Cartilage injury treatment preparation and use of m2 myeloid macrophage-derived exosomes in preparation of cartilage injury treatment preparation

Abstract

The application discloses a cartilage injury treatment preparation and application of M2 myeloid macrophage-derived exosomes in preparation of the cartilage injury treatment preparation. The cartilage injury treatment preparation comprises 50-100 mu g / mL of separated and purified M2 myeloid macrophage-derived exosomes and excipients. The M2 myeloid macrophage-derived exosomes can promote polarization of macrophages at a cartilage injury site to M2 myeloid macrophages, so that the cartilage injury site has more M2 myeloid macrophages to promote cartilage repair and regeneration, and the use of cells derived from an allogeneic body at the cartilage injury site is avoided, so that biocompatibility and immunogenicity problems are avoided.

Description

Technical Field

[0001] This application belongs to the field of articular cartilage repair technology, specifically relating to a cartilage injury treatment agent and the use of M2 bone marrow macrophage-derived exosomes in the preparation of the cartilage injury treatment agent. Background Technology

[0002] Cartilage is an important component of the skeletal system in humans and animals. For example, cartilage within the joint cavity can buffer stress and lubricate, protecting the joint from damage and increasing joint flexibility.

[0003] Because articular cartilage lacks blood vessels, nerves, and lymphatic tissue, its self-healing ability is extremely limited. Injury to articular cartilage can lead to the infiltration of immune cells into the joint cavity. How to improve the treatment of cartilage injury under the condition of immune cell infiltration still needs further research. Summary of the Invention

[0004] This application provides a cartilage injury treatment agent and the use of M2 bone marrow macrophage-derived exosomes in the preparation of a cartilage injury treatment agent, aiming to provide isolated and purified M2 bone marrow macrophage-derived exosomes to achieve repair and regeneration of damaged articular cartilage.

[0005] In a first aspect, embodiments of this application provide a cartilage injury treatment formulation comprising 50-100 μg / mL of isolated and purified M2 bone marrow macrophage-derived exosomes, and excipients.

[0006] According to one embodiment of this application, the excipient is a hydrogel, and the M2 bone marrow macrophage-derived exosomes are bound within a three-dimensional network of the hydrogel.

[0007] According to one embodiment of this application, the hydrogel comprises a cross-linked gel matrix polymer and water.

[0008] According to one embodiment of this application, the gel matrix polymer is selected from cross-linked gelatin and cross-linked polyethylene glycol-poly(lactic acid)-poly(lactic acid).

[0009] According to an embodiment of one aspect of this application, the cartilage injury treatment agent further includes at least one of sodium hyaluronate, platelet-rich plasma, mesenchymal stem cells, glucosamine, and chondroitin sulfate.

[0010] According to one embodiment of this application, the cartilage injury treatment formulation further includes a solvent.

[0011] According to an embodiment of one aspect of this application, the solvent is selected from triethanolamine, phosphate buffer solution, NaOH, or a combination thereof.

[0012] Secondly, this application provides a method for preparing the cartilage injury treatment agent of the first aspect, comprising the following steps:

[0013] Provides exosomes isolated and purified from M2 bone marrow macrophages;

[0014] Provide excipients and other additives;

[0015] The solvent is used to mix exosomes, excipients and other additives, followed by co-incubation to obtain a cartilage injury treatment formulation.

[0016] According to one embodiment of this application, the excipient is a hydrogel, and the method for preparing the hydrogel includes:

[0017] The gel matrix polymer dissolved in an aqueous solvent is mixed with a photoinitiator;

[0018] A photoinitiator is used to initiate a crosslinking reaction of a polymer containing a gel matrix to obtain a hydrogel.

[0019] Thirdly, embodiments of this application provide the use of isolated and purified M2 bone marrow macrophage-derived exosomes in the preparation of cartilage injury treatment agents.

[0020] The cartilage injury treatment formulation of this application includes M2 bone marrow macrophage-derived exosomes, which can promote the polarization of macrophages at the cartilage injury site into M2-type bone marrow macrophages, thereby increasing the number of M2-type bone marrow macrophages at the cartilage injury site to promote cartilage repair and regeneration. This avoids the use of allogeneic cells at the cartilage injury site, which could lead to biocompatibility and immunogenicity issues. M2 bone marrow macrophage-derived exosomes can effectively promote the proliferation, migration, and chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), while also inhibiting inflammatory responses, reducing the release of inflammatory factors, and promoting macrophage polarization into the M2 type. In vivo animal experiments have confirmed that M2D-Exo can effectively promote cartilage regeneration and repair in a rat knee osteochondral defect model. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments of this application will be briefly introduced below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The images show morphological diagrams of primary bone marrow macrophages and M2 bone marrow macrophages, as well as RT-qPCR detection results from embodiments of this application.

[0023] Figure 2 The diagram showing the identification results of M2D-Exo according to an embodiment of this application is illustrated.

[0024] Figure 3 This image shows a confocal image of bone marrow mesenchymal stem cells, primary bone marrow macrophages, and M1 bone marrow macrophages phagocytosing M2D-Exo, according to an embodiment of this application.

[0025] Figure 4 The following figure shows the effect of different concentrations of M2D-Exo on the proliferation of BMSCs according to embodiments of this application;

[0026] Figure 5 The following figure shows the effect of different concentrations of M2D-Exo on the migration of BMSCs in embodiments of this application;

[0027] Figure 6 The following figure shows the effect of M2D-Exo on chondrogenic differentiation of BMSCs according to an embodiment of this application;

[0028] Figure 7 The following figure shows the effect of M2D-Exo on inflammatory factors and macrophage polarization according to an embodiment of this application;

[0029] Figure 8 Gross images of articular cartilage defect repair in rats with simple defects and M2D-Exo treatment groups 12 weeks post-surgery, according to embodiments of this application, are shown.

[0030] Figure 9 The effects of M2D-Exo and cartilage injury treatment agents on the repair of cartilage damage in rats were demonstrated. Detailed Implementation

[0031] The features and exemplary embodiments of various aspects of this application will be described in detail below. To make the objectives, technical solutions, and advantages of this application clearer, the application will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only intended to explain this application and not to limit it. For those skilled in the art, this application can be implemented without some of these specific details. The following description of the embodiments is merely to provide a better understanding of this application by illustrating examples.

[0032] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising..." does not exclude the presence of additional identical elements in the process, method, article, or apparatus that includes said element.

[0033] Articular cartilage is a type of bone tissue lacking blood vessels, nerves, and lymphatic tissue, and its self-healing ability is extremely limited. When articular cartilage is damaged, early symptoms may include pain, while later stages may develop into osteoarthritis, joint deformities, and in severe cases, disability. Statistics show that the incidence of cartilage abnormalities after arthroscopy ranges from 61% to 63%, and articular cartilage damage affects people of all ages.

[0034] Studies have found that inflammation, aging, and trauma can lead to the degradation of articular cartilage. Furthermore, inappropriate or unscientific exercise may increase the probability of articular cartilage damage.

[0035] Traditional techniques still lack highly effective treatments for articular cartilage damage. Following articular cartilage injury, immune cells infiltrate the joint cavity. Common defensive immune cells in the body include macrophages, which play a crucial role in the development and progression of inflammation-related diseases.

[0036] Macrophages are broadly classified into three phenotypes: inactive macrophages (M0), pro-inflammatory macrophages (M1), and anti-inflammatory macrophages (M2). Studies have found that M2 macrophages possess anti-inflammatory properties and the ability to repair and regenerate cartilage. M2 macrophages not only secrete interleukin-10 (IL-10), transforming growth factor-β (TGF-β), interleukin-1 receptor antagonist (IL-1RA), chemokine (CeC motif) ligand 18 (CCL18), and other anti-inflammatory mediators, but also secrete cytokines that promote cartilage formation, such as transforming growth factor-β1 (TGF-β1), transforming growth factor-β3 (TGF-β3), insulin-like growth factor-1 (IGF-1), and insulin-like growth factor-2 (IGF-2). These biological factors alter the local immune microenvironment in vivo, making it more conducive to cartilage formation. However, due to the limited number of macrophages at the site of cartilage injury, and the fact that additional macrophages are derived from foreign bodies, there are issues such as biocompatibility and immunogenicity, which limits its application.

[0037] The inventors have discovered that the isolated and purified exosomes, as a novel "nanobiomaterial," possess excellent biological functions, exhibit biocompatibility and immunogenicity, and have a surprising effect on promoting cartilage repair and regeneration.

[0038] The inventors discovered that small molecules (including proteins and nucleic acids) in exosomes derived from M2 macrophages can regulate the function of recipient cells during internalization, thereby facilitating communication between various cells and organs. Furthermore, exosomes are ideal nanomaterials for delivering regulatory substances to targets, and can protect these substances during the process.

[0039] Furthermore, exosomes derived from M2 macrophages play a crucial role in intercellular communication by delivering miRNAs, mRNAs, and proteins to recipient cells. M2 macrophage-derived exosomes can influence gene transcription and cell proliferation, and can also carry various gene-based drugs, such as proteins, microRNAs, non-coding RNAs, and DNA, between cells. Because M2 macrophage-derived exosomes are non-tumorigenic and non-immunogenic, they avoid the risks associated with cell transplantation, and their storage and transport requirements are less stringent than those for cells, making them suitable for widespread application and promotion.

[0040] Studies have revealed that exosomes derived from M2 macrophages possess a strong ability to regulate the joint cavity microenvironment. For example, when exosomes derived from M2 macrophages were applied to micropores in a 3D-printed scaffold and used in a rat model of knee osteochondral defects, combined with subsequent intra-articular treatment, they demonstrated a stronger cartilage repair capacity. The underlying mechanism may be related to the abundance of miRNAs in exosomes derived from M2 macrophages.

[0041] Based on this, the inventors proposed a cartilage injury treatment preparation, comprising 50-100 μg / mL of isolated and purified M2 bone marrow macrophage-derived exosomes and excipients, which can achieve the purpose of cartilage repair and regeneration and achieve better therapeutic effects.

[0042] Cartilage injury treatment agents

[0043] In a first aspect, embodiments of this application provide a cartilage injury treatment formulation comprising 50-100 μg / mL of isolated and purified M2 bone marrow macrophage-derived exosomes, and excipients.

[0044] According to embodiments of this application, M2 macrophage-derived exosomes (hereinafter referred to as M2D-Exo) are small vesicles with a diameter of approximately 30-150 nm. They are small vesicles secreted by M2 macrophages after macrophages are stimulated by IL-4 to polarize into M2 macrophages. Studies have found that M2 bone marrow-derived macrophage exosomes can inhibit the release of inflammatory factors, such as iNOS, TNF-α, and IL-1β, and can promote macrophage polarization towards the M2 macrophage phenotype. These biological functions endow M2 bone marrow-derived macrophage exosomes with the ability to regulate the immune microenvironment, inhibit synovial inflammation, reduce the infiltration of inflammatory cells, protect normal chondrocytes under inflammatory conditions, and reduce chondrocyte apoptosis and extracellular matrix degradation.

[0045] Furthermore, M2 bone marrow-derived macrophage exosomes activate endogenous bone marrow mesenchymal stem cells in the joint cavity, while simultaneously delivering their own chondrogenic miRNAs to bone marrow mesenchymal stem cells. This may activate chondrogenic pathways, such as the TGF / Smad2 / Smad3 pathway, ultimately promoting the differentiation of bone marrow mesenchymal stem cells toward cartilage, thus achieving the effect of cartilage regeneration and repair.

[0046] Furthermore, exosomes derived from M2 bone marrow macrophages can promote the polarization of macrophages at the site of cartilage injury into M2-type bone marrow macrophages, resulting in a greater number of M2-type bone marrow macrophages at the cartilage injury site, thus promoting cartilage repair and regeneration. This avoids the use of allogeneic cells at the cartilage injury site, which could lead to biocompatibility and immunogenicity issues. The aforementioned cartilage injury can be trauma-induced cartilage damage.

[0047] Furthermore, the M2 bone marrow macrophages are generated by polarizing primary macrophages into M2 macrophages using IL-4. Furthermore, the primary macrophages are rat bone marrow-derived macrophages. Furthermore, the M2D-Exo is derived from the supernatant obtained from macrophage culture after IL-4 polarization for 48 hours.

[0048] According to the embodiments of this application, the concentration of isolated and purified M2 bone marrow macrophage-derived exosomes in the cartilage injury treatment preparation can be 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg / ml, or any value within the above range.

[0049] Applying exosomes at the above concentrations to cartilage repair can promote the diffusion and transfer of exosomes in the cartilage damage area, significantly improving the effective utilization rate of exosomes and enhancing the repair effect and healing ability of cartilage damage. The above-mentioned concentrations of exosomes exhibit strong activity in cartilage damage treatment agents, capable of secreting and providing biological factors that act on the cartilage damage area.

[0050] In some embodiments, M2 bone marrow macrophage-derived exosomes are obtained by gradient centrifugation, filtration, and precipitation of M2 bone marrow macrophages.

[0051] In some embodiments, M2 bone marrow macrophages are obtained by IL-4 biological factor-induced polarization of primary macrophages.

[0052] In some embodiments, M2 bone marrow-derived macrophages are cultured in IMDM medium containing 10% exosome-free fetal bovine serum.

[0053] In some embodiments, each 500 mL of the IMDM culture medium comprises the following components at concentrations: recombinant human insulin 1 mg / mL; recombinant transferrin 2.5 μg / mL; vitamin C 16.1 μg / mL; human albumin 1 mg / mL; fibroblast growth factor 10 ng / mL; adequate human platelet-derived growth factor 10 ng / mL; growth factor IGF-1 10 ng / mL; transforming growth factor TGF-β 10 ng / mL; fibronectin 10 ng / mL; fetoglobulin 1 mg / mL; β-cytokinin 10 μg / mL; β-mercaptoethanol 150 μM; and ALK inhibitor 1–3 mmol / L.

[0054] In some embodiments, the excipient is a hydrogel, and the M2 bone marrow macrophage-derived exosomes are bound within a three-dimensional network of the hydrogel. When the excipient is a hydrogel, the rate of diffusion and transfer of exosomes in the cartilage injury area can be controlled, enhancing the sustained repair effect and healing ability of cartilage injury, and effectively ensuring sustained-release repair.

[0055] In some embodiments, the hydrogel comprises a cross-linked gel matrix polymer and water. The gel matrix polymer can swell with water and, in addition, has an elastic and three-dimensional network structure, enhancing its suitability for use at cartilage injury sites.

[0056] In some embodiments, the surface of the isolated and purified M2 bone marrow macrophage-derived exosomes includes at least one of Arginase-1 (Arg-1) protein and interleukin-10 (IL-10) protein, which has the effect of inhibiting inflammation and promoting macrophage polarization to the M2 phenotype.

[0057] In some embodiments, exosomes also secrete at least one of transforming growth factor-β1 (TGF-β1) and transforming growth factor-β3 (TGF-β3), which promotes the proliferation and migration of mesenchymal stem cells and can be used to promote cartilage formation.

[0058] In some embodiments, the gel matrix polymer is selected from cross-linked gelatin and cross-linked polyethylene glycol-poly(lactic acid)-poly(lactic acid).

[0059] In some embodiments, the cartilage injury treatment formulation includes 50-100 μg / mL of isolated and purified M2 bone marrow macrophage-derived exosomes, methacrylated gelatin, and a photoinitiator.

[0060] When used, the mixture is irradiated with ultraviolet light. Under light irradiation, the photoinitiator causes the methacrylated gelatin to crosslink, and the cartilage injury treatment preparation is transformed into a solid state, which contains crosslinked gelatin and exosomes derived from M2 bone marrow macrophages. The photoinitiator is absent or present in trace amounts in the above solid cartilage injury treatment preparation.

[0061] Furthermore, methacrylamide gelatin hydrogel combines the characteristics of both natural and synthetic biomaterials, exhibiting excellent biocompatibility and cellular reactivity, providing suitable cell adhesion sites, and possessing proteolytic degradation properties. In addition, the aforementioned hydrogel possesses good mechanical properties, preventing further damage to normal cartilage at the site of cartilage injury and providing a microenvironment for cartilage repair.

[0062] In some embodiments, the photoinitiator includes a selection from 2-hydroxy-2-methyl-1-phenyl-1-propanone, 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone, or combinations thereof. The above photoinitiator can absorb energy of a certain wavelength under ultraviolet irradiation, generating free radicals, cations, etc., thereby initiating the polymerization, cross-linking, and curing of the gel matrix polymer.

[0063] In some embodiments, the cartilage injury treatment formulation includes 50-100 μg / mL of isolated and purified M2 bone marrow macrophage-derived exosomes and polyethylene glycol-poly(lactic acid) lactide (PEG-PEG). PEG-PEG has a certain ratio of hydrophobic and hydrophilic groups. Temperature changes affect the hydrophobic and hydrophilic interactions of these groups and the hydrogen bonding between molecular chains, thus altering the gel structure. When the cartilage injury treatment formulation is solid, it comprises PEG-PEG-PEG and M2 bone marrow macrophage-derived exosomes.

[0064] In some embodiments, the cartilage injury treatment formulation further includes at least one of sodium hyaluronate, platelet-rich plasma, mesenchymal stem cells, glucosamine, and chondroitin sulfate.

[0065] According to embodiments of this application, the gel matrix polymer can serve as a blank framework, in which the aforementioned components are embedded or bound. When intra-articular cartilage is damaged, these components can promote cartilage repair and regeneration. For example, doping the gel matrix polymer with chondroitin sulfate can induce mesenchymal stem cells to differentiate into chondrocytes. Furthermore, by altering the composition of the cartilage injury treatment agent, the phenotype of differentiated cells and matrix production mode can be customized according to specific regions of the articular cartilage, thereby achieving the repair and regeneration of specific areas of the articular cartilage.

[0066] In some embodiments, the stem cells are selected from synovial mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, or combinations thereof.

[0067] According to the embodiments of this application, since the stem cells are synovial mesenchymal stem cells, the resulting exosomes contain a variety of proteins and nucleic acid molecules, which can promote the production of anti-inflammatory factors, inhibit the production of matrix proteases, and promote macrophage polarization. Through their anti-inflammatory and immunomodulatory effects within the joint cavity, they enhance the repair effect of the cartilage scaffold on cartilage defects. Simultaneously, exosomes derived from synovial mesenchymal stem cells can work together with components such as chondroitin sulfate to promote articular cartilage regeneration, achieving rapid repair and complete cartilage regeneration.

[0068] In some embodiments, the cartilage injury treatment formulation further includes a solvent.

[0069] In some embodiments, the solvent is selected from triethanolamine, phosphate buffer solution, NaOH, or combinations thereof. The solvent in this embodiment can disperse the gel matrix polymer and exosomes therein, and can also provide a favorable microenvironment for the joint injury area, promoting the repair of the joint injury area.

[0070] According to the embodiments of this application, the isolated and purified M2 bone marrow macrophage-derived exosomes, bonded or dispersed in an excipient, such as a gel matrix polymer, can be used for cartilage repair and regeneration and improve joint function for a period of 2 weeks or more; and the exosomes and their biological factors are continuously delivered and released.

[0071] Secondly, this application provides a method for preparing the cartilage injury treatment agent of the first aspect, comprising the following steps:

[0072] Provides exosomes isolated and purified from M2 bone marrow macrophages;

[0073] Provide excipients and other additives;

[0074] Exosomes, excipients, and other additives are mixed in a solvent and then co-incubated to obtain a cartilage injury treatment formulation.

[0075] The solvents mentioned above are selected from triethanolamine, phosphate buffer solution, NaOH, or combinations thereof.

[0076] According to the embodiments of this application, exosomes, excipients and other additives are mixed in a solvent and then co-incubated. This preparation method is less likely to leave toxic and harmful chemical reagents, and the resulting cartilage injury treatment preparation has low biotoxicity and good biocompatibility. At the same time, it does not destroy the bioactivity of exosomes, so that exosomes and excipients work together to treat damaged cartilage. The preparation method is simple and easy to produce.

[0077] In some embodiments, mixing exosomes, excipients, and other additives in a solvent can enhance or maintain the activity of exosomes.

[0078] In some embodiments, the excipient is a hydrogel, and the method for preparing the hydrogel includes:

[0079] The gel matrix polymer dissolved in an aqueous solvent is mixed with a photoinitiator;

[0080] A photoinitiator is used to initiate a crosslinking reaction of a polymer containing a gel matrix to obtain a hydrogel.

[0081] In some embodiments, during use, as the gel matrix polymer is degraded, exosomes can be directly released into the joint cavity according to the rate of degradation, thus acting more directly and quickly on the damaged area within the joint cavity to achieve the purpose of repair and regeneration.

[0082] In some embodiments, the co-incubation conditions are 35-37°C. In some embodiments, the co-incubation conditions are 37°C under sterile conditions for 12-24 hours, preferably 24 hours.

[0083] Thirdly, embodiments of this application provide the use of isolated and purified M2 bone marrow macrophage-derived exosomes in the preparation of cartilage injury treatment agents.

[0084] According to the embodiments of this application, a new approach can be provided for the preparation of drugs for treating cartilage repair and regeneration, such as injectable hydrogels containing isolated and purified exosomes derived from M2 bone marrow macrophages, or by controlling the concentration of the hydrogel, applying the hydrogel to the site of cartilage injury.

[0085] In some embodiments, the cartilage injury treatment formulation is an injectable formulation. The injectable formulation is prepared by using extracted fresh exosomes as a solvent with enzyme-free deionized water, thereby protecting the nucleic acid substances in the exosomes from degradation. Further, the cartilage injury treatment formulation can be an injectable formulation for local intra-articular administration; the solvent in the injectable formulation is sterile, enzyme-free water; the injectable formulation can be mixed with biological materials or adsorbed onto their surface.

[0086] Furthermore, the injection can be used for intra-articular injection therapy or in combination with other drugs. The intra-articular injection concentration can be 100 μg / mL, and the injection interval can be 7 days.

[0087] In some embodiments, the cartilage injury treatment agent can be used as an implant.

[0088] Example

[0089] The following embodiments describe the disclosure of this application in more detail. These embodiments are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on weight, and all reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing, and the instruments used in the embodiments are commercially available.

[0090] Example 1: Isolation of exosomes derived from M2 macrophages (hereinafter referred to as M2D-Exo)

[0091] Prepare an IMDM culture solution comprising the following components at the following concentrations: recombinant human insulin, 1 mg / mL; recombinant transferrin, 2.5 μg / mL; vitamin C, 16.1 μg / mL; human albumin, 1 mg / mL; fibroblast growth factor, 10 ng / mL; sufficient human platelet-derived growth factor, 10 ng / mL; growth factor IGF-1, 10 ng / mL; transforming growth factor TGF-β, 10 ng / mL; fibronectin, 10 ng / mL; fetoglobulin, 1 mg / mL; β-cytokinin, 10 μg / mL; β-mercaptoethanol, 150 μM; and ALK inhibitor, 1–3 mmol / L.

[0092] Prepare complete culture medium, also called (serum) cell culture medium: add fetal bovine serum (final concentration 10%), 100 U / ml penicillin, 100 μg / ml streptomycin and 20 ng / ml IL-4 to the basal culture medium.

[0093] Extraction of M2 bone marrow macrophages: Male SD rats aged 6-8 weeks were selected and euthanized by spinal dislocation. The skin and muscle of the hind limbs were removed, and the tibia and femur were isolated. The femur and tibia were washed twice with sterile PBS solution, and then the ends of the bones were aseptically cut open. Cells were flushed from the bone marrow cavity using IMDM culture solution. The collected cells were centrifuged at 1500 rpm for 5 minutes, resuspended in erythrocyte lysis buffer, and lysed on ice for 10 minutes. The cell pellet was obtained by centrifugation. The collected cells were then centrifuged at 2 × 10⁻⁶ cells / mL. 6Cells were seeded per ml in culture flasks, and IMDM containing 1% penicillin + streptomycin, 10% fetal bovine serum, and 30 ng / ml recombinant rat macrophage colony-stimulating factor (M-CSF) was added. The cells were then differentiated into macrophages at 37°C and 5% CO2, with the culture medium changed every 48 hours. After 6-7 days of culture, mature bone marrow macrophages were collected. To induce macrophage polarization, M0 macrophages were treated with complete culture medium containing 20 ng / ml IL-4 for 24 hours, polarizing them into M2 macrophages.

[0094] Identification of M2 macrophages: M0 macrophages were observed using an Olympus inverted microscope; they were round or oval in shape (see...). Figure 1 (Figure A), while the morphology of M2 macrophages changed, exhibiting a spindle-shaped morphology (see Figure A). Figure 1 (Figure A). Using a DX-type flow cytometer to detect rat M2 macrophage-specific markers, it was found that the proportion of cells co-expressing CD68 and CD163 reached 88.3% (see Figure A). Figure 1 (Figure B), where CD68 represents macrophages and CD163 is a marker for the M1 state of macrophages.

[0095] RT-qPCR was used to detect M2 macrophage-related genes. The results showed that M2 macrophages highly expressed the M2 macrophage-related genes Arginase 1 and PPARγ (see... Figure 1 C) indicates that M2 macrophages were indeed obtained.

[0096] Figure 1 In the diagram, Figure A is an optical microscope image (10x magnification). The left image in Figure A shows primary bone marrow macrophages, and the right image shows M2 bone marrow macrophages. Figure B is a flow cytometry image of CD45, CD68, and CD163. The horizontal axis represents the fluorescence intensity values ​​of CD68 and CD163, and the vertical axis represents the fluorescence intensity values ​​of CD45 and CD68, indicating that CD68 and CD163 are expressed, and these macrophages are M2 macrophages. Figure C is an RT-qPCR image of arginase 1 and peroxisome proliferator-activated receptor γ (PPARγ). On the horizontal axis, "untreated" represents macrophages not treated with 20 ng / ml IL-4, and "IL-4" represents macrophages treated with 20 ng / ml IL-4. The vertical axis represents the relative mRNA expression level, indicating that M2 macrophages were indeed obtained.

[0097] Example 2: Isolation and Identification of M2D-Exo

[0098] Isolation of M2D-Exo: M0 macrophages were treated with complete medium containing 20 ng / ml IL-4 for 24 hours, and then the old medium was replaced with cell culture medium containing 10% exosome-free serum. The cells were cultured for another 24 hours and the supernatant was collected.

[0099] The collected supernatant was used to separate exosomes using gradient centrifugation: centrifugation (1000X g, 10 min, 4℃) was used to remove debris and dead cells from the culture medium. The supernatant was collected and filtered through a 0.2 μm filter. The supernatant was collected again and centrifuged (100000X g, 4 h, 4℃). The precipitate was collected, resuspended in PBS, centrifuged (100000x g, 20 min, 4℃) to wash the precipitate, and collected to obtain M2D-Exo. M2D-Exo was resuspended in 200 μL of sterile enzyme-free water and stored at -80℃ for later use.

[0100] The obtained M2 macrophage-derived exosomes were observed and identified using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA).

[0101] Transmission electron microscopy: The suspension of exosomes was dropped onto a copper grid and dried in air. Then, the sample was fixed with 3% glutaraldehyde for 2 hours, rinsed with PBS, stained with 2% platinum acetate for 30 seconds, and the sample was placed under TEM (HITACHI, Japan) to observe the morphology and size of the exosomes.

[0102] Nanoparticle tracking analysis: The particle size of exosomes was determined using a nanoparticle tracking analyzer (NanoSight NS500, Zetaview, UK). First, the NP100 nanopores of the measurement system were calibrated using particles of known size (CPC100 standard solution), followed by washing three times with PBS. The exosome samples were then diluted 1000-fold with PBS and added to the nanopores for measurement. The particle size and concentration of exosomes were measured and analyzed using the nanoparticle tracking analyzer and Control Suite V2.2 software.

[0103] The results show that the M2D-Exo transmission electron microscopy image is as follows: Figure 2 In Figure A, the M2D-Exo appears as a disk with a bilayer membrane structure. Nanoparticle tracking analysis results show that the exosome diameter is approximately 100 nanometers (see Figure A). Figure 2 (Figure B in the middle).

[0104]

[0105]

[0106] Western blot analysis of exosome characteristic proteins showed that the collected M2 bone marrow macrophage exosomes highly expressed exosome markers such as CD9, CD63, and TSG101, indicating that M2 bone marrow macrophage exosomes were obtained (see...). Figure 2 (Figure C in the middle).

[0107] Figure 2 The images show the identification results of M2D-Exo. Image A illustrates the morphology of exosomes under transmission electron microscopy (TEM), Image B shows the size of exosomes under nanoparticle tracking (NTA), and Image C shows the expression results of exosome characteristic proteins CD9, CD63, and TSG101 detected by Western blot.

[0108] Example 3: Different cells phagocytose M2D-Exo

[0109] PKH26 is a yellow-orange fluorescent dye with a maximum emission wavelength of 567 nm. It can be used in conjunction with other dyes for multicolor analysis. PKH26 is quite stable, and PKH26-labeled cells can be studied by microscopy or flow cytometry within 100 days after staining. It can be purchased from Beijing Sunshine Yingrui Trading Co., Ltd.

[0110] Rat bone marrow mesenchymal stem cells (BMSCs), primary M0 bone marrow macrophages (BMDMs), and M1 bone marrow macrophages (M1 BMDMs) were isolated and cultured using standard methods, and the cells were collected for subsequent experiments. M2D-Exo was labeled with PKH26, and the labeled M2D-Exo was co-cultured with rat BMSCs, primary M0 bone marrow macrophages, and M1 bone marrow macrophages for 24 hours, then fixed, followed by cytoskeleton staining, and finally observed under a Nikon confocal microscope. The confocal results showed strong red fluorescence signals in rat BMSCs, primary M0 bone marrow macrophages, and M1 bone marrow macrophages (see...). Figure 3 This indicates that M2D-Exo can be phagocytosed by the above three types of cells. Figure 3 In this context, "nucleus" represents the cell nucleus, "M2D-Exo" represents the fluorescence signal of M2D-Exo, "F-actin" represents / describes the skeletal morphology of rat BMSCs, "bright field" represents / describes the morphological characteristics of rat BMSCs under bright field observation, and "merge" represents the state of M2D-Exo being phagocytosed by cells during the fusion and merging stages.

[0111] Example 4: Effects of M2D-Exo on the biological function of rat BMSCs

[0112] Effects of M2D-Exo on rat BMSCs proliferation

[0113] Components of bovine serum albumin (BSA) working solution: BSA, PBS

[0114] The effect of different concentrations of M2D-Exo (0 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL) on the proliferation of rat BMSCs was investigated using the CCK-8 cell counting reagent from APExBIO. First, rat BMSCs were seeded at a rate of 5000 cells per well in 96-well plates. After cell attachment, the original culture medium was replaced with exosome-free serum containing different concentrations of M2D-Exo (0 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL). The control group consisted of unseeded cells. After co-culturing for 0, 24, and 48 hours, the CCK-8 reagent was added to the wells of each group, and incubation continued for 2 hours. The absorbance of each sample at 450 nm was measured using a microplate reader. The results of the CCK-8 assay clearly showed that, within 24-48 hours, rat BMSCs exhibited stronger proliferative capacity than control cells when the concentration of M2D-Exo ranged from 25 μg / ml to 100 μg / ml, and the proliferative capacity of rat BMSCs increased with increasing concentration, reaching its peak at a concentration of 100 μg / ml (see [link to CCK-8 assay]). Figure 4 (Figure A in the middle)

[0115] The effect of different concentrations of M2D-Exo (0 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL) on the proliferation of rat BMSCs was detected using an EdU detection kit from Acinetobacter. EdU staining was performed using an EdU proliferation staining kit. First, rat BMSCs were seeded onto cell slides pre-placed in 24-well plates. 6000 cells were seeded on each slide. After cell attachment, rat BMSCs were treated with different concentrations of M2D-Exo (0 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL) for 24 hours. Then, the culture medium was replaced with 10 μM EdU solution, and the cells were cultured at 37°C for another 4 hours. Next, the cells were fixed with 4% paraformaldehyde for 15 minutes. Finally, they were washed three times with bovine serum albumin (BSA) working solution. Cells were then permeated with PBS containing 0.5% Triton X-100 for 20 minutes. The cells were washed twice with BSA working solution. The reaction mixture (Click-iT reaction mix, a reagent included in an EdU detection kit) was added, and the cells were incubated at room temperature in the dark for 30 minutes. The cells were washed once with BSA working solution. Finally, the nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI) for 15 minutes. Fluorescence images of the samples were captured using a Nikon fluorescence microscope, as shown below. Figure 4 B and Figure 4 C.

[0116] The results of EdU analysis showed that the number of BMSCs in EdU-positive rats was greater than that in the control group after treatment with M2D-Exo. Furthermore, the proliferative capacity of rat BMSCs gradually increased with increasing concentration, reaching its peak at a concentration of 100 μg / ml (see [link to study]). Figure 4 (Figure B in the middle). In summary, M2D-Exo can promote the proliferation of rat BMSCs, and the proliferation capacity of rat BMSCs gradually increases with the increase of M2D-Exo concentration, with the strongest effect at a concentration of 100 μg / ml.

[0117] Figure 4 In the diagram, control represents the control group, Merge represents a combined image of proliferating cells and total cells, Edu represents / illustrates the color of proliferating cells, and DNA represents / illustrates the cell nucleus. Figure 4 The middle graph (C) represents / illustrates the percentage of proliferating cells in the total number of cells in each group, as well as the differences between each group.

[0118] The effects of M2D-Exo on rat BMSCs migration were detected by scratch assay and Transwell migration / invasion assay, and the migration mechanism was explored by RT-qPCR.

[0119] Scratch assay: Rat BMSCs were seeded into 6-well plates and cultured at 37°C with 5% CO2. The seeding density was 2 x 10⁵ cells per well. The experiment was performed when the cell density reached 80%-90%. A horizontal line was drawn on the bottom of the 6-well plate, and vertical scratches were made in the wells using a 200 μL sterile pipette tip. The scraped suspended cells were gently washed away with PBS, and the cells were further cultured in cell culture medium (2% FBS) containing different concentrations of M2D-Exo (0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL). The cells were observed and photographed under a microscope at 0 and 24 hours. Figure 5 A. Measure and analyze the size of the scratches.

[0120] Transwell migration / invasion assay: Rat BMSCs were seeded at a density of 2 × 10⁴ cells per well in the upper chamber of a 24-well Transwell plate and cultured in fresh α-MEM medium from Gibco without fetal bovine serum (FBS). Simultaneously, 600 μL of α-MEM (2% FBS) containing different concentrations of M2D-Exo (0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL) was added to the lower chamber of the 24-well Transwell plate, and the plates were cultured for another 24 hours. Then, the upper chambers were removed from the 24-well Transwell plates, and the cells on the bottom membrane were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The cells were observed and photographed under a microscope. Figure 5 As shown. Each group has 3 replicates at each time point.

[0121] Scratch and Transwell assays showed that M2D-Exo promoted the migration of rat BMSCs, and the migrating effect increased with increasing concentration (see [link to assay]). Figure 5 A, 5C, 5D).

[0122] After co-culturing BMSCs with M2D-Exo (100 μg / mL) for 3 days, BMSCs cultured in ordinary medium were used as a control. Cells were collected and subjected to RT-qPCR experiments. The results showed that, compared with the control group, the solution containing M2D-Exo (100 μg / mL) upregulated the expression of CXCR4, MMP-2, PDGFR, and UPA, suggesting that M2D-Exo can strongly promote the migration of rat BMSCs by upregulating the expression of migration-related genes (see...). Figure 5 B).

[0123] Figure 5The graph shows the effect of different concentrations (0 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml) of M2D-Exo on the migration of BMSCs. Figure A shows the results of the scratch assay, for example, the three graphs from the 0h control group. Figure 1 In the series of images, the left and right images (on either side of the yellow dashed line in each image) represent BMSCs seeded into a six-well plate, while the middle image (the area between the two yellow dashed lines) represents a blank area created using a 200 μL sterile pipette tip, used to observe the migration of BMSCs towards the blank area at different time points. Image B shows the RT-qPCR results for migration-related genes chemokine receptor 4 (CXCR4), matrix metalloproteinase-2 (MMP-2), platelet-derived growth factor receptor (PDGFR), and urokinase-type plasminogen activator (UPA). Image C shows the Transwell assay results, and image D shows the Transwell assay statistical quantification results.

[0124] Example 5: Effect of M2D-Exo on chondrogenic differentiation of rat BMSCs

[0125] Rat bone marrow mesenchymal stem cells (BMSCs) were seeded onto tissue culture plates and chondrogenic induction medium containing M2D-Exo (100 μg / mL) was added. Chondrogenic induction medium alone served as a control. After co-culturing in both media for 21 days, the expression of cartilage metabolism-related genes (Col2, ACAN, SOX9) in the culture mixture was detected by RT-qPCR. The results showed that M2D-Exo significantly upregulated the expression of chondrogenesis-specific genes (ACAN, SOX9, COL2) (see [link to relevant data]). Figure 6 ). Figure 6 The figure shows the effect of M2D-Exo (100 μg / ml) on chondrogenic differentiation of BMSCs. The figure also shows the RT-qPCR results of chondrogenic-related genes proteoglycan (ACAN), SOX9, and type II collagen (COL II).

[0126] Example 6: Effects of M2D-Exo on inflammatory factors and macrophage polarization

[0127] To investigate the effects of M2D-Exo on inflammatory factors and macrophage polarization, RT-qPCR, flow cytometry, and immunofluorescence staining were performed.

[0128] LPS group: BMDMs were treated with medium containing 100 ng / ml LPS for 24 hours; LPS / M2D-Exo group: BMDMs were treated with medium containing 100 ng / ml LPS and 100 μg / ml M2D-Exo for 24 hours. After the above treatments were completed, BMDMs were collected, total RNA was extracted from BMDMs, and then RT-qPCR analysis was performed.

[0129] The results showed that the expression of iNOS, TNF-α, and IL-1β in the LPS group and the LPS / M2D-Exo group was significantly higher than that in the negative control group (Control group), but the expression of iNOS, TNF-α, and IL-1β in the LPS / M2D-Exo group was significantly lower than that in the LPS group (see...). Figure 7 A).

[0130] Flow cytometry analysis of BMDMs treated with LPS and LPS / M2D-Exo groups revealed that CD86 expression in M1 phenotype macrophages was inhibited by M2D-Exo (see [link to flow analysis]). Figure 7 B).

[0131] BMDMs treated with LPS and LPS / M2D-Exo were labeled with CD86 (M1 label) via immunofluorescence staining. The expression of M1 macrophages in different groups was directly observed using a Nikon fluorescence microscope. The results showed that LPS induced high CD86 expression, while the addition of M2D-Exo decreased CD86 expression (see...). Figure 7 E).

[0132] The above results indicate that the addition of M2D-Exo can inhibit the release of LPS-induced inflammatory factors and play an anti-inflammatory role.

[0133] After treating BMDMs with IL-4 (20 ng / ml) and M2D-Exo (100 μg / ml) for 24 hours, with BMDMs treated with simple culture medium as a control, cells from different groups were collected, total RNA was extracted, and RT-qPCR experiments were performed. RT-qPCR analysis showed that although the expression of ARG, IL-10, PPARγ, and CD206 in the M2D-Exo group was significantly lower than that in the positive control group (IL-4 group), the expression of ARG, IL-10, PPARγ, and CD206 in the M2D-Exo group was significantly higher than that in the negative control group (Control group) (see...). Figure 7 C).

[0134] Flow cytometry analysis showed that M2D-Exo promoted the expression of M2 macrophage phenotype (CD163)-related markers (see...). Figure 7D). Immunofluorescence staining was used to analyze the expression of CD206 (M2 marker). Results showed that although CD206 expression in the M2D-Exo group was significantly lower than in the IL-4 group, it was significantly higher than in the negative control group (Control group) (see...). Figure 7 F).

[0135] Figure 7 The results show the effects of M2D-Exo (100 μg / ml) on inflammatory factors and macrophage polarization. A shows the RT-qPCR results of inflammatory factors iNOS, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). B shows the flow cytometry results of M1 macrophages. C shows the RT-qPCR results of anti-inflammatory factors arginase 1, interleukin-10 (IL-10), peroxisome proliferator-activated receptor γ (PPARγ), and the M2 macrophage marker CD206. D shows the flow cytometry results of M2 macrophages. E and F show the immunofluorescence results of macrophage polarization towards the M1 (CD86) and M2 (CD206) phenotypes, respectively. G and H show the quantitative statistical results of E and F, respectively. These results indicate that M2D-Exo can promote macrophage polarization towards the M2 phenotype in vitro.

[0136] Example 7: Effect of M2D-Exo on the repair of osteochondral defects in rats

[0137] To investigate the effect of M2D-Exo on the repair of osteochondral defects in rats, a rat knee osteochondral defect model was established. The rats were divided into two groups: a simple defect group (PBS group) and an M2D-Exo treatment group. The specific procedures were as follows: Six-week-old rats (250+50g) were anesthetized, routinely shaved, disinfected, and draped. The knee joint cavity was opened, and a 2mm diameter, 1.5mm deep osteochondral defect was created at the trochlea using a 2mm corneal burr. The joint cavity was then closed. For the simple defect group, approximately 30μl of PBS was injected into the joint cavity; for the M2D-Exo treatment group, approximately 30μl of M2D-Exo (100μg / mL) was injected into the joint cavity. The skin incision was then sutured. Injections were administered every 7 days post-surgery, for a total of four injections. At 12 weeks post-surgery, the rats were euthanized, and the knee joints were collected for biometric evaluation. Overall results showed that at 12 weeks post-surgery, the defect area in the M2D-Exo treatment group was significantly smaller than that in the PBS group, and the degree of granulation tissue filling was significantly higher in the M2D-Exo treatment group than in the PBS group (see...). Figure 8 ). Figure 8 Gross images of articular cartilage defect repair 12 weeks post-surgery in the simple defect group (PBS group) and the M2D-Exo treatment group (M2D-Exo group). These results indicate that M2D-Exo can promote the repair of osteocartilage defects in rats.

[0138] This application investigated the effects of M2D-Exo on the biological function of rat bone MSCs, finding that it significantly promoted BMSC proliferation, migration, and chondrogenic differentiation. Simultaneously, in terms of anti-inflammatory effects and macrophage polarization, M2D-Exo significantly inhibited inflammation and promoted macrophage polarization towards the M2 type. This application also investigated the effects of M2D-Exo on the repair of osteochondral defects, finding that it promoted the repair of osteochondral bone in the rat knee joint.

[0139] Example 8: Preparation of a cartilage injury treatment agent

[0140] The specific steps for preparing a cartilage injury treatment formulation containing hydrogel and M2D-Exo include:

[0141] Take a certain amount of GelMA powder and dissolve it in PBS (pH=7.0) to prepare a 200 ng / L GelMA solution;

[0142] Exosomes derived from M2 macrophages prepared in Example 2 were used;

[0143] 100 μg of exosomes derived from M2 macrophages were co-incubated with GelMA solution at 37°C for 36 h; then 0.05 g of LAP photoinitiator (final concentration 0.25%) was added, and finally, the mixture was irradiated with ultraviolet light for 10 seconds to prepare M2D-Exo GelMA hydrogel.

[0144] Exosomes derived from M2 macrophages prepared in Example 2 and the cartilage injury treatment preparation prepared in Example 8 were used to treat damaged cartilage in the knee joint of rats. Twelve weeks post-surgery, it was observed that the M2D-Exo-containing GelMA hydrogel showed better repair effects on cartilage defects in rats than M2D-Exo alone. Figure 9 .

[0145] The above description is merely a specific implementation of this application. Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the specific working processes of the systems, modules, and units described above can be referred to the corresponding processes in the foregoing method embodiments, and will not be repeated here. It should be understood that the protection scope of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the protection scope of this application.

Claims

1. The use of isolated and purified M2 bone marrow macrophage-derived exosomes in the preparation of a cartilage injury treatment agent, wherein the cartilage injury treatment agent comprises 50-100 μg / mL of isolated and purified M2 bone marrow macrophage-derived exosomes, and an excipient; wherein, The excipient is a hydrogel, and the M2 bone marrow macrophage-derived exosomes are bound in a three-dimensional network of the hydrogel. The hydrogel comprises a cross-linked gel matrix polymer and water, wherein the gel matrix polymer is selected from cross-linked gelatin and cross-linked polyethylene glycol-poly(lactic acid)-poly(lactic acid).

2. The use according to claim 1, characterized in that, The cartilage injury treatment formulation also includes at least one of sodium hyaluronate, platelet-rich plasma, mesenchymal stem cells, glucosamine, and chondroitin sulfate.

3. The use according to claim 1, characterized in that, The cartilage injury treatment formulation also includes a solvent.

4. The use according to claim 3, characterized in that, The solvent is selected from triethanolamine, phosphate buffer solution, NaOH, or a combination thereof.

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