Accumulation of senescent chondrocytes is thought to drive inflammatory processes and subsequent cartilage degeneration in age-related as well as posttraumatic osteoarthritis (OA). However, the underlying mechanisms of senescence and consequences on cartilage homeostasis are not completely understood so far. Therefore, suitable in vitro models are needed to study chondrocyte senescence. In this study, we established and evaluated a doxorubicin (Doxo)-based model of stress-induced premature senescence (SIPS) in human articular chondrocytes (hAC). Cellular senescence was determined by the investigation of various senescence associated (SA) hallmarks including β-galactosidase activity, expression of p16, p21, and SA secretory phenotype (SASP) markers (IL-6, IL-8, MMP-13), the presence of urokinase-type plasminogen activator receptor (uPAR), and cell cycle arrest. After seven days, Doxo-treated hAC displayed a SIPS-like phenotype, characterized by excessive secretion of SASP factors, enhanced uPAR-positivity, decreased proliferation rate, and increased β-galactosidase activity. This phenotype was proven to be stable seven days after the removal of Doxo. Moreover, Doxo-treated hAC exhibited increased granularity and flattened or fibroblast-like morphology. Further analysis implies that Doxo-mediated SIPS was driven by oxidative stress as demonstrated by increased ROS levels and NO release. Overall, we provide novel insights into chondrocyte senescence and present a suitable in vitro model for further studies.

Osteoporosis is a systemic metabolic skeletal disease characterized by low bone mass and strength associated with fragility fractures. Oxidative stress, which results from elevated intracellular reactive oxygen species (ROS) and arises in the aging organism, is considered one of the critical factors contributing to osteoporosis. Mitochondrial (mt)ROS, as the superoxide anion (O2−) generated during mitochondrial respiration, are eliminated in the young organism by antioxidant defense mechanisms, including superoxide dismutase 2 (SOD2), the expression and activity of which are decreased in aging mesenchymal progenitor cells, accompanied by increased mtROS production. Using a mouse model of osteoblast lineage cells with Sod2 deficiency, we observed significant bone loss in trabecular and cortical bones accompanied by decreased osteoblast activity, increased adipocyte accumulation in the bone marrow and augmented osteoclast activity, suggestive of altered mesenchymal progenitor cell differentiation and osteoclastogenesis. Furthermore, osteoblast senescence was increased. To date, there are only a few studies suggesting a causal association between mtROS and cellular senescence in tissue in vivo. Targeting SOD2 to improve redox homeostasis could represent a potential therapeutic strategy for maintaining bone health during aging.

Physical activity‐induced mechanical stimuli play a crucial role in preserving bone mass and structure by promoting bone formation. While the Wnt pathway is pivotal for mediating the osteoblast response to loading, the exact mechanisms are not fully understood. Here, we found that mechanical stimulation induces osteoblastic Wnt1 expression, resulting in an upregulation of key osteogenic marker genes, including Runx2 and Sp7 , while Wnt1 knockdown using siRNA prevented these effects. RNAseq analysis identified Plat as a major target through which Wnt1 exerts its osteogenic influence. This was corroborated by Plat depletion using siRNA, confirming its positive role in osteogenic differentiation. Moreover, we demonstrated that mechanical stimulation enhances Plat expression, which, in turn leads to increased expression of osteogenic markers like Runx2 and Sp7 . Notably, Plat depletion by siRNA prevented this effect. We have established that Wnt1 regulates Plat expression by activating β‐Catenin. Silencing Wnt1 impairs mechanically induced β‐Catenin activation, subsequently reducing Plat expression. Furthermore, our findings showed that Wnt1 is essential for osteoblasts to respond to mechanical stimulation and induce Runx2 and Sp7 expression, in part through the Wnt1/β‐Catenin/Plat signaling pathway. Additionally, we observed significantly reduced Wnt1 and Plat expression in bones from ovariectomy (OVX)‐induced and age‐related osteoporotic mouse models compared with non‐OVX and young mice, respectively. Overall, our data suggested that Wnt1 and Plat play significant roles in mechanically induced osteogenesis. Their decreased expression in bones from OVX and aged mice highlights their potential involvement in post‐menopausal and age‐related osteoporosis, respectively.