance genes such as SOX9, BMP-2, IGF-1, and ACAN induces senescence indirectly. Thus, the association between aging and/or trauma, reduces the number of healthy and functioning chondrocytes, promoting cartilage degeneration and eventually leads to osteoarthritic pathophysiology [10]. Therefore, the reduction of this cell population lends further credit to hydroxytyrosol ability to preserve chondrocytes from senescence after Hyt@tgel10 treatment.
