Preparation method of fiber-reinforced self-repairing hydrogel mediated sequential drug release scaffold for promoting vascularized osteochondral regeneration

Abstract

Aiming at the challenge of osteochondral defect repair, the invention provides a preparation method of a fiber-reinforced self-repairing hydrogel scaffold (SBPS). According to the method, phenylboronic acid modified sodium alginate, polyvinyl alcohol and fibroin nanofibers rich in beta-Sheet are utilized to form the hydrogel through dynamic boric acid ester bonds and hydrogen bonds. The SBPS hydrogel loaded with PDGF-BB is combined with the nanosheet containing LDH / ChS / TGF-beta3, so that multi-dimensional synergy of mechanical strengthening, sequential drug release (PDGF-BB is released in an early stage and TGF-beta3 / Mg < 2 + > is released in a later stage), ROS removal and endogenous repair is realized. The Mg < 2 + > concentration gradient effect further assists staged regulation (vascularized bone regeneration-cartilage matrix synthesis), and breaks through the limitation of a single factor. Experiments prove that the scaffold can effectively drive angiogenesis, stem cell recruitment and cartilage / bone differentiation in vivo and in vitro, and finally osteochondral regeneration with structure-function matching is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterial preparation, and relates to a method for preparing a fiber-reinforced self-repairing hydrogel-mediated timed drug release scaffold for promoting vascularized osteochondrocyte regeneration. Background Art

[0002] Clinical studies have shown that articular cartilage damage often extends deep into the subchondral bone, leading to the formation of osteochondral defects in the knee joint. Osteochondral regeneration is a process that is precisely regulated in time and space. Its core mechanism is that osteogenesis driven by angiogenesis is first initiated, which then triggers the cartilage formation process. In view of the multiple difficulties such as the limited regenerative capacity of cartilage tissue, the complex structure of joint defects, the lack of local stem cells, and the difficulty of existing technologies to achieve in situ regeneration of defect areas, there is an urgent need to develop a new type of biological scaffold. The scaffold should have triple functional characteristics: optimize the regenerative microenvironment by regulating angiogenesis, establish a cell recruitment channel to promote the homing of repair cells, and guide the directional differentiation of cells to achieve precise tissue regeneration, thereby activating the body's own repair potential.

[0003] For osteochondral tissue engineering, an ideal scaffold must not only possess excellent bioactivity, biocompatibility, a porous structure, and mechanical properties, but also possess morphological plasticity to conform to the three-dimensional contours of complex joint defects and self-healing properties to resist mechanical deformation caused by joint motion. However, single-material systems often struggle to integrate these complementary properties simultaneously. Self-healing hydrogels, due to their ability to autonomously repair their network structure and maintain mechanical integrity without external intervention, have shown unique value in long-term implant applications. SA-PBA / PVA hydrogels (SBPs), formed by dynamically crosslinking sodium alginate modified with phenylboronic acid (SA-PBA) and polyvinyl alcohol (PVA-1799) via boronate bonds, have shown potential in tissue repair applications. However, their limited biofunctionality and insufficient mechanical properties have limited their effectiveness in osteochondral repair. Silk protein, with its extraordinary strength exceeding that of steel, stems from its β-pleated nanocrystalline structure composed of highly conserved polyglycine-alanine (Gly-Ala) and polyalanine (Ala) domains. This property has led to its successful use as a reinforcing nanofiber in various tissue engineering applications. Therefore, the introduction of silk fibroin nanofibers (BSNFs) rich in β-pleated structure into hydrogel systems is expected to significantly improve the mechanical properties of composite materials. However, relying solely on hydrogel scaffolds is still difficult to effectively induce osteochondral regeneration.

[0004] Based on existing studies, we speculate that the combination of platelet-derived growth factor (PDGF-BB) and TGF-β3 will effectively promote angiogenesis, cell migration, and directional differentiation of stem cells. Considering that the sustained release of TGF-β3 can enhance cartilage regeneration in vivo, a strategy focusing on the sequential appearance of PDGF-BB and TGF-β3 during the repair process may be expected to achieve high-quality osteochondral regeneration. In addition, related studies have shown that low concentrations of Mg 2+ (100ppm) will upregulate osteogenic factors, thereby enhancing osteogenesis, while high concentrations of Mg 2+ (200ppm) will upregulate chondrogenic factors and promote cartilage formation. In order to design active factor-functionalized acellular scaffolds that can maintain the bioactivity of active factors and achieve optimal sequential delivery characteristics, we used Mg 2+ and Fe 3+ The 2D Mg-Fe-LDH nanosheets are used as effective carriers for drug delivery. By combining a matrix material loaded with PDGF-BB with Mg-Fe-LDH nanosheets loaded with TGF-β, a sequential release system for active factors was established, which may provide an innovative treatment path for the problem of osteochondral regeneration. Summary of the Invention

[0005] This study proposed a three-in-one design concept of "fiber reinforcement-timed drug release-ion regulation". We developed a multifunctional scaffold consisting of Mg-based nanosheets loaded with TGF-β3 and SA-PBA / PVA / BSNF hydrogel loaded with PDGF-BB, which achieved the effect of PDGF-BB and TGF-β3 / Mg 2+ Sequential release of PDGF-BB (for stem cell recruitment and angiogenesis) and TGF-β3 / Mg is achieved through the dynamic boronate network, BSNF nanofibers, and Mg-based nanosheets. 2+ This study innovatively developed a multifunctional bioscaffold that simultaneously regulates the natural repair cascade of angiogenesis, osteogenesis, and chondrogenesis, overcoming the limitations of existing osteochondral regeneration strategies. This scaffold achieves breakthroughs in the synergistic optimization of dynamic mechanical adaptability and bioactive functions, providing a novel solution for complex tissue regeneration.

[0006] The technical solution of the present invention comprises the following steps:

[0007] (1) To mimic the binding affinity of heparin with active factors in the human body and anchor TGF-β3 on the surface of LDH, we combined chondroitin sulfate (ChS) with negatively charged sulfate groups with TGF-β3 amino acid residues to form a ChS / TGF-β3 complex. Subsequently, the ChS / TGF-β3 complex attached to the surface of LDH through electrostatic interactions, forming an LDH / ChS / TGF-β3 controlled-release drug delivery system.

[0008] (2) Phenylboronic acid modified sodium alginate (SA-PBA), polyvinyl alcohol (PVA), β-sheet-rich silk fibroin nanofiber (BSNF)

[0009] Self-healing hydrogel scaffold (SBPS) is formed through the interaction of dynamic boronic acid bonds and hydrogen bonds.

[0010] (3) To functionalize the scaffold, LDH / ChS / TGF-β3 nanosheets were incorporated into SBPS hydrogel loaded with PDGF-BB to form a TGF-β3 / Mg 2+ Fiber-reinforced self-healing hydrogel scaffold (SBPSP-LT) with sustained release and burst release of PDGF-BB.

[0011] The present invention includes the following three key technical methods:

[0012] (1) Method for preparing LDH / ChS / TGF-β3 drug delivery system:

[0013] Principle: The negatively charged sulfate groups of ChS bind to TGF-β3 amino acid residues to form a ChS / TGF-β3 complex. This ChS / TGF-β3 complex attaches to the surface of LDH through electrostatic interactions, forming a stable LDH / ChS / TGF-β3 drug delivery system with controlled release of TGF-β3.

[0014] The preparation method comprises the following steps:

[0015] ① First, dissolve TGF-β3 in 100 μL sterile phosphate buffered saline (PBS) to obtain TGF-β3 solution (50

[0016] μg / mL);

[0017] ② ChS solution (0.05 mg / mL) was added to TGF-β3 solution and stirred at 4°C for 0.5 h to form ChS / TGF-β3 solution.

[0018] β3 complex;

[0019] ③ Then, Mg-Fe-LDHs solution (0.5 mg / mL) was added to the ChS / TGF-β3 solution and stirred gently at room temperature for 6 hours to form LDH / ChS / TGF-β3 suspension. After centrifugation at 9000 r / min for 3 times, LDH / ChS / TGF-β3 suspension was obtained.

[0020] β3 complex.

[0021] (2) Method for preparing self-healing hydrogel scaffold (SBPS):

[0022] Principle: Phenylboronic acid-modified sodium alginate (SA-PBA), polyvinyl alcohol (PVA), and β-sheet-rich silk fibroin nanofibers (BSNF) form a self-healing hydrogel scaffold through the interaction of dynamic boronic acid bonds and hydrogen bonds.

[0023] The preparation method comprises the following steps:

[0024] ① Synthesis of SA-PBA: Sodium alginate (2.0 g), EDC·HCl (1.92 g, 10 mmol), and 3-aminophenylboronic acid (0.78 g, 5 mmol) were dissolved in deionized water (200 mL). The mixture was stirred at room temperature for 24 hours and then dialyzed against distilled water for one week (Mw cutoff = 3500 Da). SA-PBA was obtained by freeze-drying.

[0025] ②Synthesis of BSNF: Raw silk was boiled in 0.02M Na2CO3 aqueous solution for 20 minutes and then rinsed thoroughly with distilled water to extract sericin. The extracted silk fibers were then dissolved in 9.3M LiBr solution at 60°C for 4 hours and dialyzed with distilled water (molecular cutoff of 3500) for 72 hours to remove salt. Subsequently, the solution was heated at 4°C.

[0026] The solution was centrifuged at 9000 rpm for 20 minutes to remove the silk aggregates formed during the process, yielding a transparent silk fibroin solution. The solution was slowly concentrated to approximately 20 wt% at 60°C to form metastable nanoparticles, and then diluted to 2 wt% with distilled water. The diluted solution was incubated at 60°C for 24 hours and then sonicated for 1 minute to induce nanofiber formation. The structure of the material was analyzed using FTIR and SEM.

[0027] ③ Preparation of SA-PBA / PVA hydrogel (SBP): First, dissolve 0.45g SA-PBA in 10mL DPBS and stir continuously until the reagent is completely dissolved. Add PVA into deionized water, heat to 95℃, and stir for 2 hours to obtain a 10wt% PVA solution. Use ultrasound to eliminate bubbles. Then add 4.5wt% SA-

[0028] The precursor of the SBP hydrogel was obtained by slowly stirring the PBA solution for 3 minutes. The precursor was poured into a mold, sealed with plastic wrap to prevent moisture loss, and placed at 4°C for 24 hours to remove bubbles. Subsequently, the formed hydrogel was stored in a -30°C refrigerator for 30 minutes and then thawed at room temperature. The final hydrogel was obtained after three freeze-thaw cycles. SBP hydrogels with different SA-PBA to PVA volume ratios were labeled as 8:2SBP, 7:3SBP, 6:4SBP, 5:5SBP, 4:6SBP, and 3:7SBP. The storage and loss moduli of the SBP hydrogels were measured by frequency sweep-based rheological testing to determine the optimal SA-PBA to PVA ratio.

[0029] ④ Preparation of SA-PBA / PVA / BSNF hydrogel (SBPS): 2% BSNF solution was slowly added to a 6:4 SBP solution and stirred for 3 minutes to obtain the SBPS hydrogel precursor. SBPS hydrogels with different SBP to BSNF volume ratios were labeled 9:1SBPS, 8:2SBPS, 7:3SBPS, and 6:4SBPS. Frequency sweep-based rheological tests were performed to determine the optimal SBP to BSNF ratio.

[0030] (3) Method for preparing self-healing hydrogel scaffold doped with Mg-Fe-LDH nanosheets:

[0031] Principle: SA-PBA, PVA, BSNF, and Mg-Fe-LDH interact through dynamic boric acid bonds and hydrogen bonds to form a self-healing hydrogel scaffold.

[0032] ① Preparation of LDH-doped SA-PBA / PVA / BSNF hydrogel (SBPS-L): BSNF solution containing LDH was gradually added to 6:4 SA-PBA / PVA solution while stirring continuously for 3 minutes to obtain a hydrogel precursor solution (BSNF:SBP=2:8). A series of hydrogels (denoted as SBPS, SBPS-L0.025, SBPS-L0.05, SBPS-L0.1, and SBPS-L0.2) were then prepared by incorporating Mg-Fe-LDH nanosheets into SBPS hydrogel at different loading weights (0.025, 0.05, 0.1, and 0.2 wt / vol%). The hydrogel precursor was poured into a mold, sealed with plastic wrap to prevent moisture loss, and stored at 4°C for 24 hours to eliminate bubbles. Subsequently, the formed hydrogel was stored at -30

[0033] oC in a refrigerator for 40 min and then thawed at room temperature, and this process was repeated three times to form SBPS-L hydrogels.

[0034] ② Preparation of functionalized hydrogel containing LDH / ChS / TGF-β3 complex (SPBSP-LT): LDH / ChS / TGF-

[0035] β3 was incorporated into the SPBS hydrogel containing PDGF-BB and the operation of 4(1) was repeated.

[0036] ③ Preparation of hydrogel containing TGF-β3 and PDGF-BB (abbreviated as SBPSPT-L): directly add ChS / TGF-β3 solution, PDGF-BB solution and LDHs into the hydrogel precursor solution, and repeat the operation of 4(1).

[0037] The beneficial effects of the present invention are as follows: in order to functionalize the scaffold, LDH / ChS / TGF-β3 nanosheets are incorporated into the SBPS hydrogel loaded with PDGF-BB, and the TGF-β3 / Mg 2+ The sustained release of PDGF-BB and the burst release of PDGF-BB in SBPSP-LT hydrogel were shown in vitro. 2+ The sequential release of promoted angiogenesis and enhanced stem cell recruitment and osteochondral differentiation in vitro. The developed hydrogel has the following advantages: ① The multiple cross-linking method significantly improved the overall mechanical properties of the hydrogel. ② The self-healing property enables the hydrogel to autonomously repair cracks, maintain structural integrity and mechanical properties, which is conducive to long-term use and maintain functional consistency. ③ The adaptive properties of the hydrogel enable it to adapt to irregular defects and prevent the scaffold from breaking during joint movement, which may prolong its effectiveness. ④ The hydrogel exhibits ROS scavenging ability through ROS-sensitive boronic acid bonds. The rupture of the boronic acid ester bond in the hydrogel not only enhances the adhesion of the hydrogel by exposing the hydroxyl group, but also promotes the growth of growth factors and Mg 2+ ⑤ The adhesive properties of the hydrogel can overcome some of the challenges associated with repair failure, such as scaffold detachment and translocation. ⑥ The reversible cross-linking sites in the hydrogel can be destroyed by minimal force from cell traction, allowing integrin receptors to cluster on the connecting domains in the hydrogel, leading to extensive cell-matrix interactions and accommodating cell spreading and migration. ⑦ The multifunctional hydrogel can make PDGF-BB, Mg 2+ and TGF-β3 are released successively, significantly promoting angiogenesis, stem cell recruitment and directional differentiation, and being used for in situ induced regeneration of osteochondral defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Preparation of tissue engineering bone and cartilage integrated scaffolds doped with Mg-Fe-LDH nanosheets

[0039] Figure 2 Physical properties of Mg-Fe-LDH nanosheets and hydrogels: (a) SEM crystal structure of Mg-Fe-LDH nanosheets; (b) 1H NMR spectrum of SA-PBA; (c) FTIR spectra of SF and BSNF; (d) FTIR spectra of BSNF, Mg-Fe-LDH, SA-PBA, PVA, SBPS, and SBPS-L; (e) SEM images of SBP, SBPS, and SBPS-L hydrogels; (f) SEM-EDS elemental mapping of SBPS-L hydrogel

[0040] Figure 3 Shape adaptation, self-healing and adhesion properties of hydrogels: (ac) Frequency sweep tests of SBP, SBPS and SBPS-L hydrogels; (d) Frequency sweep tests of SBP, SBPS and SBPS-L hydrogels with optimal concentrations; (e) G' and G" of SBPS-L hydrogel during strain amplitude oscillation (1 Hz, 25°C); (f) G' and G" of SBPS-L hydrogel obtained by alternating strain sweep; (g) Design of the self-healing behavior process of SBPS-L hydrogel: the hydrogel was fused for 30 minutes and then stretched without breaking; (h) Tensile fracture stress-strain curve of SBPS-L hydrogel after self-healing; (i) Lap shear test to detect the adhesion properties of hydrogel on pig skin; (j) Adhesion strength of hydrogel. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below with reference to specific embodiments.

[0042] Example 1: Preparation of LDH / ChS / TGF-β3 drug delivery system

[0043] Preparation of LDH / ChS / TGF-β3 drug delivery system: First, TGF-β3 was dissolved in 100 μL sterile phosphate buffered saline (PBS) to obtain a TGF-β3 solution (50 μg / mL). Then, ChS solution (0.05 mg / mL) was added and stirred at 4°C for 0.5 hours to form a ChS / TGF-β3 complex. Subsequently, Mg-Fe-LDHs solution (0.5 mg / mL) was added to the ChS / TGF-β3 solution and gently stirred at room temperature for 6 hours to form an LDH / ChS / TGF-β3 composite material. The LDH / ChS / TGF-β3 complex was obtained after centrifugation at 9000 r / min three times. The crystal structure of LDH was detected by SEM, XRD, FTIR and other methods.

[0044] Example 2: Synthesis of SA-PBA

[0045] Sodium alginate (2.0 g), EDC·HCl (1.92 g, 10 mmol), and 3-aminophenylboronic acid (0.78 g, 5 mmol) were dissolved in deionized water (200 mL). The mixture was stirred at room temperature for 24 hours and then dialyzed against distilled water for one week (Mw cutoff = 3500 Da). SA-PBA was obtained by freeze-drying. The grafting yield was determined by 1H NMR spectroscopy.

[0046] Example 3: Synthesis of BSNF

[0047] Raw silk was boiled in a 0.02M Na2CO3 aqueous solution for 20 minutes and then thoroughly rinsed with distilled water to extract sericin. The extracted silk fibers were then dissolved in a 9.3M LiBr solution at 60°C for 4 hours and dialyzed with distilled water (cutoff Mw = 3500) for 72 hours to remove salts. Subsequently, the solution was centrifuged at 9000 rpm at 4°C for 20 minutes to remove the silk aggregates formed in the process to obtain a transparent silk fibroin solution. The solution was slowly concentrated to about 20wt% at 60°C to form metastable nanoparticles, and then diluted to 2wt% with distilled water. The diluted solution was incubated at 60°C for 24 hours and then ultrasonically treated for 1 minute to induce nanofiber formation. The structure of the material was analyzed using FTIR and SEM.

[0048] Example 4: Preparation of SA-PBA / PVA hydrogel (SBP)

[0049] First, 0.45 g of SA-PBA was dissolved in 10 mL of DPBS and stirred continuously until the reagent was completely dissolved. PVA was added to deionized water, heated to 95°C, and stirred for 2 hours to obtain a 10 wt% PVA solution. Air bubbles were removed using ultrasound. The 10 wt% PVA solution was then added to the 4.5 wt% SA-PBA solution and stirred slowly for 3 minutes to obtain the SBP hydrogel precursor. This precursor was poured into a mold, sealed with plastic wrap to prevent water loss, and stored at 4°C for 24 hours to remove air bubbles. The resulting hydrogel was then stored in a -30°C freezer for 30 minutes and then thawed at room temperature. The final hydrogel was obtained after three freeze-thaw cycles. SBP hydrogels with different SA-PBA to PVA volume ratios were labeled 8:2SBP, 7:3SBP, 6:4SBP, 5:5SBP, 4:6SBP, and 3:7SBP. The storage and loss moduli of SBP hydrogels were examined by frequency sweep-based rheological tests to determine the optimal SA-PBA to PVA ratio.

[0050] Example 5: Preparation of SA-PBA / PVA / BSNF hydrogel (SBPS)

[0051] SBPS hydrogel precursors were obtained by slowly adding a 2% BSNF solution to a 6:4 SBP solution and stirring for 3 minutes. SBPS hydrogels with different SBP to BSNF volume ratios were labeled 9:1 SBPS, 8:2 SBPS, 7:3 SBPS, and 6:4 SBPS. Frequency sweep-based rheological tests were performed to determine the optimal SBP to BSNF ratio and examine the storage and loss moduli of the SBPS hydrogels.

[0052] Example 6: Preparation of LDH-doped SA-PBA / PVA / BSNF hydrogel (SBPS-L)

[0053] The BSNF solution containing LDH was gradually added to the 6:4 SA-PBA / PVA solution while continuously stirring for 3 minutes to obtain a hydrogel precursor solution (BSNF:SBP = 2:8). A series of hydrogels (denoted as SBPS, SBPS-L0.025, SBPS-L0.05, SBPS-L0.1, and SBPS-L0.2) were then prepared by incorporating Mg-Fe-LDH nanosheets into SBPS hydrogels at different loading weights (0.025, 0.05, 0.1, and 0.2 wt / vol%).

[0054] Example 7: Preparation of functionalized hydrogel containing LDH / ChS / TGF-β3 complex (SPBSP-LT)

[0055] As shown in Example 6, LDH / ChS / TGF-β3 was incorporated into a SPBS hydrogel containing PDGF-BB. The hydrogel precursor was poured into a mold, sealed with plastic wrap to prevent water loss, and stored at 4°C for 24 hours to eliminate air bubbles. The resulting hydrogel was then stored in a -30°C freezer for 40 minutes and then thawed at room temperature. This process was repeated three times to form an SBPSP-LT hydrogel.

[0056] Example 8: Preparation of a hydrogel containing TGF-β3 and PDGF-BB (SBPSPT-L for short)

[0057] As shown in Example 7, the ChS / TGF-β3 solution, PDGF-BB solution and LDHs were directly added to the hydrogel precursor solution and magnetically stirred to form the SBPSPT-L hydrogel.

[0058] Example 9: Testing of scaffold physical and chemical properties

[0059] Based on the ISO10993 series of standards, the scaffold's morphological structure, mechanical properties, biosafety, and degradation and absorption properties, among other physical and chemical properties, were tested using methods such as SEM, FTIR, mechanical testing, degradation testing, cytotoxicity testing, and genotoxicity testing. The test results demonstrate that the osteochondral composite tissue engineering scaffold of the present invention has the required internal spatial structure; exhibits good tissue compatibility and biosafety, facilitating cell adhesion and proliferation; possesses good mechanical strength, meeting the mechanical requirements of the implant site; and exhibits controllable degradation and absorption, with the rate of degradation and absorption being artificially adjusted to match the rate of new tissue growth in the body.

[0060] Example 10: Verification of stent biological performance in vitro and in vivo

[0061] In vitro cell experiments confirmed that the sustained release of PDGF-BB from the hydrogel matrix could stimulate the tube formation and migration of human umbilical vein endothelial cells; the hydrogel matrix sustained release of TGF-β3 and Mg through Mg-Fe-LDH nanosheets 2+ , promoting the differentiation of bone marrow mesenchymal stem cells towards osteoblasts / chondrogenesis. It is predicted that after the hydrogel is implanted into the osteochondral defect model, it provides the required stimulation, promotes angiogenesis, recruits endogenous stem cells, enhances their directional differentiation, and finally achieves satisfactory regeneration. The results of the in vivo implantation repair experiment in animals (rats) show that the engineered osteochondral composite tissue constructed using the osteochondral composite tissue engineering scaffold of the present invention is completely integrated with the surrounding normal tissue after implantation in the body, and the degradation time is basically matched with the growth rate of the surrounding new tissue; the cartilage part of the repair tissue has biological properties similar to natural articular cartilage, and the subchondral bone part can produce bony fusion with the host bone tissue in a relatively short time.

Claims

1. A method for preparing a fiber-reinforced self-repairing hydrogel-mediated timed drug release scaffold for promoting vascularized osteochondral regeneration, characterized in that The following steps are involved: (1) To mimic the binding affinity of heparin with active factors in the human body and anchor TGF-β3 on the surface of LDH, we combined chondroitin sulfate (ChS) with negatively charged sulfate groups with TGF-β3 amino acid residues to form a ChS / TGF-β3 complex. Subsequently, the ChS / TGF-β3 complex attached to the surface of LDH through electrostatic interactions, forming an LDH / ChS / TGF-β3 controlled-release drug delivery system. (2) Phenylboronic acid-modified sodium alginate (SA-PBA), polyvinyl alcohol (PVA), and β-sheet-rich silk nanofibers (BSNF) form self-healing hydrogel scaffolds (SBPS) through dynamic boronic acid bonds and hydrogen bonds. (3) To functionalize the scaffold, LDH / ChS / TGF-β3 nanosheets were incorporated into SBPS hydrogel loaded with PDGF-BB to form a TGF-β3 / Mg 2+ Fiber-reinforced self-healing hydrogel scaffold (SBPSP-LT) with sustained release and burst release of PDGF-BB.

2. The preparation method according to claim 1, wherein: The preparation of the LDH / ChS / TGF-β3 drug controlled release delivery system comprises the following steps: (1) First, TGF-β3 was dissolved in 100 μL of sterile phosphate-buffered saline (PBS) to obtain a TGF-β3 solution (50 μg / mL); (2) ChS solution (0.05 mg / mL) was added to the TGF-β3 solution and stirred at 4°C for 0.5 h to form a ChS / TGF-β3 complex; (3) Subsequently, the Mg-Fe-LDHs solution (0.5 mg / mL) was added to the ChS / TGF-β3 solution and gently stirred at room temperature for 6 hours to form an LDH / ChS / TGF-β3 suspension. The suspension was centrifuged three times at 9000 rpm to obtain the LDH / ChS / TGF-β3 complex.

3. The preparation method according to claim 1, wherein: The preparation of the self-healing hydrogel scaffold (SBPS) comprises the following steps: (1) Synthesis of SA-PBA: Sodium alginate (2.0 g), EDC·HCl (1.92 g, 10 mmol) and 3-aminophenylboronic acid (0.78 g, 5 mmol) were dissolved in deionized water (200 mL). The mixture was stirred at room temperature for 24 h and then dialyzed in distilled water for one week (cut-off Mw = 3500 Da). SA-PBA was obtained by freeze-drying. (2) Synthesis of BSNF: Raw silk was boiled in a 0.02M Na2CO3 aqueous solution for 20 minutes and then thoroughly rinsed with distilled water to extract sericin. The extracted silk fibers were then dissolved in a 9.3M LiBr solution at 60°C for 4 hours and dialyzed against distilled water (cutoff Mw = 3500) for 72 hours to remove salts. Subsequently, the solution was centrifuged at 9000 rpm at 4°C for 20 minutes to remove the silk aggregates formed during the process, resulting in a transparent silk fibroin solution. The solution was slowly concentrated to about 20 wt% at 60°C to form metastable nanoparticles and then diluted to 2 wt% with distilled water. The diluted solution was incubated at 60°C for 24 hours and then ultrasonicated for 1 minute to induce nanofiber formation. The structure of the material was analyzed using FTIR and SEM. (3) Preparation of SA-PBA / PVA hydrogel (SBP): First, 0.45 g of SA-PBA was dissolved in 10 mL of DPBS and stirred continuously until the reagent was completely dissolved. PVA was added to deionized water, heated to 95 °C, and stirred for 2 hours to obtain a 10 wt% PVA solution. Ultrasonication was used to eliminate bubbles. Then, the 10 wt% PVA solution was added to the 4.5 wt% SA-PBA solution and stirred slowly for 3 minutes to obtain the precursor of the SBP hydrogel. The precursor was poured into a mold, sealed with plastic wrap to prevent water loss, and placed at 4 °C for 24 hours to remove bubbles. Subsequently, the formed hydrogel was stored in a refrigerator at -30 °C for 30 minutes and then thawed at room temperature. The final hydrogel was obtained after three freeze-thaw cycles. SBP hydrogels with different SA-PBA to PVA volume ratios were labeled as 8:2SBP, 7:3SBP, 6:4SBP, 5:5SBP, 4:6SBP, and 3:7SBP, respectively. The storage and loss moduli of SBP hydrogels were examined by frequency sweep-based rheological tests to determine the optimal SA-PBA to PVA ratio. (4) Preparation of SA-PBA / PVA / BSNF hydrogel (SBPS): 2% BSNF solution was slowly added to a 6:4 SBP solution and stirred for 3 minutes to obtain the SBPS hydrogel precursor. SBPS hydrogels with different SBP to BSNF volume ratios were labeled as 9:1SBPS, 8:2SBPS, 7:3SBPS, and 6:4SBPS. The storage and loss moduli of the SBPS hydrogels were measured by frequency sweep-based rheological testing to determine the optimal SBP to BSNF ratio.

4. The preparation method according to claim 1, wherein: The preparation of the self-healing hydrogel scaffold doped with Mg-Fe-LDH nanosheets comprises the following steps: (1) Preparation of LDH-doped SA-PBA / PVA / BSNF hydrogel (SBPS-L): BSNF solution containing LDH was gradually added to 6:4 SA-PBA / PVA solution while continuously stirring for 3 minutes to obtain a hydrogel precursor solution (BSNF:SBP = 2:8). A series of hydrogels (denoted as SBPS, SBPS-L0.025, SBPS-L0.05, SBPS-L0.1, and SBPS-L0.2) were then prepared by incorporating Mg-Fe-LDH nanosheets into SBPS hydrogels at different loading weights (0.025, 0.05, 0.1, and 0.2 wt / vol%). The hydrogel precursor was poured into a mold, sealed with plastic wrap to prevent moisture loss, and stored at 4°C for 24 hours to eliminate bubbles. Subsequently, the formed hydrogel was stored in a refrigerator at -30°C for 40 minutes and then thawed at room temperature. This process was repeated three times to form SBPS-L hydrogel. (2) Preparation of functionalized hydrogel containing LDH / ChS / TGF-β3 complex (SPBSP-LT): LDH / ChS / TGF-β3 was incorporated into SPBS hydrogel containing PDGF-BB, and the operation of 4(1) was repeated. (3) Preparation of hydrogel containing TGF-β3 and PDGF-BB (abbreviated as SBPSPT-L): ChS / TGF-β3 solution, PDGF-BB solution and LDHs were directly added to the hydrogel precursor solution, and the operation of 4(1) was repeated.

5. The LDH / ChS / TGF-β3 drug controlled release delivery system as claimed in claim 2 has high loading and encapsulation efficiency, and can achieve the effect of TGF-β3 and Mg 2+ sustained release.

6. The SBPS as described in claim 3 has key properties such as adaptive and self-repair properties, adhesion ability, and reactive oxygen species scavenging ability, making it suitable for complex osteochondral defect repair applications.

7. The fiber-reinforced self-repairing hydrogel-mediated timed drug release scaffold for promoting vascularized osteochondral regeneration (SPBSP-LT) as claimed in claim 4, wherein TGF-β3 / Mg 2+ The sustained release of PDGF-BB and the burst release of PDGF-BB promote angiogenesis, recruit endogenous stem cells and enhance their directional differentiation to achieve efficient osteochondral regeneration.

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