DOC is a semi synthetic

DOC is a semi-synthetic taxane, one of many mitotic inhibitors that act by binding to the beta-tubulin sub-unit of micro-tubules, resulting in ryanodine arrest and apoptosis (Gueritte-Voegelein et al. 1991). In the present study, the inhibition of tumor cell proliferation was significantly greater in the DOC and DOC-MBs/LFUS groups, as evidenced by the decreased PCNA-LI, which was consistent with other studies (Kang et al. 2010; Yang et al. 2012). However, the level of cell apoptosis was not obviously different among the groups, although it was slightly higher in the DOC and DOC-MBs/LFUS groups than in the control group. Observations of tumor morphology revealed that coagulative necrosis was common in all the groups, particularly in the groups that were treated with LFUS. Therefore, the level of cell apoptosis in all the groups may not be as evident because of the large fields with obvious coagulative necrosis within the tumors, which may have contributed to the difference between our results and those reported in other studies (Kang et al. 2010; Yang et al. 2012). Meanwhile, the inhibition of tumor cell proliferation and the PCNA-LI in the LFUS and NMBs/LFUS groups were not consistent with the tumor growth IR. We hypothesized that this result may have been caused by obvious coagulative necrosis in tumor tissue due to the effects of LFUS. Generally, because of thermal and non-thermal (including cavitation and shear stress) effects, US has been widely used and developed in clinical therapy for many diseases, including tumors (Chung et al. 2012; Escoffre et al. 2013; Kotopoulis et al. 2013; Phenix et al. 2014; Tinkov et al. 2010). It is well known that high-intensity focused US can induce coagulative necrosis in tumor tissue to achieve a therapeutic effect based on the thermal effect of US (Jenne et al. 2012), but low-intensity US achieves cell killing through apoptosis, which is mainly mediated by the cavitation effect of US (Feril and Kondo 2004, 2005). However, in the present study, there were larger necrotic lesions within the tumor and higher levels of skin ulceration in the groups that were treated with LFUS. These effects might have resulted from the thermal effects of LFUS. Further investigations are needed to validate these findings.



Ultrasound contrast agents (UCAs) have been used extensively in contrast-enhanced ultrasonography (CEUS) near the bone cortex, particularly for monitoring free flaps (Chang et al. 2012; Kornmann et al. 2010; Krix et al. 2005; Winter et al. 2001), because UCAs present strong non-linear acoustic responses under insonation (de Jong et al. 2000; Goldberg et al. 2001). On the basis of the non-linear acoustic responses of UCAs, CEUS with second harmonic imaging and pulse inversion harmonic imaging, even subharmonic imaging techniques, has been used to detect dynamic microvascular perfusion, analyze the patency of microvascular anastomoses, and evaluate the microcirculation of flap tissues near the bone cortex (Forsberg et al. 2006; Geis et al. 2012; Kiessling et al. 2011; Lamby et al. 2009; Prantl et al. 2007). The strong backscattered echoes from UCAs can contribute to distinguishing the perfused regions from the biceps, forearm flexor muscles, and tibialis anterior (Krix et al. 2005). The replenishment kinetics of microcirculation near the humerus, ulna, radius, and tibia cortex have also been analyzed using CEUS (Duerschmied et al. 2006; Lamby et al. 2009). To clearly detect capillary perfusion in tissue layers near the bone cortex as mentioned above, CEUS should be applied with high resolution and better contrast-to-tissue ratios (CTRs) (Lamby et al. 2009; Prantl et al. 2007).
Contrast-enhanced ultrasound images near the bone cortex may, however, be affected by guided waves generated from the bone cortex, because the signal-to-noise ratio of acoustic signals is disturbed by guided waves that propagate in the surrounding soft tissue (Moilanen et al. 2006; Ta et al. 2009). These unique guided waves are generated from the bone cortex and leak to surrounding tissues when transmission waves hit the bone (Määttä et al. 2009; Moilanen 2008; Nicholson et al. 2002; Lee and Kuo 2006; Protopappas et al. 2006; Ta et al. 2009). They are multimodal and frequency dispersive because of their non-linear propagation (Moilanen 2008; Nicholson et al. 2002; Protopappas et al. 2006). These guided waves have also been used to detect and trap microdroplets, gas bubbles, and submicron particles in acoustic manipulations (Lindner et al. 2008; Schmitt et al. 2010; Wan et al. 2012). However, such detection is limited to the linear characteristic of guided waves (Zhang et al. 2014), and information on UCA responses to frequency-dispersive guided waves is lacking.