Low-intensity pulsed ultrasound reduces alveolar bone resorption during orthodontic treatment via Lamin A/C-Yes-associated protein axis in stem cells

BACKGROUND The bone remodeling during orthodontic treatment for malocclusion often requires a long duration of around two to three years,which also may lead to some complications such as alveolar bone resorption or tooth root resorption.Low-intensity pulsed ultrasound(LIPUS),a noninvasive physical therapy,has been shown to promote bone fracture healing.It is also reported that LIPUS could reduce the duration of orthodontic treatment;however,how LIPUS regulates the bone metabolism during the orthodontic treatment process is still unclear.AIM To investigate the effects of LIPUS on bone remodeling in an orthodontic tooth movement(OTM)model and explore the underlying mechanisms.METHODS A rat model of OTM was established,and alveolar bone remodeling and tooth movement rate were evaluated via micro-computed tomography and staining of tissue sections.In vitro,human bone marrow mesenchymal stem cells(hBMSCs)were isolated to detect their osteogenic differentiation potential under compression and LIPUS stimulation by quantitative reverse transcription-polymerase chain reaction,Western blot,alkaline phosphatase(ALP)staining,and Alizarin red staining.The expression of Yes-associated protein(YAP1),the actin cytoskeleton,and the Lamin A/C nucleoskeleton were detected with or without YAP1 small interfering RNA(siRNA)application via immunofluorescence.RESULTS The force treatment inhibited the osteogenic differentiation potential of hBMSCs;moreover,the expression of osteogenesis markers,such as type 1 collagen(COL1),runt-related transcription factor 2,ALP,and osteocalcin(OCN),decreased.LIPUS could rescue the osteogenic differentiation of hBMSCs with increased expression of osteogenic marker inhibited by force.Mechanically,the expression of LaminA/C,F-actin,and YAP1 was downregulated after force treatment,which could be rescued by LIPUS.Moreover,the osteogenic differentiation of hBMSCs increased by LIPUS could be attenuated by YAP siRNA treatment.Consistently,LIPUS increased alveolar bone density and decreased vertical bone absorption in vivo.The decreased expression of COL1,OCN,and YAP1 on the compression side of the alveolar bone was partially rescued by LIPUS.CONCLUSION LIPUS can accelerate tooth movement and reduce alveolar bone resorption by modulating the cytoskeleton-Lamin A/C-YAP axis,which may be a promising strategy to reduce the orthodontic treatment process.

Osteogenic Effect of Low Intensity Pulsed Ultrasound on Rat Adipose-derived Stem Cells in vitro

The osteogenic in vitro effect of low intensity pulsed ultrasound (LIPUS) on SD rat adi-pose-derived stem cells (ADSCs) was investigated.Rat ADSCs underwent LIPUS (intensity=100 mW/cm2) or sham exposure for 8 min per treatment once everyday in vitro,and then the alkaline phos-phatase (ALP) activity and mineralized nodule formation were assessed to evaluate the osteogenic effect of LIPUS on ADSCs.To further explore the underlying mechanism,the osteogenic-related gene mRNA expression was determined by using reverse transcriptase-polymerase chain reaction (RT-PCR) at 1st,3rd,5th,7th day after exposure repectively.Westen blot was used to evaluate the protein expression levels of two osteogenic differentiation associated genes at 7th and 14th day repectively.It was found that ALP activity was increased after LIPUS exposure and LIPUS resulted in mineralized nodule formation of ADSCs in vitro.LIPUS-treated ADSCs displayed higher mRNA expression levels of runt-related transcription factor 2 (Runx2),osteocalcin (OCN),ALP and bone sialoprotein (BSP) genes than con-trols,and the protein levels of Runx2 and BSP were also increased.The results suggested that LIPUS may induce the osteogenic differentiation of ADSCs in vitro.

Effects of low-intensity pulsed ultrasound stimulation on porous hydroxyapatite blocks for posterolateral fusion of lumbar spine in rabbits

he effects of porous hydroxyapatite blocks（PHABs） and an adjunct low-intensity pulsed ultrasound stimulation（LIPUS） on the fusion rate in a rabbit spinal posterolateral fusion（PLF） model were evaluated.Twenty rabbits underwent PLF with autograft and PHABs were randomly assigned to two groups：treated group with 20 min LIPUS daily and untreated control group for 4 weeks until euthanasia.The fused motion segments were subjected to manual palpation,gross observation,and radiographic investigation before histomorphologic and scanning electron microscopic analyses.Statistical differences between the LIPUS group and the control group are found in the fusion rate,bone density gray scale,trabecular bone formation,osteoblast-like cells,chondrocytes and positive expression of BMP-2 and TGF-β1 in the junction zone（significance level p〈0.05）.The results suggest that LIPUS can increase fusion rates and accelerate bone in-growth into PHAB.Hence,PHAB and LIPUS may be used together to increase fusion rates in a rabbit spinal fusion model with a promising extension to human application.

Effect of Low-Intensity Pulsed Ultrasound on Ischaemic Heart Disease Patients (Novel Technique)

Ischaemic Heart Disease (IHD) or Coronary heart disease means that the heart is not getting enough blood and oxygen supply through the coronary arteries. The most common cause of this disease is the process of atherosclerosis in the coronary arteries. Although significant progress has been made in the management of ischaemic heart disease (IHD) The number of severe IHD patients is increasing. The treatment options for IHD have not changed much over the last three decades, which is divided between medications, coronary Angioplasty and Coronary artery bypass surgery. Thus it was crucial to develop new, non-invasive therapeutic strategies in case of Failure of medical or interventional therapy or in case patient is not fit for surgery or angioplasty. In this study, we are pleased to reveal a novel technique that was carried out on a human model. We aimed to develop low-intensity pulsed ultrasound (LIPUS) therapy for the treatment of patients with Ischaemic Heart Disease. We have set up the inclusion and exclusion criteria, the treatment protocol of LIPUS on IHD patients. In this limited group of IHD patients, We found promising clinical results and improvement on myocardial functions.

The Effect of Material Surface Microstructure on the Enhancement of Bone-Bonding Ability of a Hydroxyapatite-Implant by Low-Intensity Pulsed Ultrasound (LIPUS)

Excellent firm bonding between the biomaterials and bone tissue (osseointegration and osteo-conductivity) has been desired for the stability in vivo of dental implants and artificial joints. Much has been learned about this concept, which has led to significant improvements in the design and surface modification of implants in the field of implant dentistry, orthopedic surgery. We have already reported that low-intensity pulsed ultrasound (LIPUS) irradiation can accelerate the bone bonding ability of the bio-conductive materials such as bioactive titanium and hydroxyapatite implant. However, it is still unclear whether the LIPUS could have same effect to different types of the bioactive-materials. Therefore, in this study, the differences of bone-like hydroxyapatite formation on some kind of hydroxyapatite surface in simulated body fluid (SBF) under the LIPUS irradiation were investigated. Two kinds of hydroxyapatite samples immersed in SBF was exposed to ultrasound waves, the bone-like apatite on the surface was analyzed by Scanning electron microscopy and X-ray diffraction. As a result, the enhancement of hydroxyapatite formation on the surface by LIPUS was confirmed, the initial epitaxial nucleation and crystal growth of apatite depended on crystal structure of the surface of matrix materials.

L-Type Calcium Channel Modulates Low-Intensity Pulsed Ultrasound-Induced Excitation in Cultured Hippocampal Neurons

As a noninvasive technique,ultrasound stimulation is known to modulate neuronal activity both in vitro and in vivo.The latest explanation of this phenomenon is that the acoustic wave can activate the ion channels and further impact the electrophysiological properties of targeted neurons.However,the underlying mechanism of low-intensity pulsed ultrasound(LIPUS)-induced neuro-modulation effects is still unclear.Here,we characterize the excitatory effects of LIPUS on spontaneous activity and the intracellular Ca^(2+)homeostasis in cultured hippocampal neurons.By whole-cell patch clamp recording,we found that 15 min of 1-MHz LIPUS boosts the frequency of both spontaneous action potentials and spontaneous excitatory synaptic currents(sEPSCs)and also increases the amplitude of sEPSCs in hippocampal neurons.This phenomenon lasts for>10 min after LIPUS exposure.Together with Ca^(2+)imaging,we clarified that LIPUS increases the[Ca^(2+)]cyto level by facilitating L-type Ca^(2+)channels(LTCCs).In addition,due to the[Ca^(2+)]cyto elevation by LIPUS exposure,the Ca^(2+)-dependent CaMKII-CREB pathway can be activated within 30 min to further regulate the gene transcription and protein expression.Our work suggests that LIPUS regulates neuronal activity in a Ca^(2+)-dependent manner via LTCCs.This may also explain the multi-activation effects of LIPUS beyond neurons.LIPUS stimulation potentiates spontaneous neuronal activity by increasing Ca^(2+)influx.

Extracellular vesicles derived from human dental mesenchymal stem cells stimulated with low-intensity pulsed ultrasound alleviate inflammation-induced bone loss in a mouse model of periodontitis

Extracellular vesicles(EVs)derived from mesenchymal stem cells(MSCs)have emerged as a new mode of intercellular crosstalk and are responsible for many of the thera-peutic effects of MSCs.To promote the application of MSC-EVs,recent studies have focused on the manipulation of MSCs to improve the production of EVs and EV-mediated activities.The current paper details an optimization method using non-invasive low-intensity pulsed ul-trasound(LIPUS)as the stimulation for improving oral MSC-EV production and effectiveness.Stem cells from apical papilla(SCAP),a type of oral mesenchymal stem cell,displayed inten-sity-dependent pro-osteogenic and anti-inflammatory responses to LIPUS without significant cytotoxicity or apoptosis.The stimuli increased the secretion of EVs by promoting the expres-sion of neutral sphingomyelinases in SCAP.In addition,EVs from LIPUS-induced SCAP exhibited stronger efficacy in promoting the osteogenic differentiation and anti-inflammation of peri-odontal ligament cells in vitro and alleviating oral inflammatory bone loss in vivo.In addition,LIPUS stimulation affected the physical characteristics and miRNA cargo of SCAP-EVs.Further investigations indicated that miR-935 is an important mediator of the pro-osteogenic and anti-inflammatory capabilities of LIPUS-induced SCAP-EVs.Taken together,these findings demonstrate that LIPUS is a simple and effective physical method to optimize SCAP-EV produc-tion and efficacy.

Effects of low-intensity pulsed ultrasound in repairing injured articular cartilage

Objective: To investigate the effects of low-intensity pulsed ultrasound in repairing injured articular cartilage. Methods: Ten adult New Zealand rabbits with bilateral full-thickness osteochondral defects on the cartilage surface of intercondylar fossas were used in this study. The wounds in the left knees were treated with low-intensity pulsed ultrasound as the experimental group. The right knees received no treatment as the control group. All the animals were killed at 8 weeks after injury and the tissues in the wounds were collected for gross appearance grading, histological grading and proteoglycan quantity. Results: The scores of the gross appearance grades, histological grades and the optical density of toluidine blue of the tissues in the experimental group were significantly higher than those of the controls at 8 weeks after injury (P<(0.05)). Conclusions: Low-intensity pulsed ultrasound can accelerate the repair of injured articular cartilage.

Low-intensity pulsed ultrasound therapy: a potential strategy to stimulate tendon-bone junction healing

Incorporation of a tendon graft within the bone tunnel represents a challenging clinical problem. Successfulanterior cruciate ligament (ACL) reconstruction requires solid healing of the tendon graft in the bone tunnel. En-hancement of graft healing to bone is important to facilitate early aggressive rehabilitation and a rapid return topre-injury activity levels. No convenient, effective or inexpensive procedures exist to enhance tendon-bone (T-B)healing after surgery. Low-intensity pulsed ultrasound (LIPUS) improves local blood perfusion and angiogenesis,stimulates cartilage maturation, enhances differentiation and proliferation of osteoblasts, and motivates osteogenic differentiation of mesenchymal stem cells (MSCs), and therefore, appears to be a potential non-invasive tool for T-Bhealing in early stage of rehabilitation of ACL reconstruction. It is conceivable that LIPUS could be used to stimulateT-B tunnel healing in the home, with the aim of accelerating rehabilitation and an earlier return to normal activities inthe near future. The purpose of this review is to demonstrate how LIPUS stimulates T-B healing at the cellular andmolecular levels, describe studies in animal models, and provide a future direction for research.

Low-intensity pulsed ultrasound for regenerating peripheral nerves:potential for penile nerve

Peripheral nerve damage,such as that found after surgery or trauma,is a substantial clinical challenge.Much research continues in attempts to improve outcomes after peripheral nerve damage and to promote nerve repair after injury.In recent years,low-intensity pulsed ultrasound(LIPUS)has been studied as a potential method of stimulating peripheral nerve regeneration.In this review,the physiology of peripheral nerve regeneration is reviewed,and the experiments employing LIPUS to improve peripheral nerve regeneration are discussed.Application of LIPUS following nerve surgery may promote nerve regeneration and improve functional outcomes through a variety of proposed mechanisms.These include an increase of neurotrophic factors,Schwann cell(SC)activation,cellular signaling activations,and induction of mitosis.We searched PubMed for articles related to these topics in both in vitro and in vivo animal research models.We found numerous studies,suggesting that LIPUS following nerve surgery promotes nerve regeneration and improves functional outcomes.Based on these findings,LIPUS could be a novel and valuable treatment for nerve injury-induced erectile dysfunction.

Improved activity of MC3T3-E1 cells by the exciting piezoelectric BaTiO_(3)/TC4 using low-intensity pulsed ultrasound

Developing bioactive materials for bone implants to enhance bone healing and bone growth has for years been the focus of clinical research.Barium titanate(BT)is an electroactive material that can generate electrical signals in response to applied mechanical forces.In this study,a BT piezoelectric ceramic coating was synthesized on the surface of a TC4 titanium alloy,forming a BT/TC4 material,and low-intensity pulsed ultrasound(LIPUS)was then applied as a mechanical stimulus.The combined effects on the biological responses of MC3T3-E1 cells were investigated.Results of scanning electron microscopy,energy-dispersive X-ray spectroscopy,and X-ray diffraction showed that an uniform nanospheres-shaped BT coating was formed on TC4 substrate.Piezoelectric behaviors were observed using piezoelectric force microscopy with the piezoelectric coefficient d_(33)of 0.42 pC/N.Electrochemical measures indicated that LIPUS-stimulated BT/TC4 materials could produce a microcurrent of approximately 10μA/cm^(2).In vitro,the greatest osteogenesis(cell adhesion,proliferation,and osteogenic differentiation)was found in MC3T3-E1 cells when BT/TC4 was stimulated using LIPUS.Furthermore,the intracellular calcium ion concentration increased in these cells,possibly because opening of the L-type calcium ion channels was promoted and expression of the Ca_(V)1.2 protein was increased.Therefore,the piezoelectric BT/TC4 material with LIPUS loading synergistically promoted osteogenesis,rending it a potential treatment for early stage formation of reliable bone-implant contact.

Low‐intensity pulsed ultrasound ameliorates angiotensin II-induced cardiac fibrosis by alleviating inflammation via a caveolin-1-dependent pathway

Objective:Cardiac hypertrophy and fibrosis are major pathological manifestations observed in left ventricular remodeling induced by angiotensin II(AngII).Low-intensity pulsed ultrasound(LIPUS)has been reported to ameliorate cardiac dysfunction and myocardial fibrosis in myocardial infarction(MI)through mechano-transduction and its downstream pathways.In this study,we aimed to investigate whether LIPUS could exert a protective effect by ameliorating AngII-induced cardiac hypertrophy and fibrosis and if so,to further elucidate the underlying molecular mechanisms.Methods:We used AngII to mimic animal and cell culture models of cardiac hypertrophy and fibrosis.LIPUS irradiation was applied in vivo for 20 min every 2 d from one week before mini-pump implantation to four weeks after mini-pump implantation,and in vitro for 20 min on each of two occasions 6 h apart.Cardiac hypertrophy and fibrosis levels were then evaluated by echocardiographic,histopathological,and molecular biological methods.Results:Our results showed that LIPUS could ameliorate left ventricular remodeling in vivo and cardiac fibrosis in vitro by reducing AngII-induced release of inflammatory cytokines,but the protective effects on cardiac hypertrophy were limited in vitro.Given that LIPUS increased the expression of caveolin-1 in response to mechanical stimulation,we inhibited caveolin-1 activity with pyrazolopyrimidine 2(pp2)in vivo and in vitro.LIPUS-induced downregulation of inflammation was reversed and the anti-fibrotic effects of LIPUS were absent.Conclusions:These results indicated that LIPUS could ameliorate AngII-induced cardiac fibrosis by alleviating inflammation via a caveolin-1-dependent pathway,providing new insights for the development of novel therapeutic apparatus in clinical practice.

Low-intensity pulsed ultrasound stimulates proliferation of stem/progenitor cells: what we need to know to translate basic science research into clinical applications

Low-intensity pulsed ultrasound (LIPUS) is a promising therapy that has been increasingly explored in basic research and clinical applications. LIPUS is an appealing therapeutic option as it is a noninvasive treatment that has many advantages, including no risk of infection or tissue damage and no known adverse reactions. LIPUS has been shown to have many benefits including promotion of tissue healing, angiogenesis, and tissue regeneration;inhibition of inflammation and pain relief;and stimulation of cell proliferation and differentiation. The biophysical mechanisms of LIPUS remain unclear and the studies are ongoing. In recent years, more and more research has focused on the relationship between LIPUS and stem/progenitor cells. A comprehensive search of the PubMed and Embase databases to July 2020 was performed. LIPUS has many effects on stem cells. Studies show that LIPUS can stimulate stem cells in vitro;promote stem cell proliferation, differentiation, and migration;maintain stem cell activity;alleviate the problems of insufficient seed cell source, differentiation, and maturation;and circumvent the low efficiency of stem cell transplantation. The mechanisms involved in the effects of LIPUS are not fully understood, but the effects demonstrated in studies thus far have been favorable. Much additional research is needed before LIPUS can progress from basic science research to large-scale clinical dissemination and application.

Low-intensity pulsed ultrasound prompts tissue-engineered bone formation after implantation surgery

Background A practical problem impeding clinical translation is the limited bone formation seen in artificial bone grafts.Low-pressure/vacuum seeding and dynamic culturing in bioreactors have led to a greater penetration into the scaffolds,enhanced production of bone marrow cells,and improved tissue-engineered bone formation.The goal of this study was to promote more extensive bone formation in the composites of porous ceramics and bone marrow stromal cells （BMSCs）.Methods BMSCs/β-tricalcium phosphate （β-TCP） composites were subcultured for 2 weeks and then subcutaneously implanted into syngeneic rats that were split into a low-intensity pulsed ultrasound （LIPUS） treatment group and a control group.These implants were harvested at 5,10,25,and 50 days after implantation.The samples were then biomechanically tested and analyzed for alkaline phosphate （ALP） activity and osteocalcin （OCN） content and were also observed by light microscopy.Results The levels of ALP activity and OCN content in the composites were significantly higher in the LIPUS group than in the control group.Histomorphometric analysis revealed a greater degree of soft tissue repair,increased blood flow,better angiogenesis,and more extensive bone formation in the LIPUS groups than in the controls.No significant difference in the compressive strength was found between the two groups.Conclusion LIPUS treatment appears to enhance bone formation and angiogenesis in the BMSCs/β3-TCP composites.

Low-Intensity Pulsed Ultrasound Promote Schwann Cells Pro-Myelination Activities and Neurite Outgrowth of Dorsal Root Ganglion Neurons

In this study,we investigate how Schwann cells and dorsal root ganglion(DRG)neurons response to direct low-intensity pulsed ultrasound(LIPUS)stimuli in vitro.Primary Schwann cells and DRG were isolated from rat sciatic nerve and spine,respectively.LIPUS with varied dose of intensity(low:250 mW/cm2,medium:500 m W/cm2,high:750 m W/cm2)were applied 5 min per time for every other day,and pro-myelination indicators of Schwann cell as well as neurite outgrowth of dorsal root ganglion neurons were analyzed.Our results demonstrated that LIPUS promoted Schwann cells activity and proliferation from day 3 at the highest intensity,and day 5 at all intensities.In addition,LIPUS boosted pro-myelination activities of Schwann cells,as evidenced by increased cell population that positive for immunohistochemical staining against S100,nerve growth factor receptor(NGFR)p75,glial fibrillary acidic protein(GFAP),myelin protein zero(P0),as well as up-regulation of GFAP,Protein 0,nerve growth factor(NGF),and brain derived neurotrophic factor(BDNF)genes.Furthermore,LIPUS significantly enhanced the neurite outgrowth of DRG,with the highest intensity exhibiting longest neurite outgrowth.Taken together,our results strongly improve the understanding of cellular mechanisms of ultrasonic therapies for peripheral nerve repair.

Non-thermal ablation of rabbit liver VX2 tumor by pulsed high intensity focused ultrasound with ultrasound contrast agent:Pathological characteristics

AIM： To investigate the pathological characteristics of non-thermal damage induced by pulsed high intensity focused ultrasound （PHIFU） combined with ultrasound contrast agent （UCA）, SonoVue （Bracco SpA, Milan, Italy） in rabbit liver VX2 tumor. METHODS： Liver VX2 tumor models were established in 20 rabbits, which were divided randomly into PHIFU combined with ultrasound contrast agent group （PHIFU ＋ UCA group） and sham group. In the PHIFU ＋ UCA group, 0.2 mL of SonoVue was injected intravenously into the tumor, followed by ultrasound exposure of Isp 5900 W/cm^2. The rabbits were sacrificed one day after ultrasound exposure. Specimens of the exposed tumor tissues were obtained and observed pathologically under light microscope and transmission electron microscope. The remaining tumor tissues were sent for 2,3,5-Triphenyltetrazolium chloride （TTC） staining. RESULTS： Before Trc staining, tumor tissues in both the sham and the PHIFU ＋ UCA groups resembled gray fish meat, After TIC staining, the tumor tissues were uniformly stained red, with a clear boundary between tumor tissue and normal tissue, Histological examination showed signs of tumor cell injury in PHIFU ＋ UCA group, with cytoplasmic vacuoles of various sizes, chromatin margination and karyopyknosis. Electron microscopic examination revealed tumor cell volume reduction, karyopyknosis, chromatin margination, intercellular space widening, the presence of high electro＇n-density apoptotic bodies and vacuoles in cytoplasm. CONCLUSION： The non-thermal effects of PHIFU combined with UCA can be used to ablate rabbit liver VX2 tumors.

Ultrasound-mediated transdermal drug delivery of fluorescent nanoparticles and hyaluronic acid into porcine skin in vitro

Transdermal drug delivery （TDD） can effectively bypass the first-pass effect. In this paper, ultrasound-facilitated TDD on fresh porcine skin was studied under various acoustic parameters, including frequency, amplitude, and exposure time. The delivery of yellow-green fluorescent nanoparticles and high molecular weight hyaluronic acid （HA） in the skin samples was observed by laser confocal microscopy and ultraviolet spectrometry, respectively. The results showed that, with the application of ultrasound exposures, the permeability of the skin to these markers （e.g., their penetration depth and concentration） could be raised above its passive diffusion permeability. Moreover, ultrasound-facilitated TDD was also tested with/without the presence of ultrasound contrast agents （UCAs）. When the ultrasound was applied without UCAs, low ultrasound frequency will give a better drug delivery effect than high frequency, but the penetration depth was less likely to exceed 200 p.m. However, with the help of the ultrasound-induced microbubble cavitation effect, both the penetration depth and concentration in the skin were significantly enhanced even more. The best ultrasound-facilitated TDD could be achieved with a drug penetration depth of over 600 p.m, and the penetration concentrations of fluorescent nanoparticles and HA increased up to about 4-5 folds. In order to get better understanding of ultrasound-facilitated TDD, scanning electron microscopy was used to examine the surface morphology of skin samples, which showed that the skin structure changed greatly under the treatment of ultrasound and UCA. The present work suggests that, for TDD applications （e.g., nanoparticle drug carriers, transdermal patches and cosmetics）, protocols and methods presented in this paper are potentially useful.

Study of characteristics of ultrasound pulse generated by a laser pulse

The one-dimension thermal conduction equation and Navier-Stokes equa tion are used to get a pulse stress wave in a scmi-infinitc medium when a rectangular pulse laser of duration t_0 is incident on its surface. For both tfansparent and opaque samples the ultrasonic stress waveforms arc discussed when the sample surface is con strained or free. The experimental results in these two different boundary conditions of an opaque sample are presented. It is shown, theoretically and experimentally,that the ultrasound pulse thermoelastically generated by a pulse laser is monopolar when the sample surface is constrained and it is bipolar when its surface is free.