Amniotic mesenchymal stem cells for treating knee arthritis, and preparation method and application thereof
Amniotic mesenchymal stem cells prepared using a specific culture medium have been used to treat knee osteoarthritis, solving the problem of cartilage repair and regeneration in knee osteoarthritis, achieving effective cartilage damage repair and immune regulation, and reducing production costs and safety risks.
Patent Information
- Application Number
- CN202511393976.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-28
- Publication Date
- 2026-02-24
- Estimated Expiration
- 2045-09-28
AI Technical Summary
Existing technologies are difficult to effectively promote cartilage repair and regeneration in the treatment of knee osteoarthritis, and there are problems with immune response and inflammatory damage.
Amniotic mesenchymal stem cells (AMSCs) were prepared and cultured in a specific culture medium (containing basic fibroblast growth factor, L-glutamine, transferrin, vitamin C, sodium selenite, insulin, shampodophyllin, and teprenone) to enhance their osteogenic differentiation capacity and were then applied in the knee joint to promote cartilage repair.
It significantly enhances the osteogenic differentiation capacity of amniotic mesenchymal stem cells, effectively inhibits joint inflammation, regulates the immune microenvironment, promotes chondrocyte repair and regeneration, reduces production costs, and improves the safety of clinical applications.
Smart Images
Figure CN120866212B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to amniotic mesenchymal stem cells for the treatment of knee osteoarthritis, their preparation method, and their application. Background Technology
[0002] Knee osteoarthritis is a degenerative joint disease characterized by progressive degeneration of articular cartilage. In the early stages, erosion and fibrosis first appear on the cartilage surface. As the disease progresses, patients experience full-thickness cartilage damage, eventually leading to bone-hard bone friction and osteophyte formation. This severely restricts knee joint mobility, causing pain and discomfort, and significantly impacting patients' quality of life. Therefore, timely intervention and treatment of cartilage damage caused by knee osteoarthritis are of great importance.
[0003] Amniotic mesenchymal stem cells (AMSCs) have become a research hotspot in tissue engineering and cell therapy due to their ease of acquisition, low ethical controversy, and strong proliferation and differentiation capabilities. Derived from placental amniotic tissue, AMSCs possess multi-lineage differentiation potential, low immunogenicity, and anti-inflammatory properties, demonstrating significant potential in regenerative medicine, immunomodulation, and tissue repair. In the treatment of knee osteoarthritis, AMSCs can differentiate into chondrocytes, promoting the repair and regeneration of damaged cartilage; simultaneously, the various bioactive factors they secrete can effectively inhibit intra-articular inflammatory responses, improve the local microenvironment, and slow down cartilage degradation. Furthermore, AMSCs also possess immunomodulatory functions, regulating the activity of immune cells such as T cells and macrophages, and reducing joint inflammation. These characteristics indicate that AMSCs have promising applications in the treatment of knee osteoarthritis.
[0004] To achieve the above objectives, the present invention provides amniotic mesenchymal stem cells for the treatment of knee osteoarthritis, their preparation method, and their application. Summary of the Invention
[0005] The primary objective of this invention is to provide a method for preparing amniotic mesenchymal stem cells for the treatment of knee osteoarthritis.
[0006] A second objective of this invention is to provide amniotic mesenchymal stem cells for the treatment of knee osteoarthritis.
[0007] A third objective of this invention is to provide the application of amniotic mesenchymal stem cells for the treatment of knee osteoarthritis in the preparation of drugs for treating knee osteoarthritis.
[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0009] A method for preparing amniotic mesenchymal stem cells for the treatment of knee osteoarthritis, the specific steps of which are as follows:
[0010] (1) Placental tissue was taken and amnion tissue was separated;
[0011] (2) Add the mixed enzyme digestion solution to the amniotic tissue obtained in step (1) for digestion, filter, centrifuge, and obtain cell precipitate; the concentration of each component in the mixed enzyme digestion solution is as follows, based on the final concentration: 1-3 U / mL neutral protease, 0.2-0.4 mg / mL deoxyribonuclease I, 1-2 mg / mL collagenase IV;
[0012] (3) The cell pellet obtained in step (2) is added to the culture medium and resuspended to obtain a cell suspension. The cell suspension is inoculated into the culture medium and cultured. Trypsin is added for digestion, then centrifuged and the supernatant is discarded. The resulting precipitate is P0 generation amniotic mesenchymal stem cells.
[0013] (4) The P0 generation amniotic mesenchymal stem cells are seeded into the culture medium for passage culture to obtain amniotic mesenchymal stem cells; the culture medium in steps (3) and (4) includes a basic culture medium and the following components added to the basic culture medium: basic fibroblast growth factor, L-glutamine, transferrin, vitamin C, sodium selenite, insulin, shamfotaside, and teprenone; the concentrations of each component in the culture medium are as follows, based on the final concentration: basic fibroblast growth factor 5-18 ng / mL, L-glutamine 1.8-2.5 mg / mL, transferrin 3-10 mg / mL, vitamin C 20-30 μg / mL, sodium selenite 12-25 μg / mL, insulin 10-15 μg / mL, shamfotaside 15-30 μg / mL, and teprenone 20-42 ng / mL.
[0014] Furthermore, in step (4), the basic culture medium is DMEM / F12 culture medium.
[0015] Furthermore, in step (3), the inoculation density is 1-3 × 10⁻⁶. 4 pcs / cm 2 The culture conditions were 37°C and a CO2 incubator with 5 v / v%.
[0016] Furthermore, in step (4), the seeding rate is 1:(3-6); the culture conditions are 37℃, cultured in a CO2 incubator with 5v / v%.
[0017] Furthermore, the amniotic mesenchymal stem cells obtained from passage culture in step (4) are P3-P5 generation amniotic mesenchymal stem cells.
[0018] The amniotic mesenchymal stem cells prepared according to the above-described method for treating knee osteoarthritis are used in the preparation of drugs for treating knee osteoarthritis.
[0019] Compared with the prior art, the main advantages of the present invention are as follows:
[0020] This invention prepares its own culture medium by adding fibroblast growth factor, L-glutamine, transferrin, vitamin C, sodium selenite, insulin, shamfotaximeline, and teprenone to a basal culture medium. Experimental results show that this culture medium can maintain the stem cell nature of amniotic mesenchymal stem cells and enhance their osteogenic differentiation capacity, thereby effectively treating knee osteoarthritis. Simultaneously, this basal culture medium does not contain fetal bovine serum, reducing production costs and avoiding the risk of introducing bacteria and viruses carried by heterologous serum, thus improving the safety of clinical applications. Attached Figure Description
[0021] Figure 1 Morphological diagram of P4 generation amniotic mesenchymal stem cells;
[0022] Figure 2 These are the results of osteogenic differentiation of amniotic mesenchymal stem cells obtained in Examples 1-3 of this invention;
[0023] Figure 3 The diagram shows the osteogenic differentiation results of amniotic mesenchymal stem cells prepared in Comparative Examples 1-4 of this invention. Detailed Implementation
[0024] The following is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.
[0025] Example 1
[0026] A method for preparing amniotic mesenchymal stem cells for the treatment of knee osteoarthritis, specifically including the following steps:
[0027] (1) Placental tissue was taken and amnion tissue was separated;
[0028] (2) Cut the amniotic tissue obtained in step (1) into pieces of about 1 mm. 2 Small pieces were added to an equal volume of mixed enzyme digestion solution and digested at 37°C for 60 minutes. The mixture was then filtered, centrifuged, and a precipitate was obtained. The concentrations of each component in the mixed enzyme digestion solution were 2 U / mL neutral protease, 0.3 mg / mL deoxyribonuclease I, and 1.5 mg / mL collagenase IV.
[0029] (3) The precipitate obtained in step (2) is added to the culture medium and resuspended to obtain a cell suspension. The cell suspension is then thawed at 2×10⁻⁶ ppm. 4 pcs / cm 2 The cells were seeded into the culture medium at a density of 100%, and cultured in a 37°C, 5v / v% CO2 incubator. After digestion with trypsin, the cells were centrifuged, the supernatant was discarded, and the resulting precipitate was P0 generation amniotic mesenchymal stem cells.
[0030] (4) P0 generation amniotic mesenchymal stem cells were seeded into the culture medium at a ratio of 1:4 and passaged in a 37℃, 5v / v% CO2 incubator to obtain P4 generation amniotic mesenchymal stem cells. The morphology of the amniotic mesenchymal stem cells was observed under a microscope, and the results are as follows: Figure 1 As shown. The culture medium in steps (3) and (4) includes DMEM / F12 medium and the following components added to the DMEM / F12 medium: basic fibroblast growth factor 11 ng / mL, L-glutamine 2.1 mg / mL, transferrin 6 mg / mL, vitamin C 25 μg / mL, sodium selenite 18 μg / mL, insulin 12 μg / mL, shampoosine 22 μg / mL, and teprenone 31 ng / mL.
[0031] An amniotic mesenchymal stem cell for the treatment of knee osteoarthritis was prepared according to the above-described preparation method.
[0032] Example 2
[0033] A method for preparing amniotic mesenchymal stem cells for the treatment of knee osteoarthritis, specifically including the following steps:
[0034] (1) Placental tissue was taken and amnion tissue was separated;
[0035] (2) Cut the amniotic tissue obtained in step (1) into pieces of about 1 mm. 2 Small pieces were added to an equal volume of mixed enzyme digestion solution and digested at 37°C for 60 minutes. The mixture was then filtered, centrifuged, and a precipitate was obtained. The concentrations of each component in the mixed enzyme digestion solution were 2 U / mL neutral protease, 0.3 mg / mL deoxyribonuclease I, and 1.5 mg / mL collagenase IV.
[0036] (3) The cell pellet obtained in step (2) is added to a culture medium and resuspended to obtain a cell suspension. The cell suspension is then thawed at 1×10⁻⁶ ppm. 4 pcs / cm 2 The cells were seeded into the culture medium at a density of 100%, and cultured in a 37°C, 5v / v% CO2 incubator. After digestion with trypsin, the cells were centrifuged, the supernatant was discarded, and the resulting precipitate was P0 generation amniotic mesenchymal stem cells.
[0037] (4) P0 generation amniotic mesenchymal stem cells were seeded into the culture medium at a seeding ratio of 1:3 and placed in a 37°C, 5v / v% CO2 incubator for passage culture to obtain P3 generation amniotic mesenchymal stem cells; the culture medium in steps (3) and (4) includes DMEM / F12 culture medium and the following components added to DMEM / F12 culture medium: basic fibroblast growth factor 5 ng / mL, L-glutamine 1.8 mg / mL, transferrin 3 mg / mL, vitamin C 20 μg / mL, sodium selenite 12 μg / mL, insulin 10 μg / mL, shampoosine 15 μg / mL, and teprenone 20 ng / mL.
[0038] An amniotic mesenchymal stem cell for the treatment of knee osteoarthritis was prepared according to the above-described preparation method.
[0039] Example 3
[0040] A method for preparing amniotic mesenchymal stem cells for the treatment of knee osteoarthritis, specifically including the following steps:
[0041] (1) Placental tissue was taken and amnion tissue was separated;
[0042] (2) Cut the amniotic tissue obtained in step (1) into pieces of about 1 mm. 2 Small pieces were added to an equal volume of mixed enzyme digestion solution and digested at 37°C for 60 minutes. The mixture was then filtered, centrifuged, and a precipitate was obtained. The concentrations of each component in the mixed enzyme digestion solution were 2 U / mL neutral protease, 0.3 mg / mL deoxyribonuclease I, and 1.5 mg / mL collagenase IV.
[0043] (3) The cell pellet obtained in step (2) is added to the culture medium and resuspended to obtain a cell suspension. The cell suspension is then thawed at 3×10⁻⁶ ppm. 4 pcs / cm 2 The cells were seeded into the culture medium at a density of 100%, and cultured in a 37°C, 5v / v% CO2 incubator. After digestion with trypsin, the cells were centrifuged, the supernatant was discarded, and the resulting precipitate was P0 generation amniotic mesenchymal stem cells.
[0044] (4) P0 generation amniotic mesenchymal stem cells were seeded into the culture medium at a seeding ratio of 1:5 and placed in a 37°C, 5% CO2 incubator for passage culture to obtain P5 generation amniotic mesenchymal stem cells; the culture medium in steps (3) and (4) includes DMEM / F12 culture medium and the following components added to DMEM / F12 culture medium: basic fibroblast growth factor 18 ng / mL, L-glutamine 2.5 mg / mL, transferrin 10 mg / mL, vitamin C 30 μg / mL, sodium selenite 25 μg / mL, insulin 15 μg / mL, shampoosine 30 μg / mL, and teprenone 42 ng / mL.
[0045] An amniotic mesenchymal stem cell for the treatment of knee osteoarthritis was prepared according to the above-described preparation method.
[0046] Comparative Example 1
[0047] The difference between Comparative Example 1 and Example 1 is that shampootinamide in the culture medium components in steps (3) and (4) is omitted, while the rest are the same as in Example 1.
[0048] Comparative Example 2
[0049] The difference between Comparative Example 2 and Example 1 is that teprenone in the culture medium components in steps (3) and (4) is omitted, while the rest are the same as in Example 1.
[0050] Comparative Example 3
[0051] The difference between Comparative Example 3 and Example 1 is that the amount of shampootinamide in the culture medium components in steps (3) and (4) was adjusted to 5 μg / mL.
[0052] Comparative Example 4
[0053] The difference between Comparative Example 4 and Example 1 is that the amount of shampoosine in the culture medium components in steps (3) and (4) was adjusted to 50 μg / mL.
[0054] Experimental Example 1
[0055] Effects of different culture media on the osteogenic differentiation capacity of amniotic mesenchymal stem cells
[0056] (1) Amniotic mesenchymal stem cells prepared in Examples 1, 2, 3, Comparative Examples 1, 2, 3, and 4 of the present invention were used at a concentration of 3 × 10⁻⁶. 4 / wells were seeded into 24-well culture plates and cultured in DMEM / F12 medium containing 10% (v / v) FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL) until the cells reached about 70% confluence. The supernatant was discarded and the cells were placed in osteogenic induction differentiation medium (DMEM / F12 medium containing dexamethasone (0.15 μmol / L), sodium β-glycerophosphate (10 mmol / L), sodium ascorbate (50 mg / L), 10% (v / v) FBS, penicillin (100 U / mL), and streptomycin (100 μg / mL)) to induce osteogenic differentiation of amniotic mesenchymal stem cells.
[0057] (2) The osteogenic induction medium was changed every 2 days. On the 10th day of osteogenic induction culture, the 24-well plate was removed from the incubator and placed in a clean bench. After removing the original medium from the wells, the plate was washed 3 times with PBS. 600 μL of paraformaldehyde solution was added for fixation for 20 min, and the fixative was discarded. The plate was then washed 3 times with PBS. According to the alkaline phosphatase kit instructions, the osteogenic differentiation cells of the obtained amniotic mesenchymal stem cells were stained. After staining, the cells were photographed under a fluorescence microscope. The osteogenic differentiation capacity of the amniotic mesenchymal stem cells was determined based on the staining results.
[0058] The results are as follows Figure 2 , Figure 3 The figures show the osteogenic differentiation capacity of amniotic mesenchymal stem cells prepared from different groups. Compared with comparative groups 1-4, the amniotic mesenchymal stem cells prepared from groups 1-3 of this invention exhibited stronger osteogenic differentiation capacity. This indicates that adding shampodoside and teprenone to the basal culture can provide the necessary nutritional support for the expansion process of amniotic mesenchymal stem cells, maintain the stemness of amniotic mesenchymal stem cells, and enhance their osteogenic differentiation capacity.
[0059] Experimental Example 2
[0060] Efficacy trial of amniotic mesenchymal stem cell therapy for knee osteoarthritis
[0061] (1) The amniotic mesenchymal stem cells prepared in Examples 1-3 and Comparative Examples 1-4 were collected into centrifuge tubes, resuspended in PBS, centrifuged at 2000 rpm for 10 min, the supernatant was removed, and the cells were resuspended in physiological saline. The cell concentration was adjusted to 3 × 10⁻⁶ cells / mL. 6 / mL, which yields a biological agent for treating knee joint problems.
[0062] (2) Eighty 6-month-old male New Zealand white rabbits were randomly divided into a control group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, and Comparative Example 4 group, with 10 rabbits in each group. The New Zealand white rabbits were in good health. During the experiment, the rabbits were anesthetized by the marginal ear vein with 3% sodium pentobarbital solution (1 mL / kg), and their fur was shaved and their right hind limb was disinfected. An incision was made along the inner edge of the patella, the medial joint capsule was cut, the joint cavity was exposed, the knee joint was hyperextended and the patella was dislocated laterally, and a 3 mm diameter hole was drilled in the center of the medial femoral condyle with a 2.0 mm Kirschner wire to create a cartilage defect model, and then the wound was sutured. One week later, each group of rabbits was injected with 1 mL of the corresponding group's knee joint treatment biological agent, and the injection was repeated once each in weeks 3, 5, and 7. At week 8, the experimental rabbits were sacrificed, and the repaired cartilage was histologically scored according to the Wakitani scoring system to evaluate the efficacy of the biological agent in each group. The Wakitani scoring criteria consist of five indicators: cell morphology, matrix staining, surface smoothness, thickness of newly formed cartilage, and integrity of connection with the recipient area. The scoring range is 0-14 points; a lower score indicates that the newly formed repair tissue is closer to normal tissue. The Wakitani scoring criteria are shown in Table 1. The histological scoring results are shown in Table 2.
[0063] Table 1 Wakitani Scoring Criteria
[0064]
[0065] Table 2 Histological Scoring Results
[0066]
[0067] The results are shown in Table 2, which illustrates the repair effects of biological agents prepared from amniotic mesenchymal stem cells in different groups on cartilage damage. As can be seen from the table, compared with the blank control group and comparative groups 1-4, the biological agents prepared from amniotic mesenchymal stem cells in Examples 1-3 of this invention significantly improved the repair effect on cartilage damage. This indicates that adding shampoosine and teprenone to the basal culture promotes the stem cell activity of amniotic mesenchymal stem cells, facilitates their efficient secretion of various bioactive factors, inhibits joint inflammation, regulates the immune microenvironment, and promotes the repair and regeneration of the host's own chondrocytes. Furthermore, these agents can more effectively differentiate into chondrocytes in the in vivo cartilage defect microenvironment, thereby synergistically achieving structural cartilage repair and thus achieving the goal of treating knee osteoarthritis.
[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.
Claims
1. A method for preparing amniotic mesenchymal stem cells for the treatment of knee osteoarthritis, characterized in that, The specific steps are as follows: (1) Placental tissue was taken and amnion tissue was separated; (2) Add the mixed enzyme digestion solution to the amniotic tissue obtained in step (1) for digestion, filter, centrifuge, and obtain cell precipitate; the concentration of each component in the mixed enzyme digestion solution is as follows, based on the final concentration: 1-3 U / mL neutral protease, 0.2-0.4 mg / mL deoxyribonuclease I, 1-2 mg / mL collagenase IV; (3) The cell pellet obtained in step (2) is added to the culture medium and resuspended to obtain a cell suspension. The cell suspension is inoculated into the culture medium and cultured. Trypsin is added for digestion, then centrifuged and the supernatant is discarded. The resulting precipitate is P0 generation amniotic mesenchymal stem cells. (4) P0 generation amniotic mesenchymal stem cells are seeded into a culture medium for passage culture to obtain amniotic mesenchymal stem cells; the amniotic mesenchymal stem cells obtained by passage culture are P3-P5 generation amniotic mesenchymal stem cells; the culture medium in steps (3) and (4) includes a basic culture medium and the following components added to the basic culture medium: basic fibroblast growth factor, L-glutamine, transferrin, vitamin C, sodium selenite, insulin, shamfotaside, and teprenone; the concentrations of each component in the culture medium are as follows, based on the final concentration: basic fibroblast growth factor 5-18 ng / mL, L-glutamine 1.8-2.5 mg / mL, transferrin 3-10 mg / mL, vitamin C 20-30 μg / mL, sodium selenite 12-25 μg / mL, insulin 10-15 μg / mL, shamfotaside 15-30 μg / mL, and teprenone 20-42 ng / mL.
2. The method for preparing amniotic mesenchymal stem cells for the treatment of knee osteoarthritis according to claim 1, characterized in that, In step (4), the basic culture medium is DMEM / F12 culture medium.
3. The method for preparing amniotic mesenchymal stem cells for the treatment of knee osteoarthritis according to claim 1, characterized in that, In step (3), the inoculation density is 1-3 × 10⁻⁶. 4 pcs / cm 2 The culture conditions were 37°C and a CO2 incubator with 5 v / v%.
4. The method for preparing amniotic mesenchymal stem cells for the treatment of knee osteoarthritis according to claim 1, characterized in that, In step (4), the seeding rate is 1:(3-6); the culture conditions are 37℃ and 5v / v% CO2 incubator.
5. The application of amniotic mesenchymal stem cells prepared by the method for preparing amniotic mesenchymal stem cells for the treatment of knee osteoarthritis according to any one of claims 1-4 in the preparation of drugs for treating knee osteoarthritis.
Citation Information
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