Ultrasound microbubble therapy has been widely utilized in clinical diagnosis to enhance the accuracy of diagnosis, besides; it gradually plays an undiscovered role in clinical treatments [9
]. It has been reported that ultrasound-targeted microbubble destruction could destroy the skeletal muscle blood capillaries, inducing local infiltration of erythrocytes and promoting cellular penetration of the endothelial physiologic barriers [10
]. Therefore, our aim was to clarify whether ultrasound-mediated microbubble destruction could enhance the therapeutic effect of intramyocardial BMSC transplantation on myocardial infarction.
Our data showed that ultrasound-mediated microbubble destruction increased the efficiency of intramyocardial BMSC transplantation, which was demonstrated by greater numbers of Prussian blue-positive cells in the ultrasound microbubble destruction group than in the cell control group. This result is consistent with the finding of Takanobu et al., who reported that low-frequency ultrasound associated with microbubble destruction increased the efficiency of BMSC transplantation into ischemic skeletal muscle [11
]. Ghanem et al. recently found that targeted endothelial adhesion and myocardial engraftment after intravascular delivery of MSCs can be enhanced with hf-UMS [12
]. These effects may arise from the biological responses to ultrasound-mediated microbubble destruction. These responses include the radiation pressure generated by ultrasound transmission through different tissues, the shear stress generated by microbubble rupture, and the cavitation effect generated by ultrasound-mediated microbubble destruction. Radiation pressure can promote the local accumulation of blood cells by pushing them to the vascular wall, whereas cavitation can induce the contraction of endothelial cells, the widening of gaps between endothelial cells and the subsequent destruction of microvessels [13
]. Moreover, shear stress can help the transplanted cells in the blood vessel to penetrate the vessel wall and enter tissues. In addition, slight damage-induced inflammatory responses that result from ultrasound-mediated microbubble destruction can also promote the production of inflammatory factors (e.g. interleukin-1) and induce the intramyocardial migration of mesenchymal stem cells. Expression of adhesion molecules is up-regulated in damaged endothelial cells, which allow the circular mesenchymal stem cells to stick to the damaged vascular endothelium via adhesion molecules and avoid blood scouring. All these biological responses facilitate the entrance of transplanted BMSCs into damaged myocardium and increase the efficiency of BMSC transplantation. Indeed, our results demonstrated that ultrasound-mediated microbubble destruction notably elevated transplantation efficiency. The integrity of vascular endothelial cells with diameters of about 6 µm was disrupted. Chromatin margination on the nuclear membrane and widened gaps between endothelial cells were also observed.
Ultrasound-mediated microbubble destruction promoted myocardial neovascularization. Immunohistochemical staining showed that the myocardial capillary density was significantly higher in the ultrasound microbubble destruction group than in the cell control group. Aseptic inflammation induced by microvascular fracture during ultrasound-mediated microbubble destruction can promote myocardial neovascularization, which may increase blood flow in normal and ischemic myocardium [15
]. It has been reported that transplanted BMSCs are able to secrete vascular endothelial growth factors and basic fibroblast growth factors under certain conditions [19
]. Moreover, the BMSCs transplanted into myocardium can differentiate into not only myocardial-like cells but also into vascular endothelial-like cells. All these biological effects can induce neovascularization in ischemic myocardium.
BMSC transplantation with ultrasound-targeted microbubble destruction can increase the colonization of stem cells in the infarct regions and the infarct border regions make more stem cells differentiate into myocardial cells, synergistically facilitate angiogenesis, and improve blood supply in both the infarct regions and the peripheral infarct regions. This transplantation strategy may improve cardiac blood perfusion, especially in hibernating and stunned myocardium, and lead to the recovery of systolic function in hibernating myocardium. It also provides the appropriate microenvironment for the survival of transplanted stem cells and their differentiation into myocardial cells. The strategy can also limit ventricular dilatation, especially in the ventricular scar region, inhibit cardiac remodelling, ameliorate cardiac systolic and diastolic functions and ultimately improve cardiac functions impaired by myocardial infarction. In conclusion, the present work suggested a new strategy for the transplantation of intramyocardial BMSCs. This approach may have widespread application in the treatment of myocardial infarction.
Especially, we used spiral CT rather than ultrasonic radiation in the present study due to the fact that the statistical analysis by ultrasonic radiation was more likely to be affected by operator subjectivity compared with by spiral CT. In addition, ultrasonic radiation may potentially produce more errors when measuring the volume of irregular-shaped ventricles, which formed under the symptoms of myocardial infarction and ventricular aneurysm. Second, ultrasonic analysis was one of the affecting factors in our study, which might increase the potential interfering factors during cardiac function analysis. Spiral CT ensures accurate positioning. The device provides accurate data regarding the left ventricular volume, quality and functional evaluation, and it is highly reproducible and less affected by operator subjectivity. Compared with MRI, spiral CT needs less scan time and has lower requirements of animal cooperation. The availability of better cardiac post-processing techniques is also a benefit. It has been reported that the LVEF is better correlated with spiral CT measurement than with MRI measurement [21
]. Therefore we chose to use spiral CT rather than MRI in the present study.
The present study had several limitations. First, the number of experimental samples was relatively small. Whether the findings in our study could be applied into clinical practice still requires further intense investigations with large sample size. Surprisingly, recent studies proposed the promising prospect of applying ultrasound microbubbles into treatment of myocardial infarction through stem cell transplantation. Song et al suggested that using US-mediated MB destruction prior to BMSCs transplantation into the infarcted myocardium improves the effectiveness of cardiac cell therapy and cardiac function in rabbits [23
]. Second, the respective functions of the ultrasound and microbubbles on the transplantation of BMSCs were not analysed in more detail. Third, the physical and molecular mechanisms by which ultrasound-mediated microbubble destruction improves cell homing ability remain unclear [24
]. In addition, the exact mechanisms by which stem cells transplantation ameliorated cardiac systolic function and the roles of extracellular matrix during this process also warrant clarification. It is still unknown whether myocardial-like cells differentiated from transplanted BMSCs have systolic and neovascularization
functions. Future investigations will focus on these issues.