Drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation and application of drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation in improvement of osteoarthritis

Abstract

The invention discloses a drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation as well as a preparation method and application thereof. By constructing the medicine-loaded mesenchymal stem cell preparation, the immunosuppression capacity of the mesenchymal stem cells is improved, the angiogenesis capacity of the mesenchymal stem cells is reduced, meanwhile, by releasing the glucocorticoid medicine at a low dosage, the side effect of single administration is avoided, M2 type macrophage polarization is induced, the inflammatory state of cartilage cells is improved, and the effect of treating the cartilage cells is achieved. The curative effect of the mesenchymal stem cells on osteoarthritis is improved in multiple aspects, and the mesenchymal stem cells are proved in animal bodies. The drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation provides a new thought for the treatment of osteoarthritis.

Description

Technical Field

[0001] The present invention belongs to the field of cell engineering, and specifically relates to a drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation, which utilizes drug-receptor activation to induce mesenchymal stem cells to enhance their anti-inflammatory ability, thereby achieving the purpose of enhancing the therapeutic effect of osteoarthritis. Background Art

[0002] Osteoarthritis (OA) is a chronic degenerative disease characterized by cartilage wear and synovial inflammation, which is caused by disorders in catabolism, anabolism and inflammatory pathways. Risk factors for OA include endogenous (genetic background, aging, inflammatory mediators and obesity) and environmental (mechanical stress). Aging is the main risk factor. Cartilage extracellular matrix components (proteoglycans and type II collagen) and chondrocytes undergo age-related metabolic changes, leading to an imbalance between matrix synthesis and degradation. Various risk factors may simultaneously change the biomechanical properties of cartilage tissue, leading to pain and joint dysfunction. Currently, the commonly used methods for treating OA include non-drug adjuvant therapy, drug therapy, surgical treatment and new regenerative therapy. However, the first three are mainly aimed at alleviating the disease and improving function, while regenerative therapy has the hope of repairing damaged cartilage. Regenerative therapy includes articular cavity injection of stem cells, cartilage tissue engineering and fracture surgery, but the latter two still require surgery to complete. Stem cell therapy is an effective alternative for the treatment of chronic knee joints and osteoarthritis. The intervention is simple, does not require surgery, can relieve pain, and significantly improve cartilage quality. It is a promising method for the treatment of osteoarthritis.

[0003] Mesenchymal stem cells (MSCs) are a type of multipotent stem cell in the human body. They have the ability to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro and in vivo, and have immunoregulatory and anti-inflammatory abilities. They are currently the most important and ideal seed cells for clinical cell therapy and tissue engineering research. Mesenchymal stem cells have good immunoregulatory activity and paracrine ability, can release synthetic cytokines and directly differentiate into specific connective tissue cells, migrate to the site of injury, stimulate progenitor cell proliferation, inhibit chondrocyte apoptosis and cartilage degeneration, promote tissue repair, and will not affect the normal physiological activities of surrounding undamaged chondrocytes. They are of great significance for the treatment of osteoarthritis and cartilage defects.

[0004] Glucocorticoids are common drugs used clinically to treat osteoarthritis. However, repeated large-dose injections of glucocorticoids have adverse effects on joint growth and the growth of chondrocytes, synoviocytes, and osteoblasts. Dexamethasone is a common glucocorticoid and also a supplement for chondrocyte culture medium. At low doses, it can promote the anabolism of immature tissues and help maintain the function of mature tissues, further improving the functional integrity of cartilage.

[0005] The prior art records the systemic delivery of glucocorticoids or their mixed use with mesenchymal stem cells, but this method makes it difficult to ensure that glucocorticoids directly enter the interior of mesenchymal stem cells, which may lead to a decrease in the effectiveness of the drug. At the same time, systemic administration may lead to widespread distribution of the drug in the body, increasing the risk of potential side effects. Moreover, the simple combination of glucocorticoids and mesenchymal stem cells does not involve the introduction of glucocorticoids into mesenchymal stem cells to induce gene reprogramming, thereby improving their anti-inflammatory and immunomodulatory abilities. Due to the lack of specific gene reprogramming of mesenchymal stem cells, unstable effects may occur in clinical treatment.

[0006] Therefore, there is an urgent need to optimize and improve the existing method of combining glucocorticoids with mesenchymal stem cells to achieve the goal of improving osteoarthritis. Summary of the invention

[0007] In response to the above problems, the present invention explores the use of low-dose encapsulated glucocorticoids to induce mesenchymal stem cell gene reprogramming, enhance their immunosuppressive effect and anti-inflammatory ability, and provide a drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation with immunomodulatory and anti-inflammatory abilities.

[0008] The present invention first provides a drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation, which is a drug-guided gene-reprogrammed cell. The surface of the mesenchymal stem cell preparation has cell membrane proteins, and the mesenchymal stem cell preparation is loaded with drug liposomes co-incubated with mesenchymal stem cells; the mesenchymal stem cell preparation is beneficial for the treatment of osteoarthritis.

[0009] Preferably, the drug liposome is a glucocorticoid-loaded drug liposome.

[0010] Preferably, the glucocorticoid drug-encapsulated liposome is dexamethasone liposome.

[0011] Preferably, the mesenchymal stem cells are derived from one of umbilical cord, placenta, bone marrow and adipose tissue.

[0012] Preferably, the mesenchymal stem cell preparation expresses higher levels of anti-inflammatory factors and lower levels of pro-angiogenic factors, and is more conducive to the treatment of osteoarthritis.

[0013] Preferably, the mesenchymal stem cell preparation induces M2 macrophage polarization.

[0014] Secondly, the present invention also provides a method for preparing the drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation according to claim 1, comprising the following steps:

[0015] (1) In vitro culture of mesenchymal stem cells;

[0016] (2) Preparation of drug liposomes;

[0017] (3) Mesenchymal stem cells are co-incubated with drug liposomes to assemble into a drug-loaded liposome mesenchymal stem cell preparation.

[0018] Preferably, step (1) comprises: chopping up a tissue block derived from one of the umbilical cord, placenta, bone marrow and fat, mixing it with a digestive enzyme in a centrifuge tube, placing it in a 37°C shaker, and shaking and digesting it for 2-5 hours; centrifuging the digested tissue fluid, discarding the supernatant, and washing it twice with PBS; resuspending the precipitate in a DF-12 culture medium containing 5-12v% fetal bovine serum and 0.5-2v% Penicillin-Streptomycin, planting it in a T75 culture bottle, and culturing it at a constant temperature of 2-7% CO2 and 37°C saturated humidity, and replacing the culture medium every 3-4 days. When the cells have covered 80-90% of the bottom of the bottle, they can be passaged.

[0019] Preferably, step (2) comprises: preparing dexamethasone liposomes by a thin film dispersion method, dissolving cholesterol and dipalmitoylphosphatidylcholine (DPPC) in a chloroform / methanol volume ratio of 2:1 at a mass ratio of 35:65, adding dexamethasone at a mass ratio of 8-12%, and rotary evaporating at 45-60° C. to form a thin film, and then drying in a vacuum drying oven for 8-16 hours; hydrating with purified water at 45-60° C. for 20-40 minutes, and ultrasonicating with a probe for 5-20 minutes; and extruding through a 100-300 nm filter membrane for 5-20 times to prepare a dexamethasone liposome preparation.

[0020] Preferably, step (3) comprises: mixing the dexamethasone liposome solution with 1×10 cells 6 The mesenchymal stem cells were incubated at 37° C. and 2-7% CO2 for 8-16 hours, the supernatant was aspirated, the cells were washed 1-3 times with PBS buffer, trypsinized for 1-3 minutes, centrifuged at 500-2000 rpm for 2-8 minutes, the supernatant was discarded, and the cells were washed 2-5 times with PBS buffer to obtain a drug-loaded cell preparation of mesenchymal stem cells loaded with dexamethasone.

[0021] Finally, the present invention also provides the use of the above-mentioned drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation in the treatment of osteoarthritis, which is injected into the bone joint cavity to achieve the therapeutic effect of improving osteoarthritis.

[0022] Compared with the solutions in the prior art, the advantages of the present invention are:

[0023] First, the present invention achieves gene reprogramming of mesenchymal stem cells by co-incubating drug liposomes encapsulating low-dose glucocorticoids with mesenchymal stem cells to prepare a drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation, which significantly enhances its immunosuppressive effect and anti-inflammatory ability. The present invention introduces drug liposomes into mesenchymal stem cells, and the drug liposomes enter the lysosomes of mesenchymal stem cells, thereby enhancing the autophagy level of mesenchymal stem cells, inhibiting cell senescence, and enhancing the release of anti-inflammatory molecules, while being able to release drug liposomes through the lysosomal cycle. At the same time, compared with mesenchymal stem cells treated with pure drugs, the mesenchymal stem cell preparation introduced by drug liposomes can release higher levels of drugs in 24 hours.

[0024] Secondly, the co-incubation method of the present invention ensures that glucocorticoids can directly enter the interior of mesenchymal stem cells, thereby improving the accuracy and effectiveness of the drug's action. Moreover, after the glucocorticoids enter the MSC cells, the co-incubation method regulates the expression of MSC-specific genes, giving the cells stronger anti-inflammatory, antioxidant and anti-fibrosis properties, thereby improving the stability and consistency of MSCs.

[0025] In addition, the drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation designed by the present invention gradually releases glucocorticoids and performs gene reprogramming in MSCs, so that the anti-inflammatory activity is maintained in vivo for a long time, thereby prolonging the therapeutic effect and reducing the frequency of administration.

[0026] Finally, the drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation designed by the present invention can be used for intra-articular administration to enhance the therapeutic effect of ordinary mesenchymal stem cells and achieve the characteristics of low toxicity, low immunogenicity, high efficiency and effectiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a schematic diagram of the mesenchymal stem cell-loaded glucocorticoid drug liposome of the present invention, wherein: 1-mesenchymal stem cell; 2-glucocorticoid drug liposome.

[0028] Figure 2 It is a transmission electron microscope schematic diagram of the glucocorticoid drug liposome of the present invention.

[0029] Figure 3 This is a laser confocal image of the mesenchymal stem cell bionic drug carrier obtained by the implementation of the present invention.

[0030] Figure 4 It is a graph showing the mRNA sequencing results of the dexamethasone liposome-loaded mesenchymal stem cell preparation obtained by the implementation of the present invention and untreated mesenchymal stem cells.

[0031] Figure 5 The laser confocal image and Western Blot image of glucocorticoid receptor nuclear transcription enhancement of the dexamethasone liposome-loaded mesenchymal stem cell preparation obtained by the implementation of the present invention.

[0032] Figure 6 This is a graph showing the therapeutic effect of the dexamethasone-loaded liposome-containing mesenchymal stem cell preparation obtained by the present invention on treating osteoarthritis.

[0033] Figure 7 It is the OARSI score of the therapeutic effect of the dexamethasone liposome-loaded mesenchymal stem cell preparation obtained by the implementation of the present invention in treating osteoarthritis.

[0034] Figure 8 The present invention is an immunohistochemical diagram of MMP13 and ACAN in bone and joint tissues of osteoarthritis treated with the dexamethasone liposome-loaded mesenchymal stem cell preparation obtained by the present invention. DETAILED DESCRIPTION

[0035] The preferred embodiments of the present invention are described in further detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art:

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] Example 1: The schematic diagram of the structure of the mesenchymal stem cell preparation loaded with dexamethasone liposomes prepared in this example is as follows Figure 1 As shown, 1 is mesenchymal stem cells and 2 is glucocorticoid drug liposomes.

[0038] The preparation and characterization of the mesenchymal stem cell preparation loaded with dexamethasone liposomes includes the following steps:

[0039] (1) Preparation of dexamethasone liposomes:

[0040] Dexamethasone liposomes were prepared by thin film dispersion method. Cholesterol and dipalmitoylphosphatidylcholine (DPPC) were dissolved in chloroform / methanol (2:1, V / V) at a mass ratio of 35:65. Dexamethasone was added at a mass ratio of 10%. The mixture was rotary evaporated at 55°C to form a thin film, which was then dried in a vacuum oven for 12 hours. The mixture was hydrated with purified water at 55°C for 30 minutes and probe ultrasonicated for 10 minutes. The mixture was extruded through a 200nm filter membrane 10 times to prepare a dexamethasone liposome preparation. Liposomes are shown in Figure 2 .

[0041] (2) Culture of mesenchymal stem cells:

[0042] Take the baby's umbilical cord, wash it with PBS buffer, cut it into small pieces, peel off the blood vessels, tear it into small strips, and cut it into tissue blocks. Mix the chopped tissue blocks with digestive enzymes in a centrifuge tube, place it in a 37°C shaker, and shake and digest for 3 hours. Centrifuge the digested tissue fluid, discard the supernatant, and wash it twice with PBS. Resuspend the precipitate in DF-12 culture medium containing 10% fetal bovine serum (FBS) and 1% (volume ratio) Penicillin-Streptomycin, and plant it in a T75 culture bottle, and culture it at a constant temperature of 5% CO2, 37°C, and saturated humidity. Change the culture medium every 3-4 days, and when the cells have grown to 80-90% of the bottom of the bottle, they can be passaged.

[0043] (3) Preparation of mesenchymal stem cell preparation loaded with dexamethasone liposomes:

[0044] The dexamethasone liposome solution was mixed with 1×10 cells. 6 The mesenchymal stem cells were incubated at 37°C and 5% CO2 for 12 hours. The supernatant was removed, the cells were washed 3 times with PBS buffer, trypsinized for 2 minutes, centrifuged at 1000 rpm for 5 minutes, the supernatant was removed, and the cells were washed 3 times with PBS buffer to obtain a drug-loaded cell preparation of mesenchymal stem cells loaded with dexamethasone.

[0045] (4) Characterization of Dexamethasone-loaded Liposome Mesenchymal Stem Cell Preparations:

[0046] Laser confocal microscopy: Dexamethasone liposomes labeled with DiD were incubated with mesenchymal cells at 37°C and 5% CO2 for 12 hours, the cells were washed 3 times with PBS buffer, fixed with 4% paraformaldehyde for 10 minutes, washed 3 times with PBS buffer, and the nuclei were stained with DAPI. Laser confocal microscopy was used to measure the 405 and 630 nm channels respectively. Figure 3 The laser confocal images show that the red fluorescence-labeled dexamethasone liposomes enter the cytoplasm of mesenchymal stem cells, and the blue fluorescence labels the nucleus of mesenchymal stem cells.

[0047] (5) Cell reprogramming of mesenchymal stem cell preparations loaded with dexamethasone liposomes:

[0048] By introducing dexamethasone-loaded liposomes into mesenchymal stem cells (MSCs), and using mRNA sequencing to compare the gene expression differences between dexamethasone-loaded liposome-treated MSCs and untreated MSCs, their gene reprogramming characteristics can be revealed. The process includes the preparation and co-incubation of dexamethasone liposomes, RNA extraction and sequencing, differential gene analysis and functional enrichment analysis, and finally characterizes the reprogramming characteristics of MSCs in terms of anti-inflammatory, immune regulation and signaling pathways. This data supports the application potential of dexamethasone-loaded liposome-treated MSCs as highly effective anti-inflammatory cell therapy agents in regenerative medicine, such as Figure 4 shown.

[0049] (6) Glucocorticoid receptor nuclear transcription and related protein expression in mesenchymal stem cell preparations loaded with dexamethasone liposomes:

[0050] The mesenchymal stem cell preparation loaded with dexamethasone liposomes characterized the nuclear transcription process of glucocorticoid receptor (GR) by confocal microscopy, showing significantly enhanced GR nuclear transcription activity, indicating that dexamethasone liposomes effectively promoted the translocation of glucocorticoid receptor to the nucleus and regulated the expression of downstream target genes; in addition, in the Western blot experiment, the expression levels of cysteine-rich secretory protein-like domain 2 (CRISPLD2), heme oxygenase 1 (HMOX1), autophagy-related protein p62, cyclin-dependent kinase inhibitor p21, microtubule-associated protein 1 light chain 3 (LC3) and glucose dehydrogenase (GD) were detected. The results showed that the preparation not only enhanced the expression of anti-inflammatory molecules, but also significantly improved the autophagy ability of the cells themselves, which helped to improve the anti-inflammatory and self-protection functions of the cells, thereby enhancing their stability and therapeutic potential in the inflammatory microenvironment, such as Figure 5 shown.

[0051] Case study 2: Drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation for the treatment of osteoarthritis in rats

[0052] (1) Establishment of rat OA model:

[0053] SD rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 0.5 mL / kg. The right knee joint was shaved and disinfected with iodine. A 0.8 cm incision was made with a scalpel next to the medial patellar tendon. The joint capsule was further cut along the patellar tendon. After the patella was dislocated, the knee joint was fully flexed. The anterior cruciate ligament was cut with microscissors, the patella was repositioned, and the anterior cruciate ligament was completely cut through the anterior drawer test. The joint capsule was sutured with absorbable sutures and the skin was sutured with surgical sutures. The surgical site was disinfected with 75% alcohol. The rats were allowed to move freely after the operation. The sham operation group rats were treated the same as the OA rats except that the anterior cruciate ligament was not cut.

[0054] (2) Experimental groups and administration methods:

[0055] The model animals were randomly divided into a blank group, a sham operation group, a mesenchymal stem cell treatment group loaded with dexamethasone liposomes, a mesenchymal stem cell treatment group, and a mesenchymal stem cell treatment group loaded with blank liposomes, with 5 animals in each group. The drugs were administered by intra-articular injection after the animal model was established (1 week after surgery). Each dose group was administered once in the first, third, and fifth weeks, for a total of three times; the sham operation combined model group was given cryopreserved cell lysate in the same way. After six weeks, the rats were euthanized.

[0056] (3) Pathological examination:

[0057] The articular surface of the medial femoral condyle of rats was fixed with a 10% volume fraction paraformaldehyde solution for 48 hours, decalcified, and longitudinally embedded in paraffin. The articular surface specimen of the medial femoral condyle was divided longitudinally into 4 regions, and the cartilage tissue of the middle 2 regions was used to make 5 mm thick slices. The slices were dewaxed, dehydrated, and HE stained step by step. An independent observer performed histological scoring of the cartilage slices according to the Mankin scoring standard. The final score of each specimen was the average of the scores of the cartilage slices in the two regions, such as Figure 6 and Figure 7 shown.

[0058] (4) Immunohistochemistry:

[0059] Detection of the expression and localization of MMP-13 and ACAN in rat articular cartilage tissue. (1) Dewaxing and hydration: Place tissue sections in a 60°C oven for 2 h of dewaxing and dehydration according to the following steps: xylene I (10 min) → xylene II (10 min) → 100% ethanol (5 min) → 100% ethanol (5 min) → 95% ethanol (3 min) → 80% ethanol (3 min) → 70% ethanol (3 min) → double distilled water (3 min), rinse with PBS; (2) Permeabilization: Permeabilize with 0.5% Triton permeabilization solution for 30 min, rinse with PBS; (3) Antigen retrieval: Add antigen retrieval solution for 10 min, rinse with PBS; (4) 3% Incubate with H2O2 for 10 min to block endogenous oxidase, then rinse with PBS; (5) Primary antibody incubation: Add type II collagen, MMP-13 and TIMP-1 antibodies, incubate at 4°C overnight; (6) Rewarm for 30 min, wash with PBS, add chelating agent, incubate at room temperature for 20 min, do not wash; (7) Add secondary antibody, incubate at room temperature for 30 min, then rinse with PBS; (8) Use DAB colorimetric solution to develop the target protein, and use hematoxylin to stain the cell nucleus; (9) Color development: The appearance of yellow or brownish-yellow particles on the cell cytoplasm and / or cell membrane indicates positive, and take photos using a microscope camera system, such as Figure 8 shown.

Claims

1. A drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation, characterized in that: The drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation is a drug-guided gene reprogrammed cell. The surface of the mesenchymal stem cell preparation has cell membrane proteins, and the mesenchymal stem cell preparation is loaded with drug liposomes co-incubated with mesenchymal stem cells. The mesenchymal stem cell preparation is beneficial for the treatment of osteoarthritis.

2. The drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation according to claim 1, characterized in that: The drug liposome is a glucocorticoid-loaded drug liposome.

3. The drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation according to claim 2, characterized in that: The glucocorticoid drug-encapsulated liposome is dexamethasone liposome.

4. The drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation according to claim 1, characterized in that: The mesenchymal stem cells are derived from one of umbilical cord, placenta, bone marrow and adipose tissue.

5. The drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation according to claim 1, characterized in that: The mesenchymal stem cell preparation expresses higher levels of anti-inflammatory factors and lower levels of pro-angiogenic factors, and is more conducive to the treatment of osteoarthritis; the mesenchymal stem cell preparation induces M2 macrophage polarization.

6. A method for preparing the drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation according to claim 1, characterized in that: The steps include: (1) In vitro culture of mesenchymal stem cells; (2) Preparation of drug liposomes; (3) Mesenchymal stem cells are co-incubated with drug liposomes to assemble into a drug-loaded liposome mesenchymal stem cell preparation.

7. The preparation method according to claim 6, characterized in that The step (1) comprises: cutting up a tissue block from one of the umbilical cord, placenta, bone marrow and fat, mixing the mixture with a digestive enzyme in a centrifuge tube, placing the mixture in a 37°C shaker, and shaking and digesting the mixture for 2-5 hours; centrifuging the digested tissue fluid, discarding the supernatant, and washing the mixture twice with PBS; resuspending the precipitate in a DF-12 culture medium containing 5-12v% fetal bovine serum and 0.5-2v% Penicillin-Streptomycin, and planting the precipitate in a T75 culture bottle, and culturing the mixture at a constant temperature under conditions of a volume concentration of 2-7% CO2 and saturated humidity at 37°C, replacing the culture medium every 3-4 days, and passage the cells when the cells have grown to 80-90% of the bottom of the bottle.

8. The preparation method according to claim 6, characterized in that Step (2) comprises: preparing dexamethasone liposomes by a thin film dispersion method, dissolving cholesterol and dipalmitoylphosphatidylcholine (DPPC) in a chloroform / methanol volume ratio of 2:1 at a mass ratio of 35:65, adding dexamethasone at a mass ratio of 8-12%, and rotary evaporating at 45-60° C. to form a thin film, and then placing it in a vacuum drying oven for 8-16 hours; hydrating with purified water at 45-60° C. for 20-40 minutes, and ultrasonicating with a probe for 5-20 minutes; and extruding through a 100-300 nm filter membrane for 5-20 times to prepare a dexamethasone liposome preparation.

9. The preparation method according to claim 6, characterized in that Step (3) comprises: mixing the dexamethasone liposome solution with 1×10 6 The mesenchymal stem cells were incubated at 37° C. and 2-7% CO2 for 8-16 hours, the supernatant was aspirated, the cells were washed 1-3 times with PBS buffer, trypsinized for 1-3 minutes, centrifuged at 500-2000 rpm for 2-8 minutes, the supernatant was discarded, the cells were washed 2-5 times with PBS buffer, and a drug-loaded cell preparation of mesenchymal stem cells loaded with dexamethasone was obtained.

10. Use of the drug-guided anti-inflammatory enhanced mesenchymal stem cell preparation according to claim 1 in the treatment of osteoarthritis, wherein the application is injected into the bone-articular cavity to achieve the therapeutic effect of improving osteoarthritis.

Images