3D HucMSCs derived extracellular vesicles enhanced therapeutic efficacy in treating intrauterine adhesions via BECN1 delivery.

Intrauterine adhesion (IUA) is a significant cause of female infertility and a critical public health concern among women of childbearing age. Emerging evidence has highlighted the therapeutic potential of mesenchymal stem cell-derived extracellular vesicles (EVs) in IUA treatment. However, conventional two-dimensional (2D) culture systems impose constraints on the yield and biological activity of EVs. In this study, we employed a three-dimensional (3D) culture platform based on gelatin methacrylate (GelMA) microspheres to culture human umbilical cord mesenchymal stem cells and isolate MSC-EVs. Comparative analysis demonstrated that 3D-EVs exhibited significantly superior therapeutic effects on restoring the proliferation, migration, viability, and alleviating fibrosis of mifepristone-injured human endometrial stromal cells (hESCs) compared with 2D-EVs in vitro. In IUA animal models, in situ treatment with GelMA-encapsulated 3D-EVs exhibited remarkable regenerative capabilities. The treatment effectively rejuvenated endometrial structure including mucosal thickness and glandular density, ameliorated endometrial fibrosis, and ultimately improved reproductive outcomes. Results from proteomic profiling and functional verification assays revealed that elevated expression of BECN1 in 3D-EVs contributes to the enhanced therapeutic effect of 3D-EVs on injured hESCs. This study establishes 3D culture-derived EVs as an innovative cell-free therapeutic approach for endometrial regeneration, addressing both the limitations of traditional EVs production methods and the clinical need for effective IUA treatments. Our findings provide a foundation for advanced regenerative strategies targeting endometrial pathologies.