Chitosan hydrogel incorporated with bone marrow mesenchymal stem cell-derived exosomal TIMP2 to inhibit angiogenesis in cholangiocarcinoma

Objective: Cholangiocarcinoma (CCA) presents a therapeutic challenge due to its aggressiveness and poor survival rates. This study introduces an approach using tissue inhibitor of metalloproteinase 2 (TIMP2)-enriched bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exo) encapsulated in chitosan hydrogels (CS), intending to provide novel insight into the CCA treatment. Methods: BMSC-Exo was characterized by using TEM, nanoparticle tracking analysis, and western blotting. Role of TIMP2 in CCA was explored using bioinformatics analysis. Therapeutic efficacy and mechanisms of BMSCExo/CS in CCA were assessed through cell viability tests and colony formation assays. Angiogenic and Wnt/ beta-catenin signaling pathways-related key factors were detected through RT-qPCR or western blotting. Results: BMSC-Exo displayed typical cup-shaped morphology and was positive for exosomal markers CD9 and TSG101, but negative for endoplasmic reticulum marker Calnexin, with a diameter of 124.6 nm. BMSC-Exo combined with CS showed synergistic anti-proliferative effects in CCA cells. High-expression TIMP2 samples indicated a better prognosis of CCA patients, and BMSC-Exo/CS increased the TIMP2 expression in CCA cells. Mechanistically, BMSC-Exo/CS TIMP2 overexpression inhibited key factors related to angiogenesis (VEGFA and VEGFR2) and Wnt/beta-catenin pathway (beta-catenin and c-Myc), thereby reducing CCA cell viability. Notably, these inhibitory effects were reversed by a Wnt signaling agonist (BML-284). Conclusion: The study validates the therapeutic potential of BMSC-Exo/CS TIMP2 in CCA treatment. This innovative approach targets angiogenesis and Wnt/beta-catenin signaling, providing a new avenue for more effective and comprehensive CCA therapies.