Low-intensity Pulsed Ultrasound Group Research Articles

Effects of Low-Intensity Pulsed Ultrasound on Myelosuppression of Rats Induced by Chemotherapy Drugs With Cell Cycle Specificity.

To explore the ameliorating effects of low-intensity pulsed ultrasound (LIPUS) on Sprague Dawley rat myelosuppression induced by cell cycle specificity drugs (docetaxel, mitotic phase sensitive; and etoposide, gap 2 phase sensitive). Rats were respectively administered docetaxel (100 mg/kg) or etoposide (110 mg/kg) by intraperitoneal injection for 4 consecutive days. Then the rats were divided randomly into a LIPUS group and a non-LIPUS group. In the LIPUS group, the right femoral metaphysis of rats was treated by LIPUS (acoustic intensity, 200 mW/cm2 ; frequency, 0.3 MHz; and duty cycle, 20%) for 20 minutes on 7 consecutive days from day 5. The rats of the non-LIPUS group were treated without ultrasound output. A blood cell count, an enzyme-linked immunosorbent assay, a real-time quantitative polymerase chain reaction, and hematoxylin-eosin staining were applied to detect the results. Low-intensity pulsed ultrasound significantly promoted the counts of bone marrow nucleated cells, white blood cells, immunoglobulin A (IgA), IgG, granulocyte colony-stimulating factor, stem cell factor, and intercellular cell adhesion molecule 1 and reduced the counts of vascular cell adhesion molecule 1 whether in the docetaxel or etoposide group (P < .05). Low-intensity pulsed ultrasound only increased the expression level of IgM in the docetaxel group but decreased the level of interleukin 6 in the etoposide group (P < .05). Low-intensity pulsed ultrasound has potential to be a noninvasive treatment for myelosuppression caused by different cell cycle-sensitive chemotherapy drugs.

A novel therapeutic approach for coronary inflammation and lymphatic vessels using non-invasive low-intensity pulsed ultrasound in a porcine model with DES-induced coronary hyperconstricting responses

Abstract Background The coronary adventitia harbors lymphatic vessels (LVs). We previously demonstrated that coronary adventitial inflammation and LV dysfunction play important roles in the pathogenesis of coronary artery spasm, including drug-eluting stent (DES)-induced coronary hyperconstricting responses, in pigs and humans. However, a direct therapeutic approach to the coronary adventitia remains to be developed. Purpose In this study, we aimed to examine whether our novel and non-invasive therapy with low-intensity pulsed ultrasound (LIPUS) ameliorates DES-induced coronary hyperconstricting responses, and if so, what mechanisms are involved. Methods An everolimus-eluting stent (EES) was implanted into the left anterior descending (LAD) coronary artery in normal male pigs. They were randomly assigned to the LIPUS or the sham therapy groups. After EES implantation, in the LIPUS group, LIPUS (32 cycles, 193 mW/cm2) was applied to the heart at 3 different levels (proximal and distal stent edges and middle portion of the stent) through X-ray fluoroscopy for 20 min at each level for every other day for 2 weeks (6 days in total) (Fig. 1A, B). The sham therapy group was treated in the same manner but without LIPUS. At 4 weeks after the procedure, we performed coronary angiography to examine coronary vasoconstricting responses to intracoronary serotonin in vivo. Finally, stented coronary vessels were harvested for immunohistochemistry of vasa vasorum (vWF), LVs (LYVE-1), vascular inflammation (CD68-positive macrophages and IL-1β expression), vascular endothelial growth factor A (VEGF-A, angiogenesis marker), VEGF-C and VEGF receptor 3 (VEGFR3, lymphangiogenesis markers). Results Coronary vasoconstricting responses to intracoronary serotonin at the DES edges in the LAD were significantly enhanced in the sham group but were significantly suppressed in the LIPUS group, while those responses were comparable at the non-DES implanted left circumflex (LCx) coronary artery between the 2 groups (Fig. 1C, D). In addition, in vivo lymph transport speed was significantly faster in the LIPUS group than in the sham group (Fig. 1E–G). In histological analysis, the number of LVs was significantly increased in the LIPUS group compared with the sham group, whereas those of CD68 and IL-1β expressions were significantly reduced in the LIPUS group compared with the sham group. In contrast, the density of vasa vasorum was comparable between the 2 groups. Mechanistically, the extents of VEGF-C and VEGFR3 expressions were increased in the LIPUS group, whereas that of VEGF-A was comparable between the 2 groups (Fig. 1G–K). Importantly, there were significant correlations among the LV-related changes and enhanced coronary vasoconstricting responses. Conclusion These results provide the first evidence that the LIPUS therapy ameliorates DES-induced coronary hyperconstricting responses in pigs in vivo through structural and functional alterations of LVs (Fig. 1L). Figure 1 Funding Acknowledgement Type of funding source: None

Amelioration of cyclophosphamide-induced myelosuppression during treatment to rats with breast cancer through low-intensity pulsed ultrasound.

To investigate the alleviating effects of low-intensity pulsed ultrasound (LIPUS) on myelosuppression of Sprague–Dawley rats with breast cancer induced by cyclophosphamide (CTX). Breast cancer in rats was triggered by intragastric gavage with 7,12-dimethylbenz[a]anthracene (150 mg/kg). Then, the rats with breast cancer were randomly allocated to the LIPUS group (n=50) and the control group (n=50). The LIPUS group was injected intraperitoneally with CTX (50 mg/kg) for 4 consecutive days and underwent LIPUS treatment at femoral metaphysis 20 min per day from the first day of injection for 7 consecutive days. The control group was injected with CTX (50 mg/kg) and treated with LIPUS without energy output. Blood, enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction, Hematoxylin and Eosin (H&E) staining, and scanning electron microscopy were applied to detect the changes. The results indicated that LIPUS significantly promoted the proliferation of bone marrow nucleated cells, white blood cells (WBCs), IgA, IgG, and IgM in the peripheral blood (P<0.05) without the damage to liver and kidney function simultaneously. The mechanisms may result from the LIPUS alleviation effect on bone marrow hematopoietic function through regulating cytokines such as LIPUS can increase the expression of granulocyte colony-stimulating factor (G-CSF), stem cell factor, transforming growth factor-β, and intercellular cell adhesion molecule-1, meanwhile LIPUS will decrease the expression of interleukin-6, tumor necrosis factor-α, and vascular cell adhesion molecule-1. LIPUS has potential to be a new adjuvant therapy method in clinic for ameliorating chemotherapy-induced myelosuppression.

Electroactive barium titanate coated titanium scaffold improves osteogenesis and osseointegration with low-intensity pulsed ultrasound for large segmental bone defects

For large segmental bone defects, porous titanium scaffolds have some advantages, however, they lack electrical activity which hinders their further use. In this study, a barium titanate (BaTiO3) piezoelectric ceramic was used to modify the surface of a porous Ti6Al4V scaffold (pTi), which was characterized by scanning electron microscopy, energy dispersive spectroscopy, X-ray photoelectron spectroscopy, and roughness and water contact angle analyses. Low intensity pulsed ultrasound (LIPUS) was applied in vitro and in vivo study. The activity of bone marrow mesenchymal stem cells, including adhesion, proliferation, and gene expression, was significantly superior in the BaTiO3/pTi, pTi + LIPUS, and BaTiO3/pTi + LIPUS groups than in the pTi group. The activity was also higher in the BaTiO3/pTi + LIPUS group than in the BaTiO3/pTi and pTi + LIPUS groups. Additionally, micro-computed tomography, the mineral apposition rate, histomorphology, and the peak pull-out load showed that these scaffold conditions significantly enhanced osteogenesis and osseointegration 6 and 12 weeks after implantation in large segmental bone defects in the radius of rabbits compared with those resulting from the pTi condition. Consequently, the improved osteogenesis and osseointegration make the BaTiO3/pTi + LIPUS a promising method to promote bone regeneration in large segmental bone defects for clinical application.

Low-intensity pulsed ultrasound activates autophagy in periodontal ligament cells in the presence or absence of lipopolysaccharide

ObjectiveThis study aims to determine if low-intensity pulsed ultrasound (LIPUS) activates autophagy in human periodontal ligament cells (PDLCs) irrespective of lipopolysaccharide. DesignSix groups were designed: control, LIPUS, lipopolysaccharide, LIPUS + lipopolysaccharide, LIPUS+3-Methyladenine, LIPUS + lipopolysaccharide+3-Methyl- adenine. LIPUS pretreated PDLCs for 2 h and lipopolysaccharide treated for different times. Real-time PCR and Western-blot were performed to evaluate mRNA and protein expression levels of autophagic genes Beclin-1 and LC3 respectively. A transmission electronic microscope was used to observe the autophagosome. ELISA was used to test interleukin-6 expression. ResultsCompared with the non-treatment, LIPUS pretreatment increased mRNA expression levels of LC3 (P < 0.05) and Beclin-1 (P < 0.05) at 4 h and 8 h, and enhanced the protein expression levels of LC3-Ⅱ at 8 h (P<0.05) and Beclin-1 at 4 h, 8 h and 16 h(P<0.05). After LIPUS pretreatment and lipopolysaccharide treatment for 8 h, LC3-Ⅱ and Beclin-1 protein expression levels were elevated (P < 0.05) compared with the control. Following further treatment by 3-Methyladenine, Beclin-1 protein expression was decreased (P < 0.05) compared with the LIPUS plus lipopolysaccharide group, but LC3-Ⅱ protein expression was not. Autophagosomes were not found in the LIPUS+3-Methyladenine and LIPUS+lipopolysaccharide+3-Methyladenine groups. After LIPUS pretreatment and lipopolysaccharide treatment for 36 h, intreleukin-6 expression was decreased (P<0.05) compared with the lipopolysaccharide group. However, after addition of 3-Methyladenine, intreleukin-6 expression was elevated (P < 0.05) compared with the LIPUS +lipopolysaccharide group. ConclusionsLIPUS can promote autophagy in PDLCs irrespective of lipopolysaccharide. Autophagy might be involved in LIPUS anti-inflammatory mechanism in PDLCS.

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.

Oddziaływania ultradźwięków o niskiej intensywności stosowanych na okolicę stawu skroniowo-żuchwowego w leczeniu czynnościowym wad zgryzu klasy II – randomizowane badanie kontrolowane

Low-intensity pulsed ultrasound (LIPUS) is considered one of the techniques used to improve the mandibular growth. Many animal studies have reported that significant results can be obtained using LIPUS therapy with functional appliances. This research aimed to evaluate the dentoskeletal changes produced by the combination of LIPUS therapy and functional treatment during the correction of skeletal class II malocclusion. Forty-five patients aged 10.5-14 years with skeletal class II division 1 malocclusion were randomly divided into 3 equal groups: the LIPUS group, treated with a Twin-Block appliance in combination with LIPUS therapy; the TB group, treated with a Twin-Block appliance only; and the control group, which was observational and received no treatment. Cephalometric changes were compared between the 3 groups using the analysis of variance (ANOVA) and Tukey's post hoc tests at p < 0.05. A greater significant decrease in the ANB (A point, nasion, B point) angle was observed in the treated groups (-2.67° for the LIPUS group and -2.11° for the TB group) as compared to the control group (p < 0.001). A greater improvement in the mandibular length and position was observed in the LIPUS group than in the TB group (p < 0.001). The changes in the control group as a result of continuing growth were minimal and clinically non-significant. The application of LIPUS therapy in combination with functional treatment can have a great effect on growth stimulation during the correction of class II malocclusion. In addition, LIPUS was effective in reducing the duration of functional treatment.

Low-intensity pulsed ultrasound enhances the rate of lateral tooth movement and compensatory bone formation in rats

Because mechanical stimulation of the periodontal ligament by low-intensity pulsed ultrasound (LIPUS) has been shown to increase the speed of bone remodeling, this study aimed to examine the effects of LIPUS stimulation on the rate of tooth movement and bone remodeling during lateral tooth movement. Twelve-week-old Wistar rats were divided into 2 groups. The LIPUS group received experimental tooth movement with LIPUS stimulation, and the tooth movement (TM) group were provided experimental tooth movement without LIPUS. For each group, the upper right first molars were moved buccally with fixed appliances. LIPUS exposure was placed in the region corresponding to the right maxillary first molar. Three days after tooth movement, tartrate-resistant acid phosphatase was examined. Fourteen days after tooth movement, the intermolar width, bone mineral content, and bone volume fraction were analyzed by micro-computed tomography, and newly formed bone was measured histomorphometrically. The number of TRAP-positive cells in the compressed region was higher in the LIPUS group. The intermolar width was significantly higher in the LIPUS group than in the TM group. The alveolar bone around the maxillary first molar showed no differences in bone mineral content and bone volume fraction between the LIPUS and TM groups. The LIPUS group exhibited a more significant amount of newly formed alveolar bone than the TM group. The present study provides evidence of the beneficial effects of LIPUS on the lateral tooth movement.

Low-intensity pulsed ultrasound promotes spinal fusion by regulating macrophage polarization

BackgroundSpinal fusion is considered the gold standard procedure for treating spinal degeneration, tumors, and trauma. An inflammatory response is an important part of bone repair. We investigated the polarization change of inflammatory macrophages (M1) and resident macrophages (M2) during low-intensity pulsed ultrasound (LIPUS) treatment. MethodsThirty male Sprague Dawley rats (age: 12 weeks; weight: 300 g) were used in the study. A rat spinal fusion model was established by surgical procedures. LIPUS treatment (20 min. d, 5 d/wk) was begun 3 days after surgery. The rats were randomly divided into a control group (5 subgroups, 3 rats in each subgroup) and LIPUS group (5 subgroups, 3 rats in each subgroup), and sacrificed on day 3, 5, 7, 10, and 14 after spinal fusion surgery for further evaluation. Bone volume was measured by micro-CT, fusion region was examined by histological analyses, types of macrophages in the fusion area were examined by immunohistochemical staining. Raw264.7 cells and bone marrow-derived macrophages (BMDM) were used in cell experiments. Cells were divided into a control group and LIPUS group. Flow cytometry was used to examine the rate of resident macrophages, and real-time PCR was used to examine the mRNA expression of anti-inflammation genes. ResultsLIPUS promoted spinal fusion and stimulated the transition of F4-80+/Mac-2+ (M1) to F4-80+/Mac-2− (M2), leading to the early appearance of resident macrophages. Cell experiments showed CD206+ macrophages (M2) were significantly increased after LIPUS treatment. M2-related genes and anti-inflammation factors (Arg-1, PPAR-γ, and IL-4) were increased after LIPUS treatment. ConclusionThe earlier transition from inflammatory to resident macrophage might be one reason for the positive effect of LIPUS on spinal fusion.

2381Low-intensity pulsed ultrasound ameliorates DES-induced coronary adventitial inflammation and hyperconstricting responses in pigs in vivo - A novel non-invasive therapy for coronary inflammation

Abstract Background We previously demonstrated that coronary adventitial inflammation plays important roles in the pathogenesis of coronary artery spasm, including drug-eluting stent (DES)-induced coronary hyperconstricting responses, in pigs and humans. Indeed, the coronary adventitia has recently attracted much attention as the important site for vascular inflammation. However, a direct therapeutic approach to the coronary adventitia remains to be developed. We have developed a non-invasive low-intensity pulsed ultrasound (LIPUS) therapy for angina, which exerts anti-inflammatory effects through improved coronary microcirculation. Purpose In this study, we aimed to examine whether our LIPUS therapy ameliorates DES-induced coronary hyperconstricting responses in pigs in vivo, and if so, what mechanisms are involved. Methods An everolimus-eluting stent (EES) was implanted into the left anterior descending (LAD) coronary artery in normal male pigs. They were randomly assigned to the LIPUS or the sham therapy groups. After EES implantation, in the LIPUS group, LIPUS (32 cycles, 193 mW/cm2) was applied to the heart at 3 different levels (proximal and distal stent edges and middle portion of the stent) through X-ray for 20 min at each level for every other day for 2 weeks (6 days in total) (Figs. 1A, 1B). The sham therapy group was treated in the same manner but without LIPUS. At 4 weeks after the procedure, we performed coronary angiography to examine coronary vasoconstricting responses to intracoronary serotonin in vivo. Finally, stented coronary vessels were harvested for immunohistochemistry of vasa vasorum, lymphatic vessels (LYVE-1), sympathetic nerve fibers (SNF), vascular inflammation (macrophages and IL-1β expression), Rho-kinase expression and activity as evaluated by phosphorylated myosin phosphatase target subunit-1 (pMYPT-1). Results Coronary vasoconstricting responses to intracoronary serotonin were significantly enhanced in the sham therapy group but were significantly suppressed in the LIPUS group at the DES edges in the LAD, whereas those responses were comparable at the non-DES implanted segments in the left circumflex (LCx) coronary arteries between the 2 groups. (Figs. 1C, 1D). Furthermore, in vivo lymph transport speed was significantly faster in the LIPUS group than in sham group (Figs. 1E–1G). Histological analysis showed that except vasa vasorum formation, the number of lymphatic vessels, adventitial inflammatory cells infiltration, Rho-kinase expression and activity were all significantly enhanced in the sham therapy group and were significantly suppressed in the LIPUS group (Figs. 1G–1K). Figure 1 Conclusion We were able to develop a non-invasive LIPUS therapy for coronary functional abnormalities caused by chronic adventitial inflammation in pigs in vivo, for which multiple beneficial effects appear to be involved (Fig. 1L).

Inhibition of myostatin signal pathway may be involved in low-intensity pulsed ultrasound promoting bone healing.

Low-intensity pulsed ultrasound (LIPUS) is effective in promoting bone healing, and a myostatin deficiency also has a positive effect on bone formation. In this study, we evaluated the effects of LIPUS on bone healing in rats in vivo and investigated the mechanisms in vitro, aiming to explore whether LIPUS promotes bone healing through inhibition of the myostatin signaling pathway. Rats with both drill-hole defects and MC3T3-E1 cells were randomly assigned to a LIPUS group and a control group. The LIPUS group received LIPUS treatment (1.5MHz, 30mW/cm2) for 20min/day. After 21days, the myostatin expression in quadriceps was significantly inhibited in the LIPUS group, and remodeling of the newly formed bone in the drill-hole site was significantly better in the LIPUS group than that in the control group, which was confirmed by micro-CT analysis. After 3days, LIPUS significantly promoted osteoblast proliferation; inhibited the expression of AcvrIIB (the myostatin receptor), Smad3, p-Smad3, and GSK-3β; and increased Wnt1 and β-catenin expression. Moreover, translocation of β-catenin from the cytolemma to the nucleus was observed in the LIPUS group. However, these effects were blocked by treatment with myostatin recombinant protein. The results indicate that LIPUS may promote bone healing through inhibition of the myostatin signal pathway.

Effects of low-intensity pulsed ultrasound on hematopoietic function in rats after combined chemotherapy with doxorubicin and cyclophosphamide

To investigate the effect of low-intensity pulsed ultrasound (LIPUS) on hematopoietic function in rats after combined chemotherapy with doxorubicin and cyclophosphamide. Eighty rats were randomized into control group and LIPUS group (n=40) for treatment with intraperitoneal injection of doxorubicin (2 mg/kg)+cyclophosphamide (20 mg/kg) for 4 consecutive days and continuous irradiation with LIPUS for 7 days following the injections, respectively. The white blood cells, red blood cells and platelets counts in each group were measured at 0, 4, 7, 9, 11, 14 and 18 days after the start of drug administration. The pathological sections of the bone marrow were examined at 0, 4 and 11 days, and the flow cytometry was performed for detecting the cell apoptosis; qPCR was performed for detecting the expressions of SCF, ICAM-1, and VCAM-1 mRNAs, and ELISA was used to detect the expressions of IL-3 and GM-CSF. The white blood cell count was significantly higher in LIPUS group than in the control group (P &lt; 0.05). Histopathological examination of the bone marrow revealed significantly increased hematopoietic tissue in LIPUS group (P &lt; 0.05). Flow cytometry demonstrated an obviously lower cell apoptosis rate in the bone marrow in LIPUS group than in the control group (P &lt; 0.05). Compared with those in the control group, the mRNA expression levels of ICAM-1 and VCAM-1 as well as the protein levels of IL-3 and GM-CSF were significantly increased in LIPUS group (P &lt; 0.05). LIPUS can alleviate the hematopoietic damage after combined chemotherapy with doxorubicin with cyclophosphamide probably by increasing the expressions of ICAM- 1, VCAM-1, IL- 3, and GM-CSF.

Low intensity pulsed ultrasound increases mandibular height and Col-II and VEGF expression in arthritic mice

ObjectiveRheumatoid arthritis (RA) is a chronic inflammatory disease involving persistent inflammation resulting in cartilage and bone damage. RA can affect the temporomandibular joint (TMJ), and damage to the TMJ condyle can lead to craniofacial developmental disturbances, causing micrognathia, malocclusion, retrognathia, and increased overjet. Current treatments of TMJ arthritis are unsatisfactory. This pilot study aimed to investigate the effect of low intensity pulsed ultrasound (LIPUS) on the mandible and TMJ condyles in an RA mouse model using micro-computed tomography (Micro-CT), histologic, and immunohistochemical analyses. MethodsMRL-lpr/lpr mice received LIPUS application to their TMJs for 20 min/day for 2 and 4 weeks. Micro-CT analysis measured condylar length and width, posterior mandibular height (P.M.H), mandibular ramus length (M.R.L), effective mandibular length (Ef.M.L), angular process length (A.P.L), mandibular plane (M.P), mandibular axis (M.Ax), and lower incisor height (L.I.H). Condylar cartilage thickness was histologically measured, and type II collagen (Col-II), vascular endothelial growth factor (VEGF), nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) expression was analyzed using immunohistochemistry. ResultsComparing the LIPUS-treated group with the control, P.M.H, M.R.L, and M.P were significantly greater in the LIPUS-treated group. Immunostaining for Col-II and VEGF was stronger in the LIPUS-treated group after 4 weeks. OPG showed slightly more expression in the LIPUS group. ConclusionsLIPUS may enhance mandibular and TMJ condylar bone formation in this RA mouse model by preventing any growth disturbances involved in inflammation. Further studies are recommended to analyze the effect of LIPUS on TMJ of RA in other animal models.

Effect of low-intensity pulsed ultrasound after intraoral vertical ramus osteotomy

The present study investigated the effect of low-intensity pulsed ultrasound (LIPUS) on long-term osseous healing of the cleavage space between bone fragments after intraoral vertical ramus osteotomy (IVRO). Patients undergoing IVRO were randomly assigned to the LIPUS group (n = 12) or the control group (n = 9) after surgery. LIPUS treatments were applied daily to the cleavage space between bone fragments for 3 weeks. We observed 3-dimensional quantitative color mapping of the whole mandible created by computed tomography (CT) data at 1 month, 6 months, and 1 year postoperatively. On the basis of CT values, the color grades were classified as D1 to D5 by using the Misch criteria. We then calculated mean CT values and rated each color grade in different selection ranges. The mean CT values of the LIPUS group were significantly higher than those of the control group at 1 month, 6 months and 1 year postoperatively (P < .01). The color grades of the cleavage between bone fragments increased from D5 to D1 over time. Our results indicated that LIPUS promoted osseous healing after IVRO, thus improving bone density and offering clinical benefits.

Synergies of accelerating differentiation of bone marrow mesenchymal stem cells induced by low intensity pulsed ultrasound, osteogenic and endothelial inductive agent

In terms to investigate the effect of low-intensity pulsed ultrasound (LIPUS) for differentiation of bone marrow mesenchymal stem cells (BMSCs) and the feasibility of simultaneously inducing into osteoblasts and vascular endothelial cells within the cell culture medium in which two inductive agents are added at the same time with or without LIPUS. Cells were divided into a non-induced group, an osteoblast-induced group, a vascular endothelial-induced group, and a bidirectional differentiation-induced group. Each group was further subdivided into LIPUS and non-LIPUS groups. The cell proliferation in each group was measured by MTT assay. Cell morphological and ultrastructural changes were observed by inverted phase contrast microscopy and transmission electron microscopy. The differentiation of BMSCs was detected by confocal microscopy, flow cytometry and quantitative RT-PCR. Results demonstrated that both osteoblast and vascular endothelial cell differentiation markers were expressed in the bidirectional differentiation induction group and early osteogenesis and angiogenesis appeared. The cell proliferation, differentiation rate and expression of osteocalcin and vWF in the LIPUS groups were all significantly higher than those in the corresponding non-LIPUS group (p < .05), suggesting LIPUS treatment can promote the differentiation efficiency and rate of BMSCs, especially in the bidirectional differentiation induction group. This study suggests the combination of LIPUS and dual-inducing agents could induce and accelerate simultaneous differentiation of BMSCs to osteoblasts and vascular endothelial cells. These findings indicate the method could be applied to research on generating vascularized bone tissue with a shape and function that mimics natural bone to accelerate early osteogenesis and angiogenesis.

Low-Intensity Pulsed Ultrasound Relieves Mild and Severe Myelosuppression Induced by Cyclophosphamide in Rabbits.

This study aimed to investigate the effect of low-intensity pulsed ultrasound (LIPUS) on cyclophosphamide (CTX)-induced rabbit myelosuppression. Rabbits (n = 90) were randomly divided into a mild myelosuppression group (n = 40), a severe myelosuppression group (n = 40), and a normal control group (group Cu28 ; n = 10). The mild and severe myelosuppression models were established by daily ear vein injection of 15- and 40-mg/kg CTX for 4 continuous days, respectively. Then they were randomly divided into LIPUS groups (Au and Bu ) and control groups (Ac and Bc ). LIPUS was applied once per day for 20 minutes for 7 (Au7 and Bu7 ) and 28 (Au28 and Bu28 ) days. Physical conditions, mortality, blood cell counts, and bone marrow proliferation were calculated. Erythropoietin interleukin 3, and granulocyte-macrophage colony-stimulating factor levels were measured by an enzyme-linked immunosorbent assay. Flow cytometry was used to detect the granulocyte phagocytosis rate. Hematoxylin-eosin staining was performed to analyze changes of skin and muscle. Compared with the control group, LIPUS improved the number of peripheral blood cells (P < .05) and bone marrow nucleated cells and reduced the mortality of rabbits with myelosuppression of different degrees. Long-term treatment for 28 days had no effect on the levels of erythropoietin, interleukin 3, and granulocyte-macrophage colony-stimulating factor and granulocyte phagocytosis (P > .05). The parts of the skin where LIPUS was applied did not show any burning marks, and the muscle tissue in the path of LIPUS acoustic channels showed no obvious pathologic changes. Low-intensity pulsed ultrasound is a safe and effective method to relieve CTX-induced myelosuppression.

Effects of Photobiomodulation and Ultrasound Applications on Orthodontically Induced Inflammatory Root Resorption; Transcriptional Alterations in OPG, RANKL, Cox-2: An Experimental Study in Rats

Objective: The aim of this study was to evaluate and compare the reparative and inhibitory effects of a light-emitting diode-mediated photobiomodulation (PBM) and of a low-intensity pulsed ultrasound (LIPUS) on orthodontically induced inflammatory root resorption (OIIRR). Materials and methods: Forty-nine Wistar rats were divided into four groups: untreated group (negative control), group treated with orthodontic appliances (positive control), PBM-treated group (wavelength: 618 nm, output power density: 20 mW/cm2), and LIPUS-treated group (frequency: 1.5% ± 5% MHz, pulse repetition ratio: 1.0% ± 10% kHz, effect area: 3.88% ± 1% cm2 and intensity: 30% ± 30% mW/cm2). OIIRR was induced experimentally in rats for 14 days with an applied force of 100g, and therapeutic approaches were performed concurrently. At the end of the experiment, upper first molar teeth of rats were prepared for genetic analysis, scanning electron microscopy, hematoxylin and eosin staining, and tartrate-resistant acid phosphatase staining. Kruskal-Wallis and post hoc Dunn's tests were performed. Results: Number of osteoclasts (p < 0.01), number of resorption lacunae and resorption area ratio (p < 0.001) decreased and number of total cells (p < 0.001) increased with the PBM and LIPUS applications when compared with the positive control group. Receptor activator of nuclear factor kappa B ligand (RANKL) levels of PBM and LIPUS groups were lower (p < 0.001), and osteoprotegerin (OPG) levels were higher (p < 0.001) than the positive control group. Cyclooxygenase-2 (Cox-2) expression significantly decreased with LIPUS and PBM administrations (p < 0.05). No significant difference was observed among PBM and LIPUS groups. Conclusions: PBM and LIPUS applications showed marked inhibitory and reparative effects on OIIRR by modulating the OPG/RANKL ratio, Cox-2 expression, and cell differentiation of osteoblasts and osteoclasts.

Study on proliferation effects of low intensity pulsed ultrasound on human bone marrow mesenchymal stem cells in vitro

Objective To investigate the effect of low intensity pulsed ultrasound (LIPUS) on the proliferation of human bone marrow mesenchymal stem cells (hBMSCs) and the effect of Cyclin D1 on the proliferation of hBMSCs induced by LIPUS. Methods The cells were inoculated on the a 12-well culture culture plate at a cell concentration of 2.0×104 cells/ml, LIPUS with radiation intensity of 0, 30, 40, 50, 60 and 80 mW/cm2 was used to stimulate for 5 min, the morphological changes of the cells were observed at 0, 24, 72 and 96 h, cell counting was performed with a Scepter 2.0 handheld cell counter, the cell cycle and value-added index were measured by flow cytometry, the cell proliferation was detected by cell counting kit-8 (CCK-8). The expression of Cyclin D1 was detected by Western blotting. The expression of Cyclin D1 mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Results Cell counts were found except for the 30 mW/cm2 (P=0.871) group that the number of cells in 40 W/cm2 (P=0.006), 50 W/cm2 (P=0.001), 60 W/cm2 (P=0.001), 80 W/cm2 (P=0.004) group was significantly higher than that in control group. Cell proliferation assay was found that the proliferation of 30 mW/cm2 (P=0.042), 40 W/cm2 (P=0.012), 50 W/cm2 (P=0.001), 60 W/cm2 (P=0.001), 80 W/cm2 (P=0.046) cells was significantly higher than that of the control group. Flow cytometry detection found that the cell proliferation index of LIPUS group was significantly higher than that of control group (P=0.001). Western blotting and RT-PCR showed that there was no significant difference between the 30 mW/cm2 group and the control group. The expression of Cyclin D1 was significantly increased in 40 W/cm2 (P=0.002), 50 W/cm2 (P=0.004), 60 W/cm2 (P=0.023), 80 W/cm2 (P=0.012) groups. The expression of Cycling D1 mRNA was significantly different from that of control group (P=0.001). Conclusion LIPUS can significantly promote the proliferation of hBMSCs, and promote the proliferation of hBMSCs from G1 phase to S phase by up-regulation of Cyclin D1 expression. Key words: Low intensity pulsed ultrasound; Human bone marrow mesenchymal stem cells; Proliferation; Cyclin D1

Low Intensity Pulsed Ultrasound Influences the Myogenic Differentiation of Muscle Satellite Cells in a Stress Urinary Incontinence Rat Model

ObjectiveTo investigate the therapeutic effect of low intensity pulsed ultrasound (LIPUS) in a stress urinary incontinence (SUI) rat model and its influence on myogenic satellite cells. MethodsFifty Sprague-Dawley rats underwent vaginal distension and bilateral ovariectomy mimicking partum injury and menopause to construct SUI models, which were further randomized into 100 mW/cm2 LIPUS, 200 mW/cm2 LIPUS, 300 mW/cm2 LIPUS, and none-treatment control subgroups with 10 rats per subgroup. Ten rats served as mock operation control. Leak point pressure and bladder capacity were recorded 1 week after LIPUS treatment. Immunofluorescence staining and Western blot were performed to examine histological changes, myodifferentiation, and signaling pathway. ResultsHere,we found the leak point pressure and bladder capacity were restored in 200 mW/cm2 LIPUS and 300 mW/cm2 LIPUS groups, but not in 100 mW/cm2 LIPUS group. More robust striated muscle regeneration was observed in 200 mW/cm2 LIPUS group comparing with the SUI none-treatment group. Moreover, we found LIPUS activated the myodifferentiation of muscle satellite cells, which is correlated to p38 phosphorylation level. ConclusionLIPUS restored the leak point pressure and bladder capacity, and activated satellite cell myodifferentiation in SUI rat model.

Microbubble-Mediated Ultrasound Outweighs Low-Intensity Pulsed Ultrasound on Osteogenesis and Neovascularization in a Rabbit Model of Steroid-Associated Osteonecrosis.

Microbubbles magnify the acoustic pressure of low-intensity pulsed ultrasound (LIPUS) and may enhance its bioeffect for diagnostic and therapeutic purposes. This study compared the effect of this novel microbubble-mediated ultrasound (MUS) with that of the traditional LIPUS on osteogenesis and neovascularization in a rabbit model of steroid-associated osteonecrosis. We hypothesized that MUS might outweigh LIPUS on promoting osteogenesis and neovascularization in steroid-associated osteonecrosis. The bilateral femoral head necrosis was induced by lipopolysaccharide and methylprednisolone in the rabbits. The indices of bone mineral density (BMD), trabecular number, maximal loading strength, and mineral apposition rate were analyzed, demonstrating that the animal model of steroid-associated osteonecrosis was successfully established. Both the MUS group (GM) and the LIPUS group (GL) were insonated 20 min daily for six weeks. GM received an extra intracapsular injection of microbubbles before insonation every other day. Fluorescence bone labeling, Micro-CT Analysis, biomechanical test, quantitative real-time PCR, Western blot analysis, and histological evaluation were performed for comparing GM with GL. The results demonstrated a 39% higher mineral apposition rate in GM compared with GL. The BMD and the maximal loading strength of femoral head of GM increased by 4.3% and 27.8% compared to those of GL, respectively. The mRNA and protein expression of BMP-2 and VEGF were also significantly higher in GM. The number of blood vessels of GM was 65% greater than that of GL. MUS is more potent than LIPUS in enhancing osteogenesis, neovascularization, and biomechanical strength of femoral head in the animal model of steroid-associated osteonecrosis. Without increasing the intensity of insonation or the risk of tissue damage, MUS is better for inhibiting the process of steroid-associated osteonecrosis.