Engineered hsa‐miR‐455‐3p‐Abundant Extracellular Vesicles Derived from Three‐Dimensional‐Cultured Adipose Mesenchymal Stem Cells for Tissue‐engineering Hyaline Cartilage Regeneration

Abstract

Efforts are made to enhance the inherent potential of extracellular vesicles (EVs) by utilizing three‐dimensional (3D) culture platforms and engineered strategies for functional cargo‐loading. We successfully isolate three distinct types of adipose mesenchymal stem cells‐derived EVs (ADSCs‐EVs) utilizing 3D culture platforms consisting of porous gelatin methacryloyl (PG), PG combined with sericin methacryloyl (PG/SerMA), or PG combined with chondroitin sulfate methacryloyl (PG/ChSMA). These correspond to PG‐EVs, PG/SerMA‐EVs, and PG/ChSMA‐EVs, respectively. We observe unique miRNA profiles in each type of ADSCs‐EVs. Notably, PG‐EVs encapsulate higher levels of hsa‐miR‐455‐3p and deliver more hsa‐miR‐455‐3p to chondrocytes, which results in the activation of the hsa‐miR‐455‐3p/PAK2/Smad2/3 axis and the subsequent hyaline cartilage regeneration. Furthermore, we optimize the functionality of PG‐EVs through engineered strategies, including agomir/lentivirus transfection, electroporation, and Exo‐Fect transfection. These strategies, referred to as Agomir‐EVs, Lentivirus‐EVs, Electroporation‐EVs, and Exo‐Fect‐EVs, respectively, are ranked based on their efficacy in encapsulating hsa‐miR‐455‐3p, delivering hsa‐miR‐455‐3p to chondrocytes, and promoting cartilage formation via the hsa‐miR‐455‐3p/PAK2/Smad2/3 axis. Notably, Exo‐Fect‐EVs exhibit the highest efficiency. Collectively, the 3D culture conditions and engineered strategies have an impact on the miRNA profiles and cartilage regeneration capabilities of ADSCs‐EVs. Our findings provide valuable insights into the mechanisms underlying the promotion of cartilage regeneration by ADSCs‐EVs.

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