A poly-peptide biomimetic nanofiber scaffold with osteoinductive activity and a preparation method and application thereof

Abstract

The application belongs to the technical field of medical biomaterials, and particularly relates to a kind of poly-peptide biomimetic nanofiber scaffold with bone inductive activity and its preparation method and application.The application uses synthetic poly-peptide, and PBLG is grafted on bismuth-substituted hydroxyapatite by surface-initiated polymerization, to solve the problem of particle agglomeration and interface;Double bond-containing copolymer P[BLG co ‑(LG g ‑APEG)] is introduced, and 3D printing is used to crosslink and form covalent network to enhance mechanical properties;Combining with phase separation, ECM-like nanofiber structure is obtained.The scaffold obtained by the application has macro-pore and micro-biomimetic fiber network, and organic-inorganic hybrid components provide mineralization interface, and bismuth ion release promotes bone formation, to realize bone defect repair.

Description

Technical Field

[0001] This invention belongs to the field of medical biomaterials technology, and in particular relates to a polypeptide biomimetic nanofiber scaffold with osteoinductive activity, its preparation method and application. Background Technology

[0002] Nonunion, malunion, bone tumors, and ischemic necrosis can all lead to large-scale bone defects. When the defect exceeds a critical size, the bone tissue's own repair capacity is insufficient to meet the needs of structural reconstruction and functional recovery. Surgical intervention and bone grafting are usually required to induce bone regeneration. Therefore, the repair of large-scale bone defects has become a significant challenge in the field of clinical bone repair. With the increasing aging population and rising incidence of bone-related diseases, the number of fractures and bone grafts worldwide continues to increase, making bone tissue one of the most in-demand transplant tissues. Fractures in weight-bearing areas and large-segment bone defects, in particular, not only have long treatment cycles and high risks of complications but also significantly impact patients' long-term quality of life and impose a heavy medical and economic burden. In recent years, the development of bone tissue engineering strategies has provided new treatment methods for bone defect repair, and bone tissue engineering scaffolds hold promise for solving the problems faced by traditional clinical bone grafting.

[0003] Ideal bone tissue engineering scaffolds not only need nanofiber structures that mimic the natural extracellular matrix (ECM) to provide cell attachment sites, but also need to possess excellent osteoinductive activity to actively promote osteogenic differentiation, matrix mineralization, and new bone formation in the defect area. Nanofiber scaffolds can not only achieve physical filling and osteoconduction, but also guide cell migration, provide cell adhesion sites, and regulate cell activity by mimicking the ECM. Therefore, constructing nanofiber scaffolds that combine biomimetic structure and bioactivity is of significant scientific importance for simulating the natural bone tissue microenvironment and achieving bone defect repair.

[0004] Currently, polymer nanofiber scaffolds are mainly constructed using methods such as electrospinning, phase separation, self-assembly, and 3D printing. Among these, phase separation can construct a continuous three-dimensional nanofiber network in situ within the polymer system, which is beneficial for nutrient transport, cell migration, and tissue ingrowth. Self-assembly can form ordered nanostructures at the molecular scale, offering advantages in simulating collagen fiber formation and ECM topology. 3D printing can construct three-dimensional scaffolds with regular channels and certain mechanical support capabilities based on bone defect morphology, thus attracting considerable attention from researchers. Chinese invention patent CN104324418B discloses a method for preparing polylactic acid-based nanofiber osteochondral repair scaffolds using thermally induced phase separation technology. Although phase separation can successfully construct three-dimensional nanofiber networks with high specific surface area, providing abundant attachment and proliferation sites for cells, it often struggles to form large, interconnected pores, limiting its suitability for matching critical bone defects. In addition, the polyester materials commonly used in the phase separation method will produce acidic accumulation during the degradation process, which can easily cause local aseptic inflammation, and this process makes it difficult to build a stable sustained-release microenvironment for drugs inside the fiber.

[0005] Chinese invention patent CN105169474B discloses a method for forming a three-dimensional nanofiber network hydrogel using peptide molecules under physiological conditions through self-assembly. Self-assembly technology enables precise molecular arrangement at the micro- and nanoscale from the bottom up, resulting in a fiber network exhibiting excellent structural stability and biocompatibility. However, nanofiber scaffolds formed solely through non-covalent bond-driven self-assembly typically exhibit extremely low macroscopic mechanical strength, making it difficult to meet the mechanical support requirements of scaffolds in bone tissue engineering. Therefore, a single preparation method often cannot simultaneously satisfy the multiple requirements of bone repair scaffolds regarding macroscopic shape, interconnected pores, and biomimetic nanofiber structures. Thus, constructing a scaffold structure that integrates macroscopic, microscopic, and nanoscale elements through the synergistic integration of multiple processes is an important development direction for nanofiber bone repair scaffolds.

[0006] To impart osteogenic activity to scaffold materials, osteogenic drugs, growth factors, or metal ions are typically introduced. Hydroxyapatite (HA), a natural component of bone tissue, possesses good biocompatibility and osteogenic induction. Utilizing the synergistic effect of metal ions with HA, more promising bone regeneration materials can be prepared. In recent years, bismuth ions (Bi... 3+Due to its osteogenic regulatory potential, antibacterial activity, and X-ray impermeability, hydroxyapatite has been gradually introduced into calcium-phosphorus-based bone repair material systems. Chinese invention patent CN107929807A discloses a method for preparing 3D-printed composite bone scaffolds by physically blending strontium-doped nano-hydroxyapatite with polycaprolactone. By introducing substituted hydroxyapatite containing osteogenic inducing metal ions (strontium ions), this method can impart osteoconductive properties to the scaffold to a certain extent and enhance osteoinductive activity through the release of metal ions. However, traditional physical blending easily leads to severe aggregation of inorganic nanoparticles with high surface energy in a highly hydrophobic polymer matrix, resulting in weak bonding at the organic-inorganic interface. This not only easily causes stress concentration under stress, leading to scaffold fracture, but also makes it easy for physically encapsulated inorganic components to peel off under the flushing of body fluids, hindering the effective delivery of osteogenic metal ions or drugs. In contrast, organic-inorganic hybrid materials prepared by chemical grafting are beneficial for improving the dispersion stability of the inorganic phase in the polymer matrix and improving the interfacial structure of the scaffold and the release process of active ions.

[0007] Therefore, how to simultaneously construct macroscopic interconnected macropores and microscopic biomimetic nanofiber structures in the same scaffold system, while taking into account good biocompatibility, mechanical properties and organic-inorganic hybrid characteristics, remains an important challenge in the design of bone tissue engineering scaffolds. Summary of the Invention

[0008] Given the current lack of materials in the existing technology that simultaneously possess macroscopic interconnected macropores and microscopic biomimetic nanofiber structures within the same scaffold system, while also exhibiting good biocompatibility, mechanical properties, and organic-inorganic hybrid component characteristics, this invention provides a peptide biomimetic nanofiber scaffold with osteoinductive activity, its preparation method, and its application.

[0009] This invention, through the synergistic design of structural and component biomimicry, combined with integrated and controllable fabrication technology, enables the scaffold to meet the requirements of cell adhesion, migration, nutrient transport, and tissue ingrowth while possessing a stable biomimetic structure and sustained bioactivity, thereby better adapting to the application needs of clinical bone defect repair.

[0010] One of the objectives of this invention is to overcome the shortcomings of the prior art and provide a polypeptide biomimetic nanofiber scaffold with osteoinductive activity.

[0011] A second objective of this invention is to provide a method for preparing the aforementioned stent.

[0012] The second objective of this invention is to provide the application of the scaffold in the preparation of materials for repairing skull defects.

[0013] To achieve the above objectives, the present invention adopts the following technical solution: The objective of this invention can be achieved through the following technical solutions: This invention first provides a method for preparing a polypeptide biomimetic nanofiber scaffold with osteoinductive activity, comprising the following steps: (1) Preparation of bismuth-substituted hydroxyapatite grafted peptides: Bi-HA-NH2 nanoparticles with surface amination modified by bismuth-substituted hydroxyapatite (Bi-HA) were prepared by modifying bismuth with a silane coupling agent; the amino groups on the surface of the Bi-HA-NH2 nanoparticles were then used as the initiator. γ β-Benzyl-L-glutamate-N-carboxycyclic intracyclic anhydride (BLG-NCA) is subjected to ring-opening polymerization on the surface of Bi-HA to graft poly-L-glutamate benzyl ester (PBLG) molecular chains, resulting in bismuth-substituted hydroxyapatite-grafted poly-L-glutamate benzyl ester (Bi-HA-PBLG). (2) Preparation of hydrophilically modified photocrosslinkable peptides: Carboxylation modification of poly(L-glutamic acid benzyl ester) (PBLG) yields poly( γ -Benzyl-L-glutamate- what -L-glutamic acid) (P(BLG- what -LG); then grafted with allyl polyethylene glycol (APEG) to prepare a hydrophilically modified photocrosslinkable poly[ γ -Benzyl-L-glutamate- what -(L-glutamic acid- g -allyl polyethylene glycol)](P[BLG- what -(LG- g -APEG)]); (3) Formulating cross-linkable 3D printing ink: The Bi-HA-PBLG prepared in step (1) and the P[BLG-] prepared in step (2) were used. what -(LG- g The photoinitiator and APEG were dissolved together in the 1,4-dioxane system, ultrasonically dispersed and stirred evenly to obtain a crosslinkable 3D printing ink with shear-thinning properties. (4) 3D printing to construct cross-linked hybrid nanofiber scaffolds: Using an extrusion 3D printing device, the ink obtained in step (3) is extruded layer by layer in a support bath according to a preset macroscopic model. Solvent-induced phase separation induces the self-assembly of the polymer matrix to form a three-dimensional nanofiber microstructure. After printing, a specific light source is used to irradiate the P[BLG- what -(LG- g The double bonds in [-APEG)] undergo free radical polymerization and photocrosslinking to construct a three-dimensional covalent polymer network; after washing and freeze-drying, a peptide biomimetic nanofiber scaffold with osteoinductive activity is obtained.

[0014] In one embodiment of the present invention, the poly(L-glutamic acid benzyl ester) (PBLG) may also be replaced by other polypeptides, such as poly(L-glutamic acid methyl ester) (PMLG) or poly(L-glutamic acid ethyl ester) (PELG). When the polypeptide is determined, the corresponding polypeptide monomer is replaced accordingly.

[0015] In one embodiment of the present invention, the preparation of Bi-HA-PBLG in step (1) specifically includes the following steps: (1-1) Preparation of Bi-HA: Ca(OH)2 was dispersed in water, and Bi(NO3)3·5H2O was dispersed in H3PO4. Under mechanical stirring, the Bi(NO3)3·5H2O H3PO4 suspension was added dropwise to the Ca(OH)2 suspension. After the addition was complete, the pH was adjusted with NaOH aqueous solution, heated, aged, washed three times with deionized water and anhydrous ethanol, centrifuged, and dried to obtain white solid Bi-HA. (1-2) Preparation of Bi-HA-NH2: The silane coupling agent was added to a mixed solvent of ethanol / water and dissolved by mechanical stirring. Bi-HA was then added. The mixture was stirred at room temperature, and the pH of the mixture was adjusted with ammonia. After the reaction was complete, the supernatant was discarded, and the mixture was washed with deionized water and anhydrous ethanol, centrifuged, and vacuum dried to obtain Bi-HA-NH2 nanoparticles. (1-3) Preparation of Bi-HA-PBLG: Under an inert atmosphere, BLG-NCA and Bi-HA-NH2 were added to anhydrous 1,4-dioxane in proportion, and the mixture was stirred at room temperature under N2 atmosphere until it became a viscous liquid. After the reaction was completed, the reaction solution was poured into methyl tert-butyl ether for precipitation, washing, and vacuum drying to obtain Bi-HA-PBLG solid.

[0016] In one embodiment of the present invention, in step (1-1), the concentration of Ca(OH)2 dispersed in water is 0.1-0.3 M.

[0017] In one embodiment of the present invention, in step (1-1), the concentration of the H3PO4 solution is 0.1-0.2 M.

[0018] In one embodiment of the present invention, in step (1-1), the bismuth / calcium molar ratio in the obtained bismuth-substituted hydroxyapatite is 5-10 mol.

[0019] In one embodiment of the present invention, in step (1-1), the dropping rate of the Bi(NO3)3·5H2O H3PO4 solution is 1-2 mL / min.

[0020] In one embodiment of the present invention, in step (1-1), the pH of the NaOH aqueous solution is adjusted to 10-11.

[0021] In one embodiment of the present invention, in step (1-1), the heating temperature is 80-85°C.

[0022] In one embodiment of the present invention, in step (1-1), the aging time is 24-48 h.

[0023] In one embodiment of the present invention, in step (1-1), the sample is washed 3-5 times each with deionized water and anhydrous ethanol.

[0024] In one embodiment of the present invention, in step (1-1), the centrifugal speed is 8000-10000 rpm.

[0025] In one embodiment of the present invention, in step (1-1), the drying temperature is 60-70°C and the drying time is 24-36 h.

[0026] In one embodiment of the present invention, in step (1-2), the volume ratio of the ethanol / water mixed solvent is 8-9:1.

[0027] In one embodiment of the present invention, in step (1-2), the molar ratio of Bi-HA to silane coupling agent is 1-2:1.

[0028] In one embodiment of the present invention, in steps (1-2), the silane coupling agent is either 3-aminopropyltriethoxysilane (APTES) or 3-aminopropyltrimethoxysilane (APTMS).

[0029] In one embodiment of the present invention, in steps (1-2), the concentration of Bi-HA in the ethanol / water mixed solvent is 0.01-0.02 wt%.

[0030] In one embodiment of the present invention, in step (1-2), ammonia water is used to adjust the pH of the mixture to 10-11.

[0031] In one embodiment of the present invention, the reaction time in step (1-2) is 3-5 h.

[0032] In one embodiment of the present invention, in step (1-2), the sample is washed 3-5 times each with deionized water and anhydrous ethanol.

[0033] In one embodiment of the present invention, in steps (1-3), the mass ratio of BLG-NCA to Bi-HA-NH2 is 20-5:1.

[0034] In one embodiment of the present invention, in steps (1-3), the ratio (m / v) of BLG-NCA to anhydrous 1,4-dioxane is 1:3-5.

[0035] In one embodiment of the present invention, in steps (1-3), the number of settling times is 3-5 times.

[0036] In one embodiment of the present invention, step (2) involves the hydrophilic modification of photocrosslinkable P[BLG- what -(LG- g The preparation of [-APEG] specifically includes the following steps: (2-1)P(BLG- what Preparation of -LG): PBLG was dissolved in dichloroacetic acid, and then hydrobromic acid-acetic acid solution (HBr / HAc, preferably 33 wt%) was added. The reaction was carried out at 30 °C for 1-1.5 h. The reaction solution was poured into anhydrous diethyl ether while stirring, and filamentous products precipitated out. The resulting precipitate was dissolved in anhydrous N,N-dimethylformamide and transferred to a dialysis bag (preferably MWCO 7000 Da). Dialysis was performed in deionized water for three days. The dialysate was freeze-dried to obtain P(BLG- what -LG) polymer; (2-2)P[BLG- what -(LG- g Preparation of [-APEG]: P(BLG-) was dissolved in anhydrous N,N-dimethylformamide. what -LG), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) was added as an activator and 4-dimethylaminopyridine (DMAP) as a stabilizer, followed by the addition of allyl polyethylene glycol (APEG), and the reaction was carried out at 30 °C. After the reaction was completed, the reaction solution was placed in a dialysis bag (MWCO 7000 Da) and dialyzed thoroughly to remove unreacted small molecules, activators, and organic solvents. The product was freeze-dried to obtain polymer P[BLG- what -(LG- g -APEG).

[0037] In one embodiment of the present invention, in step (2-1), the molecular weight Mn of PBLG is 4.0 × 10⁻⁶. 4 ~18×10 4 .

[0038] In one embodiment of the present invention, in step (2-1), the ratio (m / v) of PBLG to dichloroacetic acid is 5wt%.

[0039] In one embodiment of the present invention, in step (2-1), the ratio (m / v) of PBLG to hydrobromic acid acetic acid solution (HBr / HAc, 33wt%) is 1:0.25-0.75.

[0040] In one embodiment of the present invention, in step (2-1), the ratio of the reaction solution to anhydrous diethyl ether is 1:10-25.

[0041] In one embodiment of the present invention, in step (2-1), the ratio (m / v) of PBLG to anhydrous N,N-dimethylformamide is 1:15-20.

[0042] In one embodiment of the present invention, in step (2-1), the obtained P(BLG- what The degree of carboxylation modification of (-LG) is 10-30 mol%.

[0043] In one embodiment of the present invention, in step (2-2), P(BLG- what The ratio (m / v) of 1-LG to anhydrous N,N-dimethylformamide is 1:10-20.

[0044] In one embodiment of the present invention, in step (2-2), P(BLG- what The molar ratio of carboxyl groups to EDC·HCl in -LG is 1:1-2.

[0045] In one embodiment of the present invention, in step (2-2), P(BLG- what The molar ratio of carboxyl groups to DMAP in (-LG) is 1:0.5-1.

[0046] In one embodiment of the present invention, in step (2-2), allyl polyethylene glycol Mn=410-2000.

[0047] In one embodiment of the present invention, in step (2-2), the reaction time is 24-72 h.

[0048] In one embodiment of the present invention, in step (2-2), the obtained P[BLG- what -(LG- g The grafting rate of [-APEG] was 10-20 mol%.

[0049] In one embodiment of the present invention, in step (3), the photoinitiator is either I2959 or LAP.

[0050] In one embodiment of the present invention, in step (3), the photoinitiator accounts for 0.0005-0.001 wt% of the 1,4-dioxane system.

[0051] In one embodiment of the present invention, in step (3), Bi-HA-PBLG and P[BLG- what -(LG- g The ratio (w / w) of [-APEG)] is (90-50):(10-50), preferably one of 90:10, 70:30 or 50:50.

[0052] In one embodiment of the present invention, in step (3), the solid content of the 3D printing ink is 5-9 wt%.

[0053] In one embodiment of the present invention, in step (4), the printing temperature of the extrusion 3D printing equipment is 15-25°C.

[0054] In one embodiment of the present invention, in step (4), the diameter of the extruder head is 300-510 µm.

[0055] In one embodiment of the present invention, in step (4), the extrusion speed is 90-250 mm / min.

[0056] In one embodiment of the present invention, in step (4), the extrusion pressure is 100-250 kPa.

[0057] In one embodiment of the present invention, in step (4), the support bath is an ethanol bath or an ethanol gel bath.

[0058] In one embodiment of the present invention, in step (4), the specific light source is blue light or ultraviolet light.

[0059] In one embodiment of the present invention, in step (4), the washing method is to wash with gradient ethanol and then wash with deionized water 3-5 times.

[0060] The present invention further provides a polypeptide biomimetic nanofiber scaffold with osteoinductive activity prepared based on the above method.

[0061] In one embodiment of the present invention, the scaffold is obtained by extrusion 3D printing, with an extrusion strip diameter of 200-400 μm and a scaffold pore size of 300-600 μm; the micro-nano structure of the scaffold is obtained by solvation-induced phase separation and peptide self-assembly, with the micro-nanofiber diameter of 80-200 nm; the mechanical compressive modulus of the scaffold reaches 600-800 kPa.

[0062] The present invention further provides the application of the osteoinductively active peptide biomimetic nanofiber scaffold in the preparation of skull defect repair materials.

[0063] This invention utilizes a synthetic peptide material system to prepare a bone repair scaffold. First, through surface-initiated polymerization technology, bismuth-substituted hydroxyapatite (Bi-HA) with osteogenic induction activity is in-situ grafted with peptides. γ The β-benzyl-L-glutamate (PBLG) molecular chain forms a stable chemical bond structure, thereby effectively overcoming the problems of uneven dispersion, easy aggregation, and weak interphase bonding of inorganic nanoparticles in the hydrophobic polymer matrix in traditional physical blending processes. Furthermore, this invention innovatively introduces a copolymer P[BLG-... what -(LG- g [-APEG] is used to construct a robust three-dimensional covalent polymer network through cross-linking reactions during extrusion 3D printing, significantly improving the macroscopic mechanical strength of the scaffold and enabling it to withstand the complex mechanical loads of bone defects in vivo. Combined with phase separation technology, the polymer matrix is ​​induced to self-assemble into a three-dimensional continuous nanofiber microstructure. This structure highly mimics the natural extracellular matrix (ECM), providing a favorable microenvironment for osteoblast adhesion, proliferation, and migration.

[0064] The biomimetic nanofiber scaffold prepared in this invention possesses both macroscopically interconnected macropores and a microscopic ECM-mimicking nanofiber structure: the macroscopic macropores provide a spatial basis for cell migration, nutrient transport, and the ingrowth of new tissue; the microscopic nanofiber network simulates the extracellular matrix structure of natural bone tissue. Simultaneously, the organic-inorganic hybrid components in the scaffold provide a bone-like mineralization interface and further promote osteogenic processes by releasing bismuth ions with osteoinductive activity, thereby achieving excellent bone defect repair effects.

[0065] In summary, this invention provides a polypeptide biomimetic nanofiber scaffold with good mechanical properties, degradation safety, biocompatibility, and synergistic osteogenic activity, as well as its preparation method and application.

[0066] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. This invention avoids the problem that traditional aliphatic polyesters (such as polylactic acid, polycaprolactone, etc.) are prone to producing acidic degradation products and may induce local inflammatory reactions during the degradation process, and has good biocompatibility and degradation safety.

[0067] 2. This invention modifies the surface of bismuth-substituted hydroxyapatite by silane coupling agent and further initiates the ring-opening polymerization of BLG-NCA, so that the inorganic phase and the polypeptide chain segments form a stable organic-inorganic hybrid structure by chemical grafting, which overcomes the defects of easy agglomeration of hydroxyapatite and poor interfacial compatibility in traditional physical blending processes.

[0068] 3. This invention combines 3D printing, photocrosslinking curing, and solvent-induced phase separation-driven molecular self-assembly to construct a microscopic ECM-like nanofiber network in situ during the macroscopic scaffold formation process. This effectively improves the problems of insufficient macroscopic mechanical strength and poor structural stability of traditional self-assembled or phase-separated nanofiber scaffolds. The process is simple and controllable, and the scaffold morphology, pore structure, and component ratio are easily adjusted, exhibiting good applicability and reproducibility in preparation.

[0069] 4. This invention constructs a bone defect repair scaffold that integrates a macroscopic interconnected macroporous structure, a microscopic biomimetic nanofiber network, an organic-inorganic hybrid composition, and bismuth-substituted hydroxyapatite to promote osteogenic function. It can simultaneously meet multiple needs such as cell migration, nutrient transport, tissue ingrowth, mechanical support, and osteogenic induction, providing a novel functional biomimetic scaffold strategy for bone defect repair. Attached Figure Description

[0070] Figure 1 These are macroscopic morphological photographs of the polypeptide biomimetic nanofiber scaffold of Embodiment 1 of the present invention, showing the overall morphology of the scaffold, its clamping state, and the macroscopic through-pore structure on the side.

[0071] Figure 2 These are scanning electron microscope images of the surface and internal structure of the polypeptide biomimetic nanofiber scaffold in Embodiment 1 of the present invention; wherein (A-A2) are nanofiber structures on the scaffold surface at different magnifications, and (B-B2) are nanofiber structures inside the scaffold at different magnifications.

[0072] Figure 3 This is a diagram showing the diameter distribution of nanofibers on the surface and inside the polypeptide biomimetic nanofiber scaffold of the present invention, according to Embodiment 1 of the present invention; wherein (A) is the diameter distribution of nanofibers on the surface of the scaffold, and (B) is the diameter distribution of nanofibers inside the scaffold.

[0073] Figure 4 This is an in vivo evaluation image of the polypeptide biomimetic nanofiber scaffold used for skull defect repair at 4 weeks, according to Embodiment 1 of the present invention; wherein (A) is a gross photograph of the skull defect area 4 weeks after scaffold implantation, and (B) is a Micro-CT reconstruction image of the skull defect area 4 weeks after scaffold implantation. Detailed Implementation

[0074] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0075] Example 1: This embodiment provides a method for preparing a polypeptide biomimetic nanofiber scaffold with osteoinductive activity, comprising the following steps: Ca(OH)₂ was dispersed in water (0.1 mmol / mL), and Bi(NO₃)₃·5H₂O was dispersed in 0.1 M H₃PO₄ solution. Under mechanical stirring, the solution was added dropwise to the Ca(OH)₂ suspension at a rate of 1.5 mL / min. The pH was adjusted to 10.5 with NaOH solution, and the mixture was aged at 80°C for 24 h. After washing three times each with deionized water and anhydrous ethanol, the mixture was centrifuged at 8000 rpm and dried at 60°C for 24 h to obtain Bi-HA. A 9:1 volume ratio of ethanol to water was prepared, and the silane coupling agent APTES was added and stirred until dissolved. Bi-HA (0.01 wt%, molar ratio to coupling agent 2:1) was then added, stirred at room temperature, and the pH was adjusted to 10.5 with ammonia. After reacting for 4 h, the mixture was washed and dried to obtain Bi-HA-NH₂. Under a nitrogen atmosphere, BLG-NCA, Bi-HA-NH2 (mass ratio 10:1), and anhydrous 1,4-dioxane (w / v = 1:4) were mixed and stirred at room temperature until viscous. The mixture was then precipitated three times in methyl tert-butyl ether and dried to obtain Bi-HA-PBLG. Mn = 8.0 × 10⁻⁶ 4 PBLG was dissolved in dichloroacetic acid (5 wt%), and HBr / HAc solution (w / v = 1:0.5) was added. After reacting at 30°C for 1 h, it was precipitated in anhydrous diethyl ether (1:20), dissolved in anhydrous DMF (1:15), dialyzed for three days, and lyophilized to obtain P(BLG- what -LG). The product was dissolved in DMF, and EDC·HCl (molar ratio 1:2), DMAP (molar ratio 1:1), and APEG with Mn=410 were added sequentially. The reaction was carried out at 30°C for 48 h, and the product was dialyzed and lyophilized to obtain P[BLG- what -(LG- g -APEG). Take Bi-HA-PBLG and P[BLG- what -(LG- g [-APEG] (mass ratio 70:30) was dissolved in 1,4-dioxane containing 0.001 wt% LAP photoinitiator to prepare a 3D printing ink with a solid content of 7 wt%. At 20°C, using a needle with an inner diameter of 0.52 mm, the ink was extruded into an anhydrous ethanol bath at a speed of 150 mm / min and a pressure of 150 kPa for phase separation and molding. After printing, the ink was crosslinked by blue light irradiation for 30 min, washed three times each with gradient ethanol and deionized water, and then freeze-dried to obtain a biomimetic nanofiber scaffold.

[0076] like Figure 1 As shown, the peptide biomimetic nanofiber scaffold prepared in this embodiment has a complete macroscopic morphology, maintains a certain structural stability under clamping conditions, and a regularly interconnected macroporous structure can be observed on the side. The obtained scaffold is as follows. Figure 2As shown, the polypeptide biomimetic nanofiber scaffold prepared in this embodiment forms a continuous nanofiber network structure on both the surface and inside. Statistical results are as follows: Figure 3 As shown, the average diameter of the outer surface fibers is 91.93 nm, and the average diameter of the inner fibers is 119.18 nm. Four weeks after implantation of the peptide-inspired bionic nanofiber scaffold prepared in this embodiment into the skull defect area, as... Figure 4 As shown, Figure 4 The blank group in the paper stated that no scaffold material was placed, and obvious new bone repair was observed at the defect site, indicating that the scaffold has good in vivo bone defect repair capabilities.

[0077] Example 2: This embodiment provides a method for preparing a polypeptide biomimetic nanofiber scaffold with osteoinductive activity.

[0078] The preparation method of this embodiment is basically the same as that of Example 1, except that in step (3), Bi-HA-PBLG and P[BLG- what -(LG- g The mass ratio of [-APEG] was adjusted to 90:10.

[0079] The average diameter of the outer surface fibers of the scaffold obtained in this embodiment is 215.62 nm, and the average diameter of the inner fibers is 288.11 nm.

[0080] Example 3: This embodiment provides a method for preparing a biomimetic nanofiber scaffold with osteoinductive activity and drug-eluting peptide loading.

[0081] The preparation method of this embodiment is basically the same as that of Example 1, except that in step (3), Bi-HA-PBLG and P[BLG- what -(LG- g- The mass ratio of APEG was adjusted to 50:50.

[0082] The average diameter of the outer surface fibers of the scaffold obtained in this embodiment is 148.86 nm, and the average diameter of the inner fibers is 232.34 nm.

[0083] Example 4: This embodiment provides a method for preparing a biomimetic nanofiber scaffold with osteoinductive activity and drug-eluting peptide loading.

[0084] The preparation method of this embodiment is basically the same as that of Example 1. The difference is that in step (3), the solid content of the prepared 3D printing ink is increased to 9 wt%; in step (4), the inner diameter of the needle is adjusted to 300µm and the extrusion pressure is increased to 250kPa.

[0085] The mechanical compressive modulus of the stent obtained in this embodiment is 584.81 kPa.

[0086] Example 5: This embodiment provides a method for preparing a biomimetic nanofiber scaffold with osteoinductive activity and drug-eluting peptide loading.

[0087] The preparation method of this embodiment is basically the same as that of Example 1, except that: in step (3), the photoinitiator is replaced with I2959; in step (4), the specific light source used for crosslinking is replaced with ultraviolet light.

[0088] Example 6: This embodiment provides a method for preparing a biomimetic nanofiber scaffold with osteoinductive activity and drug-eluting peptide loading.

[0089] The preparation method of this embodiment is basically the same as that of Example 1, except that: in step (1), the mass ratio of BLG-NCA and Bi-HA-NH2 is adjusted to 20:1; in step (2), the Mn of the selected allyl polyethylene glycol (APEG) is 2000.

[0090] Examples 2-6 above changed the preparation conditions of the peptide biomimetic nanofiber scaffold. The resulting peptide biomimetic nanofiber scaffold also has macroscopic interconnected macropores and microscopic ECM-like nanofiber structure, and also has good mechanical properties, degradation safety, biocompatibility and synergistic osteogenic activity.

[0091] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing a polypeptide biomimetic nanofiber scaffold with osteoinductive activity, characterized in that, Includes the following steps: (1) Preparation of bismuth-substituted hydroxyapatite grafted peptides: Bi-HA-NH2 nanoparticles with surface amination modified by bismuth-substituted hydroxyapatite were prepared by modifying bismuth with a silane coupling agent; the amino groups on the surface of the Bi-HA-NH2 nanoparticles were then used as the initiator. γ -Benzyl-L-glutamate-N-carboxylic acid anhydride undergoes ring-opening polymerization on the surface of Bi-HA to graft poly-L-glutamate benzyl ester molecular chains, resulting in bismuth-substituted hydroxyapatite-grafted poly-L-glutamate benzyl ester, namely Bi-HA-PBLG. (2) Preparation of hydrophilically modified photocrosslinkable peptides: Carboxylation modification of poly(L-glutamic acid benzyl ester) (PBLG) yields poly( γ -Benzyl-L-glutamate- co -L-glutamic acid), i.e., P(BLG- co -LG); then grafted with allyl polyethylene glycol (APEG) to prepare a hydrophilically modified photocrosslinkable poly[ γ -Benzyl-L-glutamate- co -(L-glutamic acid- g -allyl polyethylene glycol)], i.e. P[BLG- co -(LG- g -APEG)]; (3) Formulating cross-linkable 3D printing ink: The Bi-HA-PBLG prepared in step (1) and the P[BLG-] prepared in step (2) were used. co -(LG- g The photoinitiator and APEG were dissolved together in the 1,4-dioxane system, ultrasonically dispersed and stirred evenly to obtain a crosslinkable 3D printing ink with shear-thinning properties. (4) 3D printing to construct cross-linked hybrid nanofiber scaffolds: Using an extrusion 3D printing device, the ink obtained in step (3) is extruded layer by layer in an ethanol bath to form a three-dimensional nanofiber microstructure by inducing polymer matrix self-assembly through solvent-induced phase separation; after printing, P[BLG- co -(LG- g The double bonds in [-APEG)] undergo free radical polymerization and photocrosslinking to construct a three-dimensional covalent polymer network; after washing and freeze-drying, a peptide biomimetic nanofiber scaffold with osteoinductive activity is obtained.

2. The method for preparing a osteoinductively active peptide-based biomimetic nanofiber scaffold according to claim 1, characterized in that, The poly(L-glutamic acid benzyl ester) may be replaced by either poly(L-glutamic acid methyl ester) or poly(L-glutamic acid ethyl ester).

3. The method for preparing a osteoinductively active peptide-based biomimetic nanofiber scaffold according to claim 1, characterized in that, The preparation of Bi-HA-PBLG in step (1) specifically includes the following steps: (1-1) Preparation of Bi-HA: Ca(OH)2 was dispersed in water, and Bi(NO3)3·5H2O was dispersed in H3PO4. Under mechanical stirring, the Bi(NO3)3·5H2O H3PO4 suspension was added dropwise to the Ca(OH)2 suspension. After the addition was complete, the pH was adjusted with NaOH aqueous solution, heated, aged, washed three times with deionized water and anhydrous ethanol, centrifuged, and dried to obtain white solid Bi-HA. (1-2) Preparation of Bi-HA-NH2: The silane coupling agent was added to a mixed solvent of ethanol / water and dissolved by mechanical stirring. Bi-HA was then added. The mixture was stirred at room temperature, and the pH of the mixture was adjusted with ammonia. After the reaction was complete, the supernatant was discarded, and the mixture was washed with deionized water and anhydrous ethanol, centrifuged, and vacuum dried to obtain Bi-HA-NH2 nanoparticles. (1-3) Preparation of Bi-HA-PBLG: Under an inert atmosphere, BLG-NCA and Bi-HA-NH2 were added to anhydrous 1,4-dioxane in proportion, and the mixture was stirred at room temperature under N2 atmosphere until it became a viscous liquid. After the reaction was completed, the reaction solution was poured into methyl tert-butyl ether for precipitation, washing, and vacuum drying to obtain Bi-HA-PBLG solid.

4. The method for preparing a osteoinductively active peptide-based biomimetic nanofiber scaffold according to claim 3, characterized in that, Choose one or more of the following conditions: In step (1-1), the concentration of Ca(OH)2 after dispersion in water is 0.1-0.3 M; In step (1-1), the concentration of the H3PO4 solution is 0.1-0.2 M; In step (1-1), the bismuth / calcium molar ratio in the obtained bismuth-substituted hydroxyapatite is 5-10 mol%; In step (1-1), the dropping rate of the Bi(NO3)3·5H2O H3PO4 solution is 1-2 mL / min; In step (1-1), the pH of the NaOH aqueous solution is adjusted to 10-11; In step (1-1), the heating temperature is 80-85℃; In step (1-1), the aging time is 24-48 hours; In step (1-1), wash with deionized water and anhydrous ethanol 3-5 times each; In step (1-1), the centrifugation speed is 8000-10000 rpm; In step (1-1), the drying temperature is 60-70℃ and the drying time is 24-36 h; In steps (1-2), the volume ratio of the ethanol / water mixed solvent is 8-9:1; In steps (1-2), the molar ratio of Bi-HA to silane coupling agent is 1-2:1; In steps (1-2), the silane coupling agent is either 3-aminopropyltriethoxysilane or 3-aminopropyltrimethoxysilane; In steps (1-2), the concentration of Bi-HA in the ethanol / water mixed solvent is 0.01-0.02 wt%; In steps (1-2), ammonia is used to adjust the pH of the mixture to 10-11; In steps (1-2), the reaction time is 3-5 hours; In steps (1-2), wash with deionized water and anhydrous ethanol 3-5 times each; In steps (1-3), the mass ratio of BLG-NCA to Bi-HA-NH2 is 20-5:1; In steps (1-3), the ratio (m / v) of BLG-NCA to anhydrous 1,4-dioxane is 1:3-5; In steps (1-3), the settling process is repeated 3-5 times.

5. The method for preparing a osteoinductively active peptide-based biomimetic nanofiber scaffold according to claim 1, characterized in that, Step (2) describes the hydrophilic modification and photocrosslinkable P[BLG- co -(LG- g The preparation of [-APEG] specifically includes the following steps: (2-1)P(BLG- co Preparation of -LG): PBLG was dissolved in dichloroacetic acid, and then hydrobromic acid-acetic acid solution was added. The reaction was allowed to proceed for 1-1.5 h. The reaction mixture was then poured into anhydrous diethyl ether while stirring, resulting in the precipitation of filamentous products. The precipitate was dissolved in anhydrous N,N-dimethylformamide and transferred to a dialysis bag. Dialysis was performed in deionized water for three days. The dialysate was then freeze-dried to obtain P(BLG- co -LG) polymer; (2-2)P[BLG- co -(LG- g Preparation of [-APEG]: P(BLG-) was dissolved in anhydrous N,N-dimethylformamide. co Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) as an activator and 4-dimethylaminopyridine (DMAP) as a stabilizer, then add allyl polyethylene glycol (APEG) and react. After the reaction is complete, the reaction solution is placed in a dialysis bag (MWCO 7000 Da) and dialyzed thoroughly to remove unreacted small molecules, activators, and organic solvents. The product is freeze-dried to obtain polymer P[BLG- co -(LG- g -APEG).

6. The method for preparing a osteoinductively active peptide-based biomimetic nanofiber scaffold according to claim 5, characterized in that, Choose one or more of the following conditions: In step (2-1), the molecular weight of PBLG is Mn = 4.0 × 10⁻⁶. 4 ~18×10 4 ; In step (2-1), the ratio (m / v) of PBLG to dichloroacetic acid is 5 wt%; In step (2-1), the ratio (m / v) of PBLG to hydrobromic acid acetic acid solution is 1:0.25-1; In step (2-1), the ratio of the reaction solution to anhydrous diethyl ether is 1:10-25; In step (2-1), the ratio (m / v) of PBLG to anhydrous N,N-dimethylformamide is 1:15-20; In step (2-1), the obtained P(BLG- co The degree of carboxylation modification of (-LG) is 10-30 mol%; In step (2-2), P(BLG- co The ratio (m / v) of -LG) to anhydrous N,N-dimethylformamide is 1:20-40; In step (2-2), P(BLG- co The molar ratio of carboxyl groups to EDC·HCl in -LG is 1:1-2; In step (2-2), P(BLG- co The molar ratio of carboxyl groups to DMAP in -LG is 1:0.5-1; In step (2-2), the weight-average molecular weight of allyl polyethylene glycol is 300-2000; In step (2-2), the reaction time is 24-72 h; In step (2-2), the obtained P[BLG- co -(LG- g The grafting rate of [-APEG] was 10-20 mol%.

7. The method for preparing a osteoinductively active peptide-based biomimetic nanofiber scaffold according to claim 1, characterized in that, In steps (3) and (4), select one or more of the following conditions: The photoinitiator is either LAP or I2959; In step (3), the photoinitiator accounts for 0.0005-0.001 wt% of the 1,4-dioxane system. In step (3), Bi-HA-PBLG and P[BLG- co -(LG- g The ratio (w / w) of [-APEG] is (90-50): (10-50); In step (3), the solid content of the 3D printing ink is 5-9 wt%; In step (4), the printing temperature of the extrusion 3D printing equipment is 15-25℃; In step (4), the diameter of the extruder head is 300-510 µm; In step (4), the extrusion speed is 90-250 mm / min; In step (4), the extrusion pressure is 100-250 kPa; In step (4), the support bath is an ethanol bath or an ethanol gel bath; In step (4), the specific light source is ultraviolet light or blue light; In step (4), the washing method is to wash with gradient ethanol and then wash with deionized water 3-5 times.

8. A polypeptide biomimetic nanofiber scaffold with osteoinductive activity prepared by the method according to any one of claims 1-7.

9. The osteoinductively active peptide-based biomimetic nanofiber scaffold according to claim 8, characterized in that, The scaffold is obtained by extrusion 3D printing, with an extrusion strip diameter of 200-400 μm and a scaffold pore size of 300-600 μm; the micro-nano structure of the scaffold is obtained by solvation-induced phase separation and peptide self-assembly, with the micro-nanofiber diameter of 80-400 nm; the mechanical compressive modulus of the scaffold reaches 500-800 kPa.

10. The application of the osteoinductively active peptide biomimetic nanofiber scaffold as described in claim 8 or 9 in the preparation of cranial defect repair materials.

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