Injectable hydrogel as well as preparation method and application thereof

Abstract

The invention discloses injectable hydrogel as well as a preparation method and application thereof. The injectable hydrogel is formed by self-assembling a natural plant active component, a framework material and an inorganic nano material, the natural plant active component comprises any one of puerarin, resveratrol, curcumin and quercetin; the framework material comprises any one of chitosan, gelatin, sodium alginate, hyaluronic acid and a polylactic acid-glycolic acid copolymer; the inorganic nano material comprises any one of mesoporous silica, calcium phosphate nano particles, zinc oxide nano particles and hydroxyapatite. The injectable hydrogel composed of puerarin, chitosan and mesoporous silica realizes the synergy of local immune regulation and tissue regeneration functions, and can be used for skeletal muscle defect repair.

Description

Technical Field

[0001] The present invention relates to the field of hydrogel preparation, and in particular to an injectable hydrogel, a preparation method and application thereof. Background Art

[0002] Volumetric muscle defect (VML) is a serious injury that exceeds the self-regeneration capacity of skeletal muscle. Currently, clinical repair mainly relies on autologous skeletal muscle transplantation, but there are the following significant defects: (1) Donor site damage and limited tissue sources: Autologous transplantation requires materials from the patient himself, which often causes donor site dysfunction, and the amount of available tissue is limited; (2) Poor tissue integration: The graft and the defect area are not fused well, and fibrosis and tissue necrosis are prone to occur; (3) Lack of immune regulation function: Most existing synthetic scaffolds and biomaterials are inert structures, which are difficult to intervene in the local immune microenvironment, especially in the context of chronic inflammation. The effect is limited; (4) Low repair efficiency and unsatisfactory functional recovery: There is a lack of multifunctional materials that can promote myogenesis and regulate immunity at the same time, resulting in the repair of intact tissue structure but insufficient functionality. Summary of the Invention

[0003] The purpose of the present invention is to overcome the defect of the prior art in lacking effective drugs and methods for treating skeletal muscle defects and repairing them.

[0004] In order to achieve the above object, the present invention provides an injectable hydrogel, which is formed by self-assembly of natural plant active ingredients, skeleton materials, and inorganic nanomaterials;

[0005] Wherein, the natural plant active ingredient comprises any one of puerarin, resveratrol, curcumin, and quercetin;

[0006] The skeleton material comprises any one of chitosan, gelatin, sodium alginate, hyaluronic acid, and polylactic acid-glycolic acid copolymer;

[0007] The inorganic nanomaterial comprises any one of mesoporous silica, calcium phosphate nanoparticles, zinc oxide nanoparticles, and hydroxyapatite.

[0008] Optionally, the injectable hydrogel is formed by self-assembly of puerarin, chitosan, and mesoporous silica.

[0009] Optionally, the particle size of the mesoporous silica is 90 nm to 110 nm.

[0010] Optionally, the injectable hydrogel realizes a transition from a non-gel state to a gel state at a shear strain of 4% to 8%.

[0011] Optionally, the pore size of the injectable hydrogel is 60 μm to 150 μm.

[0012] The present invention also provides a method for preparing the injectable hydrogel as described above, comprising the following steps:

[0013] S1, dissolving the framework material in an acid solution to prepare a solution with a mass concentration of 2% to 4%;

[0014] S2, dissolving the natural plant active ingredient in ethanol and slowly adding the solution in S1 dropwise to obtain a mixed solution;

[0015] S3, adding inorganic nanomaterials to the mixed solution in S2, and forming an injectable hydrogel in situ after sonication;

[0016] Wherein, the natural plant active ingredient comprises any one of puerarin, resveratrol, curcumin, and quercetin;

[0017] The skeleton material comprises any one of chitosan, gelatin, sodium alginate, hyaluronic acid, and polylactic acid-glycolic acid copolymer;

[0018] The inorganic nanomaterial comprises any one of mesoporous silica, calcium phosphate nanoparticles, zinc oxide nanoparticles, and hydroxyapatite.

[0019] Optionally, the natural plant active ingredient is puerarin, the skeleton material is chitosan, and the inorganic nanomaterial is mesoporous silica.

[0020] Optionally, the mass ratio of puerarin: mesoporous silica: chitosan is (10-15): (1.5-2.5): (10-15).

[0021] The present invention also provides a use of the injectable hydrogel as described above, wherein the injectable hydrogel is used to prepare a tissue regeneration and repair material.

[0022] Optionally, the injectable hydrogel is used to prepare skeletal muscle defect repair material.

[0023] Compared with the prior art, the beneficial effects of the present invention include at least:

[0024] (1) The injectable hydrogel of the present invention is self-assembled by chitosan, mesoporous silica, and puerarin, and puerarin upregulates Pdpn +The proportion of M2 macrophages is increased to achieve anti-inflammatory regulation. Mesoporous silica activates myogenic precursor cells (MuSCs) by slowly releasing silicon ions. Chitosan provides a positively charged microenvironment and an injectable gelling platform, which is beneficial to cell adhesion and tissue plasticity. They have the characteristics of immune regulation, myogenesis induction, and structural support, respectively, forming a synergistic enhancement effect in the system, realizing the synergistic integration of local immune regulation and tissue regeneration function. The injectable hydrogel of the present invention significantly promotes the differentiation of skeletal muscle stem cells towards myogenesis, enhances the speed and quality of tissue regeneration; and has long-lasting, slow-release immune regulation capabilities, effectively reducing postoperative inflammation.

[0025] (2) The injectable hydrogel system is stable, has a short in situ gelation time, and is easy to use.

[0026] (3) Injectable hydrogels are biodegradable, non-toxic, and have good tissue compatibility, making them suitable for various soft tissue defect repair scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 a is a schematic diagram of the construction of injectable hydrogel SiCP@InjGel.

[0028] Figure 1 b is the surface morphology and element distribution of mesoporous silica.

[0029] Figure 1 Figure c is a schematic diagram of the sol-gel phase transition process of the injectable hydrogel SiCP@InjGel under temperature changes.

[0030] Figure 1 Figure d is the injection performance test diagram of the injectable hydrogel SiCP@InjGel.

[0031] Figure 1 e is a transmission electron microscope image of mesoporous silica.

[0032] Figure 1 f is the X-ray diffraction (XRD) pattern of mesoporous silica, which shows a typical broad scattering peak, indicating that the material has an amorphous mesoporous structure without obvious crystalline characteristic peaks.

[0033] Figure 1 g is the X-ray photoelectron spectroscopy (XPS) graph of mesoporous silica.

[0034] Figure 1 h is the nitrogen adsorption-desorption curve and specific surface area (BET) analysis diagram of mesoporous silica, showing that it has a high specific surface area (> 500m 2 / g), and the mesopore size distribution is concentrated, which is conducive to the loading and controlled release of puerarin.

[0035] Figure 1i is the cross-sectional SEM image and EDS analysis diagram of the injectable hydrogel SiCP@InjGel after freeze-drying.

[0036] Figure 1 Figure j is the XPS spectrum of the injectable hydrogel SiCP@InjGel. The peaks correspond to Si 2p, C 1s, and N1s, confirming the presence of mesoporous silica, chitosan, and puerarin in the hydrogel, and showing typical functional group signals such as CN, C=O, and Si-O-Si.

[0037] Figure 1 k is the Fourier transform infrared (FTIR) spectrum (2000–400 cm) of the injectable hydrogel SiCP@InjGel. -1 ), absorption peaks such as -OH, -NH2, C=O and Si-O were detected, verifying the existence of hydrogen bonds and other interactions between the components of the hydrogel, providing support for physical self-assembly.

[0038] Figure 1 Figure 1: Rheological test results, showing the storage modulus (G′) and loss modulus (G″) changing with shear strain. The crossover point appears between 4% and 8%, indicating that the material transitions from a sol state to a gel state within this strain range.

[0039] Figure 1 Figure 3 is the release curve of silicon ions in the injectable hydrogel SiCP@InjGel. It was continuously released for 14 days in PBS, and the release rate showed a gradually decreasing trend, indicating that the material has good ion sustained release ability, supporting its application in tissue repair.

[0040] Figure 1 The adhesion ability of the injectable hydrogel SiCP@InjGel on different substrate surfaces is demonstrated.

[0041] Figure 2 a is the tissue appearance after surgery.

[0042] Figure 2 b is the tissue volume measurement and area recovery histogram.

[0043] Figure 2 c is a schematic diagram of H&E staining.

[0044] Figure 2 d is the Masson staining picture.

[0045] Figure 2 e is an immunofluorescence labeled image of MuSCs (Pax7+).

[0046] Figure 2 f is an image of immunofluorescence labeled MyoG+ myotube cells.

[0047] Figure 2 g is an image of immunolabeled Pdpn+M2 macrophages.

[0048] Figure 2 h is the statistical histogram of the number of fluorescent-labeled cells in each group.

[0049] Figure 2 i is the gait behavior heat map.

[0050] Figure 2 J is the motor function behavior analysis diagram.

[0051] Figure 2 k is the motion tracking path graph.

[0052] Figure 2 l is a comparison chart of gait circular heat maps.

[0053] Figure 3 a is the experimental flow chart of the porcine VML model.

[0054] Figure 3 b (b1–b8) Longitudinal and transverse MRI images of damaged skeletal muscle. High signal areas reflect inflammation, edema, and material degradation. b1–b2: sham surgery group; b3–b4: 7 days postoperatively; b5–b6: 1 month postoperatively; b7–b8: 3 months postoperatively.

[0055] Figure 3 Skeletal muscle ultrasound images in C (C1-C8) show defects and material observed 7 days, 1 month, or 3 months after surgery. Signal noise reflects inflammatory edema. C1–C2: sham surgery, C3–C4: 7 days, C5–C6: 1 month, and C7–C8: 3 months.

[0056] Figure 3 d Cross-sectional and longitudinal images of damaged skeletal muscle stained with HE and Masson staining, 6 months after surgery. Scale bar = 200 μm.

[0057] Figure 3 e–f Path diagrams of pigs during the minefield experiment under normal and stress conditions, reflecting the pigs' movement paths over time. Testing was performed 7 days, 1 month, or 3 months after surgery.

[0058] Figure 3 Gait analysis of pigs (g–h). The trajectory reflects the force applied to the injured side and gait variation. The test was performed 1 month after surgery.

[0059] Figure 4 a is a general photo.

[0060] Figure 4 b is a schematic diagram of HE staining results, scale bar = 200 μm. DETAILED DESCRIPTION

[0061] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0062] Hydrogel is a polymer material with a three-dimensional network structure that can absorb a large amount of water. Its properties are highly similar to those of the extracellular matrix (ECM), so it is often used in tissue engineering to simulate the natural microenvironment. Its core functions include:

[0063] (1) Cell carrier function:

[0064] Encapsulate seed cells (such as skeletal muscle stem cells, mesenchymal stem cells, etc.), slowly release cells through material degradation, and promote cell colonization and differentiation in the defect area.

[0065] (2) Sustained release of growth factors:

[0066] Loaded with vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), etc., they are continuously released to induce angiogenesis, myofiber regeneration and reduce fibrosis.

[0067] (3) Mechanical support and guidance:

[0068] Provide physical support, guide the directional growth of muscle fibers, and avoid excessive filling of the defect area by scar tissue.

[0069] (4) Regulating the immune microenvironment:

[0070] By modifying materials (such as adding anti-inflammatory ingredients), the inflammatory response can be regulated to promote the transition of the damaged area from the inflammatory phase to the repair phase.

[0071] Although hydrogels are currently used to treat skeletal muscle defects and repair, there are the following problems:

[0072] (1) Insufficient mechanical matching:

[0073] Natural skeletal muscle has high tensile strength and elasticity, and the mechanical properties of existing hydrogels (especially long-term stability) still need to be improved, otherwise it may affect the maturation and functional recovery of muscle fibers.

[0074] (2) Low vascularization efficiency:

[0075] Repair of large skeletal muscle defects requires the rapid formation of a vascular network. The vascular induction ability of simple hydrogels is limited and needs to be combined with endothelial cell co-transplantation or angiogenic factor gradient design.

[0076] (3) Immunogenicity and degradation products:

[0077] The long-term safety of synthetic materials (such as the toxicity of degradation products) and the immunogenicity of natural materials (such as the allergic risk of xenogeneic collagen) need further verification. The active ingredients are single and the targets are limited, making it difficult to achieve the combined mechanism of "immune regulation + stem cell activation".

[0078] (4) Bottlenecks in clinical translation:

[0079] From animal experiments to human applications, problems such as large-scale production, sterilization process, and cost control need to be solved. Currently, there are no approved hydrogel products for skeletal muscle defect repair.

[0080] The present invention provides a self-assembling injectable hydrogel with good biocompatibility and immunomodulatory function. The injectable hydrogel is formed by physical cross-linking of puerarin, chitosan, and mesoporous silica. Puerarin interacts with chitosan through non-covalent bonds (hydrogen bonds, electrostatic interactions, etc.), and mesoporous silica is embedded therein, and the in situ self-assembly of the hydrogel is achieved by a thermosensitive trigger mechanism. The chitosan of the present invention serves as a hydrogel skeleton, providing good biocompatibility, injectability, and gel-forming ability, and is the basis for forming a three-dimensional scaffold structure. Puerarin is the core immune component of the present invention, promoting macrophage M2 polarization, exerting anti-inflammatory and pro-repair effects, and mesoporous silica nanoparticles provide a high specific surface area structural platform, allowing a large amount of puerarin to be loaded inside it, and through the sustained release of silicon ions, assisting skeletal muscle stem cell activation and tissue reconstruction. The three components work synergistically, namely, puerarin regulates immunity, mesoporous silica releases silicon ions to promote myogenesis, and chitosan constructs an injectable structural platform. After rapid gelation under body temperature conditions (about 37°C), local release produces dual immune-myogenesis effects, realizing the integrated coupling of the "immune-stem cell-structure" triple mechanism.

[0081] 1. Preparation of injectable hydrogel SiCP@InjGel

[0082] 1. Synthesis of Mesoporous Silica MSNs:

[0083] 0.91 g of hexadecyltrimethylammonium bromide (CTAB, Sinopharm Group) and 1.5 g of ammonium fluoride (NH4F, Sinopharm Group) were dissolved in 250 mL of ultrapure water and stirred at 80°C. 4.5 mL of tetraethoxysilane (TEOS, Sinopharm Group) was added dropwise, and the reaction was stirred for 2 hours. The precipitate was collected by centrifugation (8000 rpm, 15 minutes), washed three times with ultrapure water and anhydrous ethanol, dried, and calcined in air at 600°C for 6 hours to produce MSN powder with a particle size of approximately 90 to 110 nm.

[0084] 2. Solution preparation:

[0085] Weigh 100 mg of low-viscosity chitosan and 100 mg of medium-viscosity chitosan (deacetylation ≥95%, Aladdin) and dissolve them in 10 mL of 0.6% glacial acetic acid. Stir for 12 hours to prepare a 2% chitosan solution. Separately, weigh 0.1 g of β-glycerophosphate disodium pentahydrate (TCL, CAS: 13408-09-8) and dissolve it in 0.5 mL of ultrapure water. Add 17.5 mg of puerarin (Sinopharm, CAS: 3681-99-0) and sonicate for 10 minutes to form a homogeneous composite solution.

[0086] 3. Mixing ratio:

[0087] The composite solution was added dropwise to 2.5 mL of chitosan solution and stirred thoroughly. 35 mg of the prepared MSNs (particle size approximately 100 nm) were then added and dispersed evenly. 0.5 mL of 2% hydroxyethyl cellulose (HEC) solution (TCL) was then added and allowed to react at 37°C for 5-10 minutes to form a milky white gel, SiCP@InjGel. The mass ratio of the components was: chitosan: puerarin: β-glycerophosphate disodium pentahydrate: MSNs = (10-15): (0.5-1.5): (1.5-2.5): (1.5-2.5).

[0088] like Figure 1 As shown in the figure, the prepared injectable hydrogel was characterized by material characterization. The gelation time of the hydrogel was 2 to 3 minutes, and it could be smoothly injected through a 27G injection needle. It had good shear thinning properties and could achieve a transition from non-gel to gel state at a shear strain of 4% to 8%. After freeze-drying, the pore size ranged from 60μm to 150μm, and a three-dimensional porous network structure was formed inside, which was suitable for cell adhesion and nutrient exchange. It had good biocompatibility and adhesion properties, and could firmly adhere to the surfaces of various materials such as glass, skin, and metal.

[0089] Application of the injectable hydrogel SiCP@InjGel in the mouse VML model

[0090] 1. Animal Modeling and Material Injection

[0091] Eight-week-old male C57BL / 6 mice were anesthetized with 2% isoflurane inhalation and then fixed to a sterile operating table. The hair on the right thigh was trimmed with surgical scissors and disinfected with iodine and alcohol. A 1-cm longitudinal incision was made along the anterolateral aspect of the thigh to fully expose the quadriceps femoris belly. A muscle defect model approximately 6 mm in diameter and 2 mm deep was created in the center of the muscle using a sterile scalpel. The treatment group received an injection of 20 μL of SiCP@InjGel into the defect, while the control group received injections of PBS or blank hydrogel. The skin was then sutured with 6-0 absorbable sutures, and the mice were disinfected and placed on a constant-temperature heating pad for recovery.

[0092] 2. Tissue Regeneration Observation

[0093] The animals were sacrificed on the 28th day after surgery, and muscle tissues were obtained for histological and functional evaluations.

[0094] like Figure 2 As shown in a, the muscle defect area in the SiCP@InjGel treatment group healed well and was completely filled, while the control group had obvious tissue collapse and depression. Figure 2 b shows that the muscle tissue recovery rate of the SiCP@InjGel treatment group was significantly improved. Figure 2 Figure c shows that the muscle fibers in the SiCP@InjGel treatment group were arranged regularly and had a continuous structure, while the muscle fibers in the control group were broken and had severe inflammatory infiltration. Figure 2 d shows that the fibrosis area in the SiCP@InjGel treatment group was significantly reduced, while the control group showed obvious collagen fiber deposition (increased blue). Figure 2 e showed that the number of MuSCs in the SiCP@InjGel treatment group was significantly upregulated. Figure 2 f shows that the number of myotubes formed in the SiCP@InjGel treatment group increased. Figure 2 g shows that the M2 polarization of the SiCP@InjGel treatment group is enhanced. Figure 2 h further quantified the changing trends of MuSCs and M2 macrophages, Figure 2 The i shows that the gait disorder of the control group was obvious after injection of PBS, while the trajectory of the SiCP@InjGel treatment group was complete and continuous. Figure 2 J~ Figure 2 The results showed that the range of motion of the SiCP@InjGel-treated group recovered well, the activity trajectory covered a wide area, and the motor behavior was significantly improved.

[0095] It can be seen that the SiCP@InjGel treatment group can effectively promote muscle fiber regeneration in the VML area, reduce collagen deposition and scar formation, activate the recruitment of myogenic stem cells and M2 macrophages, and significantly improve motor behavior and muscle function recovery.

[0096] 3. Long-term evaluation in large animal models

[0097] 1. Pig Model Establishment and Material Injection

[0098] Guangxi Bama miniature pigs (1 year old, weighing about 40 kg, male, healthy in appearance) were selected and divided into a control group, a model group and a SiCP@InjGel treatment group according to animal ethics requirements, with 3 pigs in each group. Fasting for 12 hours before surgery, subcutaneous injection of atropine (0.8 mL / kg) and Sutent-50 (0.5 mL / kg) for combined anesthesia, and surgical operation was performed after there was no limb movement and blink reflex. The pig was placed on the operating table, hair was clipped, iodine and alcohol disinfected, and sterile surgical towels were laid. An incision of about 10 cm long was made at a 30° angle 2 cm below the patella, and the tissue was separated layer by layer to fully expose the peroneus tertius muscle. A sterile scalpel was used to cut about 3×3×0.7 cm from the muscle belly. 3 (Volume about 6cm 3 , weighing approximately 4.5g) of skeletal muscle tissue. The treatment group was injected with 10mL of SiCP@InjGel at the defect, the model group was injected with PBS, and the sham group did not treat the muscle tissue. The subcutaneous tissue was then sutured and the skin was sutured with non-absorbable sutures. After the operation, the patients were disinfected with iodine tincture and bandaged with gauze, and ceftiofur (5mg / kg) was injected for 7 consecutive days to prevent infection. A follow-up observation period of 6 months was set up after the operation, during which MRI and functional tests were performed regularly to evaluate the degradation of the hydrogel and tissue regeneration.

[0099] 2. Long-term Histological Compatibility and Degradation Analysis

[0100] like Figure 3 As shown in b, it shows that SiCP@InjGel can well fill the muscle defect area. Figure 3 Figure c shows that the signal of the SiCP@InjGel treatment group was stable in the first 3 months and completely degraded in 6 months, while the control group showed discontinuous signal. Figure 3 d shows that the muscle fibers in the SiCP@InjGel treatment group are arranged continuously and neatly, while the control group has visible hollow areas. Figure 3 The e showed that the collagen deposition in the SiCP@InjGel treatment group was significantly reduced, and the fibrosis area was lower than that in the control group. Figure 3 f shows that the muscle reconstruction in the SiCP@InjGel treatment group was obvious. Figure 3 The g showed that there was almost no positive apoptosis signal in the SiCP@InjGel treatment group. Figure 3 h showed that M2 macrophages in the SiCP@InjGel treatment group significantly aggregated. Figure 3 The i showed that the indicators of the SiCP@InjGel treatment group were significantly better than those of the model group. Figure 3 The j shows that the SiCP@InjGel treatment group is close to the natural level of Sham, Figure 3 The k showed that the recovery rate of various parameters in the SiCP@InjGel treatment group reached more than 85%.

[0101] It can be seen that the SiCP@InjGel treatment group showed good biodegradability, biocompatibility and tissue regeneration promotion in the miniature pig VML model. After 6 months of treatment, the muscle tissue structure was significantly reconstructed, there was no inflammatory response, and the functional recovery rate was close to the normal control level.

[0102] IV. In vivo degradability and non-toxicity

[0103] Subcutaneous injection in rats Figure 4 As shown, it was found that SiCP@InjGel could be gradually degraded in the tissue and could be completely absorbed within 2 weeks without causing inflammation, foreign body reaction or deposition of toxic byproducts.

[0104] It is understandable that the injectable hydrogel of the present invention can also be applied to the regeneration and repair of other tissue types, such as skin burn wounds, tendon rupture repair, and support materials after myocardial infarction.

[0105] In summary, the injectable hydrogel of the present invention is self-assembled by chitosan, mesoporous silica, and puerarin, and puerarin upregulates Pdpn + The proportion of M2 macrophages is increased to achieve anti-inflammatory regulation. Mesoporous silica activates myogenic precursor cells (MuSCs) by slowly releasing silicon ions. Chitosan provides a positively charged microenvironment and an injectable gelling platform, which is beneficial to cell adhesion and tissue plasticity. They have the characteristics of immune regulation, myogenesis induction, and structural support, respectively, forming a synergistic enhancement effect in the system, realizing the synergistic integration of local immune regulation and tissue regeneration function. The injectable hydrogel of the present invention significantly promotes the differentiation of skeletal muscle stem cells towards myogenesis, enhances the speed and quality of tissue regeneration; and has long-lasting, slow-release immune regulation capabilities, effectively reducing postoperative inflammation.

[0106] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. An injectable hydrogel, characterized in that The injectable hydrogel is formed by self-assembly of natural plant active ingredients, skeleton materials, and inorganic nanomaterials; Wherein, the natural plant active ingredient comprises any one of puerarin, resveratrol, curcumin, and quercetin; The skeleton material comprises any one of chitosan, gelatin, sodium alginate, hyaluronic acid, and polylactic acid-glycolic acid copolymer; The inorganic nanomaterial comprises any one of mesoporous silica, calcium phosphate nanoparticles, zinc oxide nanoparticles, and hydroxyapatite.

2. The injectable hydrogel according to claim 1, wherein The injectable hydrogel is formed by self-assembly of puerarin, chitosan and mesoporous silica.

3. The injectable hydrogel according to claim 2, wherein The particle size of the mesoporous silica is 90 nm to 110 nm.

4. The injectable hydrogel according to claim 1, wherein The injectable hydrogel realizes the transition from a non-gel state to a gel state at a shear strain of 4% to 8%.

5. The injectable hydrogel according to claim 1, wherein The pore size of the injectable hydrogel is 60 μm to 150 μm.

6. A method for preparing an injectable hydrogel according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1, dissolving the framework material in an acid solution to prepare a solution with a mass concentration of 2% to 4%; S2, dissolving the natural plant active ingredient in ethanol and slowly adding the solution in S1 dropwise to obtain a mixed solution; S3, adding inorganic nanomaterials to the mixed solution in S2, and forming an injectable hydrogel in situ after sonication; Wherein, the natural plant active ingredient comprises any one of puerarin, resveratrol, curcumin, and quercetin; The skeleton material comprises any one of chitosan, gelatin, sodium alginate, hyaluronic acid, and polylactic acid-glycolic acid copolymer; The inorganic nanomaterial comprises any one of mesoporous silica, calcium phosphate nanoparticles, zinc oxide nanoparticles, and hydroxyapatite.

7. The preparation method according to claim 6, wherein The natural plant active ingredient is puerarin, the skeleton material is chitosan, and the inorganic nanomaterial is mesoporous silica.

8. The preparation method according to claim 6, wherein Calculated by mass ratio, the puerarin: mesoporous silica: chitosan = (0.5-1.5): (1.5-2.5): (10-15).

9. Use of the injectable hydrogel according to any one of claims 1 to 5, characterized in that: The injectable hydrogel is used for preparing tissue regeneration and repair materials.

10. The use according to claim 9, characterized in that The injectable hydrogel is used for preparing skeletal muscle defect repairing materials.

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