Abstract

Adult articular cartilage chondrocytes have a limited capacity to divide compared to juvenile cells, but the mechanisms behind this decline remain unclear. This study investigates metabolic changes associated with the cessation of chondrocyte proliferation in mouse articular cartilage. Using 5–ethynyl–2′–deoxyuridine (EdU) labeling, the postnatal decline in proliferation was tracked. Label-free fluorescence-lifetime imaging microscopy (FLIM) method, combined with artificial intelligence (AI)-assisted image segmentation, was applied to live cartilage sections to analyze metabolic parameters. Results showed that 1-month-old articular cartilage chondrocytes enter quiescence with significant changes in FLIM fluorescence decay parameters across cartilage zones compared to juvenile chondrocytes. Chondroprogenitors in the superficial zone showed a gradual decrease in citrate synthase content, while glycolytic activity increased with tissue depth. These findings reveal metabolic reprogramming that enables chondrocytes to adapt their metabolism despite limited oxygen availability to meet functional demands. Investigating these chondrocyte adaptations provides key insights for identifying metabolic targets and improving the design of durable, well-integrated tissue-engineered cartilage.