Bioactive macroporous hydrogel as well as preparation method and application thereof

Abstract

The invention discloses bioactive macroporous hydrogel as well as a preparation method and application thereof. The bioactive macroporous hydrogel comprises hydrogel particles and a bioactive load loaded on the hydrogel particles, the hydrogel particles are obtained by mechanically extruding a hydrogel matrix, and the hydrogel matrix is formed by compounding sodium alginate, agarose and methacrylated hyaluronic acid through hydrogen bond crosslinking and photo-crosslinking; the bioactive load comprises: a) small extracellular vesicles of which the surfaces are modified with macrophage targeted CRV peptides; and b) a PLGA (poly (lactic-co-glycolic acid)) microsphere loaded with a cartilage inducer Kartogenin. The bioactive macroporous hydrogel disclosed by the invention has an internally communicated macroporous network, is beneficial to cell infiltration, blood vessel ingrowth and nutrient substance diffusion, and overcomes the defects that the traditional hydrogel is small in pore size and limits cell behaviors. According to the bioactive macroporous hydrogel disclosed by the invention, dual bioactive loads are adopted to synergistically promote tendon-bone healing, so that the treatment effect is better.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a bioactive macroporous hydrogel, its preparation method, and its applications. Background Technology

[0002] The tendon-bone interface (enthesis) is a complex tissue with a functional gradient, connecting tendons and bones, and playing a crucial role in stress transmission during movement. This interface typically consists of four continuous regions: pure tendon tissue, unmineralized fibrocartilage, mineralized fibrocartilage, and bone tissue. This complex gradient structure makes true structural regeneration extremely difficult after trauma or degenerative changes (such as rotator cuff tears). Current standard clinical treatments primarily rely on surgical suture anchoring, but postoperative re-tear rates and limited functional recovery are often due to fibrous scar formation, poor interface integration, and insufficient mechanical properties of regenerated tissue.

[0003] Tissue-engineered hydrogels offer a potential strategy for addressing this challenge, but traditional hydrogels are often limited in application due to their small pore size, poor mechanical properties, lack of bioactivity guidance, and difficulty in matching complex interfacial microenvironments. In particular, there is a lack of multifunctional scaffolds capable of simultaneously regulating the immune microenvironment, promoting directed stem cell differentiation, and supporting cell ingrowth and tissue integration. In recent years, injectable microparticle-assembled hydrogels have attracted attention due to their macroporous structure, customizable rheological properties, and good biocompatibility; however, their application in tendon-bone repair is still in its early stages, and there is an urgent need to develop novel hydrogel systems with multiple bioactivities that can precisely regulate the repair process. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a bioactive macroporous hydrogel, its preparation method, and its applications.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] The first aspect is to provide a bioactive macroporous hydrogel, comprising hydrogel microparticles and a bioactive loading material supported on the hydrogel microparticles;

[0007] The hydrogel microparticles are obtained by mechanical extrusion of a hydrogel matrix, which is formed by hydrogen bonding and photocrosslinking of sodium alginate, agarose and methacrylamide hyaluronic acid.

[0008] The bioactive loading material includes:

[0009] a) Small extracellular vesicles with a surface modified with macrophage-targeting CRV peptides, denoted as CRV-sEVs; and

[0010] b) PLGA microspheres loaded with the chondroitin inducer Kartogenin, denoted as PLGA-KGN microspheres.

[0011] Furthermore, the hydrogel matrix is ​​formed by cross-linking a mixture of sodium alginate mother liquor, agarose mother liquor, and methacrylamide hyaluronic acid mother liquor containing photoinitiator in a volume ratio of (45-55):(40-45):(5-10).

[0012] More preferably, the concentration of the sodium alginate mother liquor is 1.5%–2.5% (w / v), the concentration of the agarose mother liquor is 2.0%–2.5% (w / v), and the concentration of the methacrylamide hyaluronic acid mother liquor is 1.0%–2.0% (w / v); the photoinitiator is LAP, and its concentration in the methacrylamide hyaluronic acid mother liquor is 0.1%–0.2% (w / v).

[0013] Furthermore, the sequence of the macrophage-targeting CRV peptide is CRVLRSGSC.

[0014] Furthermore, the concentration of the small extracellular vesicles is 1×10^9 to 8×10^9 particles / mL hydrogel.

[0015] Furthermore, the concentration of the PLGA microspheres in the hydrogel is 1–5 mg / mL; the loading of Kartogenin in the PLGA microspheres is 3%–5% (w / w).

[0016] The second aspect is to provide a method for preparing the aforementioned bioactive macroporous hydrogel, including the following steps:

[0017] Step 1: Preparation of bioactive loading material:

[0018] a) PLGA-KGN microspheres were prepared using a W / O / W dual emulsion-solvent evaporation method;

[0019] b) Small extracellular vesicles were isolated from the culture supernatant of human adipose-derived stem cells by differential ultracentrifugation and incubated with DSPE-PEG-CRV targeting molecules to prepare CRV-sEVs.

[0020] Step 2, constructing a macroporous hydrogel scaffold:

[0021] a) The sodium alginate mother liquor, agarose mother liquor, and methacrylamide hyaluronic acid mother liquor containing photoinitiator were rapidly mixed using a multi-channel mixing device, and after cooling, a hydrogel matrix was formed for later use.

[0022] b) The hydrogel matrix is ​​mechanically extruded through a sieve to obtain hydrogel microparticles of uniform size;

[0023] c) Physically mix the PLGA-KGN microspheres and the CRV-sEVs with the hydrogel microparticles, and add a methacrylamide hyaluronic acid solution to obtain a hydrogel precursor mixture;

[0024] d) Apply ultraviolet light to the mixed system to initiate a photocrosslinking reaction of methacrylamide hyaluronic acid, causing the hydrogel particles to covalently bond at the contact points and form an internally interconnected macroporous network structure to obtain the bioactive macroporous hydrogel.

[0025] Furthermore, the aperture of the screen is 0.250 mm.

[0026] Furthermore, the wavelength of the ultraviolet light irradiation is 365 nm, and the light intensity is 3-10 mW / cm². 2 The irradiation time is 30-90 seconds; the concentration of the methacrylamide hyaluronic acid solution is 1%-2% (w / v).

[0027] The third aspect is the application of the aforementioned precursor mixture of bioactive macroporous hydrogels in the preparation of medicaments for promoting tendon-bone interface repair, said precursor mixture comprising hydrogel microparticles, PLGA-KGN microspheres, and CRV-sEVs.

[0028] The specific method used is a minimally invasive in-situ gelation repair strategy:

[0029] Step 1), Surgical repair: In standard surgery, the torn tendon (such as the supraspinatus tendon) is first reattached to its anatomical attachment point on the bone (such as the greater tuberosity of the humerus) using anchors or sutures.

[0030] Step 2), hydrogel implantation: The prepared hydrogel precursor mixture that has not yet undergone secondary cross-linking is precisely injected and filled into the repaired tendon-bone interface using a syringe;

[0031] Step 3), in-situ crosslinking: using ultraviolet light (365nm, 5mW / cm). 2 The implantation site is irradiated for 60 seconds to trigger secondary cross-linking of the hydrogel, which enables it to rapidly form a stable three-dimensional macroporous scaffold in situ at the site of injury.

[0032] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0033] The bioactive macroporous hydrogel of the present invention has an internally interconnected macroporous network, which is conducive to cell infiltration, blood vessel ingrowth and nutrient diffusion, and overcomes the drawbacks of traditional hydrogels with small pore size and limited cell behavior.

[0034] The bioactive macroporous hydrogel of this invention employs dual bioactive loading materials to synergistically promote tendon-bone healing: CRV-sEVs regulate the immune microenvironment by targeting macrophages, reducing inflammatory responses and promoting reparative M2 phenotypic polarization, providing a favorable environment for tissue regeneration; PLGA-KGN microspheres continuously release the chondrogenic agent Kartogenin, promoting the differentiation of stem cells into chondrocytes and aiding in the regeneration and functional reconstruction of the fibrocartilage layer.

[0035] The hydrogel precursor mixture of the present invention can be precisely injected into the damaged interface through a syringe, making it suitable for minimally invasive surgical scenarios such as arthroscopy, reducing surgical trauma and improving operational precision; ultraviolet light triggers secondary cross-linking, enabling the hydrogel to solidify rapidly at the implantation site, preventing migration, ensuring that bioactive factors continue to play a role in the target area, and improving treatment efficiency. Attached Figure Description

[0036] Figure 1 (A) H&E staining of major organs (heart, liver, spleen, lung, and kidney) 8 weeks post-surgery; (B) cumulative release curves of CRV-sEVs and KGN in the hydrogel of this invention.

[0037] Figure 2 Immunofluorescence staining and semi-quantitative analysis of the M1 biomarker iNOS and the M2 biomarker CD206 at the TBI site in the entire treatment group were shown 2 weeks post-surgery.

[0038] Figure 3 (A) shows SG staining and semi-quantitative analysis at 2 and 8 weeks post-surgery; (B) shows COLII immunohistochemical staining and semi-quantitative analysis at 2 and 8 weeks post-surgery.

[0039] Figure 4 (A) shows the immunohistochemical staining and semi-quantitative analysis of COLI and COLIII at 2 and 8 weeks post-surgery; (B) shows the Masson staining results and semi-quantitative analysis at 2 and 8 weeks post-surgery.

[0040] Figure 5 The results of maximum load testing at 2 and 8 weeks post-surgery are shown. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the invention. It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of the present invention can be combined with each other.

[0042] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions or as recommended by the manufacturer. Unless otherwise stated, all reagents and materials used in the following examples were commercially available.

[0043] Example 1

[0044] This embodiment provides an injectable bioactive macroporous hydrogel assembled from microparticles, comprising hydrogel microparticles and a bioactive loading material loaded on the hydrogel microparticles.

[0045] The above-mentioned hydrogel microparticles were obtained by mechanical extrusion of a hydrogel matrix, which was formed by ionic crosslinking and photocrosslinking of sodium alginate, agarose, and methacryloyl hyaluronic acid.

[0046] The above-mentioned bioactive loading materials include:

[0047] a) Small extracellular vesicles with a surface modified with macrophage-targeting CRV peptides; and

[0048] b) PLGA microspheres loaded with the chondroitin inducer Kartogenin.

[0049] The specific preparation method of the above-mentioned bioactive macroporous hydrogel is as follows:

[0050] Step 1: Preparation of bioactive loading material:

[0051] a) PLGA microspheres loaded with the chondroitin inducer Kartogenin (denoted as PLGA-KGN microspheres) were prepared using a W / O / W dual emulsion-solvent evaporation method, specifically as follows:

[0052] KGN aqueous solution (inner aqueous phase W1) was emulsified in PLGA dichloromethane solution (oil phase O) to form W / O primary emulsion; then it was added to polyvinyl alcohol (PVA) aqueous solution (outer aqueous phase W2) for homogenization to form W / O / W dual emulsion; the microspheres were solidified by evaporating organic solvent by stirring, and finally collected, washed and freeze-dried.

[0053] b) Preparation of small extracellular vesicles (denoted as CRV-sEVs) with surface-modified macrophage-targeting CRV peptides:

[0054] sEVs were isolated and purified from the culture supernatant of human adipose-derived stem cells (ADSCs) by differential ultracentrifugation; the synthesized CRV peptide was reacted with DSPE-PEG-NHS to prepare DSPE-PEG-

[0055] CRV targeting molecules: DSPE-PEG-CRV and sEVs suspension were incubated at 37°C for 1 hour. The targeting molecules were anchored onto the lipid bilayer of sEVs by the "post-insertion method". Finally, unbound molecules were removed by ultracentrifugation to obtain CRV-sEVs.

[0056] Step 2, constructing a macroporous hydrogel scaffold:

[0057] a) Sodium alginate stock solution, agarose stock solution, and methacrylamide hyaluronic acid stock solution containing photoinitiator were rapidly mixed using a multi-channel mixing device at a volume ratio of 50:43:7. After cooling, a hydrogel matrix was formed for later use. The concentration of the sodium alginate stock solution was 2% (w / v), the concentration of the agarose stock solution was 2.3% (w / v), and the concentration of the methacrylamide hyaluronic acid stock solution was 1.6% (w / v). The photoinitiator was LAP, and its concentration in the methacrylamide hyaluronic acid stock solution was 0.16% (w / v).

[0058] b) The above hydrogel matrix was mechanically extruded through a 60-gauge (0.250 mm aperture) stainless steel mesh to obtain hydrogel microparticles of uniform size.

[0059] c) Gently mix PLGA-KGN microspheres and CRV-sEVs with the above hydrogel microparticles, and add a methacrylamide hyaluronic acid solution (concentration 1.6 w / v%) to obtain a hydrogel precursor mixture.

[0060] The concentration of small extracellular vesicles was 5 × 10^9 particles / mL hydrogel; the concentration of PLGA microspheres in the hydrogel was 3 mg / mL; and the loading of Kartogenin in the PLGA microspheres was 3.8% (w / w).

[0061] d) Apply ultraviolet light (365nm, 5mW / cm²) to the mixed system. 2 Irradiation for 60 seconds triggers a photocrosslinking reaction of methacrylamide hyaluronic acid, causing the hydrogel microparticles to covalently bond at the contact points, forming a stable, internally interconnected macroporous network structure, ultimately yielding a bioactive macroporous hydrogel (denoted as MMH+CRV-sEVs+PLGA-KGN).

[0062] Example 2

[0063] This embodiment provides an injectable bioactive macroporous hydrogel assembled from microparticles, comprising hydrogel microparticles and a bioactive loading material loaded on the hydrogel microparticles.

[0064] The above-mentioned hydrogel microparticles were obtained by mechanical extrusion of a hydrogel matrix, which was formed by ionic crosslinking and photocrosslinking of sodium alginate, agarose, and methacryloyl hyaluronic acid.

[0065] The above-mentioned bioactive loading materials include:

[0066] a) Small extracellular vesicles with a surface modified with macrophage-targeting CRV peptides; and

[0067] b) PLGA microspheres loaded with the chondroitin inducer Kartogenin.

[0068] The specific preparation method of the above-mentioned bioactive macroporous hydrogel is as follows:

[0069] Step 1: Preparation of bioactive support (same as Example 1):

[0070] Step 2, constructing a macroporous hydrogel scaffold:

[0071] a) Sodium alginate stock solution, agarose stock solution, and methacrylamide hyaluronic acid stock solution containing photoinitiator were rapidly mixed using a multi-channel mixing device at a volume ratio of 55:45:10. After cooling, a hydrogel matrix was formed for later use. The concentration of the sodium alginate stock solution was 2.3% (w / v), the concentration of the agarose stock solution was 2.5% (w / v), and the concentration of the methacrylamide hyaluronic acid stock solution was 1.9% (w / v). The photoinitiator was LAP, and its concentration in the methacrylamide hyaluronic acid stock solution was 0.2% (w / v).

[0072] b) The above hydrogel matrix was mechanically extruded through a 60-gauge (0.250 mm aperture) stainless steel mesh to obtain hydrogel microparticles of uniform size.

[0073] c) Gently mix PLGA-KGN microspheres and CRV-sEVs with the above hydrogel microparticles, and add a methacrylamide hyaluronic acid solution (concentration of 2 w / v%) to obtain a hydrogel precursor mixture.

[0074] The concentration of small extracellular vesicles was 7 × 10^9 particles / mL hydrogel; the concentration of PLGA microspheres in the hydrogel was 5 mg / mL; and the loading of Kartogenin in the PLGA microspheres was 4.5% (w / w).

[0075] d) Apply ultraviolet light (365nm, 5mW / cm²) to the mixed system. 2 Irradiation for 60 seconds triggers a photocrosslinking reaction of methacrylamide hyaluronic acid, causing the hydrogel microparticles to covalently bond at the contact points, forming a stable, internally interconnected macroporous network structure, ultimately yielding a bioactive macroporous hydrogel (denoted as MMH+CRV-sEVs+PLGA-KGN).

[0076] Example 3: In vivo efficacy verification

[0077] To verify the actual effect of the bioactive macroporous hydrogel prepared in Example 1 of this invention (hereinafter referred to as "the hydrogel of this invention") on promoting tendon-bone healing in vivo, we designed and implemented the following animal experiments.

[0078] 1. Construction of experimental animal models

[0079] Laboratory animals: Male Sprague-Dawley (SD) rats aged 12 weeks and weighing 250-300 grams were selected. All animal experiments were conducted in strict accordance with relevant animal ethics and usage guidelines.

[0080] Establishment of a rotator cuff injury and repair model: 1) Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg / kg), and the shoulder area was prepared and sterilized; 2) A longitudinal skin incision was made along the shoulder joint, the deltoid muscle was bluntly dissected, and the supraspinatus tendon was exposed; 3) The supraspinatus tendon was completely severed at its insertion point at the greater tubercle of the humerus; 4) A bone tunnel was created at the greater tubercle of the humerus using a 0.5 mm diameter Kirschner wire; 5) 4-0 absorbable sutures were passed through the bone tunnel, and the severed end of the supraspinatus tendon was re-sutured and fixed to its anatomical footprint area to complete the surgical repair.

[0081] The rats that completed the above rotator cuff injury repair model were randomly divided into the following two groups (each group contained 3 animals at each time point):

[0082] Control group: Standard surgical suturing and repair were performed without implanting any materials at the repair site.

[0083] Treatment group: After surgical suturing and repair, the “MMH+CRV-sEVs+PLGA-KGN” hydrogel described in this invention was implanted at the tendon-bone interface.

[0084] 2. Details of hydrogel implantation

[0085] For the animals in the treatment group, the following procedures were performed immediately after the tendon suturing was completed:

[0086] Injection implantation: Using a syringe, approximately 100 μL of the hydrogel precursor mixture of the present invention (containing MMH microparticles, CRV-sEVs and PLGA-KGN microspheres) is carefully injected and filled into the gaps at the tendon-bone repair interface.

[0087] In-situ crosslinking: Subsequently, immediately use a wavelength of 365nm and a power of 5mW / cm 2 The ultraviolet lamp irradiates the implantation area for 60 seconds to trigger secondary cross-linking of the hydrogel, allowing it to form a stable three-dimensional scaffold in situ at the site of injury.

[0088] Wound closure: After confirming the hydrogel has hardened, suture the muscle and skin layer by layer. Provide the animal with pain relief for three consecutive days post-surgery.

[0089] 3. Observation time points and evaluation indicators

[0090] Animals in each group were sacrificed at two key time points, 2 weeks and 8 weeks post-surgery, and rotator cuff-humerus complex specimens were collected for analysis.

[0091] Evaluation indicators include:

[0092] 1) Histological evaluation (2 weeks and 8 weeks): H&E staining was used to assess tissue morphology and healing quality; Safranin O-Fix Green (SG) staining and type II collagen (COL II) immunohistochemistry were used to assess the regeneration of the fibrocartilage layer; and type I (COL I) and type III (COL III) collagen immunohistochemistry and Masson staining were used to assess the remodeling and maturation of the tendon matrix.

[0093] 2) Evaluation of the immune microenvironment (2 weeks): The expression of macrophage M1 marker (iNOS) and M2 marker (CD206) was detected by immunofluorescence staining to evaluate the early anti-inflammatory and immunomodulatory effects of the hydrogel.

[0094] 3) Biomechanical performance evaluation (2 weeks and 8 weeks): The ultimate failure load (N) of the tendon-bone complex after repair was detected by uniaxial tensile testing to quantify the mechanical strength after healing.

[0095] 4. Experimental Results

[0096] 1) The hydrogel of this invention has good biocompatibility and structural properties.

[0097] No systemic toxicity: Eight weeks post-surgery, H&E staining of major organs (heart, liver, spleen, lungs, and kidneys) showed no pathological abnormalities. Figure 1 A).

[0098] Sequential drug delivery: In vitro release profiles show that CRV-sEVs rapidly release approximately 60% within the first week (for early anti-inflammatory purposes), while KGN provides sustained release over 30 days (for long-term regeneration), achieving sequential drug delivery. Figure 1 B).

[0099] 2) The hydrogel of this invention has good immunomodulatory capabilities and can reshape the regenerative microenvironment.

[0100] Inhibition of M1 macrophage polarization: In a rat rotator cuff injury (RCR) model, 2 weeks post-surgery, the expression level of the M1 marker iNOS at the TBI site in the full treatment group was significantly lower than that in the control group. Figure 2 ).

[0101] Promotes M2 macrophage polarization: Correspondingly, the expression level of the M2 marker CD206 was significantly higher than that in all control groups. Figure 2 ).

[0102] 3) The hydrogel of this invention can promote fibrocartilage regeneration.

[0103] Reconstruction of the fibrocartilage layer: SG staining shows ( Figure 3A) At 2 and 8 weeks post-surgery, the entire treatment group formed a continuous and significantly thickened fibrocartilage layer (red stained area), while the control group had less cartilage formation.

[0104] Promotes cartilage matrix formation: Immunohistochemical results show ( Figure 3 B) The expression of type II collagen (COLII, the key matrix of cartilage) in the TBI site of the whole treatment group was significantly higher than that in the control group at both 2 weeks and 8 weeks.

[0105] 4) The hydrogel of this invention can improve tendon matrix remodeling and maturation.

[0106] Optimizing collagen type promotes collagen fiber maturation: The expression of mature type I collagen (COLI) was significantly higher in the full treatment group, while the expression of scar-associated type III collagen (COL III) was significantly lower, indicating that the repair progresses towards regenerative healing rather than fibrotic scar formation. Figure 4 A).

[0107] Increased collagen density: Masson staining shows ( Figure 4 B) The whole treatment group formed dense, well-arranged collagen bundles at 8 weeks, and its relative collagen content was significantly higher than that of the control group.

[0108] 5) The hydrogel of this invention can restore the biomechanical strength of the tendon-bone interface and limb function.

[0109] Significantly improved mechanical properties: At 8 weeks post-surgery, the ultimate failure load of TBI repaired in the entire treatment group was significantly higher than that in the control group. Figure 5 ).

[0110] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the content and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A bioactive macroporous hydrogel, characterized in that, Includes hydrogel microparticles and bioactive loadings on the hydrogel microparticles; The hydrogel microparticles are obtained by mechanical extrusion of a hydrogel matrix, which is formed by hydrogen bonding and photocrosslinking of sodium alginate, agarose and methacrylamide hyaluronic acid. The bioactive loading material includes: a) Small extracellular vesicles with macrophage-targeting CRV peptides on their surface are denoted as CRV-sEVs; and b) PLGA microspheres loaded with the chondroitin inducer Kartogenin, denoted as PLGA-KGN microspheres.

2. The bioactive macroporous hydrogel according to claim 1, characterized in that, The hydrogel matrix is ​​formed by cross-linking a mixture of sodium alginate mother liquor, agarose mother liquor, and methacryloyl hyaluronic acid mother liquor containing photoinitiator in a volume ratio of (45-55):(40-45):(5-10).

3. The bioactive macroporous hydrogel according to claim 2, characterized in that, The concentration of the sodium alginate mother liquor is 1.5%–2.5% (w / v), the concentration of the agarose mother liquor is 2.0%–2.5% (w / v), and the concentration of the methacrylamide hyaluronic acid mother liquor is 1.0%–2.0% (w / v); the photoinitiator is LAP, and its concentration in the methacrylamide hyaluronic acid mother liquor is 0.1%–0.2% (w / v).

4. The bioactive macroporous hydrogel according to claim 1, characterized in that, The sequence of the macrophage-targeting CRV peptide is CRVLRSGSC.

5. The bioactive macroporous hydrogel according to claim 1, characterized in that, The concentration of the small extracellular vesicles is 1×10^9 to 8×10^9 particles / mL hydrogel.

6. The bioactive macroporous hydrogel according to claim 1, characterized in that, The concentration of the PLGA microspheres in the hydrogel is 1–5 mg / mL; the loading of Kartogenin in the PLGA microspheres is 3%–5% (w / w).

7. A method for preparing a bioactive macroporous hydrogel as described in any one of claims 1-6, characterized in that, Includes the following steps: Step 1: Preparation of bioactive loading material: a) PLGA-KGN microspheres were prepared using a W / O / W dual emulsion-solvent evaporation method; b) Small extracellular vesicles were isolated from the culture supernatant of human adipose-derived stem cells by differential ultracentrifugation and incubated with DSPE-PEG-CRV targeting molecules to prepare CRV-sEVs. Step 2, constructing a macroporous hydrogel scaffold: a) The sodium alginate mother liquor, agarose mother liquor, and methacrylamide hyaluronic acid mother liquor containing photoinitiator were rapidly mixed using a multi-channel mixing device, and after cooling, a hydrogel matrix was formed for later use. b) The hydrogel matrix is ​​mechanically extruded through a sieve to obtain hydrogel microparticles of uniform size; c) Physically mix the PLGA-KGN microspheres and the CRV-sEVs with the hydrogel microparticles, and add a methacrylamide hyaluronic acid solution to obtain a hydrogel precursor mixture; d) Apply ultraviolet light to the mixed system to initiate a photocrosslinking reaction of methacrylamide hyaluronic acid, causing the hydrogel particles to covalently bond at the contact points and form an internally interconnected macroporous network structure to obtain the bioactive macroporous hydrogel.

8. The preparation method according to claim 7, characterized in that, The mesh size of the screen is 0.250 mm.

9. The preparation method according to claim 7, characterized in that, The ultraviolet light irradiation has a wavelength of 365nm and a light intensity of 3-10mW / cm². 2 The irradiation time is 30-90 seconds; the concentration of the methacrylamide hyaluronic acid solution is 1%-2% (w / v).

10. The use of the precursor mixture of the bioactive macroporous hydrogel according to any one of claims 1-6 in the preparation of a medicament for promoting tendon-bone interface repair, characterized in that, The precursor mixture includes hydrogel microparticles, PLGA-KGN microspheres, and CRV-sEVs.

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