Pulsed Ultrasound Research Articles

Pulmonary Capillary Hemorrhage Induced by Fixed-Beam Pulsed Ultrasound

The induction of pulmonary capillary hemorrhage (PCH) by pulsed ultrasound was discovered 25 y ago, but early research used fixed-beam systems rather than actual diagnostic ultrasound machines. In this study, results of exposure of rats to fixed-beam focused ultrasound for 5 min at 1.5 and 7.5 MHz were compared with recent research on diagnostic ultrasound. One exposure condition at each frequency used 10-μs pulses delivered at 25-ms intervals. Three conditions involved Gaussian modulation of the pulse amplitudes at 25-ms intervals to simulate diagnostic scanning: 7.5 MHz with 0.3- and 1.5-μs pulses at 100- and 500-μs pulse repetition periods, respectively, and 1.5 MHz with 1.7-μs pulses at 500-μs repetition periods. Four groups were tested for each condition to assess PCH areas at different exposure levels and to determine occurrence thresholds. The conditions with identical pulse timing resulted in smaller PCH areas for the smaller 7.5-MHz beam, but both had thresholds of 0.69–0.75 MPa in situ peak rarefactional pressure amplitude. The Gaussian modulation conditions for both 7.5 MHz with 0.3-μs pulses and 1.5 MHz with 1.7-μs pulses had thresholds of 1.12–1.20 MPa peak rarefactional pressure amplitude, although the relatively long 1.5-μs pulses at 7.5 MHz yielded a threshold of 0.75 MPa. The fixed-beam pulsed ultrasound exposures produced lower thresholds than diagnostic ultrasound. There was no clear tendency for thresholds to increase with increasing ultrasonic frequency when pulse timing conditions were similar.

Low‐intensity Ultrasound Enhances Histon Acetylation and Improves Anti‐inflammatory Effects by Short Chain Fatty Acid Treatments in Human Dermal Fibroblasts

Short‐chain fatty acids such as butyrate (BT) with the emergence of prebiotics and probiotics improve colonic and systemic health. Especially BT has anti‐inflammatory effects and mediated by the inhibition of histone deacetylase (HDAC). However, the effect appeared to be concentration‐dependent, and lower concentrations of BT had no effect. Therefore, the facilitatory effect of low‐intensity ultrasound (US) on the anti‐inflammatory effects by BT was investigated in the present study. Human dermal fibroblasts were treated with 1 mM BT for 3 h with 20 min irradiation of 0.1 W/cm2 US at the beginning of the BT treatments. Whereas BT could not increase histone acetylation, the combined with BT and US treatments significantly increased histone acetylation in fibroblasts. In addition, this combined treatment significantly suppressed IL‐6 mRNA expression by lipopolysaccharide stimulation for 1 h after the combined treatments, but the treatment of BT alone could not decrease IL‐6 expression in fibroblasts. These effects were achieved with both of 20% pulsed and continuous US, and not observed at the treatments by US without BT. These results suggest the promotional effects of low‐intensity US on the physiological actions of BT as HDAC inhibitor, and the potential applicability of this combination in dermal fibroproliferative disorders. Supported by Grants‐in‐Aid for Scientific Research (247005300) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

A Combination of Low-Intensity Pulsed Ultrasound and Nanohydroxyapatite Concordantly Enhances Osteogenesis of Adipose-Derived Stem Cells From Buccal Fat Pad.

The osteogenic induction of adipose-derived stem cells (ADSCs) has been regarded as an important step in bone tissue engineering. In the present study, we focused on the buccal fat pad (BFP) as a source of adipose tissue, since BFPs are encapsulated by adipose tissue and are often coextirpated during oral surgery. Low-intensity pulsed ultrasound (LIPUS) is effective in the treatment of fractures, and nanohydroxyapatite (NHA) is known as a bone substitute material. Here we investigated the synergistic effects of LIPUS and NHA in the osteogenesis of ADSCs. A combination of LIPUS irritation and NHA as a scaffold significantly increased the osteogenic differentiation of ADSCs in vitro, and in our in vivo study in which ADSCs were transplanted into calvarial bone defects of nude mice, the combinational effect greatly enhanced the new bone formation of the margin of the defects. These results demonstrate that synergistic effects of LIPUS and NHA are capable of effectively inducing the differentiation of ADSCs into osteoblasts, and they suggest a novel therapeutic strategy for bone regeneration by the autotransplantation of ADSCs.

Effect of Low-Intensity Pulsed Ultrasound on Quality of Life of Knee Osteoarthritis

Background: Knee osteoarthritis (OA) is a common disease worldwide and causes disability in elderly. Recent studies showed that therapeutic ultrasound (US) can reduced pain in various musculoskeletal disorders. Objective: To determined the effect of low-intensity pulsed US on quality of life and reduction of pain in grade 1 to 3 knee OA. Material and methods: We conducted a randomized control trial before and after therapy in 61 knee OA patients. We applied 12 sessions of low-intensity pulsed US, 20 minutes each, with frequency 1 MHz, and intensity of 0.2 W/cm2, duty cycle of 20% to 28 patients. Thirty three patients without therapy were categorized as control group. Reduction of pain by a visual analog scale and Quality of life by The Western Ontario and McMaster Universities (WOMAC) questionnaire, were obtained before and after 12 sessions. We used the Wilcoxon test to compare the median and the Mann-Whitney U test for the difference between groups with a p value of 0.05 was considering as significant. Results: We observed a reduction of pain in therapy group (VAS before 3.5 (2/7); after 2.0 (0/5); p=0.001) and also in control group (VAS before 5.1 (2/8); after 4.0 (2/7); p=0.001), but the reduction was significantly more prominent in therapy group (p=0.004). There was a better quality of life in therapy group (WOMAC before 21.35 (4.2/52.1); after 11.45 (0.0/47.9); p=0.001) and also in control group (WOMAC before 25.00 (5.2/60.4); after 20.83 (3.1/55.2); p=0.001), but the score was significantly better in therapy group (p=0.001). Conclusions: Twelve sessions of low-intensity pulsed US has a benefit effect over pain and quality of life in knee OA patients.

Ultrasound v. sham ultrasound for experimentally induced delayed-onset muscle soreness: A double-blind, randomised controlled trial

Background. Therapeutic ultrasound (US) is an electrophysical therapy that is commonly used by sports physiotherapists, but its mechanismof action is unclear. There is little evidence that US therapy is more effective than sham US therapy, and any clinical benefits may be dueto a placebo effect.Objective. To investigate whether US has a specific effect that renders it effective in its own right, or whether its effect is placebo driven.Methods. In a double-blind controlled trial, delayed-onset muscle soreness (DOMS) was experimentally induced in both bicep musclesof 15 females. Sham US was applied to one bicep (n=15 biceps) and pulsed active US to the other bicep (n=15 biceps) of each participant,48 and 72 h after induction of DOMS. Primary and secondary outcomes were pain reported on the McGill Pain Questionnaire (MPQ) andrange of movement (ROM) (elbow extension) measured by goniometry, respectively.Results. Results showed significant improvements in pain and ROM over the intervention periods, but there was no difference betweeninterventions.Conclusion. US and sham US therapy improve pain equally when treating DOMS of the biceps in the context of a therapeutic encounter.This analgesic effect is placebo driven. Clinicians can influence the analgesic effect of US by managing the therapeutic context. Managementof patients’ anxiety may also boost the analgesic effect of US.

Effectiveness of continuous and pulsed ultrasound for the management of knee osteoarthritis: a systematic review and network meta-analysis

To investigate the efficacy of continuous and pulsed ultrasound (US) in the management of knee osteoarthritis (OA). This systematic review and network meta-analysis covered 12 trials in total. Electronic databases including MEDLINE, Embase and Cochrane Library were searched through to identify randomized controlled trials comparing the two modes of US with control interventions (sham or blank) or with each other. Bayesian network meta-analysis was used to integrate both the direct and indirect evidences on treatment effectiveness. Pulsed US (PUS) is more effective in both pain relief and function improvement when compared with the control group; but for continuous US (CUS), there is only a significant difference in pain relief in comparison with the control group. In addition, no matter in terms of pain intensity or function at the last follow-up time point, PUS always exhibited a greater probability of being the preferred mode. However, the evidence of heterogeneity and the limitation in sample size of some studies could be a potential threat to the validity of results. Our findings indicated that PUS, with a greater probability of being the preferred mode, is more effective in both pain relief and function improvement when compared with the control group. However, CUS could only be considered as a pain relief treatment in the management of knee OA. The findings also confirmed that none of these modes is dangerous. Level II, systematic review and network meta-analysis of randomized controlled trials.

Low-intensity pulsed ultrasound induces osteogenic differentiation of human periodontal ligament cells through activation of bone morphogenetic protein-smad signaling.

Low-intensity pulsed ultrasound (US) can accelerate fracture healing and osteogenic differentiation. The aim of this study was to investigate the osteogenic effect of low-intensity pulsed US on human periodontal ligament cells and to determine whether bone morphogenetic protein (BMP)-Smad signaling was involved. Human periodontal ligament cells were exposed to low-intensity pulsed US at a frequency of 1.5 MHz and intensity of 90 mW/cm(2) for 20 min/d. Osteogenic differentiation was determined by assaying alkaline phosphatase (ALP) and calcium deposition. Expression of BMP-2, BMP-6, and BMP-9 was detected by real-time polymerase chain reaction analysis. Phosphorylated Smad was detected by western blotting; Smad in the cells was labeled by an immunofluorescent antibody and observed by laser-scanning confocal microscopy. The optical density of ALP stimulated by US at 1.5 MHz and 90 mW/cm(2) for 20 min/d was significantly higher than in other groups (P < .01); therefore, this dosage was considered optimal for promoting osteogenic differentiation. After 13 days of US exposure, ALP increased gradually after 5 days, peaked at 11 days, and decreased at 13 days, with a significant difference compared with the control group (P < .05). Osteocalcin production increased from 9 to 13 days and peaked at 15 days, with a significant difference compared with the control group (P < .05). BMP-2 and BMP-6 increased dynamically after exposure for 13 days. BMP-2 increased 6.07-fold at 3 days, 6.39-fold at 11 days, and 5.97-fold at 13 days. BMP-6 expression increased 6.82-fold at 1 day and 51.5-fold at 3 days and decreased thereafter. BMP-9 was not expressed. Phospho-Smad1/5/8 expression was significantly increased after exposure (P< .05) and transferred from the cytoplasm into the nuclei. Low-intensity pulsed US effectively induced osteogenic differentiation of human periodontal ligament cells, and the BMP-Smad signaling pathway was involved in the mechanism.

Efficacy of Therapeutic Ultrasound for the Management of Knee Osteoarthritis

The aim of this study was to compare whether the effectiveness of continuous ultrasound (US) was superior against pulsed US and against sham US in knee osteoarthritis. A randomized controlled study was carried out on 60 patients diagnosed with knee osteoarthritis according to American College of Rheumatology. The patients were randomized into the following three treatments: (1) continuous US (at a frequency of 1 MHz with intensity of 1 W/cm), (2) pulse US (same frequency and intensity on 1:4 pulse ratio), and (3) sham US. All treatments were applied with 5-cm head US device five times a week for 2 weeks in addition to home exercise program including quadriceps isometric exercise, muscle strength exercises, and stretching exercises of the lower extremity muscles for at least three times per week. Assessments were performed at baseline, at the end of the treatment, and at the end of the treatments and at the sixth month using the following measurements: Western Ontario and McMaster University Osteoarthritis Index-pain, stiffness, function, visual analog scale-pain at rest, visual analog scale-pain on movement, visual analog scale-disease severity, and 20-m walking time. Among these parameters, the Western Ontario and McMaster University Osteoarthritis Index-pain was the primary outcome. All groups showed a significant improvement in all parameters in both following visits (P < 0.05). However, there was no significant difference between the groups. Although the mean reduction percent in Western Ontario and McMaster University Osteoarthritis Index-pain was significantly higher in group I (continuous US) when compared to sham group (46.5% vs 28.9%, P < 0.05) at the end of the treatment, this result was not found in other pain parameters. The present study demonstrated that all assessment parameters significantly improved in all groups without a significant difference. This result suggested that therapeutic US provided no additional benefit in improving pain and functions in addition to exercise training.

Ultrasound Enhanced PEI-Mediated Gene Delivery Through Increasing the Intracellular Calcium Level and PKC-δ Protein Expression

Polyethylenimine (PEI), a cationic polymer, has been shown to aggregate plasmid DNA and facilitate its internalization. It has also been shown that combining ultrasound (US) with PEI could enhance and prolong in vitro and in vivo transgene expression. However, the role US in the enhancement of PEI uptake is poorly understood. This study investigates the impact of US on PEI-mediated gene transfection. Specific endocytosis pathway siRNA, including clathrin HC siRNA, caveolin-1 siRNA and protein kinase C-delta (PKC-δ) siRNA, are used to block the corresponding endocytosis pathways prior to the transfection of luciferase DNA/PEI polyplexes to cultured cells by 1-MHz pulsed US with ultrasound contrast agent SonoVue®. Transgene expression was found not to be enhanced by US treatment in the presence of the PKC-δ siRNA. We further demonstrated that PKC-δ protein could be enhanced at 6h after US exposure. Moreover, intracellular calcium levels were found to be significantly increased at 3h after US exposure, while transgene expressions were significantly reduced in the presence of calcium channel blockers both in vitro and in vivo. Our results suggest that US enhanced PEI-mediated gene transfection specifically by increasing PKC-δ related fluid phase endocytosis, which was induced by increasing the intracellular calcium levels.

The Non-Thermal Effects of Pulsed Ultrasound Irradiation on the Development of Disuse Muscle Atrophy in Rat Gastrocnemius Muscle

This study examined the effects of therapeutic pulsed ultrasound (US) on the development of disuse muscle atrophy in rat gastrocnemius muscle. Male Wistar rats were randomly distributed into control, immobilization (Im), sham US, and US groups. In the Im, sham US and US groups, the bilateral ankle joints of each rat were immobilized in full plantar flexion with a plaster cast for a 4-wk period. The pulsed US (frequency, 1 MHz; intensity, 1.0 W/cm2; pulsed mode 1:4; 15 min) was irradiated to the gastrocnemius muscle in the US group over a 4-wk immobilization period. The pulsed US irradiation delivered only non-thermal effects to the muscle. In conjunction with US irradiation, 5-bromo-2’-deoxyuridine (BrdU) was injected subcutaneously to label the nuclei of proliferating satellite cells 1 h before each pulsed US irradiation. Immobilization resulted in significant decreases in the mean diameters of type I, IIA and IIB muscle fibers of the gastrocnemius muscle in the Im, sham US and US groups compared with the control group. However, the degrees of muscle fiber atrophy for all types were significantly lower in the US group compared with the Im and sham US groups. Although the number of capillaries and the concentrations of insulin-like growth factor and basic fibroblast growth factor did not change in the muscle, the number of BrdU-positive nuclei in the muscle was significantly increased by pulsed US irradiation in the US group. The results of this study suggest that pulsed US irradiation inhibits the development of disuse muscle atrophy partly via activation of satellite cells.

&lt;b&gt;Effects of therapeutic ultrasound on haematological dynamics and fibrinogen during the inflammatory phase after muscle injury in rats &lt;/b&gt;

This study investigated the effects of ultrasound therapy on haematological dynamics and plasma fibrinogen during the inflammatory phase of muscle injury. Forty-eight male Wistar rats were divided into control group (CG), continuous ultrasound treated group (CTU) and pulsed ultrasound (PTU). Animals were subjected to surgical incision. A transverse lesion was made in the biceps femoris muscle (50%). CTU (1.0 MHz) was applied at 12-hour intervals on the lesion, for three days, with 0.4 W cm -2 and three minutes of duration (six applications in the total). PTU was applied in the pulsed mode 20% (2 ms on/8 ms off), maintaining the other parameters. Fibrinogen, white and red blood cells were analyzed in the 24 th , 48 th and 72 nd hour after the injury. PTU has reduced fibrinogen levels by 20% at the 24 th h and by 30% at the 48 th h (p < 0.001) and haemoglobin reduction at the 72 nd hour (p < 0.001), which had already occurred during the 2 nd collection in the other groups. CTU favoured erythrocyte reduction at the 48 th h (p = 0.003). PTU presented an anti-inflammatory effect due to plasma fibrinogen reduction, and CTU favored haemorrhage due to the reduction of erythrocytes when applied in the first 72 hours after muscle injury.

Contribution of Inertial Cavitation in the Enhancement of In Vitro Transscleral Drug Delivery

In ocular drug delivery, the sclera is a promising pathway for administering drugs to both the anterior and posterior segments of the eye. Due to the low permeability of the sclera, however, efficient drug delivery is challenging. In this study, pulsed ultrasound (US) was investigated as a potential method for enhancing drug delivery to the eye through the sclera. The permeability of rabbit scleral tissue to a model drug compound, sodium fluorescein, was measured after US-irradiation at 1.1 MHz using time-averaged acoustic powers of 0.5–5.4 W (6.8–12.8 MPa peak negative pressure), with a fixed duty cycle of 2.5% for two different pulse repetition frequencies of 100 and 1000 Hz. Acoustic cavitation activity was measured during exposures using a passive cavitation detector and was used to quantify the level of bubble activity. A correlation between the amount of cavitation activity and the enhancement of scleral permeability was demonstrated with a significant enhancement in permeability of US exposed samples compared to controls. Transmission electron microscopy showed no evidence of significant alteration in viability of tissue exposed to US exposures. A pulsed US protocol designed to maximum cavitation activity may therefore be a viable method for enhancing drug delivery to the eye.

Pulsed and Continuous Ultrasound Increase Chondrogenesis through the Increase of Heat Shock Protein 70 Expression in Rat Articular Cartilage.

[Purpose] The present study was aimed to investigate the effects of pulsed and continuous ultrasound (US) irradiation on heat shock protein (HSP) 70 and mRNA levels of chondrogenesis-related gene expression in rat tibial articular cartilage. [Subjects and Methods] Forty-eight rats with body weights of 200−250 g were randomly divided into three groups. In the control (CON) group, three rats were treated with sham sonication. The pulsed US irradiation group was irradiated with a pulse rate of 20%, a frequency of 1 MHz, and an intensity of 1.5 W/cm2 for 10 minutes. The continuous US irradiation group was continuously with a frequency of 1 MHz and an intensity of 1.5 W/cm2 for 10 minutes. Immunohistochemistry for evaluation of HSP 70 and RT-PCR for expression of the chondrogenesis-related mRNA were used. [Results] The expression of HSP70 protein was increased in the pulsed and continuous US groups. The increase in the continuous US group was more prominent than in the pulsed US group. In addition, pulsed and continuous US irradiation increased the expression of Mustn1 and Sox9. [Conclusion] The results of this study show that US increases chondrogenesis via the increase of HSP 70 and chondrogenesis-related mRNA expressions in rat articular cartilage.

Short-term sensory and cutaneous vascular responses to therapeutic ultrasound in the forearms of healthy volunteers.

BackgroundTherapeutic ultrasound (US) is used for a variety of clinical pathologies and is thought to accelerate tissue repair and help with pain reduction via its thermal and nonthermal effects. The evidence on physiological effects of US on both sensory and vascular functions in humans is incomplete. Hence, the purpose of this study was to determine the short-term impact of two doses of US (3 MHz, 1:4, 0.25 W/cm2, 5 min; 1 MHz, continuous, 0.8 W/cm2, 3 min), on sensory and vascular responses in the healthy forearms.MethodsTwenty healthy subjects were recruited (mean age, 29.6 ± 8.8 years) for the study. Superficial blood flow (SBF) in the distal forearms was determined using the tissue viability imaging system. Sensory perception thresholds (SPT) were determined from ring finger (C7, C8) to assess A-beta (at 2,000 Hz) and C fiber function (at 5 Hz), using a Neurometer CPT/C device. Subject’s two hands were randomly allocated to group order (AB/BA). Scores were obtained before and immediately after the application of US and control. Differences in these were analyzed using repeated measures.ResultsBoth 3 MHz pulsed US and 1 MHz continuous US showed small to moderate (effect size = 0.12 to 0.68), statistically significant reductions in SBF (3 MHz, mean change = 2.8 AU and 1 MHz, mean change = 3.9 AU, p < 0.05 respectively), skin temperature (2.5°C and 1.1°C, p < 0.05), and SPT at 5 Hz (1.3 and 1 mA, p < 0.05) across time. SPT at 2,000 Hz remained unaltered by all three conditions (p > 0.05). Age and gender also had no effect on all outcome measures (p > 0.05).ConclusionThis study demonstrated minor reductions in skin blood flow, skin temperatures, and C fiber perception thresholds immediately after 3 MHz, and 1 MHz US. The responses observed may have been due to a thermo-cooling effect of the gel or due to the direct effect of US on C fibers of median and ulnar nerves. US had a negligible effect on A-beta fibres. This would suggest that future studies looking at physiological effects of US should move towards investigating larger dosages and study the effects in patient populations.

Pulsed ultrasound modulated optical tomography utilizing the harmonic response of lock-in detection

Ultrasound modulated optical tomography (USMOT) can image the optical properties of a scattering medium at a spatial resolution approaching that of ultrasound (US). A lock-in parallel speckle detection technique is proposed to detect pulsed US modulated light using a multipixel detector. The frequency components of the pass band match those of the US pulse train and provide efficient detection. The modulation depth is extracted by taking the difference between a pair of speckle patterns modulated by a pair of phase-inversed US bursts. Modification to pulse inversion mode enables the second harmonic US modulation due to nonlinear US propagation to be detected.

Low-intensity pulsed ultrasound enhances cell killing induced by X-irradiation

To determine the effect of pulsed ultrasound (US) on radiation-induced cell killing, U937 and Molt-4 cell lines were exposed to 1.0MHz US with 50% of duty factor at 0.3W/cm2 and pulsed at 1Hz immediately after exposure to X-rays at 0, 0.5, 2.5 and 5Gy. The cells were assayed 24h after the treatments. The result showed significant enhancement of cell killing in the combined treatments. However, the ratio of apoptotic cells induced either by X-rays or US alone did not significantly change. These findings suggest that pulsed US can enhance the anticancer effect of X-irradiation due to US streaming under non-inertial cavitational condition. This combined treatment can potentially enhance the therapeutic effect of radiation therapy.

Opening of the blood-brain barrier with an unfocused ultrasound device in rabbits

The blood-brain barrier (BBB) is a major impediment to the intracerebral diffusion of drugs used in the treatment of gliomas. Previous studies have demonstrated that pulsed focused ultrasound (US) in conjunction with a microbubble contrast agent can be used to open the BBB. To apply the US-induced opening of the BBB in clinical practice, the authors designed an innovative unfocused US device that can be implanted in the skull and used to transiently and repeatedly open the BBB during a standard chemotherapy protocol. The goal of this preliminary work was to study the opening of the BBB induced by the authors' small unfocused US transducer and to evaluate the effects of the sonications on brain parenchyma. Craniectomy was performed in 16 healthy New Zealand White rabbits; epidural application of a single-element planar ultrasonic transducer operating at 1 MHz was then used with a pulse-repetition frequency of 1 Hz, pulse lengths of 10-35 msec, in situ acoustic pressure levels of 0.3-0.8 MPa, and sonication for 60-120 seconds. SonoVue was intravenously injected during the US applications, and opening of the BBB was determined by detecting extravasation of Evans blue dye (EBD) in brain tissues, quantitative measurement of EBD with UV-visible spectrophotometry, and contrast enhancement after Gd injection in 4.7-T MRI. A histological study was performed to determine adverse effects. An opening of the BBB was observed over a large extent of the US beam in the brain corresponding to in situ pressures of greater than 0.2 MPa. The BBB opening observed was highly significant for both EBD (p < 0.01) and MRI Gd enhancement (p < 0.0001). The BBB opening was associated with minor adverse effects that included perivascular red blood cell extravasations that were less than 150 μm in size and not visible on MR images. Moderate edema was visible on FLAIR sequences and limited to the extent of the sonication field. The results demonstrate that the BBB can be opened in large areas of the brain in rabbits with low-power, pulsed, and unfocused US with limited damage to healthy tissue.

Effect of Low‐Intensity Pulsed Ultrasound on Bone Morphogenetic Protein 7–Induced Osteogenic Differentiation of Human Nonunion Tissue‐Derived Cells In Vitro

Low-intensity pulsed ultrasound (US) has been shown to have positive effects on the healing of nonunions, and bone morphogenetic protein 7 (BMP-7) is known to be a strong stimulator of osteogenic differentiation. Recently, we showed that nonunion tissue contains multilineage mesenchymal progenitor cells, suggesting that nonunion tissue-derived cells may play an important role during the healing process of nonunions. In this study, we investigated whether low-intensity pulsed US promoted BMP-7-induced osteogenic differentiation of nonunion tissue-derived cells in vitro. Nonunion tissue-derived cells were isolated from 7 patients. The cells were divided into two groups: (1) BMP-7 alone, consisting of nonunion tissue-derived cells cultured in osteogenic medium containing BMP-7 without low-intensity pulsed US treatment; and (2) BMP-7 + low-intensity pulsed US, consisting of nonunion tissue-derived cells cultured in osteogenic medium containing BMP-7 with low-intensity pulsed US treatment. The osteogenic differentiation potential and proliferation of nonunion tissue-derived cells were compared between the two groups. The alkaline phosphatase activity, gene expression levels of alkaline phosphatase and runt-related transcription factor 2, and mineralization were higher in the BMP-7 + low-intensity pulsed US group than in the BMP-7-alone group. There was no significant difference in cell proliferation between the two groups. These findings show a significant effect of low-intensity pulsed US on the osteogenic differentiation of nonunion tissue-derived cells induced by BMP-7. This study may provide substantial evidence for the clinical combined application of BMP-7 and low-intensity pulsed US for nonunion treatment.

Palladium N‐Heterocyclic Carbene Catalysts for the Ultrasound‐Promoted Suzuki–Miyaura Reaction in Glycerol

AbstractNovel palladium N‐heterocyclic‐carbene (NHC)‐based complexes with 3,4,5‐trimethoxybenzyl, alkyl and sulfonate N‐substituents were obtained and fully characterized. The new complexes were used as pre‐catalysts in the Suzuki–Miyaura coupling of various aryl halides/boron sources in glycerol under pulsed‐ultrasound (P‐US) activation. High yields were obtained under mild reaction conditions, without formation of undesired by‐products. The pure final cross‐coupling products were easily recovered without column chromatography and the catalytic/solvent system could be recycled. TEM (transmission electron microscopy) and XPS (X‐ray photoelectron spectroscopy) were used to characterize the nanoparticles and to investigate the fate of the catalysts.

Quantitative Evaluation of Ultrasound-Mediated Cellular Uptake of a Fluorescent Model Drug

This study aims to quantitatively analyze cellular uptake following local ultrasound (US)-mediated cell permeabilization. A 2μM cell-impermeable dye Sytox Green was co-injected with 3 × 10(7) microbubbles in the presence of C6 rat glioblastoma cell monolayer in total volume of 10ml. A 5.8-mm diameter mono-element US transducer was positioned at a distance of 8mm to the Opticell® membrane. Acoustical pressure of pulsed US was varied from 0.62MPa peak-to-peak (p-p) to 1.25MPa p-p. Large field of view (FOV = 15 × 15mm) 22 × 22 mosaic acquisitions were done under epifluorescence Leica DMR microscope and analyzed in Metamorph software to evaluate cell density as well as model drug uptake percentage. The size of acoustical field of the transducer closely matches the spatial pattern of the model drug internalized into the cells by US. Maximum of uptake percentage (42 ± 15%) was found at 0.88MPa p-p. Spatial aspect of US-mediated model drug uptake has been quantitatively evaluated on adherent cells using robust 2D-mapping approach.