Temporal Transcriptomics Leads From Discovery to in Vivo Validation: COL4A3/COL4A6/ COL4A5 and ITGA8 as Novel Arthrofibrosis Biomarkers in Post-traumatic Joint Contracture

Background: Post-traumatic joint contracture (PTJC), characterized by progressive fibrosis, is a frequent complication following joint injury. Nevertheless, the molecular mechanisms driving its progression remain elusive. To address this, our study aims to delineate the dynamic transcriptomic alterations during arthrofibrosis development and to identify potential biomarkers associated with PTJC. Methods: A rat model of PTJC was established, and RNA sequencing was performed on joint capsules at 0, 3, 7, 14, and 28 days post injury. We then applied Mfuzz time-series clustering, WGCNA, KEGG pathway enrichment analysis, and PPI networks to pinpoint key genes and pathways involved. Cross-species validation was conducted using human transcriptomic data from the GSE135854 dataset. Results: Progressive joint stiffness and fibrosis were observed, peaking at 28 days post-injury. Transcriptome analysis identified 1385 differentially expressed genes (DEGs). KEGG analysis revealed significant enrichment in processes such as "extracellular matrix (ECM)-receptor interaction," "focal adhesion," "cyclic Guanosine Monophosphate - Protein Kinase G (cGMP-PKG) signaling," and "vascular smooth muscle contraction". By integrating rat and human data, we identified COL4A3, COL4A6, COL4A5, and ITGA8 as consistently upregulated hub genes. Their elevated expression in fibrotic joint tissues was further validated using RT-qPCR and immunohistochemistry (IHC). Conclusion: This study provides a comprehensive transcriptomic landscape of PTJC progression and highlights COL4A3/COL4A6/COL4A5 and ITGA8 as promising biomarkers and therapeutic targets for arthrofibrosis.