Exosome delivery system and application thereof in treatment of temporomandibular arthritis

Abstract

The invention relates to an exosome delivery system and application of the exosome delivery system in treatment of temporomandibular arthritis, the exosome delivery system comprises an injectable self-repairing positively charged carrier and an exosome mixture, the positively charged carrier comprises hydrogel with gelatin as a substrate, and the hydrogel with gelatin as the substrate is a hydrogel with gelatin as the substrate. The delivery system is prepared by sufficiently mixing an exosome with a positively charged carrier, and the exosome is prepared by a specific method. The exosome delivery system provided by the invention has the characteristic of long-term stable slow release of the exosome, and can continuously release the exosome in a local microenvironment, effectively maintain high concentration and improve the targeted delivery efficiency, so that the cell uptake rate and the biological activity are enhanced, the treatment action time is prolonged, and the treatment effect is further optimized.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to an exosome delivery system and its application in the treatment of temporomandibular arthritis. Background Art

[0002] The temporomandibular joint, also known as the temporomandibular joint or craniomandibular joint, is one of the most complex joints in the body in terms of both anatomical morphology and physiological function. Temporomandibular joint osteoarthritis (TMD) is a group of diseases affecting the temporomandibular joint, masticatory muscles, and related structures. It is a functional disorder syndrome (TMD). Clinical manifestations include pain in the joint area and surrounding muscles, joint clicking during movement, and mandibular movement disorders. Currently, the incidence of temporomandibular joint osteoarthritis is high in my country, but there is still a lack of very effective treatments. Existing treatments include medication, physical therapy, and surgical intervention, but these methods often have limited efficacy, are accompanied by significant side effects, and have high treatment costs.

[0003] With the rise of tissue engineering technology, cell therapy strategies have been gradually applied to clinical treatment. Stem cells derived from sources such as bone marrow, adipose tissue, and umbilical cord demonstrate potential for anti-inflammatory, differentiation-promoting, and tissue regeneration, making them promising seed cells for regenerative therapy strategies. However, potential risks of cell therapy, such as immune response and tumorigenesis, have hindered widespread clinical application. Exosomes, considered the primary mediators of cellular function, possess advantages such as carrying bioactive substances (such as proteins, nucleic acids, and lipids), low immunogenicity, and high biocompatibility, making them a research hotspot for cell-free therapeutic strategies. However, limitations, such as unstable delivery efficiency and susceptibility to degradation, have limited their clinical translation. Therefore, there is an urgent need to explore new and more effective treatments for temporomandibular joint osteoarthritis. These include developing engineered exosome-based delivery systems, optimizing the stem cell culture microenvironment to enhance therapeutic efficacy, and combining biomaterials to construct biomimetic scaffolds to promote precise repair and functional restoration of the temporomandibular joint. Summary of the Invention

[0004] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes an exosome delivery system and its application in the treatment of temporomandibular joint arthritis. The exosomes in this delivery system can be used to prepare a product for treating subchondral bone damage in temporomandibular joint osteoarthritis, effectively providing an innovative treatment for cartilage degeneration and regulating the proliferation and repair of chondrocytes.

[0005] According to a first aspect of the present application, an exosome delivery system is provided, comprising an injectable, self-repairing, positively charged carrier and negatively charged exosomes, wherein the delivery system is obtained by fully mixing the exosomes with the positively charged carrier and achieving self-assembly through electrostatic adsorption. Wherein, the exosomes are prepared by the following method: The cells were cultured using a complete medium consisting of MEM-α medium and 10% by volume of fetal bovine serum. When the cell confluence reached 70%-80%, the medium was replaced with a medium containing exosome-free serum and cultured for 24h-72h. The supernatant was then collected and separated, and then purified by size exclusion chromatography to obtain the exosomes.

[0006] According to the exosome delivery system of the embodiment of the present application, it has the characteristics of long-term stable sustained release of exosomes, can continuously release exosomes in the local microenvironment, effectively maintain high concentration, improve targeted delivery efficiency, thereby enhancing cellular uptake rate and biological activity, prolonging the therapeutic effect time, and further optimizing the therapeutic effect. In addition, the system can effectively avoid rapid degradation or clearance of exosomes in the body, improve the stability and bioavailability of exosomes, achieve precise delivery and long-term effect of exosomes, ensure the persistence and controllability of the therapeutic effect, and provide efficient and safe exosome delivery solutions for fields such as regenerative medicine, tumor treatment, and inflammation regulation. In addition, the method can enhance the interaction between exosomes and carriers, improve the loading efficiency and stability of exosomes, prevent premature release or degradation of exosomes during the delivery process, thereby achieving controlled release of exosomes, prolonging their action time in target tissues, and improving the therapeutic effect.

[0007] Furthermore, during the exosome preparation process, cells are cultured in complete culture medium until the cell confluence reaches 70%-80%, and then replaced with culture medium containing exosome-free serum. After continued culture, the supernatant is collected and separated and purified to obtain the exosomes in the delivery system of this application. This simple preparation process does not require the use of complex culture medium, is low-cost, and is suitable for large-scale batch applications. Furthermore, this method can effectively increase the yield of exosomes, reduce the interference of background impurities, enhance the purity and biological activity of exosomes, and ensure their structural integrity and functional stability, providing reliable technical support for the subsequent industrial application of clinical translation agents.

[0008] It can be understood that under the condition of pH 7.4, the hydrogel zeta potential shows positive charge, and after mixing with the negatively charged exosomes, self-assembly is achieved through electrostatic adsorption.

[0009] In some embodiments, the elastic modulus G' of the hydrogel is 30 Pa-10 KPa, preferably 1 KPa-2 KPa, and the viscous modulus G" is 1 Pa-500 Pa, preferably 20 Pa-100 Pa.

[0010] According to some embodiments of the present application, the injectable self-repairing and positively charged carrier comprises one or more of chitosan hydrogel, gelatin hydrogel, agarose hydrogel, and hyaluronic acid hydrogel.

[0011] It should be noted that the present application chose an injectable carrier because, compared to non-injectable carriers (such as preformed scaffolds), injectable carriers such as hydrogels can be precisely delivered to the confined temporomandibular joint cavity through a minimally invasive approach. This ensures the retention and sustained release of exosomes at the lesion site, preventing the rapid loss of exosomes in the joint cavity after injection, while also avoiding the tissue damage associated with open surgery. Furthermore, the introduction of a self-healing function further addresses the vulnerability of traditional hydrogels to rupture under dynamic loads. This property not only maintains the structural integrity of the carrier to prevent the sudden release of exosomes, but also improves the material's stability by continuously repairing mechanical damage. This injectable and self-healing dual-functional hydrogel system overcomes the difficulties of implanting non-injectable carriers and the lack of mechanical stability of conventional hydrogels, providing an optimized solution for the treatment of temporomandibular joint osteoarthritis that combines minimal invasiveness, mechanical compatibility, and long-term controlled drug release. The exosome delivery system of the present application utilizes an injectable, self-healing, and positively charged carrier, creating an electrostatic force between the carrier and the exosomes, enabling long-term sustained release of exosomes, effectively maintaining high therapeutic concentrations, and improving targeted delivery efficiency.

[0012] According to some embodiments of the present application, the purification is performed by size exclusion chromatography, which specifically comprises the following steps: The crude exosome extract obtained after separation was loaded onto a chromatographic column and eluted with PBS or HEPES buffer as the mobile phase, and the exosomes were collected by fraction.

[0013] It should be noted that during the preparation of exosomes, after collecting the supernatant and separating it, the exosomes are purified by size exclusion chromatography, which can effectively remove non-exosomal components such as proteins, cell debris, and small molecule contaminants, improve the purity and homogeneity of the exosomes, and ensure their stability and biological function in subsequent applications. In addition, the exosome-enriched fraction is usually located in the 7th to 10th fraction.

[0014] According to some embodiments of the present application, the exosome-free serum culture medium comprises MEM-α complete culture medium and 10% by volume of exosome-free FBS.

[0015] It is worth noting that after the cell confluence reaches 70%-80%, the operation of replacing the culture medium with exosome-free serum is as follows: discard the original culture medium, wash the cells twice with PBS, each time for 60 seconds, and replace with culture medium containing exosome-free serum and continue culturing for 24h~72h, wherein the culture medium containing exosome-free serum is MEM-α medium + 10% volume ratio of exosome-free FBS.

[0016] In some embodiments, the above process may include: isolating cells at 8×104 / cm 2The cells were seeded into cell culture flasks and cultured with complete medium MEM-α + 10% (V / V) FBS for 2 days. When the cells grew to 80%, the medium was replaced with FBS without exosomes and cultured for another 48 hours. The supernatant was collected and the exosomes were isolated.

[0017] The cells of this embodiment are human umbilical cord mesenchymal stem cells. The mesenchymal stem cells are obtained by the following operation: removing umbilical cord tissue, cutting it into 3-5 mm 3 The tissue blocks were washed several times with sterile saline / phosphate buffered saline. The blocks were spread flat, added with MEM-α medium (containing 10% FBS), and cultured in an incubator with a carbon dioxide volume fraction of 5% and a temperature of 37°C. After the tissue blocks attached, the culture medium was changed every 2-3 days. After about 7-10 days, cells with a mesenchymal stem cell phenotype could be isolated.

[0018] In one embodiment, the procedure for obtaining exosome-free FBS is as follows: ultracentrifuging FBS at 120,000 g for 12 h, taking the supernatant, and filtering it with a 0.22 μm filter.

[0019] According to some embodiments of the present application, the exosomes are stem cell-derived exosomes. Specifically, the stem cell-derived exosomes may include, but are not limited to, any one or more of: umbilical cord-derived mesenchymal stem cell exosomes, bone marrow-derived mesenchymal stem cell exosomes, adipose-derived mesenchymal stem cell exosomes, macrophage-derived exosomes, and / or osteoclast-derived exosomes.

[0020] According to some embodiments of the present application, the separation is performed from the supernatant by differential centrifugation, ultrafiltration, polymer precipitation or magnetic bead method.

[0021] In a specific embodiment, the desired exosomes can be isolated from the supernatant by the following operation: the obtained supernatant is centrifuged (4°C, 300×g, 5 minutes), the supernatant is taken and centrifuged again (4°C, 2000×g, 15 minutes), the supernatant is taken and centrifuged again (4°C, 10000×g, 30 minutes), and then the supernatant is further centrifuged (4°C, 120,000×g, 70 minutes), the precipitate is collected and resuspended with PBS, the supernatant is filtered with a 0.22μm filter, and centrifuged again (4°C, 120,000×g, 70 minutes) to collect the precipitate, which is the desired exosomes.

[0022] In some embodiments, the protein concentration of the exosomes is 0.01 ug / ml-50 mg / ml, preferably 50 ug / ml-1 mg / ml.

[0023] According to a second aspect of the present application, there is provided an application of an exosome delivery system in the treatment of temporomandibular arthritis, which comprises the above-mentioned exosome delivery system.

[0024] It should be noted that the exosome delivery system of the present application can be used to treat temporomandibular joint arthritis by injection. The electrostatic force enables the long-term sustained release of exosomes between the exosomes and the carrier, effectively maintaining a high therapeutic concentration, improving the efficiency of targeted delivery, and effectively promoting the repair of cartilage and subchondral bone, inhibiting subchondral bone destruction, and thus providing effective support for the condylar cartilage layer. In addition, the exosome delivery system can be used to treat temporomandibular joint osteoarthritis, reduce the excessive absorption of subchondral bone and bone loss, and promote chondrocyte proliferation and matrix synthesis, thereby enhancing cartilage regeneration and repair. The exosomes of the present application can improve their stability and sustained release ability in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present application is further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 Nanoparticle tracking analysis (NTA) diagram of exosomes.

[0026] Figure 2 Transmission electron microscopy (TEM) images of exosomes.

[0027] Figure 3 Schematic diagram of the assembly of the exosome delivery system.

[0028] Figure 4 Zeta potential of exosomes, hydrogels, and exosome delivery systems.

[0029] Figure 5 Rheological properties of the exosome delivery system.

[0030] Figure 6 This is the in vitro sustained release effect of the exosome delivery system.

[0031] Figure 7 In vivo imaging of the sustained release effect of the exosome delivery system.

[0032] Figure 8 Schematic diagram of exosome uptake by macrophages and osteoclasts.

[0033] Figure 9 Schematic diagram of tartrate-resistant acid phosphatase staining of osteoclast differentiation.

[0034] Figure 10 Related gene expression during osteoclast differentiation.

[0035] Figure 11Micro-CT image of the effect of exosome delivery system in treating temporomandibular joint osteoarthritis in rats.

[0036] Figure 12 Histological analysis of the exosome delivery system in treating temporomandibular joint osteoarthritis in rats. DETAILED DESCRIPTION

[0037] The following will clearly and completely describe the concept and technical effects of this application in conjunction with the embodiments to fully understand the purpose, features and effects of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments of this application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of this application.

[0038] In the description of this application, reference to the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples.

[0039] The specific embodiments of the present application are described in detail below.

[0040] In the specific examples, FBS was purchased from Cellmax, penicillin-streptomycin solution (double antibody, PS) was purchased from Yuanye Bio, DMSO was purchased from Solebao, Dil, DAPI, and phalloidin were all purchased from Beyotime, and 8-week-old SD rats were purchased from Guangdong Provincial Laboratory Animal Center.

[0041] Example 1 Isolation and identification of umbilical cord mesenchymal stem cells 1) Umbilical cord harvesting The umbilical cord was obtained during delivery with the consent of the patient and her family.

[0042] 2) Isolation of umbilical cord mesenchymal stem cells In terms of cell extraction, the tissue block adherence method can be used to separate umbilical cord mesenchymal stem cells: Umbilical cord tissue is removed and cut into small pieces of 3-5 mm³. Wash the pieces several times with sterile saline / phosphate buffered saline. Spread the pieces flat, add MEM-α culture medium (containing 10% FBS), and culture in an incubator with a 5% CO₂ volume fraction and 37°C. After the pieces attach, the culture medium is changed every 2-3 days. After approximately 7-10 days, cells with a mesenchymal stem cell phenotype can be isolated. These cells are typical fibroblast-like cells, with a spindle or stellate shape and arranged in parallel or spiral patterns. 3) Cell passaging The cells were passaged when they grew to 80% of the culture flask, and the third to sixth generation cells were used for subsequent experiments.

[0043] Example 2 Acquisition and identification of exosomes 1) Isolation of exosomes Umbilical cord mesenchymal stem cells were cultured and, when cell proliferation reached 80%, the medium was replaced with blood-free medium for 48 h. The supernatant was collected and centrifuged (4°C, 300 × g, 5 minutes). The supernatant was removed and centrifuged again (4°C, 2000 × g, 15 minutes). The supernatant was centrifuged again (4°C, 10,000 × g, 30 minutes), and then the supernatant was centrifuged again (4°C, 120,000 × g, 70 minutes). The precipitate was collected and resuspended in PBS. The supernatant was filtered through a 0.22 μm filter and centrifuged again (4°C, 120,000 × g, 70 minutes). The precipitate, which is the desired exosomes, was collected. The exosomes were then purified by size exclusion chromatography (SEC) using a pre-equilibrated SEC column with PBS or HEPES buffer as the mobile phase. The flow rate was optimized (0.5-1.0 mL / min) to improve the recovery rate and purity of the exosomes. The culture supernatant, concentrated by ultracentrifugation or ultrafiltration, is loaded onto a chromatographic column, and the eluted fractions are fractionated to identify the exosome-enriched fraction (typically fractions 7-10). To optimize purification, nanoparticle tracking analysis (NTA) is used to monitor the size distribution of exosomes, and identification is performed by transmission electron microscopy (TEM) and Western blot analysis for markers such as CD9, CD63, and CD81. Furthermore, exosome storage conditions are optimized: short-term storage (<24 hours) at 4°C and long-term storage at -80°C to avoid freeze-thaw cycles to ensure exosome stability and bioactivity.

[0044] 2) Identification of exosomes The prepared exosomes were subjected to NTA and transmission electron microscopy to obtain Figure 1 and Figure 2 .

[0045] Depend on Figure 1NTA showed that the particles had a complete lipid bilayer membrane structure with a diameter of approximately 130 nm (range: 100–150 nm), which fully met the recognized size standard of exosomes (30–200 nm). To verify the structure of exosomes, we used transmission electron microscopy (TEM) to observe ( Figure 2 The results showed that a clear cup-shaped morphology was visible in the high-magnification TEM image, which is a typical feature of exosomes caused by dehydration and shrinkage during the electron microscopy sample preparation process, further confirming their identity.

[0046] Example 3 Preparation of exosome hydrogel delivery system The exosomes obtained above were mixed with a carrier, such as Figure 3 , a hydrogel delivery system loaded with exosomes can be obtained. The zeta potential before and after mixing shows the potential change of the mixed material, such as Figure 4 , indicating that the electrostatic network sustained-release system was successfully prepared.

[0047] Example 4 Rheological properties testing of exosome delivery system A rotational rheometer with a 40 mm diameter rotational rheometer parallel plate fixture DHR (TA, USA) was used, the test temperature was 25 °C, and the operating gap distance was 1000 µm. The viscoelastic properties of different groups of PCH were characterized by frequency sweep (0.1-50 Hz, constant strain of 0.5%). The self-healing properties of the composite scaffolds were quantitatively evaluated by monitoring the evolution of the storage modulus (G') and loss modulus (G'') during the destructive shear (oscillatory strain sweep, strain range from 0.1% to 500%, constant frequency of 1 Hz) and recovery (strain of 0.5%, frequency of 1 Hz, lasting 300 s) cycles. Figure 5 As shown in the last small picture in the group, after shear failure, the storage modulus (G') and loss modulus (G'') quickly recovered to their initial states, showing good self-healing or self-repair performance.

[0048] After selecting the hydrogel concentration, the exosomes were mixed in and the above steps were repeated. Figure 5 A. Figure 5 B. Figure 5 C. Figure 5 D and Figure 5 The significantly increased modulus (E) demonstrates enhanced interactions within the mixture. The elastic modulus (G') and viscous modulus (G'') indicate an increasing trend in internal interactions within the assembled PCH-EVs. Electrostatic forces increase the forces within the delivery system, making the entire system more stable and providing a foundation for the long-term sustained release of exosomes.

[0049] Example 5 Evaluation of sustained-release performance of exosome delivery system Negatively charged exosomes labeled with the fluorescent dye DiD were mixed with positively charged PCH hydrogel and uncharged GelMA hydrogel at a ratio of 7.5%, transferred to a 96-well plate, and 100 μL of PBS (pH 7.4) was added to each well to cover the complex. The complex was stored at 4°C in the dark. The PBS was replaced every 3 days and the old PBS was collected. The fluorescence intensity of DiD was measured using a fluorescence microplate reader. The results are shown in Figure 2. Figure 6 Exosome release from the PCH hydrogel persisted for at least 28 days, with daily release rates maintained between 2% and 4%. No bursts of exosome release were observed, and the release rate remained essentially constant throughout the experiment. The uncharged GelMA hydrogel, on the other hand, completely released exosomes within ten days, demonstrating that electrostatic adsorption can significantly mitigate the phenomenon of rapid exosome release.

[0050] like Figure 7 This result was also confirmed by an in vivo sustained-release experiment. The hydrogel delivery system was injected into the temporomandibular joint of rats, along with an equal amount of fluorescently labeled exosomes as a control. The results showed that the fluorescence of the pure exosome group completely disappeared after 9 days, while the exosome delivery system group maintained sustained release for up to 29 days, demonstrating the excellent sustained-release properties of the exosome delivery system.

[0051] Example 6 Uptake of EVs by osteoclasts and macrophages Uptake experiments are designed to verify the uptake capacity of exosomes by target cells and determine whether exosomes can be internalized by specific cells to exert their biological functions. Fluorescent labeling and detection of intracellular fluorescence signals using fluorescence microscopy provide a visual assessment of EV uptake efficiency and intracellular distribution. The specific method is as follows: First, prepare the fluorescent dye DiD according to the manufacturer's instructions. Dissolve 10 mg of the powder in 1 mL of DMSO to a 10 mM storage solution. Store at -20°C, protected from light, and maintain for one year. Upon use, dilute the solution again at a 1:1000 ratio to a final concentration of 10 μM. In this experiment, 100 µL of exosomes with a protein concentration of 1 mg / mL were thoroughly mixed with 1 µL of the 1 mM dye. The mixture was incubated at 4°C for 25 minutes, centrifuged at 120,000 g for 70 minutes to remove free dye, and then resuspended in 50 µL of PBS and stored at 4°C, protected from light, until ready for use. Macrophages were seeded onto confocal microscopy dishes and cultured at 37°C, 5% CO2, until adherent. All fluorescently labeled EVs were added to the culture medium. After a 6-hour incubation, cells were washed three times with PBS, fixed, and nuclei stained with DAPI. After a 5-10-minute incubation, cells were washed with PBS. Exosome uptake was observed using confocal microscopy. Osteoclast uptake was assessed using osteoclasts predifferentiated on day 4 of confocal microscopy. After a 6-hour incubation with the dye, cells were fixed, nuclei stained, and observed under a microscope.

[0052] Fluorescence microscopy showed that ( Figure 8 ), red fluorescence-labeled exosomes (DiD-labeled EVs) were obviously aggregated in the cytoplasm after incubation, appearing as granules or clusters, and had no overlap with the DAPI-stained cell nucleus (blue fluorescence), indicating that the exosomes did not enter the cell nucleus. The above results proved that the exosomes were successfully taken up by the cells.

[0053] Example 7 Study on the mechanism of exosomes alleviating subchondral bone destruction in the temporomandibular joint Osteoclasts are usually identified by TRAP staining and quantitative determination of TRAP activity in cell supernatants (e.g. Figure 9 After RANKL stimulation, macrophages significantly differentiated into osteoclasts, displaying typical morphological features, including enhanced TRAP activity and multinucleated cell structure, demonstrating successful osteoclast induction. In contrast, the number of TRAP-positive multinucleated osteoclasts and TRAP activity were significantly reduced in the EV-treated group, and the osteoclast size was significantly smaller than in the RANKL-induced group. These results suggest that EVs can effectively inhibit osteoclast differentiation.

[0054] Real-time quantitative PCR (qRT-PCR) was used to compare gene expression in osteoclasts before and after exosome treatment. Total RNA was first extracted using the TRIzol method. RNA concentration and purity were then assessed using Nanodrop; the A260 / A280 ratio should be between 1.8 and 2.0. A 10 μL reverse transcription reaction was prepared by adding 1 μg of RNA to 1 μL of gDNA Eraser, which was then filled to 10 μL with RNase-free H2O. The reaction was incubated according to the manufacturer's instructions to eliminate residual gDNA. Prior to reverse transcription, an equal volume of 2× SPARKscript II RT Plus Master Mix was added. The reaction was incubated at 50°C for 15 minutes, followed by heating at 85°C for 5 seconds to inactivate the gDNA Eraser. The reaction was then placed on ice. Fluorescence quantitative PCR was performed using 2× SYBR Green qPCR Mix. The total reaction volume per well was 20 µL, consisting of 10 µL of qPCR Mix (including dNTPs, buffer, enzyme, MgCl2, and SYBR Green dye), 1 µL of cDNA template, and 0.4 µL of each forward and reverse primer, with a final concentration of 0.2 µM. 0.4 µL of ROX Reference Dye II was also added to ensure uniformity and accuracy between wells. Finally, the reaction volume was brought up to 20 µL with RNase-free double-distilled water.

[0055] like Figure 10 The results showed that RANKL-induced osteoclast mRNA expression levels of CTSK, TRAP, NFATc1, DC-STAMP, and CTR were significantly upregulated, while EV treatment significantly decreased the expression of these genes. This suggests that EVs may inhibit osteoclast differentiation and maturation, thereby reducing their bone resorption activity, by regulating RANKL-mediated signaling pathways.

[0056] Example 8 Therapeutic effect of exosome delivery system on temporomandibular joint osteoarthritis in rats 1) Sodium iodoacetate (MIA) was injected into the temporomandibular joint of 8-week-old male rats for two weeks to establish the model.

[0057] 2) Two weeks later, the patients were injected with an exosome delivery system, with an exosome protein concentration of 1 mg / ml and a gelatin hydrogel concentration of 7.5%.

[0058] 3) In the fourth week, the experimental animals were collected. Micro-CT scanning of the temporomandibular joint samples of rats was performed and data analysis was performed to obtain Figure 11The results showed that injection of this exosome / hydrogel system can effectively promote the reconstruction of subchondral bone.

[0059] 4) Analysis of tissue sections Figure 12 It can be seen that at the early time point of four weeks, significant differences were observed in the condylar height and cartilage thickness, cell number and cell loss, proteoglycan deposition and reconstruction of subchondral bone between the PCH-EVs group and the Ctrl group. Compared with the Sham group, the proteoglycan observed in the Ctrl group was significantly reduced, and the therapeutic effect of the experimental group was obvious, but there was no significant difference with the EVs group. HE results showed that the cell number and cell loss were also well reversed in the experimental group, the cartilage layer was thicker, the surface was smoother, and the bone loss of the subchondral bone was also treated. In summary, the exosome delivery system of the present application can repair temporomandibular joint osteoarthritis in many aspects.

[0060] In combination with specific embodiments, the exosome delivery system prepared by the exosome delivery system preparation method of the present invention can effectively promote inflammation regulation and matrix reconstruction of temporomandibular joint osteoarthritis, and is used for the treatment of temporomandibular joint osteoarthritis.

[0061] The embodiments of the present application have been described in detail above in conjunction with specific implementation methods. However, the present application is not limited to the above embodiments. Various changes can be made within the scope of knowledge possessed by ordinary technicians in the relevant technical field without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other unless there is a conflict.

Claims

1. An exosome delivery system, characterized in that The exosome delivery system comprises an injectable, self-repairing, and positively charged carrier and exosomes. The delivery system is obtained by fully mixing the exosomes with the positively charged carrier and achieving self-assembly through electrostatic adsorption. Wherein, the exosomes are prepared by the following method: The cells were cultured using a complete medium consisting of MEM-α medium and 10% by volume of fetal bovine serum. When the cell confluence reached 70%-80%, the medium was replaced with a medium containing exosome-free serum and cultured for 24h-72h. The supernatant was then collected and separated, and then purified by size exclusion chromatography to obtain the exosomes.

2. The exosome delivery system according to claim 1, wherein The injectable, self-repairing and positively charged carrier comprises one or more of chitosan hydrogel, gelatin hydrogel, agarose hydrogel and hyaluronic acid hydrogel.

3. The exosome delivery system according to claim 1, wherein The purification is performed by size exclusion chromatography, which specifically comprises the following steps: The crude exosome extract obtained after separation was loaded onto a chromatographic column and eluted with PBS or HEPES buffer as the mobile phase, and the exosomes were collected by fraction.

4. The exosome delivery system according to claim 1, wherein The exosome-free serum culture medium comprises MEM-α complete culture medium and 10% by volume of exosome-free FBS.

5. The exosome delivery system according to any one of claims 1 to 4, characterized in that The exosomes are stem cell-derived exosomes.

6. The exosome delivery system according to any one of claims 1 to 4, characterized in that The separation is carried out by separating the supernatant through differential centrifugation, ultrafiltration, polymer precipitation or magnetic bead method.

7. Application of an exosome delivery system in the treatment of temporomandibular arthritis, characterized in that: Comprising the exosome delivery system according to any one of claims 1 to 6.

Images