A method for inducing mesenchymal stem cells to differentiate into chondrocytes, a composition for promoting regeneration of articular cartilage and application thereof

Abstract

The application relates to a method for inducing mesenchymal stem cells to differentiate into chondroblasts and a composition for promoting regeneration of articular cartilage and application, and belongs to the technical field of biomedical materials. The method for inducing mesenchymal stem cells to differentiate into chondroblasts comprises the following steps: inoculating and culturing on a substrate, wherein the surface of the substrate is provided with a plurality of convex blocks, the plurality of convex blocks are arranged at equal intervals along any extension direction of the surface of the substrate; the height of the convex blocks is not greater than 2 mu m, the ratio of the width of the convex blocks to the interval between adjacent two convex blocks is 1:0.8-1.2, and the interval between the adjacent two convex blocks is not less than 25 mu m; the method further comprises the following steps: spreading mesenchymal stem cells to the surface of the substrate for culture to obtain mesenchymal stem cells with mechanical memory of chondroblast differentiation; and the surface of the substrate comprises the surface of the convex blocks and the surface of the concave parts between the convex blocks. The method can promote the chondroblast differentiation of mesenchymal stem cells, make the subsequently formed repair tissue more close to the original cartilage, and has the advantages of simple operation, high cell survival rate and stable effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, and particularly relates to a method for inducing mesenchymal stem cells to differentiate into chondrocytes, a composition for promoting regeneration of articular cartilage and application thereof. BACKGROUND

[0002] Articular cartilage, often described as "hyaline" cartilage, promotes the load transmission of bone and smooth joints more durably than any synthetic analog, but has limited regenerative capacity. Therefore, cartilage defects often fail to heal and progress to induce degenerative changes, eventually leading to osteoarthritis (OA). A sufficient and effective method for cartilage injury regeneration is crucial for preventing or delaying the occurrence of OA. Despite extensive research and development, no surgical, material-based, cell-based or drug-based treatment can 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 disease progression. Although there are common treatment methods such as microfracture and autologous chondrocyte implantation in clinic, these methods still have many limitations. For example, although microfracture can stimulate cartilage repair, the repair tissue formed often has differences in biomechanical properties from the original cartilage; although autologous chondrocyte implantation can achieve ideal cartilage regeneration, it is complex to operate, high in cost, has low cell survival rate and unstable effect, etc. SUMMARY

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

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

[0006] The application is to culture mesenchymal stem cells by whole paving inoculation on the substrate surface with certain bump structure. The boundary height difference between the bump and the recess produces stable mechanical stimulation on the mesenchymal stem cells, which can simulate the biomechanical properties of natural chondrocytes arranged along the collagen fibers, so that the mesenchymal stem cells obtain corresponding mechanical properties, produce mechanical memory, promote chondrogenic differentiation, and make the subsequent repair tissue formed more close to the original cartilage. At the same time, by adjusting the distance between the adjacent two bumps, the mechanical stimulation produced by the mesenchymal stem cells can be precisely controlled. In addition, by using the substrate with certain bump structure to produce stable mechanical stimulation on the mesenchymal stem cells, there is no need for additional mechanical loading of external force, which simplifies the establishment of the in vitro mechanical loading model of cells, and also reduces the risk of bacterial contamination in the in vitro mechanical loading process of cells.

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

[0008] By setting the distance between the adjacent two bumps within a suitable range, the application can produce suitable mechanical stimulation on the mesenchymal stem cells, thereby promoting chondrogenic differentiation of the mesenchymal stem cells.

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

[0010] By further setting the distance between the adjacent two bumps within a more suitable range, the application can produce more suitable mechanical stimulation on the mesenchymal stem cells, thereby more promoting chondrogenic differentiation of the mesenchymal stem cells.

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

[0012] By using the cross-linked product of gelatin and transglutaminase as the substrate, the application is conducive to culturing mesenchymal stem cells and producing suitable mechanical stimulation on the mesenchymal stem cells.

[0013] In some embodiments of the application, the mesenchymal stem cells are paved on the substrate surface and cultured at 37℃ in a 5% CO2 environment for 3-6 days.

[0014] By paving the mesenchymal stem cells on the substrate surface and culturing them under suitable conditions for a suitable time, the application promotes chondrogenic differentiation of the mesenchymal stem cells.

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

[0016] The application inoculates mesenchymal stem cells of a suitable density on the surface of the substrate to lay on the surface of the substrate, which is conducive to the stable growth and chondrogenic differentiation of the mesenchymal stem cells.

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

[0018] In some embodiments of the application, the composition further comprises a hydrogel or a chondrogenic differentiation promoter, and the concentration of the mesenchymal stem cells with chondrogenic differentiation mechanical memory in the composition is 8x10 3 -12x10 3 / μL.

[0019] The application ensures that the composition contains a suitable concentration of mesenchymal stem cells with chondrogenic differentiation mechanical memory, which is conducive to promoting the regeneration of articular cartilage when subsequently contacted with articular cartilage.

[0020] In some embodiments of the application, the chondrogenic differentiation promoter comprises at least one of KGN, psoralen, and vitamin C.

[0021] The application can synergistically promote the regeneration of articular cartilage by adding a chondrogenic differentiation promoter to the composition and the mesenchymal stem cells with chondrogenic differentiation mechanical memory.

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

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

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the application, and therefore should not be considered as a limitation to the scope. For those skilled in the art, other related drawings can also be obtained without creative labor.

[0025] Figure 1 The schematic diagram of the substrate provided in the embodiments of the application.

[0026] Figure legend: 1-substrate; 11-bump; 12-recess.

[0027] Figure 2 A microscope detection chart of mesenchymal stem cells provided for Test Example 1 of the present application.

[0028] Figure 3 A qPCR detection result chart of specific markers Col II, Sox 9, and ACAN of mesenchymal stem cells in the differentiation culture period provided for Test Example 2 of the present application; wherein, PS- control group, Flat- Comparative Example 1, 50 μm- Example 1.

[0029] Figure 4 An ACAN immunofluorescence staining chart of mesenchymal stem cells at the end of differentiation provided for Test 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 An ACAN content chart of mesenchymal stem cells at the end of differentiation provided for Test 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 more clear, the technical scheme in the embodiments of the present application will be described clearly and completely below. If specific conditions are not specified in the embodiments, conventional conditions or manufacturer recommended conditions are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be purchased on the market.

[0032] In order to improve the differences in biomechanical properties between the repaired tissue formed by the current technology and the original cartilage, the complexity of the operation, the high cost, the low cell survival rate, and the unstable effect, etc. The embodiments of the present application provide a method for inducing mesenchymal stem cells to differentiate into chondrocytes, as shown in Figure 1 The method comprises seeding and culturing on a substrate 1, the surface of the substrate 1 has a plurality of protrusions 11, along any extension direction of the surface of the substrate 1, the plurality of protrusions 11 are arranged at equal intervals; the height of the protrusion 11 is not greater than 2 μm, the ratio of the width of the protrusion 11 to the interval between the adjacent two protrusions 11 is 1:0.8-1.2, and the interval between the adjacent two protrusions 11 is not less than 25 μm; the method further comprises: spreading the mesenchymal stem cells to the surface of the substrate 1 for culture to obtain mesenchymal stem cells with chondrogenic differentiation mechanical memory; wherein, the surface of the substrate 1 includes the surface of the protrusion 11 and the surface of the recess 12 between the protrusions 11.

[0033] The application is to culture mesenchymal stem cells by whole paving inoculation on the surface of the substrate 1 with certain bump 11 structure. The boundary height difference between the bump 11 and the recess 12 produces stable mechanical stimulation to the mesenchymal stem cells. The structure of the substrate 1 can simulate the mechanical environment of the surface type chondrocytes in the body cartilage tissue, change the environment of the mesenchymal stem cells, make the mesenchymal stem cells obtain corresponding mechanical properties, produce mechanical memory, directly affect the adhesion, morphology, migration and proliferation of the mesenchymal stem cells, thereby promoting the chondrogenic differentiation of the mesenchymal stem cells, and making the subsequent repair tissue formed by promoting closer to the original cartilage. At the same time, by adjusting the distance between the two adjacent bumps 11, the mechanical stimulation to the mesenchymal stem cells can be precisely controlled. In addition, the substrate 1 with certain bump 11 structure produces stable mechanical stimulation to the mesenchymal stem cells, without the need for additional mechanical loading of external force, simplifying the establishment of the in vitro mechanical loading model of the cells, and reducing the risk of bacterial contamination of the cells in the in vitro mechanical loading process.

[0034] For example, the ratio of the width of the bump 11 to the distance between the two bumps 11 can be but not limited to 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2. The width of the bump 11 and the distance between the two bumps 11 are basically close, which can ensure that the mesenchymal stem cells produce stable mechanical stimulation.

[0035] In some embodiments of the application, the distance between the two adjacent bumps 11 is 25-200 μm. Further, the distance between the two adjacent bumps 11 is 50-100 μm. For example, the distance between the two adjacent bumps 11 can be but 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. The distance between the two adjacent bumps 11 is set in a suitable range, which can produce suitable mechanical stimulation to the mesenchymal stem cells, thereby promoting the chondrogenic differentiation of the mesenchymal stem cells.

[0036] In some embodiments of the application, the preparation method of the substrate 1 comprises:

[0037] (1) Mould preparation: the glass substrate is cleaned by soaking in an arowana solution (sulfuric acid and 30% hydrogen peroxide solution in a ratio of 3-5:1), dried, coated with hexamethyldisilane (HMDS) by vapor deposition for 5-10 min, then coated with photoresist AZ5214 on the HMDS-treated glass substrate by spin coating (3000-3500 rpm for 30-40 s), placed on a hot table at 90-95°C for 2-5 min of pre-baking to remove the solvent, exposed to ultraviolet light through a designed mask, and the exposed areas of the photoresist are removed in a developer, then the glass substrate is rinsed with deionized water and dried with compressed air to obtain a mould with a bump 11 structure; then the PDMS is cast on the mould at 70-80°C for 2-4 h, and after the mould is removed, the PDMS mould with a groove structure is obtained;

[0038] (2) Preparation of substrate 1: the low-adhesion tape with a thickness of 50-60 μm is attached to the edges of the cleaned glass substrate to control the thickness of the substrate 1 to be uniform, a 10% gelatin solution and a 10% transglutaminase (MTG) solution are prepared in a water bath at 55-65°C, the solutions are filtered through a 0.22 μm filter, the gelatin solution and the MTG solution are mixed in a volume ratio of 8-12:1 to obtain a mixed solution, 100-150 μL of the mixed solution is dropped on the center of the glass substrate, and the PDMS mould with a groove structure is covered, and the crosslinking and curing are carried out at room temperature for 30-50 min, and then the PDMS mould is peeled off to obtain the substrate 1 with a certain bump 11 structure, which is subjected to ultraviolet sterilization for 30-40 min for standby use.

[0039] For example, the volume ratio of gelatin to transglutaminase can be, but is not limited to, 8:1, 9:1, 10:1, 11:1, and 12:1. The crosslinking product of gelatin and transglutaminase as the substrate 1 is beneficial to the culture of mesenchymal stem cells and produces appropriate mechanical stimulation to the mesenchymal stem cells.

[0040] In some embodiments of the present application, the mesenchymal stem cells are plated on the surface of the substrate 1 and cultured at 37°C in a 5% CO2 environment for 3-6 days. For example, the mesenchymal stem cells plated on the surface of the substrate 1 can be, but are not limited to, cultured for 3 days or 6 days. The passage is carried out every 3 days. The mesenchymal stem cells plated on the surface of the substrate 1 are cultured under appropriate conditions for an appropriate time to promote the chondrogenic differentiation of the mesenchymal stem cells.

[0041] In some embodiments of the present application, the seeding density of the mesenchymal stem cells plated on the surface of the substrate 1 is 5×10 3 -10×10 3 / cm 2. The mesenchymal stem cells are inoculated on the surface of the substrate 1 at a suitable density, which is conducive to the stable growth and chondrogenic differentiation of the mesenchymal stem cells.

[0042] The embodiment of the present application provides a composition for promoting regeneration of articular cartilage, which comprises the mesenchymal stem cells with chondrogenic differentiation mechanical memory obtained by the method.

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

[0044] In some embodiments of the present application, the composition further comprises a hydrogel or a chondrogenic differentiation promoter, and the concentration of the mesenchymal stem cells with chondrogenic differentiation mechanical memory in the composition is 8x10 3 -12x10 3 / μL. The composition is ensured to contain the mesenchymal stem cells with chondrogenic differentiation mechanical memory at a suitable concentration, which is conducive to promoting regeneration of articular cartilage when subsequently contacted with the articular cartilage.

[0045] In some embodiments of the present application, the chondrogenic differentiation promoter comprises at least one of KGN, psoralen and vitamin C. The addition of the chondrogenic differentiation promoter in the composition can synergistically promote regeneration of articular cartilage with the mesenchymal stem cells with chondrogenic differentiation mechanical memory.

[0046] The embodiment of the present application provides application of the composition in preparation of a medicine for promoting regeneration of articular cartilage. The mesenchymal stem cells with chondrogenic differentiation mechanical memory in the composition of the present application can effectively differentiate into chondrocytes, thereby effectively promoting regeneration of articular cartilage. When applied in preparation of a medicine for promoting regeneration of articular cartilage, the composition can effectively promote regeneration of articular cartilage.

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

[0048] Embodiment 1

[0049] The embodiment provides a method for inducing chondrogenic differentiation of mesenchymal stem cells, comprising: inoculating mesenchymal stem cells on a substrate at a seeding density of 10 4 / cm 2 , the surface of the substrate has 200 bumps, and the 200 bumps are arranged at equal intervals along any extension direction of the surface of the substrate; the height of the bumps is 1.3 μm, the ratio of the width of the bumps to the interval between adjacent two bumps is 1:1, and the interval between the adjacent two bumps is 50 μm; the mesenchymal stem cells are plated on the surfaces of the three bumps and the concave parts between the bumps of the substrate, and are cultured at 37℃ in a 5% CO2 environment for 6 days to obtain the mesenchymal stem cells with chondrogenic differentiation mechanical memory.

[0050] Example 2

[0051] The present example provides a method for inducing mesenchymal stem cells to differentiate into chondrocytes, comprising: inoculating mesenchymal stem cells onto a substrate at a seeding density of 10 4 / cm 2 , the surface of the substrate has 200 bumps, the 200 bumps are equidistantly arranged along any extension direction of the surface of the substrate; the height of the bumps is 1.3 μm, the ratio of the width of the bumps to the interval between adjacent two bumps is 1:1, and the interval between adjacent two bumps is 100 μm; the mesenchymal stem cells are spread on the surface of 3 bumps and the concave part between the bumps of the substrate, and are cultured at 37°C in a 5% CO2 environment for 6 days to obtain mesenchymal stem cells with mechanical memory of chondrocyte differentiation.

[0052] Example 3

[0053] The present example provides a method for inducing mesenchymal stem cells to differentiate into chondrocytes, comprising: inoculating mesenchymal stem cells onto a substrate at a seeding density of 10 4 / cm 2 , the surface of the substrate has 200 bumps, the 200 bumps are equidistantly arranged along any extension direction of the surface of the substrate; the height of the bumps is 1.3 μm, the ratio of the width of the bumps to the interval between adjacent two bumps is 1:1, and the interval between adjacent two bumps is 200 μm; the mesenchymal stem cells are spread on the surface of 3 bumps and the concave part between the bumps of the substrate, and are cultured at 37°C in a 5% CO2 environment for 6 days to obtain mesenchymal stem cells with mechanical memory of chondrocyte differentiation.

[0054] Comparative Example 1

[0055] The present comparative example provides a method for inducing mesenchymal stem cells to differentiate into chondrocytes, which is different from Example 1 in that the surface of the substrate is a plane.

[0056] Comparative Example 2

[0057] The present comparative example provides a method for inducing mesenchymal stem cells to differentiate into chondrocytes, which is different from Example 1 in that the interval between adjacent two bumps on the surface of the substrate is 20 μm.

[0058] Comparative Example 3

[0059] The present comparative example provides a method for inducing mesenchymal stem cells to differentiate into chondrocytes, which is different from Example 1 in that the height of the bumps on the surface of the substrate is 10 μm, and the mesenchymal stem cells are inoculated on the surface of the concave part between the bumps.

[0060] Test Example 1

[0061] This experimental example involved microscopic examination and imaging of the chondrogenic mesenchymal stem cells cultured in Example 1 and Comparative Example 1. Figure 2 As shown.

[0062] Depend on Figure 2 As can be seen, in Example 1 of this application, mesenchymal stem cells were cultured on a matrix 1 with a certain bump 11 structure. The mesenchymal stem cells were arranged orderly along the edge of the bump 11 of the matrix 1 and were elongated and expanded. In Comparative Example 1, the mesenchymal stem cells cultured on a planar matrix 1 were disordered and showed no morphological change.

[0063] Experimental Example 2

[0064] In this experiment, mesenchymal stem cells obtained from Example 1 (50 μm) and Comparative Example 1 (Flat) were seeded into 12-well plates at a seeding density of 10-1. 4 / cm 2 Mesenchymal stem cells (MSCs) were cultured and differentiated continuously for 14 days at 37°C and 5% CO2 using DMEM high-glucose medium (FBS 10%, PS 1%, dexamethasone 1 μM, sodium pyruvate 100 μg / mL, proline 40 μg / mL, ascorbic acid 50 μg / mL, TGF-beta3 10 μg / mL, ITS-G 50 mg / mL). Mesenchymal stem cells cultured in conventional culture dishes according to the method in Example 1 were used as a control group (PS) and continuously cultured and differentiated as described above. qPCR was used to detect the specific markers Col II, Sox9, and ACAN of MSCs at different differentiation stages: early (3 days), middle (7 days), and late (14 days).

[0065] The qPCR detection method was as follows: Total RNA was extracted from mesenchymal stem cells at various differentiation stages, and the RNA was reverse transcribed into cDNA. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using a real-time PCR system to analyze cDNA expression. The cycling parameters were as follows: 95℃ pre-denaturation for 10 min; 95℃ denaturation for 15 s, 60℃ annealing and extension for 1 min, for a total of 40 cycles. Relative mRNA expression levels were normalized using GAPDH as an internal reference gene and analyzed using 2... –ΔΔCt The method was used for quantification. The results are as follows: Figure 3 As shown.

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

[0067] Experimental Example 3

[0068] In this experiment, mesenchymal stem cells obtained from Examples 1-3 and Comparative Examples 1-2 were cultured and differentiated continuously for 14 days according to the method in Example 2. The differentiated cells were then subjected to ACAN immunofluorescence staining, imaged using confocal fluorescence microscopy, and modeled using Imaris. The volume of the ACAN cells was calculated, and the total volume was used as the cell number for quantification. Mesenchymal stem cells cultured in conventional culture dishes according to the method in Example 1 were also cultured and differentiated continuously for 14 days according to the method in Example 2. The differentiated cells served as the control group. The results are as follows: Figures 4-5 As shown.

[0069] Depend on Figures 4-5 As the results show, the mesenchymal stem cells cultured on matrix 1 with a certain bump 11 structure in Examples 1-3 of this application showed a significant increase in ACAN content in the cells at the end of differentiation, indicating that the mesenchymal stem cells cultured on matrix 1 with a certain bump 11 structure in this application can effectively differentiate into chondrocytes.

[0070] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A method for inducing chondrogenic differentiation of mesenchymal stem cells, characterized by, The method comprises: seeding and culturing on a substrate, a surface of the substrate having a plurality of protrusions, the protrusions being equidistantly arranged along any extension direction of the surface of the substrate; the height of the protrusions being not more than 2 μm, the ratio of the width of the protrusions to the interval between two adjacent protrusions being 1:0.8-1.2, the interval between two adjacent protrusions being 25-200 μm; the material of the substrate comprising a cross-linked product of gelatin and transglutaminase; the volume ratio of the gelatin to the transglutaminase being 8-12:1; the method further comprises: plating mesenchymal stem cells on the surface of the substrate for culturing to obtain mesenchymal stem cells with chondrogenic differentiation mechanical memory; wherein the surface of the substrate comprises the surface of the protrusions and the surface of the recesses between the protrusions.

2. The method of claim 1, wherein, the interval between two adjacent protrusions is 50-100 μm.

3. The method of claim 1, wherein, the plating of the mesenchymal stem cells on the surface of the substrate is carried out at 37°C in a 5% CO2 environment for 3-6 days.

4. The method of claim 1, wherein, The mesenchymal stem cells are plated at a seeding density of 5 x 10 3 -10 x 10 3 / cm 2 .

5. A composition for promoting articular cartilage regeneration, characterized by, mesenchymal stem cells with chondrogenic differentiation mechanical memory obtained by the method according to any one of claims 1-4.

6. The composition of claim 5, wherein, The composition also comprises a hydrogel or chondrogenic differentiation promoting substance, the concentration of the mesenchymal stem cells having chondrogenic differentiation mechanical memory in the composition being 8 x 10 3 -12 x 10 3 / μL.

7. The composition of claim 6, wherein, the chondrogenic differentiation promoter comprises at least one of KGN, psoralen and vitamin C.

8. Use of the composition according to any one of claims 5-7 in the preparation of a drug for promoting the regeneration of articular cartilage.

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