Preparation method of drug sustained-release hydrogel scaffold and application of hiPSCs in three-dimensional osteogenic induction

Abstract

The application discloses a preparation method of a drug sustained-release hydrogel support and a three-dimensional osteogenic induction application of hiPSCs. Firstly, a CHIR99021@BSA-DEX@PLGA time-sequential double sustained-release composite microsphere of a core-shell structure is prepared, then the freeze-dried GelMA powder is dissolved in a PBS solution containing a photoinitiator LAP, the CHIR99021@BSA-DEX@PLGA time-sequential double sustained-release composite microsphere is added after stirring and cooling, and a drug-loaded hydrogel precursor is obtained; the precursor is injected into a mold and irradiated by a specific wavelength light source to initiate free radical polymerization of a methacryl group into glue, and a drug-loaded GelMA porous hydrogel support is obtained after demolding, freezing and drying; the drug-loaded GelMA porous support is covalently grafted with VN polypeptide through EDC / NHS chemical coupling to obtain a VN-GelMA drug-loaded porous hydrogel support. The application simulates the signal time sequence regulation law in the embryonic bone formation process and significantly improves the osteogenic differentiation efficiency and mineralization quality by constructing a biomimetic three-dimensional osteogenic microenvironment. The VN polypeptide enhances the specific recognition of stem cells and significantly improves the three-dimensional cell compatibility. The application can reduce the dependence on exogenous induction factors and is safe, low in cost and stable in induction.

Description

Technical Field

[0001] This invention relates to the field of clinical bone defect repair technology, specifically a method for preparing a drug-release hydrogel scaffold and the application of hiPSCs in three-dimensional osteogenic induction. Background Technology

[0002] Clinical bone defect repair still faces key challenges, including limited autologous bone sources, risks of immune rejection and infection associated with allogeneic bone, and insufficient bioactivity of traditional artificial materials. Bone tissue engineering, through a combination of scaffold materials, seed cells, and bioactive factors, achieves functional bone regeneration and has become the most promising therapeutic strategy for clinical translation. Human induced pluripotent stem cells (hiPSCs) possess unlimited proliferative capacity and tri-germ layer differentiation potential, making them ideal seed cells for bone regeneration. Three-dimensional culture systems more closely resemble the in vivo physiological microenvironment, significantly improving the osteogenic differentiation efficiency and functional maturity of stem cells. Methacrylamide gelatin (GelMA) is a commonly used three-dimensional cell culture scaffold.

[0003] From the perspective of embryonic development, osteogenesis is a continuous cascade reaction process synergistically regulated by multiple signaling networks in time and space, which can be divided into three consecutive temporal stages: the early stage, in which pluripotent stem cells complete mesodermal lineage commitment under the activation of signals such as Wnt; the middle stage, in which precursor cells proliferate under the regulation of factors such as BMP (bone morphogenetic protein) and FGF (fibroblast growth factor); and the late stage, in which they secrete extracellular matrix and complete mineralization under the action of maturation-promoting signals such as glucocorticoids.

[0004] Currently, existing scaffold materials used for osteogenic guidance have the following drawbacks: (1) The limitations of two-dimensional culture are obvious: In the traditional two-dimensional stem cell induction system, the cell morphology, proliferation, differentiation and in vivo microenvironment are very different, which cannot simulate the three-dimensional growth state in vivo, resulting in low osteogenic differentiation efficiency and insufficient functional expression of stem cells. (2) Simple hydrogel scaffolds have limited functions: simple GelMA hydrogels have low bioactivity, weak osteogenic induction ability, and lack specific cell adhesion sites, and cannot provide a stable, biomimetic, and efficient three-dimensional osteogenic induction microenvironment for hiPSCs. (3) Lack of temporal signal regulation: Most existing osteogenic induction systems use static multi-factor mixed culture media, which differs significantly from the temporal regulation logic of alternating signals during bone development in vivo. Wnt signaling requires high-level activation in the early differentiation stage to drive osteogenic lineage formation, but continuous activation in the middle and late stages can inhibit terminal cell maturation and mineralization; glucocorticoids (such as dexamethasone DEX) can promote mineralization in the middle and late differentiation stages, but premature exposure may inhibit precursor cell proliferation; (4) Insufficient integration of composite scaffold functions: The reported composite scaffold materials have single functions and cannot simultaneously achieve multiple functions such as controllable drug loading, stable hydrogel structure, efficient adhesion and proliferation of stem cells, directed osteogenic differentiation, and real-time fluorescence tracing. (5) Strong dependence on exogenous inducing factors: Most osteogenic induction systems rely on exogenous inducing factors, which have defects such as high cost, high safety risk and unstable induction effect. There is a lack of safe, efficient and low-toxic integrated material systems. Summary of the Invention

[0005] To overcome the shortcomings of the existing technology, the present invention provides a method for preparing a drug-release hydrogel scaffold (VN-GelMA drug-loaded porous hydrogel scaffold) and the application of the drug-release hydrogel scaffold in three-dimensional osteogenic induction of hiPSCs.

[0006] This invention provides the following technical solution: A method for preparing a drug-release hydrogel scaffold, the method comprising the following steps: (1) A core-shell structured CHIR99021@BSA-DEX@PLGA time-release composite microsphere was prepared by using a single emulsion-solvent evaporation method combined with protein self-assembly technology; (2) The lyophilized GelMA powder was dissolved in PBS solution containing 0.125% w / v photoinitiator LAP at a concentration of 7.5% w / v. The solution was stirred in a water bath at 50°C in the dark until completely dissolved. The pH was adjusted to 7.0~7.4. After cooling to room temperature, the CHIR99021@BSA-DEX@PLGA time-dependent dual sustained-release composite microspheres were added and mixed thoroughly to obtain the drug-loaded hydrogel precursor solution. (3) The drug-loaded hydrogel precursor solution was injected into a sterile mold and vertically irradiated with a 405-410nm visible LED light source for 8-12 seconds to initiate the free radical polymerization of methacryloyl groups into a gel; after demolding, it was pre-frozen at -80°C for 2 hours and freeze-dried for 48 hours. A three-dimensional interconnected porous network was constructed by ice crystal sublimation to obtain a drug-loaded GelMA porous hydrogel scaffold. (4) Prepare MES buffer containing EDC and NHS, filter it through a 0.22 μm filter membrane and set it aside; place the lyophilized drug-loaded GelMA porous hydrogel scaffold in a well plate, add the prepared MES buffer, and shake at room temperature to activate the carboxyl groups of the gel side chains; aspirate the liquid, wash twice with sterile PBS, then add a VN polypeptide aqueous solution with a concentration of 50 μg / mL, seal with a sealing film, and incubate at 37°C for 120 minutes; aspirate the excess polypeptide solution, add F12 culture medium and place at 4°C for 12 hours to block unreacted active sites; wash thoroughly three times with deionized water, freeze dry, and obtain the VN-GelMA drug-loaded porous hydrogel scaffold.

[0007] Preferably, in step (1), the preparation of the CHIR99021@BSA-DEX@PLGA time-sequential dual sustained-release composite microspheres includes the following steps: (1-1) Preparation of DEX@PLGA core microspheres: PLGA in a 50:50 ratio was dissolved in acetone, and dexamethasone (DEX) was added and fully dissolved to form the oil phase. A 1% w / v PVA aqueous solution was prepared as the aqueous phase. The oil phase was slowly added dropwise to the aqueous phase under an ice-water bath, and ultrasonically broken up to form a homogeneous O / W emulsion. The O / W emulsion was transferred to a magnetic stirrer and stirred at 500 rpm at room temperature in the dark for 24 hours to allow the organic solvent to evaporate completely. The emulsion was centrifuged at 9500×g for 10 minutes at 4°C, the precipitate was collected, washed three times with deionized water, and freeze-dried to obtain DEX@PLGA core microspheres. (1-2) BSA shell coating: DEX@PLGA core microspheres were dissolved in deionized water by ultrasonic dispersion, and then the aqueous solution was added dropwise to a BSA solution with a concentration of 4% w / v. The mixture was stirred slowly at 300 rpm for 1 to 3 hours to make BSA uniformly coat the surface of DEX@PLGA core microspheres and form a BSA shell. (1-3) CHIR99021 loading: CHIR99021-DMSO stock solution with a concentration of 20mM was slowly added dropwise to the system, placed in an ice-water bath and protected from light with aluminum foil, and stirred continuously at 300rpm for 12-16h to allow CHIR99021 to be fully adsorbed in the BSA shell. (1-4) Crosslinking and purification: Glutaraldehyde solution was slowly added dropwise to the system to a final concentration of 0.2% v / v. The crosslinking reaction was carried out at room temperature for 2 hours. After the reaction was completed, the mixture was centrifuged at 4°C and 13,800×g for 15 minutes to remove the supernatant. The mixture was washed three times with deionized water to remove unreacted crosslinking agent and free drug. The precipitate was pre-frozen at -80°C and freeze-dried for 48 hours to obtain CHIR99021@BSA-DEX@PLGA time-dependent dual sustained-release composite microsphere powder.

[0008] Preferably, in step (1-1), the mass-to-volume ratio of the PLGA, acetone, dexamethasone, and PVA aqueous solution is 100 mg: 2 mL: 10 mg: 10 mL.

[0009] Preferably, in step (1-2), the mass-to-volume ratio of the DEX@PLGA microspheres, deionized water, and BSA solution is 50 mg: 2 mL: 10 mL.

[0010] Preferably, in step (2), the CHIR99021@BSA-DEX@PLGA time-dependent dual sustained-release composite microspheres are added to the reaction system at a mass / volume ratio of 20 mg / mL.

[0011] Preferably, in step (2), the preparation of GelMA includes the following steps: adding type A porcine gelatin to PBS buffer preheated to 50°C and stirring until completely dissolved to form a gelatin solution with a concentration of 10% w / v; slowly adding methacrylic anhydride dropwise under vigorous stirring, maintaining the pH at 7.5~8.0 during the dropwise addition; reacting for 3 hours in the dark at 50°C and 300 rpm; after the reaction is completed, adding PBS buffer preheated to 40°C to dilute and terminate the reaction, transferring the solution to a dialysis bag with a molecular weight cutoff of 12~14 kDa, and dialyzing in deionized water at 40°C for 7 days; after dialysis, pre-freezing and vacuum freeze-drying for 48 hours to obtain GelMA solid powder.

[0012] Preferably, in the preparation of the GelMA, the mass-to-volume ratio of type A porcine skin gelatin to 50°C PBS buffer is 10 g: 100 mL; and the molar ratio of methacrylic anhydride to gelatin solution is 10:1.

[0013] Preferably, in step (4), the concentrations of EDC, NHS and MES in the MES buffer containing EDC and NHS are 0.1M, 0.05M and 0.1M, respectively.

[0014] This invention further discloses the VN-GelMA drug-loaded porous hydrogel scaffold prepared by the above method.

[0015] This invention further discloses the application of the above-mentioned VN-GelMA drug-loaded porous hydrogel scaffold in three-dimensional osteogenic induction of hiPSCs.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) Breaking through the limitations of traditional two-dimensional culture, constructing a highly biomimetic three-dimensional osteogenic microenvironment: This invention uses a porous three-dimensional hydrogel scaffold to simulate the microenvironment of bone tissue growth in vivo. A highly interconnected porous structure with a porosity of about 75% and an average pore size of 120~150μm is prepared by photocrosslinking combined with freeze-drying process. This effectively improves the morphology, proliferation and differentiation behavior of stem cells, significantly enhances the osteogenic differentiation efficiency and functional maturity of hiPSCs, and solves the problem of large differences between the two-dimensional system and the physiological state in vivo and insufficient osteogenic effect.

[0017] (2) Innovative time-sequential dual sustained-release design to accurately simulate the signal regulation rhythm of bone development: This invention utilizes the intrinsic difference in degradation kinetics between BSA and PLGA to integrate the Wnt agonist CHIR99021 (outer BSA phase) and dexamethasone DEX (core PLGA phase) into the same core-shell structure microsphere, realizing the spontaneous time-sequential release of "early pulsed Wnt activation → late continuous glucocorticoid maturation promotion". It can simulate the signal time-sequential regulation law in the process of embryonic bone formation without artificial intervention, and solves the key problem of mismatch between the existing static induction system and the developmental time sequence.

[0018] (3) VN peptide functionalization enhances stem cell specific recognition and significantly improves three-dimensional cell compatibility: This invention covalently grafts viscosin-derived functional peptides (VN) onto the scaffold pore wall through EDC / NHS chemical coupling, which improves the ability of GelMA scaffold to recognize, adhere to, proliferate and deeply infiltrate hiPSCs, and promotes the transformation of cells from the pluripotency maintenance state to the subsequent differentiation state, overcoming the technical defects of simple GelMA lacking specific stem cell recognition sites and having low cell adhesion efficiency.

[0019] (4) The time-sequential dual sustained-release synergistic effect significantly improves osteogenic differentiation efficiency and mineralization quality: In this invention, the early release of CHIR99021 can effectively drive the orientation of the mesodermal lineage, while the continuous release of DEX in the middle and late stages further promotes osteogenic maturation. The two form an effective synergistic induction effect in time sequence.

[0020] (5) Reduced dependence on exogenous inducing factors, safe and low cost, stable induction: This invention relies on the material's own temporal signal regulation to achieve efficient osteogenic induction, without the need to add high-cost exogenous growth factors, thus reducing cost and biosafety risks; the induction process is spontaneously driven by the material degradation kinetics, stable and repeatable, and more suitable for practical applications and clinical translation of bone tissue engineering.

[0021] (6) The preparation process is mild, green, and scalable, with great potential for clinical translation: In this invention, the synthesis of GelMA adopts a mild acylation reaction with controllable conditions and adjustable degree of substitution; the microsphere preparation adopts a single emulsion-solvent evaporation method combined with protein self-assembly technology, which is a mature process; the hydrogel is cross-linked and formed by visible light initiation, which is simple to operate and has low cytotoxicity; the overall process is easy to scale up for production and has good scientific research value and clinical translation prospects. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the technical principle of the present invention.

[0023] Figure 2Characterization of the materials; where a: schematic diagram of the preparation of CHIR / BSA and DEX / PLGA composite GelMA scaffold; b: appearance of GelMA precursor solution and scaffold after gelation; cd: scanning electron microscope (SEM) image of GelMA scaffold, inset is a magnified view; e: Fourier transform infrared spectrum of GelMA; f: ¹H NMR spectrum of gelatin and GelMA; g: Fourier transform infrared spectrum of PLGA, PLGA@DEX, PLGA-BSA@CHIR and PLGA@DEX-BSA@CHIR.

[0024] Figure 3 In the figures, ab: Scanning electron microscope (SEM) images of PLGA and CHIR99021@BSA-DEX@PLGA particles; c: Microsphere size characterization results of PLGA and CHIR99021@BSA-DEX@PLGA; de: Porosity and swelling ratio of scaffolds with different concentrations of GelMA (5%, 7.5%, 10%); f: Rheological properties of GelMA hydrogels; gh: Tensile property analysis of scaffolds after GelMA, EDC / NHS treatment and with added sustained-release particles, including fracture strain and stress-strain curves; i: In vitro sustained-release curves of DEX and CHIR99021.

[0025] Figure 4 In the figures, a: Live / Dead staining images and laser confocal 3D reconstruction images of the VN− and VN+ groups on days 1, 3, 5, and 7 of culture, showing live cells, dead cells, merged images, and XYZ, XZ, and Depth views, respectively; b: Scanning electron microscope (SEM) images of cell adhesion, growth, and spreading on the scaffold well wall surface on day 7; c: Cell viability analysis of the VN− and VN+ groups at different culture times; de: Relative expression levels of stemness-related genes Nanog and OCT4 in the VN− and VN+ groups on days 1, 3, and 5 of culture.

[0026] Figure 5 Immunofluorescence staining of OCT4, a cell stem cell marker, under different induction conditions; where ad represents the OCT4 immunofluorescence staining images of the Blank group, CHIR99021+ group, DEX+ group, and CHIR+DEX+ group on days 1, 3, 5, and 7 of culture, respectively. Cell nuclei are stained with DAPI (blue), and OCT4 is green. The composite image shows the changes in OCT4 expression in each group of cells at different culture times.

[0027] Figure 6To evaluate cell lineage differentiation and osteogenic correlation under different induction conditions; where ae: relative expression levels of lineage-related genes T, MSX1, GATA4, AFP3, and SOX17 in the Blank group, CHIR99021+ group, DEX+ group, and CHIR99021+DEX+ group on days 3 and 5 of induction; f: relative expression level of stemness marker gene OCT4 in each group on days 7, 14, 21, and 28 of induction; gj: relative expression levels of osteogenic related genes RUNX2, ALP, COL1A1, and OCN in each group on days 7, 14, 21, and 28 of induction; k: Alizarin Red staining images of each group at different induction time points; l: quantitative analysis results of Alizarin Red staining (490 nm).

[0028] Figure 7 This section presents the experimental and histological evaluation of ectopic osteogenicity using composite GelMA scaffolds. A: Schematic diagram of the ectopic osteogenicity experimental procedure, including cell aggregation, embryoid body (EB) formation, scaffold seeding, osteogenic induction, in vivo implantation, and sample evaluation; b: Gross appearance of samples removed after implantation from the Blank, Control, and Composite GelMA groups; c: Masson staining images of samples from each group; d: HE staining images of samples from each group. In c and d, the left image is a low-power image, and the right image is a high-power image.

[0029] Figure 8 This study evaluates the repair efficacy and in vivo safety of the GelMA scaffold in a skull defect model. Specifically, ab: gross appearance images of specimens taken at 4 and 8 weeks post-surgery from the Blank, Control, and Composite GelMA groups, with the defect area indicated by a red box; c: Micro-CT reconstructed images and cross-sectional views of each group at 4 and 8 weeks post-surgery; d, fh: quantitative analysis of bone morphological parameters extracted from Micro-CT images, including structural model index (SMI), bone surface area / bone volume ratio (BS / BV), bone surface area / tissue volume ratio (BS / TV), and trabecular bone number (Tb.N); il: blood routine index analysis of the Blank, Control, and Composite GelMA groups, including red blood cell count (RBC), white blood cell count (WBC), platelet count (PLT), and hemoglobin (HGB). Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] like Figure 1 and Figure 2As shown in Figure a, the composite system constructed in this invention consists of a CHIR99021@BSA shell and a DEX@PLGA core, which are further composited into a VN-GelMA porous hydrogel scaffold. The construction steps are as follows: constructing a DEX@PLGA core → forming a BSA shell on the surface of the DEX@PLGA core to form BSA-DEX@PLGA particles → loading CHIR99021 into the BSA shell to form CHIR99021@BSA-DEX@PLGA particles → composite the CHIR99021@BSA-DEX@PLGA particles into a GelMA gel to form a GelMA composite scaffold → covalently grafting VN peptides onto the GelMA composite scaffold to form a VN-GelMA drug-loaded composite porous scaffold.

[0032] Example 1: Synthesis of GelMA (methacrylamide gelatin) GelMA was synthesized from type A porcine skin gelatin using the methacrylic anhydride acylation method.

[0033] Specific method: Add 10g of type A porcine skin gelatin (gel strength 300 bloom) to 100mL of PBS (phosphate buffer, pH=7.4) preheated to 50°C, and stir at 300rpm for about 1 hour until completely dissolved to form a 10% (w / v) gelatin solution; under vigorous stirring (500rpm), slowly add 8mL of methacrylic anhydride (MA) dropwise at a rate of 0.5mL / min, with a molar ratio of MA to gelatin of approximately 10:1 (relative to lysine residues). During the addition process, the pH was continuously monitored and maintained at 7.5-8.0 with 5M NaOH. After the MA addition was completed, the reaction was continued at 50°C and 300rpm for 3 hours in the dark. After the reaction was completed, 500mL of PBS preheated to 40°C was added to dilute and terminate the reaction. The mixture was then transferred to a dialysis bag with a molecular weight cutoff of 12-14kDa and dialyzed in deionized water at 40°C for 7 days, with the dialysate changed 2-3 times a day. After dialysis, the mixture was pre-frozen and freeze-dried under vacuum for 48 hours to obtain GelMA solid powder, which was stored at -20°C for later use.

[0034] Example 2: Preparation of CHIR99021@BSA-DEX@PLGA time-sequential dual sustained-release composite microspheres A time-dependent dual-release composite microsphere with a core-shell structure was prepared by using a single emulsion-solvent evaporation method combined with protein self-assembly technology.

[0035] (1) Preparation of DEX@PLGA core microspheres: 100 mg of PLGA (polylactic acid-glycolic acid copolymer) in a 50:50 ratio was accurately weighed and dissolved in 2 mL of acetone. 10 mg of dexamethasone (DEX) was added and dissolved completely to form the oil phase. 10 mL of 1% (w / v) PVA (polyvinyl alcohol) aqueous solution was prepared as the aqueous phase. The oil phase was slowly added dropwise to the aqueous phase under an ice-water bath, while the mixture was treated with an ultrasonic disruptor (70% amplitude, 5 seconds of sonication followed by a 3-second pause) for 5 minutes to form a homogeneous O / W emulsion. The O / W emulsion was then transferred to a magnetic stirrer and stirred at 500 rpm at room temperature in the dark for 24 hours to allow the organic solvent to evaporate completely. The emulsion was centrifuged at 9500 × g for 10 minutes at 4°C, the precipitate was collected, washed three times with deionized water, and freeze-dried to obtain DEX@PLGA core microspheres.

[0036] (2) BSA (bovine serum albumin) shell coating: Prepare 10 mL of 4% (w / v) BSA solution in 0.01 M Tris-HCl buffer at pH 8.5 and treat it at 37°C and 100 rpm for 12 hours; weigh 50 mg of DEX@PLGA core microspheres and sonicate them with 2 mL of deionized water for 1 minute (100 W, 40 kHz), then add them dropwise to the above BSA solution and stir slowly at 300 rpm for 2 hours to make BSA uniformly coat the surface of DEX@PLGA core microspheres and form a BSA shell.

[0037] (3) CHIR99021 loading: Slowly add 100 μL of 20 mM CHIR99021-DMSO (dimethyl sulfoxide) stock solution (each drop is 10 seconds apart) to the above system. Place the reaction system in an ice-water bath (0~4°C) with aluminum foil to protect it from light. Stir continuously at 300 rpm overnight (12~16 hours) to allow CHIR99021 to be fully adsorbed in the BSA shell.

[0038] (4) Crosslinking and purification: Glutaraldehyde solution was slowly added dropwise to the system until the final concentration was 0.2% (v / v), and the crosslinking reaction was continued at room temperature for 2 hours. After the reaction was completed, the mixture was centrifuged at 13800×g for 15 minutes at 4°C to remove the supernatant. The mixture was washed three times by centrifugation with deionized water to remove unreacted crosslinking agent and free drug. The precipitate was pre-frozen at -80°C overnight and freeze-dried for 48 hours to obtain CHIR99021@BSA-DEX@PLGA time-dependent dual sustained-release composite microsphere powder, which was stored at -20°C in the dark for later use.

[0039] Example 3: Preparation of VN-GelMA drug-loaded porous hydrogel scaffold (1) Preparation of precursor solution: Accurately weigh the lyophilized GelMA powder and dissolve it in PBS solution containing 0.125% (w / v) photoinitiator LAP (lithium phenol-2,4,6-trimethylbenzoylphosphonate) at a concentration of 7.5% (w / v). Stir in a 50°C water bath in the dark until completely dissolved; adjust the pH to 7.0~7.4 and cool to room temperature; add CHIR99021@BSA-DEX@PLGA time-dependent dual sustained-release composite microsphere powder (20mg / mL) and mix thoroughly to obtain the drug-loaded hydrogel precursor solution.

[0040] (2) Photocuring and Porosification: The drug-loaded hydrogel precursor solution was injected into a sterile cylindrical mold (5 mm in diameter and 2 mm in height), and vertically irradiated for 10 seconds with a 405~410 nm visible light LED light source (light intensity 6.9 mW / cm²) (total light dose of about 69 mJ / cm²) to initiate the free radical polymerization of methacryloyl groups into a gel; after demolding, it was placed at -80°C for 2 hours for pre-freezing and freeze-drying for 48 hours. A three-dimensional interconnected porous network was constructed by ice crystal sublimation to obtain a drug-loaded GelMA porous hydrogel scaffold.

[0041] (3) Covalent grafting of VN peptides: Prepare a 0.1M MES (2-(N-morpholino)ethanesulfonic acid) buffer (pH=5.5) containing 0.1M EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 0.05M NHS (N-hydroxysuccinimide), filter it through a 0.22μm filter membrane and set it aside; place the lyophilized drug-loaded GelMA porous hydrogel scaffold in a 24-well plate, add 1mL of the above activation solution to each well, and shake at room temperature for 30 minutes to activate the carboxyl groups of the gel side chains; discard the liquid and wash twice with sterile PBS; then add 1mL of 50μg / mL VN peptide aqueous solution to each well, seal with sealing film, and incubate at 37°C for 120 minutes; discard the excess peptide solution, and add 1mL of F12 (Ham's F-12 Nutrient) to each well. Mixture culture was performed at 4°C for 12 hours to block unreacted active sites; the mixture was thoroughly washed three times with deionized water and freeze-dried to obtain the VN-GelMA drug-loaded porous hydrogel scaffold.

[0042] Example 4: Material Characterization (1) Characterization of DEX@PLGA core microspheres: SEM (scanning electron microscopy) and DLS (dynamic light scattering) particle size distribution; (2) Characterization of CHIR99021@BSA-DEX@PLGA time-series dual sustained-release composite microspheres: SEM, DLS particle size distribution, FT-IR (Fourier transform infrared spectroscopy), drug loading and encapsulation efficiency (UV-Vis, ultraviolet-visible spectrophotometry), in vitro time-series sustained-release curves of CHIR99021 and DEX. (3) Characterization of GelMA porous hydrogel scaffold and VN-GelMA drug-loaded porous hydrogel scaffold: FT-IR, ¹H-NMR (hydrogen nuclear magnetic resonance spectroscopy) (substitution degree calculation), SEM (pore morphology and pore size distribution), porosity, compressive modulus, rheology (G' and G''), swelling ratio, in vitro degradation performance, FITC (fluorescein isothiocyanate)-VN fluorescence tracer (confocal Z-axis tomography to verify the uniformity of VN peptide distribution).

[0043] Material characterization results as follows Figure 2 , Figure 3 As shown. Specifically, as Figure 2 As shown in b, the GelMA precursor solution can form a morphologically complete and structurally stable hydrogel block after photocrosslinking; such as Figure 2 As shown in cd, the freeze-dried GelMA hydrogel scaffold forms a continuous, interconnected porous network with rough pore walls, which facilitates microsphere embedding, nutrient exchange, and cell adhesion. Figure 2 As shown in e, the Fourier transform infrared spectrum of GelMA exhibits an absorption peak for the —OH / —NH stretching vibration at approximately 3441 cm⁻¹ and a C=O / C=C related absorption peak at approximately 1635 cm⁻¹; as... Figure 2 As shown in f, the 1H NMR spectrum of GelMA shows a characteristic peak of methacryloyl group around 5.3–5.7 ppm, proving that the gelatin molecule successfully incorporates photocrosslinkable methacryloyl groups; Figure 2 As shown in g, the FT-IR spectra of PLGA, PLGA@DEX, PLGA-BSA@CHIR and PLGA@DEX-BSA@CHIR show the C=O absorption peak of the PLGA ester group and the changes in the characteristic peaks of BSA, CHIR99021 and DEX, indicating that DEX, BSA shell and CHIR99021 have been successfully integrated into the composite microsphere system.

[0044] Depend on Figure 3 The SEM images shown in ab indicate that the PLGA microspheres are nearly spherical and relatively uniformly distributed, and that the loaded composite microspheres can be dispersed within the pore walls and porous structure of GelMA; Figure 3 The DLS results shown in c indicate that the effective particle size of the microspheres after BSA coating and CHIR99021 loading is larger than that of the PLGA core, suggesting the formation of a core-shell composite structure; Figure 3 As shown in de, all GelMA scaffolds with different concentrations maintained high porosity, with the 7.5% concentration GelMA scaffold exhibiting both high porosity and moderate swelling capacity; Figure 3 The rheological results shown in f indicate that the storage modulus G' is consistently higher than the loss modulus G'', suggesting that the GelMA hydrogel forms a stable elastic network. Figure 3As shown in gh, after EDC / NHS treatment and slow-release particle reinforcement, the tensile properties of the GelMA scaffold are improved, and both the fracture strain and stress-strain curves show improved mechanical stability; Figure 3 The in vitro release curves shown in figure i indicate that CHIR99021 was released rapidly in the early stages, reaching a release level of approximately 81.5 μM around day 4; DEX, on the other hand, exhibited a sustained and slow release, accumulating to approximately 277.6 μM by day 32. This release behavior aligns with the design requirement of "early Wnt signal activation followed by sustained maturation promotion by glucocorticoids in the later stages."

[0045] Experimental results show that, based on the above material characterization results, this invention successfully prepared a VN-GelMA drug-loaded composite porous hydrogel scaffold that combines an interconnected porous structure, stable mechanical properties, and programmed dual-drug sustained-release capability; this system can provide a stable material basis for the three-dimensional adhesion, growth, and subsequent time-sequential osteogenic induction of hiPSCs.

[0046] Example 5: Biosafety Evaluation (1) CCK-8 (Cell Counting Kit-8) cell viability: to detect the compatibility and proliferation activity of different scaffold groups (VN-, VN+, and each drug loading group) on hiPSCs cells; (2) Live / dead cell staining: The three-dimensional survival distribution and Z-axis infiltration of hiPSCs inside the VN-GelMA drug-loaded composite porous scaffold were observed using laser confocal scanning microscopy; (3) Blood routine test: Peripheral blood was collected at 4 and 8 weeks after surgery in rat skull defect model to test WBC (white blood cell count), RBC (red blood cell count), HGB (hemoglobin), and PLT (platelet count) to evaluate the in vivo and systemic safety of the composite hydrogel system after implantation.

[0047] Experimental results are as follows Figure 4 As shown, hiPSCs were predominantly viable cells during culture in the VN-GelMA scaffold. In Live / Dead staining, the green viable cell signal gradually increased with prolonged culture time, while the red dead cell signal was less. CCK-8 results showed that cell viability generally increased with culture time, and the VN-modified group was higher than the unmodified GelMA group on D1, D3, D5, and D7, suggesting that the scaffold has good cell compatibility and can support continuous cell proliferation.

[0048] Blood routine test results after implantation Figure 8 As shown, there were no significant differences in RBC, WBC, PLT, and HGB levels among the Blank group, Control group, Composite GelMA group, and ectopic GelMA-related group. No significant systemic toxicity or inflammatory response was observed, such as elevated white blood cell count, anemia, or platelet abnormalities.

[0049] Experimental results show that the composite hydrogel scaffold system of the present invention can maintain good survival and proliferation of hiPSCs in vitro, and does not cause significant systemic hematological abnormalities after implantation in vivo, indicating that the material system has good biosafety and in vivo application tolerance.

[0050] Example 6: Three-dimensional culture and osteogenic induction of hiPSCs The experiment was divided into four groups, all based on the VN-GelMA porous scaffold: Blank group (no microspheres), CHIR99021+ group (only CHIR@BSA shell microspheres), DEX+ group (only DEX@PLGA core microspheres), and CHIR+DEX+ group (with CHIR99021@BSA-DEX@PLGA complete dual sustained-release microspheres).

[0051] (1) Preparation of embryoid bodies (EBs): hiPSCs (cell line hNF-C1) in the logarithmic growth phase were collected, digested with 0.5 mM EDTA (ethylenediaminetetraacetic acid), centrifuged, and resuspended in PSCeasy medium to 1×10⁻⁶. 6 cells / mL; the cell suspension was added to AggreWell™ 400 microwell array culture plates (approximately 1500 cells / well) that had been rinsed with low-adhesion solution, and incubated statically at 37°C and 5% CO2 for 24 hours to allow hiPSCs to spontaneously aggregate and form uniform EBs with a diameter of approximately 150~200μm.

[0052] (2) EBs seeded into three-dimensional scaffolds (defined as day 0, D0): each group of scaffolds was placed in a 24-well plate (1 scaffold per well, 5 mm in diameter and 2 mm in height), and washed 3 times with PBS; about 200 μL of EB suspension (containing 10-15 EBs) was slowly dripped onto the upper surface of the scaffold, and the EBs were made to penetrate into the pores of the scaffold by capillary action; after standing in a clean bench for 30 minutes, 800 μL of PSCeasy medium was added.

[0053] (3) Early culture stage (D0~D5): PSCeasy medium was used, and the medium was completely changed every 48 hours. During this stage, the CHIR99021 released by the drug-loaded microspheres provided Wnt signals to the cells, driving them to differentiate into the mesodermal lineage.

[0054] (4) Culture medium change (D5): Remove all PSCeasy medium, rinse the scaffold 3 times with α-MEM (5 minutes each time), and replace with osteogenic induction medium (α-MEM (α-minimum essential medium) + 10% FBS (fetal bovine serum) + 50 μg / mL L-ascorbic acid-2-phosphate + 10mM β-glycerophosphate sodium + 0.1mM β-mercaptoethanol, without additional DEX, supplied by continuous slow release from microspheres in the scaffold).

[0055] (5) Osteogenesis induction stage (D5~D28): Change the medium daily from D5 to D12, and change the medium every 48 hours from D12 to D28; detect germ layer genes on D3 and D5, and detect osteogenic related indicators on D7, D14, D21 and D28.

[0056] Experimental results are as follows Figure 5 As shown, hiPSCs, after low adhesion aggregation, can form relatively uniform EBs. After being seeded onto a VN-GelMA porous scaffold, they can enter the scaffold pores and continue to be cultured in a three-dimensional environment. OCT4 immunofluorescence results showed that positive OCT4 signals were observed in the Blank group, CHIR99021+ group, DEX+ group, and CHIR+DEX+ group at D1. These signals gradually weakened with increasing culture time, and the OCT4 signal significantly decreased from D5 to D7, suggesting that the cells gradually exited the pluripotent state and entered the induced differentiation process. Figure 7 The process shown in step a further demonstrates that EB formation, scaffold seeding, in vitro osteogenic induction, in vivo implantation, and tissue evaluation can form a continuous operational chain, indicating that the three-dimensional culture and induction process established in this embodiment has good executability and stability.

[0057] Experimental results show that the three-dimensional induction system can support the migration and growth of hiPSCs from the EB state into the scaffold, and gradually reduce the expression of stem markers under timed drug release conditions, laying the foundation for subsequent evaluation of mesodermal commitment, osteogenic differentiation and in vivo bone repair.

[0058] Example 7: Study on the effect of VN peptide on three-dimensional adhesion and proliferation of hiPSCs Using unmodified GelMA scaffolds (VN-) and VN peptide-modified GelMA scaffolds (VN+) as controls, the scaffolds were cultured in PSCeasy medium, and the following indicators were measured on D1, D3, D5, and D7: (1) The CCK-8 assay was used to quantitatively detect cell viability and proliferation activity in the two scaffolds and to evaluate the effect of VN modification on cell survival. (2) Calcein-AM / PI live / dead cell double staining combined with laser confocal scanning microscope Z-axis layer scanning was used to observe the three-dimensional spatial distribution, survival rate and deep infiltration of cells inside the scaffold; (3) RT-qPCR was used to detect the expression changes of the D1, D3, and D5 pluripotency marker genes OCT4 and Nanog to evaluate the effect of VN modification on the pluripotency status of hiPSCs.

[0059] Experimental results are as follows Figure 4 As shown, compared with the VN− group, the VN+ group exhibited more viable cell signals and a more pronounced three-dimensional distribution during days 1 to 7. Confocal Z-axis reconstruction results showed that cells in the VN+ group were not only distributed on the scaffold surface but also infiltrated deeper into the scaffold, exhibiting a more continuous spatial distribution in both XZ and Depth views; the VN− group had relatively fewer cells and insufficient deep cell distribution. SEM results on day 7 showed cell adhesion, spreading, and pseudopodia-like structures on the pore walls of the VN+ scaffold, with cells forming close contact with the rough pore walls, suggesting that the VN peptide provided effective adhesion sites for hiPSCs. CCK-8 quantification results showed that cell viability in the VN+ group was higher than that in the VN− group on days 1, 3, 5, and 7, and the differences were statistically significant. RT-qPCR results showed that Nanog and OCT4 expression in the VN+ group was lower than that in the VN− group on days 1, 3, and 5, suggesting that VN modification, while enhancing cell adhesion and proliferation, also facilitates the gradual exit of cells from a pluripotent state.

[0060] Experimental results show that VN peptide modification significantly improves the ability of GelMA scaffold to recognize, adhere to, proliferate and deeply infiltrate hiPSCs, and promotes the transition of cells from a pluripotency maintenance state to a subsequent differentiation state. It is an important functionalization step in constructing an efficient three-dimensional osteogenic induction microenvironment.

[0061] Example 8: Study on the promoting effect of time-sequential release of CHIR99021 on mesoderm-directed differentiation of hiPSCs Based on the results of Example 6, subsequent osteogenic induction experiments all used the VN-GelMA scaffold, and the promoting effect of early temporal CHIR99021 release on mesodermal directional differentiation was evaluated on days 3 and 5 using the following methods: (1) RT-qPCR was used to detect the expression of mesoderm marker genes T (Brachyury) and MSX1, as well as the expression of endoderm marker genes GATA4, AFP3 and SOX17, to evaluate the selective regulation of differentiation direction of each germ layer by CHIR99021. (2) The cell viability of each group in D1, D3, D5 and D7 was detected by CCK-8 assay to evaluate the effect of early CHIR99021 release on cell proliferation.

[0062] Experimental results are as follows Figure 6As shown in Figure ae, after early release of CHIR99021, the mesodermal marker genes T and MSX1 were significantly upregulated on days 3 and 5. The CHIR99021+ group showed the highest expression of T and MSX1 on day 5, while the CHIR+DEX+ group showed a significant mesodermal-related gene response as early as day 3, indicating that early Wnt signaling activation can effectively drive hiPSCs to commit to the mesodermal lineage. In contrast, the DEX+ group had a weaker promoting effect on T and MSX1, suggesting that early DEX exposure alone is insufficient to drive significant mesodermal induction. Endoderm-related genes GATA4, AFP3, and SOX17 showed transient fluctuations in some treatment groups and time points, but did not show a sustained dominant upregulation on day 5. Combined with the significant enhancement of T and MSX1, this indicates that the time-sequential release of CHIR99021 mainly plays an early mesodermal induction role, rather than simply inducing non-directional germline differentiation.

[0063] Experimental results show that early release of CHIR99021 can mimic the Wnt signal activation process in the early stage of bone development, preferentially promoting the transformation of hiPSCs from a pluripotent state to a mesodermal state, providing a precursor cell basis for subsequent DEX-mediated osteogenic maturation and mineralization.

[0064] Example 9: Study on the regulation of three-dimensional osteogenic differentiation of hiPSCs by a time-sequential dual-release system On days 7, 14, 21, and 28 of osteogenic induction, the following methods were used to systematically evaluate the promoting effect of a time-dependent dual-release system on three-dimensional osteogenic differentiation of hiPSCs: (1) The cell viability of each group was detected by CCK-8 assay to evaluate the cell compatibility of the dual sustained-release system during long-term culture. (2) RT-qPCR was used to detect the temporal expression changes of the pluripotency gene OCT4 and osteogenic-related genes ALP, RUNX2, COL1A1 and OCN; (3) Immunofluorescence staining (laser confocal scanning microscope) was used to detect the expression levels and distribution of osteogenic-related proteins RUNX2, ALP, COL1A1, and OCN; (4) Alizarin Red S staining (ARS) combined with hexadecylpyridine chloride (CPC) elution quantification (OD490) was used to assess the level of extracellular matrix mineralization deposition and compare the effects of different drug administration strategies on osteogenic mineralization.

[0065] Experimental results are as follows Figure 6As shown in fj, OCT4 expression in all groups decreased rapidly with increasing induction time, remaining at extremely low levels from D14 to D28, indicating that the three-dimensional induction system can effectively inhibit the maintenance of pluripotency. Regarding osteogenic-related genes, ALP showed high expression at D7, suggesting the initiation of early osteogenic differentiation; RUNX2 gradually increased after D14 and remained at high levels from D21 to D28; COL1A1 and OCN gradually increased with increasing induction time, especially in the CHIR+DEX+ group, where they were more pronounced at D28, suggesting that the dual-release system can promote extracellular matrix formation and late-stage osteogenic maturation.

[0066] Experimental results are as follows Figure 6 KL and Alizarin Red S staining results showed that the number of mineralized nodules in each group gradually increased with the extension of induction time, and the CHIR+DEX+ group formed stronger red mineralized deposits on D21 and D28. CPC elution quantification results were consistent with the staining observations. The OD490 value of the CHIR+DEX+ group continued to increase from D7 to D28, reaching the highest level on D28, which was significantly higher than that of the Blank group and most single-drug treatment groups.

[0067] Experimental results show that the time-sequential dual sustained-release system has a synergistic effect by promoting lineage commitment in the early stage through CHIR99021 and promoting osteogenic maturation in the middle and late stages through DEX. It can significantly improve the three-dimensional osteogenic differentiation efficiency, extracellular matrix generation capacity and mineralization quality of hiPSCs, which is superior to single drug release or no drug scaffold.

[0068] Example 10: In vivo evaluation of the bone repair capacity of hiPSC-loaded composite hydrogel (1) Subcutaneous ectopic osteogenic model in nude mice: 6-8 week old BALB / c nude mice were randomly divided into 3 groups (n=4): Matrigel-cell group (uninduced hiPSCs suspension, positive control), Control group (pure drug-loaded VN-GelMA scaffold, without cells), and Composite GelMA group (hiPSCs-loaded composite scaffold for in vitro osteogenic induction for 28 days); a cyst was prepared subcutaneously in the hind leg and the corresponding materials were implanted. The tissue was harvested 4 weeks after the operation; the formation of teratomas was observed grossly, and collagen deposition and osteoid formation were evaluated by H&E staining and Masson trichrome staining.

[0069] Experimental results are as follows Figure 7As shown, in the nude mouse subcutaneous ectopic osteogenic model, more pronounced tissue-like structures were observed after implant removal in the Composite GelMA group. Masson staining revealed richer collagen deposition and osteoid matrix formation in the Composite GelMA group, while H&E staining showed areas of cell infiltration and tissue remodeling. The Blank group mainly exhibited scaffold or residual material structures, while the Control group showed some tissue ingrowth but limited osteoid matrix formation. No typical teratoma-like tissue structures were observed in any group.

[0070] (2) Critical-size bone defect model of rat skull: 8-10 week old SD rats were randomly divided into 3 groups (n=6): Blank group (modeling only, no implantation), Control group (pure drug-loaded VN-GelMA scaffold), and Composite GelMA group (hiPSCs composite scaffold). A 5 mm diameter full-thickness circular defect was prepared in the parietal bone using a 5.0 mm outer diameter trephine, and the corresponding material was implanted. Samples were taken at 4 and 8 weeks after surgery (3 rats in each group each time). Peripheral blood was collected at the same time as the sample collection for routine blood tests (WBC, RBC, HGB, PLT) to evaluate systemic safety. The defect area was reconstructed in three dimensions using high-resolution Micro-CT, and bone morphological parameters such as SMI, BS / BV, BS / TV and Tb.N were quantitatively extracted. The tissue specimens were decalcified, paraffin embedded, and sectioned. H&E staining and Masson trichrome staining were performed to evaluate material degradation, host cell infiltration, angiogenesis, new bone formation and bone bridging from the histological level.

[0071] Experimental results are as follows Figure 8 As shown, in a rat model of critical-size skull defects, the defect area was still relatively obvious in the Blank group at 4 weeks post-surgery, while the Control group showed some new tissue or mineralization deposition. The Composite GelMA group had more obvious new bone coverage in the defect area. At 8 weeks post-surgery, the bone filling in the defect area of ​​the Composite GelMA group further increased. Micro-CT three-dimensional reconstruction and cross-sectional images showed that its bone bridging and defect closure were better than those of the Blank and Control groups. Micro-CT quantitative results showed that the Composite GelMA group had better bone microstructure parameters at both 4 and 8 weeks: decreased SMI, indicating a transformation of the new bone structure from loose rod-like to a more mature plate-like or continuous structure; increased BS / BV, BS / TV, and Tb.N, indicating an increase in new bone surface area, degree of bone tissue filling, and number of trabeculae. Blood routine results showed no statistically significant differences in RBC, WBC, PLT, and HGB among the groups, indicating that the material implantation did not cause significant systemic inflammation or hematological toxicity.

[0072] Experimental results show that the hiPSC-loaded composite hydrogel scaffold has good safety in vivo and can significantly promote the formation of ectopic osteoid tissue and the repair of critical-size defects in the skull. Its repair advantage comes from the synergistic effect of the VN-GelMA three-dimensional scaffold, hiPSC osteogenic progenitor cells, and CHIR99021 / DEX time-dependent dual sustained-release signaling.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method of preparing a drug releasing hydrogel scaffold, characterized in that, The method includes the following steps: (1) A core-shell structured CHIR99021@BSA-DEX@PLGA time-release composite microsphere was prepared by using a single emulsion-solvent evaporation method combined with protein self-assembly technology; (2) The lyophilized GelMA powder was dissolved in PBS solution containing 0.125% w / v photoinitiator LAP at a concentration of 7.5% w / v. The solution was stirred in a water bath at 50°C in the dark until completely dissolved. The pH was adjusted to 7.0~7.

4. After cooling to room temperature, the CHIR99021@BSA-DEX@PLGA time-dependent dual sustained-release composite microspheres were added and mixed thoroughly to obtain the drug-loaded hydrogel precursor solution. (3) The drug-loaded hydrogel precursor solution was injected into a sterile mold and vertically irradiated with a 405-410nm visible LED light source for 8-12 seconds to initiate the free radical polymerization of methacryloyl groups into a gel; after demolding, it was placed at -80°C for 2 hours and freeze-dried for 48 hours to obtain a drug-loaded GelMA porous hydrogel scaffold. (4) Prepare MES buffer containing EDC and NHS, filter it through a 0.22 μm filter membrane and set it aside; place the lyophilized drug-loaded GelMA porous hydrogel scaffold in a well plate, add the MES buffer, and shake at room temperature to activate the carboxyl groups of the gel side chains; aspirate the liquid, wash twice with sterile PBS, then add a 50 μg / mL VN polypeptide aqueous solution, seal with a sealing film, and incubate at 37°C for 120 minutes; aspirate the excess polypeptide solution, add F12 culture medium and place at 4°C for 12 hours; wash thoroughly three times with deionized water, freeze dry, and obtain the VN-GelMA drug-loaded porous hydrogel scaffold.

2. The method according to claim 1, characterized in that, In step (1), the preparation of the CHIR99021@BSA-DEX@PLGA time-dependent dual sustained-release composite microspheres includes the following steps: (1-1) PLGA in a 50:50 ratio was dissolved in acetone, and dexamethasone was added and fully dissolved to form the oil phase. A 1% w / v PVA aqueous solution was prepared as the aqueous phase. The oil phase was slowly added dropwise to the aqueous phase under an ice-water bath, and ultrasonically broken up to form a homogeneous O / W emulsion. The O / W emulsion was transferred to a magnetic stirrer and stirred at 500 rpm at room temperature in the dark for 24 hours to allow the organic solvent to evaporate completely. The emulsion was centrifuged at 9500×g for 10 minutes at 4°C, the precipitate was collected, washed three times with deionized water, and freeze-dried to obtain DEX@PLGA core microspheres. (1-2) The DEX@PLGA core microspheres were dissolved in deionized water by ultrasonic dispersion. Then the aqueous solution was added dropwise to a BSA solution with a concentration of 4% w / v. The solution was stirred slowly at 300 rpm for 1 to 3 hours to make BSA uniformly coat the surface of the DEX@PLGA core microspheres and form a BSA shell. (1-3) Slowly add CHIR99021-DMSO stock solution with a concentration of 20mM to the system, place it in an ice-water bath and protect it from light with aluminum foil, and stir continuously at 300rpm for 12-16h to allow CHIR99021 to be fully adsorbed in the BSA shell. (1-4) Glutaraldehyde solution was slowly added dropwise to the system to a final concentration of 0.2% v / v. The cross-linking reaction was carried out at room temperature for 2 hours. After the reaction was completed, the mixture was centrifuged at 4°C and 13,800×g for 15 minutes to remove the supernatant. The mixture was washed three times by centrifugation with deionized water to remove unreacted cross-linking agent and free drug. The precipitate was pre-frozen at -80°C and freeze-dried for 48 hours to obtain CHIR99021@BSA-DEX@PLGA time-dependent dual sustained-release composite microsphere powder.

3. The method according to claim 2, characterized in that, In step (1-1), the mass-volume ratio of PLGA, acetone, dexamethasone, and PVA aqueous solution is 100mg:2mL:10mg:10mL.

4. The method according to claim 3, characterized in that, In steps (1-2), the mass-to-volume ratio of the DEX@PLGA microspheres, deionized water, and BSA solution is 50 mg: 2 mL: 10 mL.

5. The method according to claim 4, characterized in that, In step (2), the CHIR99021@BSA-DEX@PLGA time-dependent dual sustained-release composite microspheres are added to the reaction system at a mass / volume ratio of 20 mg / mL.

6. The method according to claim 1, characterized in that, In step (2), the preparation of the GelMA includes the following steps: Type A porcine gelatin was added to PBS buffer preheated to 50°C and stirred until completely dissolved to form a 10% w / v gelatin solution. Methacrylic anhydride was slowly added dropwise to the gelatin solution under vigorous stirring, maintaining the pH at 7.5–8.0 during the addition. The reaction was carried out in the dark at 50°C and 300 rpm for 3 hours. After the reaction was completed, PBS buffer preheated to 40°C was added to dilute and terminate the reaction. The solution was then transferred to a dialysis bag with a molecular weight cutoff of 12–14 kDa and dialyzed in deionized water at 40°C for 7 days. After dialysis, the solution was pre-frozen and then freeze-dried under vacuum for 48 hours to obtain GelMA solid powder.

7. The method according to claim 6, characterized in that, In the preparation of the GelMA, the mass-to-volume ratio of type A porcine skin gelatin to 50°C PBS buffer was 10 g: 100 mL; the molar ratio of methacrylic anhydride to gelatin solution was 10:

1.

8. The method according to claim 1, characterized in that, In step (4), the concentrations of EDC, NHS and MES in the MES buffer containing EDC and NHS are 0.1M, 0.05M and 0.1M, respectively.

9. The VN-GelMA drug-loaded porous hydrogel scaffold prepared by the method according to any one of claims 1 to 8.

10. The application of the VN-GelMA drug-loaded porous hydrogel scaffold according to claim 9 in three-dimensional osteogenic induction of hiPSCs.

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