Fixing device using water as medium to load low-intensity pulsed ultrasound and application of fixing device in promoting bone marrow mesenchymal stem cell osteogenic differentiation

Abstract

The invention relates to a fixing device for loading low-intensity pulsed ultrasound by taking water as a medium and application of the fixing device in promoting osteogenic differentiation of mesenchymal stem cells. According to the device, a double-base type probe fixing structure consisting of a main body fixing base and an accessory fixing base is arranged in a main body structure; and a culture vessel fixing structure on the inner wall of the main body structure is matched, so that the problems that a probe is easy to shift, the heights of culture vessels are inconsistent and the like in the prior art are effectively solved, and a standardized and high-repeatability loading environment is provided for an LIPUS in-vitro experiment. The device is further based on the device, water is used as an ultrasonic conduction medium to be applied to osteogenic differentiation induction of bone marrow mesenchymal stem cells (BMSCs), the efficient and stable osteogenic stimulation effect can be achieved, and the core defects that in the prior art, a probe makes direct contact with a culture vessel, consequently, cells fall off and are curled, morphological damage is caused, and sound field distribution is uneven are overcome; and meanwhile, the stability and repeatability of an experimental result are remarkably improved.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and in particular to a fixation device for loading low-intensity pulsed ultrasound with water as a medium and its application in promoting osteogenic differentiation of bone marrow mesenchymal stem cells. Background Technology

[0002] Bone tissue can experience repair impairments under various conditions, including trauma, infection, tumor resection, and degenerative diseases. Its regeneration process relies on the coordinated regulation of multiple processes, such as osteoblast differentiation, matrix deposition, and bone remodeling. However, under conditions of inflammatory stimulation, microenvironmental imbalance, or insufficient physiological regulation, endogenous osteogenic activity often exhibits characteristics such as slow repair, reduced bone deposition, or decreased remodeling efficiency. To further elucidate the regulatory mechanisms of bone formation and explore effective strategies to enhance osteogenic capacity, research is increasingly emphasizing the analysis of cellular-level biological behaviors and key signaling events to establish more controllable and reproducible experimental systems.

[0003] Among various in vitro research models, bone marrow-derived mesenchymal stem cells (BMSCs) have become an ideal cell model for simulating the osteogenic process due to their osteogenic differentiation potential, high proliferative capacity, and strong responsiveness to exogenous stimuli. Their molecular and phenotypic changes can be used to evaluate the effects of physical or chemical factors on osteogenic differentiation, providing a foundation for constructing a quantitatively analyzable osteogenic research platform.

[0004] Low-intensity pulsed ultrasound (LIPUS), as a non-invasive and energy-controlled physical stimulation method, has shown potential application value in fracture healing and tissue repair. Based on this, LIPUS is increasingly being applied to in vitro experimental systems to elucidate its regulatory mechanism on the osteogenic differentiation process of bone marrow mesenchymal stem cells (BMSCs). However, existing in vitro LIPUS loading methods mostly employ direct contact between the probe and the vessel, which easily leads to cell detachment, morphological damage, and uneven sound field distribution, resulting in poor osteogenic differentiation effects and insufficient stability of BMSCs. Furthermore, the lack of standardization in loading distance, media environment, and parameter settings leads to poor reproducibility of experimental results, making it difficult to form a stable model for mechanistic research or methodological validation. Therefore, it is still necessary to construct a stable, controllable, and parameter-defined LIPUS loading method for cell culture systems to systematically evaluate its osteogenic promoting effect and provide a reproducible experimental basis for subsequent application research. Summary of the Invention

[0005] The purpose of this invention is to provide a fixation device for loading low-intensity pulsed ultrasound with water as a medium and its application in promoting osteogenic differentiation of bone marrow mesenchymal stem cells. The device effectively solves the problems of easy probe displacement, inconsistent height of culture dishes, easy cell curling and detachment in the prior art, and can be applied to osteogenic differentiation of BMSCs to achieve efficient and stable induction, and improve the stability and reproducibility of experimental results.

[0006] The objective of this invention can be achieved through the following technical solutions: One objective of this invention is to provide a fixation device for loading low-intensity pulsed ultrasound with water as a medium, comprising a main structure for containing the water medium, the main structure having an opening, a probe fixing structure being provided on the bottom inner surface of the main structure, and a culture vessel fixing structure being provided on the inner wall of the main structure, with the culture vessel fixing structure located above the probe fixing structure. The probe fixing structure includes a main body fixing base and an accessory fixing base, which are arranged opposite to each other. An accessory structure is detachably provided on the accessory fixing base. A first probe fixing cavity is opened on the side of the main body fixing base facing the accessory structure, and a second probe fixing cavity is opened on the side of the accessory structure facing the main body fixing base, which corresponds to the position and shape of the first probe fixing cavity. The fixation device is configured to fix the ultrasound probe and the culture dish. When promoting osteogenic differentiation of bone marrow mesenchymal stem cells, the vertical distance between the ultrasound probe and the bottom of the culture dish is 2-5 cm. The irradiation parameters of the ultrasound probe are set as follows: spatial average and temporal average sound intensity range of 30-60 mW / cm². 2 The frequency is 1.0-2.0MHz, the irradiation frequency is once a day, the duration of a single irradiation is 20-30 minutes, and the continuous irradiation cycle is 5-9 days.

[0007] Preferably, the accessory fixing base includes two first positioning posts spaced apart, and four second positioning posts are provided on each side of the bottom of the accessory structure. The four second positioning posts on each side are evenly distributed in two columns and two rows, and the four second positioning posts on each side enclose a positioning cavity. The positions and shapes of the two positioning cavities correspond one-to-one with the positions and shapes of the two first positioning posts.

[0008] Preferably, the first probe fixing cavity is formed along the height direction of the main body fixing base, and the shape and size of the first probe fixing cavity correspond to the shape and size of the probe.

[0009] Preferably, the second probe fixing cavity is opened along the height direction of the accessory structure, and the shape and size of the second probe fixing cavity correspond to the shape and size of the probe.

[0010] More preferably, the main structure of the main body fixing base and accessory structure is a cuboid structure.

[0011] More preferably, the accessory fixing base further includes a portal-shaped base, the opening of which faces the main fixing base, and the two first positioning posts are disposed within the opening of the portal-shaped base.

[0012] More preferably, the two first positioning posts are spaced apart along the length of the portal base.

[0013] In this invention, the main body fixing base, accessory structure and corresponding probe fixing cavity opened along the height direction can effectively prevent the probe from easily shifting or tilting, thereby making the sound field act vertically and uniformly on the cultivation area.

[0014] Preferably, the culture vessel fixing structure is a support rod structure.

[0015] Preferably, the culture vessel fixing structure includes a plurality of support rods, which are arranged in a grid pattern and vertically intersecting to form a support plane for supporting the culture vessel.

[0016] Preferably, four support rods are provided, and the four support rods are arranged in pairs perpendicularly to each other. The central support surface formed by the four support rods corresponds to the position of the probe fixing structure, and the central support surface is located directly above the probe fixing structure.

[0017] More preferably, the central support surface formed by the four support rods has a square structure.

[0018] More preferably, the culture vessel includes a standard multi-well cell culture plate or a culture dish.

[0019] More preferably, the area of ​​the central support surface needs to be large enough to stably support commonly used standard culture vessels, while also being able to achieve a positioning function, and the central support surface can serve as a positioning reference line.

[0020] In this invention, the culture vessel fixing structure possesses excellent versatility, adaptable to various sizes of culture vessels (such as culture vessels of different diameters, multi-well cell culture plates with different numbers of wells, etc.). In actual use, simply place the culture vessel stably on the fixing structure, and then finely adjust its position so that the cell culture area to be irradiated by ultrasound (such as the target well in a multi-well plate, or a specific cell growth area in a culture vessel) is located within the central support surface and precisely aligned with the ultrasound probe below. This allows for rapid and accurate positioning of the culture vessel. This design ensures that the ultrasound field acts perpendicularly on the target culture area, avoiding uneven irradiation caused by sound field tilt, further guaranteeing the stability and accuracy of the experiment.

[0021] Preferably, both ends of the support rod are connected to the inner wall of the main structure, and the height of the support rod on the inner wall is flush with the liquid level of the medium water contained inside the main structure.

[0022] More preferably, the height of the support rod is lower than the opening in the main structure to prevent the medium water from overflowing.

[0023] Preferably, the culture vessel fixing structure is detachably mounted on the inner wall of the main structure, and the inner wall is provided with a plurality of support grooves, the shape of which matches the shape of the support rod.

[0024] Preferably, the bottom outer surface of the main structure is provided with a plurality of support legs, which are evenly spaced along the bottom circumference of the main structure.

[0025] Preferably, the support legs are provided in six parts to ensure that the device is placed stably.

[0026] Preferably, the main structure is a cylindrical container.

[0027] More preferably, the main structure is made of PETG (polyethylene terephthalate), which has a much higher strength than foam box material and good chemical resistance and water resistance.

[0028] More preferably, the fixation device is used in conjunction with the Osteotron IV ultrasound therapy device.

[0029] More preferably, the probe fixing structure is used to position the ultrasonic probe of the Osteotron IV.

[0030] More preferably, the ultrasonic probe is connected to the main unit by a cable.

[0031] More preferably, the main body fixing base and accessory structure have reserved cable receiving cavities for cable passage, which can make the ultrasonic probe stable.

[0032] More preferably, the shapes of the first probe fixing cavity and the second probe fixing cavity match the contour shape of the ultrasonic probe of Osteotron Ⅳ, and can fit closely to the outer edge of the probe.

[0033] In this invention, the main structure features an open top design with six evenly distributed support legs around the bottom perimeter to ensure stable placement of the device. The interior of the main structure can hold water, serving as the ultrasonic propagation medium, providing a stable carrier for ultrasonic signal transmission. Four vertically intersecting support rods are located at the top opening of the main structure. These four rods together form a rigid support surface slightly lower than the upper edge of the main structure, used to suspend and support standard multi-well cell culture plates or culture dishes. This ensures that the bottom surface of the culture dish is precisely flush with the water level inside the main structure, preventing inconsistent heights of the culture dishes from affecting the ultrasonic loading effect.

[0034] The main structure of this invention has two bases with different functions inside, which together constitute the probe fixing structure: the main fixing base is used for the initial positioning and placement of the ultrasonic probe, and can provide basic support and centering for the probe during initial placement; the accessory fixing base is used in conjunction with the accessory structure and is located next to the main fixing base. The accessory fixing base is provided with a first positioning post, which is used to precisely fit with the positioning cavity formed by the second positioning post at the bottom of the accessory structure, thereby realizing the rapid positioning and locking of the accessory structure.

[0035] In this invention, the first probe fixing cavity on the main fixing base and the second probe fixing cavity on the accessory structure enable the probe fixing structure to quickly and uniformly press and fix the ultrasonic probe, forming a rigid constraint and effectively reducing the drift or tilting of the probe during operation. Simultaneously, the cooperation between the first positioning post and the positioning cavity ensures that the accessory structure will not loosen due to vibration or external force after pressing the probe, firmly locking the ultrasonic probe in the preset position; it also retains a suitable adjustment margin, overcoming the limitations of traditional drilling fixing methods. If drilling is used for fixing, the accessory can only be fixed in a single position, and after long-term use, wear can easily lead to loosening due to insufficient fit.

[0036] In this invention, a dual-base design (main body fixing base and accessory fixing base) and a support rod culture dish fixing structure design (the support surface formed by the support rod corresponds to the probe fixing structure formed by the dual base) are used to jointly form a spatial coordinate system that can simultaneously and accurately position the ultrasound probe and the culture dish. This is used to stably and repeatedly lock the spatial relationship between the probe and the cell culture dish, thereby improving the reliability and stability of LIPUS in vitro experiments.

[0037] This invention is simple and quick to operate, requiring no complex debugging to position the probe and culture vessel, significantly improving experimental efficiency, repeatability, and reliability. The device is robust and durable, reducing long-term experimental costs. It also has good versatility and expandability; a single device can meet the needs of most LIPUS in vitro experiments. There is no need to manufacture multiple fixing devices for different vessels. Simply place the required irradiation area of ​​different cell plates in the center of the square area enclosed by the support rod to achieve precise irradiation, improving equipment utilization and return on investment.

[0038] The second objective of this invention is to provide an application of the aforementioned fixation device in promoting osteogenic differentiation of bone marrow mesenchymal stem cells.

[0039] Preferably, the application includes the following steps: S1. The ultrasonic probe is fixed by the probe fixing structure so that the radiation surface of the ultrasonic probe is in a horizontal state. S2. Inject water into the main structure so that the water level is flush with the supporting plane of the culture vessel fixing structure. S3. Place the culture dish inoculated with bone marrow mesenchymal stem cells on the support plane of the culture dish fixing structure, so that the water surface submerges the bottom of the culture dish, and align the target irradiation area with the sound field area directly above the ultrasound probe. S4. Remove air bubbles from the surface of the ultrasonic probe and the bottom of the culture dish to ensure the integrity of the sound wave propagation path; S5. Connect the ultrasound probe to the low-intensity pulse ultrasound instrument host, set the irradiation parameters, and start the ultrasound host to irradiate the bone marrow mesenchymal stem cells in the culture dish with ultrasound.

[0040] Preferably, in step S1, the specific steps for fixing the ultrasonic probe are as follows: the ultrasonic probe is placed stably in the first probe fixing cavity of the main body fixing base to complete the initial centering and positioning; then, the accessory structure is taken out, and the two positioning cavities at its bottom are precisely aligned with the two first positioning posts on the accessory fixing base, and slowly inserted to achieve fitting, thereby fixing the ultrasonic probe.

[0041] Preferably, in step S2, the medium water is sterilized purified water or deionized water (dd water).

[0042] More preferably, in step S3, the liquid level of the medium water is level with the height of the supporting plane of the culture vessel fixing structure, while the medium water submerges the bottom of the culture vessel.

[0043] More preferably, in step S3, the vertical distance between the ultrasonic probe and the bottom of the culture vessel is 3 cm.

[0044] More preferably, in step S3, the 3cm is the vertical distance between the ultrasound probe's radiating surface and the cell culture surface.

[0045] More preferably, in step S3, the culture vessel refers to a standard multi-well culture plate.

[0046] More preferably, in step S5, the irradiation parameters are: spatially averaged time-averaged sound intensity of 45 mW / cm². 2 The frequency is 1.5MHz, the irradiation frequency is once a day, the duration of a single irradiation is 20 minutes, and the continuous irradiation cycle is 7 days.

[0047] More preferably, the application specifically includes the following steps: (1) Device preparation and probe positioning: Securely install the ultrasound probe on the probe fixing structure of the fixing device, precisely adjust it so that its radiation surface is horizontal, and ensure that there is a fixed distance of 3 cm between the probe radiation surface and the cell culture surface.

[0048] (2) Medium injection: Inject pure water or deionized water into the main structure up to the upper edge of the support rod.

[0049] (3) Cell sample positioning: Place the culture plate inoculated with BMSCs on the support rod of the device, so that the water surface submerges the bottom of the plate, and finely adjust its position so that the target well is precisely aligned with the sound field area directly above the probe.

[0050] (4) Bubble control: Thoroughly remove air bubbles from the probe surface and the bottom of the culture plate to ensure the integrity of the sound wave propagation path.

[0051] (5) Ultrasonic processing: Connect the LIPUS host (low-intensity pulse ultrasound instrument host) and set the output parameters as follows: spatial average and time average sound intensity are 30-60 mW / cm. 2 The frequency was 1.5 MHz. Then the ultrasound machine was turned on to irradiate the cells once a day for 20 minutes each time, for 7 consecutive days.

[0052] In summary, this invention addresses the core shortcomings of existing in vitro low-intensity pulsed ultrasound (LIPUS) loading techniques, including loading instability, unclear acoustic intensity parameters, poor repeatability of experimental results, and the tendency for direct contact between the probe and culture vessel to lead to cell detachment, morphological damage, and uneven sound field distribution. It proposes a water-based fixation device for loading low-intensity pulsed ultrasound and its application in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). This invention uses water as the ultrasound transmission medium, combined with a dedicated fixation device that provides probe positioning and fixation, to achieve stable and controllable transmission of ultrasound energy in the cell culture system. Furthermore, through systematic in vitro experiments, the effective acoustic intensity range (30-60 mW / cm²) for promoting osteogenic differentiation of BMSCs has been optimized and clarified. 2 ) and the optimal spatial average time average sound intensity parameter (45mW / cm) 2 This research aims to achieve efficient and stable induction of osteogenic differentiation of bone mesenchymal stem cells (BMSCs), construct a standardized in vitro model for studying the osteogenic phenotypes and molecular regulatory mechanisms of BMSCs by LIPUS, provide a unified experimental platform for comparing the effects of different experimental conditions, materials, or stimulation strategies, and provide reliable methodological support for the analysis of the biological mechanisms of LIPUS-regulated osteogenic differentiation, parameter optimization, and basic research and application development in the field of bone regeneration.

[0053] The application (method) provided by this invention does not directly target living human bodies and is not for therapeutic purposes.

[0054] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention provides a fixation device for low-intensity pulsed ultrasound using water as a medium and its application in promoting osteogenic differentiation of bone marrow mesenchymal stem cells. The device effectively avoids the problems of easy displacement of ultrasound probe and inconsistent placement height of culture vessels in the prior art by setting a double-base probe fixation structure consisting of a main fixation base and an accessory fixation base in the main structure, and combining it with the culture vessel fixation structure on the inner wall of the main structure. Based on this device, the application of water as an ultrasound transmission medium to induce osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) can achieve efficient and stable osteogenic stimulation effect, and significantly improve the stability and reproducibility of experimental results.

[0055] (2) In the fixing device provided by the present invention, the cooperation between the accessory fixing base and the main fixing base can quickly and evenly press and fix the ultrasonic probe, prevent the probe from drifting or tilting during operation, and stabilize the ultrasonic emission direction; at the same time, the positioning cavity formed by the four second positioning posts at the bottom of the accessory structure and the cooperation of the first positioning post can give the accessory structure a small rotation adjustment space, which can not only meet the assembly requirements of the probe, but also compensate for the size error through micro-adjustment, avoid the fixing effect due to poor fit, and achieve stable clamping of the probe.

[0056] (3) In the application of the fixation device provided by the present invention to promote osteogenic differentiation of bone marrow mesenchymal stem cells, water is used as the ultrasonic transmission medium, which completely avoids direct contact between the ultrasonic probe and the culture vessel, solves the problem of adherent cell detachment, edge curling and morphological damage caused by direct transmission of mechanical vibration, and can maintain the stability of cell growth state, providing a good basic condition for long-term osteogenic induction experiments.

[0057] (4) In this invention, the culture vessel fixing structure adopts a detachable support rod design, which is assembled on the inner wall of the main structure. By placing the support rod in the support grooves at different heights, the immersion depth of the culture vessel can be adjusted. Combined with the dual-base probe fixing structure for precise fixing and positioning of the ultrasonic probe, the vertical distance between the ultrasonic probe and the bottom of the culture vessel can be stably controlled (the optimal value is 3cm). When in use, keeping the support plane of the support rod flush with the liquid surface of the medium water in the main structure can ensure that the bottom of the culture vessel is always parallel to the liquid surface and at a uniform height. This design allows standard culture vessels or multi-well plates of different specifications to be suspended and fixed at a uniform height, realizing the standardization of culture vessel placement, greatly improving the uniformity of the sound field distribution, effectively reducing the data deviation caused by differences in experimental conditions, and significantly improving the standardization of experimental operations and the repeatability of results.

[0058] (5) This invention is the first to determine the effective acoustic intensity range for promoting osteogenic differentiation of BMSCs under aqueous media conditions through systematic in vitro experiments, and clarifies that the spatially and temporally averaged acoustic intensity is 45 mW / cm². 2 The biological effect is most significant at this time. This parameter can achieve stable and efficient osteogenic stimulation without completely avoiding cell damage, providing a standardized parameter standard that can be directly referenced for subsequent experiments and solving the problem of ambiguous parameters in existing technologies.

[0059] (6) Under the synergistic effect of stable loading system and optimized parameters, the present invention combines LIPUS with osteogenic induction culture system, which can significantly improve key indicators such as cell activity, expression of osteogenic genes (BMP2, RUNX2, COLIα1, OCN, OPN, ALP, OSX, etc.) and alkaline phosphatase activity, mineralized nodule formation, and osteogenic promotion effect is better than single chemical induction method.

[0060] (7) The standardized LIPUS treatment scheme established in this invention provides an in vitro osteogenic differentiation model with clear phenotype and consistent response, providing a unified experimental platform for the study of osteogenic-related mechanisms and the comparison of the effects of different stimulation strategies, reducing the research threshold and improving the comparability of research results.

[0061] (8) Based on the high osteogenic potential BMSCs prepared by the present invention, they can be used as core active ingredients to develop cell therapy products or cell-scaffold composite biomaterials related to bone defect repair. This provides a new technical path to solve the key bottleneck problem of high-quality seed cell source in bone regeneration medicine and lays a reliable foundation for subsequent process scale-up and clinical translation. Attached Figure Description

[0062] Figure 1 This is a schematic diagram of the fixing device of the present invention; Figure 2 This is a schematic diagram of the main structure of the fixing device of the present invention; Figure 3 This is a schematic diagram of the component structure and the component fixing base of the fixing device of the present invention after disassembly; Figure 4 This is a schematic diagram of the fixing device of the present invention, with the fixing structure of the culture vessel hidden. Figures 5-6 This is a schematic diagram of the component structure of the fixing device of the present invention; In the diagram, 1-main structure; 2-probe fixing structure; 21-main body fixing base; 22-accessory fixing base; 221-first positioning post; 23-accessory structure; 231-second positioning post; 3-culture vessel fixing structure; 31-support rod; 4-support leg; Figure 7 This is a diagram showing the results of alkaline phosphatase staining using the BCIP / NBT alkaline phosphatase staining kit after the fixation device of this invention was used to promote osteogenic differentiation of bone marrow mesenchymal stem cells (in the diagram, A represents 0 mW / cm²). 2 B is 30 mW / cm 2 C is 45 mW / cm 2 D is 60 mW / cm 2 ); Figure 8This is a diagram showing the results of Alizarin Red staining after the fixation device of the present invention was used to promote osteogenic differentiation of bone marrow mesenchymal stem cells (in the diagram, A represents 0 mW / cm). 2 B is 30 mW / cm 2 C is 45 mW / cm 2 D is 60 mW / cm 2 ); Figure 9 This is a graph showing the expression levels of osteogenic-related genes BMP2, RUNX2, COLIα1, OCN, OPN, ALP, and OSX after the fixation device of the present invention is used to promote osteogenic differentiation of bone marrow mesenchymal stem cells (in the graph, A is BMP2, B is RUNX2, C is COLIα1, D is OCN, E is OPN, F is ALP, and G is OSX). Figure 10 Microscopic comparison of bone marrow mesenchymal stem cells under direct coupling irradiation and irradiation with an aqueous medium using the fixation device of the present invention (A: direct coupling irradiation; B: aqueous medium irradiation). Detailed Implementation

[0063] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0064] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0065] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0066] Unless otherwise specified, the functional components or structures in the following embodiments or examples are conventional components or structures used in the art to achieve the corresponding functions.

[0067] Unless otherwise specified, the reagents, methods, instruments, and equipment used in this invention are conventional in the art. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0068] Example 1 A fixation device that uses water as a medium to apply low-intensity pulsed ultrasound, such as Figure 1 , Figure 3 As shown, the specific structure is as follows: The fixing device includes a main structure 1 for containing water as the ultrasonic propagation medium. The top of the main structure 1 has an opening to facilitate the injection of the medium water and the placement and removal of the ultrasonic probe and the culture vessel. On the bottom inner surface of the main structure 1, a probe fixing structure 2 for fixing the ultrasonic probe is assembled. At the same time, on the inner wall of the main structure 1, a culture vessel fixing structure 3 for supporting the culture vessel or porous culture plate is assembled, and the culture vessel fixing structure 3 is located above the probe fixing structure 2 in spatial position.

[0069] In this embodiment, the probe fixing structure 2 consists of a main fixing base 21 and an accessory fixing base 22. The main fixing base 21 and the accessory fixing base 22 are arranged opposite to each other on the bottom inner surface of the main structure 1. An accessory structure 23 is detachably installed on the accessory fixing base 22. The ultrasonic probe is clamped and fixed by the cooperation between the accessory structure 23 and the main fixing base 21. To adapt to the shape of the ultrasonic probe, a first probe fixing cavity is provided on the side of the main fixing base 21 facing the accessory structure 23. Correspondingly, a second probe fixing cavity is provided on the side of the accessory structure 23 facing the main fixing base 21. The second probe fixing cavity and the first probe fixing cavity are aligned with each other and matched in shape, forming a complete fixing space for embedding the ultrasonic probe.

[0070] In this embodiment, the culture vessel fixing structure 3 adopts a support rod structure to form a support plane for suspending and supporting the culture vessel.

[0071] In this embodiment, the fixation device is configured to fix the ultrasound probe and the culture dish. When promoting osteogenic differentiation of bone marrow mesenchymal stem cells, the vertical distance between the ultrasound probe and the bottom of the culture dish is 2-5 cm. The irradiation parameters of the ultrasound probe are set as follows: spatial average and temporal average sound intensity range of 30-60 mW / cm². 2 The frequency is 1.0-2.0MHz, the irradiation frequency is once a day, the duration of a single irradiation is 20-30 minutes, and the continuous irradiation cycle is 5-9 days.

[0072] Example 2 A fixation device that uses water as a medium to apply low-intensity pulsed ultrasound, in this embodiment, such as... Figures 3-6 As shown, the accessory fixing base 22 of the probe fixing structure 2 is provided with two spaced-apart first positioning posts 221; correspondingly, on both sides of the bottom of the accessory structure 23, there are four second positioning posts 231 on each side. The four second positioning posts 231 on each side are evenly spaced in a two-column, two-row arrangement, and the four second positioning posts 231 on each side together form a positioning cavity. The two positioning cavities correspond one-to-one with the two first positioning posts 221 in position and are adapted to each other in shape. During assembly, the fitting of the positioning posts and the positioning cavities achieves precise docking between the accessory structure 23 and the accessory fixing base 22, while ensuring the stability of the connection.

[0073] In this embodiment, the first probe fixing cavity on the main body fixing base 21 is opened along the height direction of the main body fixing base 21. The shape and inner size of the first probe fixing cavity correspond completely with the outer shape and outer size of the ultrasonic probe to be fixed, ensuring that the probe can be initially fitted and fixed after being embedded. The second probe fixing cavity on the accessory structure 23 is also opened along the height direction of the accessory structure 23. Its shape and inner size match the outer shape and outer size of the ultrasonic probe. When the accessory structure 23 is connected to the accessory fixing base 22, the second probe fixing cavity and the first probe fixing cavity together wrap the probe, achieving tight fixation of the probe.

[0074] In this embodiment, as Figure 2 As shown, a plurality of support legs 4 are provided on the bottom outer surface of the main structure 1. The support legs 4 are evenly spaced along the circumferential direction of the bottom of the main structure 1 to ensure the stability of the main structure 1. In this embodiment, the number of support legs 4 is specifically set to six, which can effectively prevent the device from tilting or shaking during the experiment.

[0075] In this embodiment, the main structure 1 adopts a cylindrical container structure with an opening to facilitate the injection and replacement of the medium water.

[0076] In this embodiment, the main structure 1 is made of PETG (polyethylene terephthalate) and is integrally molded.

[0077] In this embodiment, the culture vessel fixing structure 3 includes several support rods 31, which are arranged in a mesh-like vertical cross pattern.

[0078] Example 3 A fixation device that uses water as a medium to apply low-intensity pulsed ultrasound. In this embodiment, the culture vessel fixing structure 3 is a support rod structure, specifically comprising four support rods 31 arranged in pairs perpendicularly to form a square central support surface. The area of ​​this central support surface is adapted to the bottom area of ​​commonly used standard culture vessels such as well-dipped culture plates and petri dishes, ensuring stable support for the culture vessel. Furthermore, the central support surface corresponds spatially to the probe fixing structure 2, ensuring that the ultrasonic signal emitted by the probe can act perpendicularly on the sample inside the culture vessel.

[0079] Meanwhile, both ends of the four support rods 31 are connected to the inner wall of the main structure 1, and the installation height of the support rods 31 on the inner wall is precisely designed to be flush with the liquid level of the medium water contained inside the main structure 1, so that after the culture vessel is placed, its bottom can be just submerged in the medium water, ensuring the effective transmission of ultrasonic signals.

[0080] Example 4 A fixing device for loading low-intensity pulsed ultrasound using water as a medium is provided in this embodiment. To improve the flexibility of use, the culture vessel fixing structure 3 is detachably connected to the inner wall of the main structure 1. Specifically, several support grooves are provided on the inner wall of the main structure 1. The cross-sectional shape of the support grooves matches the cross-sectional shape of the support rods 31. During assembly, the two ends of each support rod 31 of the culture vessel fixing structure 3 are inserted into the corresponding support grooves to complete the fixing. During disassembly, it can be directly removed.

[0081] Example 5 The application of a water-based fixation device for loading low-intensity pulsed ultrasound in promoting osteogenic differentiation of bone marrow mesenchymal stem cells includes the following steps: (1) Fix the ultrasonic probe by probe fixing structure 2 so that the radiation surface of the ultrasonic probe is in a horizontal state; (2) Inject medium water into the main structure 1 so that the liquid level of the medium water is flush with the supporting plane of the culture vessel fixing structure 3; (3) Place the culture dish inoculated with bone marrow mesenchymal stem cells on the support plane of the culture dish fixing structure 3, so that the liquid surface of the medium water is submerged in the bottom of the culture dish, and align the target culture area with the sound field area directly above the ultrasound probe. (4) Remove air bubbles from the surface of the ultrasonic probe and the bottom of the culture dish; (5) Connect the ultrasound probe to the low-intensity pulse ultrasound instrument host, set the irradiation parameters and start the ultrasound host to irradiate the bone marrow mesenchymal stem cells in the culture dish.

[0082] In this embodiment, the vertical distance between the ultrasonic probe and the bottom of the culture dish is 2-5 cm; the irradiation parameters are: spatially averaged and temporally averaged sound intensity range of 30-60 mW / cm². 2The frequency is 1.0-2.0MHz, the irradiation frequency is once a day, the duration of a single irradiation is 20-30 minutes, and the continuous irradiation cycle is 5-9 days.

[0083] In this embodiment, the medium water is sterilized purified water or deionized water (dd water).

[0084] Example 6 The application of a water-based fixation device for loading low-intensity pulsed ultrasound in promoting osteogenic differentiation of bone marrow mesenchymal stem cells is described below: (1) Ultrasonic probe fixing operation: First, place the ultrasonic probe stably in the first probe fixing cavity of the main fixing base 21 to complete the initial centering and positioning; then, take out the accessory structure 23 and accurately align the two positioning cavities at its bottom with the two first positioning posts 221 on the accessory fixing base 22, and slowly insert it to achieve fitting. At this time, the second probe fixing cavity of the accessory structure 23 will fit tightly against the outer wall of the probe from the side, and the clamping force generated by the fitting of the structure will firmly lock the probe in the preset position, effectively avoiding problems such as probe displacement or tilting during subsequent operations or experiments, and ensuring the stability of the ultrasonic emission direction.

[0085] (2) Water medium injection operation: Slowly inject sterilized pure water or deionized water (dd water) into the main structure 1. Control the water flow rate during the injection process to prevent the water flow from impacting and causing the probe to shift or generating a large number of air bubbles. The water volume needs to be precisely controlled: the water medium level should just cover the support plane formed by the top support rod 31 and be lower than the upper edge of the container. This water level height can ensure that the bottom of the subsequent placed culture dish is in full contact with the water medium, and can also provide a stable and uniform propagation path for the ultrasonic signal, avoiding overflow due to excessively high liquid level or incomplete propagation path due to excessively low liquid level.

[0086] (3) Placement of culture vessels: Place the standard multi-well culture plate pre-seeded with bone marrow mesenchymal stem cells stably on the culture vessel fixing structure 3. Place the vessel gently to ensure it is level and stable, avoiding any impact that could cause the water medium to shake or generate air bubbles. After placement, carefully check to ensure there are no air bubbles remaining between the bottom of the culture vessel and the surface of the water medium, to avoid interference and attenuation of ultrasonic energy propagation, which could affect the accuracy of experimental data.

[0087] (4) Positioning and Experiment Start-up: Finely adjust the position of the culture vessel on the culture vessel fixing structure 3 so that the specific area in the culture vessel that needs to be irradiated by ultrasound (such as the target well position of the multi-well plate or the specific cell seeding area of ​​the culture vessel) is located in the central support surface and precisely aligned with the underwater ultrasound probe directly above it, ensuring that the area is within the core range of the sound field output, and ensuring the targeting and effectiveness of ultrasound loading. (5) After the alignment is completed, connect the ultrasound probe to the main unit of the low-intensity pulsed ultrasound (LIPUS) instrument. Set the predetermined parameters on the main unit according to the experimental plan. After the parameters are set correctly, start the instrument to start the ultrasound loading experiment and irradiate the bone marrow mesenchymal stem cells in the culture dish with ultrasound.

[0088] In this embodiment, the vertical distance between the ultrasonic probe and the bottom of the culture dish is 2-5 cm; the irradiation parameters are: spatially averaged and temporally averaged sound intensity range of 30-60 mW / cm². 2 The frequency is 1.0-2.0MHz, the irradiation frequency is once a day, the duration of a single irradiation is 20-30 minutes, and the continuous irradiation cycle is 5-9 days.

[0089] In this embodiment, the medium water is sterilized purified water or deionized water (dd water).

[0090] Example 7 An application of a water-based low-intensity pulsed ultrasound fixation device in promoting osteogenic differentiation of bone marrow mesenchymal stem cells, based on Example 6, can be used in conjunction with the Osteotron IV ultrasound therapy device. The specific steps for use are as follows: BMSCs were seeded into cell culture plates and cultured in αMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. Once the cells reached approximately 60% confluence, the medium was replaced with osteogenic induction medium for further culture. After the medium change, LIPUS treatment was initiated. During LIPUS loading, the probe was mounted and horizontally fixed to the probe mounting structure 2, adjusting it to maintain a vertical distance of approximately 3 cm between the probe's irradiation surface and the cell culture surface. Pure water was added to the main structure 1 to the set water level, and air bubbles in the acoustic path were removed. The cell-containing culture plate was placed on the support device, ensuring the target wells were located within the probe's irradiation area.

[0091] Based on different LiPUS intensities, they are divided into 4 groups: 0 mW / cm 2 Group (orifice plate placed on the mounting device but LIPUS not powered on); 30 mW / cm 2 45 mW / cm 2 60 mW / cm 2 The experimental groups had ultrasonic spatial and temporal average intensity parameters set to 0, 30, 45, and 60 mW / cm, respectively. 2 The frequency was 1.5 MHz, and the irradiation was 20 minutes per day for 7 consecutive days.

[0092] 1) Alkaline phosphatase staining Seven days after LIPUS treatment and seven days after osteogenic induction, alkaline phosphatase staining was performed using the BCIP / NBT alkaline phosphatase staining kit (Beyotime, China) (results are shown in Figure 1). Figure 7 As shown in the figure, A is 0 mW / cm². 2 B is 30 mW / cm 2 C is 45 mW / cm 2 D is 60 mW / cm 2 ).

[0093] The result showed 30 mW / cm 2 45 mW / cm 2 60 mW / cm 2 All experimental groups showed improved ALP expression in BMSCs, thus promoting osteogenic differentiation of BMSCs. Furthermore, the expression level was 45 mW / cm². 2 The group with the deepest ALP staining indicates the most significant effect in promoting osteogenic differentiation of BMSCs.

[0094] 2) Alizarin Red staining After 7 days of LIPUS treatment and 21 days of osteogenic induction, 2 mL of 4% (v / v) neutral formaldehyde solution (Biosharp, China) was used for fixation at room temperature for 30 minutes. Alizarin Red staining solution was then added for staining. The staining effect was observed under a microscope and images were acquired (results are shown in the figure). Figure 8 As shown in the figure, A is 0 mW / cm². 2 B is 30 mW / cm 2 C is 45 mW / cm 2 D is 60mW / cm 2 ).

[0095] The result showed 30 mW / cm 2 45 mW / cm 2 60 mW / cm 2 The experimental group formed more than 0 mW / cm 2 The control group showed that LIPUS can promote osteogenic differentiation of BMSCs. And 45 mW / cm 2 and 60 mW / cm 2 The group with the most mineralized nodules indicates that it has the most significant effect on promoting osteogenic differentiation of BMSCs.

[0096] 3) Quantitative Real-Time-Polymerase Chain Reaction (qRT-PCR) Replace the osteogenic induction medium with fresh medium every 3 days, 0 mW / cm². 2Group, 30 mW / cm 2 45 mW / cm 2 60mW / cm 2 Total RNA was extracted from cells 7 days after osteogenic induction using Trizol reagent (Takara, Japan); it was then reverse transcribed into cDNA using the PrimeScrip™ RT kit (Takara, Japan). Using GAPDH as an internal control, quantitative polymerase chain reaction (PCR) was performed using SYBR reagent (YEASEN, China); mRNA expression of osteogenic-related genes was detected using 2... -△△Ct Quantitative analysis was performed using this method (results as follows) Figure 9 As shown in the figure, A is BMP2, B is RUNX2, C is COLIα1, D is OCN, E is OPN, F is ALP, and G is OSX.

[0097] pass Figure 9 It was found that LIPUS significantly upregulated the expression levels of osteogenic-related genes BMP2 (p < 0.0001), RUNX2 (p < 0.0001), COLIα1 (p < 0.0001), OCN (p < 0.0001), OPN (p < 0.01), ALP (p < 0.01), and OSX (p < 0.001) compared to the control group, and the expression levels at 45 mW / cm² were significantly higher. 2 The group with the highest expression of osteogenic-related genes had the most significant effect on promoting osteogenic differentiation of BMSCs.

[0098] 4) Morphological observation of BMSCs Seven days after LIPUS treatment, BMSCs in the irradiated area of ​​the culture plate were observed under an optical microscope. It was found that BMSCs directly irradiated by LIPUS through a coupling agent exhibited large-area curling and detachment, with senescent and shrunken cell morphology. However, when LIPUS was irradiated using the fixation device of this invention with water as the medium, the BMSCs showed normal morphology and no curling or detachment. (Results are as follows) Figure 10 As shown in the figure, A represents direct coupled irradiation; B represents water-medium irradiation.

[0099] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A fixation device for applying low-intensity pulsed ultrasound using water as a medium, characterized in that, It includes a main structure (1) for containing water medium, the main structure (1) has an opening, a probe fixing structure (2) is provided on the bottom inner surface of the main structure (1), a culture vessel fixing structure (3) is provided on the inner wall of the main structure (1), and the culture vessel fixing structure (3) is located above the probe fixing structure (2). The probe fixing structure (2) includes a main body fixing base (21) and an accessory fixing base (22). The main body fixing base (21) and the accessory fixing base (22) are arranged opposite to each other. An accessory structure (23) is detachably provided on the accessory fixing base (22). A first probe fixing cavity is opened on the side of the main body fixing base (21) facing the accessory structure (23). A second probe fixing cavity is opened on the side of the accessory structure (23) facing the main body fixing base (21), which corresponds to the position and shape of the first probe fixing cavity. The fixation device is configured to fix the ultrasound probe and the culture dish. When promoting osteogenic differentiation of bone marrow mesenchymal stem cells, the vertical distance between the ultrasound probe and the bottom of the culture dish is 2-5 cm. The irradiation parameters of the ultrasound probe are set as follows: spatial average and temporal average sound intensity range of 30-60 mW / cm². 2 The frequency is 1.0-2.0MHz, the irradiation frequency is once a day, the duration of a single irradiation is 20-30 minutes, and the continuous irradiation cycle is 5-9 days.

2. The fixation device for applying low-intensity pulsed ultrasound using water as a medium according to claim 1, characterized in that, The accessory fixing base (22) includes two first positioning posts (221) spaced apart. The accessory structure (23) has four second positioning posts (231) on each side of its bottom. The four second positioning posts (231) on each side are evenly spaced in two columns and two rows. The four second positioning posts (231) on each side enclose a positioning cavity. The positions and shapes of the two positioning cavities correspond one-to-one with the positions and shapes of the two first positioning posts (221).

3. The fixation device for applying low-intensity pulsed ultrasound using water as a medium according to claim 1, characterized in that, The first probe fixing cavity is opened along the height direction of the main body fixing base (21), and the shape and size of the first probe fixing cavity correspond to the shape and size of the probe; the second probe fixing cavity is opened along the height direction of the accessory structure (23), and the shape and size of the second probe fixing cavity correspond to the shape and size of the probe.

4. The fixation device for applying low-intensity pulsed ultrasound using water as a medium according to claim 1, characterized in that, The culture vessel fixing structure (3) includes several support rods (31), which are arranged in a grid pattern and vertically intersecting to form a support plane for supporting the culture vessel.

5. The fixation device for applying low-intensity pulsed ultrasound using water as a medium according to claim 4, characterized in that, The support rod (31) is provided in four parts, and the four support rods (31) are arranged in pairs perpendicularly. The central support surface formed by the four support rods (31) corresponds to the position of the probe fixing structure (2). The central support surface is located directly above the probe fixing structure (2). Both ends of the support rod (31) are connected to the inner wall of the main structure (1), and the height of the support rod (31) on the inner wall is flush with the liquid level of the medium water contained inside the main structure (1).

6. The fixation device for applying low-intensity pulsed ultrasound using water as a medium according to claim 4, characterized in that, The culture vessel fixing structure (3) is detachably mounted on the inner wall of the main structure (1). The inner wall is provided with several support grooves, the shape of which matches the shape of the support rod (31).

7. The application of a fixation device for loading low-intensity pulsed ultrasound with water as a medium, as described in any one of claims 1-6, in promoting osteogenic differentiation of bone marrow mesenchymal stem cells.

8. The application according to claim 7, characterized in that, Includes the following steps: S1. Fix the ultrasonic probe using the probe fixing structure (2) so that the radiation surface of the ultrasonic probe is in a horizontal state. S2. Inject medium water into the main structure (1) so that the liquid level of the medium water is flush with the supporting plane of the culture vessel fixing structure (3); S3. Place the culture dish inoculated with bone marrow mesenchymal stem cells on the support plane of the culture dish fixing structure (3), so that the liquid surface of the medium water is submerged in the bottom of the culture dish, and the target irradiation area is aligned with the sound field area directly above the ultrasound probe. S4. Remove air bubbles from the surface of the ultrasonic probe and the bottom of the culture dish; S5. Connect the ultrasound probe to the low-intensity pulse ultrasound instrument host, set the irradiation parameters, and start the ultrasound host to irradiate the bone marrow mesenchymal stem cells in the culture dish with ultrasound.

9. The application according to claim 8, characterized in that, The vertical distance between the ultrasonic probe and the bottom of the culture dish is 3 cm; the irradiation parameters are: spatial average and temporal average sound intensity of 45 mW / cm². 2 The frequency is 1.5MHz, the irradiation frequency is once a day, the duration of a single irradiation is 20 minutes, and the continuous irradiation cycle is 7 days.

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