Low-intensity pulsed ultrasound system for improving osteoporosis and application of LIPUS in promoting osteogenic differentiation of BMSCs

Abstract

The invention discloses a low-intensity pulsed ultrasound system which is used for improving osteoporosis of a patient and further assisting treatment of the osteoporosis, and at least comprises a low-intensity pulsed ultrasound device which is at least provided with an ultrasonic transducer, the ultrasonic transducer is placed at the osteoporosis part of the patient, and the low-intensity pulsed ultrasound device is used for treating the osteoporosis of the patient. The ultrasonic treatment module is used for treating a patient through output low-intensity pulse ultrasonic waves; the invention also discloses an application of the LIPUS or the system in improving senescence type osteoporosis, and an application in promoting osteogenic differentiation of senescence mesenchymal stem cells and / or promoting MRTF-A nucleation of the senescence mesenchymal stem cells. According to the application disclosed by the invention, LIPUS can promote osteogenesis of the aged BMSCs and play a role through MRTF-A, and a molecular regulation mechanism of LIPUS in a process of regulating differentiation of the aged BMSCs towards the osteogenesis direction is defined, so that a deeper theoretical basis and a more effective treatment strategy are provided for treatment of osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a low-intensity pulsed ultrasound system for improving osteoporosis and application of LIPUS in promoting osteogenic differentiation of BMSCs. Background Art

[0002] The incidence of various degenerative diseases increases with the gradual aging of the body, and osteoporosis is one of the more common diseases. The disease is characterized by decreased bone density, degenerative changes in the fiber structure of bone tissue, thinning of trabeculae in cancellous bone, thinning of cortical bone thickness, and a significantly increased risk of fractures. It is a systemic, metabolic disease that affects the bones of the whole body.

[0003] The gradual functional abnormality of bone marrow mesenchymal stem cells (BMSCs) during aging is an important part of the pathological process of osteoporosis, which may hinder the bone formation process and damage the bone microstructure. These cells cannot respond appropriately to mechanical stimulation, which leads to a weakened osteogenic differentiation ability. Myocardin-related transcription factor-A (MRTF-A) is mechanosensitive and its transcriptional activity is regulated by actin dynamics. Studies have shown that MRTF-A may play a key role in regulating the balance between adipogenic and osteogenic differentiation of BMSCs.

[0004] The efficacy of drugs such as bisphosphonates and selective estrogen receptor modulators for the treatment of osteoporosis has certain limitations, and long-term use may cause many adverse reactions. As a convenient and non-invasive treatment method, low-intensity pulsed ultrasound (LIPUS) is increasingly showing its potential in the treatment of diseases such as fractures and osteoarthritis. However, there are few studies on LIPUS in senile osteoporosis, and its mechanism is still unclear. Summary of the invention

[0005] The purpose of the present invention is to provide a low-intensity pulsed ultrasound system for improving osteoporosis and the application of LIPUS in osteoporosis in view of the above problems.

[0006] In order to achieve its purpose, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a low-intensity pulsed ultrasound system for improving aging-related osteoporosis, which is used to improve osteoporosis in patients and further assist in the treatment of osteoporosis, and at least comprises:

[0008] A low-intensity pulsed ultrasound device having at least an ultrasound transducer,

[0009] The ultrasonic transducer is placed at the osteoporotic site of the patient and is used to treat the patient by outputting low-intensity pulsed ultrasound.

[0010] The osteoporotic sites include the lumbar vertebrae, femur, tibia, and radius.

[0011] The low-intensity pulsed ultrasound device also has an ultrasound control unit, a parameter input unit, an ultrasound communication unit and an ultrasound display unit; the parameter input unit is used to input low-intensity pulsed ultrasound parameters, the ultrasound display unit is used to display the waveform of the output low-intensity pulsed ultrasound wave and the numerical value of the low-intensity pulsed ultrasound parameter, and the ultrasound communication unit is used to exchange data.

[0012] The second aspect of the present invention provides the use of low-intensity pulsed ultrasound or the above-mentioned low-intensity pulsed ultrasound system in improving aging-related osteoporosis.

[0013] The third aspect of the present invention provides the use of low-intensity pulsed ultrasound or the above-mentioned low-intensity pulsed ultrasound system in promoting osteogenic differentiation of aged bone marrow mesenchymal stem cells and / or promoting MRTF-A nuclear translocation of aged bone marrow mesenchymal stem cells.

[0014] In the above-mentioned application technology solution, the therapeutic intensity of the low-intensity pulsed ultrasound is 100-150 mW / cm 2 .

[0015] In the above-mentioned application technology solution, the treatment time of the low-intensity pulsed ultrasound is 20 to 30 minutes.

[0016] In the above application technical solution, the frequency of the low-intensity pulsed ultrasound device is set to 3 MHz, with a duty cycle of 50%.

[0017] In the above-mentioned application technology scheme, low-intensity pulsed ultrasound regulates the remodeling of cell microfilaments through mechanical action, promotes the nuclear entry of MRTF-A, and thus improves the osteogenic differentiation ability of aged BMSCs.

[0018] The beneficial effects of the present invention are as follows: the present invention study proves that LIPUS can improve osteoporosis in aged mice, and the nuclear translocation of MRTF-A is the key regulatory point for LIPUS to promote osteogenic differentiation of aged BMSCs. The present invention study confirms the ability of LIPUS to promote osteogenic differentiation of aged bone marrow mesenchymal stem cells (BMSCs), and the mechanism of action of myocardin-related transcription factor-A (MRTF-A), clarifies that LIPUS can promote osteogenesis of aged BMSCs and exerts its effects through MRTF-A, clarifies the molecular regulatory mechanism of LIPUS in regulating the differentiation of aged BMSCs towards osteogenic direction, and provides a deeper theoretical basis and more effective treatment strategy for the treatment of osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effect of LIPUS on osteoporosis in elderly mice is shown: A. Schematic diagram of animal experiment design; B. Micro CT detection image of mouse femur after 8 weeks of LIPUS irradiation; C. Micro CT quantitative analysis (n=5; *P<0.05, **P<0.01); D. H&E staining of mouse femur (arrows indicate trabeculae).

[0020] Figure 2 The effect of LIPUS on osteogenic differentiation of aged BMSCs is shown: A. ROS fluorescence staining and SA-β-Gal staining after 3 days of H2O2 induction; B. Western blot results; C. ALP staining images; D. ALP staining quantitative analysis (n=3; ****P<0.00001); E. Alizarin red staining images; F. Alizarin red staining quantitative analysis (n=3; ****P<0.0001).

[0021] Figure 3 Immunofluorescence staining and phalloidin staining were used to observe the changes of MRTF-A and cytoskeleton under H2O2 and LIPUS treatment.

[0022] Figure 4 The effects of MRTF-A inhibitor CCG-100602 on LIPUS-promoted osteogenesis of senescent BMSCs are shown: A. MRTF-A immunofluorescence staining results; B. ALP staining images; C. ALP staining quantitative analysis (n=3; *P<0.05, ****P<0.0001); D. Alizarin red staining images; E. Alizarin red staining quantitative analysis (n=3; *P<0.05, ****P<0.0001). DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0024] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0025] Example 1

[0026] 1 Materials and Methods

[0027] 1.1 Experimental Materials

[0028] 1.1.1 Experimental Animals

[0029] Five 20-month-old female C57BL / 6J mice and ten 3-4-week-old female C57BL / 6J mice were supplied by the Animal Experiment Center of Chongqing Medical University [License No.: SYXK (Yu) 2022-0016]. This study was approved by the Ethics Committee of the First Affiliated Hospital of Chongqing Medical University (2021-705).

[0030] 1.1.2 Instruments and Materials

[0031] In this study, experiments were carried out with the help of a LIPUS instrument (Chattanooga, 2776), an upright fluorescence microscope (OLYMPUS, BX53), and an inverted fluorescence microscope (Nikon, Ti2).

[0032] Fetal bovine serum and α-MEM medium were purchased from Gibco.

[0033] Phosphate buffered saline (PBS), tris buffered saline with Tween-20 (TBST), β-glycerophosphate, dexamethasone, L-ascorbic acid, H2O2, radio immunoprecipitation assay (RIPA) lysis buffer, senescence-associated β-galactosidase (SA-β-gal) staining kit, and Triton X-100 were purchased from Solarbio.

[0034] Alkaline Phosphatase (ALP) staining kit, alizarin red staining solution, phalloidin, and 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) probe were purchased from Beyotime.

[0035] CCG-100602 was purchased from MedChemExpress. 4% paraformaldehyde (Paraformaldehyde, PFA) was purchased from Servicebio.

[0036] Cetylpyridinium chloride was purchased from Macklin, Inc. PMSF was purchased from KeyGEN BioTECH.

[0037] Rabbit anti-mouse ALP and rabbit anti-mouse Runt-related transcription factor 2 (RUNX2) were purchased from Zenbio. Rabbit anti-mouse osteopontin (OPN) antibody was purchased from Abcam. Rabbit anti-mouse MRTF-A, mouse anti-mouse β-actin, and CoraLite 594 labeled goat anti-rabbit antibody were purchased from Proteintech. DAPI was purchased from Biosharp.

[0038] 1.2 Methods

[0039] 1.2.1 Animal Experiment Grouping and Treatment Methods

[0040] The right femur of the aged mice (n=5) was used as the control group, and the left femur was set as the LIPUS treatment group. The LIPUS ultrasonic therapeutic instrument transducer was placed on the left femur of the mice for 30 minutes / day. The LIPUS instrument parameters were set to 3MHz frequency, 100mW / cm 2 The transducer was placed above the right femur of the mouse for 30 min / day without LIPUS irradiation. The treatment was carried out 6 times a week for 8 weeks. After the treatment, the bilateral femurs were dissected for Micro CT analysis and hematoxylin-eosin staining (HE staining).

[0041] 1.2.2 Culture of mouse BMSCs

[0042] The femurs and tibiae of 3-4 week old female C57 BL / 6J mice were completely dissected, and the medullary cavity was avoided during the dissection. After separation, the femurs and tibiae were placed in PBS containing 2% double antibody, and the medullary cavity was rinsed with 1-2mL complete culture medium (10% fetal bovine serum + α-MEM culture medium) until it turned white. The cell suspension filtered through a 70μm cell sieve was mixed and inoculated into a culture dish, and then moved into a sterile incubator for incubation. After 24h, the cell adhesion status was checked and the culture medium was replaced, and the medium was replaced every 1 day thereafter.

[0043] 1.2.3 Modeling and staining of senescent BMSCs

[0044] The fourth-generation mouse BMSCs were seeded in a 24-well plate. When the confluence reached 70%, 200 μmol / L H2O2 was added for induction for 3 days, and then ROS fluorescence staining and SA-β-gal staining were performed.

[0045] 1.2.4 Cell experiment grouping and culture method

[0046] BMSCs were divided into control group, H2O2 group (200 μmol / L H2O2), and H2O2+LIPUS group (200 μmol / L H2O2+LIPUS). The LIPUS instrument parameters were set as frequency 3 MHz, sound intensity 100 mW / cm 2 and duty cycle 50%. On the second day, the medium was replaced with osteogenic induction medium (10% fetal bovine serum + α-MEM medium + 10mmol / L sodium β-glycerophosphate + 50mg / L L-ascorbic acid + 100nmol / L dexamethasone), and H2O2 was added to continue culture, and the medium was changed every 2 days thereafter. After 3 days of H2O2 induction, the medium was replaced with a medium without H2O2. The cells were then divided into H2O2 group (200μmol / LH2O2), H2O2+LIPUS group (200μmol / L H2O2+LIPUS), H2O2+CCG100602 group (200μmol / LH2O2+20μmol / L CCG-100602), and H2O2+CCG100602+LIPUS group (200μmol / LH2O2+20μmol / L CCG-100602+LIPUS). After 3 days, the medium was changed to one without H2O2, and CCG-100602 was added again when the osteogenic medium was changed.

[0047] 1.2.5 ALP staining

[0048] After the cells were fixed with 4% PFA, an equal amount of ALP staining working solution (containing 300 μL alkaline phosphatase color development buffer, 2 μL NBT and 3 μL BCIP) was added to each well of the 24-well plate and incubated at 37°C for 30 minutes. When the blue-purple positive staining area appeared, the staining solution was aspirated and the cells were washed with PBS and observed under a microscope.

[0049] 1.2.6 Alizarin red staining and quantification

[0050] After the cells were fixed with 4% PFA, 200 μL of Alizarin Red dye solution was added to each well of the 24-well plate and incubated at room temperature for 1 hour. When the orange-red positive staining area appeared, the dye solution was aspirated and observed under a microscope after washing with PBS. After observation, Alizarin Red precipitate containing 10% cetylpyridinium chloride was prepared. 500 μL of Alizarin Red precipitate solution was added to each well, and after reacting on a shaker for 30 minutes, the dissolved solution was aspirated to a 96-well plate (100 μL / well) and the absorbance at 562 nm was detected.

[0051] 1.2.7 Western blot detection

[0052] Total protein was extracted on ice using RIPA lysis buffer containing 1% PMSF, and protein samples were quantified using BCA. Equal amounts of samples were taken from each group for gel electrophoresis to separate proteins of different sizes in the gel, and then proteins were transferred using a methanol-activated PVDF membrane (0.45 μm). Proteins were blocked with 5% skim milk powder for 1 h, followed by incubation with ALP (1:500), RUNX2 (1:500), OPN (1:1000), and β-actin (1:10000) antibodies at 4°C overnight. After washing the strips three times with TBST, secondary antibodies were added and incubated for 1 h. After washing the strips three times with TBST, chemiluminescence imager was used for exposure and color development.

[0053] 1.2.8 ROS fluorescence staining

[0054] After the cells were incubated with the DCFH-DA probe at 37°C for 20 min, the excess DCFH-DA was washed away with serum-free cell culture medium and then observed under a microscope.

[0055] 1.2.9 SA-β-gal staining

[0056] The cells were fixed with 4% PFA and incubated with the pre-prepared SA-β-Gal staining solution overnight at 37° C. The cell samples were washed with 70% ethanol and observed under a microscope.

[0057] 1.2.10 Immunofluorescence staining

[0058] After fixation with 4% PFA for 15 minutes, cells were permeabilized with 0.2% Triton X-100 for 20 minutes, and then blocked with PBS containing 5% bovine serum albumin for 1 hour. MRTF-A antibody (1:100) was added and incubated overnight at 4°C. After washing with PBA, goat anti-rabbit (1:200) labeled with CoraLite 594 was incubated for 1 hour. Cells that need microfilament staining were incubated with phalloidin (1:200) at room temperature for 1 hour. After washing with PBS three times, DAPI was used for counterstaining for 5 minutes. After washing with PBS, the slides were sealed with anti-fluorescence quencher and observed under a microscope.

[0059] 1.2.11 Statistical analysis

[0060] GraphPad Prism 9 software was used for statistical analysis of this study. The animal experiments were compared between groups using paired t-tests, while the cell experiments were compared between groups using analysis of variance. The differences were considered statistically significant when P < 0.05.

[0061] 2 Results

[0062] 2.1 LIPUS improves osteoporosis in elderly mice

[0063] Figure 1 A is a schematic diagram illustrating the key steps involved in LIPUS treatment of aged mice and sample collection. Micro CT results showed that the number of trabecular bones in the left femur irradiated by LIPUS was increased compared with that in the untreated right femur ( Figure 1 B); bone volume to total volume (BV / TV) and trabecular number (Tb.N) showed an increasing trend; while trabecular spacing (Tb.Sp) decreased significantly ( Figure 1 C). HE staining of femoral sections showed that the left femur had fewer adipocytes in the medullary cavity and more trabeculae than the right femur ( Figure 1 D).

[0064] 2.2LIPUS promotes osteogenic differentiation of aged BMSCs

[0065] Compared with the control group, the fluorescence expression of ROS probe and the blue β-gal positive area of ​​BMSCs treated with H2O2 increased ( Figure 2 A). When compared with the normal control group, Western blot analysis results showed that the protein expression levels of ALP, RUNX2, and OPN in the H2O2 group showed a downward trend, while the protein expression levels in the H2O2+LIPUS group showed an increasing trend compared with the H2O2 group ( Figure 2 B). Compared with the normal control group, the blue-purple positive staining area of ​​ALP staining was significantly reduced in the H2O2 group ( Figure 2 C, D), and alizarin red staining showed that the orange-red mineralized nodules were significantly reduced. However, the positive staining area in the LIPUS group was significantly increased compared with the H2O2 group ( Figure 2 E, F).

[0066] 2.3LIPUS promotes MRTF-A nuclear translocation in aged BMSCs

[0067] Phalloidin staining showed that compared with the normal control group, the BMSCs treated with H2O2 had larger cell volume and irregular morphology, less rigid microfilamentous structure, and a punctate depolymerization state. 8 hours after LIPUS irradiation, the intracellular punctate structure in the H2O2+LIPUS group was less than that in the H2O2 group, and the rigid microfilaments increased. MRTF-A immunofluorescence staining results showed that the fluorescence intensity in the cytoplasm of the H2O2 group was significantly higher than that of the normal control group; the fluorescence intensity in the cytoplasm of the H2O2+LIPUS group was relatively lower than that of the H2O2 group, and the fluorescence in the nucleus was significantly enhanced ( Figure 3 ).

[0068] 2.4 Inhibition of MRTF-A nuclear translocation inhibits the effect of LIPUS on promoting osteogenic differentiation of senescent BMSCs

[0069] MRTF-A immunofluorescence staining showed that under the action of CCG-100602, an inhibitor of MRTF-A nuclear entry, the fluorescence intensity in the nucleus of the H2O2+CCG-100602+LIPUS group was weakened compared with the H2O2+LIPUS group ( Figure 4 A). At the same time, the positive staining areas of ALP and alizarin red in the H2O2+CCG-100602+LIPUS group were significantly lower than those in the H2O2+LIPUS group ( Figure 4 BE).

[0070] 3 Analysis

[0071] The incidence of various degenerative diseases increases with the gradual aging of the body, and osteoporosis is one of the more common diseases. The disease is characterized by decreased bone density, degenerative changes in the fiber structure of bone tissue, thinning of trabeculae in cancellous bone, thinning of cortical bone thickness, and a significantly increased risk of fractures. It is a systemic, metabolic disease that affects bones throughout the body. In the modern social environment, osteoporosis has become a public health issue that has attracted widespread attention, and exploring non-invasive and efficient treatment methods is a topic worthy of in-depth discussion.

[0072] As a non-invasive and easy-to-use treatment method, LIPUS mainly exerts its therapeutic effect through its mechanical action. BMSCs and osteoblast precursor cells can respond to a certain degree of mechanical stimulation and then differentiate into osteoblasts, which is beneficial to prevent osteoporosis and bone loss, and plays a vital role in bone remodeling. The present study found that non-invasive LIPUS treatment can alleviate osteoporosis in elderly mice, but how LIPUS works and how to initiate the process of bone remodeling in elderly mice, and its potential regulatory mechanism are not clear.

[0073] The decline in differentiation ability and growth stagnation caused by aging of BMSCs is one of the important factors leading to osteopenia in osteoporosis. The morphology and function of mesenchymal stem cells gradually enter an aging state with age, and their proliferation and differentiation abilities are thereby limited. In the present study, it was found that the expression of osteogenic-related proteins ALP, RUNX2 and OPN in aged BMSCs in vitro was significantly increased after LIPUS irradiation, and the blue-purple positive staining range of ALP staining and the number of orange-red mineralized nodules stained with alizarin red were significantly increased, indicating that LIPUS enhances the early and late osteogenic differentiation ability of aged BMSCs. LIPUS alleviates osteoporosis-related bone loss probably by promoting the osteogenic differentiation ability of aged BMSCs.

[0074] The decrease in estrogen levels and the effects of aging prompt osteoprogenitor cells and BMSCs to adopt a more inert mechanobiological phenotype, resulting in decreased mechanical sensitivity. Effective cell function requires cells to respond quickly to external signals and complete rapid remodeling of the cytoskeleton. The actin turnover rate in aged BMSCs decreases, and the responsiveness to biological and mechanical signals decreases, resulting in a decrease in the ability of bone tissue to renew and repair. Previous studies have found that LIPUS treatment can cause the rearrangement of BMSCs actin filaments and further promote their polymerization. In the present study, it was observed that after LIPUS treatment, the filament proteins of aged BMSCs showed an increase in filamentous structures and more regular arrangement. This suggests that LIPUS can induce cytoskeletal remodeling in aged BMSCs, leading to the occurrence of downstream effects.

[0075] MRTF-A belongs to the myocardin-related transcription factor family and acts as a downstream effector of the mechanosensitive pathway ROCK / RhoA pathway. As a mechanosensitive protein, the activation of MRTF-A transcriptional activity depends on changes in actin dynamics. The important process is that during the transformation of actin from a monomeric globular actin (G-actin) form to a filamentous actin (F-actin) form, MRTF-A is released from the site bound to G-actin, followed by nuclear translocation and transcriptional activation mediated by SRF after the formation of a transcription factor complex. In the present study, it was found that MRTF-A of aged BMSCs translocated to the nucleus after LIPUS treatment. When the nuclear translocation of MRTF-A was blocked by an inhibitor, the ability of LIPUS to promote osteogenic differentiation of aged BMSCs was significantly inhibited, suggesting that nuclear translocation of MRTF-A is a key regulatory point for LIPUS to promote osteogenic differentiation of aged BMSCs.

[0076] In summary, the present invention studies the ability of LIPUS to treat aging-related osteoporosis and its potential mechanism of action. The results show that LIPUS can improve osteoporosis in aged mice, and may regulate the remodeling of cell microfilaments through mechanical action, promote the nuclear entry of MRTF-A, and thus improve the osteogenic differentiation ability of aged BMSCs.

Claims

1. A low-intensity pulsed ultrasound system for improving aging-related osteoporosis, used to improve osteoporosis in patients and further assist in the treatment of osteoporosis, characterized in that: At least: A low-intensity pulsed ultrasound device having at least an ultrasound transducer, The ultrasonic transducer is placed at the osteoporotic site of the patient and is used to treat the patient by outputting low-intensity pulsed ultrasound.

2. The low-intensity pulsed ultrasound system according to claim 1, characterized in that: The osteoporotic sites include the lumbar vertebrae, femur, tibia, and radius.

3. The low-intensity pulsed ultrasound system according to claim 1, characterized in that: The low-intensity pulsed ultrasound device also has an ultrasound control unit, a parameter input unit, an ultrasound communication unit and an ultrasound display unit; the parameter input unit is used to input low-intensity pulsed ultrasound parameters, the ultrasound display unit is used to display the waveform of the output low-intensity pulsed ultrasound wave and the numerical value of the low-intensity pulsed ultrasound parameter, and the ultrasound communication unit is used to exchange data.

4. Use of low-intensity pulsed ultrasound or the low-intensity pulsed ultrasound system according to claim 1 in improving aging-related osteoporosis.

5. Use of low-intensity pulsed ultrasound or the low-intensity pulsed ultrasound system according to claim 1 in promoting osteogenic differentiation of aged bone marrow mesenchymal stem cells and / or promoting MRTF-A translocation into the nucleus of aged bone marrow mesenchymal stem cells.

6. The use according to claim 4 or 5, characterized in that: The therapeutic intensity of the low-intensity pulsed ultrasound is 100-150 mW / cm 2 .

7. The use according to claim 4 or 5, characterized in that: The treatment time of the low-intensity pulsed ultrasound is 20 to 30 minutes.

8. The use according to claim 4 or 5, characterized in that: The frequency of the low-intensity pulsed ultrasound device was set to 3 MHz with a duty cycle of 50%.

9. The use according to claim 4 or 5, characterized in that: Low-intensity pulsed ultrasound regulates the remodeling of cell microfilaments through mechanical action, promotes the nuclear entry of MRTF-A, and thus improves the osteogenic differentiation ability of aged BMSCs.

Images