Temperature-sensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis as well as preparation method and application of temperature-sensitive hydrogel mesenchymal stem cell composition

Abstract

The invention relates to a temperature-sensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis, a preparation method and application, the composition mainly comprises mesenchymal stem cells, a microcarrier and temperature-sensitive hydrogel, and the dosage ratio of the mesenchymal stem cells to the microcarrier is 1000-50000 million-1g of the microcarrier, and the dosage ratio of the temperature-sensitive hydrogel to the microcarrier is 10-15000 million-1g of the microcarrier. The microcarrier and the mesenchymal stem cells form the mesenchymal stem cells loaded with the microcarrier, and the mesenchymal stem cells loaded with the microcarrier and the temperature-sensitive hydrogel are mixed before being used to form the composition. According to the composition disclosed by the invention, the mesenchymal stem cells can play a slow release role, can continuously survive for more than 90 days, and can be fixed at a cartilage deletion part to stably play a cell differentiation role.

Description

Technical Field

[0001] The present application relates to the technical field of drugs for treating knee osteoarthritis, and specifically to a thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis, a preparation method and application thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of specialized adult stem cells that are widely present in various tissues. They have the ability of self-renewal and multidirectional differentiation. They also have powerful immunoregulatory and anti-inflammatory effects with no toxic side effects. Umbilical cord mesenchymal stem cells have lower immunogenicity and immunoregulatory ability because they are derived from more primitive umbilical cord tissue.

[0003] Osteoarthritis is a chronic, progressive disease characterized by a pathological imbalance in the degeneration and repair process of the entire joint and its components, accompanied by secondary inflammatory changes, especially in the synovium and articular cartilage. Currently, in addition to steroidal anti-inflammatory drugs, corticosteroids, and sodium hyaluronate, mesenchymal stem cells are also a common treatment for knee osteoarthritis and are included in the Chinese Guidelines for the Diagnosis and Treatment of Osteoarthritis (2024 edition). Although mesenchymal stem cells have cartilage regeneration and immunomodulatory abilities, when injected into the joint cavity, they have a very short survival time in the absence of sufficient vascular supply of nutrients. The presence of cells is almost undetectable within a week, and it is difficult for them to continue to function.

[0004] Mesenchymal stem cells are already a common treatment for knee osteoarthritis. The main methods currently include direct injection, combined with hyaluronic acid and PRP, etc. These treatments have a therapeutic effect on knee osteoarthritis to a certain extent. Because mesenchymal stem cells are an active drug, they cannot work continuously. If you want to obtain continuous efficacy, you can only use them frequently. However, the preparation of mesenchymal stem cells is complicated and expensive. Long-term and frequent use will inevitably bring a heavy economic burden to patients. If stem cells cannot be used for a long time, the efficacy will disappear after a period of time, and inflammation will continue to occur, making it difficult for patients to fundamentally solve the problem. Allowing mesenchymal stem cells to work for a long time and fix them in the area where cartilage is missing is a long-term problem in stem cell treatment of knee osteoarthritis. Summary of the invention

[0005] In response to the above-mentioned deficiencies in the prior art, the present application provides a thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis, which can enable mesenchymal stem cells to have a sustained release effect, can survive for more than 90 days, and can be fixed at the site of cartilage loss and stably exert cell differentiation.

[0006] In order to solve the above technical problems, the technical solution adopted in the present application is: a thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis, the composition mainly comprising mesenchymal stem cells, microcarriers and thermosensitive hydrogel, the dosage ratio of the mesenchymal stem cells and the microcarriers is: 10-50 million mesenchymal stem cells / g microcarrier (that is, 10-50 million mesenchymal stem cells are added per gram of microcarrier), the microcarriers and mesenchymal stem cells form mesenchymal stem cells loaded with microcarriers, and the mesenchymal stem cells loaded with microcarriers are mixed with the thermosensitive hydrogel before use to form the composition (that is, before injection, the two are stored separately and mixed to form a composition when needed).

[0007] Furthermore, the mesenchymal stem cells include but are not limited to at least one of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, and adipose mesenchymal stem cells.

[0008] Furthermore, the mesenchymal stem cells are umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cells have lower immunogenicity and stronger immunoregulatory ability because the umbilical cord tissue they are derived from is more primitive).

[0009] Furthermore, the thermosensitive hydrogel is a mixture of poloxamer P407 and gel preservation solution, wherein the mass concentration of poloxamer P407 is in the range of 14-20%.

[0010] Furthermore, the cost of the gel preservation solution includes: 2% sodium hyaluronate, 58% Propidium A, 5% human albumin, and 35% glucose injection; the above-mentioned components are the mass percentage of each substance in the gel preservation solution.

[0011] Furthermore, the microcarriers and mesenchymal stem cells form mesenchymal stem cells loaded with microcarriers, and the volume ratio of the mesenchymal stem cells loaded with microcarriers to the thermosensitive hydrogel is 1:0.5-1.5.

[0012] The present application also provides a method for preparing the above-mentioned thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis, the method comprising: (1) The mesenchymal stem cells and microcarriers are cultured in a sealed stirring container for 5-7 days. After the culture is completed, the mesenchymal stem cells containing the microcarriers are washed by low-speed centrifugation, and the obtained complex is placed in a cell preservation solution to obtain the mesenchymal stem cells loaded with microcarriers; (2) mixing poloxamer P407 and gel preserving solution to prepare a poloxamer P407 aqueous solution (thermosensitive hydrogel); (3) The mesenchymal stem cells loaded with microcarriers prepared in step (1) and the poloxamer P407 aqueous solution prepared in step (2) are placed at 2-8° C. and packaged for storage and transportation respectively; before injection, they are mixed to obtain a thermosensitive hydrogel composition for treating knee osteoarthritis.

[0013] Furthermore, the culture described in step (1) is cultured in a culture medium, the components of which include: 500 ml of MSC-T4 serum-free culture medium (MSC-T4 human mesenchymal stem cell serum-free culture medium) and 5% mass concentration of platelet lysate (helios), and the culture medium obtained by mixing the two is the complete mesenchymal stem cell culture medium.

[0014] Furthermore, the components of the cell preservation solution described in step (1) include: 5% human albumin, 60% compound electrolyte injection (Bopuli A), 20% 10% glucose injection (that is, in the cell preservation solution, the percentage of 10% glucose injection is 20%), and 15% compound amino acid injection; the above components are the mass percentages of each substance in the cell preservation solution.

[0015] Furthermore, the mass concentration of poloxamer P407 in the poloxamer P407 aqueous solution in step (2) is in the range of 14-20%.

[0016] Furthermore, the gel preservation solution described in step (2) includes: 2% sodium hyaluronate, 58% Propidium A, 5% human albumin, and 35% glucose injection; the above components are the mass percentages of the respective substances in the gel preservation solution.

[0017] Furthermore, the poloxamer P407 aqueous solution in step (2) is in a liquid state at 2-8°C and in a gel state at above 25°C.

[0018] Furthermore, the volume ratio of the mesenchymal stem cells loaded with microcarriers and the poloxamer P407 aqueous solution, i.e., the thermosensitive hydrogel, in step (3) is 1:0.5-1.5.

[0019] Furthermore, before use, the mesenchymal stem cells loaded with microcarriers obtained in step (1) are mixed with the poloxamer P407 aqueous solution (thermosensitive hydrogel) obtained in step (2) to obtain a cell hydrogel composition, which is then immediately injected into the joint cavity, and the joint is slowly and passively moved for 5 to 20 times. The cell hydrogel composition will quickly become a gel under the action of body temperature, so that the cell hydrogel composition can be evenly dispersed in the diseased joint cartilage and synovial surface.

[0020] The present application also provides a use of the above-mentioned thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis in the preparation of a drug for treating knee osteoarthritis.

[0021] 1. The present application combines mesenchymal stem cells with microcarriers to form a composition, wherein the microcarrier is a material made of honeycomb collagen, which can be degraded in the body and is composed of tens of thousands of elastic three-dimensional porous microcarriers with a particle size of about 500 μm and a large surface area, which can achieve high-density cell production and realize true adherent bionic culture; and after being combined with mesenchymal stem cells, the mesenchymal stem cells can grow along the honeycomb wall of the microcarrier, adhere firmly to the wall, and are difficult to digest without the use of collagenase; therefore, this composition loaded with microcarriers can well enable the mesenchymal stem cells to have a sustained release effect, can survive for more than 90 days, and can stably and stably play a cell differentiation role at the site of cartilage loss.

[0022] 2. After the mesenchymal stem cells of the present application are mixed with the microcarriers, they need to be placed in a preservation solution; the formula of the preservation solution is as follows: 5% human albumin + 60% compound electrolyte injection (Bopuli A) + 20% 10% glucose injection + 15% compound amino acid injection. The main function of this preservation solution with a specific composition is to maintain the nutrition of mesenchymal stem cells on the microcarriers, allowing the cells to grow on the microcarriers for a long time. Ordinary culture media are of cell culture grade and cannot be used for human bodies. The culture medium of the present application is prepared with pharmaceutical-grade reagents, which not only ensures cell growth, but also ensures the safety of human growth.

[0023] 3. The composition of the present application, in addition to the main substances microcarrier and mesenchymal stem cells, also provides a thermosensitive hydrogel to be mixed with the composition before being injected into the joint cavity. The thermosensitive hydrogel of the present application is in liquid state at 2-8°C and in gel state at above 25°C. According to the characteristics of the thermosensitive hydrogel, the composition is immediately injected into the joint cavity after mixing (at this time, the thermosensitive hydrogel is in liquid state at 2-8°C, and the mixture formed after mixing is also liquid for easy injection), and the joint is treated by slow passive movement for 5 to 20 minutes. Secondly, the cell suspension is evenly spread on the surface of the diseased joint cartilage and synovium; then under the action of body temperature, the body temperature is generally above 25°C, which makes the thermosensitive hydrogel form a gel state, so that it will be fixed at the diseased site, and the mesenchymal stem cells loaded in the microcarrier will grow slowly and continue to play an anti-inflammatory and tissue repair role, achieving the sustained release effect of mesenchymal stem cells, and realizing the effect of long-term continuous treatment of diseased joints, thereby overcoming the following technical problems existing in the existing use of mesenchymal stem cells to treat knee osteoarthritis: because mesenchymal stem cells are an active drug and cannot continue to work, if you want to continuously obtain therapeutic effects, you can only use them frequently, but the preparation of mesenchymal stem cells is complicated and expensive, and long-term and frequent use will inevitably bring a heavy economic burden to patients. If stem cells cannot be used for a long time, the therapeutic effect will disappear after a period of time, and inflammation will continue to occur, and it is difficult for patients to fundamentally solve the problem. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the bioreactor used in Example 1.

[0025] Figure 2 These are the mesenchymal stem cells loaded by the microcarriers obtained in Example 1.

[0026] Figure 3 This is a picture showing that the thermosensitive hydrogel prepared in Example 2 is in a liquid state at 2-8°C.

[0027] Figure 4 This is a picture of the thermosensitive hydrogel prepared in Example 2 in a gel state above 25°C.

[0028] Figure 5 The thermosensitive hydrogel before mixing in Example 3 (left side) and the mesenchymal stem cells loaded with microcarriers (right side).

[0029] Figure 6 The microcarrier of mesenchymal stem cells observed under a microscope for the mixed composition of Example 3.

[0030] Figure 7 This is a diagram of mesenchymal stem cells slowly crawling out of the gel and microcarriers in the gel state in Example 3.

[0031] Figure 8 The experimental design flow chart and animal experiment grouping for Example 4.

[0032] Fig. 9 These are the gross observation and ICRS scoring data in Example 4.

[0033] Fig.10 The histopathology and Mankin score in Example 4.

[0034] Fig.11 This is the immunohistochemical staining and scoring in Example 4.

[0035] Fig.12 This is the tissue distribution of the human specific gene SRGAP2 in each group of SD rats 90 days after injection in Example 5. DETAILED DESCRIPTION

[0036] The following will combine the embodiments and drawings to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only preferred embodiments, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0037] Case study 1: Microcarriers loaded with mesenchymal stem cells Mix 10-50 million mesenchymal stem cells at a ratio of 1g microcarrier and add the mixed suspension to the bioreactor. Adjust the bioreactor parameters: 35rpm×5min, 1pm×60min, circulate for 24h, and culture at a constant 35rpm for 3-5 days. The culture medium used is: MSC-T4 serum-free medium 500ml (Zhuhai Beso) + 5% platelet lysate (helios), which is the complete mesenchymal stem cell culture medium.

[0038] like Figure 1 The figure shows the bioreactor. After the culture is completed, samples are taken and counted. 100ul of cell suspension is added with an equal volume of AM / PI dye and stained for 30 minutes. The cell adhesion is determined under a fluorescence microscope. Figure 2 , are mesenchymal stem cells loaded on microcarriers. Take 100ul of cell suspension and lyse it with collagenase. After lysis is completed, they can be counted.

[0039] Case study 2: Preparation of thermosensitive hydrogel The purchased pharmaceutical excipient poloxamer P407 is mixed with gel preservative solution at a w / v (weight to volume ratio) to prepare an 18% poloxamer P407 aqueous solution (thermosensitive hydrogel). The 18% poloxamer P407 aqueous solution is in liquid state at 2-8°C and in gel state at above 25°C. The prepared thermosensitive hydrogel is stored at 2-8°C.

[0040] The above-mentioned gel preservation solution is prepared by 2% sodium hyaluronate + 58% Bopilin A + 5% human serum albumin + 35% glucose injection solution.

[0041] As attached Figure 3 As shown, the prepared thermosensitive hydrogel is liquid at 2-8°C. Figure 4 As shown, the prepared thermosensitive hydrogel is in a gel state above 25°C; utilizing the characteristics of this specific thermosensitive hydrogel of the present application, the morphology of the composition can be changed before and after injection, so that it is more convenient to inject when it is a liquid and is evenly distributed at the lesion site, and it is transformed into a gel state under the action of body temperature to achieve position fixation for continuous treatment of the lesion site.

[0042] Case study 3: Mixing of mesenchymal stem cells loaded with microcarriers and thermosensitive hydrogel The mesenchymal stem cells loaded with microcarriers prepared in Example 1 and the thermosensitive hydrogel prepared in Example 2 were separately packaged and stored at 2-8° C. Figure 5 As shown: the left side is the thermosensitive hydrogel, and the right side is the mesenchymal stem cells loaded with microcarriers; they are mixed before injection, and the mesenchymal stem cells loaded with microcarriers and the thermosensitive hydrogel are mixed at a volume ratio of 1:1.0.

[0043] The mixed cells were observed under a microscope to have a gelatinous coating around the microcarriers, such as Figure 6 As shown. After the mixed thermosensitive hydrogel is in the gel state, the culture medium (MSC-T4 serum-free culture medium 500ml (Zhuhai Beso) + 5% platelet lysate (helios) is the complete culture medium for mesenchymal stem cells) is added in vitro and cultured for 3-7 days. Mesenchymal stem cells can be seen crawling out slowly, proving that the cells can survive safely in the microcarrier hydrogel and have no effect on cell survival. Figure 7 shown.

[0044] Case study 4: Validation of efficacy in rabbit KOA model The rabbit KOA model was induced by anterior cruciate ligament transection and meniscectomy (ACLT+MMX) of the left knee. Six weeks after modeling, hydrogel containing MSCs microcarriers was injected into the knee joint cavity. The gross observation of the knee cartilage of each group of rabbits, examination of histological changes, and detection of the expressions of type II collagen (Collagen II) and matrix metalloproteinase 13 (MMP13) were monitored.

[0045] Experimental design and grouping: Figure 8 As shown, the experimental design flow chart and animal experiment grouping: G1 Normal control group G2 Model Group G3 solvent group (microcarrier + thermosensitive hydrogel) G4 MSCs group G5 drug administration group (MSCs-loaded microcarrier + thermosensitive hydrogel) Gross observation and ICRS score: The ICRS score of the model group was 5.33±0.828, and moderate to severe cartilage erosion was observed. The ICRS scores of the vehicle group and MSCs group were 6.0±1.10 and 6.67±1.75, respectively. Compared with the model group, although the apparent score showed an increasing trend, the cartilage erosion phenomenon was not significantly alleviated, and the difference was not statistically significant (P>0.05). The ICRS score of the drug-treated group (MSCs-loaded microcarrier + thermosensitive hydrogel) was 8.8±0.84, and the cartilage damage was significantly less than that of the model group (P<0.001), and cartilage close to normal or slight surface fibrosis was observed. These gross observations indicate that the drug-treated group (MSCs-loaded microcarrier + thermosensitive hydrogel) can slow down the progression of KOA and has a protective effect on cartilage degeneration, see Fig. 9 shown.

[0046] Histopathology and Mankin score: H&E, Safranin O Fast Green Modified Mankin scores of articular cartilage of each group of animals are shown in Fig.10 . The cartilage surface of the normal control group was regular, the expression of proteoglycan was high, the tide line was well intact, there was no obvious KOA feature, and the Modified Mankin score was low. The cartilage surface of the rabbit knee joint in the model group was irregular, cracked, the cell structure was changed, and the tide line integrity was lost. The Modified Mankin score was high, which was 0.33±0.52 and 7.83±1.60, respectively, indicating that the modeling was successful. The Modified Mankin score of the vehicle control group was 6.67±2.34, and the Modified Mankin scores of the MSCs group and the drug administration group (MSCs-loaded microcarrier + thermosensitive hydrogel) were 4.50±1.52 and 3.60±1.14, respectively. The Modified Mankin scores of the vehicle group, MSCs and drug administration group (MSCs-loaded microcarrier + thermosensitive hydrogel) were all lower than those of the Model group. Among them, the drug administration group (MSCs-loaded microcarrier + thermosensitive hydrogel) had a significant effect on the repair of cartilage in the joint, which was significantly different from the Model group (P<0.05). The results of histological evaluation showed that compared with the use of MSCs alone, the MSCs-loaded microcarrier + thermosensitive hydrogel treatment could more effectively protect the cartilage structure, reduce cartilage degenerative changes, and maintain the integrity of the cartilage matrix.

[0047] Immunohistochemistry (Collagen II and MMP13 staining): Immunohistochemical staining results Fig.11As shown in the figure, the Collagen II staining in the model group was weak, indicating that the model construction led to a significant reduction in hyaline cartilage matrix. The ratio of the Collagen II staining area to the total area was measured by Image J analysis, and it was found that the Collagen II density in the microcarrier + thermosensitive hydrogel group loaded with MSCs was significantly higher than that in the model group (P<0.01), indicating that the treatment of the microcarrier + thermosensitive hydrogel loaded with MSCs helps to maintain the content of type II collagen in cartilage. In addition, the expression of MMP13 in the model group and the vehicle control group was significantly increased, indicating that cartilage degenerative lesions persisted in the KOA model without treatment and only receiving vehicle treatment. The ratio of the MMP13 staining area to the total area was measured by Image J analysis, and it was found that the MMP13 density in the microcarrier + thermosensitive hydrogel group loaded with MSCs was significantly lower than that in the Model group (P<0.01), indicating that the treatment of the microcarrier + thermosensitive hydrogel group loaded with MSCs helps to inhibit the production of MMP13. The results of immunohistochemical staining further confirmed that the microcarrier + thermosensitive hydrogel group loaded with MSCs can effectively block the progression of KOA.

[0048] Case study 5: Tissue distribution test verification in SD rat KOA model On the 90th day of administration, the rats' hearts, livers, spleens, lungs, kidneys, whole blood, brains, testes, and knee joints (synovium, cartilage, ligaments, and muscles) were collected, and the expression of the human-specific SGRAP2 gene was detected using qPCR. The results showed that only the G5 group of animals had human umbilical cord mesenchymal stem cell genomes detected in the joint cavity synovium, cartilage, and ligaments on the 90th day, while the human umbilical cord mesenchymal stem cell genomes were not detected in the other administration groups and tissues. Fig.12 Shown is the tissue distribution of the human-specific gene SRGAP2 in each group of SD rats 90 days after injection.

[0049] The above experimental results show that the mesenchymal stem cells loaded with microcarriers of this specific composition in the present application can achieve a sustained release effect by gently mixing with a thermosensitive hydrogel of a specific composition before use, and can survive for more than 90 days, and can be fixed and stably exert cell differentiation at the site of cartilage loss.

Claims

1. A thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis, characterized in that: The composition mainly comprises mesenchymal stem cells, microcarriers and thermosensitive hydrogels. The usage ratio of the mesenchymal stem cells and the microcarriers is 10-50 million mesenchymal stem cells / g microcarriers. The microcarriers and the mesenchymal stem cells form mesenchymal stem cells loaded with microcarriers. The mesenchymal stem cells loaded with microcarriers and the thermosensitive hydrogels are mixed before use to form a composition.

2. The thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis according to claim 1, characterized in that: The mesenchymal stem cells include but are not limited to at least one of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, and adipose mesenchymal stem cells.

3. The thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis according to claim 2, characterized in that: The mesenchymal stem cells are umbilical cord mesenchymal stem cells.

4. The thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis according to claim 1, characterized in that: The thermosensitive hydrogel is a mixture of poloxamer P407 and gel preservative solution, wherein the mass concentration of the poloxamer P407 is in the range of 14-20%.

5. The thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis according to claim 4, characterized in that: The cost of the gel preservation solution includes: 2% sodium hyaluronate, 58% Promali A, 5% human albumin, and 35% glucose injection; the above-mentioned components are the mass percentages of the respective substances in the gel preservation solution.

6. The thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis according to claim 5, characterized in that: The volume ratio of the mesenchymal stem cells loaded on the microcarrier formed by the microcarrier and the thermosensitive hydrogel is 1:0.5-1.

5.

7. A method for preparing a thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis, characterized in that: The method includes: (1) Cultivating the mesenchymal stem cells and microcarriers in a sealed stirring container for 5-7 days. After the cultivation is completed, the mesenchymal stem cells containing the microcarriers are washed by low-speed centrifugation, and the obtained complex is then placed in a cell preservation solution; (2) mixing poloxamer P407 and gel preservative solution to prepare a poloxamer P407 aqueous solution; (3) The mesenchymal stem cells loaded with microcarriers prepared in step (1) and the poloxamer P407 aqueous solution prepared in step (2) are placed at 2-8° C. and packaged for storage and transportation respectively; before injection, they are mixed to obtain a thermosensitive hydrogel composition for treating knee osteoarthritis.

8. The method for preparing the thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis according to claim 7, characterized in that: The culture described in step (1) is cultured in a culture medium, wherein the components of the culture medium include: 500 ml of MSC-T4 serum-free culture medium and 5% mass concentration of platelet lysate, and the culture medium obtained by mixing the two is the complete mesenchymal stem cell culture medium.

9. The method for preparing the thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis according to claim 7, characterized in that: The components of the cell preservation solution in step (1) include: 5% human albumin, 60% compound electrolyte injection, 20% 10% glucose injection, and 15% compound amino acid injection; the above components are the mass percentages of the respective substances in the cell preservation solution.

10. The method for preparing the thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis according to claim 7, characterized in that: The mass concentration of poloxamer P407 in the poloxamer P407 aqueous solution in step (2) is in the range of 14-20%.

11. The method for preparing the thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis according to claim 7, characterized in that: The poloxamer P407 aqueous solution described in step (2) is in liquid state at 2-8°C and in gel state at above 25°C.

12. The method for preparing the thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis according to claim 7, characterized in that: The volume ratio of the mesenchymal stem cells loaded with microcarriers and the poloxamer P407 aqueous solution in step (3) is 1:0.5-1.

5.

13. The method for preparing the thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis according to claim 7, characterized in that: Before use, the mesenchymal stem cells loaded with microcarriers obtained in step (1) are mixed with the poloxamer P407 aqueous solution obtained in step (2) to obtain a cell hydrogel composition, which is then immediately injected into the joint cavity. The joint is slowly and passively moved for 5 to 20 times. The poloxamer and cell suspension will quickly gel under the action of body temperature, so that the cell hydrogel composition is evenly dispersed in the diseased joint cartilage and synovial surface.

14. Use of the thermosensitive hydrogel mesenchymal stem cell composition for treating knee osteoarthritis according to any one of claims 1 to 6 in preparing a drug for treating knee osteoarthritis.

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