Objective To investigate the effect of ultrasonic irradiation time on enhanced green fluorescent protein (EGFP) gene transfection efficiency and local tissue in bone defects using ultrasound-mediated microbubble destruction. Methods Thirty 3-month-old New Zealand rabbits (2.5-3.0 kg in weight) were randomly divided into 5 groups (n=6) and bone defect models were made on the right ulna. At 10 days after modeling, suspension of microbubbles and EGFP plasmids were locally injected (0.3 mL/kg) and then ultrasound was performed on defect at a frequency of 1 MHz, a intensity of 0.5 W/cm2, and a duty ratio of 20% for 1, 2, 3, 4, and 5 minutes respectively (in 1, 2, 3, 4, and 5 minutes groups respectively). The survival condition was observed. Rabbits were sacrificed for gross observation at 7 days after transfer. The gene expression was observed by fluorescence staining. HE staining and transmission electron microscopy were used to observe the local tissue damage. Results The animals all survived. New soft tissue formed in bone defects area at 1 week after transfer, the surrounding muscle tissue was partly filled in it. Green fluorescence expression was observed in all rabbits. The expression was the strongest in 2 minutes group, and was the weakest in 1 minute group. The absorbance (A) value showed significant differences when compared 1 minute and 2 minutes groups with other groups (P<0.05), but no significant difference was found between 3, 4, and 5 minutes groups (P>0.05). Tissue damage was observed in all groups and it was aggravated with the increase of irradiation time. Conclusion EGFP transfection efficiency in bone defect by ultrasound-mediated microbubble destruction is related to irradiation time. EGFP gene can be efficiently transfected without obvious toxicity at 1 MHz, 0.5W/cm2, and duty ratio of 20% for 2 minutes in bone defects of rabbits.
In sonoporation, the cell membrane is broken-up temporarily by ultrasound mediated microbubbles, which is promoting drug or gene into the cell. In current literatures, there are numerous studies of single microbubble dynamics in sonoporation. However till now, little studies have been focused on the sonoporation incidence caused by more than one microbubble. In this article, the dynamic model of two adjacent microbubbles in stable cavitation has been introduced. By the model, the forces including secondary Bjerknes force on cell membrane given by microbubbles and their effects on sonoporation have been numerically studied. According to the experimental parameters, we numerically studied (1) effects of the ultrasound and microbubble parameters on the secondary Bjerknes forces; (2) the forces exerted on cell membrane by microbubble, including the secondary Bjerknes force; (3) the sonoporation possibility caused by those forces produced by microbubble. In this article, the ultrasound and microbubbles’ parameters range were found to produce sonoporation by two adjacent microbubbles. Furthermore, it is the first time to found that the microbubbles’ parameters are more important than ultrasound parameters on sonoporation.