Small-molecule exosome-loaded composition based on bone marrow mesenchymal stem cells and application of small-molecule exosome-loaded composition

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a small-molecule exosome-loaded composition based on bone marrow mesenchymal stem cells and application of the small-molecule exosome-loaded composition. The invention discloses the treatment effect of squalene on osteoarthritis for the first time, and further prepares the squalene-loaded bone marrow mesenchymal stem cell exosome. An in-vitro cell experiment and a model animal experiment prove that the composition has a synergistic treatment effect on osteoarthritis. In-vitro tests prove that the squalene-loaded mesenchymal stem cell exosome can improve the survival rate of inflammatory cartilage cells, inhibit release of proinflammatory factors and restore expression of repair factors. Model animal experiments prove that the squalene-loaded mesenchymal stem cell exosome has the effects of resisting inflammation, easing pain and protecting cartilage in the treatment of osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a small molecule exosome composition based on bone marrow mesenchymal stem cells and its application. Background Technology

[0002] Osteoarthritis (OA), the most common chronic degenerative joint disease worldwide, is characterized by progressive degradation of articular cartilage, subchondral bone remodeling, synovial inflammation, and osteophyte formation. Its pathological changes are not limited to a single tissue but affect the entire joint structure, including cartilage, synovium, meniscus, ligaments, and subchondral bone, ultimately leading to joint pain, limited mobility, and even functional disability, severely impacting patients' quality of life. Epidemiologically, OA exhibits a significant age-related pattern, affecting over 300 million people globally, with a prevalence exceeding 40% in those over 70 years of age, making it one of the leading causes of disability in the elderly. Besides age, obesity, joint trauma, genetic factors, and long-term abnormal mechanical stress are also important risk factors—obesity accelerates cartilage wear by increasing joint load, joint trauma (such as anterior cruciate ligament injury) can directly undermine joint structural stability, and genetic factors may increase individual susceptibility by affecting the expression of genes related to cartilage matrix synthesis and metabolism. It is worth noting that the incidence of osteoarthritis (OA) differs between the sexes. Postmenopausal women have a significantly higher incidence of OA than men of the same age due to decreased estrogen levels and weakened cartilage protection. The disease also progresses more rapidly in women. This phenomenon is closely related to the sensitivity of chondrocytes to hormonal changes and the imbalance in the regulation of inflammatory responses.

[0003] The pathogenesis of osteoarthritis (OA) is extremely complex, involving multiple disturbances at the cellular, molecular, and tissue levels, with the core being the disruption of joint microenvironment homeostasis. Articular cartilage, a major site of OA lesions, primarily functions to cushion the mechanical impact of joint movement. It is composed of chondrocytes and an extracellular matrix (ECM) rich in type II collagen and proteoglycans. In the early stages of OA, microcracks appear on the surface of articular cartilage due to abnormal mechanical loading or damage. At this time, chondrocytes initiate a compensatory repair response, maintaining ECM homeostasis by increasing the synthesis of type II collagen and proteoglycans. However, as the damage persists or the underlying cause remains unresolved, chondrocytes gradually exhibit phenotypic changes. Chondrocytes, especially those with clonal clusters, abnormally express cytokines (such as tumor necrosis factor-α, TNF-α) and chemokine receptors. Simultaneously, they secrete large amounts of matrix metalloproteinases (MMPs, such as MMP-1, MMP-3, and MMP-13) and matrix-degrading enzymes like integrin-like metalloproteinase 5 (ADAMTS5), which contains platelet-sensitive motifs. These enzymes sequentially degrade type II collagen and proteoglycans in the ECM, leading to the destruction of the cartilage matrix structure. The degradation products of the ECM further activate the catabolic pathways of chondrocytes, creating a vicious cycle of degradation-inflammation-re-degradation. Simultaneously, the anabolic function of chondrocytes gradually declines, with reduced synthesis of type II collagen and proteoglycans, resulting in a loss of ECM repair capacity. Ultimately, this leads to fibrosis, detachment, and even full-thickness defects on the cartilage surface, causing the loss of its original smooth and elastic structure.

[0004] Besides cartilage lesions, synovial inflammation and subchondral bone remodeling also play crucial roles in the progression of osteoarthritis (OA). As the inner lining of the joint capsule, the synovium becomes congested and edematous in the early stages of OA. Activated synovial cells (such as synovial fibroblasts) secrete large amounts of pro-inflammatory factors (such as interleukin-1β and TNF-α). These factors not only directly stimulate nerve endings and cause pain but also diffuse to cartilage tissue via blood circulation or synovial fluid, accelerating chondrocyte apoptosis and ECM degradation. Macrophages in the synovium exhibit functional polarization in the inflammatory response: M1 macrophages secrete pro-inflammatory factors and degrading enzymes, exacerbating inflammatory damage; while M2 macrophages exert a repairing effect by secreting anti-inflammatory factors (such as IL-10). In OA, the M1 / M2 macrophage balance is disrupted, with the pro-inflammatory phenotype dominating, further amplifying synovial inflammation. Subchondral bone, as the supporting structure of cartilage, undergoes pathological changes earlier than visible cartilage damage. Early stages involve accelerated bone resorption, thinning and fracture of trabeculae, and decreased bone density, leading to a loss of stable support and increased susceptibility to mechanical damage. Later stages involve imbalanced bone remodeling, abnormally active bone formation, thickening and sclerosis of the subchondral bone plate, accompanied by osteophyte formation. While osteophytes attempt to maintain joint stability to some extent, they further compress the joint space, restrict joint movement, and exacerbate pain. Furthermore, the meniscus, acting as a "buffer" within the joint, undergoes fibrosis, tearing, and cell cluster formation during osteoarthritis (OA), resulting in a loss of its load-bearing distribution function, leading to localized pressure concentration in the cartilage and accelerating cartilage degeneration.

[0005] At the molecular level, the abnormal activation or inhibition of multiple signaling pathways regulates the pathological process of osteoarthritis (OA). The Wnt / β-catenin signaling pathway is crucial for cartilage development and homeostasis. Overactivation of this pathway in OA promotes the differentiation of chondrocytes into a hypertrophic phenotype, increases the expression of degradative enzymes such as MMP-13, and accelerates cartilage degeneration. The mitogen-activated protein kinase (MAPK) pathway (including p38, ERK, and JNK isoforms) can regulate the inflammatory response and apoptosis of chondrocytes by phosphorylating downstream transcription factors after being activated by inflammatory factors. The transforming growth factor-β (TGF-β) / bone morphogenetic protein (BMP) signaling pathway has a dual role. Moderate activation in the early stage can promote chondrocyte proliferation and ECM synthesis, while in the late stage, it may participate in osteophyte formation due to pathway imbalance. In addition, non-coding RNAs (such as microRNAs and long non-coding RNAs) also affect the progression of OA by regulating the expression of target genes. For example, miR-140-5p can reduce cartilage matrix degradation by inhibiting ADAMTS5 expression, while its expression level is significantly reduced in the OA state, resulting in weakened cartilage protection.

[0006] Currently, the treatment of osteoarthritis (OA) primarily focuses on relieving symptoms and slowing progression; there is no radical cure that can completely reverse the disease. Clinical treatment strategies can be divided into two main categories: non-surgical and surgical treatments. Non-surgical treatment is the first-line intervention for OA, including lifestyle interventions, medication, and physical therapy. Among lifestyle interventions, weight management reduces cartilage wear by decreasing joint load and is suitable for obese patients. Appropriate exercise therapy (such as low-impact exercises like swimming and cycling) can strengthen the muscles around the joints and improve joint stability, but exercises that increase joint load, such as long-distance running and climbing stairs, should be avoided.

[0007] Drug treatment primarily focuses on symptomatic relief. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most commonly used medications. They reduce prostaglandin synthesis by inhibiting cyclooxygenase (COX) activity, thereby relieving pain and inflammation. However, long-term use may cause gastrointestinal damage (such as gastric ulcers and bleeding) and cardiovascular risks (such as thrombosis). Acetaminophen has a relatively high gastrointestinal safety profile, but its analgesic effect is weak, making it only suitable for patients with mild pain. Intra-articular injection of sodium hyaluronate can increase the viscoelasticity of synovial fluid, improve joint lubrication, and provide short-term pain relief, but its long-term efficacy is controversial. Intra-articular injection of glucocorticoids can quickly control acute inflammatory attacks, but it inhibits chondrocyte metabolism and accelerates cartilage degeneration, making repeated use inadvisable. Physical therapy, such as ultrasound and shockwave therapy, can relieve symptoms by promoting local blood circulation and reducing inflammation and edema, but individual efficacy varies considerably.

[0008] When non-surgical treatments fail and patients experience persistent, severe pain and significant loss of joint function, surgical treatment should be considered. Cartilage repair surgeries include subchondral microfracture, autologous chondrocyte transplantation (ACI), and autologous matrix-induced chondrogenesis (AMIC). These surgeries stimulate the proliferation of endogenous repair cells or transplant in vitro cultured chondrocytes to achieve local repair of cartilage defects. However, they are only suitable for localized cartilage damage in young patients and have limited effectiveness for extensive cartilage degeneration. Joint replacement surgery (such as total knee replacement) is the ultimate treatment for end-stage osteoarthritis (OA). By implanting an artificial joint prosthesis to replace the diseased joint, it can significantly improve pain and function. However, surgery carries risks of complications such as infection, prosthesis loosening, and damage to surrounding nerves and blood vessels. Furthermore, the lifespan of the prosthesis is limited (usually 10-15 years), and young patients may face the need for revision surgery. In addition, surgical treatment presents a heavy economic burden; not only are the surgical costs high, but postoperative rehabilitation and management of potential complications place a significant burden on patients' families and the social healthcare system.

[0009] The limitations of traditional treatments have driven exploration in the field of regenerative medicine. Mesenchymal stem cells (MSCs) have become a hot topic in OA treatment research due to their multi-lineage differentiation potential and immunomodulatory properties. Early studies, using a rabbit anterior cruciate ligament transection (ACLT) model, demonstrated that intra-articular injection of adipose-derived mesenchymal stem cells (ASCs) can significantly delay OA progression. ASCs can differentiate into chondrocyte-like cells to participate in cartilage repair, while secreting growth factors such as transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein 4 (BMP-4), promoting cartilage matrix synthesis and inhibiting MMP activity. However, MSC therapy faces several bottlenecks: cell senescence and decreased genetic stability may occur during in vitro culture, increasing the risk of tumorigenesis; the retention rate of cells in the joint after infusion is low, with most cells being lost with the synovial fluid circulation; in addition, standardization of cell sources, ethical controversies, and the risk of immune rejection (although MSCs have low immunogenicity, xenografting may still trigger local immune responses) all limit its clinical translation.

[0010] As research has deepened, scholars have discovered that the therapeutic effect of MSCs does not depend on cell differentiation, but is achieved through extracellular vesicles (EVs) released by paracrine function. Among them, exosomes, as membrane vesicles with a diameter of 30-150 nm in EVs, have become a new carrier for "cell-free therapy" that can replace MSCs because they carry bioactive substances such as proteins, lipids, microRNAs (miRNAs), and long non-coding RNAs (lncRNAs).

[0011] MSC-derived exosomes (MSC-Exos) exhibit multidimensional therapeutic effects in OA treatment, with their core mechanism being the regulation of joint microenvironment homeostasis.

[0012] In terms of cartilage repair, MSC-Exos can target and regulate chondrocyte function by delivering miRNAs (such as miR-140-5p and miR-320c): miR-140-5p can inhibit ADAMTS5 expression and reduce proteoglycan degradation; miR-320c promotes chondrocyte proliferation and type II collagen synthesis by upregulating SOX9 (a key transcription factor for chondrocyte differentiation). Studies have shown that in an IL-1β-induced OA-like chondrocyte model, bone marrow MSC-Exos can increase type II collagen expression by more than 40% while reducing MMP-13 expression by 50%, significantly reversing the catabolic phenotype of chondrocytes; in a rabbit ACLT model, intra-articular injection of MSC-Exos can increase cartilage thickness, reduce the fibrosis index, and reduce the Laverty score (an indicator for assessing the degree of cartilage damage) by 30%-40% compared with the control group, and the cartilage surface exhibits a smooth, hyaline cartilage-like structure with significantly restored proteoglycan content.

[0013] In terms of anti-inflammation, MSC-Exos exerts its effects by regulating macrophage polarization. Molecules such as miR-129-5p and miR-146a carried by MSCs can inhibit the TLR4 / NF-κB signaling pathway, reducing the secretion of pro-inflammatory factors such as TNF-α and IL-1β by M1 macrophages, while simultaneously promoting M2 macrophage polarization and increasing the release of anti-inflammatory factors such as IL-10, thereby alleviating synovial inflammation. In a mouse collagenase-induced osteoarthritis (CIOA) model, the MSC-Exos treatment group showed significantly reduced synovial tissue thickening, decreased inflammatory cell infiltration, and a more than 60% reduction in TNF-α concentration in the synovial fluid compared to the model group. Furthermore, the proliferation and migration of synovial fibroblasts were significantly inhibited, effectively preventing the "spread" of synovial inflammation to cartilage tissue. In addition, MSC-Exos can regulate subchondral bone remodeling by delivering protein factors such as BMP-4, promoting trabecular bone reconstruction, restoring the supporting function of subchondral bone, and inhibiting osteoclast activity to prevent excessive bone resorption and maintain a balance between bone formation and resorption.

[0014] MSC-Exos from different sources exhibit subtle differences in OA treatment, which are closely related to the tissue characteristics of the cell origin. Bone marrow MSC-Exos (BM-MSC-Exos) are rich in miR-92a-3p and can regulate chondrocyte differentiation by targeting and inhibiting Wnt5a expression, showing significant effects in cartilage defect repair. Adipose tissue MSC-Exos (AT-MSC-Exos), carrying DKK-1 (a Wnt pathway inhibitor), are more advantageous in inhibiting osteophyte formation. Umbilical cord MSC-Exos (UC-MSC-Exos) are widely available and non-invasive to obtain. The lncRNA H19 they carry can improve early OA symptoms by promoting chondrocyte metabolism, and they have the lowest immunogenicity, making them suitable for xenograft transplantation. In addition, synovial MSC-Exos (SMSC-Exos), due to their homology with joint tissue, have a longer retention time in the joint. The miR-155-5p they carry can reduce chondrocyte differentiation into osteoblasts by inhibiting Runx2 expression, further highlighting the personalized potential of MSC-Exos source selection.

[0015] To overcome the limitations of natural MSC-Exos (such as low yield, insufficient targeting, and limited function), researchers have developed various engineering modification strategies. Regarding cargo loading, pretreatment of parental MSCs (e.g., hypoxic culture, stimulation by inflammatory factors) can regulate the contents of exosomes—exosomes secreted by MSCs cultured under hypoxic conditions show increased expression of miR-210, significantly enhancing its ability to promote chondrocyte survival and angiogenesis. Furthermore, genetic engineering techniques (such as lentiviral transfection) can overexpress specific miRNAs (e.g., miR-140-5p) in MSCs, increasing the content of this molecule in exosomes by more than 10-fold, further strengthening the chondrogenic effect. Surface modification strategies achieve precise delivery by attaching targeted ligands (such as chondrocyte-binding peptides CAP and RGD peptides) to the surface of Exos. CAP-modified AT-MSC-Exos can bind to specific receptors on the surface of chondrocytes, increasing the retention rate in the joint by 3 times and penetrating deep cartilage tissue, significantly improving the repair effect of full-thickness cartilage defects. RGD-modified Exos can target and bind to integrin αvβ3 in the synovial inflammation area, enhancing the regulatory effect on synovial macrophages. In terms of production and delivery system optimization, 3D culture technology (such as hollow fiber bioreactors) can increase the yield of MSC-Exos by 7.5 times compared with traditional 2D culture, and significantly increase the content of chondrocyte protection-related proteins (such as TGF-β1) in exosomes. Combining Exos with biomaterials (such as gelatin methacrylamide hydrogel and decellularized cartilage matrix scaffolds) can construct a "slow-release system" to avoid the rapid clearance of Exos and prolong its action time in the joint. BM-MSC-Exos loaded with GelMA hydrogel can be continuously released for 21 days, so that the cartilage repair effect can be maintained for up to 24 weeks, and the biomechanical properties of the newly formed cartilage are close to those of normal cartilage.

[0016] Looking ahead, OA treatment is shifting from "symptomatic relief" to "disease modification." MSC-Exos, with their low immunogenicity, high bioactivity, and controllability, are expected to become one of the first "disease-modified OA therapies" to achieve clinical translation. With the combination of gene editing technologies (such as CRISPR / Cas9) and exosome engineering, "customized" Exos can be constructed in the future—loaded with specific therapeutic molecules based on the patient's pathological subtype of OA (such as inflammation-dominated or cartilage degradation-dominated) to achieve precision treatment. Simultaneously, high-throughput analysis of the Exos's cargo components using artificial intelligence technology is expected to discover new therapeutic targets, providing entirely new insights into the mechanism research and treatment of OA.

[0017] Although clinical research on MSC-Exos is still in its early stages (mostly Phase I / II clinical trials), existing studies have shown that in patients with knee osteoarthritis (OA), a single injection of BM-MSC-Exos (2 mL ExoFlo product) for 6 months resulted in a reduction of over 50% in the Visual Analogue Scale (VAS) for pain and a 40% improvement in the Knee Function Outcome Scale (KOOS), without any serious adverse reactions. This lays the foundation for subsequent large-scale clinical trials. It is believed that with continuous technological breakthroughs, exosomes will eventually bring hope for reversing disease progression to OA patients, propelling OA treatment into a new era of "cell-free regeneration."

[0018] Squalene, an important natural lipid compound, has a distinctive chemical structure. Its chemical name is 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosanehexaene, and its molecular formula is C2. 30 H 50 Its molecular weight is 410.72. Its chemical structure is as follows:

[0019] .

[0020] From a molecular structure perspective, squalene belongs to the triterpenoid class of compounds. Its molecule contains six isoprene units, forming its unique carbon skeleton. Six double bonds are distributed along this carbon skeleton, all of which are in the trans configuration. This specific arrangement of double bonds not only determines the chemical stability of squalene but also significantly influences its subsequent physiological activity and chemical reactions. The linear nature of its molecular structure allows it to exhibit specific spatial conformations when interacting with other molecules, laying the structural foundation for its various functions within living organisms.

[0021] From a physicochemical perspective, squalene exhibits a series of relatively stable and easily identifiable properties. At room temperature and pressure, pure squalene is typically a colorless or pale yellow transparent oily liquid with a faint, characteristic odor, but no off-odors. Its density is relatively low, approximately 0.855-0.865 g / cm³. 3With a refractive index ranging from 1.495 to 1.505 (at 20℃), these physical parameters make squalene a valuable reference for substance identification and purity testing. Squalene exhibits good lipid solubility, dissolving in various organic solvents such as diethyl ether, chloroform, petroleum ether, and benzene, but has extremely low solubility in water and highly polar solvents like methanol and ethanol. This solubility characteristic determines its application in pharmaceuticals and cosmetics, for example, it is often used as an oil phase component in the preparation of emulsions and ointments. Regarding chemical stability, squalene, due to the presence of multiple double bonds in its molecule, is easily oxidized by factors such as oxygen, light, and high temperatures, generating peroxides, aldehydes, ketones, and other oxidation products, leading to a decline in its quality. Therefore, during the storage and processing of squalene, measures such as light protection, sealing, low temperature, and the addition of antioxidants are typically required to slow down its oxidation and ensure its quality stability.

[0022] In existing pharmacological activity studies, squalene has exhibited a variety of potential biological activities, attracting widespread attention from the scientific research and pharmaceutical industries. Firstly, squalene possesses significant antioxidant properties. It can reduce oxidative damage to cell membranes, proteins, nucleic acids, and other biomolecules by scavenging free radicals in the body, such as superoxide anion radicals and hydroxyl radicals, thereby maintaining normal cell structure and function, delaying the aging process, and playing a certain preventive and protective role against various diseases related to oxidative stress, such as cardiovascular diseases and nervous system diseases. Studies have shown that squalene can increase the activity of antioxidant enzymes in the body (such as superoxide dismutase and glutathione peroxidase), enhancing the body's own antioxidant defense system and further strengthening its antioxidant effect.

[0023] Secondly, squalene also exhibits certain pharmacological activity in regulating blood lipids. Numerous animal experiments and clinical studies have found that squalene can lower serum levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol (LDL-C, or "bad cholesterol"), while potentially increasing high-density lipoprotein cholesterol (HDL-C, or "good cholesterol"). Its mechanism of action in regulating blood lipids may involve multiple aspects, such as inhibiting cholesterol absorption in the intestines, promoting cholesterol metabolism and excretion in the liver, and affecting the activity of lipid metabolism-related enzymes. By regulating blood lipid levels, squalene helps prevent the occurrence and development of atherosclerosis and reduces the risk of cardiovascular and cerebrovascular diseases.

[0024] In addition, squalene also possesses certain immunomodulatory functions. It can act on the body's immune system, enhancing the activity and function of immune cells (such as lymphocytes and macrophages), promoting cytokine secretion, thereby improving the body's immune response and resistance to pathogens (such as bacteria and viruses). Studies have found that squalene can promote the proliferation and differentiation of T lymphocytes, enhance the phagocytic capacity of macrophages, and activate the cytotoxic effects of natural killer cells. These all contribute to improving the body's non-specific and specific immune functions, offering some adjunctive therapeutic benefits for individuals with weakened immune systems and patients with infectious diseases.

[0025] In addition, squalene has shown potential in anti-fatigue, liver protection, and wound healing. In terms of anti-fatigue, squalene can improve the body's energy metabolism, increase the efficiency of oxygen utilization in muscle tissue, and reduce the accumulation of fatigue substances such as lactic acid, thereby alleviating fatigue symptoms and improving exercise endurance. Regarding liver protection, squalene can reduce chemical liver damage (such as liver damage caused by drugs or alcohol), decrease the degree of hepatocyte degeneration and necrosis, and promote hepatocyte repair and regeneration. Its mechanism of action may be related to inhibiting lipid peroxidation, regulating liver enzyme indicators, and improving liver microcirculation. In promoting wound healing, squalene can promote fibroblast proliferation and collagen synthesis, accelerate the formation of granulation tissue, thereby shortening wound healing time, improving wound healing quality, and reducing scar tissue formation.

[0026] Although squalene possesses many of the aforementioned known biological activities, its specific therapeutic efficacy for osteoarthritis, and the synergistic effect produced when it is combined with bone marrow mesenchymal stem cell exosomes, are not revealed by existing technologies, which is precisely the innovation of this invention. Summary of the Invention

[0027] The applicant is dedicated to the research of natural products and has discovered that squalene can significantly enhance the therapeutic effect of exosomes on osteoarthritis.

[0028] This application first discloses the therapeutic effect of squalene on osteoarthritis.

[0029] This application further discloses the synergistic therapeutic effect of squalene-loaded bone marrow mesenchymal stem cell exosomes on osteoarthritis.

[0030] The osteoarthritis mentioned is knee osteoarthritis.

[0031] The method for preparing the squalene-loaded bone marrow mesenchymal stem cell exosomes is as follows:

[0032] Step 1: Preparation of squalene solution: Dissolve squalene (purity ≥95%) in dimethyl sulfoxide (DMSO) to prepare a 10 mmol / L stock solution;

[0033] Step 2: Electroporation Assisted Load

[0034] Take purified exosomes, resuspend them in electroporation buffer, add squalene stock solution, mix well, and transfer to an electroporation cup. Perform electroporation; immediately after treatment, place in an ice bath. Centrifuge to remove unloaded free squalene, resuspend the precipitate, and obtain electroporated squalene-loaded exosomes.

[0035] The electroporation buffer mentioned in step 2 is a sucrose-containing HEPES buffer, and its preparation method is as follows:

[0036] Step 1: Weigh out the sucrose and add it to sterile ultrapure water. Stir magnetically until completely dissolved.

[0037] Step 2: Add HEPES and continue stirring until dissolved.

[0038] Step 3: Adjust the pH of the solution to 7.2-7.4.

[0039] Step 4: Dilute the solution to volume with sterile ultrapure water and mix gently.

[0040] Step 5: Sterilize by filtration with a sterile filter membrane, and the product is obtained.

[0041] The squalene loading rate in the squalene-loaded bone marrow mesenchymal stem cell exosomes is greater than 40%.

[0042] The squalene-loaded bone marrow mesenchymal stem cell exosomes can improve the survival rate of inflammatory chondrocytes, inhibit the release of pro-inflammatory factors, and restore the expression of repair factors.

[0043] The squalene-loaded bone marrow mesenchymal stem cell exosomes play an anti-inflammatory, analgesic, and cartilage-protective role in the treatment of osteoarthritis.

[0044] The beneficial effects of this invention are:

[0045] This application first discloses the therapeutic effect of squalene on osteoarthritis. Furthermore, it describes the preparation of squalene-loaded bone marrow mesenchymal stem cell exosomes. In vitro cell experiments and animal model experiments verified their synergistic therapeutic effect on osteoarthritis (squalene and exosomes working together). In vitro experiments confirmed that the squalene-loaded bone marrow mesenchymal stem cell exosomes described in this application can improve the survival rate of inflammatory chondrocytes, inhibit the release of pro-inflammatory factors, and restore the expression of repair factors. Animal model experiments confirmed that the squalene-loaded bone marrow mesenchymal stem cell exosomes play an anti-inflammatory, analgesic, and cartilage-protective role in the treatment of osteoarthritis.

[0046] Instruction manual illustrations:

[0047] Appendix Figure 1 The effect of squalene-loaded exosomes on chondrocyte survival;

[0048] Appendix Figure 2 Effects of squalene-loaded exosomes on the levels of inflammatory factors in chondrocytes;

[0049] Appendix Figure 3 The effects of each experimental group's samples on pain and behavioral scores in osteoarthritis model animals.

[0050] Appendix Figure 4 The effects of samples from each experimental group on the levels of inflammatory factors in osteoarthritis model animals. Detailed Implementation

[0051] The present invention will be further described in detail below through embodiments. These embodiments are illustrative of the invention, but do not limit the invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0052] Example 1: Preparation of exosomes based on bone marrow mesenchymal stem cells

[0053] Step 1: Obtain BM-MSCs from the bone marrow of experimental animals (New Zealand rabbits):

[0054] Bone marrow fluid from the femur and tibia was obtained by bone marrow aspiration, diluted with physiological saline containing an anticoagulant (such as heparin), and the mononuclear cell layer was separated by density gradient centrifugation. The intermediate white membrane layer cells were collected. After counting the cells, residual red blood cells were removed with red blood cell lysis buffer, and the cells were washed 2-3 times with phosphate-buffered saline (PBS) to obtain the initial cell population of BM-MSCs.

[0055] Step 2: Culture medium preparation

[0056] The culture medium α-MEM (Minimum Essential Medium-α) containing basic nutrients was used, supplemented with 10% fetal bovine serum (FBS, which needs to be centrifuged at 100,000g for 12-16 hours in advance to remove endogenous exosomes and avoid contamination of exosomes prepared later), 100 μg / mL penicillin-streptomycin mixture, and 2 mmol / L glutamine; to enhance cell viability, 2% platelet-rich plasma (PGFEP) or 1 U / mL heparin was added.

[0057] Step 3: Primary and Subculture

[0058] The isolated BM-MSCs were seeded in culture dishes at a density of 4000 cells / cm² and cultured in an incubator at 37°C, 5% CO2, and saturated humidity. When the cell confluence reached 70%-80%, the cells were digested with 0.25% trypsin-EDTA and passaged. During passage, the cell density was adjusted to 2000 cells / cm², and the second-generation cells were selected for exosome preparation.

[0059] Step 4: Collection of conditioned medium

[0060] When BM-MSCs reach 80%-90% confluence, replace with exosome-free medium (α-MEM containing 3% exosome-free FBS) and continue culturing for 48-72 hours. Collect the culture supernatant (i.e., conditioned medium containing exosomes). Avoid repeatedly blowing and agitating the cells during collection to prevent cell rupture and release of impurities.

[0061] Step 5: Preliminary removal of cells and impurities

[0062] Differential centrifugation was used to pretreat the conditioned medium to remove cells, apoptotic bodies, and large debris.

[0063] Centrifuge at 300g for 10 minutes to remove intact cells; centrifuge at 2500g for 25 minutes to remove cell debris and apoptotic bodies; collect the supernatant and filter it through a 0.22μm pore size filter membrane to further remove residual large particulate impurities.

[0064] Step 6: Differential ultracentrifugation to separate exosomes

[0065] The pretreated conditioned medium was transferred to an ultracentrifuge tube and centrifuged at 18,000g for 60 minutes (4℃). The supernatant was collected. The supernatant was filtered again through a 0.22μm filter membrane to ensure the removal of residual particles. The filtered supernatant was placed in an ultracentrifuge tube and centrifuged at 100,000g for 120 minutes (4℃). After centrifugation, the supernatant was discarded, and the white precipitate at the bottom was the crude exosome. The precipitate was resuspended with a small amount of sterile PBS and centrifuged again at 100,000g for 120 minutes (4℃) to further remove protein impurities and finally obtain purified exosomes.

[0066] Example 2: Preparation of electroporation buffer solution:

[0067] Step 1: Weigh 8.55g of sucrose (molecular weight 342.3, final concentration 250mM) and add it to 80mL of sterile ultrapure water. Stir magnetically until completely dissolved.

[0068] Step 2: Add 0.238g HEPES (molecular weight 238.3, final concentration 10mM) and continue stirring until dissolved.

[0069] Step 3: Slowly adjust the pH of the solution to 7.2-7.4 using 1M NaOH or 1M HCl.

[0070] Step 4: Dilute the solution to 100 mL with sterile ultrapure water and mix gently.

[0071] Step 5: Sterilize by filtration through a 0.22μm sterile filter membrane.

[0072] Step 6: Aseptically aliquot into centrifuge tubes and store at 4°C for short-term storage (within 1 week) or at -20°C for long-term storage.

[0073] Example 3: Preparation of squalene-loaded exosomes

[0074] Step 1: Preparation of squalene solution:

[0075] Squalene (purity ≥95%) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mmol / L stock solution;

[0076] Step 2: Electroporation Assisted Load (Improving Loading Efficiency)

[0077] Take 100 μg of purified exosomes, resuspend them in 200 μL of electroporation buffer, add squalene stock solution to a final concentration of 50 μM, mix well, and transfer to an electroporation cup (0.4 cm spacing). Set the electroporation parameters: voltage 100 V, capacitance 25 μF, resistance ∞, and perform electroporation at room temperature; immediately after treatment, place in an ice bath for 10 min to restore the integrity of the exosome membrane. Centrifuge at 100,000 × g for 70 min at 4 °C to remove unloaded free squalene, and resuspend the precipitate in PBS to obtain electroporated squalene-loaded exosomes.

[0078] The squalene loading efficiency was tested to be 42.2%.

[0079] Loading rate (%) = (mass of squalene loaded into exosomes / total mass of exosomes) × 100%

[0080] Example 4: Effects of squalene-loaded exosomes on chondrocyte inflammation and apoptosis

[0081] To verify the regulatory role of squalene-loaded exosomes on interleukin-1β (IL-1β)-induced inflammatory responses (release of pro-inflammatory factors) and apoptosis (cell survival and expression of apoptosis-related proteins) in chondrocytes, and to provide cellular-level evidence for targeted therapy of knee osteoarthritis.

[0082] Cell source:

[0083] Primary culture of knee chondrocytes from newborn SD rats (1-3 days old).

[0084] Main reagents:

[0085] Inflammation inducer: IL-1β (final concentration 10 ng / mL);

[0086] Positive control drug: meloxicam (final concentration 10 μmol / L);

[0087] Culture medium: DMEM / F12 medium (containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics); serum-free DMEM / F12 medium.

[0088] Experimental steps:

[0089] Isolation and culture of primary chondrocytes:

[0090] After the newborn SD rats were sacrificed, the knee joints were aseptically dissected, muscles and ligaments were removed, cartilage tissue was cut off, and the tissue was digested with 0.25% trypsin at 37°C for 30 min, followed by digestion with 0.2% type II collagenase at 37°C with shaking for 4-6 h. Single-cell suspensions were obtained by passing the tissue through a 200-mesh sieve.

[0091] Inoculate into culture dishes and culture in DMEM / F12 medium containing 10% FBS and 1% double antibiotics in an incubator at 37°C, 5% CO2 and saturated humidity.

[0092] Change the medium every 2-3 days. When the cells reach 80%-90% confluence, digest them with 0.25% trypsin and passage them. Select P2-P3 generation cells for experiments.

[0093] After cell seeding, once the cells have adhered and fused to the culture medium to a degree of 70%-80%, they are replaced with serum-free DMEM / F12 medium and starved for 12 hours to eliminate the interference of growth factors in serum on the experimental results.

[0094] Except for the blank control group, the other 5 groups were all given IL-1β (final concentration 10ng / mL) and cultured at 37℃ and 5%CO2 for 24h to establish an inflammation model;

[0095] After the model was established, each group was given the corresponding intervention agent (Exosomes from Example 1, free squalene, meloxicam, and squalene-loaded exosomes prepared in Example 3) according to their grouping. The blank control group and the model group were given an equal volume of serum-free culture medium and cultured for another 48 hours. The specific treatment procedures are as follows:

[0096] Blank control group: Only serum-free DMEM / F12 medium was added, without inflammation induction or the addition of any drugs / exosomes, serving as a baseline control for normal chondrocytes, reflecting the levels of inflammation and apoptosis under physiological conditions.

[0097] Inflammation model group: serum-free DMEM / F12 medium + IL-1β (final concentration 10ng / mL) was added to induce inflammation. No intervention was added to establish a chondrocyte inflammation and apoptosis model, which served as the efficacy reference for each intervention group.

[0098] Exosome group: Inflammation induction (same as model group) + addition of blank exosomes (final concentration 100 μg / mL)

[0099] Squalene group: Inflammation induction (same as model group) + addition of free squalene (final concentration 50 μmol / L)

[0100] Positive control group (meloxacin): Inflammation induction (same as model group) + addition of meloxicam (final concentration 10 μmol / L), providing a reference for the efficacy of commonly used clinical drugs and verifying the effectiveness of the experimental system.

[0101] Squalene-loaded exosome group: Inflammation induction (same as model group) + addition of squalene-loaded exosomes (final exosome concentration 100 μg / mL)

[0102] After 48 hours of culture, the cell supernatant was collected (for ELISA detection of pro-inflammatory factors) and the cells were collected (for CCK-8 and apoptosis detection).

[0103] Cell viability assessment: The CCK-8 assay kit was used according to the manufacturer's instructions. 10 μl of CCK-8 reagent was added to each well, and after incubation for 2 hours, the absorbance value (450 nm) was read using a microplate reader.

[0104] Cytokine assay: The concentrations of TNF-α, IL-6, TGF-β1, and IGF-1 in the culture medium were detected using an ELISA kit (according to the manufacturer's instructions). 100 μl of sample or standard was added to the kit, incubated for 2 hours, washed, and the detection antibody was added. After another 1 hour of incubation, the sample was washed, and the substrate solution was added. After incubation in the dark for 15 minutes, the stop solution was added, and the absorbance value (450 nm) was read using a microplate reader.

[0105] Table 1. Effects of squalene-loaded exosomes on chondrocyte inflammation and apoptosis (n=6, expressed as mean).

[0106]

[0107] Cell viability directly reflects the degree of IL-1β-induced apoptosis, and the repair effects of each group showed a clear gradient:

[0108] Model control group: After IL-1β induction, the cell survival rate was only 57.2%, which was significantly lower than that of the blank control group (100.0%), confirming that the inflammation model was successfully constructed and that IL-1β can effectively induce chondrocyte apoptosis.

[0109] The repair effects of each intervention group were ranked as follows: squalene-loaded exosome group (96.8%) > positive control group (91.6%) ≈ exosome group (90.9%) > free squalene group (87.7%).

[0110] Among them, the survival rate of the drug-loaded exosome group was closest to that of the blank control group and significantly higher than that of the free drug group, indicating that exosomes as carriers can enhance the anti-apoptotic effect of squalene on chondrocytes; at the same time, the survival rate of the blank exosome group reached 90.9%, suggesting that the exosome carrier itself has a certain anti-apoptotic protective effect and good safety (no additional cytotoxicity).

[0111] TNF-α and IL-6 are core markers of chondrocyte inflammation, and the degree of reduction in their concentrations directly reflects the anti-inflammatory effect of the intervention.

[0112] In the model control group, the levels of pro-inflammatory factors were significantly increased, with TNF-α reaching 98.4 pg / ml (8.7 times that of the blank group of 11.3 pg / ml) and IL-6 reaching 188.4 pg / ml (8.8 times that of the blank group of 21.4 pg / ml), verifying that IL-1β successfully activated the inflammatory pathway.

[0113] Ranking of anti-inflammatory effects in each intervention group (taking TNF-α as an example):

[0114] Squalene-loaded exosome group (28.5 pg / ml) < exosome group (49.7 pg / ml) < positive control group (64.2 pg / ml) < free squalene group (72.7 pg / ml).

[0115] It can be seen that: ① The drug-loaded exosome group had the strongest inhibitory effect on TNF-α and IL-6, with its TNF-α concentration decreasing by 71.0% compared to the model group, and significantly lower than the positive control and free drug group, demonstrating the synergistic anti-inflammatory advantage of "drug + exosome"; ② The blank exosome group had a better anti-inflammatory effect than the clinically commonly used drug meloxicam (positive control), further confirming the anti-inflammatory potential of the exosome carrier itself; ③ Although the free squalene group could reduce pro-inflammatory factors, the effect was weaker than other intervention groups, indicating that the drug's efficacy was lower when it was not loaded with a carrier.

[0116] TGF-β1 and IGF-1 are key repair factors that maintain cartilage matrix synthesis and inhibit cell apoptosis. Their increased levels reflect the degree of recovery of chondrocyte function.

[0117] In the model control group, the expression of repair factors was significantly suppressed, with TGF-β1 decreasing to 24.3 pg / ml (a 40.0% decrease compared to the blank group of 40.5 pg / ml) and IGF-1 decreasing to 12.4 pg / ml (a 55.9% decrease compared to the blank group of 28.1 pg / ml), indicating that the self-repair ability of cartilage is impaired under inflammatory conditions.

[0118] Ranking of IGF-1 recovery effects in each intervention group:

[0119] Squalene-loaded exosome group (37.6 pg / ml) > exosome group (34.9 pg / ml) > positive control group (23.8 pg / ml) > free squalene group (17.4 pg / ml).

[0120] It is worth noting that: ① The IGF-1 concentration in the drug-loaded exosome group (37.6 pg / ml) was not only significantly higher than that in the model group, but also exceeded that in the blank control group (28.1 pg / ml), suggesting that it can not only restore cartilage repair function, but may also have additional repair enhancement effects; ② The TGF-β1 (36.8 pg / ml) and IGF-1 levels in the blank exosome group were better than those in the positive control, which further verified the repair characteristics of exosomes; ③ The free drug group had the weakest effect on the recovery of repair factors, further highlighting the value of exosome carriers in enhancing drug effects.

[0121] Exosome carriers have significant advantages: blank exosomes themselves have anti-inflammatory, anti-apoptotic and repair-promoting effects, and their effects are superior to those of meloxicam, a commonly used clinical drug, providing a new carrier option for the treatment of cartilage inflammation.

[0122] Drug-loaded exosomes exhibit outstanding synergistic effects: Squalene-loaded exosomes showed the best performance in three dimensions: "improving cell survival rate, inhibiting the release of pro-inflammatory factors, and restoring the expression of repair factors." Their effects were significantly better than those of the free drug group and the positive control group, confirming that exosomes can effectively improve the bioavailability and efficacy of squalene.

[0123] These results provide cellular-level evidence for the use of squalene-loaded exosomes for targeted therapy of knee osteoarthritis. Their combination of "carrier-borne activity + drug synergy" makes them a promising new treatment strategy superior to traditional drugs.

[0124] Example 5: The in vivo chondrogenic protective effect of squalene-loaded exosomes from Example 3 on osteoarthritis model animals.

[0125] Experimental animals: Healthy SD rats with an average weight of approximately 250 grams were selected. Under aseptic conditions, the anterior cruciate ligament (ACLT) of the right knee of the rats was surgically severed, and part of the medial meniscus was removed to establish an osteoarthritis model. After the procedure, local anesthesia was administered with 1% lidocaine. Rats were randomly assigned to groups of 10. The experimental groups and treatments are as follows:

[0126] Model control group: No treatment was performed.

[0127] Positive control group: On the second day after inducing osteoarthritis, approximately 0.1 ml of meloxicam injection was injected into the knee joint cavity (dose 2 mg / kg, prepared by dissolving meloxicam raw material in polyethylene glycol 400 to a concentration of 8 mg / ml, and the volume of administration was calculated based on the rat's body weight).

[0128] Exosome group: On the second day after induction of osteoarthritis, 0.1 ml of the exosomes prepared in Example 2 were injected into the knee joint cavity.

[0129] Squalene-loaded exosomes: On the second day after induction of osteoarthritis, 0.1 ml of the squalene-loaded exosomes prepared in Example 3 was injected into the knee joint cavity.

[0130] Pain and behavioral scores: Two weeks after the start of the experiment, rats were scored for pain and behavior (pain was scored using the Rat Osteoarthritis Pain Rating Scale, and behavior was scored using the Rat Knee Joint Function Behavior Rating Scale).

[0131] Biochemical assays: Blood samples were collected from rats two weeks after the start of the experiment. The concentrations of TNF-α, IL-6, TGF-β1, and IGF-1 in serum were measured using ELISA.

[0132] Table 2. In vivo cartilage protection effect of each experimental group on osteoarthritis in model animals (n=10, expressed as mean).

[0133] .

[0134] Pain is the core symptom of knee osteoarthritis. In the experiment, the higher the pain score, the more severe the knee pain in the rats.

[0135] The pain score in the model control group was 7.7, indicating that the rats were in a state of significant pain after modeling. The pain score in the exosome group decreased to 4.9, which was about 36.4% lower than that in the model control group, indicating that exosomes alone can effectively relieve pain caused by knee osteoarthritis. The pain score in the squalene-loaded exosome group further decreased to 4.1, which was about 46.8% lower than that in the model control group, and lower than that in the exosome group (4.9), suggesting that the combination of squalene and exosomes has a stronger effect on pain relief and can more significantly improve the pain experience of rats.

[0136] Behavioral scores reflect the knee joint function of rats; higher scores indicate less restriction of joint movement and better behavioral function.

[0137] The behavioral score of the model control group was 3.8, indicating that the rats' behavioral function was severely impaired due to joint pain and inflammation after modeling. The behavioral score of the exosome group improved to 5.8, which was about 52.6% higher than that of the model control group. This shows that exosomes can improve the rats' knee joint mobility by relieving pain and reducing inflammation, and help them restore normal behavior (such as walking and climbing). The behavioral score of the squalene-loaded exosome group further increased to 6.4, which was about 68.4% higher than that of the model control group and higher than that of the exosome group (5.8). This proves that drug-loaded exosomes have a greater advantage in improving joint function and can more effectively relieve joint movement restrictions and improve the rats' motor function.

[0138] The development of knee osteoarthritis is closely related to chronic inflammation within the joint. Tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) are core pro-inflammatory factors, and the higher their levels, the more severe the inflammatory response. Exosomes and drug-loaded exosomes can exert anti-inflammatory effects by reducing the levels of these two factors.

[0139] Regulation of TNF-α: The serum TNF-α concentration in the model control group was 158.6 pg / ml, while that in the exosome group it decreased to 61.6 pg / ml, a reduction of approximately 61.1% compared to the model control group; the squalene-loaded exosome group further decreased to 52.0 pg / ml, a reduction of approximately 67.2% compared to the model control group, and was also lower than that in the exosome group, indicating that both groups could significantly inhibit the TNF-α-mediated inflammatory response, and the drug-loaded group had a better anti-inflammatory effect.

[0140] Regulation of IL-6: The serum IL-6 concentration in the model control group was 90.9 pg / ml, while that in the exosome group it decreased to 64.4 pg / ml, a reduction of approximately 29.2% compared to the model control group; the concentration in the squalene-loaded exosome group decreased to 54.5 pg / ml, a reduction of approximately 40.0% compared to the model control group. This also demonstrates that drug-loaded exosomes have a stronger inhibitory effect on IL-6 and can more effectively reduce chronic inflammation in the joints.

[0141] Transforming growth factor-β1 (TGF-β1) and insulin-like growth factor-1 (IGF-1) are key factors that promote chondrocyte proliferation and inhibit cartilage degradation. Elevated levels of these factors help delay cartilage degeneration and promote cartilage repair.

[0142] Regulation of TGF-β1: The serum TGF-β1 concentration in the model control group was 51.9 pg / ml, while that in the exosome group increased to 75.5 pg / ml, an increase of approximately 45.5% compared to the model control group; the concentration in the squalene-loaded exosome group further increased to 83.6 pg / ml, an increase of approximately 61.1% compared to the model control group. This indicates that both groups can promote the expression of TGF-β1, providing favorable conditions for cartilage repair, and the promoting effect of the drug-loaded group is more significant.

[0143] Regulation of IGF-1: The serum IGF-1 concentration in the model control group was 50.5 pg / ml, while that in the exosome group increased to 87.6 pg / ml, an increase of approximately 73.5% compared to the model control group; the concentration in the squalene-loaded exosome group increased to 95.7 pg / ml, an increase of approximately 89.5% compared to the model control group. This indicates that both groups can significantly upregulate IGF-1 levels and enhance its protective effect on chondrocytes, while drug-loaded exosomes show better performance in activating the cartilage repair pathway.

[0144] In summary, the exosome group demonstrated a clear therapeutic effect on knee osteoarthritis in rats through multi-dimensional effects of "pain relief, functional improvement, inflammation inhibition, and repair promotion." The squalene-loaded exosome group outperformed the exosome-only group in all evaluation indicators (pain score, behavioral score, TNF-α, IL-6, TGF-β1, IGF-1), suggesting a potential synergistic effect between squalene and exosomes. Exosomes, acting as a carrier, can enhance the targeted delivery efficiency of squalene, while squalene further enhances the anti-inflammatory, analgesic, and chondrogenic effects of exosomes, ultimately achieving a superior therapeutic effect on knee osteoarthritis.

[0145] The above description of the invention and embodiments illustrates the basic principles, main features, and advantages of this patent application. Those skilled in the art should understand that this patent application is not limited to the above embodiments. The embodiments and descriptions in the specification are merely the optimal technical solutions of this patent application. Various changes and improvements may be made to this patent application without departing from the spirit and scope of this patent application, all of which fall within the scope of protection of this patent application. The scope of protection of this patent application is defined by the appended claims and their equivalents.

Claims

1. The application of squalene in the preparation of drugs for treating osteoarthritis, characterized in that, The chemical structure of the squalene is as follows: 。 2. The application of squalene-loaded bone marrow mesenchymal stem cell exosomes in the preparation of drugs for treating osteoarthritis, characterized in that, The chemical structure of the squalene is as follows: 。 3. The application as described in claim 2, characterized in that, The osteoarthritis mentioned is knee osteoarthritis.

4. The application as described in claim 2, characterized in that, The method for preparing the squalene-loaded bone marrow mesenchymal stem cell exosomes is as follows: Step 1: Preparation of squalene solution: Dissolve squalene with a purity ≥95% in dimethyl sulfoxide to prepare a 10 mmol / L stock solution; Step 2: Electroporation Assisted Load Take the purified exosomes, resuspend them in electroporation buffer, add squalene stock solution, mix well, transfer to an electroporation cup, and perform electroporation. After treatment, immediately place them in an ice bath to stand, centrifuge to remove unloaded free squalene, and resuspend the precipitate to obtain electroporated squalene-loaded exosomes.

5. The application as described in claim 4, characterized in that, The electroporation buffer mentioned in step 2 is a HEPES buffer containing sucrose.

6. The application as described in claim 5, characterized in that, The preparation method of the electroporation buffer is as follows: Step 1: Weigh out the sucrose and add it to sterile ultrapure water, then stir magnetically until completely dissolved; Step 2: Add HEPES and continue stirring until dissolved; Step 3: Adjust the pH of the solution to 7.2-7.4; Step 4: Dilute the solution to volume with sterile ultrapure water and mix gently; Step 5: Sterilize by filtration with a sterile filter membrane, and the product is obtained.

7. The application as described in any one of claims 4-6, characterized in that, The squalene loading rate in the squalene-loaded bone marrow mesenchymal stem cell exosomes is greater than 40%.

8. The application as described in any one of claims 2 or 3, characterized in that, The squalene-loaded bone marrow mesenchymal stem cell exosomes exert their effects by increasing the survival rate of inflammatory chondrocytes, inhibiting the release of pro-inflammatory factors, and restoring the expression of repair factors.

9. The application as described in any one of claims 2 or 3, characterized in that, The squalene-loaded bone marrow mesenchymal stem cell exosomes play an anti-inflammatory, analgesic, and cartilage-protective role in the treatment of osteoarthritis.

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