Ultrasound‐Mediated Biotransfection of Engineered Bone Marrow Mesenchymal Stem Cells in Treated Bone Defects through Intracellular Cavitation

The lack of stem cells and difficulty in osteogenic differentiation are the primary challenges to treating bone defects. Stem cell gene therapy can efficiently replenish the number of stem cells and facilitate bone differentiation, but its security and efficacy remain challenging. The traditional ultrasound-targeted microbubble destruction (UTMD) technology with extracellular cavitation for gene transfection is safe but inefficient. Consequently, gas vesicles extracted from Halobacterium NRC-1 are used as carriers, incorporating nuclear localization signal, polyethyleneimine, and plasmid bone morphogenetic protein 2 (pBMP2). Then followed by internalization into bone marrow mesenchymal stem cells (BMSCs) to produce engineered BMSCs, which exhibit significant capacity of lysosome escape and nuclear targeting. The permeability of the nuclear membrane is substantially enhanced by low-intensity pulsed ultrasound through intracellular cavitation, thereby increasing plasmid nuclear translocation efficiency and gene transfection efficiency by 284.7% and 131.6%, respectively, compared to conventional UTMD techniques. Besides, the expression of BMP2 is maintained for 21 days, promoting osteogenic differentiation of BMSCs and enhancing bone defect repair. In conclusion, this study provides a more secure, efficient, and regulated approach to BMSCs gene therapy for bone defects.