Higher yield and enhanced therapeutic effects of exosomes derived from MSCs in hydrogel-assisted 3D culture system for bone regeneration

Exosomes are emerging in tissue engineering as up-and-coming acellular therapeutics, circumventing common restrictions inherent to cell-based therapies. The characteristics and function of exosomes are affected by the bidirectional communication of their original cells and the local microenvironment in which the cells reside (e.g., the stem cell niche). However, mesenchymal stem cells (MSCs) are customarily cultured in a traditional two-dimensional monolayer, with mechanical microenvironments varying substantially in physiological one. Few reports have addressed the effects of the 3D microenvironment on exosomal osteoinductivity. Herein, a 3D culture model is engineered through collagen hydrogel. Exosomes derived from three-dimensional culture (3D-Exos) and the conventional monolayer culture (2D-Exos) are collected and compared. The 3D culture resulted in high yield exosomes and greatly improved the efficiency of exosomes collection. The in vitro results demonstrated that the 3D-Exos induced significant promotions in osteogenic gene and protein expression (e.g., Runx2, OCN, OPN, COL1A1, and ALP), proliferation, and migration of Human bone marrow mesenchymal stem cells (hBMSCs) and inhibited hBMSCs apoptosis. Importantly, mechanistic studies revealed that the upregulation of the YAP signaling pathway is the underlying mechanism. Moreover, the 3D-Exos resulted in enhanced new bone formation and Runx2/OPN activation in rats with alveolar bone defects. These findings proposed a novel idea of the 3D culture strategy used to enhance the osteoinductivity of MSC-derived exosomes. This study also provided valuable references for exosome-based clinical applications for the treatment and regeneration of tissue defects from the perspective of culture dimensions.