Inflammatory preconditioned synovial cell-derived vesicles, methods of making and use thereof in the treatment of osteoarthritis

Abstract

This invention discloses an inflammatory pretreatment xenogeneic synovial cell-derived vesicle, its preparation method, and its application in the treatment of osteoarthritis, belonging to the field of biomedical technology. The preparation method includes: obtaining and culturing rabbit synovial fibroblast-like cells; subjecting the cells to inflammatory pretreatment for 40-60 hours using a culture medium containing 70-80 ng / mL tumor necrosis factor-α (TNF-α); collecting the cell supernatant, and purifying it by differential centrifugation and ultracentrifugation to obtain the vesicles. The vesicles have a particle size distribution of 100-1000 nm, with a higher proportion of larger particles (>800 nm) than natural vesicles. This invention also provides a pharmaceutical composition containing these vesicles and their application in the preparation of drugs for treating osteoarthritis. Experiments show that these vesicles can significantly inhibit subchondral ossification and osteophyte formation, effectively protect articular cartilage, and have a therapeutic effect superior to natural vesicles, providing a novel cell-free therapeutic strategy for osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an inflammatory pretreatment xenogeneic synovial cell-derived vesicle, its preparation method, and its application in the treatment of osteoarthritis. Background Technology

[0003] In recent years, extracellular vesicles (EVs) have shown great potential in regenerative medicine due to their natural biocompatibility, low immunogenicity, and ability to act as intercellular signaling carriers to deliver bioactive molecules. Synovial tissue, as an important component of the joint, is rich in fibroblast-like synovial cells, making it an excellent source of EVs. Utilizing synovial cell-derived EVs (SEVs) as cell-free therapeutic agents can avoid the safety risks of tumorigenesis and immune rejection associated with direct use of live cells. However, the limited anti-inflammatory or repair molecule loading of natural SEVs restricts their ability to specifically repair the complex pathological microenvironment of osteoarthritis (OA), thus limiting their efficacy.

[0004] To enhance the therapeutic efficacy of vesicles (EVs), researchers have proposed an "inflammatory preconditioning" strategy. This involves pretreating parental cells with pro-inflammatory factors (such as TNF-α and IL-1β) to induce the secretion of engineered EVs carrying a richer array of functional molecules (such as specific miRNAs and proteins). Previous studies have shown that EVs derived from mesenchymal stem cells (MSCs) undergoing inflammatory preconditioning exhibit superior effects in regulating immune cell (such as macrophage) polarization and alleviating OA inflammation. However, current research largely focuses on MSCs, while systematic studies on the physical characteristics and biological functional changes of vesicles produced by synovial cells—direct participants and regulators of the joint microenvironment—after inflammatory preconditioning are lacking. In particular, whether engineered vesicles derived from synovial cells possess unique advantages in regulating key pathological aspects of OA, such as subchondral ossification and osteophyte formation, remains a blank. Furthermore, research indicates that neutrophils, as core effector cells of innate immunity, are recruited and activated early in synovial inflammation during the early stages of OA, making them one of the key cells driving the early pathological progression of OA. Overactivation of neutrophils not only directly damages the cartilage matrix by releasing reactive oxygen species and proteases, but also creates a vicious cycle by inducing synovial inflammation. Therefore, developing novel cell-free therapeutic strategies that can target key events in the early stages of osteoarthritis (OA), effectively intervene in pathological bone remodeling, and protect cartilage is of significant clinical importance.

[0005] In summary, the existing technology has the following shortcomings: (1) Limited source: Existing engineered EVs research is mostly focused on mesenchymal stem cells, while the physical characteristics and therapeutic potential of vesicles generated by synovial cells after inflammatory preconditioning have not been fully explored; (2) Passive function: Natural SEVs have limited efficacy in regulating core pathological links of OA (such as subchondral ossification), and there is an urgent need to enhance their therapeutic efficacy through engineering. Summary of the Invention

[0006] In view of the limitations of existing osteoarthritis treatment techniques, such as the limited efficacy of natural synovial cell vesicles and the lack of engineered cell-free therapeutic agents that can effectively inhibit subchondral ossification and osteophyte formation, this invention aims to provide an inflammatory pretreatment xenogeneic synovial cell-derived vesicle, its preparation method, and its application in the treatment of osteoarthritis.

[0007] To achieve the above objectives, the present invention employs the following technical solution: This invention provides a method for preparing vesicles derived from xenogeneic synovial cells after inflammatory pretreatment, comprising the following steps: (1) Obtain and culture rabbit synovial fibroblasts; (2) The synovial fibroblasts were subjected to inflammatory pretreatment using a culture medium containing tumor necrosis factor-α; (3) Collect the cell supernatant after pretreatment in step (2), and extract and purify it by differential centrifugation and ultracentrifugation in sequence to obtain vesicles derived from inflammatory pretreated xenogeneic animal synovial cells.

[0008] In step (1), the synovial fibroblasts of the rabbit were obtained from the synovial tissue of the knee joint of a 4-week-old male New Zealand rabbit. Four-week-old New Zealand rabbits are in their juvenile stage, and the proliferative activity and secretory function of their synovial fibroblasts are superior to those of adult or older animals, which is beneficial for obtaining a sufficient number of cells for subsequent vesicle preparation and improving production efficiency.

[0009] In step (2), the culture medium is MEMα complete culture medium, which contains 10% fetal bovine serum and 1% penicillin / streptomycin.

[0010] In step (2), the concentration of tumor necrosis factor-α in the culture medium containing tumor necrosis factor-α is 70-80 ng / mL, and the pretreatment time is 40-60 hours. The concentration range of 70-80 ng / mL has been proven to be the optimal window for both effectively activating cells and maintaining high cell viability.

[0011] Preferably, the concentration of tumor necrosis factor-α in the culture medium containing tumor necrosis factor-α is 75 ng / mL, and the pretreatment time is 48 hours. The cell viability in the 75 ng / mL TNF-α treatment group remained at approximately 150% on days 3-5, significantly higher than the control group. The expression level of RAB27A protein in synovial cells was significantly upregulated. RAB27A is a key protein regulating vesicle secretion; its upregulation indicates enhanced vesicle secretion capacity, which is beneficial for increasing subsequent vesicle production.

[0012] In step (3), the specific steps of differential centrifugation and ultracentrifugation are as follows: the cell supernatant is centrifuged at 800×g for 10 minutes and at 2000×g for 20 minutes to remove cell debris and large particles. After the supernatant is filtered through a 0.22 μm filter membrane, it is centrifuged at 16,000×g for 0.5 hours, the precipitate is collected and resuspended with PBS.

[0013] This invention provides an inflammatory pretreatment xenogeneic synovial cell-derived vesicle, which is prepared by the aforementioned preparation method.

[0014] The vesicles have a particle size of 100-1000 nm, and the proportion of larger vesicles (>800 nm) is higher than that of natural vesicles. This indicates that the vesicles prepared in this invention belong to the classic extracellular vesicle category and possess the general biological characteristics and functions of this type of vesicle. The significantly increased proportion of larger vesicles (>800 nm) in Inf-SEVs, along with this difference in distribution within the same particle size range, can serve as a detectable physical marker distinguishing Inf-SEVs from natural vesicles, facilitating product quality control and efficacy prediction. The present invention provides a pharmaceutical composition comprising the aforementioned inflammatory pretreated xenogeneic synovial cell-derived vesicles, and a pharmaceutically acceptable carrier or excipient.

[0015] The pharmaceutical composition is formulated as an intra-articular injection. Intra-articular injection delivers the drug directly to the lesion site—the knee joint cavity—allowing the vesicles to come into direct contact with target tissues such as the synovium and cartilage. This avoids the dilution effect and potential systemic side effects associated with systemic administration, and improves the local bioavailability of the drug.

[0016] The use of the inflammatory pretreatment of xenogeneic animal synovial cell-derived vesicles, or the use of the pharmaceutical composition thereof, in the preparation of a medicament for the treatment of osteoarthritis.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for preparing xenogeneic animal synovial cell-derived vesicles through inflammatory preconditioning. For the first time, this method applies an "inflammatory preconditioning" strategy to synovial fibroblast-like cells. By preconditioning rabbit synovial fibroblast-like cells with a specific concentration (70-80 ng / mL) of tumor necrosis factor-α (TNF-α) for a limited time (40-60 hours), the vesicle secretion pathway of synovial cells is successfully activated, providing a reliable technical means for obtaining engineered vesicles with enhanced biological functions. The extraction method using differential centrifugation combined with ultracentrifugation is a classic technical route in the field of extracellular vesicle research. It is simple to operate, cost-effective, and can stably obtain high-purity vesicle products, suitable for laboratory research and subsequent industrial-scale production. Using rabbits as synovial cell donors has advantages such as wide availability, low cost, and standardized culture and expansion capabilities, while avoiding the secondary trauma and ethical controversies associated with extracting autologous cells from patients, laying the foundation for the clinical application of xenogeneic extracellular vesicles.

[0018] The inflammatory pretreated vesicles (Inf-SEVs) prepared by the method of this invention exhibit detectable and regular changes in their physical properties. Compared with untreated natural vesicles (SEVs), the particle size distribution of Inf-SEVs is significantly altered, with a marked increase in the proportion of larger vesicle subpopulations with a particle size greater than 800 nm. This regular change in physical properties is direct evidence of alterations in the loading or composition of their internal bioactive molecules, distinguishing them from ordinary natural vesicles. As vesicles derived from synovial cells, they inherit the advantage of low immunogenicity of parental cells while avoiding the risks of tumorigenicity and pathogen contamination that may arise from xenogeneic live cell transplantation, making them suitable as cell-free therapeutic agents for intra-articular injection. Synovial fibroblasts have vigorous secretory function, and the yield of their derived vesicles is higher than that of various primary cells. Furthermore, as an engineered product derived from xenogeneic sources, they can be prepared in batches, with quality control and storage, providing a stable source of products for subsequent clinical applications.

[0019] The pharmaceutical composition provided by this invention combines Inf-SEVs with pharmaceutically acceptable carriers or excipients to prepare a drug formulation suitable for intra-articular injection, bridging the gap between laboratory research and clinical application. By selecting appropriate formulations, the structural integrity and bioactivity of the vesicles can be effectively maintained, extending product shelf life and improving the convenience of clinical use. This pharmaceutical composition can be used in combination with or as an alternative to commonly used intra-articular injectables such as hyaluronic acid, providing clinicians with more treatment options.

[0020] The Inf-SEVs provided by this invention exhibit remarkable therapeutic efficacy in an osteoarthritis model. Animal experimental results show that the Inf-SEVs of this invention can effectively inhibit abnormal cartilage ossification caused by the progression of osteoarthritis, significantly reduce the abnormal increase in bone volume fraction (BV / TV) and trabecular bone thickness (Tb.Th), and effectively reduce osteophyte formation. This efficacy is significantly superior to the natural vesicle (SEVs) treatment group, indicating that this invention has a unique advantage in regulating the core pathological link of OA—abnormal bone remodeling. This study is the first to validate the application value of inflammatory preconditioning of synovial cell vesicles in the treatment of osteoarthritis, providing a novel cell-free treatment strategy for early intervention of OA and potentially propelling OA treatment from "symptomatic relief" to "pathological blockade." It also successfully extends the "inflammatory preconditioning" strategy to the engineering modification of synovial cell-derived vesicles, providing new research ideas and theoretical basis for the customized design of extracellular vesicle therapies for inflammation-related diseases. Attached Figure Description

[0021] Figure 1 This study aims to screen inflammatory pretreatment concentrations in synovial cells and detect vesicle secretion-related proteins after treatment. (a) shows the Calcein AM / PI staining results of synovial cells pretreated with gradient concentrations of inflammatory pretreatment; (b) shows the experimental results of CCK8 in synovial cells pretreated with gradient concentrations of inflammatory pretreatment; (c) shows the experimental results of Western blotting for detecting RAB27A, a vesicle secretion-related protein in synovial cells after inflammatory pretreatment; and (d) shows the statistical results of (c).

[0022] Figure 2 The images show representative transmission electron microscopy (TEM) images of Inf-SEVs and SEVs, as well as the particle size distribution of vesicles detected by dynamic light scattering (DLS). a is a TEM image of SEVs, b is a TEM image of Inf-SEVs, c is a particle size distribution of SEVs, and d is a particle size distribution of Inf-SEVs. Figure 3 Images are from Micro-CT scans. (a) shows a three-dimensional reconstructed image of the knee joint of 4-week-old mice; (b) shows the statistical results of bone volume fraction (BV / TV) in 4-week-old mice; (c) shows a longitudinal section of the three-dimensional reconstructed knee joint of 4-week-old mice; and (d) shows the trabecular thickness (Tb) in 4-week-old mice. (e) Statistical results of the knee joint of 8-week-old mice; (f) 3D reconstructed image of the knee joint of 8-week-old mice, with osteophytes indicated by red arrows; (g) Statistical results of the osteophyte count of the knee joint of 8-week-old mice; (h) Longitudinal sectional image of the 3D reconstructed knee joint of 8-week-old mice; (d) Trabecular thickness (Tb) of 8-week-old mice. (i) Statistical results of Th); (ii) Statistical results of bone volume fraction (BV / TV) of mice at 8 weeks; Figure 4 The images show the tissue staining results. Image a shows representative images of H&E staining and Safranin O-Fixed Green staining in 4-week-old mice; image b shows the statistical results of OARSI scores in 4-week-old mice; image c shows representative images of H&E staining and Safranin O-Fixed Green staining in 8-week-old mice; and image d shows the statistical results of OARSI scores in 8-week-old mice. Detailed Implementation

[0023] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.

[0024] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications in the art, unless otherwise stated.

[0025] The recombinant rabbit TNF-α protein used in this invention was purchased from R&D Systems, catalog number RR-900-T05. Four-week-old male New Zealand rabbits were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were fed standard rabbit feed and drinking water under standard laboratory conditions of constant temperature (22±2℃), constant humidity (50±10%), and 12-hour light / dark cycle. The experiment was started after at least one week of acclimatization.

[0026] Example 1 (1) Isolation and culture of synovial fibroblasts (synovial cells) Synovial tissue was aseptically isolated from the knee joint of 4-week-old male New Zealand rabbits. The tissue samples were washed three times with pre-cooled PBS and then minced into 1 mm pieces. 3Fragments were collected and digested in a shaker at 37°C for 1 hour using a digestion solution containing 1 mg / mL collagenase I. After digestion, undigested tissue was removed by filtration through a 100 μm cell sieve. The filtrate was centrifuged at 1200×g for 5 minutes, the supernatant was discarded, and the cells were collected. The cells were resuspended in MEMα complete medium (containing 10% fetal bovine serum FBS and 1% penicillin / streptomycin), seeded in culture dishes, and cultured in a 37°C, 5% CO2 incubator. The medium was changed every 3 days. When the cell confluence reached 80%, the cells were passaged by digestion with 0.25% trypsin. All experiments used healthy third-generation (P3) synovial fibroblast-like cells for subsequent studies.

[0027] (2) Screening of inflammatory pretreatment concentrations To determine the optimal concentration of recombinant tumor necrosis factor-α (TNF-α) for inflammatory pretreatment of synovial cells, we used a gradient concentration screening method. P3 generation synovial cells were seeded at a uniform density in 96-well plates and cultured until adherent. The medium was then replaced with MEMα complete medium containing 0 ng / mL, 50 ng / mL, 75 ng / mL, 100 ng / mL, and 125 ng / mL of recombinant rabbit TNF-α protein. Cells were incubated for 2 hours on days 1, 3, and 5 using a CCK-8 assay kit (Dojindo, catalog number CK04, Dojin Chemical Research Institute, Japan). Afterward, the absorbance (OD value) of each well was measured at 450 nm using a microplate reader to assess cell viability and determine the optimal pretreatment concentration. Cells were stained with Calcein AM / PI and observed under a laser confocal microscope. Subsequently, Western blotting was used to detect changes in RAB27A protein in synovial cells after inflammatory pretreatment to assess vesicle secretion capacity. Results are shown in the appendix. Figure 1 As shown.

[0028] From the appendix Figure 1 Data showed that on day 1, cell viability was not different from the control group in all concentration treatment groups, indicating that short-term stimulation did not cause cytotoxicity. On day 3, cell viability in the 75 ng / mL and higher concentration groups began to show an increasing trend (100% to 150%), suggesting that cells may have entered a state of proliferative or metabolic activity. On day 5, cell viability in the 75 ng / mL group remained at 150%, while the activity in the 100 ng / mL group further increased to 200%. Considering the lack of data in the 125 ng / mL group and the potential risk of excessive stress from high concentrations, a TNF-α concentration of 75 ng / mL achieved the best balance between stimulating cell metabolic / proliferative activity and maintaining cell stability, and was therefore determined to be the optimal concentration for subsequent inflammatory pretreatment.

[0029] Pretreatment with 75 ng / mL TNF-α resulted in a high proportion of viable green cells with well-developed morphology and very few dead red cells in the field of view, further confirming the high viability of cells at this concentration.

[0030] Compared with the control group, synovial cells pretreated with 75 ng / mL TNF-α showed significantly upregulated expression levels of RAB27A protein. This result indicates that appropriate inflammatory stimulation can activate the vesicle secretion pathway in synovial cells, suggesting that they may have a stronger capacity for extracellular vesicle production and release.

[0031] In summary, through CCK-8 cell viability assays, Calcein AM / PI live / dead cell staining, and RAB27A protein expression analysis, we systematically screened and determined the optimal conditions for inflammatory pretreatment of synovial cells. Treatment with 75 ng / mL recombinant rabbit TNF-α effectively maintained high synovial cell viability and good morphology while significantly upregulating the expression of the vesicle secretion-related protein RAB27A. Therefore, this concentration was selected for pretreatment of synovial cells in subsequent experiments to obtain vesicles that may enhance biological function.

[0032] (3) Extraction, purification and identification of synovial vesicles Vesicle isolation and purification process: Extracellular vesicles derived from synovial cells were isolated using ultracentrifugation. The specific steps are as follows: Third-generation synovial cells (cultured to 80% confluence) were cultured for 48 h in MEM α complete medium containing the optimal pretreatment concentration of recombinant rabbit TNF-α protein and MEM α complete medium without recombinant rabbit TNF-α protein. Supernatants from the inflammatory treatment group and the normal group were collected, and centrifuged sequentially at 800×g for 10 min at 4°C to remove cell debris and at 2000×g for 20 min to remove large particles. The supernatant was filtered through a 0.22 μm filter and then centrifuged at 16,000×g for 0.5 h. The supernatant was discarded, and the pellet was resuspended in PBS to obtain inflammatory pretreated synovial cell-derived vesicles (Inf-SEVs) and synovial cell-derived vesicles (SEVs). Vesicle morphology was observed by transmission electron microscopy, and particle size distribution and concentration were determined by dynamic light scattering (DLS). Figure 2 (As shown).

[0033] From the appendix Figure 2Data show that both the SEVs and Inf-SEVs groups exhibited typical cup-shaped or biconcave disc-shaped morphologies under transmission electron microscopy, with a clear lipid bilayer membrane structure. There was no significant difference in basic morphology between Inf-SEVs and SEVs, consistent with the morphological characteristics of extracellular vesicles. The particle size of both types of vesicles was mainly distributed in the 100-1000 nm range, consistent with the common particle size distribution of small extracellular vesicles / microvesicles. The peak particle size distribution of SEVs was concentrated in the 500-700 nm range (cumulative abundance 43%), showing a relatively concentrated single-peak distribution. The particle size distribution of Inf-SEVs was relatively wider, with the abundance peak shifting towards larger particle sizes. Specifically, the proportion of particles in the 800-1000 nm range (cumulative 24%) was significantly higher than that of the SEVs group (cumulative 6%). DLS results indicate that inflammatory pretreatment altered the particle size distribution characteristics of synovial cell secretory vesicles, resulting in a relative increase in the proportion of larger particle sizes (>800 nm) in Inf-SEVs.

[0034] SEVs and Inf-SEVs were successfully separated and purified using classic ultracentrifugation. Transmission electron microscopy confirmed their typical vesicle morphology. Dynamic light scattering analysis further revealed that, compared with SEVs secreted under normal conditions, Inf-SEVs obtained after TNF-α inflammatory pretreatment exhibited altered particle size distribution, with an increased proportion of larger-sized vesicle subsets. This difference in physical properties is related to the different bioactive substances such as proteins and nucleic acids they carry, providing a structural basis for further investigation into the enhanced therapeutic function of Inf-SEVs in osteoarthritis.

[0035] Example 2: (1) Construction of mouse DMM osteoarthritis model Forty 8-week-old male C57BL / 6 mice were randomly divided into four groups (n=5 per group): sham-operated group (Sham, n=5), OA blank control group (DMM+PBS, n=5), negative control group (DMM+SEVs, n=5), and treatment group (DMM+Inf-SEVs, n=5). In all surgical groups, mice underwent medial meniscal instability (DMM) modeling by severing the tibial ligament of the medial meniscus under isoflurane anesthesia, followed by layer-by-layer suturing. In the sham-operated group, only the joint was exposed, without damage to the ligaments or meniscus.

[0036] (2) Intervention program and efficacy evaluation Starting on day 3 post-surgery, mice in each group received intra-articular injections once a week for a total of 4 weeks. Specifically, the Sham group and the DMM+PBS group received 10 μL of PBS; the DMM+SEVs group and the DMM+Inf-SEVs group received an equal volume of PBS solution containing 40 μg of exosomes. Animals were sacrificed at 4 and 8 weeks post-surgery, and the knee joints were harvested for Micro-CT scanning, three-dimensional reconstruction, and quantitative analysis of bone volume fraction (BV / TV) and trabecular bone thickness (Tb). Th) and the number of osteophytes. Representative images of Micro-CT 3D reconstruction and longitudinal section of the knee joint are shown below. Figure 3 As shown in the figure. The results showed that, compared with the DMM+SEVs group, the DMM+Inf-SEVs treatment group mice had lower BV / TV and Tb values ​​in the knee joint at 4 and 8 weeks post-surgery. Th levels were significantly elevated. This indicates that inflammatory pretreatment exosomes (Inf-SEVs) can more effectively inhibit DMM-induced subchondral ossification or osteophyte formation and alleviate pathological bone remodeling.

[0037] Knee joint samples were fixed in 4% paraformaldehyde for 48 hours, decalcified in 10% EDTA for 2 weeks, and then embedded in paraffin. 5 μm sagittal sections were prepared, and H&E staining was used to observe tissue structure. Safranin O-Fixed Green staining was used to assess cartilage structure and proteoglycan content. OARSI scoring (0-6 points) was performed by two researchers in a double-blind manner. Representative images of H&E staining and Safranin O-Fixed Green staining are shown below. Figure 4 As shown in the figure. Histological analysis revealed that, compared with the DMM+SCEVs group, the articular cartilage structure of the DMM+Inf-SCEVs treatment group was more intact and the surface was smoother, and Safranin O staining showed a significant reduction in proteoglycan loss. Quantitative OARSI scores further confirmed that the Inf-SCEVs treatment group had a significantly lower score than the SCEVs treatment group. These results collectively demonstrate that Inf-SCEVs can effectively maintain the homeostasis of the extracellular matrix of chondrocytes and has a clear cartilage-protective and therapeutic effect on osteoarthritis.

[0038] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for preparing vesicles derived from xenogeneic synovial cells after inflammatory pretreatment, characterized in that, Includes the following steps: (1) Obtain and culture rabbit synovial fibroblasts; (2) The synovial fibroblasts were subjected to inflammatory pretreatment using a culture medium containing tumor necrosis factor-α; (3) Collect the cell supernatant after pretreatment in step (2), and extract and purify it by differential centrifugation and ultracentrifugation in sequence to obtain vesicles derived from inflammatory pretreated xenogeneic animal synovial cells.

2. The method for preparing vesicles derived from xenogeneic synovial cells after inflammatory pretreatment according to claim 1, characterized in that, In step (1), the synovial fibroblasts of the rabbit were obtained from the synovial tissue of the knee joint of a 4-week-old male New Zealand rabbit.

3. The method for preparing vesicles derived from xenogeneic synovial cells after inflammatory pretreatment according to claim 1, characterized in that, In step (2), the culture medium is MEMα complete culture medium, which contains 10% fetal bovine serum and 1% penicillin / streptomycin.

4. The method for preparing vesicles derived from synovial cells of xenogeneic animals under inflammatory pretreatment according to claim 1, characterized in that, In step (2), the concentration of tumor necrosis factor-α in the culture medium containing tumor necrosis factor-α is 70-80 ng / mL, and the pretreatment time is 40-60 hours.

5. The method for preparing vesicles derived from synovial cells of xenogeneic animals under inflammatory pretreatment according to claim 1, characterized in that, In step (3), the specific steps of differential centrifugation and ultracentrifugation are as follows: the cell supernatant is centrifuged at 800×g for 10 minutes and at 2000×g for 20 minutes to remove cell debris and large particles. After the supernatant is filtered through a 0.22 μm filter membrane, it is centrifuged at 16000×g for 0.5 hours, the precipitate is collected and resuspended with PBS.

6. A type of inflammatory pretreatment vesicle derived from xenogeneic synovial cells, characterized in that, It is prepared by any one of the methods for preparing xenogeneic synovial cell-derived vesicles under inflammatory pretreatment as described in any one of claims 1 to 5.

7. The inflammatory pretreatment vesicle derived from xenogeneic synovial cells according to claim 6, characterized in that, The vesicle size is 100-1000 nm.

8. A pharmaceutical composition, characterized in that, It contains inflammatory pretreated xenogeneic synovial cell-derived vesicles as described in claim 6 or 7, and a pharmaceutically acceptable carrier or excipient.

9. A pharmaceutical composition according to claim 8, characterized in that, The dosage form of the pharmaceutical composition is an intra-articular injection.

10. The use of an inflammatory pretreated xenogeneic synovial cell-derived vesicle as described in claim 6 or 7, or a pharmaceutical composition as described in claim 8 or 9, in the preparation of a medicament for treating osteoarthritis.

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