Preparation method and application of stem cell preparation for repairing knee joint injury

Abstract

The invention provides a preparation method and application of a stem cell preparation for repairing knee joint injury. The preparation method of the stem cell preparation comprises the following steps: performing culture passage on mesenchymal stem cells to 3-5 generations; the method comprises the following steps: pretreating the mesenchymal stem cells through a serine metabolism regulation composition, wherein the serine metabolism regulation composition comprises L-serine, 5-methyltetrahydrofolic acid and vitamin B6; preparing a mitochondrial targeting nano system of nano particles loaded with mitochondrial targeting peptide and coenzyme Q10; mixing the mesenchymal stem cells with the mitochondrial targeting nano system to obtain a mixed solution; and adding a 4% sodium hyaluronate solution into the mixed solution, and uniformly mixing to form the hydrogel. According to the present invention, the serine metabolism regulation composition is adopted to pre-treat the mesenchymal stem cells, and the mitochondrial targeting peptide is added, such that the oxidation resistance of the mesenchymal stem cells is improved, the survival time of the mesenchymal stem cells in the knee joint injury microenvironment can be easily prolonged, the repair function can be easily enhanced, and the clinical treatment effect can be easily improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell preparations, and more particularly relates to a preparation method and application of a stem cell preparation for repairing knee joint injury. BACKGROUND

[0002] The knee joint is one of the largest and most complex joints in the human body, which bears the important functions of supporting body weight, participating in walking, running, jumping and other movements. Due to its frequent activity and greater pressure, knee joint injury is very common in daily life and sports. Common types of knee joint injury include osteoarthritis, synovitis, cartilage injury, meniscus injury and ligament injury, etc. The injured part of the patient is often accompanied by symptoms such as pain, swelling, limited activity, instability or abnormal sensation, which seriously affects the quality of life.

[0003] Stem cell therapy shows great potential in the repair of knee joint injury. Among them, mesenchymal stem cells (MSCs) have become the focus of research in this field due to their strong proliferation capacity, low immunogenicity, and easy access to materials. The mechanism of action of mesenchymal stem cells in treating knee joint injury includes the following points: they can differentiate into chondrocytes to synthesize and secrete cartilage matrix; they have immune regulation function, can inhibit the activation of inflammatory cells, reduce the secretion of inflammatory factors such as TNF-α and IL-6, and reduce the damage of inflammatory reaction to knee joint tissue; they have paracrine effect, which can regulate the local microenvironment by secreting cell activation factors, promote host cell proliferation and differentiation, and repair tissues and organs.

[0004] However, stem cell therapy technology still has limitations, which restricts its clinical conversion efficiency and efficacy stability. The microenvironment of the injured knee joint is poor, characterized by low oxygen, high lactic acid, high inflammation and oxidative stress, which leads to metabolic disorders of stem cells, mainly mitochondrial dysfunction, redox imbalance and energy supply deficiency, thereby causing the problems of short survival time of treatment stem cells in the microenvironment of knee joint injury and inhibited repair function. SUMMARY

[0005] The purpose of the embodiments of the present application is to provide a preparation method and application of a stem cell preparation for repairing knee joint injury, so as to solve the technical problems of short survival time of treatment stem cells in the microenvironment of knee joint injury and inhibited repair function in the prior art.

[0006] To achieve the above object, the technical scheme adopted by the present application is: a preparation method of a stem cell preparation for repairing knee joint injury is provided, comprising the following steps: mesenchymal stem cells are separated and extracted from human tissues or purchased commercially, and are cultured and subcultured to 3-5 generations; the mesenchymal stem cells are pretreated with a serine metabolism regulation combination component, wherein the serine metabolism regulation combination component comprises L-serine, 5-methyltetrahydrofolate and vitamin B6; a mitochondrion-targeting nano system is prepared, wherein the mitochondrion-targeting nano system is a nanoparticle loaded with a mitochondrion-targeting peptide and coenzyme Q10; the pretreated mesenchymal stem cells and the mitochondrion-targeting nano system are mixed at a ratio of 1×10 6 cells: 0.5 mg of the mitochondrion-targeting nano system, and the cell concentration is adjusted to 1-2×10 7 cells / mL to obtain a mixed solution; 4% sodium hyaluronate solution is added to the mixed solution at a ratio of 1:1 of the mixed solution to the sodium hyaluronate solution, and the mixture is uniformly mixed to form a hydrogel.

[0007] In an embodiment, the preparation method of the mitochondrion-targeting nano system comprises the following steps: 100-150 mg of polylactic acid-glycolic acid copolymer is dissolved in 5-7.5 mL of dichloromethane, 10-15 mg of coenzyme Q10 is added for ultrasonic dissolution to form a uniform oil phase solution; the oil phase solution is slowly injected into 10-15 mL of an aqueous phase containing 2% polyvinyl alcohol by mass fraction, and is subjected to ice bath ultrasonic treatment to form an emulsion; the emulsion is transferred to 50-75 mL of an aqueous phase containing 0.5% polyvinyl alcohol by mass fraction, and is stirred for 4-5 h to allow the polylactic acid-glycolic acid copolymer to solidify to form nanoparticles, thereby obtaining a nanoparticle mixture; the mitochondrion-targeting peptide is added to the nanoparticle mixture at a final concentration of 0.1-0.15 mg / mL, and is incubated for 1-2 h, and the precipitate is collected by centrifugation, thereby obtaining the mitochondrion-targeting nano system.

[0008] In an embodiment, the pretreatment of the mesenchymal stem cells with the serine metabolism regulation combination component comprises the following steps: the serine metabolism regulation combination component is added to a mesenchymal stem cell serum-free culture medium, and the serine metabolism regulation combination component comprises 50-100 μM of L-serine, 10-15 μM of 5-methyltetrahydrofolate and 5-10 μM of vitamin B6; the mesenchymal stem cells in the logarithmic growth phase are taken, and the cell density is adjusted to 4-6×10 5 cells / mL, and are inoculated into a culture vessel; after at least 24 h, the culture medium is removed, and a fresh mesenchymal stem cell serum-free culture medium containing the serine metabolism regulation combination component is added, and the mesenchymal stem cells are cultured for 24-48 h, and the mesenchymal stem cell serum-free culture medium containing the serine metabolism regulation combination component is replaced with a fresh one after at least 24 h.

[0009] In an embodiment, the mesenchymal stem cells are adipose-derived mesenchymal stem cells.

[0010] In an embodiment, the nanoparticle carrier of the mitochondria-targeting nanosystem is polylactic acid-glycolic acid copolymer.

[0011] In an embodiment, the mitochondria-targeting peptide is a fusion polypeptide consisting of a TAT peptide, an SS peptide and an RGD peptide.

[0012] According to another aspect of the present application, the present application further provides a use of a stem cell preparation for repairing knee joint injury, wherein the stem cell preparation is prepared by the preparation method of any one of the above embodiments, and the stem cell preparation is used for treating knee osteoarthritis.

[0013] In an embodiment, the dosage form of the stem cell preparation is injection.

[0014] The preparation method of the stem cell preparation for repairing knee joint injury provided by the present application can precisely deliver CoQ10 nanoparticles to mitochondria of mesenchymal stem cells through mitochondria-targeting peptides, thereby enhancing oxidative phosphorylation efficiency, improving ATP generation capacity, removing reactive oxygen species (ROS) in mitochondria, protecting mitochondrial structure, and maintaining energy metabolism stability in a low-oxygen environment of a lesion. The mesenchymal stem cells are pretreated by a combination of serine metabolism regulation components, which can improve the antioxidant capacity of the cells, maintain the proliferation capacity and genomic stability of the stem cells, strengthen the serine metabolism of the mesenchymal stem cells, and improve the stress resistance of the cells, so that the cells can quickly adapt to the harsh microenvironment of the knee joint injury site after injection. In summary, the present application helps to prolong the survival time of mesenchymal stem cells in the microenvironment of the knee joint injury and enhance the repair function of the mesenchymal stem cells. BRIEF DESCRIPTION OF DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0016] Figure 1 Statistical graph of joint swelling degree of mouse models of knee osteoarthritis before and after treatment with different stem cell preparations.

[0017] Figure 2 Statistical graph of survival rate of stem cells in different stem cell preparations under oxidative stress environment. DETAILED DESCRIPTION

[0018] In order to make the technical problems, technical solutions and beneficial effects of the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely intended to explain the present application and not to limit the present application.

[0019] It should be noted that when an element is referred to as being "fixed" or "disposed" on another element, it can be directly on the other element or indirectly on the other element. When an element is referred to as being "connected" to another element, it can be directly connected to the other element or indirectly connected to the other element.

[0020] It should be understood that the terms "length", "width", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are merely for the purpose of facilitating the description of the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application.

[0021] In addition, the terms "first", "second" are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. In the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specifically limited.

[0022] Please refer to Figure 1 and Figure 2 , the preparation method of the stem cell preparation for repairing knee joint injury provided by the embodiments of the present application and the stem cell preparation prepared will be described. The stem cell preparation for repairing knee joint injury comprises mesenchymal stem cells (MSCs), a mitochondria-targeting nano system and a preparation matrix; the mitochondria-targeting nano system is a nanoparticle loaded with a mitochondria-targeting peptide and coenzyme Q10 (CoQ10); the mesenchymal stem cells are pretreated by a serine metabolism regulation combined component, and the serine metabolism regulation combined component comprises L-serine, 5-methyltetrahydrofolic acid and vitamin B6.

[0023] Specifically, mesenchymal stem cells are a kind of multipotent stem cells with all the common characteristics of stem cells, i.e. self-renewal and multi-directional differentiation ability, and are widely used in clinical applications. Umbilical cord mesenchymal stem cells (UMSCs), adipose-derived mesenchymal stem cells (ADSCs) and bone marrow mesenchymal stem cells (BMSCs) are the three common types of mesenchymal stem cells, which have a wide range of tissue sources and relatively mature isolation and extraction techniques. The preparation matrix can be selected from sodium hyaluronate, chitosan or collagen, which are all natural polymer matrices that can simulate the extracellular matrix of tissues, provide adhesion sites for mesenchymal stem cells and reduce the loss of the preparation after injection with body fluids.

[0024] Mitochondrial-targeted nano-systems can provide mitochondrial protection for mesenchymal stem cells, especially in maintaining energy metabolism stability under low-oxygen lesion environment. Among them, the mitochondrial-targeted peptide can accurately deliver nanoparticles to the mitochondria of stem cells by recognizing the specific proteins of the mitochondrial inner membrane, avoiding the non-specific distribution of CoQ10 in cells and improving the utilization efficiency; CoQ10, as a key electron carrier of the mitochondrial respiratory chain, can enhance the efficiency of oxidative phosphorylation and improve the ATP generation capacity, while as a liposoluble antioxidant, it can remove reactive oxygen species (ROS) in mitochondria and protect the structure of mitochondria; nanoparticles are commonly used drug carriers in vivo, which can protect CoQ10 from degradation and provide sustained mitochondrial protection for stem cells through sustained-release characteristics.

[0025] Serine metabolic regulation combination ingredients can enhance the serine metabolism of mesenchymal stem cells and improve the stress resistance of cells, so that they can quickly adapt to the harsh microenvironment of the knee joint injury site after injection. L-serine is the common precursor of L-cysteine and glycine, which can directly supplement the raw materials for stem cells to synthesize glutathione (GSH), improve the antioxidant capacity of cells and reduce the damage of active oxygen to stem cells in lesions. L-serine can also provide substrates for one-carbon unit metabolism to support DNA synthesis and cell proliferation. 5-methyltetrahydrofolate and vitamin B6, as key cofactors for one-carbon unit metabolism, promote the transfer of serine-derived one-carbon units to purine and pyrimidine synthesis pathways, maintain the proliferation ability and genomic stability of stem cells; vitamin B6, as a SHMT2 coenzyme, accelerates the metabolic flux, and it also participates in amino acid metabolism to enhance the adaptation of stem cells to nutrient-deficient environments. In addition to the above-mentioned 3 substances, 3-phosphoglycerate (3-PG), apigenin and vitamin B12, etc. can also be added to enhance the serine metabolism of the ingredients.

[0026] Compared with the prior art, the stem cell preparation for repairing knee joint injury provided in the application can precisely deliver CoQ10 nanoparticles to mitochondria of mesenchymal stem cells through a mitochondrial targeting peptide, can enhance oxidative phosphorylation efficiency, can improve ATP generation capacity, can also remove reactive oxygen species (ROS) in mitochondria, can protect the structure of mitochondria, and can maintain stable energy metabolism under a low-oxygen environment of a lesion; the mesenchymal stem cells can be pretreated by a combination of serine metabolism regulation components, so as to improve the antioxidant capacity of the cells, maintain the proliferation capacity and genomic stability of the stem cells, strengthen the serine metabolism of the mesenchymal stem cells, and improve the stress resistance of the cells, so that the cells can quickly adapt to the harsh microenvironment of a knee joint injury site after injection. In summary, the application helps to prolong the survival time of mesenchymal stem cells in the microenvironment of a knee joint injury and enhance the repair function.

[0027] In an embodiment, the mesenchymal stem cells are adipose-derived mesenchymal stem cells (ADSCs).

[0028] Specifically, adipose-derived mesenchymal stem cells, as the name implies, are derived from adipose tissue, have strong plasticity, low immunogenicity, are easy to obtain, and have fast proliferation, etc. ADSCs have common characteristics of mesenchymal stem cells, including fibroblast-like morphology and adhesion to plastic surfaces of culture plates, expression of surface markers CD73, CD90 and CD105, and non-expression of CD11b, CD14, CD19, CD45 and HLA-DR. The differentiation of ADSCs is not limited to the adipocyte lineage, and ADSCs can also differentiate into osteoblasts, chondrocytes, etc., and have been widely used in the repair of bone and cartilage tissue. ADSCs can escape the recognition of allogeneic T cells and NK cells and survive in xenogeneic bodies for a long time, thus having low immunogenicity and the ability of bidirectional immune regulation. Compared with bone marrow mesenchymal stem cells, ADSCs can be easily obtained in large quantities by non-invasive surgery, and have a longer aging time. In addition, ADSCs produce various growth factors and cytokines, such as insulin-like growth factor (IGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), interleukin-6 (IL-6), interleukin-8 (IL-8) and transforming growth factor-β1 (TGF-β1), which endow them with high proliferation rate, differentiation potential and immune regulation characteristics.

[0029] In an embodiment, the nanoparticle carrier of the mitochondrial targeting nanosystem is polylactic-glycolic acid copolymer (PLGA).

[0030] Specifically, PLGA is a high molecular compound formed by polymerization of lactic acid and glycolic acid monomers, which has been approved by the US Food and Drug Administration (FDA) for various clinical preparations, such as sustained-release microspheres. PLGA can be hydrolytically degraded in vivo, and the degradation products lactic acid and glycolic acid can be metabolized to CO2 and H2O through the tricarboxylic acid cycle, avoiding accumulation in the knee joint to cause foreign body reactions, and having excellent biocompatibility and degradability. PLGA is an ideal material for preparing drug sustained-release and controlled-release systems, and its degradation rate can be precisely controlled by adjusting the ratio of lactic acid to glycolic acid, for example, when the lactic acid content is high, the PLGA degrades slowly. For the key period of knee injury repair, a PLGA carrier with a degradation period of 2-3 weeks can be selected to ensure the sustained-release efficiency of CoQ10. In addition, the surface of the PLGA nanoparticle can be easily modified with a mitochondria-targeting peptide through chemical coupling or physical adsorption.

[0031] In an embodiment, the mitochondria-targeting peptide is a fusion polypeptide consisting of a TAT peptide, an SS peptide, and an RGD peptide.

[0032] Specifically, TAT peptide is a cell-penetrating peptide derived from human immunodeficiency virus (HIV-1), which is widely used in the biomedical field due to its high transmembrane ability, especially as a drug delivery carrier. TAT peptide can enter cells by directly penetrating the cell membrane barrier or endocytosis, and modification on the surface of polymeric nanoparticles can improve the enrichment efficiency of loaded drugs at the lesion site and achieve targeted therapy. TAT peptide is usually composed of 8 to 16 amino acids, rich in positively charged arginine R, so it can bind to the negatively charged cell membrane, and its core sequence is YGRKKRRQRRR. SS peptide (Szeto-Schiller peptide, SS peptide) is a kind of polypeptide with mitochondrial targeting antioxidant function, which can specifically target the inner membrane of mitochondria, and is usually composed of 4 positively charged amino acids, mainly including SS01 (Tyr-D-Arg-Phe-Lys-NH2), SS02 (Dmt-D-Arg-Phe-Lys-NH2) and SS31 (D-Arg-Dmt-Lys-Phe-NH2). SS peptide can reduce mitochondrial ROS production, inhibit mitochondrial permeability transition pore opening, and has a significant effect in preventing oxidative stress or inhibiting apoptosis and necrosis induced by mitochondrial electron transport chain. RGD peptide is a sequence polypeptide composed of Arg-Gly-Asp (arginine-glycine-aspartic acid), which widely exists in the proteins of extracellular matrix and can be used as an integrin receptor antagonistic polypeptide, and specifically binds to 11 kinds of integrins. In this application, RGD peptide mediates the specific binding of nanoparticles and mesenchymal stem cell surface integrin α5β1. In summary, the mitochondrial targeting peptide in this application can mediate the specific binding of the mitochondrial targeting nanosystem and mesenchymal stem cells, break through the cell membrane barrier into the cytoplasm, precisely target mitochondria to release CoQ10, and improve the utilization rate of CoQ10.

[0033] The fusion polypeptide composed of TAT peptide, SS peptide and RGD peptide can be obtained by two technical paths of genetic engineering recombinant expression or chemical synthesis. For the genetic engineering recombinant expression method, an expression vector containing the fusion peptide gene sequence can be constructed, expressed in E. coli or eukaryotic cells, and purified. For the chemical synthesis method, amino acids are connected step by step by solid-phase synthesis, for example, the amino acids of SS peptide, linker, TAT peptide and RGD peptide are sequentially connected on a solid-phase carrier. Among them, the linker is a connecting peptide, which is used to connect different peptide segments. Of course, many biotechnology companies on the market can provide corresponding technical support services.

[0034] In an embodiment, the preparation matrix is sodium hyaluronate.

[0035] Specifically, sodium hyaluronate as a matrix carrier of the preparation has excellent biocompatibility, can reduce the acute inflammatory reaction after mesenchymal stem cell injection; can also simulate the viscoelasticity of synovial fluid, provide a temporary colonization scaffold for mesenchymal stem cells, and reduce the amount of mesenchymal stem cells lost with synovial fluid. In addition, sodium hyaluronate itself has the effect of lubricating the joint and inhibiting synovial inflammation, which synergizes with the paracrine effect of mesenchymal stem cells to improve the microenvironment of the damaged site.

[0036] In an embodiment, the dosage form of the stem cell preparation is an injection type.

[0037] Specifically, most of the patients with knee injury are middle-aged and elderly groups, or accompanied by underlying diseases, and have poor tolerance to invasive surgery. The trauma caused by the injection type dosage form is significantly reduced compared with open surgery, and the recovery time of the patients is shorter. Using the injection type dosage form, the patient can complete the treatment in the outpatient department without the need for complex surgical equipment, and the operation is convenient, which is conducive to clinical promotion. Moreover, the injection type dosage form supports multiple drug administration, and the dose can be dynamically adjusted according to the treatment progress of the patient.

[0038] The application also provides a preparation method of a stem cell preparation for repairing knee injury, comprising the following steps: separating and extracting or commercially purchasing mesenchymal stem cells from human tissues, and culturing and subculturing to 3-5 generations; pretreating the mesenchymal stem cells with a serine metabolism regulation combination component, wherein the serine metabolism regulation combination component comprises L-serine, 5-methyltetrahydrofolate and vitamin B6; preparing a mitochondrion-targeting nano system, wherein the mitochondrion-targeting nano system is a nanoparticle loaded with a mitochondrion-targeting peptide and coenzyme Q10; mixing the pretreated mesenchymal stem cells with the mitochondrion-targeting nano system at a ratio of 1×10 6 cells: 0.5 mg of the mitochondrion-targeting nano system, and then adjusting the cell concentration to 1-2×10 7 cells / mL to obtain a mixed solution; adding 4% sodium hyaluronate solution to the mixed solution at a volume ratio of 1:1 of the mixed solution to the sodium hyaluronate solution, and mixing to form a hydrogel.

[0039] Specifically, the bone marrow mesenchymal stem cells can be separated from human bone marrow; or the umbilical cord mesenchymal stem cells can be separated from human umbilical cord blood; or the adipose tissue mesenchymal stem cells can be separated from human adipose tissue. According to the general method for separating mesenchymal stem cells provided by the Merck website, the method comprises the following steps: An appropriate amount of fresh tissue aspirate collected under sterile conditions is taken; the cell suspension is filtered through a 70 mm sieve to remove any cell clumps; the cells are precipitated at 500 g for 5 min in a table centrifuge; the cells are resuspended and the yield and viability of the cells are determined by the trypan blue exclusion method; 25×10 6The density of cells / ml is in a T75 culture flask, the culture flask is placed in an incubator, 37℃, 5% CO2, static incubation; 3 hours later, the medium is replaced with 10 ml of fresh MSCs serum-free medium, and the non-adherent cells accumulated on the surface of the culture dish are removed. After another 8 hours of culture, the medium is replaced with 10 ml of fresh complete medium. Thereafter, the procedure is repeated every 8 hours until the initial culture of 72 hours is completed; the excess cells can be collected at 2 x 10 6 The density of cells / tube is mixed in MSCs growth medium and 10% dimethyl sulfoxide (DMSO) for cryopreservation.

[0040] The mesenchymal stem cells are passaged to 3-5 generations, and the cells of low generations may contain more impurities, and the cells of high generations may cause the function of the cells to decrease. When the preparation is performed, the concentration of the cells is adjusted to 1-2 x 10 7 The concentration of the cells is adjusted to 1-2 x 10

[0041] In an embodiment, the preparation method of the mitochondria-targeting nano system comprises the following steps: 100-150 mg of polylactic acid-glycolic acid copolymer (PLGA) is dissolved in 5-7.5 mL of dichloromethane, 10-15 mg of coenzyme Q10 is added for ultrasonic dissolution to form a uniform oil phase solution; the oil phase solution is slowly injected into 10-15 mL of an aqueous phase containing 2% by mass fraction of polyvinyl alcohol, and is subjected to ice bath ultrasonic treatment to form an emulsion; the emulsion is transferred to 50-75 mL of an aqueous phase containing 0.5% by mass fraction of polyvinyl alcohol, and is stirred for 4-5 h to allow the polylactic acid-glycolic acid copolymer to solidify to form nanoparticles, to obtain a nanoparticle mixture; mitochondria-targeting peptides are added to the nanoparticle mixture at a final concentration of 0.1-0.15 mg / mL, and are incubated for 1-2 h, and the precipitate is collected by centrifugation, to obtain the mitochondria-targeting nano system.

[0042] Specifically, PLGA is not soluble in water, but is soluble in various organic solvents, such as dichloromethane, acetone, ethyl acetate, tetrahydrofuran, DMSO, etc. Dichloromethane is a commonly used solvent for the preparation of drugs in the pharmaceutical industry, and has good solubility for lipid-soluble CoQ10. The mass ratio of PLGA to CoQ10 can ensure uniform dispersion of CoQ10, avoid crystallization, and at the same time provide a suitable high polymer concentration for subsequent nanoparticle formation. Ultrasonic dissolution can break the aggregation state of CoQ10, which is helpful for forming a uniform oil phase. The parameters of ultrasonic dissolution can be set as a power of 200-300 W and a time of 3-5 min.

[0043] Polyvinyl alcohol (PVA) is a synthetic water-soluble polymer with good biocompatibility, non-toxicity, good chemical stability and adjustable crystallinity. In the present embodiment, PVA is used as an emulsifier. Through its hydrophobic segment, PVA is adsorbed on the oil-water interface to reduce surface tension and stabilize the emulsion. Ice bath can reduce the volatilization of dichloromethane and maintain the fluidity of the oil phase. The parameters of ultrasonic can be set as power 200-300 W and time 3-5 min.

[0044] 0.5% PVA, i.e. low concentration of PVA, can reduce the excessive adsorption of PVA on the surface of nanoparticles, avoiding the hindrance of subsequent peptide segment modification. A large amount of aqueous phase promotes the diffusion and volatilization of dichloromethane, so that the PLGA macromolecular chain gradually solidifies to form nanoparticles, and the particle size is usually 100-200 nm. Stirring for 4-5 h ensures that dichloromethane is completely volatilized, so that its residual amount is <0.1%, reducing its toxic side effects on cells. Magnetic stirring device can be used during stirring, and the parameter is set as 300-500 rpm.

[0045] The surface of PLGA nanoparticles can be combined with positively charged mitochondrial targeting peptides through electrostatic adsorption or hydrophobic interaction. Incubation can be carried out at 37°C under constant temperature oscillation, which helps the mitochondrial targeting peptide to fully combine with the PLGA nanoparticle. The centrifugation parameters can be set as 4°C, 10000-12000g, 15-20min, so as to fully separate the combined mitochondrial targeting nanoparticle system from free PVA, uncombined peptide segment and nanoparticles.

[0046] In an embodiment, the pretreatment of mesenchymal stem cells with serine metabolism regulation combination components includes the following steps: adding serine metabolism regulation combination components to the mesenchymal stem cell serum-free culture medium, and the serine metabolism regulation combination components include 50-100 μM of L-serine, 10-15 μM of 5-methyltetrahydrofolate and 5-10 μM of vitamin B6; taking mesenchymal stem cells in logarithmic growth phase, adjusting the cell density to 4-6 x 10 5 cells / mL, and inoculating in a culture vessel; after at least 24 h, removing the culture medium and replacing it with MSCs serum-free culture medium containing serine metabolism regulation combination components, and culturing for 24-48 h, during which the fresh mesenchymal stem cell serum-free culture medium containing serine metabolism regulation combination components is replaced at least 24 h later.

[0047] Specifically, the amount of L-serine is controlled at 50-100 μM, which can fully meet the demand of mesenchymal stem cells for synthesizing glutathione (GSH) and one-carbon units, while avoiding osmotic stress caused by high concentration. 5-methyltetrahydrofolate and vitamin B6 act as key cofactors for one-carbon unit metabolism, and a small amount can play a role.

[0048] Unlike general cell culture, mesenchymal stem cells often use serum-free medium, i.e., without adding fetal bovine serum (FBS), to avoid the stimulation of animal components to the human body in clinical use and to avoid zoonotic infection. DMEM / F12 is a commonly used mesenchymal stem cell basic culture medium, but due to the lack of proteins, lipids and growth factors, it is necessary to add components to replace FBS before being used to culture cells. The optional components are derived from human platelets or products obtained by catalytic hydrolysis of plant raw materials such as wheat, soybean, and rice. Of course, commercial mesenchymal stem cell growth medium can also be used.

[0049] Cells in the logarithmic growth phase are metabolically active, have strong proliferation ability, and are more sensitive to changes in the nutritional environment, and can more efficiently utilize serine metabolic regulation combination components for metabolic remodeling. The cell inoculation density is 4-6 x 10 5 cells / mL, at which density the cells will neither be overcrowded to cause contact inhibition nor be sparse to secrete insufficient signals. The initial culture is 24 h, allowing mesenchymal stem cells to adhere to the wall and recover in the conventional culture medium. Since serine is rapidly consumed within a few hours in cell metabolism, and metabolic products may accumulate, timely medium replacement can maintain an effective concentration.

[0050] In an embodiment, the mesenchymal stem cells are adipose-derived mesenchymal stem cells (ADSCs).

[0051] Specifically, adipose-derived mesenchymal stem cells, as the name implies, are derived from adipose tissue, have strong plasticity, low immunogenicity, easy to obtain, and fast proliferation, etc. ADSCs have common characteristics of mesenchymal stem cells, including fibroblast-like morphology and adhesion to plastic surfaces of culture plates, expression of surface markers CD73, CD90 and CD105, and non-expression of CD11b, CD14, CD19, CD45 and HLA-DR. The differentiation of ADSCs is not limited to the adipocyte lineage, and it can also differentiate into osteoblasts, chondrocytes, etc., and has been widely used in the repair of bone and cartilage tissue. ADSCs can escape the recognition of allogeneic T cells and NK cells and survive in xenogeneic body for a long time, thus having low immunogenicity and the ability of bidirectional immune regulation. Compared with bone marrow mesenchymal stem cells, ADSCs can be easily obtained in large quantities by non-invasive surgery and have a longer aging time. In addition, ADSCs produce various growth factors and cytokines, such as insulin-like growth factor (IGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), interleukin-6 (IL-6), interleukin-8 (IL-8) and transforming growth factor-β1 (TGF-β1), endowing them with high proliferation rate, differentiation potential and immune regulation characteristics.

[0052] In one embodiment, the nanoparticle carrier of the mitochondria-targeting nanosystem is polylactic-co-glycolic acid (PLGA).

[0053] Specifically, PLGA is a high-molecular compound formed by polymerization of lactic acid and glycolic acid monomers, which has been approved by the U.S. Food and Drug Administration (FDA) for various clinical preparations, such as sustained-release microspheres. PLGA can be hydrolytically degraded in vivo, and the degradation products, lactic acid and glycolic acid, can be metabolized to CO2 and H2O through the tricarboxylic acid cycle, avoiding accumulation in the knee joint to cause foreign body reactions, and having excellent biocompatibility and biodegradability. PLGA is an ideal material for preparing drug sustained-release and controlled-release systems, and its degradation rate can be precisely controlled by adjusting the ratio of lactic acid to glycolic acid, for example, when the lactic acid content is high, the PLGA degrades slowly. For the key period of knee injury repair, a PLGA carrier with a degradation period of 2-3 weeks can be selected to ensure the sustained-release efficiency of CoQ10. In addition, the surface of the PLGA nanoparticle can be easily modified with a mitochondria-targeting peptide through chemical coupling or physical adsorption.

[0054] In one embodiment, the mitochondria-targeting peptide is a fusion polypeptide composed of TAT peptide, SS peptide, and RGD peptide.

[0055] Specifically, TAT peptide is a cell-penetrating peptide derived from human immunodeficiency virus (HIV-1), which is widely used in the biomedical field due to its high transmembrane ability, especially as a drug delivery carrier. TAT peptide can enter cells by directly penetrating the cell membrane barrier or endocytosis, and can be modified on the surface of polymeric nanoparticles to improve the enrichment efficiency of loaded drugs at the lesion site and achieve targeted therapy. TAT peptide is usually composed of 8 to 16 amino acids, rich in positively charged arginine R, so it can bind to the negatively charged cell membrane, and its core sequence is YGRKKRRQRRR. SS peptide (Szeto-Schiller peptide, SS peptide) is a kind of polypeptide with mitochondrial targeting antioxidant function, which can specifically target the inner membrane of mitochondria, and is usually composed of 4 positively charged amino acids, mainly including SS01 (Tyr-D-Arg-Phe-Lys-NH2), SS02 (Dmt-D-Arg-Phe-Lys-NH2) and SS31 (D-Arg-Dmt-Lys-Phe-NH2). SS peptide can reduce mitochondrial ROS production, inhibit mitochondrial permeability transition pore opening, and has a significant effect in preventing oxidative stress or inhibiting apoptosis and necrosis induced by mitochondrial electron transport chain. RGD peptide is a sequence polypeptide composed of Arg-Gly-Asp (arginine-glycine-aspartic acid), which widely exists in the proteins of extracellular matrix and can specifically bind to 11 kinds of integrins as an integrin receptor antagonistic polypeptide. In this application, RGD peptide mediates the specific binding of nanoparticles to mesenchymal stem cell surface integrin α5β1. In summary, the mitochondrial targeting peptide in this application can mediate the specific binding of the mitochondrial targeting nanosystem to mesenchymal stem cells, break through the cell membrane barrier into the cytoplasm, and precisely target mitochondria to release CoQ10, thereby improving the utilization rate of CoQ10.

[0056] The fusion polypeptide composed of TAT peptide, SS peptide and RGD peptide can be obtained by two technical paths of genetic engineering recombinant expression or chemical synthesis. For the genetic engineering recombinant expression method, an expression vector containing the fusion peptide gene sequence can be constructed, expressed in E. coli or eukaryotic cells, and purified. For the chemical synthesis method, the amino acids are connected step by step by solid-phase synthesis, for example, the amino acids of SS peptide, linker, TAT peptide and RGD peptide are sequentially connected on a solid-phase carrier. Among them, the linker is a connecting peptide, which is used to connect different peptide segments. Of course, many biotechnology companies on the market can provide corresponding technical support services.

[0057] According to another aspect of the present application, the application also provides the use of the above-mentioned stem cell preparation for repairing knee joint injury, which is applied to the treatment of knee osteoarthritis (KOA).

[0058] Specifically, knee osteoarthritis (KOA) is a chronic degenerative joint disease characterized by degeneration of knee joint cartilage, synovial inflammation, and hyperostosis, and is one of the most common causes of joint pain and dysfunction in the elderly. The core of the pathological mechanism of KOA is the progressive degeneration of cartilage, accompanied by a chain reaction of surrounding tissues such as synovium, subchondral bone, and ligament. In KOA, the abnormal function of chondrocytes leads to the breakage of collagen fibers and the loss of proteoglycans, resulting in the gradual thinning of cartilage, the appearance of cracks, and even the exfoliation, losing the buffering and lubricating effect. KOA can cause synovial inflammation, i.e., the stimulation of synovial membrane by cartilage fragments, leading to chronic inflammation, and the secretion of pro-inflammatory factors such as IL-1β and TNF-α by synoviocytes, further accelerating cartilage destruction and causing joint effusion and pain. KOA can also cause other tissue involvement, including meniscus wear, ligament relaxation, and joint capsule fibrosis, further damaging the stability of the knee joint and aggravating the symptoms. The stem cell preparation of the present application can treat KOA by inhibiting synovial inflammation and pain, delaying cartilage degeneration and promoting repair, and improving metabolic disorders of the joint microenvironment.

[0059] In an embodiment, the dosage form of the stem cell preparation is an injection type.

[0060] Specifically, most of the patients with knee joint injury are middle-aged and elderly groups, or accompanied by underlying diseases, and have poor tolerance to invasive surgery. The trauma caused by the injection type dosage form is significantly reduced compared with open surgery, and the recovery time of the patients is shorter. The use of injection type dosage form enables the patients to complete the treatment in outpatient department, without the need for complex surgical equipment, and the operation is highly convenient, which is beneficial to clinical promotion. Moreover, the injection type dosage form supports multiple drug administration, and the dose can be dynamically adjusted according to the treatment progress of the patient.

[0061] Embodiment 1 The stem cell preparation for repairing knee joint injury in this embodiment comprises the following components: adipose-derived mesenchymal stem cells (ADSCs), a mitochondria-targeting nano system, and sodium hyaluronate. The mitochondria-targeting nano system is a nanoparticle loaded with a mitochondria-targeting peptide and coenzyme Q10 (CoQ10), and the mitochondria-targeting peptide is a fusion polypeptide composed of TAT peptide, SS31 peptide, and RGD peptide. The nanoparticle carrier is poly (lactic-co-glycolic acid) (PLGA). The ADSCs are pretreated with serine metabolism regulation combination components, which include L-serine, 5-methyltetrahydrofolate, and vitamin B6. The dosage form of the stem cell preparation is an injection type, and is used for treating knee osteoarthritis.

[0062] The mitochondrial targeting peptide is synthesized by Nanjing Kingsrui Biotechnology Co., Ltd. The sequence is RGDfK-GGS-YGRKKRRQRRR-GGS-D-Arg-Dmt-Lys-Phe-NH2, GGS is a flexible linker, RGDfK is a common sequence of RGD peptide, YGRKKRRQRRR is the shortest active peptide sequence of TAT peptide, and D-Arg-Dmt-Lys-Phe-NH2 is the SS31 peptide sequence; CoQ10, L-serine are purchased from MedChemExpress (MCE) company, the part numbers are HY-N0111, HY-N0650 respectively, and the purity is greater than 98%; 5-methyltetrahydrofolic acid, vitamin B6 are purchased from Shanghai Yuan Ye Biotechnology Co., Ltd., the part numbers are S26122, S13026 respectively; PLGA is purchased from Zhongxin Kangming, the part number is Z-1011032.

[0063] The preparation method of the stem cell preparation for repairing knee joint injury in the embodiment comprises the following steps: 1) Isolating ADSCs from human adipose tissue: The fresh human adipose tissue collected under sterile conditions is rinsed with normal saline and then cut into a cell suspension with surgical scissors. The cell suspension is filtered through a 70 mm mesh to remove any cell clumps. The cells are precipitated at 500 g for 5 min in a table centrifuge. The cells are resuspended and the yield and viability of the cells are determined by the trypan blue exclusion method. In 10 ml of MSCs serum-free medium, 25 x 10 6 cells / ml of density in a T75 culture flask, the culture flask is placed in a incubator, 37°C, 5% CO2, and incubated at rest; 3 hours later, the medium is replaced with 10 ml of fresh MSCs serum-free medium, and the non-adherent cells accumulated on the surface of the culture dish are removed. After another 8 hours of culture, the medium is replaced with 10 ml of fresh complete medium. Thereafter, the step is repeated every 8 hours until the initial culture of 72 hours is completed; an appropriate amount of cells are taken for flow cytometric identification of cell phenotype CD90 + / CD105 + / CD34 - / CD45 - , and the purity is ≥95%.

[0064] In addition to the cells needed for subsequent use, the excess cells can be mixed into MSCs growth medium and 10% DMSO at a density of 2 x 10 6 cells / tube for cryopreservation.

[0065] The serum-free culture medium for MSCs has the following components: CTS™ StemPro MSC SFM basal medium, 2 mM L-glutamine, 10% CELLStart™ Substrate dilution solution, and 1% penicillin-streptomycin double antibiotic. Among them, the CTS™ StemPro MSC SFM basal medium and the CELLStart™ Substrate are both purchased from Thermo Fisher Scientific.

[0066] 2) Pretreatment of ADSCs with serine metabolic regulatory combination components: The serine metabolic regulatory combination components, including 50 μM of L-serine, 10 μM of 5-methyltetrahydrofolate, and 5 μM of vitamin B6, were added to the serum-free culture medium for MSCs. The ADSCs in the logarithmic growth phase were taken, and the cell density was adjusted to 4 x 10 5 cells / mL, and inoculated in the culture vessel. After 24 h, the culture medium was removed and replaced with the serum-free culture medium for MSCs containing the serine metabolic regulatory combination components, and cultured for 48 h. During the period, the serum-free culture medium for MSCs containing the serine metabolic regulatory combination components was replaced with fresh ones at 24 h.

[0067] 3) Preparation of mitochondria-targeting nano-system: 100 mg of PLGA was weighed and dissolved in 5 mL of dichloromethane, and 10 mg of CoQ10 was ultrasonically dissolved at 200 W for 3 min to form a uniform oil phase solution. The oil phase solution was slowly injected into 10 mL of an aqueous phase containing 2% polyvinyl alcohol (PVA), and ice-bath ultrasonication was performed at 200 W for 3 min to form a milk phase. The emulsion was transferred to 50 mL of an aqueous phase containing 0.5% PVA, and stirred for 4 h to allow the PLGA to solidify and form nanoparticles. Mitochondria-targeting peptides were added to the above system at a final concentration of 0.1 mg / mL, incubated for 1 h, and the precipitate was collected by centrifugation to obtain the mitochondria-targeting nano-system.

[0068] 4) Mixing the pretreated ADSCs with the mitochondria-targeting nano-system at a ratio of 1 x 10 6 cells to 0.5 mg of the mitochondria-targeting nano-system, and adjusting the cell concentration to 1 x 10 7 cells / mL to obtain a stem cell suspension component. 4% sodium hyaluronate solution was added at a volume ratio of 1:1, and mixed to form a hydrogel. That is, each mL of the stem cell preparation contains 5 x 10 6 cells of mesenchymal stem cells.

[0069] Example 2 Based on Example 1, the difference is that the dosage form of the stem cell preparation is chitosan.

[0070] Example 3 Based on Example 1, the difference is that the mitochondrial targeting peptide is only the SS peptide.

[0071] Comparative Example 1 Based on Example 1, the difference is that there is no mitochondrial targeting system.

[0072] Comparative Example 2 Based on Example 1, the difference is that the adipose-derived mesenchymal stem cells are not pretreated with the serine metabolism regulation composition.

[0073] Comparative Example 3 Based on Example 1, the difference is that there is no mitochondrial targeting system, and the adipose-derived mesenchymal stem cells are not pretreated with the serine metabolism regulation composition.

[0074] Experimental Example 1 The knee osteoarthritis mouse model is used to verify the treatment effect of the stem cell preparation.

[0075] The experiment uses 6-8 week old C57BL / 6 mice, and injects sodium monoiodoacetate (MIA) solution into the joint cavity of the left hind limb. The specific steps are as follows: (1) Group setting: set up a control group, a model group, and each of the example and comparative example groups, and randomly divide the mice into each group, 10 in each group.

[0076] (2) Anesthetize the mice with 1.25% tri bromoethanol, and when modeling and injecting, inject 0.9% normal saline into the joint cavity of the left hind limb of the control group mice, and inject sodium monoiodoacetate solution into the other group mice according to the proportion of 1 mg / 10 g, that is, inject 1 mg of sodium monoiodoacetate solution containing sodium monoiodoacetate into each 10 g of mice. The sodium monoiodoacetate solution is obtained by dissolving sodium monoiodoacetate in 0.9% normal saline, and can be prepared as a 2 mg / 10 µL stock solution, and then the injection dose is accurately adjusted according to the body weight of the mouse. After injection, the mice are put back into the cage, and sufficient food, water and activity space are ensured, and observed for 4 weeks.

[0077] (3) After 4 weeks of modeling of the mice, 0.9% normal saline is injected into the joint cavity of the left hind limb of the control group and the model group, and the other group mice are injected with 200 µL of stem cell preparation of the corresponding example and comparative example group. After injection, the mice are put back into the cage, and sufficient food, water and activity space are ensured, and observed regularly.

[0078] (4) At 4 weeks after injection, the joint swelling degree of the left hind limb of the mouse was detected by using a digital vernier caliper (accuracy 0.01 mm), and the data was obtained to calculate the joint swelling subsidence rate. The specific detection method is as follows: taking the midpoint of the connection line between the lateral condyle and the medial condyle of the left hind joint of the mouse as the measurement reference point, the two measurement jaws of the vernier caliper were gently attached to the skin on both sides of the knee joint to avoid excessive extrusion causing tissue deformation, and the value (unit: mm) was recorded after the reading was stable. Each mouse was repeatedly measured 3 times, and the average value was taken as the joint swelling degree data at that time point. The swelling subsidence rate was calculated according to the formula "swelling subsidence rate (%) = (swelling degree before injection - swelling degree at a certain time point after injection) / swelling degree before injection x 100%".

[0079] The results were analyzed as follows: Figure 1 The joint swelling degree statistics chart before and after the treatment of different stem cell preparations for the mouse model of knee osteoarthritis. As shown in Figure 1 , the swelling subsidence rate of Example 1 group was as high as 50%, which was significantly higher than that of other groups, indicating that it could effectively reduce the inflammatory response of the joint; the subsidence rate of Comparative Example 3 group was close to that of the model group, which proved that the stem cell preparation of the application was helpful to enhance the repair function of stem cells in the microenvironment of knee joint injury.

[0080] Experimental Example 2 Calcein-AM / PI double staining method was used to detect the cell survival rate of adipose tissue mesenchymal stem cells in oxidative stress environment. In this experiment, Calcein / PI cell activity and cytotoxicity detection kit was used, which was purchased from Biyun Tian, and the item number was C2015.

[0081] (1) Group setting: setting the control group, hydrogen peroxide group (oxidative stress), N-acetylcysteine group (antioxidant) of each example and comparative example, and setting 3 replicate holes for each group.

[0082] (2) The stem cell suspension components of each example and comparative example (i.e. without adding the preparation matrix sodium hyaluronate) were inoculated in a 96-well black multi-well plate, and the cell inoculation density was 2x10 3 / well, 100 μL of MSCs serum-free medium was added to each well, and it was placed in a culture box at 37°C, 5% CO2, and cultured for 24 h.

[0083] (3) Remove the culture medium and operate as follows: Control group: add 100 μL of fresh MSCs serum-free medium; Hydrogen peroxide group: add MSCs serum-free medium containing 100 μM hydrogen peroxide (H2O2); N-acetyl cysteine group: MSCs serum-free medium containing 10 μM antioxidant N-acetyl cysteine (NAC) was added for 30 min, and then replaced with MSCs serum-free medium containing 100 μM hydrogen peroxide (H2O2).

[0084] The 96-well plate was placed in the incubator for 2 h.

[0085] (4) 800xg centrifugation at room temperature for 5 min, aspirate the culture solution, and wash the cells with phosphate buffer solution (PBS) once. Add 100 μl of Calcein AM / PI detection working solution to each well, and incubate at 37°C for 30 min in the dark.

[0086] (5) After incubation, detect with a fluorescence microplate reader (Calcein AM is green fluorescence, Ex / Em = 494 / 517 nm; PI is red fluorescence, Ex / Em = 535 / 617 nm).

[0087] (6) Calculate the cell survival rate by the detection results. The results are analyzed as follows: Figure 2 The survival rate of stem cells in different stem cell preparations under oxidative stress environment is shown in the graph. Figure 2 It can be seen that the cell survival rate of all control groups is ≥95%, indicating that the initial culture of ADSCs has good activity and no obvious spontaneous apoptosis; the cell survival rate of all N-acetyl cysteine groups is higher than that of the hydrogen peroxide group, further confirming that oxidative stress is a key factor leading to ADSCs death; the cell survival rate of the hydrogen peroxide group of Example 1 is the highest, which is significantly higher than that of Comparative Examples 1-3, proving that the synergistic protective effect of the serine metabolism regulation combination component pretreatment in the stem cell preparation and the tripeptide fusion mitochondria-targeted nano system can effectively resist hydrogen peroxide-induced oxidative damage, which helps to prolong the survival time of stem cells in the microenvironment of knee joint injury.

[0088] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application. The separation and preparation method described is a method known in the art, and the cell concentration can be adjusted appropriately, which belongs to the conventional technology in the art.

Claims

1. A method for preparing a stem cell preparation for repairing knee joint injuries, comprising the following steps: Mesenchymal stem cells are isolated from human tissues or commercially purchased and cultured for 3-5 generations. Mesenchymal stem cells were pretreated with a serine metabolism regulation combination, wherein the serine metabolism regulation combination included L-serine, 5-methyltetrahydrofolate, and vitamin B6. Prepare a mitochondrial-targeting nanosystem, wherein the mitochondrial-targeting nanosystem is a nanoparticle loaded with mitochondrial-targeting peptides and coenzyme Q10. Pretreated mesenchymal stem cells were combined with a mitochondrial-targeting nanosystem at a ratio of 1×10⁻⁶. 6 Individual cells: Mix 0.5 mg of the mitochondrial-targeting nanosystem and then adjust the cell concentration to 1-2 × 10⁻⁶. 7 The volume ratio of the mixed solution to sodium hyaluronate solution was 1:1 (cells / mL) to obtain a mixed solution. 4% sodium hyaluronate solution was added to the mixed solution at a volume ratio of 1:1, and the mixture was stirred to form a hydrogel.

2. The method for preparing the stem cell preparation for repairing knee joint injuries as described in claim 1, characterized in that, The preparation method of the mitochondrial-targeted nanosystem includes the following steps: Weigh 100-150 mg of polylactic acid-glycolic acid copolymer and dissolve it in 5-7.5 mL of dichloromethane. Add 10-15 mg of coenzyme Q10 and sonicate to dissolve it to form a homogeneous oil phase solution. The oil phase solution is slowly injected into 10-15 mL of an aqueous phase containing 2% polyvinyl alcohol by mass, and then subjected to ice bath ultrasonic treatment to form an emulsion. The emulsion is then transferred to 50-75 mL of an aqueous phase containing 0.5% polyvinyl alcohol by mass, and stirred for 4-5 h to solidify the polylactic acid-glycolic acid copolymer into nanoparticles, thus obtaining a nanoparticle mixture. Add mitochondrial-targeting peptides to the nanoparticle mixture at a final concentration of 0.1-0.15 mg / mL, incubate for 1-2 hours, centrifuge to collect the precipitate, and obtain the mitochondrial-targeting nanosystem.

3. The method for preparing the stem cell preparation for repairing knee joint injuries as described in claim 1, characterized in that, The pretreatment of mesenchymal stem cells with serine metabolism-regulated components includes the following steps: Serine metabolism regulation components were added to the serum-free culture medium of mesenchymal stem cells. The serine metabolism regulation components included 50-100 μM L-serine, 10-15 μM 5-methyltetrahydrofolate, and 5-10 μM vitamin B6. Mesenchymal stem cells in the logarithmic growth phase were harvested, and the cell density was adjusted to 4-6 × 10⁻⁶. 5 MSCs were seeded at a density of 100 cells / mL in a culture dish. After at least 24 hours, the culture medium was removed and replaced with serum-free MSC culture medium containing serine metabolic regulation components. The culture was then carried out for 24-48 hours, during which time the culture medium was replaced with fresh serum-free mesenchymal stem cell culture medium containing serine metabolic regulation components after at least 24 hours.

4. The method for preparing the stem cell preparation for repairing knee joint injuries as described in claim 1, characterized in that, The mesenchymal stem cells mentioned are adipose-derived mesenchymal stem cells.

5. The method for preparing the stem cell preparation for repairing knee joint injury as described in claim 1, characterized in that, The nanoparticle carrier of the mitochondrial-targeting nanosystem is a polylactic acid-glycolic acid copolymer.

6. The method for preparing the stem cell preparation for repairing knee joint injuries as described in claim 1, characterized in that, The mitochondrial targeting peptide is a fusion polypeptide composed of TAT peptide, SS peptide and RGD peptide.

7. The application of a stem cell preparation for repairing knee joint injuries, prepared using the preparation method according to any one of claims 1-6, characterized in that, The stem cell preparation is used in the treatment of knee osteoarthritis.

8. The application of the stem cell preparation for repairing knee joint injuries as described in claim 7, characterized in that, The stem cell preparation is in the form of an injectable injection.

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