Multi-effect synergistic repair large bone defect stent and preparation method thereof

Abstract

This invention discloses a multi-effect synergistic repair scaffold for large bone defects and its preparation method. This invention relates to the field of biomedical materials technology. The scaffold comprises a three-dimensional biomimetic structure with a central tubular channel. The structure consists of a core layer, a middle layer, and an outer layer from the inside out. The core layer is a hollow structure made of shape memory polymer material, and the middle layer is a biodegradable biomimetic porous ceramic matrix loaded with a first bioactive factor. The advantages of this invention are: by constructing a hierarchical structure of core layer-middle layer-outer layer, it integrates mechanical support, osteogenic activity, vascularization, and immune regulation functions, achieving synergistic intervention throughout the entire process of repairing large bone defects. The core layer uses a shape memory polymer, which not only provides axial compressive strength but also allows the scaffold to be implanted via minimally invasive surgery in a folded state. After implantation, it automatically recovers its shape at body temperature, closely adhering to the user's bone interface.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a multi-effect synergistic repair scaffold for large bone defects and its preparation method. Background Technology

[0002] Large bone defects caused by trauma, tumor resection, infection, etc. are a major challenge in orthopedic clinics. Due to the limited availability of autologous bone and the existence of donor site complications, and the risk of immune rejection and disease transmission from allogeneic bone, there is currently no perfect solution in clinical practice.

[0003] Existing artificial bone repair materials mainly face three core challenges: slow vascularization (difficulty in establishing microcirculation after scaffold implantation, leading to necrosis in the central area and failure to achieve deep bone integration), poor mechanical adaptability (insufficient mechanical strength of traditional scaffolds, unable to provide early stability, and the degradation rate does not match bone regeneration, resulting in stress shielding effect), and lack of spatiotemporal delivery function (difficulty in releasing bioactive factors as needed at different stages of defect repair (inflammatory phase, repair phase, remodeling phase). To address these challenges, we propose a multi-effect synergistic repair scaffold for large bone defects and its preparation method. Summary of the Invention

[0004] The purpose of this invention is to provide a multi-effect synergistic repair scaffold for large bone defects and its preparation method.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a multi-effect synergistic repair scaffold for large bone defects, the scaffold as a whole comprising a three-dimensional biomimetic structure with a central tubular channel, the structure being composed of a core layer, a middle layer and an outer layer from the inside out, wherein the core layer is a hollow structure made of shape memory polymer material;

[0006] The middle layer is a biodegradable biomimetic porous ceramic matrix loaded with the first bioactive factor. Its porosity is 60%-85%, and the pore size is distributed in a gradient. The outer layer pore size is 50μm-100μm, and the inner layer pore size is 200μm-400μm.

[0007] The outer layer is a nanofiber hydrogel composite membrane layer loaded with a second bioactive factor, which is coated on the surface of the middle layer.

[0008] A method for preparing a multi-effect synergistic scaffold for repairing large bone defects includes a method for preparing the entire scaffold, the specific steps of which are as follows:

[0009] Step 1

[0010] Polymer tubular scaffolds with shape memory function were prepared using 3D printing technology. The polymer was a block copolymer of polycaprolactone and polyurethane. The printing path combined axial parallel arrangement and radial ring arrangement to form a biomimetic bone unit structure. After printing, the scaffolds were treated with low-temperature plasma to introduce amino functional groups on the surface.

[0011] Step Two

[0012] Nano-hydroxyapatite and β-tricalcium phosphate were mixed at a mass ratio of 7:3, and chitosan-gelatin microspheres containing vascular endothelial growth factor were added. The mixture was stirred evenly to form a photocurable slurry with a vascular endothelial growth factor loading of 0.5 μg / mL-5 μg / mL.

[0013] Step 3

[0014] The core layer obtained in step one is placed inside the coaxial printing device, and the outer nozzle extrudes the photocurable slurry obtained in step two. The intermediate layer is printed layer by layer on the surface of the core layer. During the printing process, it is cured by ultraviolet light irradiation. After printing, it is placed in a 37°C constant temperature oven for incubation, so that the amino functional groups on the surface of the core layer and the carboxyl groups in the intermediate layer slurry undergo an amidation reaction to form covalent bonds.

[0015] Step 4

[0016] The semi-finished product obtained in step three is immersed in a methacrylamide gelatin solution containing macrophage polarization regulator and stromal cell-derived factor-1α, and a nanofiber hydrogel composite film with a thickness of 100μm-300μm is formed through secondary cross-linking.

[0017] Step 5

[0018] The entire scaffold was prepared by sterilization using supercritical carbon dioxide drying.

[0019] As a further aspect of the present invention: the core layer is a 3D printed polymer tubular scaffold with an axially parallel arrangement and a radially annular arrangement structure, and its shape memory transition temperature is 37℃-42℃.

[0020] As a further aspect of the present invention, the porosity of the intermediate layer is 60%-85%, and the pore size is gradient-distributed.

[0021] As a further aspect of the present invention: In step one, fused deposition modeling 3D printing is used, with the printing nozzle temperature set to 90℃-110℃, the platform temperature to 40℃-50℃, and the layer thickness to 0.1mm-0.2mm, constructing a tubular structure with an outer diameter of 4mm-12mm and a wall thickness of 0.5mm-2mm, consisting of alternating stacked axial parallel fibers and radial annular fibers. After printing, the structure is sequentially ultrasonically cleaned with acetone for 10min, dehydrated with anhydrous ethanol, and vacuum dried for 12h. After completion, it is treated with a plasma treatment instrument at a power of 150W and an oxygen flow rate of 30sccm for 5min-8min, and then immersed in a 2% 3-aminopropyltriethoxysilane ethanol solution at 60℃ for 4h to introduce stable amino functional groups.

[0022] As a further aspect of the present invention: In step two, nano-hydroxyapatite with a particle size of 20nm-50nm and β-tricalcium phosphate with a particle size of 1μm-5μm are placed in a ball mill at a mass ratio of 7:3 and dry-mixed at 300r / min for 2h. Polyethylene glycol diacrylate photocurable resin is added until the solid content is 50%-60%. After stirring and dispersing evenly, chitosan-gelatin microspheres loaded with vascular endothelial growth factor prepared by emulsification crosslinking are slowly incorporated and mixed with 0.5% by mass of photoinitiator 2959.

[0023] As a further aspect of the present invention: In step three, the core layer is coaxially fixed to the inner nozzle of the three-axis linkage printing platform, and a 22G coaxial nozzle is selected as the outer nozzle. The intermediate layers are printed layer by layer under the conditions of printing air pressure of 0.2MPa-0.4MPa, printing speed of 5mm / s-10mm / s, and layer thickness of 0.3mm. After each layer is printed, a wavelength of 365nm and a light intensity of 20mW / cm are immediately applied. 2 The resin was cured by irradiation with a UV point light source for 15 seconds. After all printing was completed, it was placed in a constant temperature and humidity chamber at 37℃ and 95% relative humidity for 24 hours. During this period, a small amount of pH 7.4 phosphate buffer was sprayed every 6 hours to maintain interface moisture and promote the full amidation reaction between the amino groups of the core layer and the residual carboxyl groups of the intermediate layer photocured resin.

[0024] As a further aspect of the present invention: In step four, a 10%-15% (w / w) solution of methacrylamide gelatin with a substitution degree ≥60% is prepared. 0.25% of the photoinitiator LAP is added, followed by the sequential addition of 50 ng / mL recombinant human IL-4 and 100 ng / mL SDF-1α, with gentle stirring until completely dissolved. The composite scaffold obtained in step three is then immersed in the solution and allowed to stand for 10 minutes to allow for full penetration. Subsequently, it is removed and placed in a high-voltage electrostatic spraying device with a voltage of 15 kV and a receiving distance of 15 cm for rotary spraying to form a nanofiber membrane. Finally, a light intensity of 10 mW / cm is applied. 2Secondary cross-linking was performed by irradiating with 405nm blue light for 90s.

[0025] As a further aspect of the present invention: In step five, the finished bracket is placed in a supercritical carbon dioxide drying device, using CO2 as the medium, and the temperature is set to 35℃-37℃, the pressure to 10MPa-12MPa, and the dynamic circulation time to 2h for drying and sterilization. At the same time, the strong permeability of supercritical fluid is used to remove residual organic solvents and achieve a sterile state. After sterilization, it is immediately sealed in a sterile vacuum bag under a Class 100 laminar flow environment.

[0026] Compared with the prior art, the beneficial effects of the present invention by adopting the above technical solution are as follows:

[0027] 1. This invention integrates mechanical support, osteogenic activity, vascularization, and immune regulation functions by constructing a hierarchical structure of core layer-middle layer-outer layer, thereby achieving synergistic intervention in the entire process of repairing large bone defects. The core layer uses shape memory polymer, which not only provides axial compressive strength but also allows the scaffold to be implanted in a folded state through minimally invasive surgery. After implantation, it automatically restores its shape at body temperature and fits tightly to the user's bone interface, solving the problems of large trauma and poor fit in the implantation of large bone scaffolds.

[0028] 2. This invention utilizes microsphere encapsulation and the different degradation rates of various materials. The outer hydrogel degrades quickly, releasing immune factors in the early stage, while the middle ceramic layer degrades slowly, releasing osteogenic factors in the later stage. This achieves the spatiotemporal sequential release of bioactive factors, precisely matching the biological needs during the bone healing process, and significantly improving vascularization efficiency and the quality of new bone formation.

[0029] 3. This invention connects the core layer and the intermediate layer by chemical bonding, avoiding the interlayer delamination problem caused by different degradation rates in traditional multilayer stents in vivo, and ensuring the structural stability of the stent during long-term repair. Attached Figure Description

[0030] Figure 1 This is a flowchart illustrating the stent fabrication process in an embodiment of the present invention. Detailed Implementation

[0031] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0032] Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0033] Please see the appendix Figure 1The present invention provides a multi-effect synergistic repair scaffold for large bone defects. The scaffold as a whole includes a three-dimensional biomimetic structure with a central tubular channel. The structure consists of a core layer, a middle layer and an outer layer from the inside to the outside. The core layer is a hollow structure made of shape memory polymer material.

[0034] The middle layer is a biodegradable biomimetic porous ceramic matrix loaded with the first bioactive factor. Its porosity is 60%-85%, and the pore size is distributed in a gradient. The outer layer pore size is 50μm-100μm, and the inner layer pore size is 200μm-400μm.

[0035] The outer layer is a nanofiber hydrogel composite membrane layer loaded with a second bioactive factor, which is coated on the surface of the middle layer.

[0036] A method for preparing a multi-effect synergistic scaffold for repairing large bone defects includes a method for preparing the entire scaffold, and the specific steps of the scaffold preparation method are as follows:

[0037] Step 1

[0038] Polymer tubular scaffolds with shape memory function were prepared using 3D printing technology. The polymer was a block copolymer of polycaprolactone and polyurethane. The printing path combined axial parallel arrangement and radial ring arrangement to form a biomimetic bone unit structure. After printing, the scaffolds were treated with low-temperature plasma to introduce amino functional groups on the surface.

[0039] Step Two

[0040] Nano-hydroxyapatite and β-tricalcium phosphate were mixed at a mass ratio of 7:3, and chitosan-gelatin microspheres containing vascular endothelial growth factor were added. The mixture was stirred evenly to form a photocurable slurry with a vascular endothelial growth factor loading of 0.5 μg / mL-5 μg / mL.

[0041] Step 3

[0042] The core layer obtained in step one is placed inside the coaxial printing device, and the outer nozzle extrudes the photocurable slurry obtained in step two. The intermediate layer is printed layer by layer on the surface of the core layer. During the printing process, it is cured by ultraviolet light irradiation. After printing, it is placed in a 37°C constant temperature oven for incubation, so that the amino functional groups on the surface of the core layer and the carboxyl groups in the intermediate layer slurry undergo an amidation reaction to form covalent bonds.

[0043] Step 4

[0044] The semi-finished product obtained in step three is immersed in a methacrylamide gelatin solution containing macrophage polarization regulator and stromal cell-derived factor-1α, and a nanofiber hydrogel composite film with a thickness of 100μm-300μm is formed through secondary cross-linking.

[0045] Step 5

[0046] The entire scaffold was prepared by sterilization using supercritical carbon dioxide drying.

[0047] In one embodiment of the present invention: the core layer is a 3D printed polymer tubular scaffold with an axially parallel arrangement and a radially annular arrangement, and its shape memory transition temperature is 37℃-42℃.

[0048] In one embodiment of the present invention: the porosity of the intermediate layer is 60%-85%, and the pore size is gradient-distributed.

[0049] In one embodiment of the present invention: In step one, fused deposition modeling 3D printing is used, with the printing nozzle temperature set to 90℃-110℃, the platform temperature to 40℃-50℃, and the layer thickness to 0.1mm-0.2mm, to construct a tubular structure with an outer diameter of 4mm-12mm and a wall thickness of 0.5mm-2mm, consisting of alternating stacked axial parallel fibers and radial annular fibers. After printing, the structure is sequentially ultrasonically cleaned with acetone for 10min, dehydrated with anhydrous ethanol, and vacuum dried for 12h. After completion, it is treated with a plasma treatment instrument at a power of 150W and an oxygen flow rate of 30sccm for 5min-8min, and then immersed in a 2% 3-aminopropyltriethoxysilane ethanol solution at 60℃ for 4h to introduce stable amino functional groups.

[0050] In one embodiment of the present invention: In step two, nano-hydroxyapatite with a particle size of 20nm-50nm and β-tricalcium phosphate with a particle size of 1μm-5μm are placed in a ball mill at a mass ratio of 7:3 and dry-mixed at 300r / min for 2h. Polyethylene glycol diacrylate photocurable resin is added until the solid content is 50%-60%. After stirring and dispersing evenly, chitosan-gelatin microspheres loaded with vascular endothelial growth factor prepared by emulsification crosslinking are slowly incorporated and mixed with 0.5% by mass of photoinitiator 2959.

[0051] In one embodiment of the present invention: In step three, the core layer is coaxially fixed to the inner nozzle of the three-axis linkage printing platform, and a 22G coaxial nozzle is selected as the outer nozzle. The intermediate layers are printed layer by layer under the conditions of printing air pressure of 0.2MPa-0.4MPa, printing speed of 5mm / s-10mm / s, and layer thickness of 0.3mm. After each layer is printed, a light intensity of 20mW / cm is applied immediately. 2 The resin was cured by irradiation with a UV point light source for 15 seconds. After all printing was completed, it was placed in a constant temperature and humidity chamber at 37℃ and 95% relative humidity for 24 hours. During this period, a small amount of pH 7.4 phosphate buffer was sprayed every 6 hours to maintain interface moisture and promote the full amidation reaction between the amino groups of the core layer and the residual carboxyl groups of the intermediate layer photocured resin.

[0052] In one embodiment of the present invention: In step four, a 10%-15% (w / w) solution of methacrylamide gelatin with a substitution degree ≥60% is prepared. 0.25% of photoinitiator LAP is added, followed by the sequential addition of 50 ng / mL recombinant human IL-4 and 100 ng / mL SDF-1α, with gentle stirring until completely dissolved. The composite scaffold obtained in step three is immersed in the solution and allowed to stand for 10 minutes to allow for full penetration. Then, it is removed and placed in a high-voltage electrostatic spraying device with a voltage of 15 kV and a receiving distance of 15 cm for rotary spraying to form a nanofiber membrane. Finally, a light intensity of 10 mW / cm is applied. 2 Secondary cross-linking was performed by irradiating with 405nm blue light for 90s.

[0053] In one embodiment of the present invention: In step five, the finished bracket is placed in a supercritical carbon dioxide drying device, using CO2 as the medium, and the temperature is set to 35℃-37℃, the pressure to 10MPa-12MPa and the dynamic circulation time to 2h for drying and sterilization. At the same time, the strong permeability of supercritical fluid is used to remove residual organic solvents and achieve a sterile state. After sterilization, it is immediately sealed in a sterile vacuum bag under a Class 100 laminar flow environment.

[0054] Example 1

[0055] 1. Material and parameter selection:

[0056] Core layer: polycaprolactone-polyurethane block copolymer, molar ratio 70:30, transition temperature set at 40℃;

[0057] Intermediate layer: Nano-hydroxyapatite and β-tricalcium phosphate in a mass ratio of 7:3, with bioactive factors being vascular endothelial growth factor and BMP-2, encapsulated in chitosan-gelatin microspheres;

[0058] Outer layer: Methacrylamide gelatin, containing IL-4 and SDF-1α;

[0059] 2. Preparation process:

[0060] Step 1 (Core Layer): Fused deposition modeling 3D printing was used with a nozzle temperature of 100℃, a platform temperature of 45℃, and a layer thickness of 0.15mm to construct a tubular scaffold with an outer diameter of 6mm, an inner diameter of 3mm, and a length of 40mm. The printing path was as follows: axial fiber spacing of 0.5mm and radial annular fiber spacing of 1.0mm, with 5 layers stacked alternately. After printing, the scaffold was ultrasonically cleaned with acetone for 10min, dehydrated with anhydrous ethanol, and vacuum dried for 12h. It was then treated with a plasma treatment instrument (power 150W, oxygen flow rate 30sccm, treatment for 6min), and subsequently immersed in a 2% APTES ethanol solution at 60℃ for 4h to introduce amino functional groups.

[0061] Step 2 (Intermediate Layer Slurry): Mix nHA (particle size 30nm) and β-TCP (particle size 2μm) at a mass ratio of 7:3 by ball milling (300rpm, 2h). Add PEGDA photocurable resin to a solid content of 55%. After stirring and dispersing, slowly incorporate drug-loaded microspheres (microsphere to slurry mass ratio 1:10) under ice bath conditions. Add 0.5% photoinitiator 2959 and stir until homogeneous.

[0062] Step 3 (Integrated Molding): Fix the core layer to the coaxial printing inner needle (inner diameter 2.5mm), outer nozzle 22G, printing air pressure 0.3MPa, printing speed 8mm / s, layer thickness 0.3mm, print the intermediate layers layer by layer until the total thickness is 2mm. After each layer is printed, irradiate with 365nm ultraviolet light (20mW / cm²) for 15s to cure. After completion, place it in a constant temperature chamber at 37℃ and 95% humidity for 24h, and spray a small amount of PBS buffer every 6h.

[0063] Step 4 (Outer Layer): Prepare a 12% GelMA solution, add 0.25% LAP photoinitiator, then add IL-4 (50 ng / mL) and SDF-1α (100 ng / mL). Immerse the composite scaffold in the solution for 10 min, then remove it and place it in an electrostatic spraying device (voltage 15 kV, receiving distance 15 cm, ambient temperature 25 °C, humidity 35%) for rotary spraying to form a nanofiber film with a thickness of approximately 200 μm. Then, apply 405 nm blue light (10 mW / cm²) to the film. 2 Irradiation for 90 seconds results in secondary cross-linking;

[0064] Step 5 (sterilization and packaging): Place the bracket in a vacuum oven at 40°C and -0.08MPa for 8 hours to pre-dry, then transfer it to a supercritical CO2 drying device, set the temperature to 37°C and the pressure to 11MPa, and circulate it dynamically for 2 hours. Then, sterilely vacuum package it under Class 100 laminar flow.

[0065] 3. Structural characterization and performance testing:

[0066] Microstructure: Scanning electron microscopy shows that the pore size of the middle layer is gradient-distributed, with the inner layer (near the core layer) having a pore size of 200 μm-400 μm, the outer layer having a pore size of 50 μm-100 μm, and the outer layer nanofibers having a diameter of 80 nm-150 nm, with a porosity of approximately 85%.

[0067] Mechanical properties: The compressive strength test showed that the axial compressive strength of the scaffold was 18.3MPa±2.1MPa and the elastic modulus was 320MPa±25MPa, which matched the mechanical properties of cancellous bone.

[0068] In vitro sustained-release behavior: ELISA detection showed that vascular endothelial growth factor was rapidly released from 1 to 7 days, with a cumulative release rate of about 45%; BMP-2 was continuously released from 14 to 28 days, with a cumulative release rate of about 65%; IL-4 was completely released within 3 days; and SDF-1α was released by about 70% within 7 days, achieving the time sequence of early release of immunomodulatory factors and sequential release of angiogenic and osteogenic factors.

[0069] Animal experiments: Using a 15mm segmental femoral defect model in New Zealand rabbits, Micro-CT results at 12 weeks post-surgery showed that the defect area was completely bridged by new bone tissue, with a bone volume fraction (BV / TV) of 76.8% ± 5.2% and a vessel density of 42.6 ± 6.3 vessels / mm². 2 The results were significantly better than the control group, with a BV / TV ratio of 38.4% ± 4.1% and a vessel density of 18.2 ± 3.5 vessels / mm². 2 Biomechanical testing showed that the shear strength at the scaffold-bone interface was 11.7 MPa ± 1.8 MPa.

[0070] Example 2

[0071] 1. Material and parameter selection:

[0072] Core layer: Polycaprolactone-polyurethane block copolymer, molar ratio 60:40, transition temperature set at 38℃;

[0073] Intermediate layer: Nano-hydroxyapatite and β-tricalcium phosphate in a mass ratio of 8:2, with vascular endothelial growth factor as the bioactive factor, loaded at 4 μg / mL, encapsulated separately in chitosan-gelatin microspheres with an average particle size of 120 μm and an encapsulation rate of 88%. Additionally, BMP-2 is directly added to the slurry at a loading of 2 μg / mL to achieve biphasic release differences.

[0074] Outer layer: Methacrylated gelatin, containing IL-10 and SDF-1α;

[0075] 2. Preparation process:

[0076] Step 1 (Core Layer): Fused deposition modeling 3D printing was used with a nozzle temperature of 95°C, a platform temperature of 42°C, and a layer thickness of 0.12 mm to construct a tubular scaffold with an outer diameter of 8 mm, an inner diameter of 4 mm, and a length of 50 mm. The printing path was as follows: axial fiber spacing of 0.6 mm and radial annular fiber spacing of 1.2 mm. The plasma treatment parameters were the same as in Example 1. The grafted amino group was then ready for use.

[0077] Step 2 (Intermediate Layer Slurry): Mix nHA (particle size 50nm) and β-TCP (particle size 3μm) at a mass ratio of 8:2 by ball milling (350r / min, 1.5h). Add PEGDA photocurable resin to a solid content of 60%, add 0.6% photoinitiator 2959, and slowly mix in vascular endothelial growth factor-loaded microspheres (microsphere to slurry mass ratio 1:12) at 4℃. At the same time, add free BMP-2 (final concentration 2μg / mL) and stir until homogeneous.

[0078] Step 3 (Integrated Molding): Adjust the coaxial printing parameters to air pressure 0.35MPa, printing speed 6mm / s, layer thickness 0.35mm, print the intermediate layer to a total thickness of 2.5mm, UV curing parameters are the same as in Example 1, and the incubation conditions are adjusted to incubate in a constant temperature chamber at 37℃ and 90% humidity for 48h to enhance the degree of amidation reaction;

[0079] Step 4 (outer layer): Prepare a 15% GelMA solution, add 0.3% LAP photoinitiator, then add IL-10 (80 ng / mL) and SDF-1α (150 ng / mL). After impregnation, adjust the electrostatic spraying parameters to a voltage of 18 kV, a receiving distance of 12 cm, an ambient temperature of 22 °C, and a humidity of 40% to form a nanofiber membrane with a thickness of about 250 μm. The blue light crosslinking time is extended to 120 s.

[0080] Step 5 (sterilization and packaging): The pre-drying conditions are 45℃ and -0.08MPa for 10h. The supercritical CO2 sterilization parameters are 38℃, 12MPa, and dynamic circulation for 2.5h. The rest is the same as in Example 1.

[0081] 3. Structural characterization and performance testing:

[0082] Microstructure: The pore size of the intermediate layer is gradient-distributed, with the inner layer having a pore size of 250μm-450μm and the outer layer having a pore size of 60μm-120μm. The outer layer nanofibers have a diameter of 100nm-200nm and a porosity of approximately 82%. Energy dispersive X-ray spectroscopy (EDS) analysis shows that the carbon and nitrogen elements are continuously distributed at the interface between the core layer and the intermediate layer, confirming that the amide bonds are well formed. The interfacial bonding strength test shows that the interlayer shear strength is 2.3MPa±0.3MPa.

[0083] Mechanical properties: compressive strength is 15.6MPa±1.9MPa, elastic modulus is 280MPa±22MPa. Shape memory test shows that the stent can be compressed to 40% of its original volume in an ice-water mixture (0℃), and recovers more than 95% of its original shape within 30s when placed in 38℃ physiological saline.

[0084] In vitro sustained-release behavior: ELISA test showed that free BMP-2 was released burstily in 1-3 days with a cumulative release rate of about 60%, vascular endothelial growth factor encapsulated in microspheres was released steadily in 7-21 days with a cumulative release rate of about 72%, IL-10 was completely released in 5 days, and SDF-1α was continuously released in 14 days, achieving a synergistic effect of early osteogenic induction, mid-term angiogenesis, and continuous stem cell homing;

[0085] Animal experiments: Using a beagle dog femoral segmental defect model with a 20mm segmental defect, Micro-CT results at 16 weeks post-surgery showed complete repair of the defect area, with a bone volume fraction of 81.2% ± 4.8% and a vascular density of 51.3 ± 5.7 vessels / mm². The newly formed bone tissue integrated tightly with the scaffold interface, with no obvious fibrous capsule formation. Biomechanical testing showed that the torsional strength of the repaired femur reached 82.6% ± 6.3% of the normal side, significantly better than the control group. The PCL / nHA composite scaffold only achieved 45.2% ± 5.1% of the normal side. Histological staining revealed neovascularization and active osteoblasts within the central tubular channels of the scaffold. The degradation of the outer hydrogel layer created an immunomodulatory microenvironment rich in M2 macrophages.

[0086] The two embodiments above demonstrate the implementation effects under different material ratios, active factor combinations, and process parameters, respectively. Both achieved effective repair of large bone defects, verifying the universality and stability of the technical solution of the present invention.

[0087] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Any variations and modifications can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, any modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention, without departing from the scope of the invention, fall within the protection scope defined by the claims of the present invention.

Claims

1. A multi-functional scaffold for repairing large segmental bone defects, the scaffold comprising a three-dimensional biomimetic structure with a central tubular channel, the structure consisting of a core layer, a middle layer, and an outer layer from the inside out, characterized in that: The core layer is a hollow structure made of shape memory polymer material; The middle layer is a biodegradable biomimetic porous ceramic matrix loaded with the first bioactive factor. Its porosity is 60%-85%, and the pore size is distributed in a gradient. The outer layer pore size is 50μm-100μm, and the inner layer pore size is 200μm-400μm. The outer layer is a nanofiber hydrogel composite membrane layer loaded with a second bioactive factor, which is coated on the surface of the middle layer.

2. A method for preparing a multi-effect synergistic repair scaffold for large segmental bone defects, applicable to any one of claims 1, comprising a method for preparing the entire scaffold, characterized in that: The specific steps of the stent preparation method are as follows: Step 1 Polymer tubular scaffolds with shape memory function were prepared using 3D printing technology. The polymer was a block copolymer of polycaprolactone and polyurethane. The printing path combined axial parallel arrangement and radial ring arrangement to form a biomimetic bone unit structure. After printing, the scaffolds were treated with low-temperature plasma to introduce amino functional groups on the surface. Step Two Nano-hydroxyapatite and β-tricalcium phosphate were mixed at a mass ratio of 7:3, and chitosan-gelatin microspheres containing vascular endothelial growth factor were added. The mixture was stirred evenly to form a photocurable slurry with a vascular endothelial growth factor loading of 0.5 μg / mL-5 μg / mL. Step 3 The core layer obtained in step one is placed inside the coaxial printing device, and the outer nozzle extrudes the photocurable slurry obtained in step two. The intermediate layer is printed layer by layer on the surface of the core layer. During the printing process, it is cured by ultraviolet light irradiation. After printing, it is placed in a 37°C constant temperature oven for incubation, so that the amino functional groups on the surface of the core layer and the carboxyl groups in the intermediate layer slurry undergo an amidation reaction to form covalent bonds. Step 4 The semi-finished product obtained in step three is immersed in a methacrylamide gelatin solution containing macrophage polarization regulator and stromal cell-derived factor-1α, and a nanofiber hydrogel composite film with a thickness of 100μm-300μm is formed through secondary cross-linking. Step 5 The entire scaffold was prepared by sterilization using supercritical carbon dioxide drying.

3. The method for preparing a multi-effect synergistic repair scaffold for large bone defects according to claim 2, characterized in that: The core layer is a 3D-printed polymer tubular scaffold with an axially parallel arrangement and a radially annular arrangement, and its shape memory transition temperature is 37℃-42℃.

4. The method for preparing a multi-effect synergistic repair scaffold for large bone defects according to claim 3, characterized in that: The porosity of the intermediate layer is 60%-85%, and the pore size is gradient-distributed.

5. The method for preparing a multi-effect synergistic repair scaffold for large bone defects according to claim 4, characterized in that: In step one, fused deposition modeling (FDM) 3D printing is used, with the nozzle temperature set to 90℃-110℃, platform temperature to 40℃-50℃, and layer thickness to 0.1mm-0.2mm. This constructs a tubular structure with alternating stacked axial parallel fibers and radial annular fibers, with an outer diameter of 4mm-12mm and a wall thickness of 0.5mm-2mm. After printing, the structure is sequentially ultrasonically cleaned with acetone for 10 minutes, dehydrated with anhydrous ethanol, and vacuum dried for 12 hours. After completion, it is treated with a plasma treatment instrument at a power of 150W and an oxygen flow rate of 30sccm for 5-8 minutes. Then, it is immersed in a 2% 3-aminopropyltriethoxysilane ethanol solution and reacted at 60℃ for 4 hours to introduce stable amino functional groups.

6. The method for preparing a multi-effect synergistic repair scaffold for large bone defects according to claim 5, characterized in that: In step two, nano-hydroxyapatite with a particle size of 20nm-50nm and β-tricalcium phosphate with a particle size of 1μm-5μm are placed in a ball mill at a mass ratio of 7:3 and dry-mixed at 300r / min for 2h. Polyethylene glycol diacrylate photocurable resin is added until the solid content is 50%-60%. After stirring and dispersing evenly, chitosan-gelatin microspheres loaded with vascular endothelial growth factor prepared by emulsification crosslinking are slowly incorporated and mixed with 0.5% by mass of photoinitiator 2959.

7. The method for preparing a multi-effect synergistic repair scaffold for large bone defects according to claim 6, characterized in that: In step three, the core layer is coaxially fixed to the inner nozzle of the three-axis linkage printing platform, and a 22G coaxial nozzle is selected as the outer nozzle. The intermediate layers are printed layer by layer under the conditions of printing air pressure of 0.2MPa-0.4MPa, printing speed of 5mm / s-10mm / s, and layer thickness of 0.3mm. After each layer is printed, a light source with a wavelength of 365nm and an intensity of 20mW / cm² is immediately applied. 2 The resin was cured by irradiation with a UV point light source for 15 seconds. After all printing was completed, it was placed in a constant temperature and humidity chamber at 37℃ and 95% relative humidity for 24 hours. During this period, a small amount of pH 7.4 phosphate buffer was sprayed every 6 hours to maintain interface moisture and promote the full amidation reaction between the amino groups of the core layer and the residual carboxyl groups of the intermediate layer photocured resin.

8. The method for preparing a multi-effect synergistic repair scaffold for large bone defects according to claim 7, characterized in that: In step four, a 10%-15% (w / w) solution of methacrylamide gelatin with a substitution degree ≥60% is prepared. 0.25% of the photoinitiator LAP is added, followed by the sequential addition of 50 ng / mL recombinant human IL-4 and 100 ng / mL SDF-1α, with gentle stirring until completely dissolved. The composite scaffold obtained in step three is then immersed in the solution and allowed to stand for 10 minutes to allow for full penetration. Subsequently, it is removed and placed in a high-voltage electrostatic spraying device with a voltage of 15 kV and a receiving distance of 15 cm for rotary spraying to form a nanofiber membrane. Finally, a light intensity of 10 mW / cm is applied. 2 Secondary cross-linking was performed by irradiating with 405nm blue light for 90s.

9. The method for preparing a multi-effect synergistic repair scaffold for large bone defects according to claim 8, characterized in that: In step five, the finished bracket is placed in a supercritical carbon dioxide drying device, using CO2 as the medium, and the temperature is set to 35℃-37℃, the pressure to 10MPa-12MPa, and the dynamic circulation time to 2h for drying and sterilization. At the same time, the strong permeability of supercritical fluid is used to remove residual organic solvents and achieve a sterile state. After sterilization, it is immediately sealed in a sterile vacuum bag under a Class 100 laminar flow environment.

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