Regulation of fibroblast phenotype in osteoarthritis using CDKN1A-loaded copper sulfide nanoparticles delivered by mesenchymal stem cells

This study aimed to investigate the regulation of fibroblast phenotypes by mesenchymal stem cells (MSCs) delivering copper sulfide (CuS) nanoparticles (NPs) loaded with CDKN1A plasmids and their role in cartilage repair during osteoarthritis (OA). Single-cell RNA sequencing data from the GEO database were analyzed to identify subpopulations within the OA immune microenvironment. Quality control, filtering, principal component analysis (PCA) dimensionality reduction, and tSNE clustering were performed to obtain detailed cell subtypes. Pseudotime analysis was used to understand the developmental trajectory of fibroblasts, and GO/KEGG enrichment analyses highlighted biological processes related to fibroblast function. Transcriptomic data and WGCNA identified CDKN1A as a key regulatory gene. A biomimetic CuS@CDKN1A nanosystem was constructed and loaded into MSCs to create MSCs@CuS@CDKN1A. The characterization of this system confirmed its efficient cellular uptake by fibroblasts. In vitro experiments demonstrated that MSCs@CuS@CDKN1A significantly modulated fibroblast phenotypes and improved the structure, proliferation, reduced apoptosis, and enhanced migration of IL-1β-stimulated chondrocytes. In vivo, an OA mouse model was treated with intra-articular injections of MSCs@CuS@CDKN1A. Micro-CT scans revealed a significant reduction in osteophyte formation and improved joint space compared with control groups. Histological analysis, including H&E, Safranin O-Fast Green, and toluidine blue staining, confirmed improved cartilage integrity, whereas the International Osteoarthritis Research Society (OARSI) scoring indicated reduced disease severity. Immunofluorescence showed upregulated CDKN1A expression, decreased MMP13, and reduced α-SMA expression in fibroblast subtypes. Major organs exhibited no signs of toxicity, confirming the biocompatibility and safety of the treatment. These findings suggest that MSCs@CuS@CDKN1A can effectively regulate fibroblast activity and promote cartilage repair, providing a promising therapeutic strategy for OA treatment.<b>NEW & NOTEWORTHY</b> This study introduces MSCs@CuS@CDKN1A, a nanoengineered MSC platform that targets fibroblast phenotypes in osteoarthritis (OA). By modulating CDKN1A expression, this innovative approach not only enhances cartilage repair but also effectively mitigates fibroblast-driven inflammation, marking a significant advancement in OA therapeutics with demonstrated efficacy and biocompatibility.