Mechanosensitive miR-99b mediates the regulatory effect of matrix stiffness on bone marrow mesenchymal stem cell fate both in vitro and in vivo

Mechanical signals from extracellular matrix stiffness are important cues that regulate the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the incorporation of BMSCs into soft hydrogels and the dominance of soft matrices for BMSC growth and differentiation limit the directed differentiation of BMSCs incorporated into hydrogels for tissue engineering, especially osteogenesis. Here, we found that the expression of miR-99b increased with increasing hydrogel stiffness and that miR-99b regulated the proliferation and differentiation of BMSCs seeded on the surface of substrates with different stiffnesses. Furthermore, miR-99b significantly promoted the migration of BMSCs in 3D hydrogels. Mechanistically, we demonstrated that matrix stiffness-sensitive miR-99b targets the mammalian target of the rapamycin signaling pathway to regulate the adipogenic and osteogenic differentiation of BMSCs. In addition, by modulating the expression of miR-99b, the osteogenic differentiation of BMSCs in soft 3D hydrogels was promoted. Consistently, the flexible BMSC-GelMA hydrogel transfected with miR-99b significantly promoted bone regeneration in the rat calvarial defect area. These results suggest that miR-99b plays a key role in the mechanotransduction and phenotypic transformation of BMSCs and may inspire new tissue engineering applications with MSCs as key components.