Self-powered electrical stimulation system for enhancing exosome secretion of mesenchymal stem cells and application method of self-powered electrical stimulation system

Abstract

The invention discloses a self-powered electrical stimulation system for enhancing exosome secretion of mesenchymal stem cells and an application method of the self-powered electrical stimulation system, and relates to the technical field of wearable medical equipment. According to the method for increasing the yield of the exosomes of the electrical stimulation mesenchymal stem cells driven by the novel friction nano-generator, an external power supply or a circuit system is not needed, the yield of the exosomes in a cell culture bottle can be increased by about 3.2 times, and the problems of hydrolysis, bubble formation and the like caused by a traditional power supply are effectively avoided. A rectified ternary medium is used for rubbing a direct current signal generated by the nano generator to provide electric energy for cells. The self-powered electrical stimulation system for enhancing exosome secretion of the mesenchymal stem cells and the application method of the self-powered electrical stimulation system can be obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of wearable medical devices, and particularly to a self-powered electrical stimulation system for enhancing the secretion of mesenchymal stem cell exosomes and an application method thereof. Background Art

[0002] Exosomes are nanoscale membrane vesicles secreted by cells into the extracellular environment, with diameters typically in the range of 30 - 150 nm. They carry various bioactive substances such as RNA, proteins, and lipids, playing an important mediating role in cell - cell communication. As "messengers" of information transfer, exosomes can transfer these bioactive molecules from the parent cell to the target cell, affecting the function of the target cell by activating specific signaling pathways and regulating gene expression. Among them, exosomes derived from mesenchymal stem cells (MSCs) have attracted much attention due to their unique biological functions. MSCs exosomes can mimic the paracrine effect of MSCs and promote tissue repair and regeneration. MSCs exosomes play a therapeutic role through mechanisms such as regulating the immune response, inhibiting inflammation, and promoting angiogenesis, showing good curative effects in fields such as osteochondral repair, myocardial infarction, and wound healing.

[0003] However, the efficiency of obtaining exosomes by culturing mesenchymal stem cells is low, and the yield is limited in the natural state. In addition, the cell culture cycle is long, usually taking 48 - 72 hours to collect a sufficient amount of exosomes for subsequent research. To improve the yield of cell exosomes, researchers have explored various stimulation strategies. Datta et al. reported that stimulating MSCs with the cAMP analog 8 - Br - cAMP can increase the exosome yield by 4 times. In addition, some studies have found that by applying mechanical stimuli such as shear force, tensile force, and acoustic stimulation and other physical methods, the secretion level of exosomes can also be significantly increased, obtaining a 3 - 6 - fold increase in yield respectively. However, although these methods have improved the exosome yield to a certain extent, there are still some limitations. For example, chemical inducers may have an adverse impact on cell viability and the biological characteristics of exosomes; it is difficult to ensure the uniformity of mechanical stimuli, and excessive mechanical force will cause cell rupture, generating non - specific cell debris and increasing the difficulty of subsequent purification; acoustic stimulation is not suitable for equipment scale - up, as large - scale equipment will affect the sound wave transmission effect, and the preparation structure of high - quality acoustic equipment is also relatively complex.

[0004] In contrast, as a physical stimulation method, electrical stimulation has the advantages of simple operation and remarkable effects, and can effectively improve the secretion of extracellular vesicles. However, there are still some problems with traditional electrical stimulation. For example, at high current densities, electrode hydrolysis and bubble formation may occur in low-frequency signals and aqueous environments, which may cause damage to cells. In addition, traditional electrical stimulation devices are usually large in size and not convenient to use in a cell culture incubator, which affects their popularization and application. Therefore, developing a novel portable, self-powered, and low-power cell electrical stimulation method is of great significance for improving the yield and quality of exosomes. The triboelectric nanogenerator (TENG) is a new type of energy harvesting and conversion device. Its working principle is to utilize the contact electrification and electrostatic induction effects on the material surface to directly convert the mechanical energy (such as vibration, pressure, torsion, etc.) commonly present in the environment into electrical energy, thereby providing a continuous and stable power source for various electronic devices and systems. Compared with traditional power sources, TENG has multiple advantages: high output voltage, high energy conversion efficiency, simple structure, low cost, and low current density. Although TENG has shown good biocompatibility in multiple biomedical applications, so far, no research has reported its use for stimulating cells to produce exosomes. Therefore, developing a system based on TENG electric field stimulation to promote the secretion of exosomes by cells may provide a new and effective way to improve the yield of extracellular vesicles. Summary of the Invention

[0005] The purpose of the present invention is to solve the above technical problems and provide a self-powered electrical stimulation system for enhancing the secretion of mesenchymal stem cell exosomes and its application method.

[0006] A self-powered electrical stimulation system for enhancing the secretion of mesenchymal stem cell exosomes, the self-powered electrical stimulation system includes a triboelectric nanogenerator, an energy management module, electrodes, and a cell culture flask, and a cell culture medium and mesenchymal stem cells are provided in the cell culture flask.

[0007] An application method of a self-powered electrical stimulation system for enhancing the secretion of mesenchymal stem cell exosomes is carried out according to the following steps:

[0008] Start the rotor of the triboelectric nanogenerator, make wool contact with polytetrafluoroethylene and nylon film in sequence, and apply the electrical stimulation signal processed by the energy management module to the cell culture medium through the electrodes and transmit it to the mesenchymal stem cells, ultimately promoting the generation of exosomes and the cell proliferation of mesenchymal stem cells.

[0009] The beneficial effects of the present invention:

[0010] (1) The present invention proposes a method for enhancing the production of exosomes from mesenchymal stem cells driven by a novel triboelectric nanogenerator (TENG). This method does not require an external power source or circuit system, can increase the production of exosomes in cell culture flasks by about 3.2 times, and effectively avoids problems such as hydrolysis and bubble formation caused by traditional power sources. We use the direct current (DC) signal generated by a rectified ternary dielectric triboelectric nanogenerator (TD-TENG) to provide electrical energy for cells.

[0011] (2) The present invention's research details the effects of parameters such as current magnitude, electrical stimulation time, and number of treatment cycles on the increase in exosome production, and deeply analyzes the mechanism by which electrical stimulation enhances exosome production. The research finds that the mechanism for promoting the increase in exosome production is that electrical stimulation leads to an increase in intracellular calcium influx, which promotes the exosome secretion process.

[0012] The present invention can obtain a self-powered electrical stimulation system for enhancing the secretion of exosomes from mesenchymal stem cells and its application method. Brief Description of the Drawings

[0013] Figure 1 a shows the self-powered microneedle ES system for accelerating the healing of infectious wounds in the present invention, Figure 1 b shows the configuration of the SPMES system and the delivery of ES and the antibacterial effect of LevCDs; Figure 1 c shows the TEM (i) and HRTEM (ii) images of LevCDs, with a scale bar of 20 nm; Figure 1 d shows the photos of LevMN under daylight (i) and ultraviolet light (ii), with a scale bar of 500 μm in (i) and 200 μm in (ii); Figure 1 e shows the mechanism of the SPMES system for treating infected wounds in mice to treat infected wounds;

[0014] Figure 2 represents the dimensions of the stator and rotor of the rotary triboelectric nanogenerator in the present invention;

[0015] Figure 3 a shows the overall cell culture system in the present invention, Figure 3 b shows the energy management unit, Figure 3 c shows the designed carbon electrode, Figure 3 d shows the photo of the assembled cell culture flask; the scale bars in Figures a - d are all 1 cm;

[0016] Figure 4 a shows the physical diagram and corresponding model of the stator and rotor of the triboelectric nanogenerator in the present invention, Figure 4 b shows the equivalent circuit model of the SPES cell culture system, Figure 4 c shows the voltage response characteristics of the TENG,Figure 4 d represents the current response characteristics of the TENG, Figure 4 e represents the relationship between the voltage output and power of the TENG and the resistive load, Figure 4 f represents the charging curves of different capacitors at a rotational speed of 300 rpm, Figure 4 g represents the voltage output of the TENG during 400,000 working cycles, Figure 4 h represents the direct current flowing through the cell culture medium, Figure 4 i represents precisely regulating the magnitude of the current flowing through the cell culture medium by an external resistor;

[0017] Figure 5 a represents the effect of different treatment times of ES (25 μA) on exosome production, Figure 5 b represents the effect of ES treatment (1 hour) with different current intensities on exosome production, Figure 5 c represents the effect of ES treatment with different cycles (45 μA, 1 hour) on exosome production, Figure 5 d represents the intracellular calcium fluorescence intensity of mesenchymal stem cells treated by different methods and the corresponding exosome production, Figure 5 e represents the effect of ES (45 μA, 1 hour, 3 cycles / 24 hours) on the proliferation of mesenchymal stem cells, Figure 5 f represents the effect of electrical stimulation on cell proliferation;

[0018] Figure 6 a represents the TEM image of Exo, Figure 6 b represents the TEM image of ES@Exo. The scale bars in Figures a and b are 50 nm; Figure 6 c represents the scattering images of exosomes and ES@Exo, Figure 6 d represents the size distribution of exosomes and ES@Exo, Figure 6 e represents the zeta potential of exosomes and ES@Exo, Figure 6 f represents the Western Blot analysis of exosome-specific markers CD9 and TSG101; Figure 6 g represents the uptake of PKH26-labeled Exo and ES@Exo by HUVEC. The scale bar is 20 μm;

[0019] Figure 7 a represents the experimental procedure for treating wounds with ES@Exo, Figure 7 b represents the schematic diagram of wound formation and treatment. The scale bar is 2 mm; Figure 7 c represents the photos of the wound healing process on days 0, 3, 6, 9, and 12, Figure 7 d represents the relative wound area of different groups within 12 days; Figure 7e shows the tissue sections of different groups on the 12th day stained with hematoxylin-eosin and Masson's trichrome stain. The scale bars in the first row are all 200 μm, and the scale bars in the second and third rows are all 50 μm; Figure 7 f shows the quantitative analysis of the width of granulation tissue, Figure 7 g shows the quantitative analysis of the thickness of epithelial tissue. Detailed implementation manners

[0020] Detailed implementation manner one: A self-powered electrical stimulation system for enhancing the secretion of mesenchymal stem cell exosomes in this implementation manner. The self-powered electrical stimulation system includes a triboelectric nanogenerator, an energy management module, electrodes, and a cell culture flask. A cell culture medium and mesenchymal stem cells are provided in the cell culture flask.

[0021] Detailed implementation manner two: The difference between this implementation manner and the first detailed implementation manner is that the triboelectric nanogenerator includes a stator and a rotor.

[0022] Other steps are the same as those in the first detailed implementation manner.

[0023] Detailed implementation manner three: The difference between this implementation manner and the first or second detailed implementation manner is that the stator is composed of tin electrodes radially arranged on the surface of a circular printed circuit board (PCB) and dielectric materials provided in the gaps between the tin electrodes.

[0024] Other steps are the same as those in the first or second detailed implementation manner.

[0025] Detailed implementation manner four: The difference between this implementation manner and one of the first to third detailed implementation manners is that the dielectric material is polyester fiber.

[0026] Other steps are the same as those in the first to third detailed implementation manners.

[0027] Detailed implementation manner five: The difference between this implementation manner and one of the first to fourth detailed implementation manners is that the rotor is composed of a circular acrylic plate and polytetrafluoroethylene and nylon alternately arranged on the circular acrylic plate.

[0028] Other steps are the same as those in the first to fourth detailed implementation manners.

[0029] Detailed implementation manner six: The difference between this implementation manner and one of the first to fifth detailed implementation manners is that polytetrafluoroethylene and nylon films are alternately adhered to the surface of the rotor.

[0030] Other steps are the same as those in the first to fifth detailed implementation manners.

[0031] Detailed implementation manner seven: The difference between this implementation manner and one of the first to sixth detailed implementation manners is that the energy management module is composed of a rectifier bridge and a resistor. The rectifier bridge can convert alternating current into direct current, and the resistor is used to adjust the magnitude of the current.

[0032] The other steps are the same as those in the first to sixth specific embodiments.

[0033] Specific Embodiment 8: An application method of a self-powered electrical stimulation system for enhancing the secretion of mesenchymal stem cell exosomes is carried out according to the following steps:

[0034] Start the rotor of the triboelectric nanogenerator, make the wool contact with the polytetrafluoroethylene and nylon films successively, and apply the electrical stimulation signal processed by the energy management module to the cell culture medium through the electrode and transmit it to the mesenchymal stem cells, ultimately promoting the generation of exosomes and the cell proliferation of mesenchymal stem cells.

[0035] The following examples are used to verify the beneficial effects of the present invention:

[0036] Example 1:

[0037] In order to effectively transmit the electrical stimulation of the TENG to the mesenchymal stem cells, the present invention proposes a self-powered cell electrical stimulation system (see Figure 1 a), which includes a novel triboelectric nanogenerator (TENG), a power management module, carbon electrodes and a cell culture flask. The TENG component consists of a stator and a rotor (see Figure 1 b), and its detailed dimensions are shown in Figure 2 . Specifically, PTFE and nylon films are alternately adhered to the surface of the acrylic plate as the rotor, and the stator is a PCB board with tin electrodes radially arranged on the surface. Wool is distributed between the electrode gaps as a dielectric material for charge replenishment and transfer. The energy management module consists of a rectifier bridge and a resistor, and the current magnitude can be precisely controlled by replacing different resistance values.

[0038] The rectifier bridge is electrically connected to the tin electrodes of the stator through wires, and the rectifier bridge is successively connected in series with the resistor and two carbon electrodes in the cell culture flask through wires.

[0039] The working mechanism of the cell electrical stimulation operating system based on the TENG is as shown in Figure 1 c. Among the triboelectric materials, PTFE, wool and nylon successively have strong charge affinities, and among them, polyester fiber wool can quickly replenish the charges lost by the dielectric layer. When the rotor starts, the wool contacts the PTFE and nylon films successively. When the wool contacts the more electronegative PTFE, electrons flow to the PTFE, thereby enhancing its electronegativity. This mechanism keeps the charges on the surfaces of the nylon and PTFE films saturated, thus effectively improving the high-voltage output efficiency of the TENG. During the operation of the TENG, the electrical stimulation signal processed by the energy management module acts on the cell culture medium through the carbon electrodes and then is transmitted to the mesenchymal stem cells, which can effectively promote the influx of calcium ions, and further promote the generation of exosomes and cell proliferation ( Figure 1d). Therefore, this electrical stimulation significantly increases the yield of exosomes. When naturally produced exosomes and exosomes generated by electrical stimulation are used for wound healing, both can maintain high biological activity and effectively promote wound healing ( Figure 1 e).

[0040] 1. Fabrication of TENG:

[0041] The rotary friction nanogenerator consists of a rotor and a stator electrode. The stator uses a custom PCB electrode plate from JLCPCB Technology Group, and the electrode is a tin electrode. The dimensions are shown in Figure 2 . The surface of the rotor is alternately layered with positive and negative dielectric materials to optimize the power output. In addition, wool is strategically placed between the copper electrodes as a third dielectric material, which not only supplements the positive and negative dielectrics but also ensures soft contact between them, thereby enhancing the induced charges on the dielectric materials. This design maximizes the charge generation efficiency of the TENG during operation.

[0042] 2. Establishment of the electrical stimulation system and detection of exosomes: Two notches are prepared on the surface of a 75T cell culture flask for placing custom carbon electrodes (see Figure 2 ), and they are sealed with 3M sterile indwelling needle stickers. The overall cell culture system is shown in Figure 3 . The cells are seeded at a density of 1.5×10^6 cells / well, 15 mL of DMEM / F12 medium (containing 10% FBS) is added, and then it is placed in a cell culture incubator for culture. Next, electrical stimulation with different durations, intensities, and cycle numbers from the TENG is applied within 24 hours. After 48 hours, the cell culture medium is collected, centrifuged at 120,000 g for 2 hours using an ultracentrifuge (Optima XPN, Beckman, USA), and the exosomes are resuspended in 500 μL of PBS. Finally, the samples are processed according to the instructions of the exosome purification kit to obtain purified exosomes. The concentration of exosomes is detected using an acetylcholinesterase kit according to the instructions.

[0043] 3. Calcium ion concentration detection:

[0044] Group 1: As a control group, it is cultured using complete medium.

[0045] Group 2: In the complete medium, a current of 45 μA is applied, each treatment lasts for 1 hour, and three cycles are performed every 24 hours (once every 8 hours), for a total of 48 hours of treatment.

[0046] Group 3: The electrical stimulation protocol of Group 2 is adopted, but calcium-free medium is used.

[0047] Group 4: Cells were cultured in a calcium-free medium. After the cells adhered to the wall for 3 hours, they were successively treated with Amiloride HCl (100 μM) for 1 hour, Thapsigargin (100 nM) for 0.5 hour, and BAPTA-AM (10 μM) for 0.5 hour. Then, they were treated according to the electrical stimulation protocol of Group 2 for 48 hours.

[0048] Group 5: Cells were cultured in a complete medium. First, they were treated according to the CCBs treatment protocol of Group 4, and then they were treated according to the electrical stimulation protocol of Group 2 for 48 hours.

[0049] After the treatment, the cells were incubated with a 2 μM calcium fluorescent probe at 37 °C for 1 hour, and then the cells were digested with trypsin until they detached from the wall and were resuspended in 3 mL of PBS. Finally, a fluorometer (F97pro, Lengguang, Shanghai) was used to detect the intracellular calcium level.

[0050] 4. Characterization of exosomes:

[0051] To characterize the isolated exosomes, TEM images were obtained using a scanning transmission electron microscope (Tecnai G2, Thermo Fisher). The samples were negatively stained with 1 w / w% uranyl acetate. The size of the exosomes was measured using nanoparticle tracking analysis (NTA, ZetaView, Particle Metrix). The potential of the samples was measured using a Zeta potential analyzer (NanoPlus, Micromeritics, USA).

[0052] 5. Cell uptake:

[0053] HUVECs were cultured using Endothelial Cell Medium. To perform the cell uptake assay, exosomes were first stained with PKH26. HUVECs were seeded into 12-well glass plates and co-cultured with the labeled exosomes (50 μg / mL) for 6 hours. Subsequently, the cells were stained with a microfilament green fluorescent probe for 10 minutes, and the cell nuclei were stained with DAPI for 8 minutes. Finally, a laser confocal microscope (FV3000, Olympus) was used to observe the uptake of exosomes by HUVECs.

[0054] 6. Wound assay:

[0055] Animal experiments have been approved by the Ethics Committee of the Life Science Center of Harbin Institute of Technology (approval number: IACUC-2023056) and comply with animal welfare standards. Male Balb / c mice at 10 weeks of age were randomly assigned to the CTRL group, Exo group, and ES@Exo group, with 3 mice in each group. The mice were housed in a standard breeding environment, fed standard food, and maintained on a 12-hour light / 12-hour dark cycle. A full-thickness wound model was established using a punch (diameter: 6 mm), and ES@Exo (500 μg / mL, 10 μL) was dropped onto the wound. The control group was treated with 10 μL of PBS on the wound. From day 3 to day 12, the wound developed scabs. To ensure effective absorption of exosomes, a 1.2-mm microneedle stamp (S2, AMIPJI) was used to press the wound and then the exosome sample was dropped. The mice were euthanized on day 12, and their tissues were collected and fixed overnight in 4% paraformaldehyde solution. Tissue sections were stained with hematoxylin-eosin (H&E) and Masson's trichrome, and quantitative analysis was performed using Image J software.

[0056] Experimental results:

[0057] The physical pictures and corresponding models of the stator and rotor of the triboelectric nanogenerator (TENG) are shown as Figure 4 shown in Fig. a. To evaluate the adaptability of the TENG in cell electrical stimulation and before assessing the effect of self-powered on exosome production, we characterized the equivalent circuit of the system and the output performance of the TENG.

[0058] As Figure 4 shown in Fig. b, the cell culture medium, electrodes, and cells can be equivalent to a resistor (R), which was measured to be approximately 259.7 Ω by an electrochemical workstation. The open-circuit output performance of the TENG is significantly affected by the rotation speed. When the rotation speed increases from 50 rpm to 300 rpm, the voltage output increases from approximately 320 V to 1800 V ( Figure 4 Fig. c), and the current output increases from approximately 25 μA to 88 μA ( Figure 4 Fig. d). This phenomenon is mainly due to the increase in the friction cycle per unit time caused by the increase in the rotation speed, which accelerates the accumulation and transfer of charges. Figure 4 Fig. e shows the output performance of the TENG at a rotation speed of 300 rpm under different external load resistances. As the load resistance increases, the voltage output also increases; the power output first increases and then decreases, reaching a peak of approximately 27 mW at a load of 60 MΩ. Figure 4 Fig. f further shows the ability of the TENG to charge capacitors with different capacitances through a bridge rectifier circuit, highlighting its feasibility as an energy source for electronic devices. Figure 4g verified the durability and reliability of the TENG through 400,000 cycle tests. When the electrical output of the TENG was connected to the cell culture flask through the energy management module, since the internal resistance of the TENG was about 60 MΩ, which was about 230,000 times that of the equivalent resistance of the cell module, 259.7 Ω, according to the voltage division principle, although the output voltage of the TENG was as high as 1,800 V, the voltage acting on the cells was actually very small.

[0059] Therefore, this study focused on studying its effect on the secretion amount of extracellular vesicles through current. As Figure 4 shown in h, under different rotation speeds and with an external load of 0 (Rx = 0), the current flowing through the cell culture medium was basically the same as the open-circuit current of the TENG, and showed a relationship of doubling the frequency. By adjusting the external load, the current could be precisely controlled between 5 - 65 μA ( Figure 4 i).

[0060] Acetylcholinesterase (AChE) is a key enzyme that mainly terminates nerve signal transmission by rapidly hydrolyzing the neurotransmitter acetylcholine to maintain the normal function of the nervous system. Due to its high abundance and specificity in extracellular vesicles, acetylcholinesterase is widely used as a marker for extracellular vesicles. We measured the concentration of acetylcholinesterase by colorimetry to indirectly reflect the concentration of extracellular vesicles after purification.

[0061] The study found that when mesenchymal stem cells were treated with TENG electrical stimulation, the content of extracellular vesicles showed a trend of first increasing and then decreasing with the increase of treatment intensity. Specifically, when the current was fixed at 25 μA, with the increase of treatment time, the acetylcholinesterase concentration first increased and then decreased, reaching a maximum value of 2.2 nmol / min / mL at 1 hour of treatment ( Figure 5 a). To optimize the treatment parameters, we studied the effect of different current intensities on the concentration of extracellular vesicles. The results showed that when the current was 45 μA, the best yield of extracellular vesicles could be obtained ( Figure 5 b).

[0062] In addition, increasing the number of treatment cycles could further improve the yield of extracellular vesicles. Under the condition of using a current of 45 μA, treating for 1 hour each time, and performing three cycles every 24 hours, the yield of extracellular vesicles increased significantly, about 3.2 times that of the untreated group ( Figure 5 c). Subsequent experiments were carried out based on these optimized parameters. We deeply explored the mechanism of extracellular vesicle production. The study found that the increase in the yield of extracellular vesicles was closely related to the influx of intracellular calcium ions.

[0063] Therefore, we used calcium ion inhibitors and calcium-free culture medium to study their effects. The calcium channel blockers (CCBs) used included Amiloride HCl (which inhibits calcium entry into cells), BAPTA-AM (which binds and sequesters intracellular calcium), and Thapsigargin (which inhibits calcium entry into the endoplasmic reticulum). As Figure 5 shown in

[0064] d, the intracellular calcium content of the electrically stimulated samples (Group 2) was higher than that of the control group (Group 1).

[0065] However, calcium fluorescent reagents mainly detect calcium ions in the cytoplasm, and for calcium ions stored in organelles (such as mitochondria, endoplasmic reticulum, Golgi apparatus, etc.), calcium fluorescent reagents may not be able to directly detect them. Therefore, we removed calcium ions from the cell culture medium and then applied electrical stimulation, and found that the intracellular calcium content did not increase (Group 3). When the cell culture medium lacked calcium ions and CCBs were added, although electrical stimulation was applied, the intracellular calcium concentration still decreased significantly (Group 4).

[0066] In addition, when using normal culture medium and CCBs, the change in the intracellular calcium concentration was not significant. Therefore, the above results indicate that the increase in intracellular calcium mainly comes from the influx of calcium ions from the external environment, rather than the release of calcium ions from organelles. At the same time, the yield of exosomes showed the same change trend as the intracellular calcium concentration, indicating that calcium ion influx is the main reason for the increase in exosome yield. Figure 5

[0067] Figure 6 In addition, we also found that electrical stimulation promoted cell proliferation. As Figure 5 shown in d, compared with the untreated group, the number of mesenchymal stem cells in the electrically stimulated treatment group increased by about 1.4 times. Therefore, the increase in cell number also promoted the increase in exosome yield. Previous studies have shown that cell proliferation is closely related to calcium ion influx, further supporting the key role of calcium ions in exosome generation. Figure 6 To study the differences between exosomes produced by electrically stimulated mesenchymal stem cells (ES@Exo) and exosomes produced under natural conditions (Exo), we characterized the two types of exosomes in detail. Transmission electron microscopy (TEM) imaging results showed that both presented a typical round or cup-shaped morphology ( Figure 6 a and b), with a diameter of approximately 140 nanometers ( Figure 6 c and d), and there were no significant differences in morphology and size. Further analysis found that the Zeta potential of ES@Exo was higher than that of Exo ( Figure 6e). This difference may be due to the fact that electrical stimulation changes the distribution of ions on the cell membrane surface. It is known that electrical stimulation promotes the influx of calcium ions, thereby reducing the negative charges on the cell membrane surface. Considering that the release of exosomes requires the fusion of the plasma membrane, electrical stimulation may cause more positive ions to accumulate on the surface area of exosomes, and thus make their Zeta potential more positive.

[0068] In addition, we also found that the protein expression level of ES@Exo was significantly higher than that of Exo ( Figure 6 f). This may be related to the upregulation of calcium signaling, which enhances the synthesis of intracellular proteins, especially those involved in processes such as cellular stress response, extracellular matrix remodeling, and cell migration. To further evaluate the biological activity of ES@Exo, we selected human umbilical vein endothelial cells (HUVECs) as the main target cells of MSC exosomes and tested the absorption efficiency of 50 μg / mL exosomes. The results showed that at the same concentration, both ES@Exo and Exo groups of HUVECs exhibited strong fluorescence signals ( Figure 6 g), indicating that ES@Exo still has strong biological activity.

[0069] To further verify the biological activity of ES@Exo in vivo, we applied it to the field of wound healing. First, a full-thickness wound was created on the back of mice using a 6-mm puncher, and ES@Exo was dropped. Subsequently, the mice were randomly divided into three groups: CTRL, Exo, and ES@Exo. Considering that wound scabbing may hinder the absorption of exosomes, we used a microneedle stamp to treat the wound at the third day and subsequent time points to help exosomes be more efficiently absorbed through the microneedle puncture channels in the case of wound scabbing ( Figure 7 a and b). The dynamic observation results of wound healing showed that the wound healing rate of the Exo and ES@Exo groups was significantly faster than that of the control group ( Figure 7 c). Quantitative analysis showed that within 12 days, the relative wound areas of the CTRL, Exo, and ES@Exo groups were reduced to 19.2% ± 1.8%, 9.5% ± 1.4%, and 7.8% ± 1.8%, respectively ( Figure 7 d).

[0070] To further evaluate the wound healing situation, we performed histological analysis ( Figure 7e). Hematoxylin and eosin (H&E) staining results showed that the wounds in the CTRL group had a thicker newly formed epidermis and enlarged granulation tissue, with a relatively large number of blood vessels, but a relatively small number of skin appendages such as hair follicles and sebaceous glands. In contrast, the granulation tissue width and epithelial thickness in the Exo and ES@Exo groups were thinner, the number of new hair follicles increased, and the excess blood vessels were cleared, resulting in a decrease in the number of blood vessels. Masson's trichrome staining results indicated that there was more mature collagen deposition in the Exo and ES@Exo groups compared with the control group. Quantitative analysis results showed that the granulation tissue widths in the Exo and ES@Exo groups were 463.3 ± 71.7 μm and 374.6 ± 26.5 μm( Figure 7 f), and the epithelial thicknesses were 28.5 ± 4.7 μm and 32.0 ± 6.1 μm( Figure 7 g). Therefore, ES@Exo exhibits strong biological activity in vivo and can effectively promote wound healing.

Claims

1. A self-powered electrical stimulation system for enhancing exosome secretion from mesenchymal stem cells, characterized in that The self-powered electrical stimulation system comprises a friction nanogenerator, an energy management module, electrodes and a cell culture bottle, wherein the cell culture bottle is provided with a cell culture medium and mesenchymal stem cells.

2. A self-powered electrical stimulation system for enhancing exosome secretion of mesenchymal stem cells according to claim 1, characterized in that The friction nanogenerator comprises a stator and a rotor.

3. A self-powered electrical stimulation system for enhancing exosome secretion of mesenchymal stem cells according to claim 2, characterized in that The stator is composed of tin electrodes arranged radially on the surface of a circular PCB board and dielectric materials arranged in the gaps between the tin electrodes.

4. A self-powered electrical stimulation system for enhancing exosome secretion of mesenchymal stem cells according to claim 3, characterized in that The dielectric material is polyester fiber.

5. A self-powered electrical stimulation system for enhancing exosome secretion of mesenchymal stem cells according to claim 2, characterized in that The rotor is composed of a circular acrylic plate and polytetrafluoroethylene and nylon alternately arranged on the circular acrylic plate.

6. A self-powered electrical stimulation system for enhancing exosome secretion of mesenchymal stem cells according to claim 5, characterized in that PTFE and nylon films are alternately adhered to the surface of the rotor.

7. A self-powered electrical stimulation system for enhancing exosome secretion of mesenchymal stem cells according to claim 1, characterized in that The energy management module is composed of a rectifier bridge and a resistor.

8. The method for using the self-powered electrical stimulation system for enhancing the secretion of exosomes from mesenchymal stem cells according to any one of claims 1 to 7, characterized in that The application method is carried out in the following steps: Start the rotor of the friction nanogenerator, so that the wool comes into contact with the polytetrafluoroethylene and nylon films in turn, and the electrical stimulation signal processed by the energy management module acts on the cell culture medium through the electrodes and is transmitted to the mesenchymal stem cells, ultimately promoting the production of exosomes and the cell proliferation of mesenchymal stem cells.

Images