Application of induced treatment of osteoarthritis by pluripotent stem cells

Abstract

The invention discloses application of pluripotent stem cells to induced treatment of osteoarthritis, and belongs to the technical field of regenerative medicine. According to the research, a natural inducer is used for inducing pluripotent stem cells to be differentiated into mesenchymal stem cells, cartilage precursor cells and mature cartilage cells, and the mature cartilage cells and the mesenchymal stem cells are compounded to prepare the medicine for treating osteoarthritis to treat white rabbits with osteoarthritis, so that osteoarthritis inflammation can be remarkably eliminated, and damaged cartilage can be repaired; the curative effect is obviously superior to that of treatment by singly using mature cartilage cells or pluripotent stem cells. Through the synergistic effect of the mature cartilage cells, the human induced pluripotent stem cells, the natural inducer with the effects of diminishing inflammation and promoting cartilage regeneration, the dexamethasone and the glucosamine in combination with the collagen carrier, a microenvironment is provided for cell growth, the cell survival rate is guaranteed, inflammation is specifically inhibited, articular cartilage is repaired, and the cartilage repairing effect is improved. The invention provides a new scheme with better effect and more comprehensive mechanism for the treatment of osteoarthritis, and has important clinical transformation value.

Description

Technical Field

[0001] This invention relates to the field of regenerative medicine technology, specifically to the use of pluripotent stem cell induction therapy for osteoarthritis. Background Technology

[0002] Osteoarthritis is the most common form of arthritis. Patients with osteoarthritis experience severe pain and loss of mobility, significantly impacting their quality of life. In 2020, 595 million people worldwide suffered from osteoarthritis, placing a huge burden on society. Among all joints, the knee is the most frequently affected joint in osteoarthritis. Major risk factors for osteoarthritis include age, being overweight, and knee injuries. Recent studies have found a close correlation between meniscus damage in knee injuries and the development and progression of osteoarthritis; the meniscus plays a crucial role in maintaining knee health.

[0003] Chondrogenesis refers to the differentiation of mesenchymal stem cells into chondrocytes, a process dependent on complex interactions and cross-regulation among various regulatory factors and signaling pathways. However, currently, there are no approved drugs specifically targeting cartilage matrix regeneration for disease-modifying osteoarthritis, and existing treatments only alleviate pain and inflammation symptoms. Stem cell therapy, currently a hot research topic in disease treatment, faces two major challenges in osteoarthritis treatment: firstly, in vitro induction often relies on growth factors or chemical inducers, which significantly increases the potential risks of carcinogenesis, stem cell differentiation errors, and cytotoxicity; secondly, the joint cavity of osteoarthritis patients contains a large number of inflammatory factors and degrading enzymes, creating a highly inflammatory environment that is unfavorable for stem cell growth, proliferation, and differentiation, leading to unsatisfactory treatment outcomes. Furthermore, existing cell delivery technologies lack the ability to actively regulate the microenvironment, resulting in unsustainable and unstable treatment effects.

[0004] Therefore, there is an urgent need for a treatment that can efficiently induce stem cells to differentiate into chondrocytes and ensure cell survival in order to eliminate osteoarthritis inflammation and promote cartilage repair. Summary of the Invention

[0005] Therefore, this invention provides a method for treating osteoarthritis by inducing pluripotent stem cells, in order to solve the problems in the prior art, which are due to the focus on anti-inflammation, poor stem cell resistance to harsh environments, and insufficient carrier-mediated environmental regulation, resulting in the inability to repair cartilage, large side effects, poor stem cell differentiation ability, and low survival rate.

[0006] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the invention, there is provided the use of pluripotent stem cell-induced therapy for osteoarthritis, including the use of pluripotent stem cells in the preparation of medicaments for treating or alleviating primary or secondary osteoarthritis.

[0007] Preferably, the pluripotent stem cells are human induced pluripotent stem cells.

[0008] Furthermore, the drug for treating or alleviating primary or secondary osteoarthritis comprises a carrier and active cells, wherein the ratio of active cells to carrier is 5 × 10⁻⁶. 6 CFU cells were loaded onto a 1 mL carrier and combined with a multipotent stem cell induction therapy drug for osteoarthritis. The drug was administered as an injection.

[0009] Furthermore, the active cells are obtained by combining human induced pluripotent stem cells and mature chondrocytes prepared by differentiation of human induced pluripotent stem cells at a ratio of 3:7.

[0010] Furthermore, the method for preparing mature chondrocytes obtained from the differentiation of human induced pluripotent stem cells includes the following steps: S1. Human induced pluripotent stem cells are induced to differentiate into mesenchymal stem cells; S2, mesenchymal stem cells are induced to differentiate into chondrocyte precursor cells; S3, chondrocyte precursor cells are induced to differentiate into mature chondrocytes.

[0011] Further, step S1 includes the following steps: a. Human induced pluripotent stem cells were used at a rate of 5 × 10⁻⁶ 3 Inoculate the culture flasks at a density of CFU / cm² and incubate them in serum-free DEME / F12 medium containing 1% penicillin-streptomycin antibiotics (i.e., medium 1) at 5% CO2 and 37°C for 24 h. b. Take fresh culture medium 1, add 10 μmol / L of polysaccharide and 50 μmol / L of magnesium ascorbate phosphate to obtain culture medium 2; c. Discard culture medium 1, add culture medium 2, and incubate in a 37℃, 5% CO2 incubator for 6 days. Replace the induction culture medium with fresh medium every 2 days to obtain mesenchymal stem cells.

[0012] Further, step S2 includes the following steps: a. Mesenchymal stem cells were injected at a rate of 1×10⁻⁶. 4 Subcultured to a density of CFU / cm² into a new culture flask and cultured at 37°C for 24 h with 5% CO2. b. Add 1% insulin-transferrin-selenium, proline to a final concentration of 40 μg / mL, and 1% penicillin-streptomycin antibiotics to the high-glucose DEME medium to obtain medium 3; c. After passage, the cells were transferred to medium 3 containing 10 μmol / L curcumin, 50 μmol / L magnesium ascorbate phosphate and 5 μmol / L icariin, and cultured at 37°C for 14 days with the induction medium changed every 3 days to obtain chondrocyte precursor cells.

[0013] Further, step S3 includes the following steps: Select CD105 + / Sox9 + Double-positive chondrogenic progenitor cells, at 2×10 4 CFU / cm² was seeded and cultured at 5% CO2 and 37°C for 3 days in medium containing 10 μmol / L curcumin, 5 μmol / L tanshinone IIA, 20 μmol / L EGCG and 50 μmol / L magnesium ascorbate phosphate to obtain mature chondrocytes.

[0014] Furthermore, the carrier is a collagen carrier.

[0015] Furthermore, the preparation method of the collagen carrier includes the following steps: S1. Dissolve type O collagen and recombinant human type II collagen in serum-free DEME / F12 medium at a mass ratio of 1:1 until the final concentration of each collagen is 3 mg / mL. Stir at 4°C for 12 h and adjust the pH to 7.2-7.4 to obtain collagen solution. S2. Add dexamethasone to the collagen solution at a final concentration of 10-50 nmol / L, glucosamine at a final concentration of 5-10 mmol / L, and a trace inducer, and stir until homogeneous to obtain a collagen-trace inducer solution. S3. Dissolve gelatin in DEME / F12 medium to a final concentration of 20 mg / mL; add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the gelatin solution at a molar ratio of 1:2:1 and activate at room temperature for 10-15 min; add 3-aminophenylboronic acid to the activated solution at a molar ratio of 50 μmol:1 g and react in the dark at room temperature for 12-24 h; dialyze with deionized water for 2-3 days and then freeze-dry to obtain PBA-modified gelatin peptides; S4. Add PBA-modified gelatin peptides to the collagen-micro-inducer solution to make the final concentration of PBA-modified gelatin peptides 2-5 mg / mL, crosslink at room temperature for 15-20 min, and obtain collagen carrier.

[0016] Furthermore, the trace inducer is a mixture of curcumin, EGCG, resveratrol and polygalactoside, with final concentrations of 1-2 μmol / L, 2-4 μmol / L, 1-2 μmol / L and 1-2 μmol / L, respectively.

[0017] Furthermore, this invention provides an application for preparing a pluripotent stem cell-induced drug for the treatment of osteoarthritis, the specific steps of which include: S1. Human induced pluripotent stem cells are induced to differentiate into mesenchymal stem cells: a. Human induced pluripotent stem cells were used at a rate of 5 × 10⁻⁶ 3 Inoculate the culture flasks at a density of CFU / cm² and incubate them in serum-free DEME / F12 medium containing 1% penicillin-streptomycin antibiotics (i.e., medium 1) at 5% CO2 and 37°C for 24 h. b. Take fresh culture medium 1, add 10 μmol / L of polysaccharide and 50 μmol / L of magnesium ascorbate phosphate to obtain culture medium 2; c. Discard culture medium 1, add culture medium 2, and incubate in a 37℃, 5% CO2 incubator for 6 days. Replace the induction culture medium with fresh medium every 2 days during this period to obtain mesenchymal stem cells. S2. Mesenchymal stem cells are induced to differentiate into chondrocyte progenitor cells: a. Mesenchymal stem cells were injected at a rate of 1×10⁻⁶. 4 Subcultured to a density of CFU / cm² into a new culture flask and cultured at 37°C for 24 h with 5% CO2. b. Add 1% insulin-transferrin-selenium, proline to a final concentration of 40 μg / mL, and 1% penicillin-streptomycin antibiotics to the high-glucose DEME medium to obtain medium 3; c. After passage, the cells were transferred to medium 3 containing 10 μmol / L curcumin, 50 μmol / L magnesium ascorbate phosphate and 5 μmol / L icariin, and cultured at 37°C for 14 days with the induction medium replaced every 3 days to obtain chondrocyte precursor cells. S3. Chondroblast precursor cells are induced to differentiate into mature chondrocytes: Select CD105 + / Sox9 + Double-positive chondrogenic progenitor cells, at 2×10 4 CFU / cm² was seeded and cultured in medium containing 10 μmol / L curcumin, 5 μmol / L tanshinone IIA, 20 μmol / L EGCG and 50 μmol / L magnesium ascorbate phosphate for 3 days at 5% CO2 and 37°C to obtain mature chondrocytes. S4. Preparation of collagen carrier: a. Dissolve type O collagen and recombinant human type II collagen in serum-free DEME / F12 medium at a mass ratio of 1:1 until the final concentration of each collagen is 3 mg / mL. Stir at 4°C for 12 h and adjust the pH to 7.2-7.4 to obtain collagen solution. b. Add curcumin, EGCG, resveratrol, polydipsia glycoside, dexamethasone and glucosamine to the collagen solution to make their final concentrations 1-2 μmol / L, 2-4 μmol / L, 1-2 μmol / L, 1-2 μmol / L, 10-50 nmol / L and 5-10 mmol / L, respectively, and stir well to obtain collagen-micro-inducer solution; c. Dissolve gelatin in DEME / F12 medium to a final concentration of 20 mg / mL; add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the gelatin solution at a molar ratio of 1:2:1 (carboxyl group of gelatin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide), and activate at room temperature for 10-15 min; add 3-aminophenylboronic acid to the activated solution at a molar-mass ratio of 50 μmol:1 g (3-aminophenylboronic acid to gelatin), and react at room temperature in the dark for 12-24 h; dialyze with deionized water for 2-3 days and then lyophilize to obtain PBA-modified gelatin peptides; d. Add PBA-modified gelatin peptides to the collagen-micro-inducer solution to make the final concentration of PBA-modified gelatin peptides 2-5 mg / mL, crosslink at room temperature for 15-20 min to obtain collagen carrier; S5. Mature chondrocytes and human induced pluripotent stem cells were mixed at a cell ratio of 7:3 and then dispersed in phosphate buffer (pH 7.2-7.4) until the cell density reached 1×10⁻⁶. 6 -5×10 6 CFU / mL, based on a cell-to-carrier cell number-volume ratio of 5 × 10⁻⁶. 6 CFU: 1 mL of cells loaded onto a carrier is a drug for the induction of osteoarthritis by pluripotent stem cells. S6. Aspirate the pluripotent stem cell-induced osteoarthritis drug into a 25G syringe and inject it into the OA cartilage defect area through joint puncture within 30 minutes. The injection volume is adjusted according to the defect area, with 0.5-1 mL injected per cm² of defect. After injection, have the patient slowly move the joint 3-5 times to promote uniform drug distribution.

[0018] The present invention has the following advantages: 1. This invention uses polygalactoside, curcumin, icariin, tanshinone IIA and EGCG as inducing agents for pluripotent stem cell differentiation. All inducing agents are all-natural products, reducing the risk of carcinogenesis and cytotoxicity, and covering the entire pathway from stem cells to chondrocytes in stages. Curcumin, while inducing differentiation, inhibits the differentiation of mesenchymal stem cells into osteoblasts and adipocytes, improves the differentiation efficiency of chondrocytes, and reduces the risk of disease.

[0019] 2. This invention uses a combination of chondrocytes and pluripotent stem cells in a specific ratio. Mature chondrocytes are used to quickly fill defects, while pluripotent stem cells are reserved for long-term proliferation and differentiation, thus avoiding the problem of short-lasting repair by a single cell type.

[0020] 3. Using type O collagen and type II collagen as cell carriers, the extracellular matrix environment of chondrocytes is simulated to provide cell adhesion and nutritional support. At room temperature, it is a sol, which gels in situ after injection, fixing the cells in the cartilage defect area and preventing loss. At the same time, type II collagen is the main component of cartilage and helps the regeneration of cartilage tissue. Type O collagen improves the dispersibility of the system, which is convenient for the preparation of microgel injections.

[0021] 4. A carrier-loaded sustained-release combination of dexamethasone / glucosamine / micro-inducer. Dexamethasone is a potent anti-inflammatory agent and also acts as an inducer of chondrocytes. Micro-addition can enhance the in vivo induction efficiency of the above-mentioned inducers. Glucosamine inhibits the degradation of cartilage matrix, and the micro-inducer, as a polyphenol, has anti-inflammatory effects. It can synergistically reduce inflammation and promote cell survival, further optimizing the repair microenvironment. At the same time, it slowly promotes the further induction of stem cells in vivo, thereby improving the efficiency of cartilage regeneration.

[0022] 5. The entire system of this invention is almost entirely composed of natural products, with low antigenicity and low cytotoxicity to the human body. Type II collagen, as the main component of cartilage, can effectively supplement cartilage repair. This effectively improves the core defects of existing conventional technologies that focus on anti-inflammatory and analgesic effects but cannot repair cartilage, have large systemic side effects of conventional anti-inflammatory drugs, and have poor differentiation, low survival, and insufficient safety of traditional stem cell technology in high inflammatory environments. Attached Figure Description

[0023] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0024] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0025] Figure 1 A graph showing the decrease rate of IL-1β in the joint fluid of rabbits after 4 weeks of pluripotent stem cell drug treatment, provided by the present invention. Figure 2 A graph showing the decrease rate of TNF-α in the joint fluid of rabbits after 4 weeks of pluripotent stem cell drug treatment, as provided by this invention. Figure 3 This invention provides images showing the cartilage defect filling rate in rabbits after 12 and 24 weeks of pluripotent stem cell drug injection treatment; wherein... Figure 3 A shows the cartilage defect filling rate in rabbits 12 weeks after injection; Figure 3 B is a graph showing the cartilage defect filling rate of rabbits 24 weeks after injection; Figure 4 This invention provides a stride length and support time diagram for rabbits after 24 weeks of pluripotent stem cell drug treatment; wherein... Figure 4 A is the support time graph of the rabbits after treatment; Figure 4 B is a graph showing the stride length of the rabbits after treatment. Detailed Implementation

[0026] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] According to a first aspect of the invention, there is provided the use of pluripotent stem cell-induced therapy for osteoarthritis, including the use of pluripotent stem cells in the preparation of medicaments for treating or alleviating primary or secondary osteoarthritis.

[0028] Among them, pluripotent stem cells are human induced pluripotent stem cells.

[0029] Among them, drugs for treating or alleviating primary or secondary osteoarthritis include carriers and active cells, with an active cell to carrier ratio of 5 × 10⁻⁶. 6 CFU cells were loaded onto a 1 mL carrier and combined with a multipotent stem cell induction therapy drug for osteoarthritis. The drug was administered as an injection.

[0030] Among them, the active cells are obtained by combining human induced pluripotent stem cells and mature chondrocytes prepared by differentiation of human induced pluripotent stem cells at a ratio of 3:7.

[0031] The method for preparing mature chondrocytes obtained from the differentiation of human induced pluripotent stem cells includes the following steps: S1. Human induced pluripotent stem cells are induced to differentiate into mesenchymal stem cells; S2, mesenchymal stem cells are induced to differentiate into chondrocyte precursor cells; S3, chondrocyte precursor cells are induced to differentiate into mature chondrocytes.

[0032] Step S1 includes the following steps: a. Human induced pluripotent stem cells were used at a rate of 5 × 10⁻⁶ 3 Inoculate the culture flasks at a density of CFU / cm² and incubate them in serum-free DEME / F12 medium containing 1% penicillin-streptomycin antibiotics (i.e., medium 1) at 5% CO2 and 37°C for 24 h. b. Take fresh culture medium 1, add 10 μmol / L of polysaccharide and 50 μmol / L of magnesium ascorbate phosphate to obtain culture medium 2; c. Discard culture medium 1, add culture medium 2, and incubate in a 37℃, 5% CO2 incubator for 6 days. Replace the induction culture medium with fresh medium every 2 days to obtain mesenchymal stem cells.

[0033] Step S2 includes the following steps: a. Mesenchymal stem cells were injected at a rate of 1×10⁻⁶. 4 Subcultured to a density of CFU / cm² into a new culture flask and cultured at 37°C for 24 h with 5% CO2. b. Add 1% insulin-transferrin-selenium, proline to a final concentration of 40 μg / mL, and 1% penicillin-streptomycin antibiotics to the high-glucose DEME medium to obtain medium 3; c. After passage, the cells were transferred to medium 3 containing 10 μmol / L curcumin, 50 μmol / L magnesium ascorbate phosphate and 5 μmol / L icariin, and cultured at 37°C for 14 days with the induction medium changed every 3 days to obtain chondrocyte precursor cells.

[0034] Step S3 includes the following steps: Select CD105 + / Sox9 + Double-positive chondrogenic progenitor cells, at 2×10 4 CFU / cm² was seeded and cultured at 5% CO2 and 37°C for 3 days in medium containing 10 μmol / L curcumin, 5 μmol / L tanshinone IIA, 20 μmol / L EGCG and 50 μmol / L magnesium ascorbate phosphate to obtain mature chondrocytes.

[0035] The carrier is a collagen carrier.

[0036] The preparation method of the collagen carrier includes the following steps: S1. Dissolve type O collagen and recombinant human type II collagen in serum-free DEME / F12 medium at a mass ratio of 1:1 until the final concentration of each collagen is 3 mg / mL. Stir at 4°C for 12 h and adjust the pH to 7.2-7.4 to obtain collagen solution. S2. Add dexamethasone to the collagen solution at a final concentration of 10-50 nmol / L, glucosamine at a final concentration of 5-10 mmol / L, and a trace inducer, and stir until homogeneous to obtain a collagen-trace inducer solution. S3. Dissolve gelatin in DEME / F12 medium to a final concentration of 20 mg / mL; add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the gelatin solution at a molar ratio of 1:2:1 and activate at room temperature for 10-15 min; add 3-aminophenylboronic acid to the activated solution at a molar ratio of 50 μmol:1 g and react in the dark at room temperature for 12-24 h; dialyze with deionized water for 2-3 days and then freeze-dry to obtain PBA-modified gelatin peptides; S4. Add PBA-modified gelatin peptides to the collagen-micro-inducer solution to make the final concentration of PBA-modified gelatin peptides 2-5 mg / mL, crosslink at room temperature for 15-20 min, and obtain collagen carrier.

[0037] The trace inducers were a mixture of curcumin, EGCG, resveratrol and polygalactoside, with final concentrations of 1-2 μmol / L, 2-4 μmol / L, 1-2 μmol / L and 1-2 μmol / L, respectively.

[0038] This invention provides a method for preparing a drug for the treatment of osteoarthritis induced by pluripotent stem cells, the specific steps of which include: S1. Human induced pluripotent stem cells are induced to differentiate into mesenchymal stem cells: a. Human induced pluripotent stem cells were used at a rate of 5 × 10⁻⁶ 3 Inoculate the culture flasks at a density of CFU / cm² and incubate them in serum-free DEME / F12 medium containing 1% penicillin-streptomycin antibiotics (i.e., medium 1) at 5% CO2 and 37°C for 24 h. b. Take fresh culture medium 1, add 10 μmol / L of polysaccharide and 50 μmol / L of magnesium ascorbate phosphate to obtain culture medium 2; c. Discard culture medium 1, add culture medium 2, and incubate in a 37℃, 5% CO2 incubator for 6 days. Replace the induction culture medium with fresh medium every 2 days during this period to obtain mesenchymal stem cells. S2. Mesenchymal stem cells are induced to differentiate into chondrocyte progenitor cells: a. Mesenchymal stem cells were injected at a rate of 1×10⁻⁶.4 Subcultured to a density of CFU / cm² into a new culture flask and cultured at 37°C for 24 h with 5% CO2. b. Add 1% insulin-transferrin-selenium, proline to a final concentration of 40 μg / mL, and 1% penicillin-streptomycin antibiotics to the high-glucose DEME medium to obtain medium 3; c. After passage, the cells were transferred to medium 3 containing 10 μmol / L curcumin, 50 μmol / L magnesium ascorbate phosphate and 5 μmol / L icariin, and cultured at 37°C for 14 days with the induction medium replaced every 3 days to obtain chondrocyte precursor cells. S3. Chondroblast precursor cells are induced to differentiate into mature chondrocytes: Select CD105 + / Sox9 + Double-positive chondrogenic progenitor cells, at 2×10 4 CFU / cm² was seeded and cultured in medium containing 10 μmol / L curcumin, 5 μmol / L tanshinone IIA, 20 μmol / L EGCG and 50 μmol / L magnesium ascorbate phosphate for 3 days at 5% CO2 and 37°C to obtain mature chondrocytes. S4. Preparation of collagen carrier: a. Dissolve type O collagen and recombinant human type II collagen in serum-free DEME / F12 medium at a mass ratio of 1:1 until the final concentration of each collagen is 3 mg / mL. Stir at 4°C for 12 h and adjust the pH to 7.2-7.4 to obtain collagen solution. b. Add curcumin, EGCG, resveratrol, polydipsia glycoside, dexamethasone and glucosamine to the collagen solution to make their final concentrations 1-2 μmol / L, 2-4 μmol / L, 1-2 μmol / L, 1-2 μmol / L, 10-50 nmol / L and 5-10 mmol / L, respectively, and stir well to obtain collagen-micro-inducer solution; c. Dissolve gelatin in DEME / F12 medium to a final concentration of 20 mg / mL; add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the gelatin solution at a molar ratio of 1:2:1 (carboxyl group of gelatin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide), and activate at room temperature for 10-15 min; add 3-aminophenylboronic acid to the activated solution at a molar-mass ratio of 50 μmol:1 g (3-aminophenylboronic acid to gelatin), and react at room temperature in the dark for 12-24 h; dialyze with deionized water for 2-3 days and then lyophilize to obtain PBA-modified gelatin peptides; d. Add PBA-modified gelatin peptides to the collagen-micro-inducer solution to make the final concentration of PBA-modified gelatin peptides 2-5 mg / mL, crosslink at room temperature for 15-20 min to obtain collagen carrier; S5. Mature chondrocytes and human induced pluripotent stem cells were mixed at a cell ratio of 7:3 and then dispersed in phosphate buffer (pH 7.2-7.4) until the cell density reached 1×10⁻⁶. 6 -5×10 6 CFU / mL, based on a cell-to-carrier cell number-volume ratio of 5 × 10⁻⁶. 6 CFU: 1 mL of cells loaded onto a carrier is a drug for the induction of osteoarthritis by pluripotent stem cells. S6. Aspirate the pluripotent stem cell-induced osteoarthritis drug into a 25G syringe and inject it into the OA cartilage defect area through joint puncture within 30 minutes. The injection volume is adjusted according to the defect area, with 0.5-1 mL injected per cm² of defect. After injection, have the patient slowly move the joint 3-5 times to promote uniform drug distribution.

[0039] To better illustrate the use of pluripotent stem cell induction therapy for osteoarthritis in this invention, the following examples and comparative examples are provided.

[0040] Example 1 S1. Human induced pluripotent stem cells are induced to differentiate into mesenchymal stem cells: a. Human induced pluripotent stem cells (purchased from Hebei Beike Biotechnology Co., Ltd.) were introduced at a rate of 5 × 10⁻⁶. 3 Inoculate the culture flasks at a density of CFU / cm² and incubate them in serum-free DEME / F12 medium containing 1% penicillin-streptomycin antibiotics (i.e., medium 1) at 5% CO2 and 37°C for 24 h. b. Take 1L of fresh culture medium 1, add 10μmol of polysaccharide and 50μmol of magnesium ascorbate phosphate to obtain culture medium 2; c. Discard culture medium 1, add culture medium 2, and incubate in a 37℃, 5% CO2 incubator for 6 days. Replace the induction culture medium with fresh medium every 2 days during this period to obtain mesenchymal stem cells. S2. Mesenchymal stem cells are induced to differentiate into chondrocyte progenitor cells: a. Mesenchymal stem cells were injected at a rate of 1×10⁻⁶. 4 Subcultured to a density of CFU / cm² into a new culture flask and cultured at 37°C for 24 h with 5% CO2. b. Add 1% insulin-transferrin-selenium (purchased from Shanghai Aiyan Biotechnology Co., Ltd.), proline to a final concentration of 40 μg / mL, and 1% penicillin-streptomycin antibiotics to the high-glucose DEME medium to obtain medium 3; c. After passage, the cells were transferred to medium 3 containing 10 μmol / L curcumin, 50 μmol / L magnesium ascorbate phosphate and 5 μmol / L icariin, and cultured at 37°C for 14 days with the induction medium replaced every 3 days to obtain chondrocyte precursor cells. S3. Chondroblast precursor cells are induced to differentiate into mature chondrocytes: Select CD105 + / Sox9 + Double-positive chondrogenic progenitor cells, at 2×10 4 CFU / cm² was seeded and cultured in medium containing 10 μmol / L curcumin, 5 μmol / L tanshinone IIA, 20 μmol / L EGCG and 50 μmol / L magnesium ascorbate phosphate for 3 days at 5% CO2 and 37°C to obtain mature chondrocytes. S4. Preparation of collagen carrier: a. Dissolve 1200 mg of type O collagen and 1200 mg of recombinant human type II collagen (purchased from Chengdu Yunxi Chemical Co., Ltd.) in 400 mL of serum-free DEME / F12 medium, stir at 4℃ for 12 h, and adjust the pH to 7.2-7.4 to obtain collagen solution; b. Add 20 nmol dexamethasone, 4 mmol glucosamine, 0.8 μmol curcumin, 1.6 μmol EGCG, 0.8 μmol resveratrol and 0.8 μmol polysaccharide to the collagen solution, and stir well to obtain a collagen-micro-inducer solution; c. Dissolve 1.8 g of gelatin in 90 mL of DEME / F12 medium, add 69 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 20.7 mg of N-hydroxysuccinimide, activate at room temperature for 15 min, add 12.3 mg of 3-aminophenylboronic acid to the activated solution, and react at room temperature in the dark for 12 h; dialyze with deionized water for 3 days and then freeze-dry to obtain PBA-modified gelatin peptides; d. Add 1500 mg of PBA-modified gelatin peptide to 300 mL of collagen-micro-inducer solution, crosslink at room temperature for 20 min to obtain collagen carrier; S5. Preparation of drugs for treating osteoarthritis: a. Mature chondrocytes and human induced pluripotent stem cells were separately dispersed in phosphate buffer (pH 7.2-7.4) until the cell density reached 5 × 10⁻⁶ cells / mL. 6 CFU / mL, to obtain a dispersion of mature chondrocytes and human induced pluripotent stem cells; b. Mix 210 mL of mature chondrocyte dispersion, 90 mL of human induced pluripotent stem cell dispersion and 300 mL of collagen carrier evenly to prepare pluripotent stem cell-induced osteoarthritis drug I1.

[0041] Example 2 This embodiment is based on Example 1, except that in step S4, c, 4 nmol of dexamethasone and 2 mmol of glucosamine are added to the collagen sol, and the other specific processing parameters are the same as in Example 1, and multipotent stem cell-induced osteoarthritis drug I2 is added.

[0042] Example 3 This embodiment is based on embodiment 1, except that step S5 is as follows: a. Mature chondrocytes and human induced pluripotent stem cells were separately dispersed in phosphate buffer (pH 7.2-7.4) until the cell density reached 5 × 10⁻⁶ cells / mL. 6 CFU / mL, to obtain a dispersion of mature chondrocytes and human induced pluripotent stem cells; b. Mix 150 mL of mature chondrocyte dispersion, 150 mL of human induced pluripotent stem cell dispersion, and 300 mL of collagen carrier evenly. That is, the ratio of mature chondrocytes to human induced pluripotent stem cells is 1:1. Other specific processing parameters are the same as in Example 1, thus producing pluripotent stem cell-induced osteoarthritis treatment drug I3.

[0043] Comparative Example 1 S1. Human induced pluripotent stem cells are induced to differentiate into mesenchymal stem cells: Human induced pluripotent stem cells (purchased from Hebei Beike Biotechnology Co., Ltd.) were used at a rate of 5 × 10⁻⁶. 3 Inoculate the culture flasks at a density of CFU / cm² and incubate them in serum-free DEME / F12 medium containing 1% penicillin-streptomycin antibiotics (i.e., medium 1) at 5% CO2 and 37°C for 24 h. S2. Preparation of collagen carrier: a. Dissolve 1200 mg of type O collagen and 1200 mg of recombinant human type II collagen (purchased from Chengdu Yunxi Chemical Co., Ltd.) in 400 mL of serum-free DEME / F12 medium, stir at 4℃ for 12 h, and adjust the pH to 7.2-7.4 to obtain collagen solution; b. Add 20 nmol dexamethasone, 4 mmol glucosamine, 0.8 μmol curcumin, 1.6 μmol EGCG, 0.8 μmol resveratrol and 0.8 μmol polysaccharide to the collagen solution, and stir well to obtain a collagen-micro-inducer solution; c. Dissolve 1.8 g of gelatin in 90 mL of DEME / F12 medium, add 69 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 20.7 mg of N-hydroxysuccinimide, activate at room temperature for 15 min, add 12.3 mg of 3-aminophenylboronic acid to the activated solution, and react at room temperature in the dark for 12 h; dialyze with deionized water for 3 days and then freeze-dry to obtain PBA-modified gelatin peptides; d. Add 1500 mg of PBA-modified gelatin peptide to 300 mL of collagen-micro-inducer solution, crosslink at room temperature for 20 min to obtain collagen carrier; S3. Preparation of drugs for treating osteoarthritis: a. Disperse human induced pluripotent stem cells in phosphate buffer (pH 7.2-7.4) until the cell density reaches 5 × 10⁻⁶ cells / mL. 6 CFU / mL, obtained human induced pluripotent stem cell dispersion; b. Mix 300 mL of human induced pluripotent stem cell dispersion and 300 mL of collagen carrier evenly to prepare pluripotent stem cell induction therapy for osteoarthritis drug D1.

[0044] This comparative example is based on Example 1, except that no mature chondrocytes are added to the resulting osteoarthritis treatment drug, while the other specific processing parameters are the same as in Example 1.

[0045] Comparative Example 2 This comparative example is based on Example 1, except that step S5 is as follows: a. Disperse mature chondrocytes in phosphate buffer (pH 7.2-7.4) until the cell density reaches 5 × 10⁻⁶ cells / mL. 6 CFU / mL, to obtain a mature chondrocyte dispersion; b. Mix 300 mL of mature chondrocyte dispersion and 300 mL of collagen carrier evenly to prepare pluripotent stem cell-induced osteoarthritis drug D2. All other specific processing parameters are the same as in Example 1.

[0046] Comparative Example 3 This comparative example is based on Example 1, except that steps S4 and S5 are not performed, that is, no carrier gel is added to the obtained drug, and the other specific processing parameters are the same as in Example 1, resulting in pluripotent stem cell-induced osteoarthritis drug D3.

[0047] Comparative Example 4 This comparative example is based on Example 1, except that dexamethasone is not added in step S4,b, while the other specific processing parameters are the same as in Example 1, and the drug D4 is a pluripotent stem cell-induced therapy for osteoarthritis.

[0048] Comparative Example 5 This comparative example is based on Example 1, except that glucosamine is not added in step S4,b. The other specific processing parameters are the same as in Example 1. The drug D5 is used to induce osteoarthritis treatment with pluripotent stem cells.

[0049] Comparative Example 6 This comparative example is based on Example 1, except that no trace amounts of inducers were added to the carrier of the obtained drug, that is, no final concentrations of 2 μmol / L curcumin, 4 μmol / L EGCG, 2 μmol / L resveratrol and 2 μmol / L polygaloside were added. The other specific processing parameters were the same as in Example 1, resulting in pluripotent stem cell-induced osteoarthritis drug D6.

[0050] Comparative Example 7 This comparative example is based on Example 1, except that curcumin is not added in steps S2, S3 and S4, b, that is, curcumin is not added in the preparation of the obtained drug. The other specific processing parameters are the same as in Example 1, and the drug is D7, which is a pluripotent stem cell-induced treatment for osteoarthritis.

[0051] Test Example 1 To verify the differentiation efficacy and safety of cells at each stage, the differentiation rate and the ΔCt values ​​of Runx2 and PPARγ at each stage were measured, including the following steps: S1, induction rate of mesenchymal stem cells: The induced mesenchymal stem cells were resuspended at 1×10⁻⁶. 6 CFU / mL, add 1:100 diluted CD105-FITC and CD90-PE antibodies, incubate at 4°C in the dark for 30 min, add 7-AAD to remove dead cells, and detect CD105 by flow cytometry. + / CD90 + The proportion of double-positive cells; S2, induction rate of chondrocyte precursor cells: a. Fix the induced chondrogenic progenitor cells with 4% paraformaldehyde for 15 min, permeabilize with 0.1% Triton X-100, and then incubate with 5% BSA blocking solution for 1 h; b. Add 1:200 diluted Sox9 primary antibody and incubate overnight at 4°C; add 1:500 diluted fluorescent secondary antibody and incubate for 1 hour, then count Sox9. + Percentage of positive cells; S3, Induction rate of mature chondrocytes: Induced mature chondrocytes were stained with Col2A1-PE antibody to detect Col2A1 levels. +The proportion of positive cells was determined. Cell slides were fixed with 4% paraformaldehyde for 15 min, incubated with safranin O staining solution for 30 min, and proteoglycan secretion was observed to verify functional maturity. Determination of ΔCt values ​​for S4, Runx2, and PPARγ a. Take differentiated chondrocyte progenitor cells from S2 and extract total RNA using a total RNA extraction kit, following the instructions. b. Using a reverse transcription kit, reverse transcribe 1 μg of the extracted total RNA into cDNA according to the instructions. c. Using SYBR Green-based real-time quantitative PCR premixed reagents, with internal reference genes (such as GAPDH) as controls, the mRNA expression levels of Runx2 and PPARγ genes were detected by real-time fluorescence quantitative PCR instrument. d. According to the formula ΔCt values ​​were calculated, where Ct1 is the Ct value of the target gene and Ct2 is the Ct value of the internal reference gene. The cell induction rates and Runx2 and PPARγ ΔCt values ​​at each stage of the pluripotent stem cell-induced osteoarthritis therapy drug prepared in this invention are shown in Table 1.

[0052] Table 1. Cell induction rate and Runx2 and PPARγΔCt values ​​at different stages of pluripotent stem cell induction therapy for osteoarthritis.

[0053] As can be seen, all examples and comparative examples exhibited high mesenchymal stem cell induction rates, indicating that human induced pluripotent stem cells were successfully induced to differentiate into mesenchymal stem cells. Except for comparative example 7, which lacked curcumin during induction, the induction rates of chondrocyte progenitor cells in the other examples and comparative examples were all >87%, and the induction rates of mature chondrocytes were all >93%. Furthermore, the ΔCt values ​​of Runx2 and PPARγ were close to those of undifferentiated mesenchymal stem cells, indicating successful induction of mesenchymal stem cells and chondrocyte progenitor cells with a low risk of misdifferentiation into osteoblastic and adipocyte lineages. For group D7, the Runx2 ΔCt and PPARγ ΔCt values ​​decreased by 42.4% and 43.8% respectively compared to group I1, and the induction rates of chondrocyte progenitor cells and mature chondrocytes decreased by 26.4% and 23.0% respectively, indicating that curcumin is an important substance regulating the differentiation of pluripotent stem cells into mature chondrocytes. Simultaneously, the synergistic activation of the induction pathway by multiple inducers can effectively improve the induction rate.

[0054] Test Example 2 S1, Dexamethasone and Glucosamine Extended-Release Assay for Collagen Carriers a. Weigh 2 mL of collagen carrier, put it into a dialysis bag, immerse it in a centrifuge tube containing 10 mL of phosphate buffer solution at pH 7.2-7.4, and incubate it in a constant temperature shaker at 37°C and 100 rpm to simulate the in vivo environment; b. Collect 1 mL of dialysis fluid on days 0, 1, 3, 7, 14, 21, and 28 respectively; c. Using a mixture of 10 mL methanol and 90 mL deionized water as the mobile phase, glucosamine was detected by HPLC at a detection wavelength of 205 nm. d. Dexamethasone content was detected using an ELISA kit; the sustained-release rates of dexamethasone and gluconeamine were determined using the formula: calculate.

[0055] S2, Cell Loading Rate and Viability Measurement a. Preloading cell counts were prepared by dispersing cells in phosphate buffer to a density of 1×10⁻⁶ cells. 4 For a mixed cell suspension of CFU / mL, take 10μL of the mixed cell suspension and mix it with trypan blue at a 1:1 ratio. Count the number of viable cells using a hemocytometer and record it as N0. b. Following the steps in each embodiment, mix the cell suspension with the collagen carrier and crosslink at room temperature for 20 minutes to form a cell-loaded microgel; c. Add 10 mL of phosphate buffer to the loaded microgel, gently pipette 10 times, centrifuge at 1000 rpm for 5 min, collect the supernatant, count the number of viable cells, and record it as N1; the cell loading rate is calculated using the formula: Cell loading rate = ; d. Cell viability in the osteoarthritis drugs prepared in the various embodiments and comparative examples of the present invention was determined by flow cytometry. The sustained-release rates of glucosamine and dexamethasone, as well as cell load and viability, of the pluripotent stem cell-induced osteoarthritis drugs are shown in Table 2.

[0056] Table 2. Sustained-release rates of glucosamine and dexamethasone, cell load, and survival rate of drugs used in pluripotent stem cell induction therapy for osteoarthritis.

[0057] As can be seen, the glucosamine and dexamethasone sustained-release rates of the pluripotent stem cell-induced osteoarthritis drug prepared in this invention are both >70%, and the cell loading rate is >84%, demonstrating good sustained-release and cell loading performance of active substances. This fully confirms that the carrier can efficiently fix cells and provide them with a superior microenvironment for survival, proliferation and differentiation through sustained-release of functional components.

[0058] Regarding cell survival rates, the osteoarthritis treatment drugs prepared in Comparative Examples 6 and 7 exhibited low cell survival rates, only 85.2% and 81.4%, respectively. This indicates that the combined use of multiple inducers can further promote cell survival while activating signaling pathways during the differentiation of pluripotent stem cells into mature chondrocytes. Comparative Examples 1 and 2, which used human induced pluripotent stem cells and mature chondrocytes alone, showed the lowest cell survival rates except for Comparative Example 3, indicating that the two cell types can exert a synergistic effect in the drug, enhancing cell survival. The cell survival rate in group I1 was as high as 92.3%, while the cell survival rate in group D3 could not be determined, indicating that the collagen carrier can effectively protect cells and improve their survival rate.

[0059] Test Example 3 To verify the therapeutic effect of the pluripotent stem cell-induced osteoarthritis drug prepared in this invention, 6-month-old New Zealand white rabbits weighing 3.5-5 kg ​​were selected as model animals, and the specific steps included: New Zealand white rabbits were injected with 3% sodium pentobarbital via ear marginal injection at a dose of 1 mL / kg. They were then fixed on the operating table, and the hair around their right knee joint was shaved. After disinfection, the anterior cruciate ligament was cut and the anterior 1 / 3 of the medial meniscus was removed. After disinfection and bandaging, the rabbits were housed in individual cages. For three consecutive days, the rabbits were injected with 8 wU of penicillin daily to prevent infection and were given 0.2 mg / kg of meloxicam orally for pain relief. The rabbits were encouraged to move around freely.

[0060] Eight weeks after modeling, inflammatory factors, cartilage defect rate, and gait analysis were performed on the rabbits to verify the modeling effect, including the following steps: S1. Measurement of inflammatory factors: a. After injecting 3% sodium pentobarbital at 1 mL / kg into the ear margin, draw 0.3-0.5 mL of joint fluid from the right knee joint, centrifuge at 1000 rpm for 10 min, and collect the supernatant. b. The contents of IL-1β and TNF-α in the supernatant were determined using ELISA kits for IL-1β and TNF-α, respectively; S2. Determination of cartilage defect rate: Rabbits were euthanized with an overdose of anesthesia. The affected limb was completely amputated. After removing the muscle, the cartilage defect rate of the affected limb was observed and analyzed using μCT scanning and ImageJ software. S3. Gait analysis: a. For the first three days of the experiment, the rabbits were placed in the experimental passage and allowed to walk freely for 10 minutes each day to adapt to the environment. b. Place the rabbits in the experimental tunnel and allow them to walk freely from one end to the other under quiet, dark conditions. Record the rabbits' stride length and support time using a camera. Define the stride length as the point where the center of two consecutive paw prints on the same hind limb is located, and define the support time as the total time that hind limb is in contact with the glass floor during one gait cycle. The levels of inflammatory factors, cartilage defect rate, and gait analysis of the rabbits before and after modeling are shown in Table 3.

[0061] Table 3. Analysis of inflammatory factor levels, cartilage defect rate, and gait in rabbits before and after modeling.

[0062] It can be seen that the present invention successfully induced osteoarthritis-like model in New Zealand white rabbits, with a success rate of 91.4%.

[0063] Test Example 4 To verify the therapeutic effect of the pluripotent stem cell-induced osteoarthritis drug prepared in this invention, pluripotent stem cell-induced osteoarthritis drug was injected into model rabbits 8 weeks after modeling, including the following steps: S1. Eight weeks after modeling, the rabbits were treated with drug injection, with a single injection dose of 1 mL / cm². 2 For cartilage defects, administer injections once a week for a total of 4 weeks. S2. After injection, rabbits were housed individually. Inflammatory factors were measured again at 4 weeks post-injection, and cartilage defect rate was measured again at 12 and 24 weeks post-injection. Gait analysis was performed again at 24 weeks post-injection, and the rate of decrease in inflammatory factors and the rate of cartilage defect filling were calculated. The rate of decrease in inflammatory factors was calculated using the formula: Calculation; Cartilage defect filling rate is obtained through the formula: The calculations were performed using rabbits in the model group that did not receive drug injection as blank control groups for the determination of inflammatory factors and cartilage defects, and rabbits in the model group and rabbits without modeling as negative and positive controls for gait analysis.

[0064] Figure 1 and Figure 2The graphs show the decrease rates of IL-1β and TNF-α in the synovial fluid of rabbits after treatment with a pluripotent stem cell drug provided by this invention. It can be seen that after 4 weeks of injection of the osteoarthritis treatment drug prepared in Example 1, the IL-1β and TNF-α levels in the rabbit synovial fluid decreased by 42.3% and 45.6%, respectively, indicating that the drug effectively alleviated the inflammatory response. The drug without the added collagen carrier showed the lowest decrease rate of inflammatory factors, with IL-1β and TNF-α levels decreasing by only 15.2% and 16.5%, respectively. This indicates that mature chondrocytes and human induced pluripotent stem cells depend on the carrier to provide a suitable microenvironment to ensure cell survival. Simultaneously, the dexamethasone, glucosamine, and trace amounts of inducers loaded within the carrier promote in vivo differentiation while possessing certain anti-inflammatory capabilities. The significantly lower decrease rates of inflammatory factors in Comparative Examples 4-7 compared to Example 1 further corroborate this. Among them, group D7 showed the lowest anti-inflammatory effect, indicating that curcumin plays an important role in regulating differentiation and anti-inflammation. The decrease rates of IL-1β levels in groups D1 and D2 were 34.8% and 33.6% lower than those in group I1, respectively, and the decrease rates of TNF-α levels were 35.7% and 34.0% lower than those in group I1, respectively. This indicates that the anti-inflammatory effects of pluripotent stem cell-induced osteoarthritis drugs depend on the synergistic effect of the two cell types.

[0065] The cartilage defect filling rate of rabbits 12 and 24 weeks after injection of pluripotent stem cell drugs was as follows: Figure 3 As shown in the figure. In Example 1, the cartilage defect filling rates in rabbits were 78.5% and 86.3% after 12 and 24 weeks of drug injection, respectively, indicating that the drug not only has a rapid onset of action for the treatment of osteoarthritis but also provides stable long-term treatment. Group D3 showed the lowest filling rate, indicating that the cells depend on the extracellular matrix-like microenvironment provided by the carrier to prevent cell loss and continuously induce cell differentiation. The cartilage defect filling rates in the knee joints of rabbits treated with the drug alone using mature chondrocytes and human induced pluripotent stem cells were 53.2% and 51.6% after 12 weeks of treatment, respectively, and 60.1% and 58.4% after 24 weeks of treatment, significantly lower than in Example 1. This indicates that the synergistic effect of the two cell types is key to the drug's short-term filling and long-term repair. Although the filling rates of the groups lacking dexamethasone, glucosamine, or trace amounts of the inducer were higher than those of the group without the carrier, they were still significantly lower than those of the full-component group in Example 1. This shows that anti-inflammation, matrix protection, and continuous induction are crucial for achieving high-quality and efficient repair, and they optimize the regenerative microenvironment. For the group that did not add curcumin throughout the preparation process, the cartilage defect filling rate at 12 and 24 weeks after treatment was reduced by 38.6% and 35.6% respectively compared with Example 1, further demonstrating the synergistic effect of multiple inducers on the regulation of the differentiation process from pluripotent stem cells to mature stem cells.

[0066] Figure 4This invention provides a diagram showing the stride length and support time of rabbits after pluripotent stem cell drug treatment; wherein... Figure 4 A is the support time graph of the rabbits after treatment; Figure 4 B shows the stride length of the rabbits after treatment. It can be seen that the stride length and support time of the rabbits treated in Example 1 were close to healthy levels. The therapeutic effect of the osteoarthritis treatment drug with missing components was significantly lower than that in Example 1, indicating that the synergistic effect between the components can effectively treat or alleviate osteoarthritis symptoms.

[0067] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The application of pluripotent stem cell induction therapy for osteoarthritis, characterized in that, Use of pluripotent stem cells in the preparation of drugs for the treatment or relief of primary or secondary osteoarthritis.

2. The use of pluripotent stem cell induction therapy for osteoarthritis as described in claim 1, characterized in that, The drug for treating or alleviating primary or secondary osteoarthritis comprises a carrier and active cells, wherein the ratio of active cells to carrier is 5 × 10⁻⁶. 6 CFU cells were loaded onto a 1 mL carrier and combined with a multipotent stem cell induction therapy drug for osteoarthritis. The drug was administered as an injection.

3. The use of pluripotent stem cell induction therapy for osteoarthritis as described in claim 2, characterized in that, The active cells are obtained by combining human induced pluripotent stem cells and mature chondrocytes prepared by differentiation of human induced pluripotent stem cells at a ratio of 3:

7.

4. The use of pluripotent stem cell induction therapy for osteoarthritis as described in claim 3, characterized in that, The method for preparing mature chondrocytes obtained from the differentiation of human induced pluripotent stem cells includes the following steps: S1. Human induced pluripotent stem cells are induced to differentiate into mesenchymal stem cells; S2, mesenchymal stem cells are induced to differentiate into chondrocyte precursor cells; S3, chondrocyte precursor cells are induced to differentiate into mature chondrocytes.

5. The use of pluripotent stem cell induction therapy for osteoarthritis as described in claim 4, characterized in that, Step S1 includes the following steps: a. Human induced pluripotent stem cells were used at a rate of 5 × 10⁻⁶ 3 Inoculate the culture flasks at a density of CFU / cm² and incubate them in serum-free DEME / F12 medium containing 1% penicillin-streptomycin antibiotics (i.e., medium 1) at 5% CO2 and 37°C for 24 h. b. Take fresh culture medium 1, add 10 μmol / L of polysaccharide and 50 μmol / L of magnesium ascorbate phosphate to obtain culture medium 2; c. Discard culture medium 1, add culture medium 2, and incubate in a 37℃, 5% CO2 incubator for 6 days. Replace the induction culture medium with fresh medium every 2 days to obtain mesenchymal stem cells.

6. The use of pluripotent stem cell induction therapy for osteoarthritis as described in claim 4, characterized in that, Step S2 includes the following steps: a. Mesenchymal stem cells were injected at a rate of 1×10⁻⁶. 4 Subcultured to a density of CFU / cm² into a new culture flask and cultured at 37°C for 24 h with 5% CO2. b. Add 1% insulin-transferrin-selenium, proline to a final concentration of 40 μg / mL, and 1% penicillin-streptomycin antibiotics to the high-glucose DEME medium to obtain medium 3; c. After passage, the cells were transferred to medium 3 containing 10 μmol / L curcumin, 50 μmol / L magnesium ascorbate phosphate and 5 μmol / L icariin, and cultured at 37°C for 14 days with the induction medium changed every 3 days to obtain chondrocyte precursor cells.

7. The use of pluripotent stem cell induction therapy for osteoarthritis as described in claim 4, characterized in that, Step S3 includes the following steps: Select CD105 + / Sox9 + Double-positive chondrogenic progenitor cells, at 2×10 4 CFU / cm² was seeded and cultured at 5% CO2 and 37°C for 3 days in medium containing 10 μmol / L curcumin, 5 μmol / L tanshinone IIA, 20 μmol / L EGCG and 50 μmol / L magnesium ascorbate phosphate to obtain mature chondrocytes.

8. The use of pluripotent stem cell induction therapy for osteoarthritis as described in claim 2, characterized in that, The carrier is a collagen carrier.

9. The use of pluripotent stem cell induction therapy for osteoarthritis as described in claim 8, characterized in that, The preparation method of the collagen carrier includes the following steps: S1. Dissolve type O collagen and recombinant human type II collagen in serum-free DEME / F12 medium at a mass ratio of 1:1 until the final concentration of each collagen is 3 mg / mL. Stir at 4°C for 12 h and adjust the pH to 7.2-7.4 to obtain collagen solution. S2. Add dexamethasone to the collagen solution at a final concentration of 10-50 nmol / L, glucosamine at a final concentration of 5-10 mmol / L, and a trace inducer, and stir until homogeneous to obtain a collagen-trace inducer solution. S3. Dissolve gelatin in DEME / F12 medium to a final concentration of 20 mg / mL; add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the gelatin solution in a molar ratio of 1:2:1 and activate at room temperature for 10-15 min. 3-Aminophenylboronic acid was added to the activated solution at a molar-mass ratio of 50 μmol: 1 g, and the reaction was carried out at room temperature in the dark for 12-24 h. After dialyzing with deionized water for 2-3 days, PBA-modified gelatin peptides were obtained by freeze-drying. S4. Add PBA-modified gelatin peptides to the collagen-micro-inducer solution to make the final concentration of PBA-modified gelatin peptides 2-5 mg / mL, crosslink at room temperature for 15-20 min to obtain collagen carrier.

10. The use of pluripotent stem cell induction therapy for osteoarthritis as described in claim 9, characterized in that, The trace inducer is a mixture of curcumin, EGCG, resveratrol and polygalactoside, with final concentrations of 1-2 μmol / L, 2-4 μmol / L, 1-2 μmol / L and 1-2 μmol / L, respectively.

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