Composite hydrogels of loaded nanoszymes and engineered stem cells and methods and uses thereof

Abstract

This invention relates to the field of biomedical materials and tissue technology, specifically disclosing a composite hydrogel loaded with nanozymes and engineered stem cells, its method, and applications. The hydrogel comprises a thermosensitive hydrogel matrix and Mn3O4 nanozymes and Kartogenin-pretreated bone marrow mesenchymal stem cells loaded therein. The Mn3O4 nanozymes can scavenge reactive oxygen species and adsorb free DNA, while the Kartogenin-pretreated bone marrow mesenchymal stem cells can differentiate into chondrocytes. This invention reduces oxidative stress and inflammation through nanozyme "debridement," creating a survival space for stem cell "repair" and achieving synergistic remodeling of the immune-oxidative microenvironment. This invention also discloses a hydrothermal method for preparing Mn3O4 nanozymes, Kartogenin-induced stem cells in vitro, and a low-temperature mixing method for preparing the hydrogel. This hydrogel can be used to prepare drugs or formulations for treating osteoarthritis, significantly improving stem cell survival rates, inhibiting osteophyte formation, and promoting cartilage regeneration.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and tissue technology, and in particular to composite hydrogels loaded with nanozymes and engineered stem cells, their methods and applications. Background Technology

[0002] Currently, osteoarthritis (OA) remains a clinical challenge. Stem cell therapy (such as BMSC transplantation) is considered a promising strategy, often combined with hydrogels as carriers for intra-articular injection, such as hyaluronic acid (HA). Existing technologies have explored the use of antioxidants or anti-inflammatory drugs to improve stem cell survival. For example, Wang et al. (2024) reviewed the latest advancements in hydrogel technology for delivering MSCs to treat OA, emphasizing the crucial impact of scaffold materials on stem cell survival and function. Peng et al. (2023) further pointed out that various pathological factors in the OA microenvironment significantly affect the balance between osteogenic and chondrogenic differentiation of MSCs, leading to poor repair outcomes. Furthermore, Kim and Guilak (2022) explored the application of engineered hyaluronic acid in developing new OA treatment strategies, suggesting that ideal materials should possess both biocompatibility and microenvironment regulation capabilities. Chen et al. (2025) also summarized the research progress of injectable hydrogels in OA treatment, believing that multifunctional hydrogels represent a future development direction.

[0003] Despite the significant progress made in the aforementioned research, the existing technology still suffers from the following key shortcomings: Microenvironment incompatibility: The joint cavity of osteoarthritis (OA) contains excessive reactive oxygen species (ROS) and cell-free DNA (cfDNA), which form a vicious cycle of "immune-oxidation," leading to a large number of apoptosis or functional inactivation of transplanted stem cells. Traditional hydrogel scaffolds mostly serve only as inert carriers and lack the ability to actively remove ROS and cfDNA, thus failing to provide a suitable microenvironment for stem cell survival.

[0004] Limited therapeutic function: Existing hydrogel systems mostly focus on single cell delivery or simple structural support, failing to simultaneously achieve multiple functions such as anti-oxidation, anti-inflammation, and stem cell protection. In particular, the clearance of cfDNA, a key pro-inflammatory factor, has not yet been effectively addressed.

[0005] Stem cell fate is unstable: Under inflammatory and oxidative stress, untreated MSCs are prone to dedifferentiation or abnormal differentiation (such as differentiation into adipocytes), leading to cartilage repair failure and even exacerbating osteophyte formation. Existing in vitro expansion methods have failed to effectively "lock in" the cartilage differentiation fate of stem cells.

[0006] In summary, current technologies lack an integrated hydrogel system capable of simultaneously clearing ROS / cfDNA and directionally inducing chondrocyte differentiation from stem cells. Therefore, the development of composite hydrogels loaded with nanozymes and engineered stem cells, along with their methods and applications, is crucial. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art by proposing a composite hydrogel loaded with nanozymes and engineered stem cells, as well as its method and application.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A composite hydrogel loaded with nanozymes and engineered stem cells includes a thermosensitive hydrogel matrix and a Mn3O4 nanozyme loaded in the hydrogel matrix, and also includes Kartogenin-pretreated bone marrow mesenchymal stem cells loaded in the hydrogel matrix.

[0009] Preferably, the thermosensitive hydrogel is a composite hydrogel of hyaluronic acid and poloxamer.

[0010] Preferably, the Kartogenin-pretreated bone marrow mesenchymal stem cells are obtained by in vitro induction with Kartogenin for 3 days, and the induction concentration of Kartogenin is 1 μM.

[0011] The preparation method of the composite hydrogel loaded with nanozymes and engineered stem cells based on the above includes the following steps: Step 1: Preparation of Mn3O4 nanozymes; Step 2: Preparation of Kartogenin-pretreated bone marrow mesenchymal stem cells; Step 3: Mix the thermosensitive hydrogel matrix, Mn3O4 nanozyme, and Kartogenin-pretreated bone marrow mesenchymal stem cells to obtain the hydrogel.

[0012] Preferably, in step 1, Mn3O4 nanozymes are prepared using a hydrothermal method.

[0013] Preferably, in step 2, the induction temperature is 37°C and the CO2 concentration is 5%.

[0014] Preferably, in step 3, the thermosensitive hydrogel matrix is ​​a composite hydrogel of hyaluronic acid and poloxamer, which is prepared by dissolving 23.5% poloxamer and 0.5% hyaluronic acid by weight in ultrapure water and stirring at 4°C until completely dissolved to obtain a composite matrix solution with a total concentration of 24% by weight.

[0015] Preferably, in step 3, Mn3O4 nanozyme is added to the thermosensitive hydrogel matrix to a final concentration of 10 μg / mL; then, Kartogenin-pretreated bone marrow mesenchymal stem cells are added to it to a final density of 2 × 10⁻⁶. 6 cells / mL; after mixing, the hydrogel is obtained.

[0016] Preferably, in step 3, the hydrogel is in a liquid state at 4°C and forms a gel at 37°C.

[0017] The above describes the application of a composite hydrogel loaded with nanozymes and engineered stem cells in the preparation of drugs or formulations for treating osteoarthritis.

[0018] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention utilizes Mn3O4 nanozymes, which possess activities mimicking superoxide dismutase (SOD) and catalase (CAT), enabling cascade scavenging of O2⁻ and H2O₂, significantly reducing oxidative stress levels. Simultaneously, the Mn3O4 nanozyme exhibits a flower-like physical structure with a large specific surface area, effectively physically adsorbing cfDNA and blocking TLR9-mediated inflammatory pathways. Thus, without relying on exogenous anti-inflammatory drugs, it simultaneously achieves ROS scavenging and cfDNA adsorption, breaking the vicious cycle of "oxidation-immunity" within osteoarthritis (OA) joints and creating favorable conditions for the survival of bone marrow mesenchymal stem cells.

[0019] 2. This invention uses Kartogenin (KGN) to pretreat BMSCs in vitro for 3 days, enabling the stem cells to differentiate into chondrocyte lines before transplantation, thus locking their differentiation "fate" into chondrocyte lines. Compared to untreated stem cells, the engineered stem cells of this invention can maintain their chondrocyte differentiation potential even in harsh microenvironments, significantly reducing dedifferentiation and abnormal differentiation.

[0020] 3. This invention creates a living space for stem cells by using nanozymes for "debridement" (removing ROS and cfDNA), followed by "repair" by KGN-pretreated stem cells, i.e., secreting cartilage matrix, achieving a synergistic therapeutic effect. In vivo DMM rat model experiments confirmed that after 8 weeks of treatment, articular cartilage defects were largely repaired, and the OARSI score was significantly reduced; Micro-CT three-dimensional reconstruction showed a significant reduction in osteophyte volume; immunohistochemical staining showed decreased MMP13 (matrix-degrading enzyme) expression and increased COL2 (type II collagen) expression. These results indicate that this invention not only promotes hyaline cartilage regeneration but also effectively inhibits osteophyte formation. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 The preparation process and mechanism of action of the composite hydrogel (MNKB / HA composite hydrogel) of the present invention are shown in the following flowchart: Figure 2 : In vitro performance test data graph; Figure 3 Comparison of in vivo animal experimental results: Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0024] Unless otherwise specified, all reagents and instruments used in this invention are commercially available. The Sprague-Dawley rats used in this invention were purchased from Liaoning Changsheng Biotechnology Co., Ltd., and all animal experiments involved have been approved by the Experimental Animal Management and Use Committee of Anhui Medical University (Approval No.: LLSC20252077).

[0025] Example 1 Mn3O4 nanozymes were synthesized via a hydrothermal method. The preparation steps are as follows: 1. Solution preparation and mixing: Weigh 1.0 g of potassium permanganate (KMnO4) and dissolve it in 500 mL of deionized water. Stir for 30 minutes at room temperature until completely dissolved. Then, slowly add 10 mL of oleic acid to the above purple-red solution.

[0026] 2. Pre-emulsification: The mixed solution was stirred in a water bath at 28°C for 5 hours to fully mix and emulsify oleic acid and potassium permanganate, forming a uniform precursor suspension.

[0027] 3. Hydrothermal reaction: The pH of the precursor suspension was finely adjusted to between 5.0 and 6.0 using dilute hydrochloric acid (HCl) to obtain a mixture. The mixture was then transferred to a stainless steel high-pressure reactor lined with polytetrafluoroethylene (PTFE) (the filling degree was approximately 70%-80% of the liner volume).

[0028] Sealing and heating: Tighten the lid of the reactor and place it in an electric drying oven.

[0029] Reaction parameters: Heat to 160℃ and maintain the temperature for 10-12 hours.

[0030] Cooling: After the reaction is complete, allow the furnace to cool naturally to room temperature.

[0031] 4. Solid-liquid separation and washing: Open the reaction vessel and collect the black precipitate at the bottom. Wash it alternately with ethanol and deionized water by centrifugation 3-5 times to thoroughly remove unreacted oleic acid and byproduct ions.

[0032] 5. Drying: The washed precipitate was placed in a vacuum drying oven and dried at 60℃-80℃ for 10-12 hours to obtain black Mn3O4 nanozyme powder.

[0033] Example 2 Preparation steps of Kartogenin (KGN) pretreated BMSCs (KGN-BMSCs): 1. Primary cell extraction and expansion: Two-week-old Sprague-Dawley rats were used. The femurs were removed under aseptic conditions and analyzed using MEM α containing 10% fetal bovine serum (FBS) and 1% penicillin-α antibodies. α Bone marrow cavities were flushed with culture medium to obtain bone marrow mesenchymal stem cells (BMSCs). The cell suspension was seeded into culture flasks and incubated statically at 37°C in a 5% CO2 incubator. Fresh complete culture medium was replaced for the first time after 24 hours to remove non-adherent suspension cells; thereafter, the culture medium was replaced every 2 days.

[0034] 2. Cell passage and selection: When the primary cells (P0) reached 80%-90% confluence, they were digested and passaged using 0.25% trypsin. Third-generation (P3) BMSCs with good proliferative activity were used for subsequent experiments.

[0035] 3. KGN-induced pretreatment (key step): P3 generation BMSCs were used at 1×10 6 Seed cells at a density of 1 cell / well in 6-well plates. Once cells have adhered and grown to 70%-80% confluence, discard the original complete culture medium. Take Kartogenin (KGN) powder, dissolve it in dimethyl sulfoxide (DMSO) to prepare a stock solution, and then dilute it with complete culture medium. Add fresh complete culture medium containing 1 μM KGN to the wells (ensuring the final volume concentration of DMSO in the culture system is less than 0.1%).

[0036] 4. Culture conditions and medium changes: The 6-well plates were placed in a 37°C, 5% CO2 incubator for further induction culture for 3 days. During induction, the drug-containing medium was changed every 24 hours to maintain drug concentration and provide nutrition.

[0037] 5. Cell collection: After induction, the KGN-containing culture medium was aspirated, and the cells were washed twice with pre-cooled PBS buffer to remove residual drug. Cells were digested with 0.25% trypsin-EDTA solution, and digestion was stopped when cells were observed to become rounded and detach under a microscope. The cell suspension was transferred to centrifuge tubes and centrifuged (e.g., 1000 rpm, 5 min) to collect the cell pellet, yielding KGN-pretreated engineered BMSCs (KGN-BMSCs).

[0038] Preparation steps of HA / PF127 thermosensitive hydrogel: A cold dissolution method was used to dissolve 23.5% (w / v) poloxamer (PF127) and 0.5% (w / v) hyaluronic acid (HA) in ultrapure water. The mixture was placed in a refrigerator at 4°C and magnetically stirred for 12 hours until the polymer was completely dissolved, resulting in a clear and transparent homogeneous composite PF127 / HA matrix solution with a total concentration of 24% (w / v).

[0039] Preparation steps of MNKB / HA composite hydrogel: Matrix preparation: Using a cold dissolution method, 23.5% (w / v) of PF127 and 0.5% (w / v) of hyaluronic acid (HA) were dissolved in ultrapure water. The mixture was placed in a refrigerator at 4°C and magnetically stirred overnight until the polymer was completely dissolved, resulting in a clear and transparent homogeneous composite PF127 / HA matrix solution with a total concentration of 24% (w / v).

[0040] Nanozyme dispersion: Under ice bath conditions, Mn3O4 nanozyme powder was added to the above matrix solution and dispersed uniformly by vortexing to prepare an enzyme-containing hydrogel precursor solution with a final Mn3O4 concentration of 10 μg / mL.

[0041] Cell reconstitution: BMSCs pretreated with KGN for 3 days (KGN-BMSCs) were collected and the cell pellet was resuspended in a small amount of complete culture medium. Under ice bath conditions, the cell suspension was gently mixed into the enzyme-containing hydrogel precursor solution, adjusting the final cell density in the mixture to 2 × 10⁻⁶ cells / mL. 6 cells / mL.

[0042] Final product: Gently blow and mix (avoiding air bubbles) to obtain a thermosensitive composite hydrogel (MNKB / HA) loaded with nanozymes and engineered stem cells. This hydrogel remains in a liquid, injectable state at 4°C and undergoes a rapid sol-gel transition at 37°C.

[0043] Figure 1 This is a flowchart illustrating the preparation process and a schematic diagram illustrating the mechanism of action of the composite hydrogel (MNKB / HA composite hydrogel) of the present invention.

[0044] Example 3 1. Free radical scavenging ability test (ABTS / DPPH) Experimental methods: ABTS·⁺ Determination: ABTS (2,2′-adiazon-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (3 mg) was dissolved in 0.735 mL of distilled water to prepare a 7.4 mM solution; potassium persulfate (1 mg) was dissolved in 1.43 mL of distilled water to prepare a 2.6 mM oxidant solution. Equal volumes (0.2 mL each) of the two solutions were mixed and reacted at room temperature in the dark for 12 hours to generate ABTS·⁺ free radical cations. The resulting blue-green solution was diluted 10-20 times with distilled water to adjust its absorbance at 734 nm to approximately 0.7, thus preparing the working solution. Different concentrations of Mn3O4 nanozyme were reacted with 1 mL of ABTS·⁺ working solution at room temperature for 15 minutes, and the decrease in absorbance at 734 nm was measured.

[0045] DPPH assay: Dissolve 1 mg of DPPH in 24 mL of ethanol and sonicate for 5 minutes to ensure complete dissolution, preparing a stock solution. Dilute the stock solution with ethanol until the absorbance at 519 nm is between 0.6 and 1.0, preparing the working solution. React different concentrations of Mn3O4 nanozyme with 1 mL of DPPH working solution at room temperature in the dark for 30 minutes, and measure the absorbance at 519 nm.

[0046] Scavenging rate (%) = [(A0-A1) / A0] × 100%, where A0 is the absorbance of the control group and A1 is the absorbance of the experimental group.

[0047] result: Mn3O4 nanozymes exhibited significant dose-dependent free radical scavenging activity.

[0048] ABTS results ( Figure 2 a) As the concentration of Mn3O4 increases, the solution color gradually fades from blue-green to colorless, indicating that it can effectively scavenge ABTS·⁺ cation free radicals.

[0049] DPPH results ( Figure 2a) As the concentration of Mn3O4 increases, the solution color gradually lightens from dark purple, indicating that it can effectively scavenge DPPH free radicals.

[0050] 2. DNA Adsorption Effect Test (Gel Electrophoresis) Experimental methods: Agarose gel electrophoresis: A 1 μM solution of CpG 1826 (an immunostimulatory oligonucleotide that mimics bacterial DNA and is often used as a substitute for free DNA) was mixed with a gradient concentration of Mn3O4 nanozyme and incubated at room temperature (25°C) for 2 hours. After incubation, 10 μL of sample was spotted onto a 5% agarose gel containing SerRed nucleic acid dye. Electrophoresis was performed at a constant voltage of 120 V for 35 minutes in 1×TAE buffer. The bands were observed and recorded using a hyperspectral gel imaging system (Jena, Germany).

[0051] result( Figure 2 b, Figure 2 c) The results show that as the concentration of Mn3O4 nanozyme increases, the fluorescence intensity of the DNA band on the gel gradually decreases, indicating that Mn3O4 nanozyme can efficiently adsorb and bind DNA in solution.

[0052] 3. Quantitative analysis of apoptosis and cell activity (immunofluorescence / flow cytometry) Experimental methods: Flow cytometry quantification: Intracellular ROS levels were detected using the reactive oxygen species (ROS)-specific fluorescent probe DCFH-DA, and the fluorescence intensity and apoptosis rate of DCFH-DA were quantitatively analyzed by flow cytometry.

[0053] Immunofluorescence staining: Cells were stained with Calcein / PI (live cells were green, dead cells were red) and observed under a fluorescence microscope.

[0054] result: ROS clearance and cell protection: such as ( Figure 2 d, Figure 2 As shown in e), in the H2O2 (300 μM) induced oxidative stress model, Mn3O4 nanozyme (10 μg / mL) significantly reduced ROS levels in BMSCs, chondrocytes and RAW 264.7 macrophages.

[0055] Apoptosis inhibition: Quantitative analysis by flow cytometry ( Figure 2 f, Figure 2 g) showed that the apoptosis rate in the Mn3O4 nanozyme-treated group was significantly lower than that in the untreated group. This indicates that the nanozyme exerts a significant anti-apoptotic protective effect by scavenging ROS.

[0056] Consistent with the known effects of reactive oxygen species (ROS) on cell death, live / dead cell staining results showed that H2O2 significantly increased BMSC death; while co-incubation with Mn3O4 nanozymes significantly reduced cell death. Figure 2 h, Figure 2 i).

[0057] 4. Inflammatory factors and polarization state (ELISA / flow cytometry) Experimental methods: ELISA: The supernatant of RAW 264.7 macrophages cultured after stimulation with CpG ODN 1826 (1 μg / mL) was collected, and the secretion levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were quantitatively detected using an ELISA kit.

[0058] Flow cytometry: Cells are stained with fluorescently labeled anti-CD206 (marker of M2 macrophages) and anti-CD86 (marker of M1 macrophages), and the polarization ratio of macrophages is quantitatively analyzed by flow cytometry.

[0059] result: Inflammatory factors: Mn3O4 nanozymes significantly inhibited the secretion of IL-1β and TNF-α by macrophages. Figure 2 j, Figure 2 k).

[0060] Polarization state: Quantitative results from flow cytometry ( Figure 2 l, Figure 2 The m) showed that the expression level of CD206 on the surface of macrophages in the Mn3O4 nanozyme treatment group was significantly increased, while the expression level of CD86 was significantly decreased. This indicates that nanozymes can effectively promote the transformation of macrophages from the pro-inflammatory M1 type to the anti-inflammatory M2 type.

[0061] Example 4 Summary of in vivo animal experimental protocols and results 1. Experimental Methodology 1.1 Animal Model Construction Animal selection: Male Sprague-Dawley (SD) rats weighing 300 ± 20 g were selected.

[0062] Adaptation feeding: One week of acclimatization feeding is required before surgery.

[0063] Model building (DMM model): Surgery group (DMM): The right knee joint of rats was operated on, and part of the medial meniscus was removed after the joint capsule was opened to disrupt joint stability and induce post-traumatic osteoarthritis.

[0064] Sham surgery group: Only the joint capsule was cut open, without meniscectomy.

[0065] Postoperative care: Disinfect with povidone-iodine solution. Administer intramuscular injections of cefazolin (100 mg / kg / day) for three consecutive days postoperatively to prevent infection.

[0066] 1.2 Experimental grouping and dosing regimen Grouping: Rats that successfully developed the model were randomly divided into 6 groups: Sham group (sham surgery group) PBS group (control group) BMSCs / HA group (stem cell-only group) Mn3O4NPs / HA group (simple nanozyme group) KGN-BMSCs / HA group (pretreated stem cell group) MNKB / HA group (combination therapy group, i.e., composite hydrogel loaded with nanozymes and KGN-BMSCs) Administration method: Intra-articular injection.

[0067] Dosage: 100 μL each time.

[0068] Administration timing: Treatment begins in the 5th week after surgery, with injections once a week for 4 consecutive weeks.

[0069] 1.3 Sample Collection and Testing Observation time point: After the last administration (i.e., 9 weeks post-surgery), rats were sacrificed with sodium pentobarbital (150 mg / kg, ip), and right knee joint tissue was collected.

[0070] Micro-CT examination: Voltage 85 kV, current 200 mA, resolution 10 µm. 3D reconstruction was performed using CTVOX software.

[0071] Histological analysis: Fixation and decalcification: 4% paraformaldehyde fixation for 48 hours, 12.5% ​​EDTA decalcification for 4 weeks.

[0072] Sectioning: Paraffin-embedded, section thickness 5 µm.

[0073] Staining methods: HE staining, Safranin O-Fast Green staining.

[0074] Scoring criteria: The degree of cartilage degeneration was assessed using the OARSI histopathological scoring system.

[0075] Immunohistochemistry (IHC): Detection of the expression of ACAN, MMP13, and COL II.

[0076] 2. Experimental Results 2.1 Articular cartilage repair and matrix metabolism (histology and IHC) HE staining and safranin O staining ( Figure 3 a, Figure 3 c): The MNKB / HA treatment group showed improved cartilage structural integrity, significantly increased proteoglycan content, and decreased OARSI score, indicating that cartilage degeneration was significantly inhibited.

[0077] Semi-quantitative immunohistochemical analysis: Anabolic metabolism ( Figure 3 b、 Figure 3 c): The positive expression area of ​​COL II (type II collagen) and ACAN (aggregated proteoglycan) in the cartilage of the MNKB / HA group was significantly higher than that of other control groups, indicating that the treatment promoted the synthesis and deposition of cartilage matrix.

[0078] Catabolism ( Figure 3 b、 Figure 3 c): The expression of MMP13 (matrix metalloproteinase 13) was significantly reduced in the MNKB / HA group, indicating that the treatment effectively inhibited the degradation of cartilage matrix.

[0079] 2.2 Osteophyte formation and subchondral bone reconstruction (Micro-CT) Micro-CT 3D Reconstruction: Reconstructing 3D images of the knee joint using CTVOX software.

[0080] 3D Reconstructed Images and Quantitative Analysis Figure 3 d、 Figure 3 e) The results showed that the BMSCs treatment group alone showed significant osteophyte formation at the cartilage margin, while the MNKB / HA treatment group significantly inhibited osteophyte formation and the normal bone structure of the tibial subchondral bone was basically preserved, demonstrating a protective effect on bone structure.

[0081] 2.3 In vivo tracer and biosafety In vivo tracer ( Figure 3f): After injection of Cy5.5-labeled MNKB / HA, fluorescence imaging showed that the hydrogel had good retention in the joint cavity.

[0082] Biosafety Figure 3 g): H&E staining showed no obvious pathological damage in the major organs (heart, liver, spleen, lungs, kidneys, and brain), and the blood routine and liver and kidney function indicators were normal, proving that the material has good biosafety.

[0083] Example 5 Composite Hydrogel Usage Guide 1. Product preparation and rewarming Product removal: Remove the syringe pre-loaded with MNKB / HA composite hydrogel from a 4°C refrigerator or cryogenic storage environment.

[0084] State check: At this point, the hydrogel should be in a liquid state and have good fluidity.

[0085] Warming process: Place the syringe in a room temperature environment (approximately 25°C) for 5-10 minutes, or gently warm it by wrapping it in your palm until its temperature gradually rises to near body temperature (30-35°C). Note: Do not use high-temperature equipment (such as ovens or hot water) for direct heating, as this may damage cell viability.

[0086] 2. Syringe assembly and venting Connecting the needle: Unscrew the protective cap at the tip of the syringe and connect the sterile joint puncture needle.

[0087] Air release procedure: Gently push the plunger vertically upwards to expel air bubbles from the tip of the syringe, ensuring the needle is filled with medication until a small amount of liquid overflows from the needle tip.

[0088] 3. Joint cavity puncture and injection Disinfection of the affected knee: Expose the affected knee. Disinfect the skin with iodine solution as usual, and drape with a sterile drape.

[0089] Puncture and needle insertion: Under aseptic conditions, the needle is inserted through the upper outer or upper inner edge of the patella, passing through the skin, subcutaneous tissue and joint capsule, and entering the joint cavity.

[0090] Injecting the gel: After confirming that the needle is in the joint cavity (no fluid accumulation or loss of resistance upon aspiration), slowly push the plunger to inject 100 μL of liquid hydrogel into the joint cavity.

[0091] Key points of operation: The injection process should be gentle to avoid excessive pressure that could cause needle displacement or tissue damage.

[0092] 4. In-situ gelation and postoperative care Thixotropic recovery: After injection, remove the needle and cover the area with a sterile dressing.

[0093] In-situ gelation: When hydrogels come into contact with body temperature of 37°C, a sol-gel transition will occur within minutes, forming a gel with a three-dimensional network structure.

[0094] Functional release: The gel releases Mn3O4 nanozymes (which scavenge ROS and adsorb cfDNA) and KGN-BMSCs (which secrete nutrient factors and differentiate into chondrocytes) within the joint cavity through enzymatic degradation and physical dissolution, thereby reshaping the pathological immune oxidative microenvironment and promoting cartilage regeneration.

[0095] Example 6 Based on the teachings of Examples 1-5 above, those skilled in the art should understand that the core of this invention lies in achieving immune-oxidative microenvironment remodeling through "nanozyme debridement (removing ROS and adsorbing cfDNA) combined with KGN engineered stem cell repair". The following alternative solutions, if employing the same synergistic mechanism, can also achieve the expected effects of this invention and are equivalent embodiments of this invention: (1) Alternatives to hydrogel matrices: In the embodiments of this invention, a hyaluronic acid / poloxamer (HA / PF127) composite hydrogel is specifically used. However, other thermosensitive gelling or injectable self-healing and biocompatible hydrogel materials, such as chitosan / sodium glycerophosphate thermosensitive systems, gelatin-based hydrogels, and polylactic-co-glycolic acid copolymer (PLGA)-PEG-PLGA triblock copolymer in situ gels, can also achieve in situ gelation and sustained-release functions in the joint cavity when used to load Mn3O4 nanozymes and KGN-pretreated mesenchymal stem cells, and should be considered as equivalent matrices of this invention.

[0096] (2) Replacement of nanozymes: In the embodiments of the present invention, Mn3O4 nanozymes are preferred. Their core function is to mimic the activities of superoxide dismutase (SOD) and catalase (CAT) to cascade and remove reactive oxygen species (ROS). At the same time, they effectively adsorb free DNA (cfDNA) due to their flower-like physical structure. Other nanomaterials with SOD / CAT-like enzyme activities and nucleic acid adsorption capabilities, such as nano-cerium dioxide (CeO2) and Fe3O4 nanozymes, are equivalent to the nanozymes of the present invention if they can achieve the same ROS removal and cfDNA adsorption effects and thereby break the vicious cycle of "oxidation-immunity".

[0097] (3) Alternative sources of stem cells: The KGN engineered cells used in the embodiments of the present invention are bone marrow mesenchymal stem cells (BMSCs). However, other types of mesenchymal stem cells, such as adipose-derived mesenchymal stem cells (ADSCs), umbilical cord mesenchymal stem cells (UC-MSCs), synovial-derived mesenchymal stem cells (SDSCs), etc., are also expected to obtain similar cartilage-directed differentiation ability and microenvironment remodeling effect if they are pretreated in vitro with Kartogenin (preferably 1 μM, induced for 3 days) to lock the cartilage differentiation fate and are encapsulated in hydrogel in combination with nanozymes. These are equivalent sources of engineered stem cells in the present invention.

[0098] The above alternatives do not deviate from the technical concept of "nanozyme debridement-stem cell repair" synergistic remodeling of the immune-oxidative microenvironment of this invention. Any composite hydrogel obtained based on the above equivalent substitutions, its preparation method and application fall within the protection scope of this invention.

[0099] In the description of this specification, the references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0100] The foregoing has shown and described the basic principles, main features, and advantages of this application. Those skilled in the art should understand that this application is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of this application. Various changes and modifications can be made to this application without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the claims of this application.

Claims

1. A composite hydrogel loaded with nanozymes and engineered stem cells, characterized in that, It includes a thermosensitive hydrogel matrix and a Mn3O4 nanozyme loaded in the hydrogel matrix, as well as bone marrow mesenchymal stem cells pretreated with Kartogenin loaded in the hydrogel matrix.

2. The composite hydrogel loaded with nanozymes and engineered stem cells according to claim 1, characterized in that, The thermosensitive hydrogel is a composite hydrogel of hyaluronic acid and poloxamer.

3. The composite hydrogel loaded with nanozymes and engineered stem cells according to claim 1, characterized in that, The Kartogenin-pretreated bone marrow mesenchymal stem cells were obtained by in vitro induction with Kartogenin for 3 days at an induction concentration of 1 μM.

4. A method for preparing a composite hydrogel loaded with nanozymes and engineered stem cells according to any one of claims 1-3, characterized in that, Includes the following steps: Step 1: Preparation of Mn3O4 nanozymes; Step 2: Preparation of Kartogenin-pretreated bone marrow mesenchymal stem cells; Step 3: Mix the thermosensitive hydrogel matrix, Mn3O4 nanozyme, and Kartogenin-pretreated bone marrow mesenchymal stem cells to obtain the hydrogel.

5. The preparation method according to claim 4, characterized in that, In step 1, Mn3O4 nanozymes are prepared using a hydrothermal method.

6. The preparation method according to claim 4, characterized in that, In step 2, the induction temperature is 37°C and the CO2 concentration is 5%.

7. The preparation method according to claim 4, characterized in that, In step 3, the thermosensitive hydrogel matrix is ​​a composite hydrogel of hyaluronic acid and poloxamer. The preparation method is as follows: 23.5% by weight and 0.5% by weight and hyaluronic acid are dissolved in ultrapure water and stirred at 4°C until completely dissolved to obtain a composite matrix solution with a total concentration of 24% by weight and volume.

8. The preparation method according to claim 4, characterized in that, In step 3, Mn3O4 nanozymes were added to a thermosensitive hydrogel matrix to a final concentration of 10 μg / mL; then, Kartogenin-pretreated bone marrow mesenchymal stem cells were added to the matrix. Make its final density 2×10 6 cells / mL; after mixing, the hydrogel is obtained.

9. The preparation method according to claim 4, characterized in that, In step 3, the hydrogel is liquid at 4°C and forms a gel at 37°C.

10. The use of a composite hydrogel loaded with nanozymes and engineered stem cells according to any one of claims 1-3 in the preparation of a drug or formulation for treating osteoarthritis.

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