Method for inducing chondrogenic differentiation of mesenchymal stem cells, composition for promoting regeneration of articular cartilage and application

Abstract

The invention relates to a method for inducing chondrogenic differentiation of mesenchymal stem cells, a composition for promoting regeneration of articular cartilage and application, and belongs to the technical field of biomedical materials. A method for inducing chondrogenic differentiation of mesenchymal stem cells comprises the steps that the mesenchymal stem cells are inoculated to a matrix for culture, the surface of the matrix is provided with a plurality of protruding blocks, and the protruding blocks are arranged at equal intervals in any extension direction of the surface of the matrix; the height of each convex block is not greater than 2 microns, the ratio of the width of each convex block to the distance between two adjacent convex blocks is 1: (0.8-1.2), and the distance between two adjacent convex blocks is not less than 25 microns; the method further comprises the following steps: spreading the mesenchymal stem cells on the surface of a matrix for culturing to obtain the mesenchymal stem cells with chondrogenic differentiation mechanical memory; wherein the surface of the substrate comprises the surfaces of the convex blocks and the surfaces of the concave parts between the convex blocks. The chondrogenic differentiation of the mesenchymal stem cells can be promoted, so that the subsequently promoted repaired tissue is closer to the original cartilage; the operation is simple, the cell survival rate is high, and the effect is stable.

Description

Technical Field

[0001] The present application relates to the technical field of biomedical materials, and in particular to a method for inducing cartilage differentiation of mesenchymal stem cells and a composition and application thereof for promoting the regeneration of articular cartilage. Background Art

[0002] Articular cartilage, often described as "hyaline" cartilage, facilitates load transfer to bone and smooth joints more durably than any synthetic analog, but has limited regenerative capacity. As a result, cartilage defects often fail to heal and progressively induce degenerative changes, ultimately leading to osteoarthritis (OA). Adequate and effective methods for regenerating cartilage damage are essential to prevent or delay the onset of OA. Despite extensive research and development, no surgical, material-based, cell-based, or drug-based treatment has been able to predictably and durably restore the structure and function of hyaline cartilage.

[0003] At present, the treatment of OA mainly includes measures such as controlling pain, reducing inflammation, improving joint function and delaying the progression of the disease. Although there are common treatment methods such as microfracture and autologous chondrocyte implantation in clinical practice, these methods still have many limitations. For example, although microfracture can stimulate cartilage repair, the biomechanical properties of the repaired tissue formed are often different from those of the original cartilage; although autologous chondrocyte implantation can achieve relatively ideal cartilage regeneration, it is complicated to operate, costly, and has problems such as low cell survival rate and unstable effect. Summary of the invention

[0004] In view of the deficiencies of the prior art, the purpose of the embodiments of the present application is to improve the problems such as the difference in biomechanical properties between the repair tissue formed by the current technology and the original cartilage, complex operation, high cost, low cell survival rate, unstable effect, etc.

[0005] In a first aspect, an embodiment of the present application provides a method for inducing chondrogenic differentiation of mesenchymal stem cells, comprising: inoculating on a matrix for culturing, the surface of the matrix having a plurality of bumps, the plurality of bumps being arranged at equal intervals along any extension direction of the surface of the matrix; the height of the bump is not greater than 2 μm, the ratio of the width of the bump to the spacing between two adjacent bumps is 1:0.8-1.2, and the spacing between two adjacent bumps is not less than 25 μm; the method further comprises: spreading the mesenchymal stem cells on the surface of the matrix for culturing to obtain mesenchymal stem cells with mechanical memory of chondrogenic differentiation; wherein the surface of the matrix includes the surface of the bumps and the surface of the recesses between the bumps.

[0006] The present application cultivates mesenchymal stem cells by paving the entire surface of a matrix with a certain bump structure. The height difference between the bumps and the recesses produces a stable mechanical stimulation to the mesenchymal stem cells, which can simulate the biomechanical properties of natural chondrocytes arranged along collagen fibers, so that the mesenchymal stem cells obtain corresponding mechanical properties, produce mechanical memory, promote their cartilage differentiation, and make the repair tissue formed by the subsequent promotion closer to the original cartilage. At the same time, by adjusting the spacing between two adjacent bumps, the mechanical stimulation generated by the mesenchymal stem cells can be accurately controlled. In addition, by generating a stable mechanical stimulation to the mesenchymal stem cells through a matrix with a certain bump structure, no additional mechanical loading of external force is required, which simplifies the establishment of a mechanical loading model of the cells in vitro, and also reduces the risk of bacterial infection of the cells during the mechanical loading process in vitro.

[0007] In some embodiments of the present application, the distance between two adjacent bumps is 25-200 μm.

[0008] The present application can generate appropriate mechanical stimulation to mesenchymal stem cells by setting the distance between two adjacent protrusions within an appropriate range, thereby facilitating the differentiation of mesenchymal stem cells into cartilage.

[0009] In some embodiments of the present application, the distance between two adjacent bumps is 50-100 μm.

[0010] The present application further sets the distance between two adjacent bumps within a more suitable range, thereby producing more suitable mechanical stimulation to the mesenchymal stem cells, thereby being more conducive to promoting the cartilage differentiation of the mesenchymal stem cells.

[0011] In some embodiments of the present application, the material of the matrix includes a cross-linked product of gelatin and transglutaminase; the volume ratio of gelatin to transglutaminase is 8-12:1.

[0012] The present application uses a cross-linked product of gelatin and transglutaminase as a matrix, which is beneficial to the culture of mesenchymal stem cells and produces appropriate mechanical stimulation to the mesenchymal stem cells.

[0013] In some embodiments of the present application, mesenchymal stem cells are plated on the surface of a matrix and cultured at 37° C. and 5% CO 2 for 3-6 days.

[0014] The present application promotes the differentiation of mesenchymal stem cells into cartilage by plating mesenchymal stem cells on the surface of a matrix and culturing them under suitable conditions for a suitable period of time.

[0015] In some embodiments of the present application, the seeding density of mesenchymal stem cells plated on the matrix surface is 5×10 3 -10×10 3 / cm 2 .

[0016] The present invention is to seed the matrix surface with mesenchymal stem cells of appropriate density and spread them on the matrix surface, thereby facilitating the stable growth and cartilage differentiation of the mesenchymal stem cells.

[0017] In a second aspect, an embodiment of the present application provides a composition for promoting articular cartilage regeneration, comprising mesenchymal stem cells having mechanical memory of chondrogenic differentiation obtained by the method provided in the first aspect.

[0018] In some embodiments of the present application, the composition further comprises a hydrogel or a substance promoting cartilage differentiation, and the concentration of the mesenchymal stem cells with mechanical memory of cartilage differentiation in the composition is 8×10 3 -12×10 3 / μL.

[0019] The present application ensures that the composition contains an appropriate concentration of mesenchymal stem cells with mechanical memory of chondrogenic differentiation, thereby facilitating the regeneration of articular cartilage when it subsequently comes into contact with articular cartilage.

[0020] In some embodiments of the present application, the substance promoting cartilage differentiation includes at least one of KGN, psoralen, and vitamin C.

[0021] The present application can promote the regeneration of articular cartilage by adding a substance promoting cartilage differentiation into the composition, thereby synergistically promoting the regeneration of articular cartilage with mesenchymal stem cells having mechanical memory of chondrogenic differentiation.

[0022] In a third aspect, an embodiment of the present application provides the use of the composition provided in the second aspect in the preparation of a drug for promoting the regeneration of articular cartilage.

[0023] The mesenchymal stem cells with mechanical memory of chondrogenic differentiation in the composition of the present application can effectively differentiate into chondrocytes, thereby effectively promoting the regeneration of articular cartilage; therefore, when used in the preparation of drugs for promoting the regeneration of articular cartilage, the regeneration of articular cartilage can be effectively promoted. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 A schematic diagram of a substrate provided in an embodiment of the present application.

[0026] Icon: 1-matrix; 11-protrusion; 12-concave.

[0027] Figure 2 This is a microscopic examination image of mesenchymal stem cells provided in Experimental Example 1 of the present application.

[0028] Figure 3 This is a graph showing the qPCR detection results of the specific markers Col II, Sox 9, and ACAN during the differentiation culture period of mesenchymal stem cells provided in Experimental Example 2 of the present application; wherein, PS-control group, Flat-comparative example 1, 50μm-example 1.

[0029] Figure 4 This is the ACAN immunofluorescence staining image of the mesenchymal stem cells at the end of differentiation provided in Experimental Example 3 of the present application; wherein, Control-control group, 0-comparative example 1, 20-comparative example 2, 50-example 1, 100-example 2, 200-example 3.

[0030] Figure 5 This is a graph of ACAN content at the end of differentiation of mesenchymal stem cells provided in Experimental Example 3 of the present application; wherein, Control-control group, 0-comparative example 1, 20-comparative example 2, 50-example 1, 100-example 2, 200-example 3. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0032] In order to improve the problems of the repair tissue formed by the current technology, such as the difference in biomechanical properties between the original cartilage and the complex operation, high cost, low cell survival rate, unstable effect, etc., the present application embodiment provides a method for inducing mesenchymal stem cells to differentiate into cartilage, such as Figure 1 As shown, it includes: inoculating on a matrix 1 for culturing, the surface of the matrix 1 has a plurality of bumps 11, and along any extension direction of the surface of the matrix 1, the plurality of bumps 11 are arranged at equal intervals; the height of the bump 11 is not greater than 2 μm, the ratio of the width of the bump 11 to the spacing between two adjacent bumps 11 is 1:0.8-1.2, and the spacing between two adjacent bumps 11 is not less than 25 μm; the method also includes: spreading mesenchymal stem cells on the surface of the matrix 1 for culturing to obtain mesenchymal stem cells with mechanical memory of chondrogenic differentiation; wherein the surface of the matrix 1 includes the surface of the bump 11 and the surface of the recess 12 between the bumps 11, the bump 11, the recess 12.

[0033] The present application is to culture mesenchymal stem cells by paving the surface of a matrix 1 having a certain bump 11 structure as a whole, and the boundary height difference between the bump 11 and the recess 12 produces a stable mechanical stimulation to the mesenchymal stem cells. The structural setting of the matrix 1 can simulate the mechanical environment of the surface chondrocytes of the cartilage tissue in the body, change the environment in which the mesenchymal stem cells are located, so that the mesenchymal stem cells obtain corresponding mechanical properties, produce mechanical memory, and directly affect the adhesion, morphology, migration and proliferation of the mesenchymal stem cells, thereby promoting the differentiation of mesenchymal stem cells into cartilage, so that the repair tissue formed by the subsequent promotion is closer to the original cartilage. At the same time, by adjusting the spacing between two adjacent bumps 11, the mechanical stimulation generated by the mesenchymal stem cells can be accurately controlled. In addition, the matrix 1 with a certain bump 11 structure produces a stable mechanical stimulation to the mesenchymal stem cells, without the need for additional mechanical loading of external force, which simplifies the establishment of the in vitro mechanical loading model of the cells, and also reduces the risk of bacterial infection of the cells during the in vitro mechanical loading process.

[0034] As an example, the ratio of the width of the bump 11 to the distance between the two bumps 11 can be, but is not limited to, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2. The width of the bump 11 is substantially close to the distance between the two bumps 11, which can ensure stable mechanical stimulation of the mesenchymal stem cells.

[0035] In some embodiments of the present application, the spacing between two adjacent bumps 11 is 25-200 μm. Further, the spacing between two adjacent bumps 11 is 50-100 μm. As an example, the spacing between two adjacent bumps 11 can be, but is not limited to, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm, 50 μm, 55 μm, 60 μm, 65 μm, 70 μm, 75 μm, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm, 200 μm. By setting the distance between two adjacent protrusions 11 within a suitable range, suitable mechanical stimulation can be generated on the mesenchymal stem cells, thereby facilitating the differentiation of the mesenchymal stem cells into cartilage.

[0036] In some embodiments of the present application, the preparation method of the matrix 1 includes:

[0037] (1) Mold preparation: The glass substrate is cleaned by soaking in piranha solution (sulfuric acid and 30% hydrogen peroxide solution in a ratio of 3-5:1), and the dried glass substrate is coated with hexamethyldisilane (HMDS) by vapor deposition for 5-10 minutes, and then the photoresist AZ5214 is coated on the HMDS-treated glass substrate by spin coating (3000-3500 rpm for 30-40 seconds), and placed on a 90-95° C. hot stage for 2-5 minutes for pre-baking to remove the solvent, and the photoresist is exposed to light using an ultraviolet exposure device through a designed mask, and the exposed area of ​​the photoresist is removed by a developer, and the glass substrate is rinsed with deionized water and dried with compressed air to obtain a template with a bump 11 structure; polydimethylsiloxane (PDMS) is then cast on the template at 70-80° C. for 2-4 hours, and after removing the template, a PDMS mold with a groove structure is obtained;

[0038] (2) Preparation of substrate 1: A low-adhesion tape with a thickness of 50-60 μm is attached to the opposite sides of the cleaned glass substrate to control the thickness of substrate 1 to be consistent. A 10% gelatin solution and a 10% transglutaminase (MTG) solution are prepared in a water bath at 55-65° C., and the solutions are filtered through a 0.22 μm filter membrane. The gelatin solution and the MTG solution are mixed at a volume ratio of 8-12:1 to form a mixed solution. 100-150 μL of the mixed solution is dropped on the center of the glass substrate and covered with a PDMS mold with a groove structure. The substrate is cross-linked and cured at room temperature for 30-50 min. The PDMS mold is peeled off to obtain a substrate 1 with a certain bump 11 structure. The substrate is sterilized by ultraviolet for 30-40 min for use.

[0039] As an example, the volume ratio of gelatin to transglutaminase can be, but is not limited to, 8: 1, 9: 1, 10: 1, 11: 1, 12: 1. Using a cross-linked product of gelatin and transglutaminase as the matrix 1 is conducive to culturing mesenchymal stem cells and generating appropriate mechanical stimulation to the mesenchymal stem cells.

[0040] In some embodiments of the present application, mesenchymal stem cells are plated on the surface of the matrix 1 and cultured for 3-6 days at 37°C and 5% CO2. As an example, the mesenchymal stem cells can be plated on the surface of the matrix 1 for, but not limited to, 3 days and 6 days. Passage is performed every 3 days. The mesenchymal stem cells are plated on the surface of the matrix 1 and cultured for a suitable time under suitable conditions to promote the chondrogenic differentiation of the mesenchymal stem cells.

[0041] In some embodiments of the present application, the seeding density of mesenchymal stem cells plated on the surface of the matrix 1 is 5×10 3 -10×10 3 / cm 2Mesenchymal stem cells are seeded at an appropriate density on the surface of the matrix 1 and spread on the surface of the matrix 1, which is conducive to the stable growth and cartilage differentiation of the mesenchymal stem cells.

[0042] The embodiments of the present application provide a composition for promoting articular cartilage regeneration, comprising mesenchymal stem cells having mechanical memory of chondrogenic differentiation obtained by the above method.

[0043] In some embodiments of the present application, the composition for promoting articular cartilage regeneration contains only mesenchymal stem cells having chondrogenic differentiation mechanical memory obtained by the above method.

[0044] In some embodiments of the present application, the composition further comprises a hydrogel or a substance promoting cartilage differentiation, and the concentration of the mesenchymal stem cells with mechanical memory of cartilage differentiation in the composition is 8×10 3 -12×10 3 / μL. The composition is ensured to contain an appropriate concentration of mesenchymal stem cells with mechanical memory of chondrogenic differentiation, which is beneficial to promote the regeneration of articular cartilage when it comes into contact with articular cartilage later.

[0045] In some embodiments of the present application, the substance promoting cartilage differentiation includes at least one of KGN, psoralen, and vitamin C. Adding the substance promoting cartilage differentiation into the composition can promote the regeneration of articular cartilage in synergy with mesenchymal stem cells having mechanical memory of chondrogenic differentiation.

[0046] The present application provides an application of the above-mentioned composition in the preparation of a drug for promoting articular cartilage regeneration. The mesenchymal stem cells with chondrogenic differentiation mechanical memory in the composition of the present application can effectively differentiate into chondrocytes, thereby effectively promoting articular cartilage regeneration; when used in the preparation of a drug for promoting articular cartilage regeneration, it can effectively promote articular cartilage regeneration.

[0047] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0048] Example 1

[0049] This embodiment provides a method for inducing cartilage differentiation of mesenchymal stem cells, comprising: seeding the mesenchymal stem cells onto a matrix at a seeding density of 10 4 / cm 2 The surface of the matrix has 200 bumps, and along any extension direction of the surface of the matrix, the 200 bumps are arranged at equal intervals; the height of the bump is 1.3 μm, the ratio of the width of the bump to the distance between two adjacent bumps is 1:1, and the distance between two adjacent bumps is 50 μm; mesenchymal stem cells are spread on the three bumps of the matrix and the concave surface between the bumps, and cultured at 37°C and 5% CO2 for 6 days to obtain mesenchymal stem cells with mechanical memory of chondrogenic differentiation.

[0050] Example 2

[0051] This embodiment provides a method for inducing cartilage differentiation of mesenchymal stem cells, comprising: seeding the mesenchymal stem cells onto a matrix at a seeding density of 10 4 / cm 2 The surface of the matrix has 200 bumps, and along any extension direction of the surface of the matrix, the 200 bumps are arranged at equal intervals; the height of the bump is 1.3 μm, the ratio of the width of the bump to the distance between two adjacent bumps is 1:1, and the distance between two adjacent bumps is 100 μm; mesenchymal stem cells are spread on the three bumps of the matrix and the concave surface between the bumps, and cultured at 37°C and 5% CO2 for 6 days to obtain mesenchymal stem cells with mechanical memory of chondrogenic differentiation.

[0052] Example 3

[0053] This embodiment provides a method for inducing cartilage differentiation of mesenchymal stem cells, comprising: seeding the mesenchymal stem cells onto a matrix at a seeding density of 10 4 / cm 2 The surface of the matrix has 200 bumps, and along any extension direction of the surface of the matrix, the 200 bumps are arranged at equal intervals; the height of the bump is 1.3 μm, the ratio of the width of the bump to the distance between two adjacent bumps is 1:1, and the distance between two adjacent bumps is 200 μm; mesenchymal stem cells are spread on the three bumps of the matrix and the concave surface between the bumps, and cultured at 37°C and 5% CO2 for 6 days to obtain mesenchymal stem cells with mechanical memory of chondrogenic differentiation.

[0054] Comparative Example 1

[0055] This comparative example provides a method for inducing mesenchymal stem cells to differentiate into chondrogenic cartilage, which is different from Example 1 in that the surface of the matrix is ​​flat.

[0056] Comparative Example 2

[0057] This comparative example provides a method for inducing mesenchymal stem cells to differentiate into chondrogenic cartilage, which is different from Example 1 in that the distance between two adjacent bumps on the surface of the matrix is ​​20 μm.

[0058] Comparative Example 3

[0059] This comparative example provides a method for inducing mesenchymal stem cells to differentiate into cartilage, which is different from Example 1 in that the height of the bumps on the matrix surface is 10 μm, and the mesenchymal stem cells are seeded on the surface of the concave portions between the bumps.

[0060] Test Example 1

[0061] In this test example, the mesenchymal stem cells with chondrogenic differentiation cultured in Example 1 and Comparative Example 1 were photographed under a microscope. Figure 2 shown.

[0062] Depend on Figure 2 It can be seen that in Example 1 of the present application, mesenchymal stem cells are cultured on a substrate 1 having a certain bump 11 structure, and the mesenchymal stem cells are orderly arranged along the edges of the bumps 11 of the substrate 1, and are elongated and extended. In Comparative Example 1, the mesenchymal stem cells cultured on a flat substrate 1 are disordered and have no morphological changes.

[0063] Test Example 2

[0064] In this test example, the mesenchymal stem cells obtained by culture in Example 1 (50 μm) and Comparative Example 1 (Flat) were inoculated into 12-well plates at a density of 10 4 / cm 2 , using chondrogenic differentiation conditioned medium (DMEM high glucose medium: FBS10%, PS1%, dexamethasone 1μM, sodium pyruvate 100μg / mL, proline 40μg / mL, ascorbic acid 50μg / mL, TGF-beta3 10μg / mL, ITS-G50mg / mL) for continuous culture and differentiation at 37°C, 5% CO2 environment for 14 days. Mesenchymal stem cells cultured in a traditional culture dish according to the method of Example 1 were continuously cultured and differentiated according to the above method as a control group (PS). qPCR detection of Col II, Sox9, and ACAN, specific markers of mesenchymal stem cells at each time period, was performed in the early stage (3 days), middle stage (7 days), and late stage (14 days) of differentiation.

[0065] The qPCR detection method is as follows: extract total RNA from mesenchymal stem cells at each differentiation stage, reverse transcribe RNA into cDNA, and use a real-time PCR system to perform quantitative reverse transcription polymerase chain reaction (qRT-PCR) to analyze cDNA expression. The cycle parameters are as follows: 95°C pre-denaturation for 10 min; 95°C denaturation for 15 s, 60°C annealing and extension for 1 min, for a total of 40 cycles. The relative mRNA expression was standardized using GAPDH as an internal reference gene and was measured by 2 –ΔΔCt The results are shown in Figure 3 shown.

[0066] Depend on Figure 3 The results show that as the differentiation time increases, the Sox 9 and ACAN contents in the mesenchymal stem cells cultured on the matrix 1 having a certain bump 11 structure in Example 1 of the present application significantly increase with the differentiation time during the differentiation culture stage, indicating that the mesenchymal stem cells cultured on the matrix 1 having a certain bump 11 structure in the present application can effectively differentiate into cartilage.

[0067] Test Example 3

[0068] In this test example, the mesenchymal stem cells obtained by culture in Examples 1-3 and Comparative Examples 1-2 were continuously cultured and differentiated for 14 days according to the method of Test Example 2, and the cells that had completed differentiation were subjected to ACAN immunofluorescence staining, imaged using a confocal fluorescence microscope, modeled using Imaris, and the volume of ACAN was calculated, and quantified by the total volume ratio to the number of cells. The mesenchymal stem cells cultured in a traditional culture dish according to the method of Example 1 were continuously cultured and differentiated for 14 days according to the method of Test Example 2, and the cells that had completed differentiation were used as the control group (Control). The results are shown in FIG. Figure 4-Figure 5 shown.

[0069] Depend on Figure 4-Figure 5 As can be seen from the results, the mesenchymal stem cells cultured on the matrix 1 having a certain bump 11 structure in Examples 1-3 of the present application have a significantly increased ACAN content in the cells at the end of differentiation, indicating that the mesenchymal stem cells cultured on the matrix 1 having a certain bump 11 structure in the present application can effectively differentiate into cartilage.

[0070] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

Claims

1. A method for inducing chondrogenic differentiation of mesenchymal stem cells, characterized in that: include: The cells are inoculated on a substrate for culturing, wherein the surface of the substrate has a plurality of protrusions, and the plurality of protrusions are arranged at equal intervals along any extension direction of the surface of the substrate; the height of the protrusion is not greater than 2 μm, the ratio of the width of the protrusion to the interval between two adjacent protrusions is 1:0.8-1.2, and the interval between two adjacent protrusions is not less than 25 μm; The method further comprises: paving mesenchymal stem cells on the surface of the matrix for culturing to obtain mesenchymal stem cells with mechanical memory of chondrogenic differentiation; wherein the surface of the matrix comprises the surfaces of the bumps and the surfaces of the recesses between the bumps.

2. The method according to claim 1, characterized in that The distance between two adjacent bumps is 25-200 μm.

3. The method according to claim 2, characterized in that The distance between two adjacent bumps is 50-100 μm.

4. The method according to any one of claims 1 to 3, characterized in that: The material of the matrix includes a cross-linked product of gelatin and transglutaminase; the volume ratio of the gelatin to the transglutaminase is 8-12:

1.

5. The method according to claim 1, characterized in that The mesenchymal stem cells are plated on the surface of the matrix and cultured at 37° C. and 5% CO 2 for 3-6 days.

6. The method according to claim 1, characterized in that The seeding density of the mesenchymal stem cells plated on the surface of the matrix is ​​5×10 3 -10×10 3 / cm 2 .

7. A composition for promoting articular cartilage regeneration, characterized in that: Comprising mesenchymal stem cells having mechanical memory of chondrogenic differentiation obtained by the method according to any one of claims 1 to 6.

8. The composition according to claim 7, characterized in that The composition further comprises a hydrogel or a substance promoting cartilage differentiation, and the concentration of the mesenchymal stem cells with mechanical memory of cartilage differentiation in the composition is 8×10 3 -12×10 3 / μL.

9. The composition according to claim 8, characterized in that The substance promoting cartilage differentiation includes at least one of KGN, psoralen and vitamin C.

10. Use of the composition according to any one of claims 7 to 9 in the preparation of a medicament for promoting regeneration of articular cartilage.

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