- When fractured, bone can spontaneously heal to a certain extent. In critical-size defects, in which bone loss is severe, a bone graft is required. Faced with the shortcomings of grafts currently in use—autografts, allografts, and mineral-based bone substitutes—efforts are being made to establish new methods of bone regeneration. One promising approach involves the use of exogenous gene-modified mesenchymal stem cells (MSCs), which can rapidly repair large bone defects in animal models. Unfortunately, ex vivo culture of MSCs may add certain complexity to the advancement of this cell therapy to the clinic. Previously, we demonstrated efficient bone regeneration following direct gene delivery to endogenous MSCs that had been attracted to a fracture site. In that study, electroporation was used, but it is an invasive method of gene transfection that may result in tissue damage. Unlike electroporation, sonoporation—the use of ultrasound for gene delivery—is noninvasive, considered safer, and relevant to the clinical setting. In this study, we evaluated the feasibility of ultrasound-based gene delivery to resident MSCs that had been recruited to a fracture site in different animal models. Our results show transient (up to 21 days) expression of a reporter gene in radial, vertebral, and tibial bone defects in mice, rats, and a minipig. These results could prove beneficial for the next phase of experiments, in which osteogenic genes such as the bone morphogentic proteins (BMPs) could be utilized to achieve bone regeneration in similar challenging conditions of bone tissue loss. Copyright © 2014 John Wiley & Sons, Ltd.