Sonoporation-mediated transduction of siRNA ameliorated experimental arthritis using 3 MHz pulsed ultrasound

The goal of this feasibility study was to examine whether sonoporation assisted transduction of siRNA could be used to ameliorate arthritis locally. If successful, such approach could provide an alternative treatment for the patients that have or gradually develop adverse response to chemical drugs. Tumor necrosis factor alpha (TNF-α) produced by synovial fibroblasts has an important role in the pathology of rheumatoid arthritis, inducing inflammation and bone destruction. In this study, we injected a mixture of microbubbles and siRNA targeting TNF-α (siTNF) into the articular joints of rats, and transduced siTNF into synovial tissue by exposure to a collimated ultrasound beam, applied through a probe 6 mm in diameter with an input frequency of 3.0 MHz, an output intensity of 2.0 W/cm² (spatial average temporary peak; SATP), a pulse duty ratio of 50%, and a duration of 1 min. Sonoporation increased skin temperature from 26.8 °C to 27.3 °C, but there were no adverse effect such as burns. The mean level of TNF-α expression in siTNF-treated knee joints was 55% of those in controls. Delivery of siTNF into the knee joints every 3 days (i.e., 7, 10, 13, and 16 days after immunization) by in vivo sonoporation significantly reduced paw swelling on days 20–23 after immunization. Radiographic scores in the siTNF group were 56% of those in the CIA group and 61% of those in the siNeg group. Histological examination showed that the number of TNF-α positive cells was significantly lower in areas of pannus invasion into the ankle joints of siTNF- than of siNeg-treated rats. These results indicate that transduction of siTNF into articular synovium using sonoporation may be an effective local therapy for arthritis.     

Effect of ultrasound therapy on the repair of gastrocnemius muscle injury in rats

The aim of this study was to evaluate the effect of the pulsed ultrasound therapy (PUT) in stimulating myoregeneration and collagen deposition in an experimental model of lacerative gastrocnemius muscle lesion in 30 Wistar rats. Fifteen rats were treated (TG) daily with 1 MHz pulsed ultrasound (50%) at 0.57 W/cm² for 5 min, and 15 were control animals (CG). Muscle samples were analyzed on postoperative days 4, 7 and 14 through H&E, Picrosirius-polarization and immunohistochemistry for desmin. The lesions presented similar inflammatory responses in both treated and control groups. The areal fraction of fibrillar collagen was larger in the TG at 4 days post-operatively (17.53 ± 6.2% vs 6.79 ± 1.3%, p = 0.0491), 7 days (31.07 ± 7.45% vs 12.57 ± 3.6%, p = 0.0021) and 14 days (30.39 ± 7.3% vs 19.13 ± 3.51%, p = 0.0118); the areal fraction of myoblasts and myotubes was larger in the TG at 14 days after surgery (41.66 ± 2.97% vs 34.83 ± 3.08%, p = 0.025). Our data suggest that the PUT increases the differentiation of muscular lineage cells, what would favor tissue regeneration. On the other hand, it is also suggested that there is a larger deposition of collagenous fibers, what could mean worse functional performance. However, the percentage of fibers seems to have stabilized at day 7 in TG and kept increasing in CG. Furthermore, the collagen supramolecular organization achieved by the TG is also significant according to the Sirius red staining results.     

Targeted sonodynamic therapy using protein-modified TiO2 nanoparticles

Our previous study suggested new sonodynamic therapy for cancer cells based on the delivery of titanium dioxide (TiO₂) nanoparticles (NPs) modified with a protein specifically recognizing target cells and subsequent generation of hydroxyl radicals from TiO₂ NPs activated by external ultrasound irradiation (called TiO₂/US treatment). The present study first examined the uptake behavior of TiO₂ NPs modified with pre-S1/S2 (model protein-recognizing hepatocytes) by HepG2 cells for 24 h. It took 6 h for sufficient uptake of the TiO₂ NPs by the cells. Next, the effect of the TiO₂/US treatment on HepG2 cell growth was examined for 96 h after the 1 MHz ultrasound was irradiated (0.1 W/cm², 30 s) to the cells which incorporated the TiO₂ NPs. Apoptosis was observed at 6 h after the TiO₂/US treatment. Although no apparent cell-injury was observed until 24 h after the treatment, the viable cell concentration had deteriorated to 46% of the control at 96 h. Finally, the TiO₂/US treatment was applied to a mouse xenograft model. The pre-S1/S2-immobilized TiO₂ (0.1 mg) was directly injected into tumors, followed by 1 MHz ultrasound irradiation at 1.0 W/cm² for 60 s. As a result of the treatment repeated five times within 13 days, tumor growth could be hampered up to 28 days compared with the control conditions.     

Monitoring tissue inflammation and responses to drug treatments in early stages of mice bone fracture using 50 MHz ultrasound

Bone fracture induces moderate inflammatory responses that are regulated by cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LO) for initiating tissue repair and bone formation. Only a handful of non-invasive techniques focus on monitoring acute inflammation of injured bone currently exists. In the current study, we monitored in vivo inflammation levels during the initial 2 weeks of the inflammatory stage after mouse bone fracture utilizing 50 MHz ultrasound. The acquired ultrasonic images were correlated well with histological examinations. After the bone fracture in the tibia, dynamic changes in the soft tissue at the medial-posterior compartment near the fracture site were monitored by ultrasound on the days of 0, 2, 4, 7, and 14. The corresponding echogenicity increased on the 2nd, 4th, and 7th day, and subsequently declined to basal levels after the 14th day. An increase of cell death was identified by the positive staining of deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay and was consistent with ultrasound measurements. The increases of both COX-2 and Leukotriene B4 receptor 1 (BLT₁, 5-LO-relative receptor), which are regulators for tissue inflammation, in the immunohistochemistry staining revealed their involvement in bone fracture injury. Monitoring the inflammatory response to various non-steroidal anti-inflammatory drugs (NSAIDs) treatments was investigated by treating injured mice with a daily oral intake of aspirin (Asp), indomethacin (IND), and a selective COX-2 inhibitor (SC-236). The Asp treatment significantly reduced fracture-increased echogenicity (hyperechogenicity, p < 0.05) in ultrasound images as well as inhibited cell death, and expression of COX-2 and BLT₁. In contrast, treatment with IND or SC-236 did not reduce the hyperechogenicity, as confirmed by cell death (TUNEL) and expression levels of COX-2 or BLT₁. Taken together, the current study reports the feasibility of a non-invasive ultrasound method capable of monitoring post-fracture tissue inflammation that positively correlates with histological findings. Results of this study also suggest that this approach may be further applied to elucidate the underlying mechanisms of inflammatory processes and to develop therapeutic strategies for facilitating fracture healing.     

Ultrasound-assisted microbubbles gene transfer in tendons for gene therapy

Our study aimed at evaluating the use of ultrasound-assisted microbubbles gene transfer in mice Achilles tendons. Using a plasmid encoding luciferase gene, it was found that an efficient and stable gene expression for more than two weeks was obtained when tendons were injected with 10 μg of plasmid in the presence of 5 × 10⁵ BR14 microbubbles with the following acoustic parameters: 1 MHz, 200 kPa, 40% duty cycle and 10 min of exposure time. The rate of gene expression was 100-fold higher than that obtained with naked plasmid injected alone without ultrasound or with ultrasound in absence of microbubbles. The long term expression of transgene makes ultrasound-assisted microbubble a suitable method for gene therapy in tendons.     

Investigation of rat bone fracture healing using pulsed 1.5 MHz, 30 mW/cm burst ultrasound – Axial distance dependency

This study investigated the effect of LIPUS on fracture healing when fractures were exposed to ultrasound at three axial distances: z = 0 mm, 60 mm, and 130 mm. We applied LIPUS to rat fracture at these three axial distances mimicking the exposure condition of human fractures at different depths under the soft tissue. Measurement of LIPUS shows pressure variations in near field (nearby transducer); uniform profile was found beyond it (far field). We asked whether different positions of the fracture within the ultrasound field cause inconsistent biological effect during the healing process. Closed femoral fractured Sprague–Dawley rats were randomized into control, near-field (0 mm), mid-near field (60 mm) or far-field (130 mm) groups. Daily LIPUS treatment (plane, but apodized source, see details in the text; 2.2 cm in diameter; 1.5 MHz sine waves repeating at 1 kHz PRF; spatial average temporal average intensity, I_SATA = 30 mW/cm²) was given to fracture site at the three axial distances. Weekly radiographs and endpoint microCT, histomorphometry, and mechanical tests were performed. The results showed that the 130 mm group had the highest tissue mineral density; and significantly higher mechanical properties than control at week 4. The 60 mm and 0 mm groups had significantly higher (i.e. p < 0.05) woven bone percentage than control group in radiological, microCT and histomorphometry measurements. In general, LIPUS at far field augmented callus mineralization and mechanical properties; while near field and mid-near field enhanced woven bone formation. Our results indicated the therapeutic effect of LIPUS is dependent on the axial distance of the ultrasound beam. Therefore, the depth of fracture under the soft tissue affects the biological effect of LIPUS. Clinicians have to be aware of the fracture depth when LIPUS is applied transcutaneously.     

Low intensity pulsed ultrasound for fracture healing: a review of the clinical evidence and the associated biological mechanism of action

Low intensity pulsed ultrasound is used in the clinical treatment of fractures and other osseous defects. Level I clinical studies demonstrate the ability of a specific ultrasound signal (1.5 MHz ultrasound pulsed at 1 kHz, 20% duty cycle, 30 mW/cm² intensity (SATA)) to accelerate the healing time in fresh tibia, radius and scaphoid fractures by up to 40%. Additionally, the same ultrasound signal has been shown to be effective at resolving all types of nonunions of all ages, following a wide range of fracture types and primary fracture management techniques.Recently, significant efforts have resulted in a more comprehensive understanding of the biological mechanism of action that produces the documented clinical outcomes. Low intensity pulsed ultrasound has been demonstrated to accelerate in vivo all stages of the fracture repair process (inflammation, soft callus formation, hard callus formation). In particular, accelerated mineralisation has been demonstrated in vitro with increases in osteocalcin, alkaline phosphatase, VEGF and MMP-13 expression. Integrins, a family of mechanoreceptors present on a wide range of cells involved in the fracture healing process, have been shown to be activated by the ultrasound signal. Downstream of the integrin activation, focal adhesions occur on the surface of cells with the activation of multiple signalling pathways, including the ERK, NF-κβ, and PI3 kinase pathways. These pathways have been directly linked to the production of COX-2 and prostaglandin, which are key to the processes of mineralisation and endochondral ossification in fracture healing.     

The value of diffusion-weighted MRI to evaluate the response to radiochemotherapy for cervical cancer

Purpose

To investigate the value of apparent diffusion coefficient (ADC) to predict and monitor the therapy response for cervical cancer patients receiving concurrent radiochemotherapy, and to analyze the influence of different b-value combinations on ADC-based evaluation of treatment response.

Material and Methods

Seventy-five cervical cancer patients treated with radiochemotherapy received conventional MRI and DWI prior to therapy, after 2 weeks of therapy, after four weeks of therapy and after therapy completion. Treatment response was classified as complete response (CR, n = 35), partial response (PR, n = 22) and stable disease (SD, n = 18), which was determined according to final tumor size after 6 months of therapy completion. Dynamic changes of apparent diffusion coefficients (ADC) and tumor size in the three tumor groups were observed and compared. All the ADCs were calculated from b = 0, 600 s/mm² and b = 0, 1000 s/mm².

Results

The ADC increased percentage was higher in CR group than those in PR and SD groups after two weeks and four weeks of therapy, with significant differences in absolute ADCs between CR and PR, SD groups after therapy completion; the overall discriminatory capability for differentiation of CR and PR, SD groups was higher for high b-value combination (0, 1000 s/mm²) than for low b-value combination (0, 600 s/mm²).

Conclusion

DWI can be used as a predictive and monitoring biomarker of treatment response to radiochemotherapy in patients with cervical cancer. High b-value combination may be more reliable to evaluate the treatment response for cervical cancer.     

Parametric harmonic-to-fundamental ratio contrast echocardiography: a novel approach to identification and accurate measurement of left ventricular area under variable levels of ultrasound signal attenuation

Objectives

We introduced a harmonic-to-fundamental ratio (HFR) of the radiofrequency (RF) signals that reduces confounding effects of attenuation. We studied whether HFR analysis of RF signals received from contrast microbubbles allows accurate measurement of the left ventricular (LV) cavity area under varying levels of attenuation.

Background

Attenuation is a fundamental problem in ultrasound imaging and limits the use of clinical echocardiography.

Methods

RF data from short axis systolic and diastolic scans were obtained from 14 open-chest dogs following left-atrial bolus of Optison. Attenuation was induced by interposed silicone pads calibrated to induce 7 dB or 14 dB reductions of the backscattered RF signal. RF images were reconstructed from the RF signals, HFR values calculated for each image pixel for 0 dB, 7 dB and 14 dB attenuation conditions, and LV area obtained by summation of “LV cavity pixels”. A reference LV cavity area was obtained from endocardial border tracings in enhanced scans by experts.

Results

Correlation of the HFR-defined and reference areas at systole was R = 0.95, R = 0.94, and R = 0.91 for 0 dB, 7 dB and 14 dB levels of attenuation, respectively, and at diastole was R = 0.95 for 0 dB, 7 dB and 14 dB levels of attenuation. The mean difference from both systolic and diastolic values was <1.45 cm² (i.e. negligible) in all attenuation settings.

Conclusion

Our novel HFR method supports precise measurement of the LV cavity area in contrast images with simulated high attenuation of ultrasound signals.     

Influence of low-intensity pulsed ultrasound on osteogenic tissue regeneration in a periodontal injury model: X-ray image alterations assessed by micro-computed tomography

Objective

This study was conducted to evaluate, with micro-computed tomography, the influence of low-intensity pulsed ultrasound on wound-healing in periodontal tissues.

Methods

Periodontal disease with Class II furcation involvement was surgically produced at the bilateral mandibular premolars in 8 adult male beagle dogs. Twenty-four teeth were randomly assigned among 4 groups (G): G1, periodontal flap surgery; G2, periodontal flap surgery + low-intensity pulsed ultrasound (LIPUS); G3, guided tissue regeneration (GTR) surgery; G4, GTR surgery plus LIPUS. The affected area in the experimental group was exposed to LIPUS. At 6 and 8 weeks, the X-ray images of regenerated teeth were referred to micro-CT scanning for 3-D measurement.

Results

Bone volume (BV), bone surface (BS), and number of trabeculae (Tb) in G2 and G4 were higher than in G1 and G3 (p < 0.05). BV, BS, and Tb.N of the GTR + LIPUS group were higher than in the GTR group. BV, BS, and Tb.N of the LIPUS group were higher than in the periodontal flap surgery group.

Conclusion

LIPUS irradiation increased the number, volume, and area of new alveolar bone trabeculae. LIPUS has the potential to promote the repair of periodontal tissue, and may work effectively if combined with GTR.     

Ultrasound induces cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin

Objectives

Curcumin, a natural pigment from the traditional Chinese herb, has shown promise as an efficient enhancer of ultrasound. The present study aims to investigate ultrasound-induced cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin in vitro.

Methods

Nasopharyngeal carcinoma cell line CNE2 cells were incubated by 10 μm curcumin and then were treated by ultrasound for 8 s at the intensity of 0.46 W/cm². Cytotoxicity was evaluated using MTT assay and light microscopy. Mitochondrial damage was analyzed using a confocal laser scanning microcopy with Rhodamine 123 and ultrastructural changes were observed using a transmission electron microscopy (TEM).

Results

MTT assay showed that cytotoxicity induced by ultrasound treatment alone and curcumin treatment alone was 18.16 ± 2.37% and 24.93 ± 8.30%, respectively. The cytotoxicity induced by the combined treatment of ultrasound and curcumin significantly increased up to 86.67 ± 7.78%. TEM showed that microvillin disappearance, membrane blebbing, chromatin condensation, swollen mitochondria, and mitochondrial myelin-like body were observed in the cells treated by ultrasound and curcumin together. The significant collapse of mitochondrial membrane potential (MMP) was markedly observed in the CNE2 cells after the combined treatment of curcumin and ultrasound.

Conclusions

Our findings demonstrated that ultrasound sonication in the presence of curcumin significantly killed the CNE2 cells and induced ultrastructural damage and the dysfunction of mitochondria, suggesting that ultrasound treatment remarkably induced cellular destruction of nasopharyngeal carcinoma cells in the presence of curcumin.     

Osteogenic differentiation of rat bone marrow stromal cells by various intensities of low-intensity pulsed ultrasound

Bone growth and repair are under the control of biochemical and mechanical signals. Low-intensity pulsed ultrasound (LIPUS) stimulation at 30 mW/cm² is an established, widely used and FDA approved intervention for accelerating bone healing in fractures and non-unions. Although this LIPUS signal accelerates mineralization and bone regeneration, the actual intensity experienced by the cells at the target site might be lower, due to the possible attenuation caused by the overlying soft tissue. The aim of this study was to investigate whether LIPUS intensities below 30 mW/cm² are able to provoke phenotypic responses in bone cells. Rat bone marrow stromal cells were cultured under defined conditions and the effect of 2, 15, 30 mW/cm² and sham treatments were studied at early (cell activation), middle (differentiation into osteogenic cells) and late (biological mineralization) stages of osteogenic differentiation. We observed that not only 30 mW/cm² but also 2 and 15 mW/cm², modulated ERK1/2 and p38 intracellular signaling pathways as compared to the sham treatment. After 5 days with daily treatments of 2, 15 and 30 mW/cm², alkaline phosphatase activity, an early indicator of osteoblast differentiation, increased by 79%, 147% and 209%, respectively, compared to sham, indicating that various intensities of LIPUS were able to initiate osteogenic differentiation. While all LIPUS treatments showed higher mineralization, interestingly, the highest increase of 225% was observed in cells treated with 2 mW/cm². As the intensity increased to 15 and 30 mW/cm², the increase in the level of mineralization dropped to 120% and 82%. Our data show that LIPUS intensities lower than the current clinical standard have a positive effect on osteogenic differentiation of rat bone marrow stromal cells. Although Exogen™ at 30 mW/cm² continues to be effective and should be used as a clinical therapy for fracture healing, if confirmed in vivo, the increased mineralization at lower intensities might be the first step towards redefining the most effective LIPUS intensity for clinical use.     

Impact of thermal effects induced by ultrasound on viability of rat C6 glioma cells

In order to have consistent and repeatable effects of sonodynamic therapy (SDT) on various cancer cells or tissue lesions we should be able to control a delivered ultrasound energy and thermal effects induced. The objective of this study was to investigate viability of rat C6 glioma cells in vitro depending on the intensity of ultrasound in the region of cells and to determine the exposure time inducing temperature rise above 43 °C, which is known to be toxic for cells. For measurements a planar piezoelectric transducer with a diameter of 20 mm and a resonance frequency of 1.06 MHz was used. The transducer generated tone bursts with 94 μs duration, 0.4 duty-cycle and initial intensity I_SATA (spatial averaged, temporal averaged) varied from 0.33 W/cm² to 8 W/cm² (average acoustic power varied from 1 W to 24 W). The rat C6 glioma cells were cultured on a bottom of wells in 12-well plates, incubated for 24 h and then exposed to ultrasound with measured acoustic properties, inducing or causing no thermal effects leading to cell death. Cell viability rate was determined by MTT assay (a standard colorimetric assay for assessing cell viability) as the ratio of the optical densities of the group treated by ultrasound to the control group. Structural cellular changes and apoptosis estimation were observed under a microscope. Quantitative analysis of the obtained results allowed to determine the maximal exposure time that does not lead to the thermal effects above 43 °C in the region of cells for each initial intensity of the tone bursts used as well as the threshold intensity causing cell death after 3 min exposure to ultrasound due to thermal effects. The averaged threshold intensity was found to be about 5.7 W/cm².     

Dual-high-frequency ultrasound excitation on microbubble destruction volume

Objective and motivation

The goal of this work was to test experimentally that exposing air bubbles or ultrasound contrast agents in water to amplitude modulated wave allows control of inertial cavitation affected volume and hence could limit the undesirable bioeffects.

Methods

Focused transducer operating at the center frequency of 10 MHz and having about 65% fractional bandwidth was excited by 3 μs 8.5 and 11.5 MHz tone-bursts to produce 3 MHz envelope signal. The 3 MHz frequency was selected because it corresponds to the resonance frequency of the microbubbles used in the experiment. Another 5 MHz transducer was used as a receiver to produce B-mode image. Peak negative acoustic pressure was adjusted in the range from 0.5 to 3.5 MPa. The spectrum amplitudes obtained from the imaging of SonoVue^TM contrast agent when using the envelope and a separate 3 MHz transducer were compared to determine their cross-section at the - 6 dB level.

Results

The conventional 3 MHz tone-burst excitation resulted in the region of interest (ROI) cross-section of 2.47 mm while amplitude modulated, dual-frequency excitation with difference frequency of 3 MHz produced cross-section equal to 1.2 mm.

Conclusion

These results corroborate our hypothesis that, in addition to the considerably higher penetration depth of dual-frequency excitation due to the lower attenuation at 3 MHz than that at 8.5 and 11.5 MHz, the sample volume of dual-frequency excitation is also smaller than that of linear 3-MHz method for more spatially confined destruction of microbubbles.     

Investigation of the effect of power ultrasound on the nucleation of water during freezing of agar gel samples in tubing vials

Nucleation, as an important stage of freezing process, can be induced by the irradiation of power ultrasound. In this study, the effect of irradiation temperature (−2 °C, −3 °C, −4 °C and −5 °C), irradiation duration (0 s, 1 s, 3 s, 5 s, 10 s or 15 s) and ultrasound intensity (0.07 W cm⁻², 0.14 W cm⁻², 0.25 W cm⁻², 0.35 W cm⁻² and 0.42 W cm⁻²) on the dynamic nucleation of ice in agar gel samples was studied. The samples were frozen in an ethylene glycol-water mixture (−20 °C) in an ultrasonic bath system after putting them into tubing vials. Results indicated that ultrasound irradiation is able to initiate nucleation at different supercooled temperatures (from −5 °C to −2 °C) in agar gel if optimum intensity and duration of ultrasound were chosen. Evaluation of the effect of 0.25 W cm⁻² ultrasound intensity and different durations of ultrasound application on agar gels showed that 1 s was not long enough to induce nucleation, 3 s induced the nucleation repeatedly but longer irradiation durations resulted in the generation of heat and therefore nucleation was postponed. Investigation of the effect of ultrasound intensity revealed that higher intensities of ultrasound were effective when a shorter period of irradiation was used, while lower intensities only resulted in nucleation when a longer irradiation time was applied. In addition to this, higher intensities were not effective at longer irradiation times due to the heat generated in the samples by the heating effect of ultrasound. In conclusion, the use of ultrasound as a means to control the crystallization process offers promising application in freezing of solid foods, however, optimum conditions should be selected.     

Osteocytes exposed to far field of therapeutic ultrasound promotes osteogenic cellular activities in pre-osteoblasts through soluble factors

Low intensity pulsed ultrasound (LIPUS) was reported to accelerate the rate of fracture healing. When LIPUS is applied to fractures transcutaneously, bone tissues at different depths are exposed to different ultrasound fields. Measurement of LIPUS shows pressure variations in near field (nearby transducer); uniform profile was found beyond it (far field). Moreover, we have reported that the therapeutic effect of LIPUS is dependent on the axial distance of ultrasound beam in rat fracture model. However, the mechanisms of how different axial distances of LIPUS influence the mechanotransduction of bone cells are not understood. To understand the cellular mechanisms underlying far field LIPUS on enhanced fracture healing in rat model, the present study investigated the effect of ultrasound axial distances on (1) osteocyte, the mechanosensor, and (2) mechanotransduction between osteocyte and pre-osteoblast (bone-forming cell) through paracrine signaling. We hypothesized that far field LIPUS could enhance the osteogenic activities of osteoblasts via paracrine factors secreted from osteocytes. The objective of this study was to investigate the effect of axial distances of LIPUS on osteocytes and osteocyte–osteoblast mechanotransduction. In this study, LIPUS (plane; 2.2 cm in diameter, 1.5 MHz sine wave, I_SATA = 30 mW/cm²) was applied to osteocytes (mechanosensor) at three axial distances: 0 mm (near field), 60 mm (mid-near field) and 130 mm (far field). The conditioned medium of osteocytes (OCM) collected from these three groups were used to culture pre-osteoblasts (effector cell). In this study, (1) the direct effect of ultrasound fields on the mechanosensitivity of osteocytes; and (2) the osteogenic effect of different OCM treatments on pre-osteoblasts were assessed. The immunostaining results indicated the ultrasound beam at far field resulted in more β-catenin nuclear translocation in osteocytes than all other groups. This indicated that osteocytes could detect the acoustic differences of LIPUS at various axial distances. Furthermore, we found that the soluble factors secreted by far field LIPUS exposed osteocytes could further promote pre-osteoblasts cell migration, maturation (transition of cell proliferation into osteogenic differentiation), and matrix calcification. In summary, our results of this present study indicated that axial distance beyond near field could transmit ultrasound energy to osteocyte more efficiently. The LIPUS exposed osteocytes conveyed mechanical signals to pre-osteoblasts and regulated their osteogenic cellular activities via paracrine factors secretion. The soluble factors secreted by far field exposed osteocytes led to promotion in migration and maturation in pre-osteoblasts. This finding demonstrated the positive effects of far field LIPUS on stimulating osteocytes and promoting mechanotransduction between osteocytes and osteoblasts.     

Apoptosis of ovarian cancer cells induced by methylene blue-mediated sonodynamic action

Objective

The present study aims to investigate apoptosis of ovarian cancer cells induced by methylene blue (MB)-mediated sonodynamic therapy (SDT).

Methods

The MB concentration was kept constant at 100 μM and ovarian cancer HO-8910 cells were exposed to ultrasound therapy for 5 s with an intensity of 0.46 W/cm². The cytotoxicity was investigated 24 h after MB-mediated sonodynamic action. Apoptosis was analyzed using a flow cytometer with Annexin V-FITC and propidium iodine (PI) staining as well as fluorescence microscopy with Hoechst 33258 staining. Intracellular reactive oxygen species (ROS) level was measured by flow cytometer with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) staining.

Results

The cytotoxicity of MB-mediated SDT on HO-8910 cells after MB-mediated SDT was significantly higher than those of other treatments including ultrasound alone, MB alone and sham treatment. Flow cytometric analysis showed a significant increase in the early and late apoptotic cell populations by MB-mediated SDT of HO-8910 cells. Nuclear condensation and increased ROS levels were also found in HO-8910 cells treated by MB-mediated SDT.

Conclusions

Our findings demonstrated that MB-mediated sonodynamic action significantly induced apoptosis of HO-8910 cells and an increase in intracellular ROS level. This indicates that apoptosis is an important mechanism of cell death induced by MB-mediated SDT. Thus, MB-mediated SDT might be a potential therapeutic strategy for combating ovarian cancer.     

Sonodynamic action of pyropheophorbide-a methyl ester induces mitochondrial damage in liver cancer cells

Sonodynamic therapy with pyropheophorbide-a methyl ester (MPPa) presents a promising aspect in treating liver cancer. The present study aims to investigate the mitochondrial damage of liver cancer cells induced by MPPa-mediated sonodynamic action. Mouse hepatoma cell line H₂₂ cells were incubated with MPPa (2 μM) for 20 h and then exposed to ultrasound with an intensity of 0.97 W/cm² for 8 s. Cytotoxicity was investigated 24 h after sonodynamic action using MTT assay and light microscopy. Mitochondrial membrane potential (ΔΨm) was analyzed using flow cytometry with rhodamine 123 staining and ultrastructural changes were observed using transmission electron microscopy (TEM).The cytotoxicity of MPPa-mediated SDT on H₂₂ cell line was 73.00 ± 3.42%, greater than ultrasound treatment alone (28.12 ± 5.19%) significantly while MPPa treatment alone had no significant effect on H₂₂ cells. Moreover, after MPPa-mediated SDT cancer cells showed swollen mitochondria under TEM and a significant collapse of mitochondrial membrane potential. Our findings demonstrated that MPPa-mediated SDT could remarkably induce cell death of H₂₂ cells, and highlighted that mitochondrial damage might be an important cause of cell death induced by MPPa-mediated SDT.     

In vivo imaging of blood flow in the mouse Achilles tendon using high-frequency ultrasound

Objective

Achilles tendinitis is a common clinical problem with many treatment modalities, including physical therapy, exercise and therapeutic ultrasound. However, evaluating the effects of current therapeutic modalities and studying the therapeutic mechanism(s) in vivo remains problematic. In this study, we attempted to observe the morphology and microcirculation changes in mouse Achilles tendons between pre- and post-treatment using high-frequency (25 MHz) ultrasound imaging. A secondary aim was to assess the potential of high-frequency ultrasound in exploring therapeutic mechanisms in small-animal models in vivo.

Methods

A collagenase-induced mouse model of Achilles tendinitis was adopted, and 5 min treatment of continuous-mode low-frequency (45 kHz) ultrasound with 47 mW/cm² maximum intensity and 16.3 cm² effective beam radiating area was applied. The B-mode images showed no focal hypoechoic regions in normal Achilles tendons either pre- or post-treatment. The Doppler power energy and blood flow rate were measured within the peritendinous space of the Achilles tendon.

Conclusion

An increase in the microcirculation was observed soon after the low-frequency ultrasound treatment, which was due to immediate induction of vascular dilatation. The results suggest that applying high-frequency Doppler imaging to small-animal models will be an invaluable aid in explorations of the therapeutic mechanism(s). Our future work includes using imaging to assess microcirculation changes in tendinitis between before and after treatment over a long time period, which is expected to yield useful physiological data for future human studies.     

The targeted gene (KDRP-CD/TK) therapy of breast cancer mediated by SonoVue and ultrasound irradiation in vitro

Suicide gene therapy has become an effective therapy for breast cancer, and ultrasound targeted microbubble destruction (UTMD) has become a popular topic in the gene therapy field. In this study, MCF-7 cells with the KDR promoter and LSl74T cells without the KDR promoter were transfected with the recombinant plasmid pEGFP-KDRP-CD/TK using UTMD. The recombinant plasmid pEGFP-KDRP-CD/TK was transfected into MCF-7 and LS174T cells successfully with no significant difference in transfection efficiency (p > 0.05). By RT-PCR, the CD/TK fusion gene was shown to be expressed in MCF-7 cells but not expressed in LS174T cells. In a cytotoxicity experiment, transgenic MCF-7 cells were sensitive to the prodrugs 5-FC and GCV. When both 5-FC and GCV were administered, the rate of cellular inhibition was significantly greater than that achieved when only one of the prodrugs was administered (p < 0.001). Moreover, the inhibition rates achieved administering 5-FC, GCV and both 5-FC and GCV were all significantly greater than the gene transfection rate of 21.92 ± 3.64% (p < 0.001). However, transgenic LS174T cells were not sensitive to any prodrug. These results demonstrated that UTMD is a safe, effective and targeted gene delivery system. Also, the KDR promoter can drive expression of the CD/TK double suicide gene target in MCF-7 cells, and the targeted killing effect of the KDRP-CD/TK gene on MCF-7 cells in vitro has good synergy with expression of the CD/TK fusion gene.