Comparative phenotypic and molecular profiling of replicative and chemically-induced senescence in articular chondrocytes

TL;DR

This study compares different ways to induce cell aging in cartilage cells to better understand osteoarthritis and improve research models.

Contribution

The study identifies distinct molecular and phenotypic features of different senescence models in chondrocytes, highlighting mitochondrial dysfunction as a central mechanism.

Findings

All senescence models showed cell cycle arrest, increased SA-β-gal activity, and mitochondrial dysfunction.

DOX induced DNA damage and apoptosis, while DEX caused senescence without significant ROS or apoptosis.

Transcriptomics and proteomics revealed divergent secretory profiles and pathway alterations between models.

Abstract

Osteoarthritis (OA) is a degenerative joint disease characterized by the accumulation of senescent chondrocytes, which drive inflammation and cartilage degradation. However, in vitro models often fail to recapitulate the complexity of OA-associated senescence. This study compares three senescence induction strategies in chondrocytes—replicative senescence (HP), and stress-induced premature senescence (SIPS) via doxorubicin (DOX) and dexamethasone (DEX)—to establish a physiologically relevant in vitro model for OA research. To this end ovine chondrocytes (n = 3) were subjected to serial passaging (to P40) or exposed to optimized concentrations of DOX (50 nM) or DEX (1 µM). Low passage (P3) cells served as controls. Cellular senescence was assessed via proliferation assays, cell cycle analysis, SA-β-gal activity, telomere length, ROS levels, mitochondrial function, transcriptomic profiling (NGS), and high-resolution mass spectrometry proteomic analysis. All models induced key senescence hallmarks including cell cycle and proliferation arrest, increased SA-β-gal activity, and mitochondrial dysfunction. HP cells showed telomere shortening, ROS accumulation, ATP depletion, and SASP secretion. DOX induced strong DNA damage responses and elevated apoptosis markers, while DEX induced senescence without significant ROS or apoptosis, suggesting distinct SIPS mechanisms. Transcriptomics revealed convergent downregulation of oxidative phosphorylation and selenoamino acid metabolism pathways, implicating mitochondrial dysfunction and redox imbalance as shared features. However, HP induced broad transcriptional suppression, also of inflammatory pathways, while DOX and DEX activated immune and SASP-related pathways. Proteomics confirmed divergent secretory profiles, with DOX/DEX increasing SASP-factors and HP enriching matrix proteins. In summary, although all models recapitulate fundamental aspects of senescence, they diverge in stress responses, immune signaling, and apoptosis profiles. HP most closely mimics aging-associated senescence, whereas DOX and DEX model distinct SIPS relevant to oxidative or pharmacological stress. These findings underscore the importance of model selection in senescence-focused OA research and highlight mitochondrial dysfunction as a central mechanistic hub across senescence pathways.

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