Articular cartilage damage is a universal health problem. Despite recent progress, chondrocyte dedifferentiation has severely compromised the clinical outcomes of cell-based cartilage regeneration. Loss-of-function changes are frequently observed in chondrocyte expansion and other pathological conditions, but the characteristics and intermediate molecular mechanisms remain unclear. In this study, we demonstrate a time-lapse atlas of chondrocyte dedifferentiation to provide molecular details and informative biomarkers associated with clinical chondrocyte evaluation. We performed various assays, such as single-cell RNA sequencing (scRNA-seq), live-cell metabolic assays, and assays for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), to develop a biphasic dedifferentiation model consisting of early and late dedifferentiation stages. Early-stage chondrocytes exhibited a glycolytic phenotype with increased expression of genes involved in metabolism and antioxidation, whereas late-stage chondrocytes exhibited ultrastructural changes involving mitochondrial damage and stress-associated chromatin remodeling. Using the chemical inhibitor BTB06584, we revealed that early and late dedifferentiated chondrocytes possessed distinct recovery potentials from functional phenotype loss. Notably, this two-stage transition was also validated in human chondrocytes. An image-based approach was established for clinical use to efficiently predict chondrocyte plasticity using stage-specific biomarkers. Overall, this study lays a foundation to improve the quality of chondrocytes in clinical use and provides deep insights into chondrocyte dedifferentiation.

关节软骨损伤是一个普遍的健康问题。尽管最近取得了进展，但软骨细胞去分化严重损害了基于细胞的软骨再生的临床结果。在软骨细胞扩张和其他病理条件下经常观察到功能丧失的变化，但其特征和中间分子机制仍不清楚。在这项研究中，我们展示了软骨细胞去分化的延时图谱，以提供与临床软骨细胞评估相关的分子细节和信息生物标志物。我们进行了各种检测，例如单细胞 RNA 测序 (scRNA-seq)、活细胞代谢检测和高通量测序 (ATAC-seq) 转座酶可及染色质检测，开发由早期和晚期去分化阶段组成的双相去分化模型。早期软骨细胞表现出糖酵解表型，参与代谢和抗氧化的基因表达增加，而晚期软骨细胞表现出涉及线粒体损伤和应激相关染色质重塑的超微结构变化。使用化学抑制剂 BTB06584，我们发现早期和晚期去分化软骨细胞具有不同的功能表型丧失恢复潜力。值得注意的是，这种两阶段的转变也在人类软骨细胞中得到了验证。建立了一种用于临床的基于图像的方法，以使用特定阶段的生物标志物有效地预测软骨细胞的可塑性。全面的，