Application of dental pulp stem cell exosome in preparation of medicine for treating and preventing osteoarthritis
By preparing dental pulp stem cell exosomes and applying them to drugs, the shortcomings of existing osteoarthritis treatments have been addressed, achieving the protection and repair of chondrocytes and demonstrating the important significance of dental pulp stem cell exosomes in the treatment of osteoarthritis.
Patent Information
- Application Number
- CN202511910585.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-17
- Publication Date
- 2026-02-17
AI Technical Summary
Existing treatments for osteoarthritis cannot fundamentally repair cartilage, regulate the intra-articular environment, or stop disease progression. Furthermore, direct stem cell transplantation presents challenges such as complex procedures, poor safety profiles, and immune rejection.
By utilizing dental pulp stem cell exosomes, a primary articular chondrocyte system was constructed in vitro to prepare dental pulp stem cell exosomes and apply them to drugs. The bioactive substances secreted by these exosomes, such as cytokines, growth factors, and miRNAs, can inhibit endoplasmic reticulum stress-induced senescence of chondrocytes and protect and repair cartilage.
Dental pulp stem cell exosomes effectively inhibited IL-1β-promoted endoplasmic reticulum stress-induced senescence in chondrocytes, demonstrating strong cartilage protection and repair capabilities. They have the advantages of simple operation, high safety, and avoidance of immune rejection, and can improve the pathological process of osteoarthritis.
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Figure CN121534086A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application relates to the technical field of biological medicine, in particular to application of dental pulp stem cell exosomes in preparation of drugs for treating and preventing osteoarthritis. BACKGROUND
[0002] Osteoarthritis is a globally high-incidence degenerative joint disease, and its core pathological features are progressive degeneration of articular cartilage, subchondral bone sclerosis, synovial inflammation and osteophyte formation. The disease is particularly high in the elderly population, and according to statistics, the number of osteoarthritis patients in China has exceeded 100 million. However, the current clinical treatment of osteoarthritis is still limited to symptomatic treatment such as weight reduction, non-steroidal anti-inflammatory analgesics or intra-articular injection of glucocorticoids, and joint replacement. These methods can temporarily relieve symptoms, but cannot reverse or delay the degenerative changes of cartilage. Therefore, developing a regenerative therapy that can fundamentally repair cartilage, regulate the intra-articular environment and prevent disease progression has become an urgent need and important research direction in this field.
[0003] Under the pathological state of osteoarthritis, a large amount of released inflammatory factor IL-1β can destroy the redox balance in chondrocytes, induce ferroptosis and thus accelerate the degradation of cartilage tissue. Meanwhile, protein homeostasis imbalance caused by endoplasmic reticulum stress is a key link of cell death. As cells highly dependent on secretion function, chondrocytes are extremely susceptible to changes in endoplasmic reticulum homeostasis. The treatment method of directly transplanting stem cells has problems such as complex operation, poor safety, immune rejection and the like. SUMMARY
[0004] The application provides application of dental pulp stem cell exosomes in preparation of drugs for treating and preventing osteoarthritis, and explores the regulation of dental pulp stem cell exosomes on endoplasmic reticulum stress and the mechanism of improving chondrocyte degeneration.
[0005] The technical scheme of the application is as follows: The application provides application of dental pulp stem cell exosomes in preparation of drugs for treating and preventing osteoarthritis, and the preparation method of the dental pulp stem cell exosomes comprises the following steps: S1, dental pulp tissue is obtained from the dental pulp cavity of an isolated tooth and is stored in complete culture medium; S2, after the dental pulp tissue is pretreated, a digestive juice is added, and after digestion, centrifugation is performed, the supernatant is removed, complete culture medium containing antibiotics is added to the precipitate, filtration and centrifugation are performed, and dental pulp stem cells are obtained; S3, the dental pulp stem cells are cultured with complete culture medium, when the confluence degree of the dental pulp stem cells reaches 60% to 80%, the serum-free culture medium is replaced to continue the culture, the dental pulp stem cell conditioned medium is collected, and after differential centrifugation, dental pulp stem cell exosomes are obtained.
[0006] As a further technical solution, in step S1, the preparation method of the dental pulp tissue comprises the following steps: Through oral surgery, the patient complained of wisdom tooth extraction, fresh and healthy teeth were extracted, immediately placed in a complete culture medium containing antibiotics, washed and removed all tissues outside the teeth, i.e. impurities, then the crown was split longitudinally with a bone chisel, the dental pulp cavity was exposed, and the dental pulp tissue was taken out with a forceps.
[0007] As a further technical solution, in step S2, the dental pulp tissue pretreatment comprises the following steps: The dental pulp tissue is placed in a 3cm culture dish and cut into small pieces of 1-3mm in size 2 The complete culture medium containing antibiotics is added, and the small pieces of dental pulp tissue are transferred to a centrifuge tube with a pipette, shaken and mixed, and then centrifuged at 1000-1200rpm to obtain the pretreated dental pulp tissue.
[0008] As a further technical solution, in step S2, the digestive juice comprises one or both of collagenase digestive juice and tissue digestive juice.
[0009] As a further technical solution, the digestion is performed at 200rpm and 37℃ for 30-60min, and the filtration is performed with a 200-mesh cell sieve.
[0010] As a further technical solution, in step S3, the complete culture medium is a complete culture medium containing antibiotics.
[0011] As a further technical solution, the centrifugation is first frozen centrifugation, and then ultracentrifugation. The frozen centrifugation comprises first frozen centrifugation and second frozen centrifugation, and the centrifugal forces of the first frozen centrifugation and the second frozen centrifugation are different.
[0012] As a further technical solution, the centrifugal force of the first frozen centrifugation is 300xg, the time of the first frozen centrifugation is 10min, and the temperature of the first frozen centrifugation is 4℃.
[0013] As a further technical solution, the centrifugal force of the second frozen centrifugation is 2000xg, the time of the second frozen centrifugation is 10min, and the temperature of the second frozen centrifugation is 4℃.
[0014] As a further technical solution, the centrifugal force of the ultracentrifugation is 100000xg, and the time of the ultracentrifugation is 60min.
[0015] The working principle and beneficial effects of the present application are as follows: The application discloses application of dental pulp stem cell exosomes in preparation of drugs for treating and preventing osteoarthritis, and the dental pulp stem cell exosomes involved in the application can obtain enough joint cartilage cells by constructing a primary joint cartilage cell system in vitro, and maintain good proliferation and self-renewal capacity. The dental pulp stem cell is a kind of mesenchymal stem cell derived from dental pulp tissue, and has great potential in the field of tissue repair and regeneration due to its multi-directional differentiation potential, low immunogenicity and immune regulation characteristics. Compared with directly transplanting stem cells, the dental pulp stem cell exosomes (various biological active substances, including cytokines, growth factors, miRNAs and the like, secreted into exosomes by the dental pulp stem cell in a culture process) have the advantages of simple operation, high safety and avoidance of immune rejection. Experiments prove that IL-1beta can promote endoplasmic reticulum stress aging of chondrocytes, and the dental pulp stem cell exosomes can effectively inhibit the endoplasmic reticulum stress aging of chondrocytes promoted by IL-1beta, indicating that the dental pulp stem cell exosomes have stronger cartilage protection and repair capacity, and have important significance for the treatment of osteoarthritis. BRIEF DESCRIPTION OF DRAWINGS
[0016] The application will be described in further detail below with reference to the drawings and specific embodiments.
[0017] Figure 1 It is a morphological observation diagram of the dental pulp stem cell in Example 1 of the application; Figure 2 It is a relative mRNA expression determination result diagram of the molecular CD73 and CD90 of the dental pulp stem cell and the hematopoietic markers CD34 and CD45 in Example 1 of the application; Figure 3 It is a mouse chondrocyte cell morphology diagram in Example 2 of the application; In the diagram, P1 is a first generation chondrocyte cell, P3 is a third generation chondrocyte cell, P5 is a fifth generation chondrocyte cell, and P7 is a seventh generation chondrocyte cell; Figure 4 It is an expression amount experiment result diagram of COL II of the mouse chondrocyte cell in Example 2 of the application; In the diagram, P1 is a first generation chondrocyte cell, P3 is a third generation chondrocyte cell, P5 is a fifth generation chondrocyte cell, and P7 is a seventh generation chondrocyte cell; Figure 5 It is a chondrocyte morphology diagram after HE staining in Example 2 of the application; Figure 6 It is a chondrocyte morphology diagram under a microscope in Example 2 of the application; Figure 7 It is a chondrocyte cell morphology diagram after toluidine blue staining in Example 2 of the application; In the figure, P1 is the 1st generation of chondrocyte cells, P3 is the 3rd generation of chondrocyte cells, P5 is the 5th generation of chondrocyte cells, and P7 is the 7th generation of chondrocyte cells; Figure 8 It is a quantitative diagram of the toluidine blue staining analysis of the chondrocyte cells in Example 2 of the present application; In the figure, P1 is the 1st generation of chondrocyte cells, P3 is the 3rd generation of chondrocyte cells, P5 is the 5th generation of chondrocyte cells, and P7 is the 7th generation of chondrocyte cells; Figure 9 It is a volcano diagram of the differential gene analysis between the chondrocyte cells (control) and the IL-1β treated chondrocyte cells in Example 4 of the present application; Figure 10 It is a diagram of the enrichment analysis of the differential genes between the chondrocyte cells (control) and the IL-1β treated chondrocyte cells in Example 4 of the present application; Figure 11 It is a result diagram of the protein imprinting change of the endoplasmic reticulum stress related proteins in Example 5 of the present application; In the figure, IL-1β is the IL-1β treated chondrocyte cells, and DPSC-Exo is the dental pulp stem cell exosome incubated chondrocyte cells; Figure 12 It is the expression amount of BiP, CHOP and ATF4 in the chondrocyte cells (control) and the IL-1β treated chondrocyte cells in Example 5 of the present application; In the figure, Con is the chondrocyte cells (control), and IL-1β is the IL-1β treated chondrocyte cells; Figure 13 It is the expression amount of BiP, CHOP and ATF4 in the IL-1β treated chondrocyte cells and the dental pulp stem cell exosome incubated IL-1β treated chondrocyte cells in Example 5 of the present application; In the figure, IL-1β is the IL-1β treated chondrocyte cells, and IL-1β+DPSC-Exo is the dental pulp stem cell exosome incubated IL-1β treated chondrocyte cells; Figure 14 It is a diagram of the mitochondrial morphological change of the chondrocyte cells (control), the IL-1β treated chondrocyte cells, and the dental pulp stem cell exosome incubated IL-1β treated chondrocyte cells in Example 6 of the present application after Mito Tracker staining; In the figure, CON is the chondrocyte cells (control), IL-1β is the IL-1β treated chondrocyte cells, and IL-1β+DPSC-CM is the dental pulp stem cell exosome incubated IL-1β treated chondrocyte cells; Figure 15 It is a diagram of the mitochondrial length analysis of the chondrocyte cells (control), the IL-1β treated chondrocyte cells, and the dental pulp stem cell exosome incubated IL-1β treated chondrocyte cells in Example 6 of the present application; In the figure, CON is cartilage cells (control), IL-1β is IL-1β treated cartilage cells, IL-1β+DPSC-Exo is dental pulp stem cell exosome incubated IL-1β treated cartilage cells; Figure 16 ATP content determination for cartilage cells (control), IL-1β treated cartilage cells, dental pulp stem cell exosome incubated IL-1β treated cartilage cells in Example 6 of the present application; In the figure, CON is cartilage cells (control), IL-1β is IL-1β treated cartilage cells, IL-1β+DPSC-Exo is dental pulp stem cell exosome incubated IL-1β treated cartilage cells; Figure 17 The figure is the articular cartilage tissue hematoxylin-eosin staining section of normal mice, OA mice, OA mice injected with dental pulp stem cell exosomes in Example 7 of the present application; In the figure, WT is a normal mouse, DMM is an OA mouse, and DMM+DPSC-exo is an OA mouse injected with dental pulp stem cell exosomes into the joint cavity; Figure 18 The figure is the classification of the severity of osteoarthritis of normal mice, OA mice, OA mice injected with dental pulp stem cell exosomes into the joint cavity in Example 7 of the present application; In the figure, WT is a normal mouse, DMM is an OA mouse, and DMM+DPSC-exo is an OA mouse injected with dental pulp stem cell exosomes into the joint cavity; Figure 19 The figure is the analysis of cartilage tissue thickness of normal mice, OA mice, OA mice injected with dental pulp stem cell exosomes into the joint cavity in Example 7 of the present application; In the figure, WT is a normal mouse, DMM is an OA mouse, and DMM+DPSC-exo is an OA mouse injected with dental pulp stem cell exosomes into the joint cavity; Figure 20 The figure is the analysis of cartilage tissue thickness of normal mice, OA mice, OA mice injected with dental pulp stem cell exosomes into the joint cavity in Example 7 of the present application; In the figure, WT is a normal mouse, DMM is an OA mouse, and DMM+DPSC-exo is an OA mouse injected with dental pulp stem cell exosomes into the joint cavity. DETAILED DESCRIPTION
[0018] The technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.
[0019] Example 1 Culture and identification of dental pulp stem cells: S1, freshly extracted healthy teeth were immediately placed in complete culture medium containing antibiotics, washed and all tissues outside the teeth were removed, then the crown was split longitudinally with a bone chisel to expose the dental pulp cavity, the dental pulp tissue was removed with forceps and stored in complete culture medium; S2, the dental pulp tissue was cut into small pieces of 3mm 2 in size, 10 times the volume of 2mg / mL collagenase type I digestion solution was added, and after 50min of digestion at 200rpm and 37°C, the precipitate was mixed with complete culture medium containing antibiotics, filtered with a 200 mesh cell sieve, and then centrifuged at 1000rpm for 5min to obtain dental pulp stem cells; Microscopic observation of dental pulp stem cells was performed, as shown in Figure 1 ; The results of the determination of the relative mRNA expression of dental pulp stem cell marker molecules CD73 and CD90, and hematopoietic markers CD34 and CD45, are shown in Figure 2 .
[0020] Example 2 Culture and identification of articular chondrocytes: Five-day-old mice were selected, the articular cartilage was isolated under anesthesia, the fascia and perichondrium wrapped around the cartilage tissue were stripped, and the cartilage tissue was placed in a culture dish containing phosphate buffered saline; the isolated cartilage tissue was cut into small pieces of 0.4mm 2 in size, 10 times the volume of 0.25% trypsin was added, and after 2 hours of digestion at 37°C, 0.02% collagenase type II was added, and after 4 hours of digestion at 37°C, the filtrate was filtered with a 200 mesh sieve, and then centrifuged at 1000r / min for 5min, and the precipitate was resuspended in complete culture medium; Cell viability was detected by 0.2% trypan blue staining, and the rate of viable cells was greater than 90%, and the chondrocytes were placed in a CO2 incubator for routine culture; The morphology of chondrocytes of generations 1-7 was observed, and the morphology of chondrocytes of the first generation was spherical or ellipsoidal, and the morphology of chondrocytes gradually changed to spindle-shaped as the number of generations increased (chondrocyte cell morphologies of generations 1, 3, 5 and 7 are shown in Figure 3 ), and COL II is a marker molecule of chondrocytes, as shown in Figure 4 , it can be seen that the transcription of COL II decreases as the number of generations of chondrocytes increases. Chondrocytes were stained with HE, and the results showed that the morphology of chondrocytes after HE staining (as shown in Figure 5 ) was consistent with the morphology of chondrocytes observed under a microscope (as shown in Figure 6The main component of cartilage matrix is proteoglycan, and toluidine blue has metachromasia to proteoglycan. The 1st, 3rd, 5th and 7th generations of chondrocytes were subjected to toluidine blue staining, and the proteoglycan content in the extracellular matrix was determined according to the staining intensity of toluidine blue (the darker the staining, the higher the content of proteoglycan). Figure 7 and Figure 8 It can be obtained that the content of proteoglycan in the extracellular matrix is significantly reduced with the increase of the generation number of chondrocytes.
[0021] Example 3 Preparation of dental pulp stem cell exosomes: The dental pulp stem cells obtained in Example 1 were resuspended in complete culture medium and cultured in a culture box. When the confluence of the dental pulp stem cells reached 80%, the cells were cultured in serum-free medium (without penicillin and streptomycin) for 48 hours. Then, the cells were centrifuged at 4℃ and 300xg for 10 minutes, followed by centrifugation at 4℃ and 2000xg for 10 minutes, and then ultracentrifugation at 100000xg for 60 minutes. The obtained precipitate was dental pulp stem cell exosomes.
[0022] Example 4 Analysis of the transcriptome landscape of chondrocytes: The GSE215039 data set in the GEO database was analyzed, and the GEO2R tool was used for differential analysis of untreated chondrocytes (control) and IL-1β treated chondrocytes. The expression of differentially expressed genes between the two samples was calculated, and genes with a fold change of 1 or more were selected as differentially expressed genes (selected according to the volcano plot of differential analysis in Figure 9 ); signal pathway enrichment analysis was performed on the 279 genes; The results show that the differential genes are mainly enriched in signal pathways related to mitochondrial cytochrome P450 metabolism of exogenous substances, glutathione metabolism, oxidative stress, calcium signaling pathway, cell aging and ferroptosis (obtained according to the enrichment analysis diagram of differential genes in Figure 10 ); mitochondrial and endoplasmic reticulum homeostasis are potential targets for inflammatory-induced chondrocyte degeneration.
[0023] Example 5 Effect of the dental pulp stem cell exosomes prepared by the present application on IL-1β induced endoplasmic reticulum stress-induced aging: The potential mechanism of inflammatory-induced chondrocyte degeneration is highly related to mitochondrial and endoplasmic reticulum homeostasis. The endoplasmic reticulum (ER) is the site of protein synthesis and folding, and also the starting point of the secretion pathway. Studies have shown that increased ROS production and unfolded protein response (UPR) in the microenvironment of chondrocytes can activate ER stress, leading to mitochondrial damage and chondrocyte apoptosis. ER stress in chondrocytes plays an important role in the pathogenesis of arthritis. According to the changes in the Western blotting of endoplasmic reticulum stress marker proteins in Figure 11 and the changes in the expression of endoplasmic reticulum stress marker proteins in the chondrocytes treated with the dental pulp stem cell exosomes prepared by the present application, it can be obtained that the dental pulp stem cell exosomes prepared by the present application can effectively alleviate the endoplasmic reticulum stress-induced aging of chondrocytes.Figure 12 The expression levels of BiP, CHOP, and ATF4 in chondrocytes (control) were lower than those in IL-1β-treated chondrocytes. IL-1β-induced chondrocytes were obtained, with increased expression of endoplasmic reticulum stress markers BiP, CHOP, and ATF4. In addition, according to Figure 11 Changes in the protein imprint of endoplasmic reticulum stress marker proteins and Figure 13 When chondrocytes were treated with IL-1β, the expression levels of BiP, CHOP, and ATF4 were higher than when chondrocytes were treated with IL-1β and dental pulp stem cell exosomes simultaneously. This indicates that the dental pulp stem cell exosomes prepared in this invention can reverse the endoplasmic reticulum stress response caused by IL-1β treatment.
[0024] Example 6 The effect of dental pulp stem cell exosomes prepared in this invention on IL-1β-induced mitochondrial stress senescence: MitoTracker staining revealed that IL-1β-treated chondrocytes exhibited reduced mitochondrial length and significantly abnormal mitochondrial morphology, changing from normal rod-shaped and network-like structures to dot-shaped and fragment-like structures, indicating enhanced mitochondrial fragmentation in IL-1β-treated chondrocytes. ATP production was also significantly reduced, leading to mitochondrial dysfunction in chondrocytes. In contrast, treatment of IL-1β-induced chondrocytes with dental pulp stem cell exosomes improved these changes, as shown in Figures 14-16. The results showed that dental pulp stem cell exosomes could regulate inflammation-induced mitochondrial stress-induced senescence in chondrocytes and had a protective effect on chondrocytes; dental pulp stem cell exosomes could inhibit IL-1β-induced mitochondrial and endoplasmic reticulum stress; and dental pulp stem cell exosomes could inhibit endoplasmic reticulum stress-induced senescence and degeneration of cartilage by blocking mitochondrial dysfunction.
[0025] Example 7 The ameliorative effect of dental pulp stem cell exosomes prepared in this invention on the pathological changes of articular cartilage in OA mice: An in vivo mouse model of osteoarthritis (OA) was established. Dental pulp stem cell exosomes were injected intra-articularly to examine their ameliorative effect on the pathological changes of articular cartilage in OA mice. Figure 17 Osteoarthritis severity grading of articular cartilage tissue sections stained with Safranbon-Fix Green from normal mice, OA mice, and OA mice injected intra-articularly with dental pulp stem cell exosomes (e.g., grading of osteoarthritis severity). Figure 18 As shown in the figure, it can be seen that OA mice injected with dental pulp stem cell exosomes into the joint cavity can alleviate the severity of osteoarthritis, and their joint morphology is close to that of normal mice. Figure 19 , Figure 20It was found that intra-articular injection of dental pulp stem cell exosomes could increase the thickness and quantity of articular cartilage in OA mice and restore them to near-normal levels, indicating that dental pulp stem cell exosomes can improve the pathological process in OA mice. Through the above in vitro studies, it was determined that the dental pulp stem cells prepared in this invention can regulate chondrocyte inflammatory response, mitochondrial stress and aging through exocrine mechanisms, thereby affecting senescent degeneration of chondrocytes.
[0026] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. The application of dental pulp stem cell exosomes in the preparation of drugs for the treatment and prevention of osteoarthritis, characterized in that, The method for preparing the dental pulp stem cell exosomes includes the following steps: S1. Obtain dental pulp tissue from the pulp cavity of an extracted tooth and preserve it in a complete culture medium; S2. After pretreating the dental pulp tissue, add digestive solution, digest, centrifuge, discard the supernatant, add complete culture medium containing antibiotics to the precipitate, mix, filter, and centrifuge to obtain dental pulp stem cells. S3. Culterate dental pulp stem cells in complete culture medium. When the confluence of dental pulp stem cells reaches 60%~80%, replace with serum-free culture medium and continue culturing. Collect the conditioned medium of dental pulp stem cells and obtain dental pulp stem cell exosomes after differential centrifugation.
2. The application of dental pulp stem cell exosomes according to claim 1 in the preparation of drugs for treating and preventing osteoarthritis, characterized in that, In step S1, the method for preparing dental pulp tissue includes the following steps: Freshly extracted healthy teeth are immediately placed in a complete culture medium containing antibiotics to clean and remove all tissues and impurities outside the tooth. Then, the crown is split longitudinally with a bone chisel to expose the pulp cavity, and the pulp tissue is removed with forceps.
3. The application of dental pulp stem cell exosomes according to claim 1 in the preparation of drugs for treating and preventing osteoarthritis, characterized in that, In step S2, the pretreatment of the dental pulp tissue includes the following steps: The dental pulp tissue was placed in a 3cm culture dish and minced into pieces of 1-3mm size. 2 Small pieces of dental pulp tissue were added to a complete culture medium containing antibiotics, transferred to a centrifuge tube using a pipette, vortexed to mix, and centrifuged at 1000-1200 rpm to obtain pretreated dental pulp tissue.
4. The application of dental pulp stem cell exosomes according to claim 1 in the preparation of drugs for treating and preventing osteoarthritis, characterized in that, In step S2, the digestive fluid includes one or both of collagenase digestive fluid and tissue digestive fluid.
5. The application of the dental pulp stem cell exosomes according to claim 1 in the preparation of drugs for treating and preventing osteoarthritis, characterized in that, In step S2, the digestion is carried out at 200 rpm and 37°C for 30-60 minutes; the filtration is carried out using a 200-mesh cell sieve.
6. The application of dental pulp stem cell exosomes according to claim 1 in the preparation of drugs for treating and preventing osteoarthritis, characterized in that, In step S3, the complete culture medium is a complete culture medium containing antibiotics.
7. The application of dental pulp stem cell exosomes according to claim 1 in the preparation of drugs for treating and preventing osteoarthritis, characterized in that, In step S3, the differential centrifugation involves first performing refrigerated centrifugation and then ultracentrifugation. The refrigerated centrifugation includes a first refrigerated centrifugation and a second refrigerated centrifugation, and the centrifugal forces of the first refrigerated centrifugation and the second refrigerated centrifugation are different.
8. The application of the dental pulp stem cell exosomes according to claim 7 in the preparation of drugs for treating and preventing osteoarthritis, characterized in that, The centrifugal force of the first freeze centrifugation was 300×g, the time of the first freeze centrifugation was 10min, and the temperature of the first freeze centrifugation was 4℃.
9. The application of the dental pulp stem cell exosomes according to claim 7 in the preparation of drugs for treating and preventing osteoarthritis, characterized in that, The centrifugal force of the second freeze centrifugation is 2000×g, the time of the second freeze centrifugation is 10min, and the temperature of the second freeze centrifugation is 4℃.
10. The application of the dental pulp stem cell exosomes according to claim 7 in the preparation of drugs for treating and preventing osteoarthritis, characterized in that, The centrifugal force of the ultracentrifugation is 100,000 × g, and the ultracentrifugation time is 60 min.