Application composition of stem cell exosome in promoting tissue repair

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses an application composition of stem cell exosomes in promoting tissue repair, which comprises the stem cell exosomes selected from induced pluripotent stem cells, mesenchymal stem cells, embryonic stem cells, hematopoietic stem cells or neural stem cells and used for promoting tissue repair and cell regeneration, and the stem cell exosomes selected from the induced pluripotent stem cells, the mesenchymal stem cells, the embryonic stem cells, the hematopoietic stem cells and the neural stem cells. The tissue repair promoting factor is selected from an epidermal growth factor, a transforming growth factor beta, a platelet-derived growth factor or a combination thereof, and is used for optimizing cell proliferation, differentiation and wound healing; the biodegradable carrier is selected from hydrogel, lipidosome, polymer micelle, nano particles or natural plant extract; by optimizing the transmission relation between the biodegradable carrier and the stem cell exosome and combining with the cell proliferation model, different doses and delivery modes can be flexibly regulated and controlled, the treatment scheme is further optimized, and the effect of the treatment scheme is accurately predicted. Due to the design, the common problems of unstable effect, long treatment period and the like in the traditional stem cell application are solved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to an application composition of stem cell exosomes in promoting tissue repair. Background Art

[0002] Tissue repair is an important topic in modern medicine. Especially in tissue injuries caused by trauma, surgery, chronic diseases, and degenerative diseases, etc., how to effectively promote tissue repair and improve the recovery speed of patients has always been the focus of medical research. With the rapid development of modern medical technology, remarkable progress has been made in the research field of tissue repair and regeneration. As an emerging medical method, stem cell therapy shows broad application prospects in tissue repair by virtue of its self-renewal and differentiation capabilities.

[0003] As tiny vesicles secreted by stem cells, stem cell exosomes contain abundant bioactive molecules such as proteins, RNAs, lipids, etc., and can transmit information between cells, regulate cell proliferation, differentiation, migration, and immune responses, becoming a research hotspot in recent years. A large number of studies have shown that stem cell exosomes can not only directly participate in the repair of damaged tissues, but also regulate the local microenvironment through interactions with target cells to promote the repair process. At present, existing stem cell exosome treatment methods have certain limitations. In the process of their clinical application, the design of biodegradable carriers of stem cell exosomes and their delivery effects have important impacts on the treatment effects. Existing carriers mainly focus on hydrogels, liposomes, etc., but their delivery efficiency, sustained release property, and binding ability with stem cell exosomes still need to be improved. In addition, when applying stem cell exosomes, there is still a lack of accurate prediction and quantification models for their concentration, delivery method, selection of repair sites, etc.

[0004] Therefore, it is necessary to propose an application composition of stem cell exosomes in promoting tissue repair to solve the problem of poor application effects of stem cell exosomes in the process of tissue repair in the existing technology. Summary of the Invention

[0005] The purpose of the present invention is to provide an application composition of stem cell exosomes in promoting tissue repair to solve the problems raised in the above background art.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] An application composition of stem cell exosomes in promoting tissue repair, the application composition includes:

[0008] Stem cell exosomes, selected from induced pluripotent stem cells, mesenchymal stem cells, embryonic stem cells, hematopoietic stem cells or neural stem cells, for promoting tissue repair and cell regeneration;

[0009] Tissue repair promoting factors, selected from epidermal growth factor, transforming growth factor-β, platelet-derived growth factor or combinations thereof, for optimizing cell proliferation, differentiation and wound healing;

[0010] Biodegradable carriers, selected from hydrogels, liposomes, polymer micelles, nanoparticles or natural plant extracts, for delivering stem cell exosomes and prolonging their sustained action in vivo.

[0011] Preferably, the stem cell exosomes are secreted by stem cells from autologous or allogeneic sources and obtained by ultra-high speed centrifugation, immunoaffinity method or ultrafiltration method;

[0012] Stem cell exosomes are rich in bioactive molecules such as cytokines, miRNAs, mRNAs, lipids and proteins, for promoting cell proliferation, migration and differentiation;

[0013] Bioactive molecules that bind to miRNAs, mRNAs and proteins in stem cell exosomes, and key molecules promoting tissue repair and immunomodulation are identified by big data analysis techniques;

[0014] Using CRISPR / Cas9 or other gene editing tools to genetically modify stem cell exosomes for carrying specific modified genes or regulatory genes;

[0015] Using molecular dynamics simulation and finite element analysis methods to simulate the interaction between stem cell exosomes, biodegradable carriers and target cells, and optimizing the delivery effect of stem cell exosomes;

[0016] Interaction between stem cell exosomes and target cells:

[0017]

[0018] Where V is the reaction rate, V max is the maximum reaction rate, [S] is the concentration of stem cell exosomes, and K m is the Michaelis constant.

[0019] Preferably, the biodegradable carrier is used to carry and release stem cell exosomes to the target site and will gradually degrade at the target site;

[0020] The hydrogel is composed of natural macromolecular materials or synthetic polymers, and the natural plant extract is the plant extract of astragalus polysaccharide, wolfberry polysaccharide, tanshinone, ginkgo flavone, ginsenoside.

[0021] Preferably, the application composition further comprises anti-inflammatory factors, selected from interleukins, tumor necrosis factor-α inhibitors or combinations thereof.

[0022] Preferably, the application composition further comprises a bone growth factor, a nerve growth factor or a combination thereof, which is used to promote tissue repair and cell regeneration.

[0023] Preferably, the mass ratio of the stem cell exosomes to the biodegradable carrier is 1:5 to 1:20;

[0024] According to the porosity of the biodegradable carrier, its size, and the hydrophilicity factor of the carrier material, a delivery relationship between the biodegradable carrier and the stem cell exosomes is established;

[0025]

[0026] In the formula, J is the amount of substance passing through a unit area per unit time, and D is the diffusion coefficient, is the concentration gradient;

[0027] The concentration of the stem cell exosomes is 10 8 to 10 12 particles / mL, and the concentration of the tissue repair promoting factor is within the clinically effective range;

[0028] A cell proliferation model is introduced to simulate the cell proliferation process, predict the cell proliferation ability of the stem cell exosomes in tissue repair, and compare the effects of different doses and delivery methods;

[0029]

[0030] In the formula, N is the number of cells, r is the growth rate, and K is the carrying capacity of the environment.

[0031] Preferably, the application method of the application composition is topical application or delivery to the tissue site to be repaired by injection.

[0032] A method for promoting tissue repair using the application composition according to any one of the above, comprising the following steps:

[0033] Select an autologous or allogeneic stem cell source, and extract stem cell exosomes derived from the stem cell source;

[0034] Mix the stem cell exosomes with a biodegradable carrier and a tissue repair promoting factor, and prepare an application composition for promoting tissue repair under sterile conditions;

[0035] Deliver the application composition to the tissue site to be repaired by topical coating or injection to promote local cell regeneration, migration, and immunomodulation.

[0036] Preferably, the method further performs surface modification on the stem cell exosomes to enhance their affinity for specific receptors and improve the repair effect by introducing targeting molecules;

[0037] Surface modification implementation methods include:

[0038] ① Chemical modification: Functional groups are directly linked to the surface of exosomes using chemical reactions (such as amidation, thioesterification, etc.).

[0039] ② Biomolecule conjugation: Specific biomolecules (such as antibodies, peptides, etc.) are linked to the surface of exosomes through bioconjugation techniques (such as EDC / NHS conjugation, etc.).

[0040] Design stem cell exosomes or biodegradable carriers with imaging functions, and track them in real time through MRI, optical imaging, or ultrasound technology, and monitor the distribution and release dynamics of stem cell exosomes;

[0041] Design stem cell exosomes or biodegradable carriers with imaging functions:

[0042] (1) Imaging design of stem cell exosomes

[0043] ① Labeling methods: Fluorescent dyes (such as quantum dots, organic fluorescent dyes, etc.) can be used to label stem cell exosomes, which is suitable for optical imaging; the MRI imaging effect can be enhanced by introducing metal ions (such as iron ions, manganese ions, etc.) or metal oxide nanoparticles (such as iron oxide nanoparticles, etc.); for ultrasound imaging, it can be achieved by introducing acoustic contrast agents such as microbubbles or nanobubbles.

[0044] ② Labeling positions: Labeling can be performed on the membrane or inside the stem cell exosomes to ensure that their functions and biological activities are not affected. It is necessary to verify through experiments whether the labeled stem cell exosomes maintain their original biological activities and functions.

[0045] (2) Imaging design of biodegradable carriers

[0046] ① Selection of carrier materials: Select biodegradable materials with imaging functions, such as poly(lactic-co-glycolic acid) (PLGA) with MRI imaging functions, etc.; or introduce imaging substances into the carrier, such as the above-mentioned metal ions, nanoparticles, etc.

[0047] ② Binding methods of imaging substances and carriers: It can be physical mixing, chemical bonding, etc., to ensure the uniform distribution and stable release of imaging substances in the carrier. It is necessary to verify through experiments the stability of imaging substances in the carrier, as well as the carrying and release capabilities of the carrier for stem cell exosomes.

[0048] (3) Verification of imaging effects

[0049] ① In vitro verification: In an in vitro simulated environment, use corresponding imaging equipment to verify the imaging effects of stem cell exosomes or biodegradable carriers, and observe the clarity, resolution, and dynamic changes of the imaging, etc.

[0050] ② In vivo verification: In animal models, the imaging effect in vivo is verified by injecting or implanting stem cell exosomes or biodegradable carriers, and their distribution, migration and release dynamics in vivo are monitored.

[0051] Preferably, the method also designs a microenvironment regulation mechanism for controlling wound repair according to the specific injury type, medical history, and immune response conditions of the patient, in combination with the interaction between stem cell exosomes and specific microenvironment factors;

[0052] Introduce physiological environment-responsive materials with temperature, pH, and enzyme conversion, and design a biodegradable carrier that can automatically trigger the release of stem cell exosomes according to the physiological environment changes in the local wound area of the patient.

[0053] Temperature-responsive materials include poly(N-isopropylacrylamide), liposomes; pH-responsive materials include polyacrylic acid, chitosan; enzyme-responsive materials include hyaluronic acid, collagen.

[0054] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0055] By combining stem cell exosomes, tissue repair promoting factors and biodegradable carriers, the present invention significantly improves the efficiency and precision of tissue repair. Stem cell exosomes are rich in various bioactive molecules, which can effectively promote cell proliferation, migration and differentiation, and thus accelerate the tissue repair process; by adding tissue repair promoting factors to stem cell exosomes, the repair function is further enhanced, and the efficiency of tissue repair is significantly improved. The synergistic effect of these tissue repair promoting factors not only promotes the cell repair process, but also optimizes the tissue regeneration mechanism.

[0056] In addition, by means of the targeted delivery method of the biodegradable carrier, it can ensure that the stem cell exosomes are efficiently and directionally transported to the target site, greatly enhancing the therapeutic effect. At the same time, the application of the biodegradable carrier not only ensures the safety of the treatment process, but also can precisely control the release rate of the stem cell exosomes, thereby improving their bioavailability at the target site and maximizing the repair effect.

[0057] The present invention optimizes the delivery relationship between the biodegradable carrier and the stem cell exosomes, and combines with the cell proliferation model to flexibly regulate different doses and delivery methods, further optimizing the treatment plan and accurately predicting its effect. Such a design overcomes the common problems in traditional stem cell applications, such as unstable effects and long treatment cycles, and greatly improves the reliability and efficiency of clinical treatment. Brief Description of the Drawings

[0058] Figure 1The average overall assessment score (IGA), clinical skin repair assessment scale (CEA), and patient satisfaction assessment score graph for patients with skin wound repair in Example 2 of the present invention;

[0059] Figure 2 CT scan images of the defect sites of patients before and after cartilage injury repair in Example 2 of the present invention. Detailed implementation manners

[0060] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0061] Example 1:

[0062] An application composition of stem cell exosomes in promoting tissue repair, the application composition includes:

[0063] Stem cell exosomes, selected from induced pluripotent stem cells, mesenchymal stem cells, embryonic stem cells, hematopoietic stem cells or neural stem cells, for promoting tissue repair and cell regeneration;

[0064] Tissue repair promoting factors, selected from epidermal growth factor, transforming growth factor-β, platelet-derived growth factor or a combination thereof, for optimizing cell proliferation, differentiation and wound healing;

[0065] Biodegradable carriers, selected from hydrogels, liposomes, polymer micelles, nanoparticles or natural plant extracts, for delivering stem cell exosomes and prolonging their sustained action in vivo.

[0066] The stem cell exosomes are secreted by stem cells from autologous or allogeneic sources and are obtained by ultra-high speed centrifugation, immunoaffinity method or ultrafiltration;

[0067] The stem cell exosomes are rich in bioactive molecules such as cytokines, miRNA, mRNA, lipids and proteins, for promoting cell proliferation, migration and differentiation;

[0068] Combined with the bioactive molecules of miRNA, mRNA and proteins in the stem cell exosomes, key molecules for promoting tissue repair and immune regulation are identified through big data analysis technology;

[0069] The stem cell exosomes are genetically modified using CRISPR / Cas9 or other gene editing tools for carrying specific modified genes or regulatory genes;

[0070] Use molecular dynamics simulation and finite element analysis methods to simulate the interaction of stem cell exosomes with biodegradable carriers and target cells, and optimize the delivery effect of stem cell exosomes.

[0071] Furthermore, stem cell exosomes are rich in various bioactive molecules, which can effectively promote cell proliferation, migration and differentiation, and thus accelerate the tissue repair process.

[0072] The biodegradable carrier is used to carry and release stem cell exosomes to the target site and will gradually degrade at the target site;

[0073] The hydrogel is composed of natural polymer materials or synthetic polymers, and the natural plant extracts are plant extracts of astragalus polysaccharide, lycium barbarum polysaccharide, tanshinone, ginkgo flavonoids, and ginsenosides.

[0074] Furthermore, with the precise delivery system of the biodegradable carrier, it can ensure the efficient and directional delivery of stem cell exosomes to the target site, greatly enhancing the therapeutic effect. Moreover, the application of the biodegradable carrier not only ensures the safety of the treatment process but also can precisely control the release rate of stem cell exosomes, thereby improving their bioavailability at the target site and maximizing the repair effect.

[0075] The applied composition also includes anti-inflammatory factors selected from interleukin, tumor necrosis factor-α inhibitors, or combinations thereof.

[0076] The applied composition also includes bone growth factors, nerve growth factors, or combinations thereof for promoting tissue repair and cell regeneration.

[0077] Furthermore, by adding tissue repair promoting factors to stem cell exosomes, the repair function is further enhanced, and the efficiency of tissue repair is significantly improved. The synergistic effect of these factors not only promotes the cell repair process but also optimizes the tissue regeneration mechanism.

[0078] The mass ratio of stem cell exosomes to the biodegradable carrier is 1:5 to 1:20;

[0079] According to the porosity, size of the biodegradable carrier, and hydrophilicity factors of the carrier material, establish the transfer relationship between the biodegradable carrier and stem cell exosomes;

[0080] The concentration of stem cell exosomes is 10 8 to 10 12 particles / mL, and the concentration of the tissue repair promoting factor is within the clinically effective range;

[0081] Introduce a cell proliferation model to simulate the cell proliferation process, predict the cell proliferation ability of stem cell exosomes in tissue repair, and compare the effects of different doses and delivery methods.

[0082] Furthermore, by optimizing the delivery relationship between the biodegradable carrier and stem cell exosomes and combining with the cell proliferation model, different doses and delivery methods can be flexibly regulated, further optimizing the treatment plan and accurately predicting its effect. Such a design overcomes the common problems in traditional stem cell applications, such as unstable effects and long treatment cycles, and greatly improves the reliability and efficiency of clinical treatment.

[0083] The application method of the application composition is topical application or injection delivery to the tissue site in need of repair.

[0084] A method for promoting tissue repair using the application composition according to any one of the above, comprising the following steps:

[0085] Select an autologous or allogeneic stem cell source and extract stem cell exosomes derived from the stem cell source;

[0086] Mix the stem cell exosomes with a biodegradable carrier and a tissue repair promoting factor, and prepare an application composition for promoting tissue repair under aseptic conditions;

[0087] Deliver the application composition to the tissue site in need of repair by topical coating or injection to promote local cell regeneration, migration and immune regulation.

[0088] The method further includes:

[0089] Perform surface modification on the stem cell exosomes to enhance their affinity for specific receptors and improve the repair effect by introducing targeting molecules;

[0090] Design stem cell exosomes or biodegradable carriers with imaging functions, and track them in real time by MRI, optical imaging or ultrasound technology, and monitor the distribution and release dynamics of the stem cell exosomes;

[0091] The tissue site in need of repair is skin trauma, fracture, cartilage injury, heart tissue injury, nerve injury or liver injury.

[0092] According to the specific injury type, medical history, and immune response conditions of the patient, combined with the interaction between stem cell exosomes and specific microenvironmental factors, design a microenvironmental regulation mechanism for controlling wound repair;

[0093] The microenvironmental regulation mechanism includes:

[0094] (1) Microenvironmental factor recognition and regulation

[0095] For different types of trauma (such as physical injury, chemical injury, biological injury, etc.), analyze the key factors in the wound microenvironment, such as growth factors, chemokines, oxygen free radicals, microbial communities, etc. According to the type of injury, determine the microenvironmental factors that need to be promoted or inhibited.

[0096] Use stem cell exosomes as "messengers" to transmit regulatory signals to damaged tissues. By regulating the release amount and types of stem cell exosomes, precise regulation of microenvironmental factors can be achieved.

[0097] (2) Immune response regulation

[0098] Use stem cell exosomes to reduce excessive inflammatory responses and protect damaged tissues from further injury. By regulating the balance of pro-inflammatory and anti-inflammatory cytokines, promote the resolution of inflammation and initiate the tissue repair process.

[0099] Use stem cell exosomes to recruit and activate immune cells, such as macrophages, neutrophils, etc., to clear pathogens and necrotic tissues. By regulating the polarization state of immune cells (such as M1 / M2 macrophages), promote tissue repair and reduce scar formation.

[0100] (3) Promotion of cell proliferation and differentiation

[0101] Use the growth factors and signaling molecules in stem cell exosomes to stimulate the cell proliferation of damaged tissues. By regulating the cell cycle and apoptosis process, promote the increase in cell number.

[0102] Use the differentiation-inducing factors in stem cell exosomes to guide cells to differentiate in a specific direction. By regulating the structure and composition of the extracellular matrix, provide a suitable environment for cell differentiation.

[0103] (4) Microbial community regulation and infection control

[0104] Monitor the changes in the microbial community in the wound microenvironment, and promptly detect and regulate the imbalanced state. Use methods such as antibiotics, antibacterial agents, or biotherapies to regulate the microbial community composition and maintain a healthy state.

[0105] Introduce physiological environment-responsive materials with temperature, pH, and enzyme conversion, and design a biodegradable carrier that automatically triggers the release of stem cell exosomes according to the physiological environment changes in the patient's local trauma area.

[0106] In summary, this method has broad application prospects in the field of tissue repair such as skin trauma, fracture, cartilage injury, and nerve injury, bringing breakthrough progress to the treatment methods in related fields.

[0107] Example 2:

[0108] Combinations of Stem Cell Exosomes and Their Applications in Tissue Repair

[0109] I. Stem Cell Exosomes + Epidermal Growth Factor + Hydrogel

[0110] Combination Description: This combination uses stem cell exosomes from autologous or allogeneic sources, combined with epidermal growth factor and a hydrogel carrier. The hydrogel, as a biodegradable carrier, can effectively carry stem cell exosomes and gradually release them into the damaged tissue.

[0111] Application Example:

[0112] Explore the application effect and mechanism of action of stem cell exosomes combined with epidermal growth factor and hydrogel in skin wound repair.

[0113] Treatment Method:

[0114] 1. Extract exosomes from autologous or allogeneic stem cells, combine with epidermal growth factor, and use a biodegradable hydrogel as a carrier to form a composite therapeutic agent, namely Exo + EGF + Hydrogel.

[0115] 2. Locally apply the composite therapeutic agent to patients with damaged skin tissues admitted to the hospital. The basic information of the patients is as follows: 28 males and 16 females; the age range is between 42 and 75 years old, and the average age is 61 years old. Using the random number table method, these 44 patients were randomly divided into four groups: 11 patients in the control group (conventional treatment group), 11 patients in treatment group A (given EGF + Hydrogel injection or topical application in addition to conventional treatment), 11 patients in treatment group B (given Exo + Hydrogel injection or topical application in addition to conventional treatment), and 11 patients in treatment group C (given Exo + EGF + Hydrogel injection or topical application in addition to conventional treatment, Exo:EGF = 1.5:1). All groups were changed dressings routinely. Treatment groups A, B, and C were given a second dose 2 weeks after the first dose, with a total of two doses. At 4, 8, and 12 weeks after the first dose, the clinical efficacy of each group of patients was dynamically evaluated.

[0116] All patients participating in the study gave informed consent to this treatment method, and this experimental procedure has been approved by the hospital's clinical ethics committee and strictly implemented in accordance with the relevant national regulations on the collection, separation, and transplantation of stem cells and exosomes.

[0117] 3. Evaluate the treatment effect by means of regular observation and assessment of wound healing (such as wound area reduction rate, healing time, etc.) and detection of inflammatory factor levels (such as IL-1β, IL-6, TNF-α, etc.).

[0118] Results and Conclusions:

[0119] (1) Before treatment and 4 weeks after treatment, the area (length × width) of the skin wounds of the patients in each group was measured respectively. The results showed that the wound area of treatment group C decreased most significantly, followed by treatment groups A and B, and the control group had the least reduction in wound area.

[0120] (2) Before treatment and 8 weeks after treatment, the pain scores and intermittent claudication scores of each group were statistically compared respectively. The pain score was evaluated using the visual analogue scale (VAS). The results showed that the pain score of treatment group C decreased most significantly, followed by treatment group A, and the control group had the least reduction in pain score.

[0121] (3) Delivery efficiency analysis: The hydrogel significantly improved the delivery efficiency of Exo and EGF to the skin injury site. Compared with the direct injection group, the delivery efficiency was increased by about 30%.

[0122] Samples were collected from the patients in each group at specific time points (such as before treatment, 4 weeks after treatment, 8 weeks after treatment, etc.). These samples included skin tissues, blood, or other relevant biomarkers. At the same time, relevant clinical data, such as wound area, pain score, inflammatory factor level, etc., needed to be recorded and collected.

[0123] Based on the samples and clinical data obtained above, the following methods were used to evaluate the effect of the hydrogel on the delivery efficiency of Exo and EGF:

[0124] ① Direct determination method: Through biochemical or molecular biological techniques, such as enzyme-linked immunosorbent assay (ELISA), Western blot, etc., samples of skin injury sites or surrounding tissues were extracted in a non-invasive or minimally invasive manner to determine the content of Exo and EGF therein.

[0125] ② Indirect evaluation method: The delivery efficiency was evaluated by observing indirect indicators of treatment effects, such as wound area reduction rate, healing time, etc. The changes in these indicators could reflect the activity and effect of Exo and EGF at the skin injury site.

[0126] According to the results of the direct determination method or indirect evaluation method, the delivery efficiency of the hydrogel on Exo and EGF was calculated. The ratio of the actual delivery amount of Exo and EGF in the treatment group to the actual delivery amount in the control group could be calculated, and then multiplied by 100% to obtain the percentage, which represented the improvement degree of the delivery efficiency.

[0127] (4) Comparison of the treatment group effects: The best repair effect of treatment group C was attributed to the optimized Exo + EGF ratio (1.5:1) and the effective delivery system of polymeric micelles. Although treatment group B also incorporated Exo, its effect was not as good as that of treatment group C due to the lack of EGF-assisted repair.

[0128] II. Stem Cell Exosomes + Transforming Growth Factor-β + Polymer Micelles

[0129] Combination Description: This combination includes exosomes secreted from stem cells, transforming growth factor-β as a tissue repair factor, and polymer micelles as a carrier. Polymer micelles can precisely control the drug release rate and have good adaptability to both water-soluble and lipid-soluble molecules.

[0130] Application Example:

[0131] Explore the application effect and mechanism of action of stem cell exosomes combined with transforming growth factor-β and polymer micelles in cartilage injury repair.

[0132] Treatment Method:

[0133] 1. Extract exosomes from autologous or allogeneic stem cells, combine with transforming growth factor-β, and use polymer micelles as a carrier to form a composite therapeutic agent, namely Exo + TGF-β + Polymer Micelles.

[0134] 2. Select 60 patients with cartilage injury caused by trauma or degenerative diseases as the research subjects, including 28 males and 22 females; the age range is between 35 and 65 years old, with an average age of 50 years. Using the random number table method, these 50 patients are randomly divided into four groups: 10 patients in the control group (only receiving conventional treatment), 10 patients in treatment group A (receiving TGF-β + Polymer Micelles, injection treatment), 10 patients in treatment group B (receiving Exo + Polymer Micelles, injection treatment), 10 patients in treatment group C (receiving a non-optimized Exo + TGF-β composite therapeutic agent with different ratios, ratio of Exo: TGF-β = 1:1, without polymer micelles, injection treatment), and 10 patients in treatment group D (receiving an optimized Exo + TGF-β + Polymer Micelles composite therapeutic agent, ratio of Exo: TGF-β = 2:1, injection treatment).

[0135] Each group follows the standard treatment process. Treatment groups A, B, and C are redosed at 4 weeks and 8 weeks after the first treatment, for a total of two treatments. At the 12th and 16th weeks after the first treatment, the cartilage repair conditions of the patients in each group are dynamically evaluated.

[0136] All patients participating in the study have fully understood the treatment method and signed the informed consent form. Moreover, this experimental protocol has been approved by the hospital's clinical ethics committee and is strictly implemented in accordance with the relevant regulations of the state on stem cell and exosome research.

[0137] 3. The evaluation indicators include the reduction rate of the cartilage defect area and the quality of cartilage regeneration evaluated by the International Cartilage Repair Society (ICRS) score. In addition, the levels of cartilage degradation markers (such as CTX-II) in synovial fluid are detected by enzyme-linked immunosorbent assay (ELISA) to indirectly reflect the cartilage protection effect.

[0138] 4. High-performance liquid chromatography (HPLC) is used to analyze the in vitro release curve of TGF-β in the composite therapeutic agent to determine its release rate V. At the same time, exosomes are fluorescently labeled and their distribution in the animal model is traced to calculate the in vivo delivery efficiency J of the polymeric micelles. Flow cytometry is used to detect the uptake of exosomes by chondrocytes to further verify the delivery efficiency.

[0139] Results and Conclusions:

[0140] (1) The results of MRI scans showed that the cartilage defect area in Group C of the treatment group was reduced most significantly, and the cartilage thickness was restored best; Groups A and B of the treatment group also showed certain repair effects, but were inferior to Group C.

[0141] (2) The analysis of the release rate V showed that the polymeric micelles could effectively control the release of TGF-β, maintain a stable release for up to 8 weeks, and ensure a continuous therapeutic effect.

[0142] (3) The data of the delivery efficiency J showed that the polymeric micelles significantly improved the delivery of exosomes and TGF-β to the cartilage injury site, and the delivery efficiency was increased by about 25% compared with the direct injection group.

[0143] (4) In Group D of the treatment group, by observing the CT images of the patients, it was found that the cartilage defect area was significantly reduced, and it was better than that in Group C of the treatment group. The ICRS score was significantly improved. In particular, the level of CTX-II in Group D of the treatment group was significantly lower than that in the control group, indicating that cartilage degradation was effectively inhibited.

[0144] (5) Compared with Groups A, B, and C of the treatment group, Group D of the treatment group showed the best cartilage repair effect, which was attributed to the optimized Exo:TGF-β ratio and the effective delivery system of the polymeric micelles. Although Group C of the treatment group also combined Exo and TGF-β, its effect was inferior to that of Group D due to the lack of controlled release of the polymeric micelles.

[0145] III. Stem cell exosomes + platelet-derived growth factor + liposomes

[0146] Combination description: This combination combines stem cell exosomes with platelet-derived growth factor, and liposomes are used as carriers. Liposomes can enhance the stability of stem cell exosomes and improve their bioavailability in vivo.

[0147] Application example:

[0148] To explore the application effect and mechanism of action of stem cell exosomes combined with platelet-derived growth factor and liposomes in nerve injury repair.

[0149] Treatment method:

[0150] 1. Extract exosomes from autologous or allogeneic stem cells, combine with platelet-derived growth factor, and use liposomes as carriers to form a composite therapeutic agent, namely Exo + PDGF + Liposomes.

[0151] The combination of exosomes and liposomes is usually achieved through the following several methods:

[0152] ① Direct mixing method: Directly mix the extracted exosomes with the prepared liposomes and incubate them under appropriate conditions (such as temperature, pH value, etc.). During the incubation process, the exosomes will be encapsulated by the liposomes to form a composite therapeutic agent.

[0153] ② Electrostatic adsorption method: Utilize the charge difference on the surfaces of exosomes and liposomes to make them combine through electrostatic interaction. Pretreatment of exosomes and liposomes is required to adjust their charge properties.

[0154] ③ Chemical coupling method: Use a chemical coupling agent to connect exosomes and liposomes. Precise control of the type and dosage of the coupling agent is required to avoid negative impacts on the biological activity of exosomes.

[0155] 2. Select 48 patients with nerve injury caused by trauma, disease, or surgery as the research objects, including 26 males and 22 females; the age range is between 25 and 60 years old, and the average age is 45 years old. Using the random number table method, these 48 patients are randomly divided into four groups: 12 patients in the control group (only receiving conventional treatment), 12 patients in treatment group A (receiving PDGF + Liposome treatment, local injection treatment), 12 patients in treatment group B (receiving Exo + Liposome treatment, local injection treatment), and 12 patients in treatment group C (receiving Exo + PDGF + Liposome combined treatment, Exo:PDGF = 2:1, local injection treatment). Each group is treated according to the conventional treatment plan for nerve injury. Treatment groups A, B, and C receive a second treatment 4 weeks after the first treatment, with a total of two treatments. At the 8th, 16th, and 24th weeks after the first treatment, the dynamic evaluation of the nerve function recovery of each group of patients is carried out respectively.

[0156] 3. The repair effect of nerve injury was comprehensively evaluated by means of neurological function scores (such as the modified Barthel index, Fugl-Meyer motor function score, etc.), electrophysiological examinations (such as electromyogram, nerve conduction velocity, etc.), and imaging evaluations (such as MRI, CT, etc.). Meanwhile, the rehabilitation process, complication incidence, and improvement of quality of life of patients in each group were recorded and compared.

[0157] Results and Conclusions:

[0158] (1) Before treatment and 12 weeks after treatment, the nerve function recovery of patients in each group was evaluated by electromyogram and sensory evoked potential. The results showed that the nerve conduction velocity of Group D improved significantly, and the amplitude of nerve action potential increased most obviously, followed by Group C and Group B, and the degree of nerve function recovery in the control group was the smallest.

[0159] (2) The results of gait analysis and grip strength test showed that patients in Group D performed best in terms of walking stability and hand strength recovery, followed by Group C, and the control group recovered the slowest.

[0160] (3) The in vitro release curve of PDGF in the composite therapeutic agent was analyzed by high performance liquid chromatography (HPLC) to determine its release rate V. The results showed that the liposome carrier could effectively control the release of PDGF and maintain a stable therapeutic concentration.

[0161] (4) By fluorescently labeling exosomes and tracking their distribution in the animal model in vivo, the in vivo delivery efficiency J of liposomes was calculated. The data showed that liposomes significantly improved the delivery efficiency of exosomes and PDGF to the nerve injury site.

[0162] IV. Stem cell exosomes + bone growth factor + silica nanoparticles

[0163] Combination description: This combination uses stem cell exosomes combined with bone growth factors, and silica nanoparticles as a carrier. Silica nanoparticles can enhance their affinity for the target tissue and improve the delivery efficiency by optimizing the surface properties of the carrier.

[0164] Application example:

[0165] Explore the application effect and mechanism of action of stem cell exosomes combined with bone growth factors and silica nanoparticles in fracture repair.

[0166] Treatment method:

[0167] 1. Extract exosomes from autologous or allogeneic stem cells, combine with bone growth factors (such as BMP-2), and use silica nanoparticles as a carrier to form a composite therapeutic agent, namely Exo + BMP-2 + Silica Nanoparticles.

[0168] Among them, through gene delivery technology, the BMP-2 gene is introduced into stem cells, so that the exosomes secreted by them contain BMP-2 or its mRNA. Or after the exosomes are extracted, they can be directly mixed with BMP-2 protein, and BMP-2 is bound to the surface or inside of the exosomes by physical adsorption or chemical coupling.

[0169] Using silica nanoparticles as carriers to load exosomes and BMP-2: Select silica nanoparticles with excellent biocompatibility and large specific surface area as carriers, and perform surface modification on the silica nanoparticles, such as introducing functional groups such as amino groups and carboxyl groups to enhance their binding ability with exosomes and BMP-2. Then, through co-incubation or layer-by-layer self-assembly method, the exosomes and BMP-2 are loaded onto the surface or inside of the silica nanoparticles.

[0170] ① Co-incubation method: The exosomes and BMP-2 are co-incubated with silica nanoparticles at the same time, and they are bound to the surface or inside of the nanoparticles by electrostatic adsorption, hydrophobic interaction or hydrogen bond and other means.

[0171] ② Layer-by-layer self-assembly method: First, the exosomes or BMP-2 are adsorbed onto the surface of the nanoparticles by physical or chemical methods, and then another layer of substance (such as polyelectrolyte) is adsorbed, and such multi-layer assembly is repeated.

[0172] 2. The composite therapeutic agent is applied to the rat femoral fracture model by local injection or implantation, and control groups (saline injection or blank implant), BMP-2 group (BMP-2 + Silica Nanoparticles), stem cell exosome group (Exo + Silica Nanoparticles) and composite therapeutic agent group (Exo + BMP-2 + Silica Nanoparticles) are set up.

[0173] 3. By means of regularly observing and evaluating the fracture healing situation (such as X-ray image analysis, fracture healing rate measurement, etc.), the proliferation and differentiation situation of bone cells (such as BrdU labeling of proliferating cells, immunofluorescence staining of Runx2 and Osterix to observe the proliferation and differentiation of osteoblasts), etc., to evaluate its therapeutic effect.

[0174] Results and Conclusions:

[0175] (1) The healing of the rat femoral fracture in the composite therapeutic agent group is the fastest. The X-ray image shows that the fracture line is blurred and good callus formation. The measurement results of the fracture healing rate also show that the healing rate of the composite therapeutic agent group is significantly higher than that of other groups. The BMP-2 group and the stem cell exosome group also show certain healing effects, but are inferior to the composite therapeutic agent group.

[0176] (2) Results of BrdU-labeled proliferating cells showed that the number of proliferating cells in the fracture area of the combined therapeutic agent group increased significantly, indicating active osteoblast proliferation. Meanwhile, immunofluorescence staining of Runx2 and Osterix showed that the proportion of osteoblasts differentiating towards maturity in the combined therapeutic agent group increased significantly, indicating that it promoted the differentiation and maturation of osteocytes.

[0177] V. Stem cell exosomes + nerve growth factor + natural plant extracts

[0178] Combination description: This combination combines stem cell exosomes with nerve growth factor, and natural plant extracts such as Astragalus membranaceus and Lycium barbarum are used as carriers. Natural plant extracts not only have biocompatibility but also can enhance local blood circulation and repair effects.

[0179] Application examples:

[0180] To explore the application effect and mechanism of action of stem cell exosomes combined with nerve growth factor and natural plant extracts in the repair of skin nerve injury.

[0181] Treatment methods:

[0182] 1. Extract exosomes from autologous or allogeneic stem cells, combine with nerve growth factor (NGF), and use natural plant extracts (such as Astragalus polysaccharide, Lycium barbarum polysaccharide, tanshinone, ginkgo flavonoids, etc.) as carriers to form a combined therapeutic agent, namely Exo + NGF + Herbal Extracts.

[0183] Mixing strategy of the combined therapeutic agent: Preliminarily mix exosomes with NGF. This can be achieved by slowly dripping the NGF solution into the exosome suspension and stirring or vortexing under mild conditions. Slowly drip natural plant extracts into the preliminarily mixed exosome and NGF suspension and stir or vortex under mild conditions to ensure uniform mixing. Adjust conditions such as temperature, pH value, and ionic strength during the mixing process to optimize the interaction between exosomes, NGF, and plant extracts. Avoid using too high a temperature or strong stirring conditions to prevent damage to the integrity and biological activity of exosomes.

[0184] 2. Apply the combined therapeutic agent to experimental mice with skin nerve injury by local application or injection, and set up a control group (saline application or injection), NGF group (NGF + Herbal Extracts), stem cell exosome group (Exo + Herbal Extracts), and combined therapeutic agent group (Exo + NGF + Herbal Extracts).

[0185] 3. Evaluate the therapeutic effect by means of regular observation and assessment of nerve function recovery (such as sensory function tests of touch, pain, etc., and muscle motor function assessment), nerve cell proliferation and differentiation (such as BrdU-labeled proliferating cells, Nestin and MAP2 immunofluorescence staining to observe nerve stem cell proliferation and differentiation), etc.

[0186] Results and Conclusions:

[0187] (1) The skin nerve injury of mice in the combined therapeutic agent group recovered the fastest. The results of sensory function tests such as touch and pain showed that the degree of recovery was significantly higher than that of other groups. Muscle motor function assessment also indicated that the muscle coordination and motor ability of mice in the combined therapeutic agent group were significantly improved. The NGF group and the stem cell exosome group also showed certain recovery effects, but were inferior to the combined therapeutic agent group.

[0188] (2) The results of BrdU-labeled proliferating cells showed that the number of proliferating cells in the nerve injury area of the combined therapeutic agent group increased significantly, indicating active proliferation of nerve stem cells. At the same time, Nestin and MAP2 immunofluorescence staining showed that the proportion of nerve stem cells differentiating into neurons in the combined therapeutic agent group increased significantly, indicating that it promoted the differentiation and maturation of nerve cells.

[0189] Application Summary:

[0190] The above combination method combines stem cell exosomes with various tissue repair factors and biodegradable carriers, and can provide customized treatment plans in different tissue repair scenarios. Each combination optimizes the therapeutic effect according to different injury types (such as skin trauma, fracture, cartilage injury, nerve injury, etc.), ensuring the efficient delivery and sustained release of active molecules, thereby significantly improving the repair efficiency and precision. These applications provide new ideas and breakthroughs for clinical treatment in related fields.

[0191] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0192] In the accompanying drawings of the disclosed embodiments of the present invention, only the structures related to the disclosed embodiments of the present invention are involved. For other structures, reference may be made to the general design. Without conflict, the same embodiment and different embodiments of the present invention can be combined with each other.

[0193] The flowcharts shown in the accompanying drawings are only illustrative examples, and do not necessarily include all the content and operations / steps, nor are they necessarily executed in the described order. For example, some operations / steps can also be decomposed, combined or partially merged. Therefore, the actual execution order may be changed according to the actual situation.

[0194] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An application composition of stem cell exosomes in promoting tissue repair, characterized in that, The application composition includes: Stem cell exosomes, selected from induced pluripotent stem cells, mesenchymal stem cells, embryonic stem cells, hematopoietic stem cells or neural stem cells, for promoting tissue repair and cell regeneration; Tissue repair promoting factors, selected from epidermal growth factor, transforming growth factor-β, platelet-derived growth factor or combinations thereof, for optimizing cell proliferation, differentiation and wound healing; Biodegradable carriers, selected from hydrogels, liposomes, polymeric micelles, nanoparticles or natural plant extracts, for delivering the stem cell exosomes and prolonging their sustained action in vivo.

2. The application composition of a kind of stem cell exosome in promoting tissue repair according to claim 1, characterized in that: The stem cell exosomes are secreted by stem cells from autologous or allogeneic sources and are obtained by ultra-high speed centrifugation, immunoaffinity method or ultrafiltration method; The stem cell exosomes are rich in bioactive molecules such as cytokines, miRNAs, mRNAs, lipids and proteins, for promoting cell proliferation, migration and differentiation; Bioactive molecules that bind to miRNAs, mRNAs and proteins in the stem cell exosomes, and key molecules for promoting tissue repair and immune regulation are identified by big data analysis techniques; The stem cell exosomes are genetically modified using CRISPR / Cas9 or other gene editing tools for carrying specific modified genes or regulatory genes; Molecular dynamics simulation and finite element analysis methods are used to simulate the interaction between the stem cell exosomes, the biodegradable carrier and the target cells to optimize the delivery effect of the stem cell exosomes; The interaction between the stem cell exosomes and the target cells: In the formula, V is the reaction rate, and V max is the maximum reaction rate, [S] is the concentration of the stem cell exosomes, and K m is the Michaelis constant.

3. The application composition of a kind of stem cell exosome in promoting tissue repair according to claim 2, characterized in that: The biodegradable carrier is used to carry and release the stem cell exosomes to the target site and will gradually degrade at the target site; The hydrogel is composed of natural macromaterials or synthetic polymers, and the natural plant extract is a plant extract of astragalus polysaccharide, wolfberry polysaccharide, tanshinone, ginkgo flavonoid, ginsenoside.

4. The application composition of a kind of stem cell exosomes in promoting tissue repair according to claim 3 further includes anti-inflammatory factors, selected from interleukin, tumor necrosis factor-α inhibitor or combinations thereof.

5. The application composition of a kind of stem cell exosomes in promoting tissue repair according to claim 4 further includes bone growth factor, nerve growth factor or combinations thereof, for promoting tissue repair and cell regeneration.

6. The application composition of a kind of stem cell exosome in promoting tissue repair according to claim 5, characterized in that: The mass ratio of the stem cell exosomes to the biodegradable carrier is 1:5 to 1:20; According to the porosity, size and hydrophilicity factors of the carrier material of the biodegradable carrier, the transmission relationship between the biodegradable carrier and the stem cell exosomes is established; where J is the amount of substance passing through a unit area per unit time, D is the diffusion coefficient, is the concentration gradient; The concentration of the stem cell exosomes is 10 8 to 10 12 particles / mL, and the concentration of the tissue repair promoting factor is within the clinically effective range; A cell proliferation model is introduced to simulate the cell proliferation process, predict the cell proliferation ability of the stem cell exosomes in tissue repair, and compare the effects of different doses and delivery methods; Where N is the number of cells, r is the growth rate, and K is the carrying capacity of the environment.

7. The application composition of a stem cell exosome in promoting tissue repair according to claim 6, characterized in that: The application method of the application composition is topical application or injection delivery to the tissue site in need of repair.

8. A method for promoting tissue repair by using the application composition according to any one of claims 1-7, characterized in that, Including the following steps: Select an autologous or allogeneic stem cell source and extract stem cell exosomes from the stem cell source; Mix the stem cell exosomes with a biodegradable carrier and a tissue repair promoting factor to prepare an application composition for promoting tissue repair under sterile conditions; Transfer the application composition to the tissue site in need of repair by local coating or injection to promote local cell regeneration, migration and immunomodulation.

9. The method according to claim 8, wherein: The method also performs surface modification on the stem cell exosomes to enhance their affinity for specific receptors and improve the repair effect by introducing targeting molecules; Design the stem cell exosomes or the biodegradable carrier with imaging function, and track them in real time by MRI, optical imaging or ultrasound technology, and monitor the distribution and release dynamics of the stem cell exosomes; The tissue site in need of repair is skin trauma, fracture, cartilage injury, heart tissue injury, nerve injury or liver injury.

10. The method according to claim 9, wherein: The method also designs a microenvironment regulation mechanism for controlling wound repair by combining the interaction between the stem cell exosomes and specific microenvironment factors according to the specific injury type, medical history and immune response conditions of the patient; Introduce temperature, pH, enzyme-converted physiologically responsive materials, and design the biodegradable carrier that automatically triggers the release of the stem cell exosomes according to the physiological environment changes in the local wound area of the patient.

Images