Bionic stent for treating osteoporotic bone defect

Abstract

The invention discloses a bionic scaffold for treating osteoporotic bone defect, and relates to a bionic material, the scaffold is composed of three parts, namely melatonin-loaded PLGA sustained release microspheres, a silk fibroin / nano-hydroxyapatite (SF / n-HA) composite scaffold and methacrylic anhydride gelatin (GelMA) hydrogel wrapping bone marrow mesenchymal stem cells. The preparation method comprises the following steps: firstly, preparing PLGA sustained-release microspheres by an emulsion solvent evaporation method, and encapsulating melatonin in a PLGA matrix; secondly, mixing SF and n-HA according to a specific proportion, and freeze-drying to form a porous scaffold; then, the microspheres are compounded in pores of the stent through vacuum adsorption; and finally, performing negative pressure perfusion on the stem cell-containing GelMA hydrogel, and performing ultraviolet light crosslinking and curing on the surface of the composite scaffold. According to the scheme, the synergistic effect of drug sustained release, bone conduction and stem cell osteogenesis activity is achieved through layered structure design, and the problems that in the prior art, drug release is unstable, cell distribution is uneven, and stent degradation and bone regeneration are not matched are solved.

Description

Technical Field

[0001] The present invention relates to biomimetic materials, and particularly to a biomimetic scaffold for treating osteoporotic bone defects. Background Art

[0002] Currently, the treatment of osteoporotic bone defects mainly relies on autologous bone transplantation, allogeneic bone transplantation, and artificial bone repair materials. Autologous bone transplantation has problems such as donor site injury and limited bone mass; allogeneic bone faces challenges such as immune rejection, disease transmission risk, and donor scarcity. Among artificial bone repair materials, hydroxyapatite (HA)-based scaffolds have osteoconductivity, but are brittle and have a degradation rate that does not match bone regeneration; single polymer scaffolds (such as PLGA) are degradable, but lack osteoinductive activity and cannot synergistically treat osteoporosis.

[0003] To solve the problem of drug sustained release, the prior art uses PLGA microspheres loaded with drugs (such as alendronate sodium), but systemic administration requires a high dose and is prone to side effects; local sustained release also has problems such as drug burst release or uneven distribution due to the immature composite process of microspheres and scaffolds. In addition, in stem cell therapy, the survival rate of directly inoculating bone marrow mesenchymal stem cells (BMSCs) on traditional scaffolds (such as collagen sponges) is low (<70%), and the cell distribution is uneven, making it difficult to effectively promote bone regeneration. Existing biomimetic scaffolds (such as SF / HA composites) simulate bone components, but do not integrate drug sustained release and stem cell activity protection functions, and cannot simultaneously meet the dual requirements of "bone filling + bone metabolism regulation" for osteoporotic bone defects. Summary of the Invention

[0004] Aiming at the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a biomimetic scaffold for treating osteoporotic bone defects to solve one or more problems in the prior art.

[0005] To achieve the above purpose, the technical solution of the present invention is as follows: A biomimetic scaffold for treating osteoporotic bone defects is prepared by the following method: (1) Prepare PLGA sustained-release microspheres loaded with melatonin by the emulsion solvent evaporation method, where the mass ratio of PLGA to melatonin is 4:1 to 6:1; (2) Mix silk fibroin (SF) and nano-hydroxyapatite (n-HA) in a mass ratio of 6.5:3.5 to 7.5:2.5, and form an SF / n-HA scaffold with a porosity >80% through freeze-drying; (3) Composite the PLGA sustained-release microspheres in step (1) into the pores of the SF / n-HA scaffold in step (2) by vacuum adsorption; (4) The methacrylated gelatin hydrogel (GelMA-BMSCs) encapsulating bone marrow mesenchymal stem cells was solidified on the surface of the SF / n-HA scaffold incorporated with PLGA microspheres through negative pressure perfusion and ultraviolet light crosslinking.

[0006] Further, in step (1), the particle size of the PLGA sustained-release microspheres is 10-20 μm, and the melatonin encapsulation rate is ≥85%.

[0007] Further, the emulsion solvent evaporation method of step (1) includes: PLGA is dissolved in dichloromethane, and melatonin is dissolved in absolute methanol; The mixture is homogenously emulsified at a rotation speed of 10,000-12,000 rpm for 1.5-2.5 min; The emulsion is added dropwise to a ~5% polyvinyl alcohol (PVA) solution, and secondarily emulsified at 10,000-14,000 rpm for 4-6 min; The solvent is volatilized by stirring at room temperature for 10-14 h, and the microspheres are collected by centrifugation.

[0008] Further, it is pre-frozen at -35°C to -45°C for 5-7 h and main-frozen at -75°C to -85°C for 22-26 h.

[0009] Further, the vacuum adsorption parameters in step (3) are -0.09 MPa to -0.11 MPa, and the action time is 25-35 min.

[0010] Further, in step (4), the concentration of the GelMA hydrogel is 4%-6% w / v, and the density of bone marrow mesenchymal stem cells is 8×10 6 ~1.2×10 7 cells / mL.

[0011] Further, in step (4), the vacuum degree of negative pressure perfusion is -0.07 MPa to -0.09 MPa, and the action time is 8-12 min.

[0012] Further, in step (4), the wavelength of ultraviolet light crosslinking is 400-410 nm, the intensity is 8-12 mW / cm 2 , and the curing time is 35-45 s.

[0013] Further, the SF / n-HA scaffold is a cylinder with a diameter of 4.5-5.5 mm and a height of 5.5-6.5 mm.

[0014] Further, the scaffold is sterilized by cobalt-60 irradiation, and the dose is 22-28 kGy.

[0015] Compared with the prior art, the beneficial technical effects of the present invention are as follows: (1) Through the vacuum adsorption and composite of PLGA sustained-release microspheres and SF / n-HA scaffolds, the synergistic effect of precise drug positioning and long-acting release is achieved. Melatonin-loaded PLGA microspheres are evenly distributed in the pores of the SF / n-HA scaffold through vacuum adsorption to form a local drug reservoir. The combination of the degradation characteristics of the microspheres and the osteoconductivity of the scaffold enables the sustained release of melatonin in the osteoporosis area, which not only inhibits the activity of osteoclasts but also avoids the side effects of systemic drug administration. At the same time, the inorganic-organic ratio of the scaffold simulates the natural bone structure, providing a mechanical support and degradation-matching environment for bone regeneration.

[0016] (2) The high-porosity structure of the SF / n-HA scaffold and the negative-pressure perfusion-photocrosslinking curing of GelMA-BMSCs synergistically optimize the distribution and activity of stem cells. The SF / n-HA scaffold with a high porosity (>80%) enables the GelMA hydrogel encapsulating BMSCs to be fully infiltrated into the pores through negative-pressure perfusion, and in-situ curing is achieved by ultraviolet photocrosslinking. This combination not only avoids cell loss but also maintains the three-dimensional growth microenvironment of stem cells through the physical protection of the hydrogel, significantly improving cell survival rate and osteogenic differentiation efficiency, and overcoming the problem of uneven distribution in traditional inoculation.

[0017] (3) The multi-component functional hierarchical integration realizes the bidirectional regulation of bone metabolism. The spatial hierarchical design of PLGA microspheres (melatonin sustained release), SF / n-HA scaffolds (osteoconduction and degradation regulation), and GelMA-BMSCs (stem cell osteogenic induction) forms a synergistic mechanism: the microspheres regulate the bone metabolism balance from the inside, the scaffold provides the basis for structural regeneration, and the stem cells actively promote new bone formation from the surface layer. This hierarchical integration targets both the "inhibiting bone loss" and "promoting bone formation" dual pathways of osteoporosis, breaking through the limitations of single-functional materials.

[0018] (4) The full-process process integration ensures the stable adaptation of bionic functions. From the preparation of microspheres with controllable sizes by the emulsion evaporation method, freeze-drying to construct the pores of the bionic scaffold, to the precise composite of vacuum adsorption and negative-pressure photocrosslinking, the whole process guarantees the stability of drug release kinetics, stem cell activity protection, and the mechanical properties of the scaffold. The close connection of each step enables the final scaffold to maintain structural integrity and functional persistence in a complex osteoporosis environment, meeting the clinical repair requirements. Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the steps of the preparation method of the bionic scaffold in the present invention. Detailed Embodiments

[0020] To make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and exemplary illustrations. It should be noted that the structures, proportions, sizes, etc. shown in the drawings of this specification are only used to cooperate with the content disclosed in the specification for those skilled in this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they do not have substantial technical significance. Any modification of the structure, change in the proportional relationship or adjustment of the size, without affecting the efficacy that the present invention can produce and the objectives that can be achieved, should still fall within the scope covered by the technical content disclosed in the present invention.

[0021] Application Overview Currently, the treatment of osteoporotic bone defects in the industry mainly relies on three types of solutions: One is autologous or allogeneic bone transplantation. Although it has biocompatibility, it faces bottlenecks such as donor site injury, immune rejection, and donor scarcity. The second is artificial bone repair materials (such as hydroxyapatite or polymer scaffolds). Although they can fill bone defects, single materials lack the function of regulating bone metabolism, and the degradation rate does not match the bone regeneration cycle. The third is the drug sustained-release system (such as PLGA microspheres loaded with anti-osteoporosis drugs). Although it can administer drugs locally, due to the immature composite process of microspheres and scaffolds, it often leads to sudden drug release or uneven distribution, and it cannot cooperate with stem cells for repair.

[0022] These conventional solutions have significant defects: Functional fragmentation: The integration of drug sustained-release, scaffold filling, and stem cell repair has not been achieved, and the dual needs of "bone structure reconstruction" and "bone metabolism balance" for osteoporotic bone defects cannot be solved synchronously. Activity loss: The traditional way of inoculating stem cells results in uneven cell distribution and low survival rate, weakening the osteogenic efficacy. Insufficient environmental adaptation: The mechanical properties of existing scaffolds degrade under osteoporotic conditions, and there is a lack of a synergistic mechanism for targeting osteoclast inhibition and osteoblast activation.

[0023] This solution breaks through the above limitations through the innovative integration of a bionic scaffold structure design, a multi-stage sustained-release system, and a stem cell activity protection process, providing an integrated solution path for osteoporotic bone defects.

[0024] Comprehensive Description 1. Preparation of PLGA Sustained-Release Microspheres Loaded with Melatonin The PLGA sustained-release microspheres loaded with melatonin are prepared by the emulsion solvent evaporation method. The specific steps are as follows: Solution Preparation: Dissolve PLGA (molecular weight 50 kDa) in dichloromethane to form a solution with a concentration of 40 mg / mL; dissolve melatonin in anhydrous methanol with a concentration of 40 mg / mL. The mass ratio of PLGA to melatonin is 4:1 to 6:1.

[0025] Emulsification process: Dropwise add the melatonin solution to the PLGA solution and homogenize and emulsify it at a rotation speed of 10,000–12,000 rpm for 1.5 to 2.5 min to form a primary emulsion. Subsequently, add the primary emulsion dropwise to a 5% aqueous solution of polyvinyl alcohol (PVA) and perform secondary emulsification at a rotation speed of 10,000–14,000 rpm for 4 to 6 min to form an oil-in-water emulsion.

[0026] Solvent evaporation and microsphere collection: Stir the emulsion at room temperature for 10 to 14 h to completely evaporate the organic solvent. Centrifuge (8,000 rpm, 10 min) to collect the microspheres, wash them 3 times with deionized water, and obtain PLGA sustained-release microspheres with a particle size of 10–20 μm and a melatonin encapsulation efficiency of ≥85% after freeze-drying.

[0027] 2. Construction of SF / n-HA biomimetic scaffold Raw material mixing: Mix silk fibroin (SF) and nano-hydroxyapatite (n-HA) at a mass ratio of 6.5:3.5 to 7.5:2.5, add deionized water to prepare a slurry, and perform ultrasonic dispersion (200 W, 30 min) to ensure uniformity.

[0028] Freeze-drying and forming: Inject the slurry into a cylindrical mold (diameter 4.5–5.5 mm, height 5.5–6.5 mm), pre-freeze it at -35°C to -45°C for 5 to 7 h, and then perform main freezing at -75°C to -85°C for 22 to 26 h to form a porous scaffold with a porosity >80%.

[0029] Sterilization treatment: Sterilize the scaffold by cobalt-60 irradiation with a dose of 22–28 kGy for standby.

[0030] 3. Composite of PLGA microspheres and scaffold Uniformly disperse the PLGA sustained-release microspheres in the pores of the SF / n-HA scaffold, place it in a vacuum chamber (vacuum degree -0.09 MPa to -0.11 MPa) for adsorption for 25 to 35 min to ensure that the microspheres are stably anchored in the pores.

[0031] 4. Preparation and composite of GelMA-BMSCs hydrogel Hydrogel preparation: Dissolve methacrylic anhydride gelatin (GelMA) powder in deionized water to prepare a solution with a concentration of 4% to 6% w / v. Add the photoinitiator LAP (final concentration 0.25% w / v) and sterilize through a 0.22 μm filter membrane.

[0032] Stem cell encapsulation: Resuspend bone marrow mesenchymal stem cells (BMSCs) at a density of 8×10 6 to 1.2×10 7 cells / mL in the GelMA solution and gently mix.

[0033] Negative pressure perfusion and photocrosslinking: Place the scaffold with composite PLGA microspheres in a custom mold, inject the GelMA-BMSCs suspension to submerge the scaffold, and perform negative pressure perfusion at a vacuum of -0.07 MPa to -0.09 MPa for 8 to 12 minutes to allow the gel to fully infiltrate the pores. Subsequently, irradiate with ultraviolet light at a wavelength of 400–410 nm and an intensity of 8–12 mW / cm 2 for 35 to 45 seconds to achieve in-situ crosslinking and curing of the hydrogel, forming the final biomimetic scaffold.

[0034] Aseptic operation: Complete the whole process in a laminar flow hood and sterilize key materials by irradiation.

[0035] Experimental design: Verification of the technical effects of key parameters of the biomimetic scaffold 1. Experimental purpose To verify the practical significance of the defined ranges of the three core parameters in this scheme for the scaffold performance, a control experiment was designed: PLGA:melatonin mass ratio (variable A, range: 4:1–6:1): Affects drug encapsulation efficiency and sustained-release kinetics; SF:n-HA mass ratio (variable B, range: 6.5:3.5–7.5:2.5): Determines the mechanical strength and degradation compatibility of the scaffold; Main freezing temperature of lyophilization (variable C, range: -75°C to -85°C): Controls the pore structure and uniformity.

[0036] 2. Test standards and methods (ISO standards) Drug sustained-release efficiency (ISO 10993-12:2021) Operation procedure: ① Immerse the scaffold in PBS buffer (pH 7.4) at 37°C; ② Take 1 mL of samples at 1 h, 6 h, 24 h, 3 d, 7 d, and 14 d at regular intervals; ③ Add an equal amount of fresh PBS; ④ The samples were filtered through a 0.22 μm filter membrane, and the melatonin concentration was detected by HPLC (mobile phase: methanol: water = 60:40, detection wavelength: 278 nm); ⑤ Calculate the cumulative release rate (%).

[0037] Cell viability (ISO 10993-5:2009) Operation procedure: ① BMSCs were seeded on the scaffolds at a density of 1×10 4 cells / well; ② After culturing for 72 h, they were washed with PBS; ③ A mixed staining solution of Calcein-AM (2 μM) / PI (4.5 μM) was added and incubated at 37 °C in the dark for 30 min; ④ The number of live cells (green) and dead cells (red) was counted under a fluorescence microscope (excitation at 488 nm); ⑤ Viability = number of live cells / total number of cells × 100%.

[0038] Degradation rate of the scaffold (ISO 13781:2017) Operation procedure: ① The initial mass (W0) of the scaffold was accurately weighed; ② It was immersed in simulated body fluid (SBF, 37 °C), and the liquid was changed weekly; ③ After 4 weeks, the scaffold was taken out, freeze-dried and weighed (W t ); ④ Degradation rate = (W0 - W t ) / W0 × 100%.

[0039] 3. Experimental grouping design (10 groups)

[0040] Description of controlled variables: The basic processes of all groups were the same: emulsion evaporation method (homogenization speed 12,000 rpm), vacuum adsorption (-0.1 MPa), negative pressure photocrosslinking (405 nm, 10 mW / cm 2 ); The cell density was uniformly 1×10 7 cells / mL, and the GelMA concentration was 5% w / v.

[0041] 4. Experimental results and data analysis

[0042] Comprehensive scoring formula: Score = 0.4 × Sustained Release Efficiency + 0.4 × Cell Survival Rate + 0.2 × (100 - Degradation Rate × 10) (Note: The weight of the degradation rate is calculated inversely, and the lower the rate, the higher the score).

[0043] 5. Key Conclusions Parameter Synergistic Effect: The comprehensive score of Group 3 (A: 5.5:1, B: 7.2:2.8, C: -82°C) is the highest (93.15), which verifies the existence of a non-linear relationship between parameters: A medium-high proportion of PLGA (5.5:1) improves the encapsulation rate, but an excessive proportion (6.5:1, Group 9) leads to loose microsphere structure; When SF:n-HA is 7.2:2.8, the porosity and degradation rate are optimal (degradation rate of 3.76% / week); The main freezing temperature of -82°C makes the ice crystal size uniform and avoids local collapse.

[0044] Necessity of Range Limitation: The performance of the conventional groups (1–5) is comprehensively better than that of the control groups (6–9): Sustained Release Efficiency: Conventional groups > 84% vs. Control groups < 81%; Cell Survival Rate: Conventional groups > 91% vs. Control groups < 87%; Degradation Rate: Conventional groups < 5% / week vs. Control groups > 6% / week.

[0045] The performance of the blank group (10) lags significantly behind: The cell survival rate is only 68.75%, and the degradation is too fast (9.87% / week).

[0046] Verification of Technical Advantages: The parameter combination of this scheme (such as Group 3) achieves: Long-term Sustained Release (Release rate at 14 days > 89%, conforming to first-order kinetics); High Cell Activity (Survival rate > 96%, significant hydrogel protection effect); Degradation Matches Bone Regeneration (Degradation rate at 4 weeks < 15%, superior to the blank group by 40%).

[0047] Explanation of Data Reliability: All data are based on ISO standards, repeated 3 times, and the average value is taken, with an error range < 5%; The comprehensive score design reflects the core performance weights (sustained release and survival rate account for 80%), meeting the priority of clinical needs.

[0048] Performance Trend Analysis at the Molecular Level 1. Influence of the Proportion of PLGA:Melatonin (Variable A) on Sustained Release Efficiency Molecular Mechanism: The degradation rate of PLGA (poly(lactic-co-glycolic acid)) is determined by the hydrolysis rate of the ester bond. When the mass ratio of PLGA:melatonin is 5.5:1 (Group 3): The hydrophobicity of PLGA molecular chain is enhanced, which delays the penetration of water molecules and slows down the hydrolysis of ester bonds (degradation rate↓); Melatonin molecules are embedded in the hydrophobic region of PLGA through hydrophobic interactions, forming a stable entrapment (encapsulation efficiency ↑); When the ratio is unbalanced (such as 6.5:1, Group 9): Excessive PLGA leads to loose stacking of molecular chains, increased porosity of microspheres, and intensified drug burst release (sustained release efficiency↓).

[0049] Data association: The sustained-release efficiency of group 3 (89.47%) was significantly higher than that of group 9 (72.94%), because the hydrophobic effect and the dense structure synergistically delayed drug diffusion.

[0050] 2. Effect of SF:n-HA ratio (variable B) on cell survival and degradation rate Molecular mechanism: The β-folded conformation of SF (silk fibroin) is cross-linked with the calcium phosphate lattice of n-HA (nanohydroxyapatite) through hydrogen bonds: SF:n-HA = 7.2:2.8 (Group 3): The glycine-alanine repeating sequence of SF directionally wraps n-HA to form a collagen-like mineralized network (pore uniformity↑); Ca on n-HA surface 2+ Form ionic bonds with SF carboxyl groups to enhance the scaffold's compressive strength (degradation rate↓); When the ratio is unbalanced (such as 8.5:1.5, group 9): Excessive n-HA leads to agglomeration, destroys the continuity of SF molecular chains, and produces microcracks (degradation rate ↑); Rough surface damages cell membrane integrity (survival rate↓).

[0051] Data association: The cell survival rate (96.38%) and low degradation rate (3.76% / week) of group 3 were due to the biomimetic mineralized network; the survival rate (79.61%) and degradation rate (8.43% / week) of group 9 deteriorated due to structural defects.

[0052] 3. Effect of freezing temperature (variable C) on pore structure and cell activity Molecular mechanism: -82℃ main freezing temperature (Group 3): Slow ice crystal growth causes water molecules to be orderly arranged in the gaps of SF peptide chains, forming through micropores (pore diameter 100–300 μm); Low temperature inhibits ice crystal coarsening and avoids mechanical stress from tearing the SF molecular chains (porosity > 80%); Too high temperature (-70 °C, group 8): Rapid freezing results in uneven ice crystal sizes, and local macropores damage the three-dimensional network permeability of the GelMA hydrogel (hindered nutrient diffusion → decreased survival rate).

[0053] Data correlation: The pore uniformity of group 3 supports the uniform infiltration of GelMA, and the cell survival rate (96.38%) is higher than that of group 8 (87.35%).

[0054] 4. Multicomponent synergistic effect (reason for the peak in group 3) Molecular cascade reaction: Sustained release of melatonin from PLGA microspheres → activation of the MT1 receptor of BMSCs and upregulation of osteogenic genes (Runx2); The uniform pores of the SF / n-HA scaffold → promotion of the anchoring of integrin (α5β1) in GelMA and inhibition of cell apoptosis; Low-temperature stable cross-linking → protection of the activity of the RGD peptide sequence of GelMA and enhancement of cell adhesion.

[0055] Root cause of the non-linear relationship: When the parameters of the three are in the middle (A: 5.5:1, B: 7.2:2.8, C: -82 °C), the best kinetic balance at the molecular level is formed: Melatonin release rate ≈ cell osteogenic differentiation rate; Scaffold degradation rate ≈ new bone deposition rate.

[0056] Conclusion The performance trend of the experimental data is dominated by molecular interactions and kinetic matching: Hydrophobic interaction and hydrogen bond regulate drug sustained release; Ionic bond and mineralization network stabilize the scaffold structure; Ice crystal growth kinetics determines pore uniformity; Receptor-ligand signaling pathway activates cell activity.

[0057] The parameter combination of group 3 achieves the synergistic optimum at the molecular level, verifying the scientific necessity of the parameter range limitation.

[0058] Exemplary illustration Example 1 Preparation method: Preparation of PLGA sustained-release microspheres: Dissolve PLGA (200 mg) in 5 mL of dichloromethane and melatonin (40 mg) in 1 mL of absolute methanol. The mass ratio of PLGA to melatonin is 5.0:1. Homogenize and emulsify the mixture at 12,000 rpm for 2 min. Drop the emulsion into a 5% polyvinyl alcohol (PVA) solution and perform secondary emulsification at 12,000 rpm for 5 min. Stir at room temperature for 12 h to evaporate the solvent, collect the microspheres by centrifugation, and freeze-dry.

[0059] Construction of SF / n-HA scaffold: Mix silk fibroin (SF) and nano-hydroxyapatite (n-HA) at a mass ratio of 7.0:3.0. Inject the slurry into a mold (diameter 5.0 mm, height 6.0 mm), pre-freeze at -40°C for 6 h, and then main-freeze at -80°C for 24 h. Sterilize the scaffold by cobalt-60 irradiation at a dose of 25 kGy.

[0060] Composite of microspheres and scaffold: Composite PLGA microspheres into the pores of the scaffold by vacuum adsorption (-0.10 MPa, action time 30 min).

[0061] Composite of GelMA-BMSCs: The concentration of GelMA hydrogel is 5.0% w / v, and the density of bone marrow mesenchymal stem cells is 1.0×10 7 cells / mL; Inject the mixture into a mold containing the scaffold and perform negative pressure perfusion (-0.08 MPa, action time 10 min). Crosslink with ultraviolet light (wavelength 405 nm, intensity 10 mW / cm 2 , curing time 40 s).

[0062] Examples 2 to 9 are basically the same as Example 1, except for the specific implementation parameters, as shown in the following table: Table of example parameters

[0063] Example 10 (blank control group) Preparation method: Construction of SF scaffold: Dissolve silk fibroin (SF) in deionized water to prepare a 10% w / v solution. The slurry was injected into a mold (diameter 5.0 mm, height 6.0 mm) and freeze-dried at -80 °C for 24 h; The scaffolds were sterilized by cobalt-60 irradiation at a dose of 25 kGy.

[0064] Note: No PLGA microspheres, melatonin, or stem cells were added; No vacuum adsorption, negative pressure perfusion, or photo-crosslinking steps were performed.

[0065] Specific working process The PLGA sustained-release microspheres loaded with melatonin were prepared by the emulsion solvent evaporation method. The melatonin solution was dropped into the PLGA solution and homogenized at high speed to form a primary emulsion, which was then transferred to an aqueous polyvinyl alcohol solution for secondary emulsification to form an oil-in-water emulsion. The microspheres were obtained by solvent evaporation, centrifugal washing, and freeze-drying.

[0066] The mixed slurry of silk fibroin and nano-hydroxyapatite was ultrasonically dispersed and then injected into a mold. A porous scaffold was formed by freeze-drying and then sterilized by irradiation. The PLGA sustained-release microspheres were uniformly dispersed in the pores of the scaffold by vacuum adsorption to form a drug-scaffold complex.

[0067] The methacrylated gelatin was dissolved and mixed with bone marrow mesenchymal stem cells to form a cell suspension, which was injected into a mold containing the composite scaffold. Negative pressure perfusion was used to fully infiltrate the pores of the scaffold with the gel. Subsequently, photo-crosslinking curing was triggered by ultraviolet light irradiation to stably bind the hydrogel encapsulating the stem cells to the scaffold, ultimately forming a bionic bone tissue engineering scaffold.

[0068] The technical features described in the above exemplary description can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above exemplary description are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

Claims

1. A bionic scaffold for treating osteoporotic bone defects, characterized in that, Prepared by the following method: (1) Prepare PLGA sustained-release microspheres loaded with melatonin by the emulsion solvent evaporation method, where the mass ratio of PLGA to melatonin is 4:1 to 6:1; (2) Mix silk fibroin (SF) and nano-hydroxyapatite (n-HA) at a mass ratio of 6.5:3.5 to 7.5:2.5, and form an SF / n-HA scaffold with a porosity > 80% by freeze-drying; (3) Composite the PLGA sustained-release microspheres in step (1) into the pores of the SF / n-HA scaffold in step (2) by vacuum adsorption; (4) Fix the gelatin methacrylate hydrogel (GelMA-BMSCs) encapsulating bone marrow mesenchymal stem cells on the surface of the SF / n-HA scaffold composite with PLGA microspheres by negative pressure perfusion and ultraviolet light cross-linking curing.

2. The bionic scaffold for treating osteoporotic bone defects according to claim 1, wherein: In step (1), the particle size of the PLGA sustained-release microspheres is 10 - 20 μm, and the encapsulation rate of melatonin is ≥ 85%.

3. The bionic scaffold for treating osteoporotic bone defects according to claim 1, wherein The emulsion solvent evaporation method in step (1) includes: Dissolve PLGA in dichloromethane and dissolve melatonin in absolute methanol; Homogenize and emulsify the mixture at a rotation speed of 10,000 - 12,000 rpm for 1.5 - 2.5 min; Drop the emulsion into a ~5% polyvinyl alcohol (PVA) solution and perform secondary emulsification at 10,000 - 14,000 rpm for 4 - 6 min; Stir at room temperature for 10 - 14 h to volatilize the solvent, and collect the microspheres by centrifugation.

4. The bionic scaffold for treating osteoporotic bone defects according to claim 1, characterized in that, Pre-freeze at -35°C to -45°C for 5 - 7 h and main-freeze at -75°C to -85°C for 22 - 26 h.

5. The bionic scaffold for treating osteoporotic bone defects according to claim 1, characterized in that: The vacuum adsorption parameters in step (3) are -0.09 MPa to -0.11 MPa, and the action time is 25 - 35 min.

6. The bionic scaffold for treating osteoporotic bone defects according to claim 1, characterized in that: In step (4), the GelMA hydrogel concentration is 4% - 6% w / v, and the density of bone marrow mesenchymal stem cells is 8×10 6 ~1.2×10 7 cells / mL.

7. The bionic scaffold for treating osteoporotic bone defects according to claim 1, characterized in that: The vacuum degree of negative pressure perfusion in step (4) is -0.07 MPa to -0.09 MPa, and the action time is 8 - 12 min.

8. The bionic scaffold for treating osteoporotic bone defects according to claim 1, characterized in that: In step (4), the wavelength of the ultraviolet light crosslinking is 400~410 nm, and the intensity is 8~12 mW / cm 2 , and the curing time is 35~45 s.

9. The bionic scaffold for treating osteoporotic bone defects according to claim 1, wherein: The SF / n-HA scaffold is a cylinder with a diameter of 4.5 - 5.5 mm and a height of 5.5 - 6.5 mm.

10. The bionic scaffold for treating osteoporotic bone defects according to claim 1, wherein: The scaffold is sterilized by cobalt-60 irradiation, and the dose is 22 - 28 kGy.

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