Small extracellular vesicle composite collagen hydrogel injection

Abstract

The invention provides a small extracellular vesicle composite collagen hydrogel injection, and belongs to the technical field of osteoarthritis, and the small extracellular vesicle composite collagen hydrogel injection is prepared by the following steps: S1, mixing a sodium alginate solution with a type I collagen solution to obtain a mixed solution, adding a CaCl2 solution into the mixed solution to form gel, adding an FGF18 solution into the gel, and mixing to obtain FGF18 sodium alginate / collagen hydrogel; s2, transfecting a Lenti-IGF-1-EGF lentiviral vector to P3 generation human umbilical cord mesenchymal stem cells, culturing to obtain P5 generation IGF-1 overexpressed MSCs cells, namely P5 generation IGF-1-MSCs cells, co-culturing with the P5 generation IGF-1-MSCs cells by using curcumin, and separating and extracting a small extracellular vesicle solution rich in curcumin; and S3, mixing the small extracellular vesicle solution rich in curcumin with the FGF18 sodium alginate / collagen hydrogel, so as to obtain the small extracellular vesicle composite collagen hydrogel injection. The invention provides a small extracellular vesicle composite collagen hydrogel injection, which achieves the purposes of relieving inflammatory injury of osteoarthritis and enhancing the treatment effect of osteoarthritis.

Description

Technical Field

[0001] The present invention relates to the technical field of osteoarthritis, and particularly relates to a small extracellular vesicle composite collagen hydrogel injection. Background Art

[0002] Knee osteoarthritis is mainly characterized by the degeneration of knee joint cartilage and the hyperplasia of subchondral bone, and the main clinical manifestations are knee joint pain, swelling, and limited movement, which seriously affect the quality of life of patients and have a high disability rate. At present, in the surgical treatment of osteoarthritis in clinical practice, it usually includes total knee arthroplasty (TKA), knee osteotomy, arthroscopic debridement surgery, autologous chondrocyte implantation (ACI), etc. Usually, patients with advanced osteoarthritis can be treated by artificial knee joint replacement, but artificial knee joint replacement has problems such as high treatment costs and many complications. Therefore, patients usually prefer non-surgical treatment methods. Non-surgical treatment includes physical therapy mainly based on appropriate exercise, weight loss, etc. and drug therapy mainly based on drugs such as non-steroidal anti-inflammatory drugs and painkillers. The current drug delivery method for drug treatment is mainly oral administration, but there are corresponding problems. For example, oral non-steroidal anti-inflammatory drugs will affect the gastrointestinal tract and cardiovascular functions and have potential safety hazards. Therefore, non-steroidal anti-inflammatory drugs are suitable for short-term use in small doses; opioid analgesics such as tramadol are restricted in clinical application due to their easy addiction with long-term use.

[0003] Intra-articular injection is one of the most attractive methods for treating osteoarthritis, which can significantly increase the local concentration of drugs and reduce the occurrence of systemic adverse reactions. Chinese Patent with publication number CN118403006A and title "Use of cross-linked sodium hyaluronate injection for treating osteoarthritis" discloses the use of cross-linked sodium hyaluronate injection for treating osteoarthritis, characterized in that: in the cross-linked sodium hyaluronate injection, divinyl sulfone is used as the only cross-linking agent. Although the above technical solution reduces the number of drug administrations compared with the existing injection and reduces the incidence of side effects such as inflammation and infection caused by repeated intra-articular injections, there is still a lack of an injection for increasing the treatment effect of osteoarthritis. Summary of the Invention

[0004] In view of this, the present invention provides a small extracellular vesicle composite collagen hydrogel injection to achieve the purpose of alleviating the inflammatory damage of osteoarthritis and enhancing the treatment effect of osteoarthritis.

[0005] To achieve the above purpose, the present invention provides a small extracellular vesicle composite collagen hydrogel injection, including the following steps: S1. Take a sodium alginate solution and a type I collagen solution and mix them to obtain a mixed solution. Add a CaCl2 solution to the mixed solution to form a gel, and add an FGF18 solution to the gel and mix to obtain an FGF18 sodium alginate / collagen hydrogel; S2. Transfect the Lenti-IGF-1-EGF lentiviral vector into passage 3 human umbilical cord mesenchymal stem cells. After culturing, passage 5 MSCs cells overexpressing IGF-1 are obtained, namely passage 5 IGF-1-MSCs cells. Co-culture curcumin with passage 5 IGF-1-MSCs cells, and isolate and extract a small extracellular vesicle solution rich in curcumin. S3. Mix the small extracellular vesicle solution rich in curcumin with FGF18 sodium alginate / collagen hydrogel to obtain a small extracellular vesicle composite collagen hydrogel injection.

[0006] Optionally, the sodium alginate solution is obtained by dissolving sodium alginate in deionized water, and the mass concentration of the sodium alginate solution is 10 - 15 g / L; the type I collagen solution is obtained by dissolving type I collagen in an acetate buffer solution, and the mass concentration of the type I collagen solution is 2 - 5 g / L; the volume ratio of the sodium alginate solution to the type I collagen solution is 2:1.

[0007] Optionally, the concentration of the CaCl2 solution is 0.3 - 0.5 mol / L.

[0008] Optionally, adding the FGF18 solution to the gel includes sequentially adding FGF18 solutions with different concentrations to the gel.

[0009] Optionally, adding the FGF18 solution to the gel includes sequentially adding FGF18 solutions with concentrations of 20 ug / ml, 40 ug / ml, and 80 ug / ml to the gel respectively.

[0010] Optionally, the culturing method of the passage 5 IGF-1-MSCs cells includes the following steps: Inoculate passage 3 human umbilical cord mesenchymal stem cells into a T25 culture flask. When the cell density grows to 70 - 80%, add the Lenti-IGF-1-EGF lentivirus with a multiplicity of infection MOI-40, mix well and culture, and replace with fresh complete medium for mesenchymal stem cells. Continuously culture and passage until passage 5.

[0011] Optionally, when co-culturing curcumin with passage 5 IGF-1-MSCs cells, the concentration of curcumin is 4 - 5 µmmol / L.

[0012] Optionally, when mixing the small extracellular vesicle solution rich in curcumin with FGF18 sodium alginate / collagen hydrogel, the concentration ratio of the small extracellular vesicle solution rich in curcumin to FGF18 sodium alginate / collagen hydrogel is 1:2.

[0013] Optionally, the concentration of the small extracellular vesicle solution rich in curcumin is 20 ug / ml, and the concentration of the FGF18 sodium alginate / collagen hydrogel is 40 ug / ml.

[0014] The above technical solutions of the present invention have at least the following beneficial effects: In the technical solution of the present invention, the combination of sodium alginate and collagen is based on the fact that the injectable hydrogel of sodium alginate has similarity with the natural extracellular matrix, good porosity and biodegradability, and is widely used in tissue engineering. And collagen is the main component of the extracellular matrix of bone and cartilage. Therefore, the composite hydrogel of sodium alginate and collagen can simulate the chondrocyte microenvironment and provide an excellent drug delivery carrier for intra-articular injection.

[0015] In addition, FGF18 is a synthetic growth factor. In addition to participating in articular cartilage repair, it is also related to chondrogenesis and osteogenesis; recombinant human FGF-18 can significantly reduce the loss of cartilage thickness and volume and the narrowing of joint space width; FGF-18 shows good OA treatment effects in in vitro and in vivo experiments and is expected to become a new type of OA treatment method. Chondrocytes can proliferate and synthesize extracellular matrix under the action of IGF-1. Chondrocytes are also regulated by IGF-1 while secreting IGF-1. IGF-1 can significantly accelerate the synthesis of proteoglycans and promote cartilage repair.

[0016] Curcumin is a polyphenolic drug extracted from turmeric rhizomes and has anti-inflammatory, anti-tumor and other effects. However, due to the disadvantages of poor water solubility and poor permeability of curcumin, the medicinal value of curcumin is greatly reduced. In recent years, the research on exosomes as a drug delivery system to improve the efficacy of traditional Chinese medicine has attracted much attention.

[0017] Small extracellular vesicles can play an important role as drug carriers in a variety of diseases. They are not only the main effective components for MSC to repair cartilage damage, but also have the advantages of easy collection and storage, good stability, strong targeting and low immune rejection, and have great application potential in the field of tissue repair.

[0018] In summary, the present invention combines and applies the above-mentioned cytokine FGF-18 with potential effects on osteoarthritis as a basic drug and adds it into the sodium alginate and collagen hydrogel to form a functional hydrogel. Then, the IGF-1 gene is transferred into human umbilical cord mesenchymal stem cells in vitro to make IGF-1 highly expressed. Then, curcumin is used to stimulate and culture the IGF-1 overexpressing human umbilical cord mesenchymal stem cells in vitro to obtain a small extracellular vesicle solution rich in curcumin and IGF-1. Finally, the small extracellular vesicle solution is integrated into the FGF-18 sodium alginate / collagen hydrogel as an injectable preparation with good efficacy and high safety for osteoarthritis and can replace traditional treatments. Brief Description of the Drawings

[0019] Figure 1This is the CCK8 proliferation activity graph of FGF18 sodium alginate / collagen hydrogel in the embodiments of the present invention; Figure 2 This is the chondrocyte ACAN level expression graph of umbilical cord mesenchymal stem cells in FGF18 sodium alginate / collagen hydrogel in the embodiments of the present invention; Figure 3 This is the chondrocyte COL2A1 level expression graph of umbilical cord mesenchymal stem cells in FGF18 sodium alginate / collagen hydrogel in the embodiments of the present invention; Figure 4 This is the chondrocyte SOX9 level expression graph of umbilical cord mesenchymal stem cells in FGF18 sodium alginate / collagen hydrogel in the embodiments of the present invention; Figure 5 This is the mRNA expression level graph of IGF-1 in the embodiments of the present invention; Figure 6 This is one of the surface marker expression graphs of IGF-1-MSCs in the embodiments of the present invention; Figure 7 This is one of the surface marker expression graphs of IGF-1-MSCs in the embodiments of the present invention; Figure 8 This is the histological evaluation (20X, 100X) graph of articular cartilage tissue under safranin-fast green staining in the embodiments of the present invention. Detailed implementation manners

[0020] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the described embodiments of the present invention fall within the scope of protection of the present invention.

[0021] Embodiments 1. Preparation of FGF18 sodium alginate / collagen hydrogel Dissolve sodium alginate in deionized water to prepare a sodium alginate solution with a concentration of 10-15 g / L; dissolve type I collagen in acetic acid buffer to prepare a type I collagen solution with a concentration of 2-5 g / L. Mix the sodium alginate solution and the type I collagen solution at a volume ratio of 2:1, and slowly add a 0.4 mol / L CaCl2 solution to the above-mentioned sodium alginate / collagen mixed solution containing it, so that the mixed solution crosslinks to form a gel. Subsequently, add FGF18 solutions with concentrations of 20 μg / ml, 40 μg / ml, and 80 μg / ml to the above-mentioned gel in sequence for mixing to form FGF18 sodium alginate / collagen hydrogel.

[0022] 2. Biocompatibility evaluation of FGF18 sodium alginate / collagen hydrogel 2.1 Cell proliferation activity detection Umbilical cord mesenchymal cells were seeded into 24-well plates at a density of 1×10 4 / well, and 0.5 ml of sodium alginate / collagen hydrogel (control group), 20 μg / ml FGF18 sodium alginate / collagen hydrogel (experimental group 1), 40 μg / ml sodium alginate / collagen hydrogel (experimental group 2), and 80 μg / ml sodium alginate / collagen hydrogel (experimental group 3) were added to each well. Cells cultured alone were used as the blank control. After co-culturing for 1 day, 3 days, 5 days, and 7 days, 10% of the medium volume of CCK-8 reagent was added to each well and incubated for 2 h, and the absorbance value was measured at 450 nm. The detection results are shown in Figure 1 .

[0023] 2.2 Cartilage differentiation ability detection Umbilical cord mesenchymal cells were seeded into 6-well plates at a density of 5×10 5 / well, and 1 ml of sodium alginate / collagen hydrogel (control group), 20 μg / ml FGF18 sodium alginate / collagen hydrogel (experimental group 1), 40 μg / ml sodium alginate / collagen hydrogel (experimental group 2), and 80 μg / ml FGF18 sodium alginate / collagen hydrogel (experimental group 3) were added to each well. After culturing for 14 days, the cells were harvested, total RNA was extracted, and the mRNA levels of SOX-9, COLⅡ, and proteoglycan Aggrecan were detected. The detection results are shown in Figure 2 , Figure 3 , Figure 4 .

[0024] 3. Preparation and phenotypic identification of IGF-1 overexpressing human umbilical cord mesenchymal stem cells (IGF-1-MSCs) P3 human umbilical cord mesenchymal stem cells (HucMSCs) were seeded into T25 culture flasks. When the cell density grew to 70 - 80%, Lenti-IGF-1-EGF lentivirus with a multiplicity of infection MOI-40 was added, mixed and cultured, and then fresh complete mesenchymal stem cell medium was replaced. After culturing for 48 h, when the cell transfection efficiency reached 98% under a fluorescence microscope, the cells were collected and seeded into 6-well plates. After culturing for 48 h, they were divided into MSCs group, vector-MSCs group, and IGF-1-MSCs group, and the expression of IGF-1 gene in the cells was detected by real-time quantitative PCR. The detection results are shown in Figure 5 ; .

[0025] Take the above-mentioned P5 IGF-1-MSCs cells with stable growth, centrifuge at 500 g for 5 min to discard the supernatant, wash twice with PBS and then resuspend, and adjust the cell density to 1×10 6cells / mL. Take 200 μL of the cell suspension and aliquot it into flow cytometry tubes. Add human monoclonal antibodies FITC-CD73, PE-CD90, PE-CD105, PC5.5-CD34, PC5.5-CD19, APC-CD45, APC-CD11b, and FITC-HLA-DR to each tube for labeling. After mixing, incubate in the dark at room temperature for 15 min, wash with PBS, resuspend in 500 μL of PBS, and then detect by flow cytometry. Analyze the results using Cell-Quest software. The test results are shown in Figure 6 , Figure 7 .

[0026] 4. Preparation of Curcumin-Induced Small Extracellular Vesicle Composite FGF18 Alginate / Collagen Hydrogel After co-culturing 6 μmmol / L curcumin with P5 IGF-1-MSCs for 72 h, isolate and extract the simple small extracellular vesicle solution (IGF-1-MSCs-EV) and the small extracellular vesicle solution rich in curcumin (curcumin-IGF-1-MSCs-EV). Mix the above two groups of exosome solutions with a concentration of 20 μg / ml with FGF18 alginate / collagen hydrogel with a concentration of 40 μg / ml to form FGF18 alginate / collagen hydrogel of simple IGF-1-MSCs-EV (experimental group 5) and FGF18 collagen hydrogel of curcumin-induced IGF-1-MSCs-EV (experimental group 6).

[0027] 5. Efficacy Evaluation of Small Extracellular Vesicle Collagen Hydrogel in an Osteoarthritis Animal Model 5.1 Construction and Grouping of a Rabbit Knee Osteoarthritis Animal Model Select 20 healthy adult New Zealand white rabbits. Randomly divide the experimental animals into a blank group, a model control group, experimental group 5, and experimental group 6, with 5 rabbits in each group. Anesthetize the rabbits by intravenous injection of 3% sodium pentobarbital at a dose of 30 mg / kg through the marginal ear vein. Routinely prepare the surgical area by shaving and disinfecting. Make a longitudinal incision about 2 cm long on the medial side of both knees, cut through the skin and joint capsule to enter the joint cavity, retract the patella laterally, fully flex the knee joint to expose the knee joint, cut the anterior cruciate ligament and perform an anterior drawer test to confirm complete cutting. Pay attention to protecting the articular cartilage surface during the operation. Irrigate the joint cavity with normal saline and suture the joint capsule and skin layer by layer. After suturing, intramuscularly inject penicillin (400,000 U / rabbit / day for 3 days after surgery) to prevent infection. Do not fix the injured limb after surgery, and let the rabbits move freely.

[0028] 4.2 Administration and Treatment by Intra-Articular Injection in Experimental Animals Arthroscopic cavity injection was performed 2 weeks after model establishment. The animals were taken out, placed on a fixing rack for fixation, the wound condition of the animals was observed, the wound was routinely disinfected with iodophor, and arthroscopic cavity injection was performed. In the experimental group, 0.4 ml of the exosome hydrogel solution obtained from the above experimental groups 5 and 6 was injected into each knee joint, and the model group was given an equal amount of normal saline. After 8 weeks of feeding, the animals were sacrificed, and articular cartilage tissue and synovial tissue were taken.

[0029] 4.3 Histopathological observation Safranin O-fast green staining was performed on the femoral condyle and tibial plateau. The femoral condyle and tibial plateau tissues were first soaked in 10% EDTA for decalcification treatment (about 8 - 10 weeks) until the tissue could be easily pierced with a needle. After being examined by an X-ray machine and no calcium remained, they were then dehydrated by conventional gradient and embedded in paraffin. 5-μm thick continuous sections were made on a microtome for further staining analysis. The test results are shown in Figure 8 .

[0030] 4.4 ELISA detection of the expression levels of inflammatory factors The synovial tissue of the animals was extracted, weighed, and a certain amount of PBS (pH 7.4) was added. The specimens were homogenized thoroughly by hand or with a homogenizer. Centrifuge for about 20 minutes (2000 - 3000 revolutions per minute), and carefully collect the supernatant. One portion was reserved for testing after aliquoting, and the rest were frozen for standby. According to the ELISA kit instructions for IL-6, IL-10, TNF-α, and TGF-β, the inflammatory factor levels in the synovial tissue were detected, and the test results are shown in Table 1.

[0031] Table 1 ELISA assessment of the expression levels of inflammatory factors TNF-α, TGF-β, IL-6, and IL-10

[0032] As shown in Table 1, compared with normal rabbits, the levels of pro-inflammatory factors TNF-α and IL-6 in the synovial tissue of the model group were significantly increased (P < 0.01), while the levels of anti-inflammatory factors TGF-β and IL-10 were significantly decreased, indicating that the establishment of the degenerative knee osteoarthritis model was successful. After treatment with stem cell exosome hydrogel after model establishment, the levels of TNF-α and IL-6 in the synovial tissue of the two experimental groups decreased (P < 0.01), and the levels of TGF-β and IL-10 increased. Among them, the levels of TNF-α and IL-6 in experimental group 6 were significantly lower than those in experimental group 5, and the expression levels of TGF-β and IL-10 were significantly higher than those in experimental group 5. It was proved that the sodium alginate / collagen hydrogel prepared by the small extracellular vesicles derived from human umbilical cord mesenchymal stem cells with overexpression of IGF-1 induced by curcumin combined with 40 μg / ml FGF18 could significantly reduce the levels of TNF-α and IL-6 in the joint, promote the secretion of TGF-β and IL-10, thereby alleviating the inflammatory injury of the bone joint and delaying the progression of the course of knee osteoarthritis.

[0033] As shown by Figure 1 it can be seen that all groups have a certain promoting effect on the growth of umbilical cord mesenchymal stem cells. Compared with the control group, there is no obvious trend in experimental group 1, there is a significant upward trend in experimental group 2, and there is a downward trend in experimental group 3. Among them, the upward trend of experimental group 2 is more obvious than the other two groups on the 5th and 7th days, indicating that compared with the simple sodium alginate / collagen hydrogel, the sodium alginate / collagen hydrogel added with 20 μg / ml of FGF18 has no obvious promoting effect on the growth of umbilical cord mesenchymal stem cells, the sodium alginate / collagen hydrogel added with 80 μg / ml of FGF18 has a certain inhibitory effect on the growth of umbilical cord mesenchymal stem cells, while the sodium alginate / collagen hydrogel with 40 μg / ml of FGF18 has a very significant promoting proliferation activity on umbilical cord mesenchymal stem cells.

[0034] As shown by Figure 2 and 3 and Figure 4, it can be seen that all experimental groups have good chondrogenic differentiation induction ability compared with the control group. Among them, the relative mRNA expression level of the chondrogenic differentiation surface marker gene ANCN in experimental group 2 (10.1) is significantly higher than that in experimental group 1 (3.05) and experimental group 3 (2.6), its relative mRNA expression level of COL2A1 (12.9) is much higher than that in experimental group 1 (2.7) and experimental group 3 (3.05), and its relative mRNA expression level of SOX9 (15.4) is significantly higher than that in experimental group 1 (4.05) and experimental group 3 (3.85); the results show that the sodium alginate / collagen hydrogel synthesized with 40 μg / ml of FGF18 has the best effect on the chondrogenic differentiation induction ability of umbilical cord mesenchymal stem cells.

[0035] As shown by Figure 5 it can be seen that there are significant differences in the IGF-1 gene expression between the MSCs group and the vector-MSCs group compared with the IGF-1-MSCs group; as shown by Figure 6 and 7 it can be seen that the expression levels of the positive markers of IGF-1-MSCs are all higher than 95%, and the expression levels of its negative markers are all lower than 2%, indicating that human umbilical cord mesenchymal stem cells with overexpressed IGF-1 are successfully constructed.

[0036] As shown by Figure 8It can be seen that compared with the blank group, typical OA changes occurred in the femoral condyle and tibial plateau in the model group. Safranin-fast green staining showed that in the model group, the staining depth and area of the cartilage matrix decreased significantly, the tidemark disappeared, and acellularization occurred; in experimental group 5, the staining depth and area of the cartilage matrix increased, and the tidemark was visible; in experimental group 6, compared with experimental group 5, the staining depth and area of the cartilage matrix increased significantly, the tidemark was clearly visible, the surface was regular, and the chondrocytes were arranged in columns. It can be seen that the chondrogenic ability of the sodium alginate / collagen hydrogel prepared with small extracellular vesicles overexpressing IGF-1 induced by curcumin combined with FGF18 is higher than that of the sodium alginate / collagen hydrogel prepared with small extracellular vesicles overexpressing IGF-1 combined with FGF18.

[0037] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art of the present technology, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A small extracellular vesicle composite collagen hydrogel injection, characterized in that, It includes the following steps: S1. Take a sodium alginate solution and a type I collagen solution and mix them to obtain a mixed solution. Add a CaCl2 solution to the mixed solution to form a gel, and add an FGF18 solution to the gel and mix to obtain an FGF18 sodium alginate / collagen hydrogel; S2. Transfect the Lenti-IGF-1-EGF lentiviral vector into passage 3 human umbilical cord mesenchymal stem cells, and after culturing, obtain passage 5 MSCs cells overexpressing IGF-1, that is, passage 5 IGF-1-MSCs cells. Use curcumin to co-culture with passage 5 IGF-1-MSCs cells, and isolate and extract a small extracellular vesicle solution rich in curcumin; S3. Mix the small extracellular vesicle solution rich in curcumin with the FGF18 sodium alginate / collagen hydrogel to obtain a small extracellular vesicle composite collagen hydrogel injection.

2. The small extracellular vesicle composite collagen hydrogel injection according to claim 1, wherein The sodium alginate solution is obtained by dissolving sodium alginate in deionized water, and the mass concentration of the sodium alginate solution is 10-15 g / L; the type I collagen solution is obtained by dissolving type I collagen in an acetate buffer solution, and the mass concentration of the type I collagen solution is 2-5 g / L; the volume ratio of the sodium alginate solution to the type I collagen solution is 2:

1.

3. The small extracellular vesicle composite collagen hydrogel injection according to claim 1, characterized in that, The concentration of the CaCl2 solution is 0.3-0.5 mol / L.

4. The small extracellular vesicle composite collagen hydrogel injection according to claim 1, wherein Adding the FGF18 solution to the gel includes sequentially adding FGF18 solutions with different concentrations to the gel.

5. The small extracellular vesicle composite collagen hydrogel injection according to claim 4, wherein Adding the FGF18 solution to the gel includes sequentially adding FGF18 solutions with concentrations of 20 ug / ml, 40 ug / ml, and 80 ug / ml to the gel respectively.

6. The small extracellular vesicle composite collagen hydrogel injection according to claim 1, wherein The culture method of the passage 5 IGF-1-MSCs cells includes the following steps: Take passage 3 human umbilical cord mesenchymal stem cells and inoculate them into a T25 culture flask. When the cell density grows to 70-80%, add the Lenti-IGF-1-EGF lentivirus with a multiplicity of infection MOI-40, mix and culture, and replace with fresh complete medium for mesenchymal stem cells, and continuously culture and passage to passage 5.

7. The small extracellular vesicle composite collagen hydrogel injection according to claim 1, characterized in that When using curcumin to co-culture with passage 5 IGF-1-MSCs cells, the concentration of curcumin is 4-5 µmmol / L.

8. The small extracellular vesicle composite collagen hydrogel injection according to claim 1, wherein When mixing the small extracellular vesicle solution rich in curcumin with the FGF18 sodium alginate / collagen hydrogel, the concentration ratio of the small extracellular vesicle solution rich in curcumin to the FGF18 sodium alginate / collagen hydrogel is 1:

2.

9. The small extracellular vesicle composite collagen hydrogel injection according to claim 8, wherein The concentration of the small extracellular vesicle solution rich in curcumin is 20 ug / ml, and the concentration of the FGF18 sodium alginate / collagen hydrogel is 40 ug / ml.

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