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.     

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.     

Physical mechanisms of the therapeutic effect of ultrasound (a review)

Therapeutic ultrasound is an emerging field with many medical applications. High intensity focused ultrasound (HIFU) provides the ability to localize the deposition of acoustic energy within the body, which can cause tissue necrosis and hemostasis. Similarly, shock waves from a lithotripter penetrate the body to comminute kidney stones, and transcutaneous ultrasound enhances the transport of chemotherapy agents. New medical applications have required advances in transducer design and advances in numerical and experimental studies of the interaction of sound with biological tissues and fluids. The primary physical mechanism in HIFU is the conversion of acoustic energy into heat, which is often enhanced by nonlinear acoustic propagation and nonlinear scattering from bubbles. Other mechanical effects from ultrasound appear to stimulate an immune response, and bubble dynamics play an important role in lithotripsy and ultrasound-enhanced drug delivery. A dramatic shift to understand and exploit these nonlinear and mechanical mechanisms has occurred over the last few years. Specific challenges remain, such as treatment protocol planning and real-time treatment monitoring. An improved understanding of the physical mechanisms is essential to meet these challenges and to further advance therapeutic ultrasound.     

Elimination of standing wave effects in ultrasound radiation force excitation in air using random carrier frequency packets

The ultrasound radiation force has been used for noncontact excitation of devices ranging from microcantilevers to acoustic guitars. For ultrasound radiation force excitation, one challenge is formation of standing waves between the ultrasound transducer and the device under test. Standing waves result in constructive/destructive interference causing significant variations in the intensity of the ultrasound field. The standing-wave induced intensity variations in the radiation force can result from minor changes in the transducer position, carrier frequency, or changes in the speed of sound due to changes in ambient temperature. The current study demonstrates that by randomly varying the ultrasound carrier frequency in packets, it is possible to eliminate the negative consequences resulting from the formation of standing waves. A converging ultrasound transducer with a central frequency of 550 kHz was focused onto a brass cantilever. The 267 Hz resonance was excited with the ultrasound radiation force with a carrier frequency that randomly varied between 525 kHz to 575 kHz in packets of 10 cycles. Because each packet had a different carrier frequency, the amplitude of standing wave artifacts was reduced by a factor of 20 compared to a constant frequency excitation of 550 kHz.     

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.     

Effects of nonlinear propagation, cavitation, and boiling in lesion formation by high intensity focused ultrasound in a gel phantom

The importance of nonlinear acoustic wave propagation and ultrasound-induced cavitation in the acceleration of thermal lesion production by high intensity focused ultrasound was investigated experimentally and theoretically in a transparent protein-containing gel. A numerical model that accounted for nonlinear acoustic propagation was used to simulate experimental conditions. Various exposure regimes with equal total ultrasound energy but variable peak acoustic pressure were studied for single lesions and lesion stripes obtained by moving the transducer. Static overpressure was applied to suppress cavitation. Strong enhancement of lesion production was observed for high amplitude waves and was supported by modeling. Through overpressure experiments it was shown that both nonlinear propagation and cavitation mechanisms participate in accelerating lesion inception and growth. Using B-mode ultrasound, cavitation was observed at normal ambient pressure as weakly enhanced echogenicity in the focal region, but was not detected with overpressure. Formation of tadpole-shaped lesions, shifted toward the transducer, was always observed to be due to boiling. Boiling bubbles were visible in the gel and were evident as strongly echogenic regions in B-mode images. These experiments indicate that nonlinear propagation and cavitation accelerate heating, but no lesion displacement or distortion was observed in the absence of boiling.     

Generation and detection of nano ultrasound waves with a multiple strained layer structure

In this paper a multiple strained layer structure with multiple quantum wells as a piezoelectric transducer is proposed for generating and detecting nano ultrasound waves with nanometer wavelength and tera hertz frequency. By inducing femtosecond optical pulses at this strained structure, internal piezoelectric field is changed. As a result longitudinal acoustic phonon oscillations can be treated as nano acoustic waves. It could be noticed in simulated cases that detection of nano ultrasound waves can be used in non destructive testing and high accuracy measurements with this structure. It is also shown that the MQW structure design how influences in generated nano acoustic waves.     

Readout-segmented echo-planar diffusion-weighted MR at 3.0 T for the evaluation the effect of low-intensity transcranial ultrasound on stroke in a rat model

ObjectiveTo investigate the feasibility of the relative apparent diffusion coefficient (rADC) values based on readout-segmented echo-planar diffusion sequence in evaluation the effects of low-intensity transcranial ultrasound (LIPUS) on ischemic stroke in rat models at different onset times.MethodsSixty Sprague-Dawley rats (weight, 250 ± 10 g) were divided randomly into six groups (n = 10) to establish a permanent model of distal middle cerebral artery occlusion (dMCAO). The rats were stimulated with ultrasound for 10 min in the LIPUS groups at 0.5 h, 1 h, 3 h, 6 h, and 9 h after dMCAO (denoted as the LIPUS group (0.5 h), LIPUS group (1 h), LIPUS group (3 h), LIPUS group (6 h), LIPUS group (9 h), and Control group without ultrasound stimulation). Magnetic resonance imaging was acquired at 0.5 h and 1 h after dMCAO and then at 1-hour intervals until 12 h. The rADC values were then measured and calculated. The pathological results from the rat brains were obtained after the experiment.ResultsWhen LIPUS was applied 0.5 h after dMCAO, the rate of decline of rADC values in the early stages were significantly reduced and the final rADC values were significantly increased. When LIPUS were applied at 1 h, 3 h, and 6 h, the final rADC values only increased slightly and did not prevent early declines. No significant difference of ultrasound stimulation at 9 h was found.ConclusionThe rADC values demonstrated difference at different time points after dMCAO between with and without LIPUS groups.     

Experimental study on subharmonic and ultraharmonic acoustic waves in water-saturated sandy sediment

Experimental observations of the subharmonic and ultraharmonic acoustic waves in water-saturated sandy sediment are reported in this paper. Acoustic pressures of both nonlinear acoustic waves strongly depend on the driving acoustic pressure at a transducer. The first ultraharmonic wave reaches a saturation value as the driving acoustic pressure increases. The acoustic pressure levels of both nonlinear acoustic waves exhibit some fluctuations in comparison with that of the primary acoustic wave as the receiving distance of hydrophone increases in sediment. The subharmonic and the ultraharmonic phenomena in this study show close resemblance to those produced in bubbly water.     

Acoustic attenuation in the layers of a microwave transducer at the excitation of longitudinal and shear elastic waves by a piezoelectric of the 6<Emphasis Type="Italic">mm</Emphasis> symmetry class

An electroacoustic transducer in the form of a piezoelectric of the 6mm symmetry class with an arbitrary orientation of the sixfold axis and with two finite-thickness metal electrodes is considered taking into account the acoustic attenuation in the transducer layers. A system of equations is obtained to determine the impedance of the transducer, the radiation resistances for shear and longitudinal waves, the power ratio of these waves in the acoustic line, and the transformation factors for transverse and longitudinal waves. The effect of attenuation on the characteristics of a specific transducer operating in the 15-GHz frequency range is numerically analyzed.     

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.     

经颅超声聚焦方法和声场特性研究

基于高分辨的CT数据建立了非均匀颅骨仿真模型,该模型引入了颅骨的声衰减系数,深入研究和分析了声波时间反转法和超声相控阵法在颅脑中的聚焦方法及效果。颅骨具有较强的声波衰减特性,使用时间反转聚焦时需要进行幅度补偿,对于0.7MHz的频率信号,幅度补偿后的时间反转聚焦声场主瓣宽度窄、旁瓣低,焦点处声场比无幅度补偿的时间反转法提高8.86dB,比超声相控阵聚焦法提高7.89dB,具有很好的空间聚焦精度和聚焦效率。研究了颅骨衰减系数、声场焦点位置、声波频率、换能器阵列位置和方位等参数对聚焦声场的影响,结果表明,幅度补偿时间反转法比相控阵法具有更低的旁瓣,且高频时的聚焦效果比相控阵好,相控阵聚焦对换能器阵列的位置和方位比较敏感,而时间反转经颅超声聚焦对声传播路径和入射角具有更高的鲁棒性。      

低强度脉冲超声的骨骼肌修复效应及生物学机制研究*

骨骼肌是保持人体姿态和运动机能的重要组织器官。大量研究采用低强度脉冲超声治疗骨折、骨不连、肌腱和韧带损伤等并取得了较好的效果。然而应用低强度脉冲超声进行肌肉相关疾病治疗的研究相对较少,详细的机制仍不确定。该文综合相关研究报道,介绍了应用低强度脉冲超声进行肌肉修复的生物效应和细胞、肌细胞因子和细胞内信号转导通路的超声作用机制的研究现状。对现有研究存在的问题做了分析,并对肌肉组织损伤的超声治疗和对抗的研究方向进行了展望。     

Flow waves of hierarchical pattern formation induced by chemical waves: The birth, growth and death of hydrodynamic structures

We introduce a short review of chemically driven convection together with a series of our experiments on hydrodynamic instabilities induced by chemical waves excited in the batch reactor of a Belousov-Zhabotinsky reaction. Several unresolved phenomena are picked out and possible mechanisms are discussed extensively. Interesting features of these phenomena can be summarized as being caused by the ‘global and dynamic hydrodynamic pattern induced by chemical waves’. These chemically induced global pattern of hydrodynamic phenomena may not be simply explained by the reaction-diffusion-convection model based on Marangoni instability (surface tension-driven convection), which produces only a localized structure of the convection pattern. Observed flow waves show global and dynamic patterns of convection that generate a functional structure associated with hierarchical patterns appearing in the reaction-diffusion-convection system. In particular, we clarify the existence of a continuous stream of hydrodynamic flow with growing amplitude and its rotating direction. We find that the flow does not stabilize to a motionless state until the system has self-collapsed. This new picture of the flow waves requires a revision of the reaction-diffusion-convection model. The established flow structure can be regarded as a mixing and/or transport process to supply the substrate from the peripheral region to the centre of the chemical waves to sustain the reaction. This characteristic may be a function of the hierarchical structure. A new mechanism for the viscous-elastic feature of the gas-liquid interface is discussed in order to understand these curious phenomena of interest.     

Three-dimensional acoustic Fabry-Perot interferometer of α-quartz as converter of acoustic modes

A cube of α-quartz with polished flat and parallel sides was used as an acoustic three-dimensional Fabry-Perot interferometer. The sides of the cube are perpendicular to the crystallographic axesx, y, z of the quartz. By means of a transducer, transverse elastic waves were injected along thez-axis. Because of anisotropy, this produces a diffuse acoustic field in the cube. From this diffuse field, the interferometer selects pure longitudinal acoustic waves along thex andz axis, which were identified by means of the different velocities of propagation.     

Investigation of mass transfer intensification under power ultrasound irradiation using 3D computational simulation: A comparative analysis

This paper aims at investigating the influence of acoustic streaming induced by low-frequency (24 kHz) ultrasound irradiation on mass transfer in a two-phase system. The main objective is to discuss the possible mass transfer improvements under ultrasound irradiation. Three analyses were conducted: i) experimental analysis of mass transfer under ultrasound irradiation; ii) comparative analysis between the results of the ultrasound assisted mass transfer with that obtained from mechanically stirring; and iii) computational analysis of the systems using 3D CFD simulation. In the experimental part, the interactive effects of liquid rheological properties, ultrasound power and superficial gas velocity on mass transfer were investigated in two different sonicators. The results were then compared with that of mechanical stirring. In the computational part, the results were illustrated as a function of acoustic streaming behaviour, fluid flow pattern, gas/liquid volume fraction and turbulence in the two-phase system and finally the mass transfer coefficient was specified. It was found that additional turbulence created by ultrasound played the most important role on intensifying the mass transfer phenomena compared to that in stirred vessel. Furthermore, long residence time which depends on geometrical parameters is another key for mass transfer. The results obtained in the present study would help researchers understand the role of ultrasound as an energy source and acoustic streaming as one of the most important of ultrasound waves on intensifying gas-liquid mass transfer in a two-phase system and can be a breakthrough in the design procedure as no similar studies were found in the existing literature.     

Lens multielement acoustic microscope in the mode for measuring the parameters of layered objects

An acoustic microscope with a cylindrical lens and ultrasound transducer have been considered, as well as the method based on it for the measuring of longitudinal and transverse wave velocities, the thickness and density of the investigated layer. A theoretical model of the microscope has been constructed, and the relation between the spatiotemporal output signal of the transducer and the angular dependence of the sample reflection coefficient has been found. It has been shown that the velocities of body waves and the thickness can be determined by the delays of ultrasound responses reflected from the layer boundaries measured by the transducer elements, and the density, by the amplitudes of these responses. The method was tested experimentally using a 20-element transducer with a central frequency of 15 MHz and a period of 0.8 mm. The example of a duralumin plate has shown that the error in measuring the thickness and velocity of longitudinal waves error does not exceed 1%; the velocity of transverse waves, 2%; and the density can be estimated with an accuracy of about 5%.     

Energetic study of ultrasonic wettability enhancement

Many industrial and biological interfacial processes, such as welding and breathing depend directly on wettability and surface tension phenomena. The most common methods to control the wettability are based on modifying the properties of the fluid or the substrate. The present work focuses on the use of high-frequency acoustic waves (ultrasound) for the same purpose. It is well known that ultrasound can effectively clean a surface by acoustic cavitation, hence ultrasonic cleaning technology. Besides the cleaning process itself, many authors have observed an important wettability enhancement when liquids are exposed to low and high (ultrasonic) frequency vibration. Ultrasound goes one step further as it can instantly adjust the contact angle by tuning the vibration amplitude, but there is still a lack of comprehension about the physical principles that explain this phenomenon. To shed light on it, a thermodynamic model describing how ultrasound decreases the contact angle in a three-phase wetting system has been developed. Moreover, an analytical and experimental research has been carried out in order to demonstrate that ultrasound is an important competitor to surfactants in terms of energy efficiency and environmental friendliness.     

低强度脉冲超声激发的声微流增强藻酸钙凝胶支架材料的孔隙率和通透性研究

低强度脉冲超声(LIPUS)激发的声微流场所产生的剪切应力可作用于细胞膜表面,从而显著增强细胞膜的通透性。构建了三维藻酸钙凝胶支架培养系统,来模拟有利于细胞生长的营养供给和新陈代谢体内微环境;基于扫描电子显微镜、体内荧光图像和激光共聚焦图像观测技术,对LIPUS增强三维藻酸钙凝胶支架材料的孔隙率和通透性的作用机制和参数相关性进行了系统的研究。结果表明,三维藻酸钙凝胶支架材料的孔隙率和通透性可随着LIPUS的驱动声压的升高而显著增强。此外,通过对三维支架材料内的细胞增殖情况分析,发现在适当的LIPUS驱动声压(如P-=0.055 MPa)下,HeLa细胞在LIPUS作用下的三维藻酸钙凝胶支架材料中可获得更高的增殖率。      

Gigahertz modulators using bulk acousto-optic interactions in thin-film waveguides

Abstract

When bulk acoustic waves are applied to an optical waveguide, several modulation effects are observed, depending on the type of wave (longitudinal or shear). Longitudinal sound waves frequency-shift the guided light, thus providing a means of modulating light in a wide variety of waveguide materials. Using thin-film mosaic acoustic transducer technology, we have demonstrated such modulation at frequencies in the gigahertz region. By segmenting the acoustic transducer electrodes, the same arrangement can be used for deflecting the light since, with this arrangement, the acoustic field sets up a time-varying grating whose spatial frequency is set by the segment spacing. Theoretical frequency limitations on these devices do not appear to be important until approximately 30 GHz is reached. Thus, they are potentially useful for extremely wide-band data links. Experiments at 1.5 GHz show 30% bandwidth of acoustic modulation using optical heterodyne detection.