Integrative Transcriptomic Profiling Delineates LTBP 4‐Driven Fibroblast Reprogramming Across the Ossification Continuum of the Ligamentum Flavum

To delineate the dynamic transcriptomic landscape of ossification of the ligamentum flavum (OLF) across normal, immature, and mature stages, identify core genes, and screen stage‐specific drug candidates. Normal, immature, and mature OLF tissues (nine samples) from three patients with multilevel OLF were collected for RNA‐seq. Differential expression, functional enrichment, and dynamic trend analyses were performed. Key genes were validated using an external GEO dataset. Single‐cell and spatial transcriptomics were integrated to determine cellular origin and spatial localization. AI‐based drug prediction and molecular docking were conducted. Between normal versus immature, 1026 DEGs were identified; between immature versus mature, 713 DEGs; and between normal versus mature, 1285 DEGs. Upregulated genes were enriched in RNA processing and ribosome biogenesis, whereas downregulated genes were enriched in ECM‐receptor interaction. LTBP4 showed progressive downregulation during ossification (AUC = 0.88). Single‐cell transcriptomics and spatial transcriptomic analysis of a public multi‐omics dataset (GSE255942) revealed that LTBP4 is expressed in fibroblasts, with lower expression in ossified than normal tissues. Virtual knockout of Ltbp4 in fibroblasts recapitulated the OLF transcriptomic signature. AI‐based drug prediction identified stage‐specific candidates; BRD‐K59831625 exhibited the lowest binding energy with LTBP4 (−8.9 kcal/mol). Sustained LTBP4 downregulation drives OLF progression, with fibroblasts as core effector cells. Stage‐specific drug candidates were identified. These findings provide new targets for molecular diagnosis and precision treatment of OLF.