Single-cell transcriptomics reveals cellular heterogeneity of condylar chondrocytes in SD rat temporomandibular joint osteoarthritis

The differentiation of condylar chondrocytes plays a pivotal role in the pathogenesis of temporomandibular joint osteoarthritis (TMJOA). However, the characteristics of condylar chondrocyte subsets and their functional roles in TMJOA remain poorly defined. This study aims to investigate the heterogeneity and functional characteristics of condylar chondrocyte subsets in TMJOA. A TMJOA model was established in SD rats via intra-articular injection of complete Freund’s adjuvant (CFA) into the temporomandibular joint (TMJ). Micro-CT was performed to evaluate the extent of damage and destruction in the subchondral bone of the condylar cartilage. HE staining, Safranin O–Fast Green staining, modified Mankin scoring and immunohistochemical analysis of IL-1β, MMP13, Aggrecan and COL2A1 were conducted to evaluate joint pathology. Subsequently, single-cell RNA sequencing (scRNA-seq) was employed to construct a cellular atlas of condylar cartilage. The analysis identified multiple cell types, including distinct chondrocyte subsets. Furthermore, a systematic characterization of the transcriptomic features, biological functions, differentiation trajectories and intercellular communication patterns of the chondrocyte subsets was conducted. Finally, the expression of marker genes was validated by WB, qRT-PCR, IHC and IF. Micro-CT, histological and immunohistochemical staining confirmed successful establishment of the TMJOA model in CFA-induced SD rats. scRNA-seq identified 13 major cell types in the condylar cartilage and characterized three distinct chondrocyte subsets: homeostatic chondrocytes (HomCs), pre-inflammatory chondrocytes (preInfCs) and inflammatory chondrocytes (InfCs). Among them, InfCs exhibited high expression of S100a9 and CAMP, which were markedly upregulated at the terminal stage of InfC differentiation. Western blotting and qRT-PCR confirmed their elevated expression, while IHC and IF demonstrated strong co-localization of these markers in InfCs, predominantly located in the pre-hypertrophic and hypertrophic layers of the cartilage. This study reveals the heterogeneity of chondrocytes in TMJOA and identifies three distinct subsets: HomCs, preInfCs and InfCs. S100a9⁺CAMP⁺ InfCs are primarily localized in the pre-hypertrophic and hypertrophic layers of condylar cartilage, where they are involved in signaling pathways related to cartilage matrix degradation. These findings enhance our understanding of the pathological mechanisms underlying TMJOA and provide a theoretical basis for the identification of potential therapeutic targets.