A mussel-mucin-based multistage slow-release biomimetic bone repair scaffold and a preparation method thereof

Abstract

This invention relates to the field of bone repair scaffold technology, specifically to a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein and its preparation method. This invention organically integrates the strong wet adhesion of recombinant mussel adhesive protein, the excellent mechanical properties of biomimetic mineralized collagen, the programmed drug release capability of drug-loaded PLGA-PEG biphase sustained-release microspheres, and the 4D shape memory characteristics of thermosensitive poly(N-isopropylacrylamide) hydrogel to achieve multi-functional synergy. Then, low-temperature deposition 3D printing combined with thermosensitive materials is used to achieve 4D printing of the scaffold, resulting in a scaffold with a complex external morphology and a precise internal porous structure. The mechanical strength reaches the level of cortical bone, and it can actively adapt to the defect morphology after implantation, reducing intraoperative trimming time by more than 50%.

Description

Technical Field

[0001] This invention relates to the field of bone repair scaffold technology, specifically to a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein and its preparation method. Background Technology

[0002] Bone defect repair is a major clinical challenge, especially for large bone defects in weight-bearing areas. Ideal bone repair materials need to possess good biocompatibility, match the mechanical properties of the host bone, exhibit osteoinductive activity, and controllable drug release. However, current technologies face several bottlenecks: traditional calcium phosphate, hydroxyapatite, or collagen-based scaffolds often have compressive strengths below 20 MPa, making them insufficient for load-bearing requirements; conventional drug delivery systems such as poly(lactic-co-glycolic acid) microspheres exhibit significant initial burst release effects (release exceeding 40% within 24 hours); most bioadhesives show adhesion strength degradation exceeding 50% in humid environments; existing scaffolds are mostly static structures, unable to adaptively match irregular bone defects; and they often lack effective pro-angiogenic designs.

[0003] Chinese patent CN105457085A discloses a collagen / dopamine composite hydrogel, but its adhesive strength is limited and it lacks drug sustained-release function. European patent EP3618881A1 modifies polypropylene fumarate scaffolds through a thiol-ene click chemistry reaction, but the material lacks dynamic responsiveness. Chinese patent CN104491934A relates to collagen-coated cardiovascular scaffolds, but it does not solve the problem of controlled drug release in bone repair.

[0004] Therefore, it is of great significance to develop an intelligent bone repair scaffold that integrates strong wet adhesion, bone-like mechanical properties, programmed drug release, and morphological adaptation. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein, which has high compressive strength, stable drug release, strong wet adhesion, and can adaptively match the morphology of bone defects, as well as its preparation method.

[0006] The objective of this invention is achieved through the following technical solution: a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein, manufactured by printing from the following raw materials by weight percentage: Recombinant mussel adhesive protein 20-35% Biomimetic mineralized collagen 40-60% 10-25% drug-loaded PLGA-PEG biphasic sustained-release microspheres Thermosensitive poly(N-isopropylacrylamide) hydrogel 5-15%.

[0007] Preferably, the recombinant mussel adhesive protein is recombinant mussel adhesive protein Mfp-151.

[0008] Preferably, the biomimetic mineralized collagen has a Ca / P molar ratio of 1.60-1.70, a crystallinity of 65-75%, and a BET specific surface area of ​​35-60 m² / g.

[0009] Preferably, the core of the drug-loaded PLGA-PEG biphasic sustained-release microspheres is PLGA encapsulating a hydrophobic drug; the outer shell of the drug-loaded PLGA-PEG biphasic sustained-release microspheres is a composite layer of recombinant mussel adhesive protein Mfp-151 and polyethylene glycol encapsulating a hydrophilic drug.

[0010] Preferably, the drug-loaded PLGA-PEG biphase sustained-release microspheres have a particle size of 50 μm to 200 μm and a polydispersity index of less than 0.15.

[0011] Preferably, the thermosensitive poly(N-isopropylacrylamide) hydrogel is prepared by dissolving poly(N-isopropylacrylamide) powder with a molecular weight of 30,000-50,000 Da in deionized water to prepare a 15-25 wt% solution, and adding 0.3-0.6 wt% of photoinitiator Irgacure 2959.

[0012] A method for preparing a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein, comprising the following process steps: Step (1) Mix the formulated amounts of recombinant mussel adhesive protein, biomimetic mineralized collagen, drug-loaded PLGA-PEG biphase sustained-release microspheres, and thermosensitive poly(N-isopropylacrylamide) hydrogel, and homogenize to obtain printing ink. Step (2) Using low-temperature deposition 3D printing technology, the printing ink is printed into a preset three-dimensional structure on a printing platform at a temperature 15°C lower than the phase transition temperature of the temperature-sensitive poly(N-isopropylacrylamide) hydrogel, thus obtaining the molded scaffold. Step (3) The molded scaffold is freeze-dried, cross-linked and sterilized to obtain a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein.

[0013] Preferably, in step (2), the printing platform temperature is maintained at 10°C to 15°C, and the printing nozzle diameter is 200 μm to 400 μm; In step (3), the freeze-drying process specifically involves freezing the molded support at -80℃ for 20-28 hours, and then freeze-drying it at -50℃ and 0.1 mbar for 40-60 hours to obtain a porous dried support. The crosslinking in step (3) is as follows: the dried scaffold is immersed in a 0.05 wt% sodium periodate aqueous solution and crosslinked for 2-3 hours at 4°C in the dark, and then washed with a pH=7.4 phosphate buffer solution.

[0014] Preferably, the preparation method of the biomimetic mineralized collagen is as follows: type I collagen is dissolved in a 0.3-0.7M acid solution to prepare a collagen solution of 5-15 mg / mL, and the pH is adjusted to 7.2-7.6; 10 times the concentration of simulated body fluid is added dropwise at 2-8℃ to maintain pH stability, and the solution is transferred to 35-40℃ for shaking mineralization for 24-72 hours; the precipitate is collected, washed, and freeze-dried to obtain biomimetic mineralized collagen.

[0015] More preferably, the type I collagen is rat tail collagen with a purity ≥95%; the acid solution is glacial acetic acid solution, with pH adjusted using 1M sodium hydroxide solution; the simulated body fluid is SBF (Simulated Body Fluid); the washing endpoint is a supernatant conductivity <5 μS / cm; and the freeze-drying process includes: pre-freezing at -80℃ to -20℃ for 6-24 hours, followed by drying at -60℃ to -30℃ and 0.05-0.5 mbar for 24-72 hours.

[0016] Preferably, the preparation method of the drug-loaded PLGA-PEG biphasic sustained-release microspheres includes the following steps: (A) Dissolve PLGA and a hydrophobic drug in an organic solvent to prepare an oil phase solution; wherein the concentration of PLGA is 40-70 mg / mL and the concentration of the hydrophobic drug is 15-25% of the mass of PLGA; (B) The recombinant mussel adhesive protein and the hydrophilic drug are dissolved in an aqueous solvent containing an emulsifier to prepare an aqueous solution; wherein the concentration of the recombinant mussel adhesive protein is 0.3-0.8 mg / mL and the concentration of the hydrophilic drug is 0.1-0.4 mg / mL. (C) W / O / W type double emulsion droplets were prepared by using an oil phase solution as the inner phase and an aqueous phase solution as the outer phase via a coaxial microfluidic device; wherein the flow rate of the inner phase was 0.1-0.4 mL / h and the flow rate of the outer phase was 10-15 mL / h. (D) The W / O / W type double emulsion droplets prepared in step (C) are transferred to a curing solution containing 0.05-0.2 wt% polyvinyl alcohol, stirred for 2-6 hours, and the microspheres are collected, washed and dried to obtain drug-loaded PLGA-PEG biphase sustained-release microspheres.

[0017] More preferably, in step (A), the PLGA has a lactic acid to glycolic acid molar ratio of 50:50 to 85:15 and a molecular weight of 30,000-80,000 Da, and the organic solvent is dichloromethane; in step (B), the aqueous solvent is a phosphate buffer solution with a pH of 6.0-7.4, and the emulsifier is composed of 0.8-1.2 wt% vinyl alcohol and 0.2-0.6 wt% sodium dodecyl sulfate.

[0018] This invention is the first to introduce recombinant mussel adhesive protein Mfp-151 as a key component into a bone repair scaffold. Mussel adhesive protein is rich in dopa groups, enabling it to form strong underwater adhesion to the bone defect interface in a physiologically moist environment through hydrogen bonding, metal coordination bonds, and π-π stacking interactions. Simultaneously, through a sodium periodate-mediated cross-linking reaction, a stable cross-linked network is formed between mussel adhesive protein molecules, significantly enhancing the initial fixation strength between the scaffold and the host bone. This overcomes the defect of rapid adhesion decay in traditional hydrogels or bone cements under blood / body fluid infiltration, ensuring the mechanical stability of the scaffold in the early stages of implantation.

[0019] This invention uses biomimetic mineralized collagen as the main scaffold material. Its Ca / P molar ratio (1.60-1.70), crystallinity (65-75%), and high specific surface area (35-60 m² / g) highly simulate the composite characteristics of hydroxyapatite and collagen fibers in human bone tissue. Combined with low-temperature deposition 3D printing technology, a scaffold with a porous and interconnected structure can be precisely constructed. This not only provides a biomimetic microenvironment for cell ingrowth and vascularization, but also enhances the compressive strength of the scaffold by controlling the ratio of mineralized collagen to thermosensitive hydrogel, thus meeting the mechanical requirements for repairing bone defects in non-load-bearing and partially load-bearing areas.

[0020] This invention presents a unique "core-shell" biphasic sustained-release microsphere designed based on microfluidic technology. The core consists of a hydrophobic drug encapsulated in PLGA, while the shell consists of a hydrophilic drug encapsulated in a composite layer of recombinant mussel adhesive protein and polyethylene glycol. This structure combines monodisperse microspheres (particle size 50-200 μm, PDI < 0.15) prepared using coaxial microfluidic technology, enabling multi-stage drug release: the hydrophilic drug in the shell is rapidly released in the early stages of implantation, exerting anti-inflammatory or pro-angiogenic effects; the hydrophobic drug in the core is slowly and continuously released as the PLGA degrades, achieving long-term bone induction. This design effectively solves the problem of initial burst release caused by conventional PLGA microspheres where the drug is concentrated on the core surface or a single structure (the release rate can be controlled below 20% within 24 hours), ensuring stable and long-lasting drug release.

[0021] This invention introduces a thermosensitive poly(N-isopropylacrylamide) hydrogel with a phase transition temperature (approximately 32°C) slightly lower than body temperature. During the low-temperature printing and freeze-drying stages, the scaffold maintains a stable shape. After implantation, the hydrogel undergoes volume shrinkage or modulus changes at body temperature, allowing the scaffold to adaptively and closely conform to irregular bone defect morphologies. Simultaneously, this thermosensitive property endows the scaffold with potential for injectability. Combined with 3D printing technology, it enables personalized customization of the scaffold's macroscopic morphology and precise control of its microstructure, meeting the clinical needs for treating complex bone defects.

[0022] This invention employs low-temperature deposition 3D printing technology. The entire printing process, including subsequent cross-linking and drying, is conducted under mild conditions (printing platform 10-20°C, cross-linking 2-8°C), avoiding the damage to recombinant mussel adhesive proteins and growth factors caused by harsh conditions such as high temperatures, strong acids, and strong alkalis. Simultaneously, the freeze-drying process preserves the interconnected porous structure within the scaffold, resulting in high porosity and uniform pore size distribution, which facilitates cell infiltration, nutrient exchange, and the removal of metabolic waste.

[0023] The beneficial effects of this invention are as follows: This invention organically integrates the strong wet adhesion of recombinant mussel adhesive protein, the excellent mechanical properties of biomimetic mineralized collagen, the programmed drug release capability of drug-loaded PLGA-PEG biphasic sustained-release microspheres, and the 4D shape memory characteristics of thermosensitive poly(N-isopropylacrylamide) hydrogel to achieve multifunctional synergy. Then, by using low-temperature deposition 3D printing combined with thermosensitive materials, a scaffold is 4D printed to obtain a scaffold with a complex external morphology and a precise internal porous structure. The mechanical strength reaches the level of cortical bone, and it can actively adapt to the defect morphology after implantation, reducing intraoperative trimming time by more than 50%. The multi-level structure and component design of the scaffold simultaneously provide osteoconduction, osteoinduction, and vascularization signals, realizing the synergistic regulation of multiple biological processes in bone regeneration and significantly improving repair efficiency. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein, as per the present invention.

[0025] Figure Labels 1---Recombinant mussel adhesive protein adhesion layer; 2---Bionic mineralized collagen mechanical layer; 3---Drug-loaded microsphere sustained-release layer; 4---Thermosensitive hydrogel support layer. Detailed Implementation

[0026] The present invention will be further described in conjunction with the following embodiments.

[0027] Example 1 A multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein, manufactured by printing from the following raw materials by weight percentage: Composition: 20% recombinant mussel adhesive protein, 60% biomimetic mineralized collagen, 10% drug-loaded PLGA-PEG biphasic sustained-release microspheres, and 10% thermosensitive poly(N-isopropylacrylamide) hydrogel. The recombinant mussel adhesive protein is recombinant mussel adhesive protein Mfp-151.

[0028] The biomimetic mineralized collagen has a Ca / P molar ratio of 1.60, a crystallinity of 65%, and a BET specific surface area of ​​35 m² / g.

[0029] The core of the drug-loaded PLGA-PEG biphasic sustained-release microspheres is PLGA encapsulating a hydrophobic drug; the outer shell of the drug-loaded PLGA-PEG biphasic sustained-release microspheres is a composite layer of recombinant mussel adhesive protein Mfp-151 and polyethylene glycol encapsulating a hydrophilic drug.

[0030] The drug-loaded PLGA-PEG biphase sustained-release microspheres have a particle size of 50 μm and a polydispersity index of 0.12.

[0031] The thermosensitive poly(N-isopropylacrylamide) hydrogel is prepared by dissolving poly(N-isopropylacrylamide) powder with a molecular weight of 30,000 Da in deionized water to prepare a 15 wt% solution, and adding 0.3 wt% of photoinitiator Irgacure 2959.

[0032] A method for preparing a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein, comprising the following process steps: Step (1) Mix the formulated amounts of recombinant mussel adhesive protein, biomimetic mineralized collagen, drug-loaded PLGA-PEG biphase sustained-release microspheres, and thermosensitive poly(N-isopropylacrylamide) hydrogel, and homogenize to obtain printing ink. Step (2) Using low-temperature deposition 3D printing technology, the printing ink is printed into a preset three-dimensional structure on a printing platform below the phase transition temperature of the temperature-sensitive poly(N-isopropylacrylamide) hydrogel to obtain the molded scaffold. Step (3) The molded scaffold is freeze-dried, cross-linked and sterilized to obtain a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein.

[0033] Preferably, in step (2), the printing platform temperature is maintained at 10°C, the printing nozzle diameter is 200 μm, the extrusion pressure is set to 25 kPa, the printing speed is 8 mm / s, and the layer thickness is 200 μm. Based on the three-dimensional model (a porous cylinder with a diameter of 6 mm, a height of 5 mm, and a porosity of 80%) reconstructed from the Micro-CT data of the rabbit femoral condyle defect, the printing path is planned and the scaffold is printed layer by layer.

[0034] In step (3), the freeze-drying process specifically involves freezing the molded support at -80°C for 20 hours, and then freeze-drying it at -50°C and 0.1 mbar for 40 hours to obtain a porous dried support. The crosslinking in step (3) is as follows: the dried scaffold is immersed in a 0.05 wt% sodium periodate aqueous solution and crosslinked for 2 hours at 4°C in the dark, and then washed with a pH=7.4 phosphate buffer solution.

[0035] The preparation method of the biomimetic mineralized collagen is as follows: Type I collagen is dissolved in a 0.3M acid solution to prepare a collagen solution of 5 mg / mL, and the pH is adjusted to 7.2; 10 times the concentration of simulated body fluid is added dropwise at 2℃ to maintain pH stability, and the solution is transferred to 35℃ for shaking mineralization for 24 hours; the precipitate is collected, washed, and freeze-dried to obtain biomimetic mineralized collagen.

[0036] The type I collagen is rat tail collagen with a purity ≥95%; the acid solution is glacial acetic acid solution, with pH adjusted using 1M sodium hydroxide solution; the simulated body fluid is SBF (Simulated Body Fluid); the washing endpoint is a supernatant conductivity <5 μS / cm; the freeze-drying process includes: pre-freezing at -80℃ for 6 hours, followed by drying at -60℃ and 0.05 mbar for 24 hours.

[0037] The preparation method of the drug-loaded PLGA-PEG biphasic sustained-release microspheres includes the following steps: (A) PLGA and the hydrophobic drug dexamethasone (purity >98%) were dissolved in an organic solvent to prepare an oil phase solution; wherein the concentration of PLGA was 40 mg / mL and the concentration of the hydrophobic drug was 15% of the mass of PLGA; (B) Recombinant mussel adhesive protein and hydrophilic drug recombinant human VEGF (activity >95%) were dissolved in an aqueous solvent containing an emulsifier to prepare an aqueous solution; wherein the concentration of recombinant mussel adhesive protein was 0.3 mg / mL and the concentration of hydrophilic drug was 0.1 mg / mL. (C) W / O / W type double emulsion droplets were prepared by using an oil phase solution as the inner phase and an aqueous phase solution as the outer phase via a coaxial microfluidic device; wherein the inner phase flow rate was 0.1 mL / h and the outer phase flow rate was 10 mL / h. (D) The W / O / W type double emulsion droplets prepared in step (C) are transferred to a curing solution containing 0.05 wt% polyvinyl alcohol, stirred for 2 hours, and the microspheres are collected, washed and dried to obtain drug-loaded PLGA-PEG biphase sustained-release microspheres.

[0038] In step (A), the PLGA has a lactic acid to glycolic acid molar ratio of 50:50 and a molecular weight of 30,000 Da, and the organic solvent is dichloromethane; in step (B), the aqueous solvent is a phosphate buffer solution with a pH of 6.0, and the emulsifier consists of 0.8 wt% polyvinyl alcohol and 0.2 wt% sodium dodecyl sulfate.

[0039] Example 2 A multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein, manufactured by printing from the following raw materials by weight percentage: The formula consists of 27.5% recombinant mussel adhesive protein, 47.5% biomimetic mineralized collagen, 17.5% drug-loaded PLGA-PEG biphasic sustained-release microspheres, and 7.5% thermosensitive poly(N-isopropylacrylamide) hydrogel. The recombinant mussel adhesive protein is recombinant mussel adhesive protein Mfp-151.

[0040] The biomimetic mineralized collagen has a Ca / P molar ratio of 1.65, a crystallinity of 70%, and a BET specific surface area of ​​47.5 m² / g.

[0041] The core of the drug-loaded PLGA-PEG biphasic sustained-release microspheres is PLGA encapsulating a hydrophobic drug; the outer shell of the drug-loaded PLGA-PEG biphasic sustained-release microspheres is a composite layer of recombinant mussel adhesive protein Mfp-151 and polyethylene glycol encapsulating a hydrophilic drug.

[0042] The drug-loaded PLGA-PEG biphase sustained-release microspheres have a particle size of 125 μm and a polydispersity index of 0.10.

[0043] The thermosensitive poly(N-isopropylacrylamide) hydrogel is prepared by dissolving poly(N-isopropylacrylamide) powder with a molecular weight of 40,000 Da in deionized water to prepare a 20 wt% solution, and adding 0.45 wt% of photoinitiator Irgacure 2959.

[0044] A method for preparing a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein, comprising the following process steps: Step (1) Mix the formulated amounts of recombinant mussel adhesive protein, biomimetic mineralized collagen, drug-loaded PLGA-PEG biphase sustained-release microspheres, and thermosensitive poly(N-isopropylacrylamide) hydrogel, and homogenize to obtain printing ink. Step (2) Using low-temperature deposition 3D printing technology, the printing ink is printed into a preset three-dimensional structure on a printing platform below the phase transition temperature of the temperature-sensitive poly(N-isopropylacrylamide) hydrogel to obtain the molded scaffold. Step (3) The molded scaffold is freeze-dried, cross-linked and sterilized to obtain a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein.

[0045] Preferably, in step (2), the printing platform temperature is maintained at 12°C, the printing nozzle diameter is 300 μm, the extrusion pressure is set to 25 kPa, the printing speed is 8 mm / s, and the layer thickness is 200 μm. Based on the three-dimensional model (a porous cylinder with a diameter of 6 mm, a height of 5 mm, and a porosity of 80%) reconstructed from the Micro-CT data of the rabbit femoral condyle defect, the printing path is planned and the scaffold is printed layer by layer.

[0046] In step (3), the freeze-drying process specifically involves freezing the molded support at -80°C for 24 hours, and then freeze-drying it at -50°C and 0.1 mbar for 50 hours to obtain a porous dried support. The crosslinking process in step (3) is as follows: the dried scaffold is immersed in a 0.05 wt% sodium periodate aqueous solution and crosslinked for 2.5 hours at 4°C in the dark, and then washed with a pH=7.4 phosphate buffer solution.

[0047] The preparation method of the biomimetic mineralized collagen is as follows: Type I collagen is dissolved in a 0.5M acid solution to prepare a collagen solution of 10 mg / mL, and the pH is adjusted to 7.4; 10 times the concentration of simulated body fluid is added dropwise at 5℃ to maintain pH stability, and the solution is transferred to 37.5℃ for shaking mineralization for 48 hours; the precipitate is collected, washed, and freeze-dried to obtain biomimetic mineralized collagen.

[0048] The type I collagen is rat tail collagen with a purity ≥95%; the acid solution is glacial acetic acid solution, with pH adjusted using 1M sodium hydroxide solution; the simulated body fluid is SBF (Simulated Body Fluid); the washing endpoint is a supernatant conductivity <5 μS / cm; the freeze-drying process includes: pre-freezing at -50℃ for 15 hours, followed by drying at -45℃ and 0.275 mbar for 48 hours.

[0049] The preparation method of the drug-loaded PLGA-PEG biphasic sustained-release microspheres includes the following steps: (A) PLGA and the hydrophobic drug dexamethasone (purity >98%) were dissolved in an organic solvent to prepare an oil phase solution; wherein the concentration of PLGA was 55 mg / mL and the concentration of the hydrophobic drug was 20% of the mass of PLGA; (B) The recombinant mussel adhesive protein and the hydrophilic drug recombinant human VEGF (activity >95%) were dissolved in an aqueous solvent containing an emulsifier to prepare an aqueous solution; wherein the concentration of the recombinant mussel adhesive protein was 0.55 mg / mL and the concentration of the hydrophilic drug was 0.25 mg / mL. (C) W / O / W type double emulsion droplets were prepared by using an oil phase solution as the inner phase and an aqueous phase solution as the outer phase via a coaxial microfluidic device; wherein the inner phase flow rate was 0.25 mL / h and the outer phase flow rate was 12.5 mL / h. (D) The W / O / W type double emulsion droplets prepared in step (C) are transferred to a curing solution containing 0.125 wt% polyvinyl alcohol, stirred for 4 hours, and the microspheres are collected, washed and dried to obtain drug-loaded PLGA-PEG biphase sustained-release microspheres.

[0050] In step (A), the PLGA has a lactic acid to glycolic acid molar ratio of 67.5:32.5 and a molecular weight of 55,000 Da, and the organic solvent is dichloromethane; in step (B), the aqueous solvent is a phosphate buffer solution with a pH of 6.7, and the emulsifier consists of 1.0 wt% polyvinyl alcohol and 0.4 wt% sodium dodecyl sulfate.

[0051] Example 3 A multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein, manufactured by printing from the following raw materials by weight percentage: Composition: 35% recombinant mussel adhesive protein, 40% biomimetic mineralized collagen, 15% drug-loaded PLGA-PEG biphasic sustained-release microspheres, and 10% thermosensitive poly(N-isopropylacrylamide) hydrogel. The recombinant mussel adhesive protein is recombinant mussel adhesive protein Mfp-151.

[0052] The biomimetic mineralized collagen has a Ca / P molar ratio of 1.70, a crystallinity of 75%, and a BET specific surface area of ​​60 m² / g.

[0053] The core of the drug-loaded PLGA-PEG biphasic sustained-release microspheres is PLGA encapsulating a hydrophobic drug; the outer shell of the drug-loaded PLGA-PEG biphasic sustained-release microspheres is a composite layer of recombinant mussel adhesive protein Mfp-151 and polyethylene glycol encapsulating a hydrophilic drug.

[0054] The drug-loaded PLGA-PEG biphase sustained-release microspheres have a particle size of 200 μm and a polydispersity index of 0.08.

[0055] The thermosensitive poly(N-isopropylacrylamide) hydrogel is prepared by dissolving poly(N-isopropylacrylamide) powder with a molecular weight of 50,000 Da in deionized water to prepare a 25 wt% solution, and adding 0.6 wt% of photoinitiator Irgacure 2959.

[0056] A method for preparing a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein, comprising the following process steps: Step (1) Mix the formulated amounts of recombinant mussel adhesive protein, biomimetic mineralized collagen, drug-loaded PLGA-PEG biphase sustained-release microspheres, and thermosensitive poly(N-isopropylacrylamide) hydrogel, and homogenize to obtain printing ink. Step (2) Using low-temperature deposition 3D printing technology, the printing ink is printed into a preset three-dimensional structure on a printing platform below the phase transition temperature of the temperature-sensitive poly(N-isopropylacrylamide) hydrogel to obtain the molded scaffold. Step (3) The molded scaffold is freeze-dried, cross-linked and sterilized to obtain a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein.

[0057] Preferably, in step (2), the printing platform temperature is maintained at 15°C, the printing nozzle diameter is 350 μm, the extrusion pressure is set to 25 kPa, the printing speed is 8 mm / s, and the layer thickness is 200 μm. Based on the three-dimensional model (a porous cylinder with a diameter of 6 mm, a height of 5 mm, and a porosity of 80%) reconstructed from the Micro-CT data of the rabbit femoral condyle defect, the printing path is planned and the scaffold is printed layer by layer.

[0058] In step (3), the freeze-drying process specifically involves freezing the molded support at -80°C for 28 hours, and then freeze-drying it at -50°C and 0.1 mbar for 60 hours to obtain a porous dried support. The crosslinking in step (3) is as follows: the dried support is immersed in a 0.05 wt% sodium periodate aqueous solution and crosslinked for 3 hours at 4°C in the dark, and then washed with a pH=7.4 phosphate buffer solution.

[0059] The preparation method of the biomimetic mineralized collagen is as follows: Type I collagen is dissolved in a 0.7M acid solution to prepare a collagen solution of 15 mg / mL, and the pH is adjusted to 7.6; 10 times the concentration of simulated body fluid is added dropwise at 8℃ to maintain pH stability, and the solution is transferred to 40℃ for shaking mineralization for 72 hours; the precipitate is collected, washed, and freeze-dried to obtain biomimetic mineralized collagen.

[0060] The type I collagen is rat tail collagen with a purity ≥95%; the acid solution is glacial acetic acid solution, with pH adjusted using 1M sodium hydroxide solution; the simulated body fluid is SBF (Simulated Body Fluid); the washing endpoint is a supernatant conductivity <5 μS / cm; the freeze-drying process includes: pre-freezing at -20℃ for 24 hours, followed by drying at -30℃ and 0.5 mbar for 72 hours.

[0061] The preparation method of the drug-loaded PLGA-PEG biphasic sustained-release microspheres includes the following steps: (A) PLGA and the hydrophobic drug dexamethasone (purity >98%) were dissolved in an organic solvent to prepare an oil phase solution; wherein the concentration of PLGA was 70 mg / mL and the concentration of the hydrophobic drug was 25% of the mass of PLGA; (B) The recombinant mussel adhesive protein and the hydrophilic drug recombinant human VEGF (activity >95%) were dissolved in an aqueous solvent containing an emulsifier to prepare an aqueous solution; wherein the concentration of the recombinant mussel adhesive protein was 0.8 mg / mL and the concentration of the hydrophilic drug was 0.4 mg / mL. (C) W / O / W type double emulsion droplets were prepared by using an oil phase solution as the inner phase and an aqueous phase solution as the outer phase via a coaxial microfluidic device; wherein the inner phase flow rate was 0.4 mL / h and the outer phase flow rate was 15 mL / h. (D) The W / O / W type double emulsion droplets prepared in step (C) were transferred to a curing solution containing 0.2 wt% polyvinyl alcohol, stirred for 6 hours, and the microspheres were collected, washed and dried to obtain drug-loaded PLGA-PEG biphase sustained-release microspheres.

[0062] In step (A), the PLGA has a lactic acid to glycolic acid molar ratio of 85:15 and a molecular weight of 80,000 Da, and the organic solvent is dichloromethane; in step (B), the aqueous solvent is a phosphate buffer solution with a pH of 7.4, and the emulsifier consists of 1.2 wt% polyvinyl alcohol and 0.6 wt% sodium dodecyl sulfate.

[0063] Comparative Example 1 The difference between this comparative example and Example 2 is that the recombinant mussel adhesive protein Mfp-151 is replaced with naturally extracted mussel adhesive protein.

[0064] Comparative Example 2 The difference between this comparative example and Example 2 is that the biomimetic mineralized collagen is replaced with ordinary hydroxyapatite (HA), while everything else remains the same.

[0065] Comparative Example 3 The difference between this comparative example and Example 2 is that the drug-loaded PLGA-PEG biphasic sustained-release microspheres are replaced with ordinary PLGA monophasic microspheres (only encapsulated with dexamethasone, without shell protein and VEGF). The preparation method of ordinary PLGA monophasic microspheres includes the following steps: (A) PLGA and the hydrophobic drug dexamethasone (purity >98%) were dissolved in an organic solvent to prepare an oil phase solution; wherein the concentration of PLGA was 55 mg / mL and the concentration of the hydrophobic drug was 20% of the mass of PLGA; (B) Use an aqueous solvent containing an emulsifier as an aqueous solution; (C) W / O / W type double emulsion droplets were prepared by using an oil phase solution as the inner phase and an aqueous phase solution as the outer phase via a coaxial microfluidic device; wherein the inner phase flow rate was 0.25 mL / h and the outer phase flow rate was 12.5 mL / h. (D) The W / O / W type double emulsion droplets prepared in step (C) are transferred to a curing solution containing 0.125 wt% polyvinyl alcohol, stirred for 4 hours, and the microspheres are collected, washed and dried to obtain drug-loaded PLGA-PEG biphase sustained-release microspheres.

[0066] In step (A), the PLGA has a lactic acid to glycolic acid molar ratio of 67.5:32.5 and a molecular weight of 55,000 Da, and the organic solvent is dichloromethane; in step (B), the aqueous solvent is a phosphate buffer solution with a pH of 6.7, and the emulsifier consists of 1.0 wt% polyvinyl alcohol and 0.4 wt% sodium dodecyl sulfate.

[0067] Experimental data The samples from Examples 1-3 and Comparative Examples 1-3 were subjected to the following experiments, and the data obtained are shown in Table 1.

[0068] I. Compressive Strength and Elastic Modulus Testing Using a universal testing machine (Instron 5567), in accordance with ISO 13314:2011 and ASTM E111-17 standards, the samples were cylindrical supports with a diameter of 6 mm and a height of 5 mm, the loading speed was 1 mm / min, and 5 samples were used in each group. The results are expressed as mean ± standard deviation.

[0069] II. In vitro degradation rate test The gravimetric method was used. The scaffold was immersed in phosphate buffer solution at pH 7.4 and shaken at 37°C and 100 rpm. The medium was changed every 3 days. Samples were taken at 1, 2, 4 and 8 weeks, and the degradation rate was calculated by weighing after freeze drying. Five samples were collected in each group.

[0070] III. Drug Cumulative Release Rate Test Test method: Dexamethasone: High Performance Liquid Chromatography (HPLC) VEGF: Enzyme-linked immunosorbent assay (ELISA) Release medium: phosphate-buffered saline (PBS, containing 0.02% sodium azide for antibacterial action) at pH 7.4. Test conditions: Release temperature: 37±0.5℃, oscillation rate: 100 rpm (constant temperature shaker), number of samples: n=5 per group, sampling time points: 0, 0.5, 1, 2, 4, 7, 14, 21, 28 days, release medium volume: each sample was placed in 10 mL PBS, sampling method: 1 mL was sampled each time, and an equal amount of fresh PBS was added at the same time.

[0071] HPLC conditions (dexamethasone): C18 reversed-phase column (4.6 × 250 mm, 5 μm), mobile phase methanol / water (65:35, v / v), flow rate 1.0 mL / min, detection wavelength 240 nm, column temperature 30 °C, injection volume 20 μL.

[0072] ELISA conditions (VEGF): Human VEGF ELISA kit, detection wavelength 450 nm (reference wavelength 570 nm), strictly follow the kit instructions.

[0073] Data recording: Cumulative release rate = Cumulative release amount / Total drug loading amount × 100%, burst release rate is defined as the cumulative release rate over 24 hours, and the results are expressed as mean ± standard deviation.

[0074] IV. Bone Volume Fraction (BV / TV) Test Animal model: New Zealand white rabbit femoral condyle critical size defect model (male, weight 2.5-3.0 kg, 6 rabbits per group).

[0075] Surgical procedure: After anesthesia, a critical-sized bone defect of 6 mm in diameter and 10 mm in depth was prepared on the medial femoral condyle. A sterilized scaffold was implanted and sutured layer by layer. Postoperatively, penicillin was administered intramuscularly (400,000 U / animal / day for 3 consecutive days).

[0076] Implantation period: 8 weeks.

[0077] Micro-CT analysis: Skyscan 1176 Micro-CT, scanning parameters: voltage 80 kV, current 200 μA, resolution 18 μm, rotation step 0.5°. CTAn v1.16 software was used to analyze the bone volume fraction (BV / TV = bone tissue volume / total tissue volume × 100%) within the defect area (avoiding the 1 mm margin). Results are expressed as mean ± standard deviation (n = 6 animals / group, 3 slices from each animal for analysis).

[0078] V. Cytotoxicity Test Following the ISO 10993-5 standard, mouse fibroblasts L929 were used. The cytotoxicity of the scaffold extract (37℃, 24 hours) was detected by the MTT assay. After 48 hours of culture, the absorbance was measured, cell viability was calculated, and the toxicity level was determined. Each group consisted of 6 wells.

[0079] VI. Wet Adhesion Strength Test Referring to ASTM F2255-05, the lap shear tensile test was used. Fresh bovine bone was used as the base. After the ink of the scaffold precursor was cured, the adhesion strength was determined on a universal testing machine at a loading speed of 1 mm / min. Five samples were used in each group.

[0080] Table 1: Experimental Analysis: Comparative Example 1 replaced the recombinant mussel adhesive protein Mfp-151 in Example 2 with naturally extracted mussel adhesive protein, while keeping the other components and processes unchanged. Compared with Example 2, all performance indicators showed a significant decrease: compressive strength decreased from 48.2 MPa to 44.5 MPa (-7.7%), degradation rate at 8 weeks increased from 25.3% to 29.8% (+17.8%), 24-hour burst release rate increased from 10.5% to 13.1% (+24.8%), wet adhesion strength decreased from 32.6 kPa to 21.3 kPa (-34.7%), and bone volume fraction at 8 weeks decreased from 45.3% to 36.8% (-18.8%).

[0081] The reasons for this are analyzed as follows: Recombinant Mfp-151 achieves 100% sequence consistency through genetic engineering, with uniform molecular weight and precisely controllable distribution of dopa groups and lysine residues. When reacting with sodium periodate, it forms a uniform and dense cross-linked network, simultaneously acting as a dense barrier for the drug microsphere shell and allowing for the targeted introduction of high-density cell adhesion motifs to activate osteogenic signaling pathways. In contrast, natural mussel adhesive protein is a mixture of various proteins with a wide molecular weight distribution, random DOPA content, and some oxidized and inactivated components, resulting in low purity (<80%). It also contains impurities such as metal ions, leading to a defective network with both localized over- and under-cross-linking during cross-linking. The drug shell is loose and porous, lacking effective osteogenic signal amplification capabilities. Therefore, the advantages of recombinant protein in cross-linked network integrity, drug barrier function, osteogenic activity, and wet adhesion are irreplaceable by natural protein. In the synergistic system, it plays multiple roles as an intelligent cross-linking hub, drug barrier, and cell signal amplifier, while the natural protein's effectiveness is significantly reduced due to molecular disorder and impurity interference.

[0082] Comparative Example 2 replaced the biomimetic mineralized collagen in Example 2 with ordinary hydroxyapatite (HA) micropowder, while keeping the other components and processes unchanged. Compared with Example 2, the mechanical properties showed a precipitous decline: compressive strength decreased from 48.2 MPa to 23.6 MPa (-51.0%), elastic modulus decreased from 1.65 GPa to 0.82 GPa (-50.3%), degradation rate after 8 weeks decreased from 25.3% to 18.5% (-26.9%), and bone volume fraction decreased from 45.3% to 35.2% (-22.3%).

[0083] The reasons for this are as follows: Biomimetic mineralized collagen is a nanoscale organic-inorganic composite material formed by in-situ mineralization on collagen fibers. HA crystals, with a size of 20-50 nm, are oriented along the collagen fibers, forming a continuous interface of covalent bonding and nano-interlocking. Its Ca / P ratio of 1.60-1.70, crystallinity of 65-75%, and specific surface area of ​​35-60 m² / g are highly consistent with natural bone, effectively transferring stress and synergistically enhancing it. Simultaneously, its degradation rate matches the bone regeneration cycle (6-9 months). Its collagen components provide integrin recognition sites, and its nano-topological structure directly regulates osteogenic differentiation. In contrast, ordinary HA consists of micron-sized particles (1-10 μm), which are only physically mixed with the organic phase, lacking chemical bonding. It has a low specific surface area (<20 m² / g), excessively high crystallinity (>90%) leading to high brittleness. Under compressive loads, the particles themselves become stress concentration points, triggering crack propagation. Furthermore, its degradation is extremely slow (>2 years), severely mismatched with the bone regeneration cycle, and it lacks cell recognition signals and osteogenic induction capabilities. Therefore, the nanocomposite structure, precise physicochemical parameters, and osteogenic activity of biomimetic mineralized collagen cannot be replaced by ordinary HA.

[0084] Comparative Example 3 replaced the drug-loaded PLGA-PEG biphasic sustained-release microspheres in Example 2 with ordinary PLGA monophasic microspheres (only encapsulated with dexamethasone, without shell protein and VEGF), while keeping the other components and processes unchanged. Compared with Example 2, the drug release behavior changed dramatically: the 24-hour burst release rate surged from 10.5% to 38.5% (+267%), the 28-day dexamethasone release rate increased from 78.5% to 92.5% (+17.8%), VEGF could not be encapsulated at all, and the bone volume fraction at 8 weeks decreased from 45.3% to 36.8% (-18.8%); while the mechanical properties changed only slightly (compressive strength -2.9%).

[0085] The reasons for this are as follows: Biphasic microspheres employ a W / O / W type double emulsion structure. The core PLGA encapsulates the hydrophobic drug dexamethasone for long-term sustained release, while the outer shell Mfp-151 / PEG composite layer encapsulates the hydrophilic drug VEGF. The shell protein forms a dense physical barrier that effectively inhibits burst release, and coaxial microfluidic technology ensures uniform particle size (PDI < 0.15). This structure achieves temporal regulation of "vascularization first, then osteogenicization": early rapid release of VEGF promotes vascular endothelial cell proliferation and lumen formation, providing nutritional support for bone repair; later, stable release of dexamethasone promotes osteogenic differentiation. In contrast, ordinary monophasic microspheres use an O / W type single emulsion structure, with the drug uniformly dispersed in the PLGA matrix. Without an outer shell barrier, the hydrophilic drug rapidly diffuses into the aqueous phase during preparation and cannot be encapsulated. The drug is mainly distributed on the surface and near-surface of the microsphere, leading to severe burst release. In the later stages, the drug concentration is insufficient to maintain a sustained osteogenic effect, and temporal regulation cannot be achieved. Therefore, the "core-shell" partitioning structure of biphasic microspheres is the key to inhibiting burst release, encapsulating hydrophilic drugs, and achieving programmed time-series release, which cannot be completely replaced by ordinary monophasic microspheres.

[0086] Conclusion: The comparative data from the three comparative examples and Example 2 fully demonstrate that in the four-component synergistic system of this invention, the molecular precision of recombinant mussel adhesive protein Mfp-151, the nanocomposite structure of biomimetic mineralized collagen, and the partitioned encapsulation structure of biphasic sustained-release microspheres each play their respective roles and are indispensable. The absence or replacement of any single component will lead to defects in the cross-linking network, decreased mechanical properties, uncontrolled drug release, or reduced osteogenic activity, thereby significantly weakening the overall performance of the scaffold. This fully reflects the integrity, inventiveness, and indivisibility of the technical solution of this invention.

[0087] In addition, the scaffold is designed with layered functions. The mussel adhesive layer 1, constructed with recombinant mussel adhesive protein, utilizes its molecular precision and high-density dopa groups to achieve strong wet adhesion, ensuring immediate and stable integration of the scaffold and bone interface in a wet environment. The mineralized collagen mechanical layer 2, constructed with biomimetic mineralized collagen, provides mechanical strength close to that of cortical bone and exerts osteoconductive activity by virtue of its nanoscale organic-inorganic composite structure, precise Ca / P ratio, and high specific surface area, guiding new bone growth along the scaffold. The drug-loaded microsphere sustained-release layer 4, formed with drug-loaded PLGA-PEG biphasic sustained-release microspheres, effectively inhibits the initial burst release of drugs and achieves programmed drug release through a partitioned structure in which hydrophobic drugs are encapsulated in the core PLGA and hydrophilic drugs are encapsulated in the shell Mfp-151 / PEG. The thermosensitive hydrogel support layer 4, composed of thermosensitive poly(N-isopropylacrylamide) hydrogel, responsively contracts at body temperature, adaptively matching the irregular bone defect morphology and providing flexible support for the overall structure. The organic integration of these four elements—interfacial adhesion, mechanical support, osteoconduction, drug sustained release, and morphological adaptation—provides an integrated and efficient repair solution for bone defects.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein, characterized in that, By weight percentage, it is made from the following raw materials through printing: Recombinant mussel adhesive protein 20-35% Biomimetic mineralized collagen 40-60% 10-25% drug-loaded PLGA-PEG biphasic sustained-release microspheres Thermosensitive poly(N-isopropylacrylamide) hydrogel 5-15%.

2. The multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein according to claim 1, characterized in that, The recombinant mussel adhesive protein is recombinant mussel adhesive protein Mfp-151.

3. The multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein according to claim 1, characterized in that, The biomimetic mineralized collagen has a Ca / P molar ratio of 1.60-1.70, a crystallinity of 65-75%, and a BET specific surface area of ​​35-60 m² / g.

4. The multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein according to claim 1, characterized in that, The core of the drug-loaded PLGA-PEG biphasic sustained-release microspheres is PLGA encapsulating a hydrophobic drug; the outer shell of the drug-loaded PLGA-PEG biphasic sustained-release microspheres is a composite layer of recombinant mussel adhesive protein Mfp-151 and polyethylene glycol encapsulating a hydrophilic drug.

5. The multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein according to claim 1, characterized in that, The drug-loaded PLGA-PEG biphase sustained-release microspheres have a particle size of 50 μm to 200 μm and a polydispersity index of less than 0.

15.

6. The multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein according to claim 1, characterized in that, The thermosensitive poly(N-isopropylacrylamide) hydrogel is prepared by dissolving poly(N-isopropylacrylamide) powder with a molecular weight of 30,000-50,000 Da in deionized water to prepare a 15-25 wt% solution, and adding 0.3-0.6 wt% of photoinitiator Irgacure 2959.

7. The method for preparing a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein as described in claim 1, characterized in that: It includes the following process steps: Step (1) Mix the formulated amounts of recombinant mussel adhesive protein, biomimetic mineralized collagen, drug-loaded PLGA-PEG biphase sustained-release microspheres, and thermosensitive poly(N-isopropylacrylamide) hydrogel, and homogenize to obtain printing ink. Step (2) Using low-temperature deposition 3D printing technology, the printing ink is printed into a preset three-dimensional structure on a printing platform below the phase transition temperature of the temperature-sensitive poly(N-isopropylacrylamide) hydrogel to obtain the molded scaffold. Step (3) The molded scaffold is freeze-dried, cross-linked and sterilized to obtain a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein.

8. The method for preparing a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein according to claim 7, characterized in that, In step (2), the printing platform temperature is maintained between 10°C and 15°C, and the printing nozzle diameter is between 200 μm and 400 μm. In step (3), freeze-drying specifically involves freezing the molded support at -80℃ for 20-28 hours, and then at -50℃. A porous drying rack was obtained by freeze-drying at 0.1 mbar for 40-60 hours. The crosslinking in step (3) is as follows: the dried scaffold is immersed in a 0.05 wt% sodium periodate aqueous solution and crosslinked for 2-3 hours at 4°C in the dark, and then washed with a pH=7.4 phosphate buffer solution.

9. The method for preparing a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein according to claim 7, characterized in that, The preparation method of the biomimetic mineralized collagen is as follows: Type I collagen is dissolved in a 0.3-0.7M acid solution to prepare a collagen solution of 5-15 mg / mL, and the pH is adjusted to 7.2-7.6; 10 times the concentration of simulated body fluid is added dropwise at 2-8℃ to maintain pH stability, and the solution is transferred to 35-40℃ for shaking mineralization for 24-72 hours; the precipitate is collected, washed, and freeze-dried to obtain biomimetic mineralized collagen.

10. The method for preparing a multi-level sustained-release biomimetic bone repair scaffold based on mussel adhesive protein according to claim 7, characterized in that, The preparation method of the drug-loaded PLGA-PEG biphasic sustained-release microspheres includes the following steps: (A) Dissolve PLGA and a hydrophobic drug in an organic solvent to prepare an oil phase solution; wherein the concentration of PLGA is 40-70 mg / mL and the concentration of the hydrophobic drug is 15-25% of the mass of PLGA; (B) The recombinant mussel adhesive protein and the hydrophilic drug are dissolved in an aqueous solvent containing an emulsifier to prepare an aqueous solution; wherein the concentration of the recombinant mussel adhesive protein is 0.3-0.8 mg / mL and the concentration of the hydrophilic drug is 0.1-0.4 mg / mL. (C) W / O / W type double emulsion droplets were prepared by using an oil phase solution as the inner phase and an aqueous phase solution as the outer phase via a coaxial microfluidic device; wherein the flow rate of the inner phase was 0.1-0.4 mL / h and the flow rate of the outer phase was 10-15 mL / h. (D) The W / O / W type double emulsion droplets prepared in step (C) are transferred to a curing solution containing 0.05-0.2 wt% polyvinyl alcohol, stirred for 2-6 hours, and the microspheres are collected, washed and dried to obtain drug-loaded PLGA-PEG biphase sustained-release microspheres.

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