Integrative Single-Cell and Spatial Transcriptomics Identifies CRIP1⁺ Prehypertrophic Chondrocytes as Inflammatory...

Osteoarthritis (OA) is a prevalent degenerative joint disease characterized by progressive cartilage destruction, yet the heterogeneity of transitional chondrocyte states and their contributions to pathogenesis remain incompletely understood. In this study, we constructed a single-cell transcriptomic atlas of human knee cartilage from 16 OA samples and 2 non-OA normal donors. High-resolution sub-clustering of the prehypertrophic chondrocytes (preHTC) compartment revealed eight transcriptionally distinct subtypes, among which CRIP1⁺ preHTC emerged as the most significantly expanded population in OA, particularly in weight-bearing regions. Pseudotemporal trajectory inference and regulon analysis identified EGR3 as a candidate key regulator for CRIP1⁺ preHTC. High-dimensional weighted gene coexpression network analysis (hdWGCNA) demonstrated matrix-remodeling and proteostasis-stress transcriptional programs were highly active in CRIP1⁺ preHTC. Cell-cell communication analysis uncovered that CRIP1⁺ preHTC acts as a central signaling hub in OA, with markedly enhanced FN1 signaling pathway. Spatial transcriptomics confirmed that CRIP1⁺ preHTC co-localizes with prefibroblasts (preFC) in pathological niches and interacts with other cells in the OA microenvironment. Finally, a machine-learning-based 10-gene classifier (JUN, MCOLN3, RHOC, AKR7A2, HSPG2, AK4, B4GALT2, CLSTN1, LRRC41, and GADD45A) derived from CRIP1⁺ preHTC-associated hub genes accurately discriminated OA from normal tissues in two independent bulk RNA-seq cohorts. Collectively, these findings suggest that CRIP1⁺ preHTC may represent a pathogenic cell state in OA, provide a multi-layered molecular framework that links a specific chondrocyte transitional subset to cartilage degradation, and offer a potentially useful gene signature for OA diagnosis.