Ultrasound‐mediated transgene expression in endogenous stem cells recruited to bone injury sites

Abstract

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.

Multiparameter evaluation of in vivo gene delivery using ultrasound-guided, microbubble-enhanced sonoporation

“…Luciferase expression in the thigh muscle was detected and recorded at different time points using a BLI system, as described previously [27, 28]. Briefly, 10 min before BLI of the mice commenced, the animals were given an intraperitoneal injection of luciferin diluted with PBS (126 mg/kg body weight; Promega, Madison, WI, USA).…”

In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs

“…Although that study combined exogenous cells within the scaffold, only a small amount of new bone was generated in vivo. In our two-step method, the recruitment of endogenous progenitor cells is an important factor that occurs over the course of at least 2 weeks, as we have previously shown in rodents (14, 24). Hence, implanting scaffolds containing plasmids and micro-bubbles would require long-term protection of these components because naked DNA is degraded by deoxyribonucleases, and microbubbles have a short half-life of about 10 min when injected locally.…”

Gene Transfection for Stem Cell Therapy

“…Instead, sonoporation and various microfluidic devices have been developed to improve physical gene transfection. In the case of sonoporation, several studies have reported that ultrasound-mediated microbubble burst increases gene transfection efficiency both in vitro and in vivo [78,79]. Microfabricated fluidic devices have also been used in order to overcome limitations with gene transfection.…”