Spatial Average Intensity Research Articles

Low-intensity pulsed ultrasound activated the anti-tumor immunity by irradiating the spleen of mice in 4 T-1 breast cancer

Tumor immunotherapy is booming around the world. However, strategies to activate the immune system and alleviate the immunosuppression still need to be refined. Here, we demonstrate for the first time that low-intensity pulsed ultrasound (LIPUS, spatial average time average intensity (Isata) is 200 mW/cm2, frequency is 0.3 MHz, repetition frequency is 1 kHz, and duty cycle is 20%) triggers the immune system and further reverses the immunosuppressive state in the mouse models of breast cancer by irradiating the spleen of mice. LIPUS inhibited tumor growth and extended survival in mice with 4 T-1 tumors. Further studies had previously shown that LIPUS enhanced the activation of CD4+ and CD8+ T cells in the spleen and led to significant changes in cytokines, as well as induced upregulation of mRNA levels involved in multiple immune regulatory pathways in the spleen. In addition, LIPUS promoted tumor-infiltrating lymphocyte accumulation and CD8+ T cell activation and improved the dynamics of cytokines/chemokines in the tumor microenvironment, resulting in a reversal of the immunosuppressive state of the tumor microenvironment. These results suggest a novel approach to activate the immune response by irradiating the spleen with LIPUS.

Low-Intensity Pulsed Ultrasound Promotes the Repair of Achilles Tendinopathy by Downregulating the JAK/STAT Signaling Pathway in Rabbits.

Tendinopathy is a complex tendon injury or pathology outcome, potentially leading to permanent impairment. Low-intensity pulsed ultrasound (LIPUS) is emerging as a treatment modality for tendon disorders. However, the optimal treatment duration and its effect on tendons remain unclear. This study aims to investigate the efficacy of LIPUS in treating injured tendons, delineate the appropriate treatment duration, and elucidate the underlying treatment mechanisms through animal experiments. Ninety-six three-month-old New Zealand white rabbits were divided into normal control (NC) and model groups. The model group received Prostaglandin E2 (PGE2) injections to induce Achilles tendinopathy. They were then divided into model control (MC) and LIPUS treatment (LT) groups. LT received LIPUS intervention with a 1-MHz frequency, a pulse repetition frequency (PRF) of 1 kHz, and spatial average temporal average sound intensity ( [Formula: see text]) of 100 mW/cm2. MC underwent a sham ultrasound, and NC received no treatment. Assessments on 1, 4, 7, 14, and 28 days after LT included shear wave elastography (SWE), mechanical testing, histologic evaluation, ribonucleic acid sequencing (RNA-seq), polymerase chain reaction (PCR), and western blot (WB) analysis. SWE results showed that the shear modulus in the LT group was significantly higher than that in the MC group after LT for seven days. Histological results demonstrated improved tendon tissue alignment and fibroblast distribution after LT. Molecular analyses suggested that LIPUS may downregulate the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway and regulate inflammatory and matrix-related factors. We concluded that LT enhanced injured tendon elasticity and accelerated Achilles tendon healing. The study highlighted the JAK/STAT signaling pathway as a potential therapeutic target for LT of Achilles tendinopathy, guiding future research.

Low-intensity pulsed ultrasound enhances neurite growth in serum-starved human neuroblastoma cells.

Low-intensity pulsed ultrasound (LIPUS) is a recognized tool for promoting nerve regeneration and repair; however, the intracellular mechanisms of LIPUS stimulation remain underexplored. The present study delves into the effects of varying LIPUS parameters, namely duty cycle, spatial average-temporal average (SATA) intensity, and ultrasound amplitude, on the therapeutic efficacy using SK-N-SH cells cultured in serum-starved conditions. Four distinct LIPUS settings were employed: (A) 50 mW/cm2, 40%, (B) 25 mW/cm2, 10%, (C) 50 mW/cm2, 20%, and (D) 25 mW/cm2, 10%. Immunochemistry analysis exhibited neurite outgrowth promotion in all LIPUS-treated groups except for Group D. Further, LIPUS treatment was found to successfully promote brain-derived neurotrophic factor (BDNF) expression and enhance the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, protein kinase B (Akt), and mammalian target of rapamycin (mTOR) signaling pathways, as evidenced by western blot analysis. The study suggests that the parameter combination of LIPUS determines the therapeutic efficacy of LIPUS. Future investigations should aim to optimize these parameters for different cell types and settings and delve deeper into the cellular response mechanism to LIPUS treatment. Such advancements may aid in tailoring LIPUS treatment strategies to specific therapeutic needs.

Thermal analysis and SNR comparison of CMUT and PZT transducers using coded excitation

Coded excitation (CE) has the ability to enhance image quality and penetration depth by improving the signal-to-noise ratio (SNR). Their usefulness has been extensively proven in the literature, however, there are very few publications that have discussed the practicality of using CEs, as they can increase the operating temperature of a transducer beyond the safety limits. In this paper, the potential for capacitive micromachined ultrasonic transducers (CMUTs) to handle CEs is investigated and compared to a geometrically similar Lead Zirconate Titanate (PZT) probe. It is hypothesized that CMUTs are comparatively advantageous for CE and can generate CE signals more effectively while operating within the safety limits of temperature specified by the International Electrotechnical Commission (IEC). Simple chirp signals were used for excitation and the temperature of the transducer assembly was measured under two different test conditions following the IEC standards. The still air test showed that at the pulse repetition frequency of 4 kHz and a signal duration of 8μs, the PZT probe reached the safe limit of 27 °C within 3 min of acquisition, whereas the CMUT probe showed an increase of only 4–5 °C over 30 min of scan time. The phantom experiments showed that for the maximum temperature rise allowed at the object-transducer interface by the IEC, the gain in SNR possible for the CMUT probe was 13–14 dB and the increase in penetration depth was 30 mm, whereas no gain in SNR and penetration depth was achieved for the PZT probe. The peak mechanical index and the derated spatial peak temporal averaged intensity were found to be less than half the FDA limits for both probes in all cases, proving temperature to be the first limiting factor when using CE. Therefore, the CMUT has shown to be thermally more efficient than the PZT probe and a good candidate for CE imaging.

The Fifth Bioelectronic Medicine Summit Hosted by the Feinstein Institutes for Medical Research (Manhasset, NY) and Columbia Engineering (NY, NY) at The Garden City Hotel, Garden City, NY 11530 on October 11-12th, 2022- Bioelectronic Medicine: Today’s Tools, Tomorrow’s Therapies

The Fifth Bioelectronic Medicine Summit Hosted by the Feinstein Institutes for Medical Research (Manhasset, NY) and Columbia Engineering (NY, NY) at The Garden City Hotel, Garden City, NY 11530 on October 11-12th, 2022- Bioelectronic Medicine: Today’s Tools, Tomorrow’s Therapies

Monitoring of in-vitro ultrasonic stimulation of cells by numerical modeling

Ultrasound stimulation of living tissues is a promising technique that can be safely applied for regenerative treatments. However, the ultrasound-induced mechanotransduction is still not well understood because of the large number of parameters involved at different scales and their difficult experimental accessibility. In this context, in-vitro studies may help to gain insight into the interaction between ultrasound and cells. Nevertheless, to conduct a reliable analysis of ultrasound effects on cell culture, the monitoring of the acoustic intensity delivered to the cells is of prime interest. Thanks to the development of an innovative custom experimental set-up inspired from ultrasound stimulation of bone regeneration conditions, major disturbing phenomena such as multiple reflections and standing wave formation inside the Petri dish are eliminated. Thus, the level of ultrasound stimulation, especially, in terms of spatial average temporal average intensity (ISATA), delivered to the cells can be monitored. Then, to properly estimate the level of ultrasound stimulation, a finite element model representing the experimental in-vitro configuration is developed. The numerical model manages on capturing the characteristics of the experimentally measured acoustic intensity distribution as illustrated by the experimental and numerical ISATA values of 42.3 and 45.8 mW/cm2 respectively, i.e. a relative difference of 8%. The numerical model would therefore allow exploring data inaccessible to experimental measurement and parametric studies to be carried out and facilitates the investigation of different virtual experimental configurations.

A Spatial Multitarget Ultrasound Neuromodulation System Using High-Powered 2-D Array Transducer.

Transcranial focused ultrasound (tFUS) is increasingly used in experimental neuroscience due to its neuromodulatory effectiveness in animal studies. However, achieving multitarget tFUS in small animals is typically limited by transducer size, energy transfer efficiency, and brain volume. The objective of this work was to construct an ultrasound system for multitarget neuromodulation in small animals. First, a miniaturized high-powered 2-D array transducer was developed. The phase delay of each array element was calculated based on the multifocal time-reversal method, generating multiple foci simultaneously in a 3-D field. The effects of the axial focal length, interfocus spacing (lateral distance between the two focal centers), and the number of foci on the focal properties of the pressure field were examined through numerical simulations. In-vitro ultrasonic measurements and transcranial simulations on a rat skull were conducted. The minimum interfocus spacing separating two -6-dB foci and the peak full-width at half-maximum were positively correlated with axial focal length; the relative relationship between the interfocus spacing and pressure field properties was similar for each axial focal length. The maximum acoustic pressure and spatial average intensity at focus in deionized water were 2.21 MPa and 133 W/cm2, respectively. The simulated and experimental results were compared, demonstrating agreement in both peak position and focus shape. The ultrasound system can provide a neuroscientific platform for evaluating the feasibility of multitarget ultrasound stimulation treatment protocols, thus improving the understanding of functional neuroanatomy.

Low-Intensity Pulsed Ultrasound Promotes Osteogenic Differentiation of Reamer-Irrigator-Aspirator Graft-Derived Cells in Vitro.

Recently, reamer-irrigator-aspirator (RIA) systems have been increasingly used to harvest autologous bone grafts. RIA graft materials contain bone marrow, which provides a viable source to derive large numbers of mesenchymal stem cells. Low-intensity pulsed ultrasound (LIPUS) significantly accelerates the differentiation of stem cells derived from bone marrow. This in vitro study investigated the effect of LIPUS on the osteogenic activity and differentiation of RIA graft-derived cells. A small amount of RIA graft was obtained from seven patients. After the cells derived from RIA grafts were cultured, they were divided into two groups: the LIPUS and control groups. LIPUS was applied once daily for 20 min (1.5 MHz, pulse duration: 200 µs, pulse repetition rate: 1 kHz, spatial average-temporal average intensity: 30 mW/cm2). Alkaline phosphatase activity (113.4% and 130.1% on days 7 and 14), expression of osteoblast-related genes (ALP, Runx2) and mineralization (135.2% on day 21) of the RIA graft-derived cells were significantly higher in the LIPUS group than in the control group. However, LIPUS did not affect the cell proliferation of RIA graft-derived cells. This study indicates that LIPUS may enhance the healing of non-union and critical bone defects treated by autologous bone grafting using the RIA system.

Polarization-based intensity correlation of a depolarized speckle pattern

A different kind of intensity correlation, denoted as polarization-based intensity correlation (PBIC), is proposed and demonstrated to investigate the correlation between different polarizations of a depolarized speckle pattern (DSP), which has non-uniform spatial polarization distribution. It is shown both theoretically and experimentally that the range of the PBIC for any polarization of the DSP depends on the spatial average intensity of the speckles corresponding to that particular polarization. The experimentally determined nature of the change of the range of the PBIC for different polarization components, due to the variation in the average intensity, is found to be matching well with the theoretical prediction. The existence of non-zero correlation between two orthogonally polarized speckle patterns, filtered from a partially DSP, is also observed. This study may be useful in exploiting the PBIC for different applications such as speckle cryptography.

Effects of sub-atmospheric pressure and dissolved oxygen concentration on lesions generated in ex vivo tissues by high intensity focused ultrasound

BackgroundAcoustic cavitation plays an important role in the medical treatment using high-intensity focused ultrasound (HIFU), but unnecessarily strong cavitation also could deform the morphology and enlarge the size of lesions. It is known that the increase of ambient hydrostatic pressure (Pstat) can control the acoustic cavitation. But the question of how the decrease of Pstat and dissolved oxygen concentration (DOC) influence the strength of cavitation has not been thoroughly answered. In this study, we aimed to investigate the relationship among the Pstat, DOC and the strength of cavitation.MethodsEx vivo bovine liver tissues were immersed in degassed water with different DOC of 1.0 mg/L, 1.5 mg/L and 2.0 mg/L. Ultrasound (US) of 1 MHz and the spatial and temporal average intensity (Isata) of 6500 W/cm2 was used to expose two groups of in vitro bovine livers for 2 s; one group was under atmospheric pressure (Pstat = 1 bar) and the other was under sub-atmospheric pressure (Pstat = 0.1 bar). Acoustic cavitation was detected by a passive cavitation detector (PCD) during the exposure process. Echo signals at the focal zone of HIFU were monitored by B-mode ultrasound imaging before and after exposure. The difference between two pressure groups was tested using paired sample t-test. The difference among different DOC groups was evaluated by one-way analysis of variance (ANOVA).ResultsThe results demonstrated a significant difference of broadband acoustic emissions from the cavitation bubbles, echo signals on B-mode image, morphology of lesions under various conditions of ambient pressure and DOC. The lesion volume in tissue was increased with the increase of ambient pressure and DOC.ConclusionCavitation could be suppressed through sub-atmospheric pressure and low DOC level in liver tissue, which could provide a method of controlling cavitation in HIFU treatment to avoid unpredictable lesions.

Ultrasound combined with microbubbles enhances the renoprotective effects of methylprednisolone in rats with adriamycin-induced nephropathy

The purpose of this study was to investigate the effect of ultrasound combined with microbbules (SonoVueTM) on the potency of methylprednisolone in attenuating the renal injury induced by adriamycin in rats. Animal model was established by two intravenous injections of 4 mg/kg adriamycin with a 2-week interval in rats. One week later, the adriamycin injected rats were randomly divided into 7 groups, receiving various treatments daily for 2 weeks. Two doses of methylprednisolone (20 or 40 mg/kg) were administrated alone or 20 mg/kg methylprednisolone and 100 µL SonoVueTM microbbules (1–5 × 108 bubbles/mL; mean diameter of bubbles: 2.5 µm) was co-administrated by intravenous injections from the tail vein. The ultrasound was applied at a frequency of 0.8 MHz and a spatial average temporal average intensity of 2.79 W/cm2 for 5 min at a 50% duty cycle (1 s on 1 s off) on the back skin of the anatomic position of the kidney in rats of two groups combined with ultrasound. Renal injury were analyzed using immunohistochemical staining, real-time PCR, light and transmission electron microcopies. The kidney function related biochemical indexes were measured by automatic biochemistry analyzer. The results showed that adriamycin induced a typical renal injury and 40 mg/kg methylprednisolone injection significantly ameliorated the abnormality of key parameters such as proteinuria, renal mRNA and protein expression levels of nephrin, collagens III and IV as well as podocyte impairment, glomerulosclerosis and tubulointerstitial injury indexes. However, a sub-dose of methylprednisolone at 20 mg/kg was ineffective when administered intravenously, but its potency at this dosage was enhanced by co-administration with 100 µL SonoVueTM microbubbles plus ultrasound irradiation. In conclusion, ultrasound combined with microbubbles can significantly increase local renal drug delivery leading to enhanced therapeutic effect of low dose methylprednisolone in ameliorating adriamycin-induced nephropathy in rats.

Shortening of Overall Orthodontic Treatment Duration with Low-Intensity Pulsed Ultrasound (LIPUS).

The aim of this retrospective clinical study was to determine if there is a reduction in the overall treatment duration in orthodontic patients using low-intensity pulsed ultrasound (LIPUS) and Invisalign SmartTrack® clear aligners. Data were collected from the first thirty-four patients (9 males, 25 females; average age 41.37 ± 15.02) who finished their orthodontic treatment using an intraoral LIPUS device and Invisalign clear aligners in a private clinic. The LIPUS parameters used by patients at home for 20 min/day were: ultrasonic frequency 1.5 MHz, pulse duration 200µs, pulse repetition rate 1 kHz, and spatial average-temporal average intensity 30mW/cm2. A control group (11 males, 23 females; average age 31.36 ± 14.41) matching for the same malocclusions was randomly selected from finished treatment cases of the same clinician. The date of first Invisalign attachment placement and first use of LIPUS application was recorded as T0, and the date of retainer delivery was recorded as T1. The treatment duration (T1–T0) and treatment reduction percentage with LIPUS device were collected and analyzed using two-sample t-test in Microsoft Excel. Treatment duration was significantly reduced in the LIPUS group (541.44 ± 192.23 days) compared to control group (1061.05 ± 455.64 days) (p < 0.05). The LIPUS group showed on average 49% reduction in the overall treatment time as compared to the control group. The average compliance of the patients using LIPUS was 66.02%. Patients who used LIPUS showed a clinically significant reduction in the overall orthodontic treatment duration compared to the control group who used Invisalign clear aligners only.

Effect of Pulsed Focused Ultrasound on the Native Pancreas

Pulsed focused ultrasound (pFUS) utilizes short cycles of sound waves to mechanically shake cells within tissues which, in turn, causes transient local increases in cytokines, growth factors and cell adhesion molecules. Although the effect of pFUS has been investigated in several different organs including the kidney, muscle and heart, its effect on the pancreas has not been investigated. In the present work, we applied pFUS to the rodent pancreas with the following parameters: 1.1-MHz frequency, 5-Hz pulse repetition frequency, 5% duty cycle, 10-ms pulse length, 160-s duration. Low-intensity pFUS had a spatial average temporal average intensity of 11.5 W/cm2 and a negative peak pressure of 3 MPa; high-intensity pFUS had a spatial average temporal average intensity of 18.5 W/cm2 and negative peak pressure of 4 MPa. Here we found that pFUS changed the expression of several cytokines while having no effect on the underlying tissue histology or health of pancreatic cells (as reflected by no significant change in plasma levels of amylase and lipase). Furthermore, we found that this effect on cytokine expression in the pancreas was acoustic intensity dependent; while pFUS at low intensities turned off the expression of several cytokines, at high intensities it had the opposite effect and turned on the expression of these cytokines. The ability to non-invasively manipulate the microenvironment of the pancreas using sound waves could have profound implications for priming and modulating this organ for the application of cellular therapies in the context of both regenerative medicine (i.e., diabetes and pancreatitis) and oncology (i.e., pancreatic cancer).

Sonoporation as an Approach for siRNA delivery into T cells

Delivery of small interfering RNAs (siRNAs) into primary T cells is quite challenging because they are non-proliferating cells and are difficult to transfect with non-viral approaches. Because sonoporation is independent of the proliferation status of cells and siRNA acts in the cell cytoplasm, we investigated whether sonoporation could be used to deliver siRNA into mouse and human T cells. Cells mixed with Definity microbubbles and siRNA were sonicated with a non-focused transducer of center frequency 2.20 MHz producing ultrasound at a 10% duty cycle, pulse repetition frequency of 2.20 kHz and spatial average temporal average ultrasound intensity of 1.29 W/cm2 for 5 s and then examined for siRNA fluorescence by flow cytometry analysis. These sonoporation conditions resulted in high-efficiency transfection of siRNA in mouse and human T cells. Further, the efficacy of siRNA delivery by sonoporation was illustrated by the successful visualization of decreased methylation-controlled J protein expression in mouse and human CD8 T cells via Western blot analysis. The results provide the first evidence that sonoporation is a novel approach to delivery of siRNA into fresh isolated mouse and human T cells in vitro, and might be used for in vivo studies in the future.

Acoustic Streaming in a Soft Tissue Microenvironment

We demonstrated that sound can push fluid through a tissue-mimicking material. Although acoustic streaming in tissue has been proposed as a mechanism for biomedical ultrasound applications, such as neuromodulation and enhanced drug penetration, streaming in tissue or acoustic phantoms has not been directly observed. We developed a material that mimics the porous structure of tissue and used a dye and a video camera to track fluid movement. When applied above an acoustic intensity threshold, a continuous focused ultrasound beam (spatial peak time average intensity: 238 W/cm2, centre frequency: 5 MHz) was found to push the dye axially, that is, in the direction of wave propagation and in the radial direction. Dye clearance increased with ultrasound intensity and was modelled using an adapted version of Eckart's acoustic streaming velocity equation. No microstructural changes were observed in the sonicated region when assessed using scanning electron microscopy. Our study indicates that acoustic streaming can occur in soft porous materials and provides a mechanistic basis for future use of streaming for therapeutic or diagnostic purposes.

The optimal conditions of therapeutic ultrasound on muscle stiffness of medial gastrocnemius muscle in human: A shear-wave elastography study

Therapeutic ultrasound (TU) is most widely used as biophysical agents to treat contracture in orthopedics rehabilitation. Its effect is to increase range of motion by increasing tissue extensibility. However, no studies have reported the effect of TU on human muscle stiffness (MS). Also, the optimal conditions including frequency, intensity, and duty cycle of TU remain unclear. This study aimed to clarify the optimal TU conditions on MS using ultrasonic shear-wave elastography (SWE). This study was a randomized cross-over trial. All of the following TU conditions were applied on ten healthy males (mean ± SD, age 25 ± 2.9 y.o., height 171.2 ± 4.7 cm, weight 63.1 ± 5.1 kg); frequency (1 and 3 MHz), intensity (continued mode: spatial average-temporal average (SATA) intensity of 0.5, 1.0, 1.5, and 2.0 W/cm 2 ; pulsed mode: 1.0, 2.0, and 3.0 W/cm 2 with 50% duty cycle (i.e., 0.5, 1.0, and 1.5 W/cm 2 SATA )) for 10 minutes. Outcome was the change rate of MS in medial gastrocnemius (MG) muscle by using SWE. Statistical analysis was performed using one-way analysis of variance followed by Bonferroni post-hoc test. MS showed significant differences between the different frequency, intensity, and duty cycle. MS significantly decreased with 1.0, 1.5, and 2.0 W/cm 2 SATA than 0.5 W/cm 2 SATA at continued mode, and with 1.0, and 1.5 W/cm 2 SATA than 0.5 W/cm 2 SATA at pulsed mode at both 1 MHz and 3 MHz. There was no significant difference in MS between the above five SATA intensities, i.e., 1.0, 1.5, 2.0 W/cm 2 SATA at continued mode and 1.0, 1.5 W/cm 2 SATA at pulsed mode. The optimal conditions of TU to decrease MS in MG were over 1.0 W/cm 2 SATA of intensity for both continued mode and pulsed mode at both 1 MHz and 3 MHz of frequency.

Effect of rainfall spatial distribution and duration on minimum spatial resolution of rainfall data for accurate surface runoff prediction

The effect of rainfall spatial distribution and duration on determining rainfall spatial resolution for runoff discharge prediction and equilibrium time estimation was studied. We simulated hydrographs and equilibrium times of storms which have the same spatial averaged rainfall intensity with wide range of spatial distributions and durations under stationary storm conditions on an idealized plane. The equilibrium times under various rainfall conditions were estimated with an approximate solution based on the kinematic wave assumption. The runoff hydrographs were simulated using a kinematic wave runoff model. The approximate and computed results were used to investigate the relationship between the spatial–temporal rainfall characteristics and rainfall spatial resolution. From the simulated results, it was found that (i) A required rainfall spatial resolution to achieve threshold accuracy depended on rainfall spatial distribution. For the simulated cases, the critical rainfall spatial resolution was higher as the slope of rainfall spatial distribution was steeper. (ii) The rainfall duration, as well as rainfall spatial distribution, was an important factor for determining a required rainfall spatial resolution for accurate flood prediction. The required rainfall spatial resolution to achieve threshold prediction accuracy was higher as the rainfall duration was shorter. (iii) The rainfall spatial distribution and duration which were traditionally neglected need to be considered in lumped runoff modelling or measurement as an important factor. Although limited to the ideal test cases, the maximum errors due to the low rainfall spatial resolution for the equilibrium time and peak discharge were about 25% and 35%, respectively.

Cell damage of hepatoma-22 cells exposed to continuous wave ultrasound.

The cellular response of hepatoma-22 cells to ultrasonic irradiation and the potential cause for the action were evaluated. Hepatoma-22 cells were subjected to ultrasound irradiation at a frequency of 2.17 MHz and a spatial average intensity of 1.6 W/cm2 for variable periods of time, and several biological parameters were analyzed. The terephthalic acid (TA) dosimetry method was used to evaluate the efficacies of irradiation parameters on the acoustic cavitation activity by monitoring hydroxyl radical (OH) production. Lactate dehydrogenase (LDH) leakage was assayed to investigate cell membrane integrity. The polarization value of fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was measured to monitor plasma membrane fluidity. The malonaldehyde content in cells was determined to reflect lipid peroxidation. Trypan blue exclusion was used to detect cell viability. Additionally, electron microscopy was used to observe morphological changes. The generation of intracellular reactive oxygen species, mitochondria swelling and the loss of mitochondria membrane potential were also investigated. The results showed that 1) the concentration of ·OH production by ultrasonic irradiation in air-saturated cell suspensions increased as ultrasound exposure time increased; 2) compared with control, lactate dehydrogenase leakage, the polarization value of 1,6-diphenyl-1,3,5-hexatriene, malonaldehyde content and cell lysis were significantly elevated when cells were treated by ultrasound for 60 s; 3) cytotoxicity by ultrasound irradiation was also accompanied by an increase in production of intracellular reactive oxygen species and dissipation of mitochondria membrane potential as well as by mitochondria swelling. Presently available information indicates that the plasma membrane and mitochondria are the main targets for ultrasound treatment, and free radicals formation such as ·OH due to ultrasound cavitation may play an important role in mediating these cellular response processes. Moreover the mechanical effect might also be involved in inducing cell damage because there was significant mitochondria membrane potential loss and no visible ROS detection when cells were exposed to ultrasound for 30 s.

Effect of Low-Intensity Pulsed Ultrasound on the Expression of Calcium Ion Transport-Related Proteins during Tertiary Dentin Formation

Low-intensity pulsed ultrasound (LIPUS) is known for its positive effect on bone healing and reparative regeneration. This study investigated whether LIPUS affects reparative progression of the tooth and the expression of calcium ion transport-related proteins in odontoblasts and dental pulp cells using a rat dentin–pulp complex injury model. Forty male adult Sprague-Dawley rats underwent cavity preparation in the right maxillary first molar: 20 received LIPUS irradiation on the cavity-prepared tooth; 20 received LIPUS irradiation on the left maxillary first molar. Rats were randomly allocated into four groups: blank control group, LIPUS group, cavity-prepared group, cavity-prepared + LIPUS group. LIPUS irradiation (frequency: 1.5 MHz, 200-µs pulse width, 1-kHz pulse repetition frequency, 30 mW/cm2 spatial averaged temporal averaged intensity) was administered individually for 20 min daily. Rats were sacrificed 1, 3, 7 and 14 d post-operation. The histopathological and cellular morphologic changes in the dentin–pulp complex were detected with hematoxylin and eosin staining. Expression of calcium ion transport-related proteins (Cav1.2, NCX1 and TRPV1) was determined with immunohistochemical staining and imaging analysis. Histopathological analysis revealed obvious reparative dentin formation at day 14 in the cavity-prepared + LIPUS group compared with the other groups. Expression levels of Cav1.2, NCX1 and TRPV1 increased significantly by 22%, 53% and 23%, respectively, at day 1 and increased significantly by 23%, 27% and 22%, respectively, at day 3 in the cavity-prepared + LIPUS group (p < 0.05) compared with the cavity-prepared group. LIPUS has a positive effect on the expression of calcium transport-related proteins during early-stage dentin injury and facilitates tertiary dentin formation; the mechanism for this likely relates to the inflammatory reaction and a mechanical effect.

Effect of low intensity pulsed ultrasound on expression of TGF-β1 and Smads during dentin injury and repair

To explore the effect of low intensity pulsed ultrasound (LIPUS) on TGF-β1/Smad 2, 3 signal pathway during the dentin injury and repair. Methods: Among 25 Sprague-Dawley rats, 5 rats served as a blank control group without treatment. The remaining 20 rats received modified caries preparation inbilateral maxillary first molars to establish a model of dentin-pulp injury and repair. The right maxillary first molars served as a LIPUS group, which received LIPUS irradiation (frequency: 1.5 MHz, pulse width: 200 μs, pulse repetition frequency: 1 kHz, spatial averaged temporal averaged intensity: 30 mW/cm2, 20 min/d), and the left maxillary first molars served as a cavity-prepared group, which received fake LIPUS irradiation. The rats were sacrificed at 1, 3, 5, 7 and 14 days after LIPUS irradiation. Immunohistochemical staining and Image-pro plus 6.0 were applied to detect the expression and distribution of transforming growth factor-beta1 (TGF-β1) and small mothers against decapentaplegic 2/3(Smad 2 and Smad 3). Results: Immunohistochemical staining showed that the expression of TGF-β1 and Smad 2, 3 were low innormal pulp, but they were increased in different degree after dentin injury. The result of image analysis showed that the expression of TGF-β1 in the cavity-prepared group gradually increased at the first day and peaked at day 5, and then it returned to normal level at day 14. However, the expression of TGF-β1 in the LIPUS group were significantly higher than that in the cavity-prepared group at day 3 and 5 (both P<0.05). The expressions of Smad 2,3 in both the LIPUS group and the cavity-prepared group were consistently increased all the way, but the expressions in the LIPUS group were higher compared with that in the cavity-prepared group (P<0.05). Conclusion: The TGF-β1/Smad 2, 3 signal pathway can be activated during the dentin injury and repair. LIPUS can up-regulate the expression of TGF-β1 and Smad 2, 3 in the early period, which may take part in the dentin-pulp complex injury and repair process.