Multi-stage structure bioactive three-dimensional scaffold for multi-cell interaction and neurotized bone regeneration as well as preparation method and application of multi-stage structure bioactive three-dimensional scaffold

Abstract

The invention relates to a multilevel-structure bioactive three-dimensional scaffold for multi-cell interaction and neurotized bone regeneration as well as a preparation method and application of the multilevel-structure bioactive three-dimensional scaffold. The multi-stage structure bioactive three-dimensional scaffold comprises a bioactive scaffold base body with a macroscopic channel structure and a microcosmic directional topological structure filled in the base body, the macroscopic channel structure has a three-dimensional controllable diversified structure, and the microcosmic directional topological structure is a directional micron-sized channel filled in the microcosmic directional topological structure; the multi-level structure bioactive three-dimensional scaffold provided by the invention can be used as a'space bridge 'for coordinating space-time interaction of multiple cells, and has a beneficial promotion effect on mutual migration, energy metabolism level and specific differentiation behavior of multiple cells in a three-dimensional space; the multi-stage structure bioactive three-dimensional scaffold constructed by the invention is a novel bone repair biological material with a multi-stage structure and biological activity, and a new strategy is provided for large-section bone repair and other complex tissue reconstruction.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomaterial preparation, and particularly relates to a multi-level structure bioactive three-dimensional scaffold for multi-cell interaction and neuralized bone regeneration, and a preparation method and application thereof. BACKGROUND

[0002] Bone is a dynamic and complex organ tissue with multiple physiological functions, and the repair and regeneration of bone requires the synergistic participation of multiple cells, growth factors and signaling molecules. Studies have found that the nerve fiber network in bone can regulate the activity and function of bone-related cells by secreting neurotransmitters such as NGF and CGRP, and thus plays an important role in the growth and repair and regeneration of bone. In addition, the introduction of nerve cells, neurotrophic factors or nerve-derived exosomes can simulate the nerve microenvironment, thereby accelerating bone repair. However, these studies only focus on the one-way regulation of nerves on bone. In fact, there is a close two-way communication between bone and nerves, and the regulation of bone on the nerve in bone is also crucial. Bone can secrete biologically active signaling factors and provide mechanical stimulation for nerves to affect the activity of nerves in bone. For example, the prostaglandin E2 (PGE2) secreted by osteoblasts can act as a nerve modulator, bind to receptor 4 (EP4) on the surface of sensory nerves, and thus help to inhibit the activity of sympathetic nerves. In addition, the microstructure characteristics of bone, including thickness, porosity and pore size, also affect the growth of nerves. Therefore, exploring the two-way interaction between nerves and bone is of great significance for designing bone regeneration biomaterials and ultimately promoting functional bone regeneration.

[0003] Natural bone tissue has a multi-level directional channel structure from macro to micro, which endows the bone matrix with good biological activity and excellent mechanical properties and supports the distribution of nerve fiber networks. Cells are very sensitive to micro-nano topological structures, and anisotropic topological structures at the micro-nano scale can guide cell proliferation, differentiation and directional migration. However, the current mainstream 3D printing technology can only construct three-dimensional structures at the level of one or two hundred microns, and it is difficult to realize the controllable preparation of tens of micrometer topological structures. Ice template method is a method that uses temperature gradient to affect the movement process of ice freezing elements to form anisotropic microchannel structures, which is widely used in the biomedical field to prepare bioactive scaffolds. Inspired by this, a bioactive scaffold with both macroscopic channel structure and microscopic topological structure is designed to explore the interaction between bone cells and nerve cells, which has important scientific significance and clinical value. SUMMARY

[0004] In view of the above technical problems, the present application aims to provide a multi-level structure bioactive three-dimensional scaffold for multi-cell interaction and neuralized bone regeneration, and a preparation method and application thereof.

[0005] In a first aspect, the present invention provides a multi-level structured bioactive three-dimensional scaffold for multi-cell interaction and neural bone regeneration, the multi-level structured bioactive three-dimensional scaffold comprising: a bioactive scaffold matrix having a macroscopic channel structure, and a microscopic oriented topology filling the interior of the matrix; the microscopic oriented topology comprising ordered oriented micro- and nano-scale channels; The aforementioned multi-level bioactive three-dimensional scaffold can serve as a "spatial bridge" for coordinating the spatiotemporal interactions of multiple cells, and has a beneficial promoting effect on the mutual migration, energy metabolism level and specific differentiation behavior of multiple cells in three-dimensional space.

[0006] Preferably, the material of the multi-level structured bioactive three-dimensional scaffold matrix is ​​selected from at least one of bioceramics, bioactive glass, biometals, metal oxides, biopolymers, and carbon-based nanomaterials, and is preferably bioceramics; more preferably, the bioceramic material includes β-tricalcium phosphate or magnesium feldspar.

[0007] Preferably, the macroscopic channel structure of the bioactive scaffold matrix includes at least one of the following: a directional channel structure formed by 3D printing, a branched channel, and a cross-stacked structure; Preferably, the diameter of the bioactive scaffold matrix is ​​0.2–5 mm and the height is 1–10 mm; Preferably, the diameter of the micro-nano channel is 5–200 μm.

[0008] Secondly, the present invention provides a method for preparing the above-mentioned multi-level structured bioactive three-dimensional scaffold. The preparation method includes the following steps: using a precursor solution for electrospinning, electric / magnetic field induced self-assembly, or directional freezing ice template method to form ordered and directionally arranged micro-nano channels inside a bioactive scaffold matrix with a macroscopic channel structure formed by 3D printing, thereby obtaining the multi-level structured bioactive three-dimensional scaffold.

[0009] Preferably, the preparation process of the precursor solution includes: taking 200 μL of 2-6% initiator solution and 2-10 μL of catalyst and adding them to 20 mL of 0.1-20% hydrogel solution to form the precursor solution.

[0010] Preferably, the initiator includes at least one of glutaraldehyde, genipin, ammonium persulfate, and lithium phenyl (2,4,6-trimethylbenzoyl)phosphate; The catalyst includes tetramethylethylenediamine; The hydrogel includes at least one of gelatin, methacrylamide gelatin, chitosan, sodium alginate, polyvinyl alcohol, silk fibroin, and polyethylene glycol diacrylate.

[0011] Preferably, the process of the directional freezing ice template method includes: in a directional freezing mold, injecting a precursor solution into the interior of a bioceramic scaffold until it is submerged, then placing the entire system in an ultra-low temperature environment to induce the directional growth of ice crystals to form directionally arranged micro-nano channels, then transferring the entire scaffold system to a low temperature environment for chemical cross-linking, and freeze-drying to remove the ice crystals to obtain the multi-level structured bioactive three-dimensional scaffold.

[0012] Preferably, the induced ice crystal directional growth is carried out in a liquid nitrogen environment at a temperature of -80 to -196°C for a time of 20 minutes or more, preferably 20 to 40 minutes.

[0013] Preferably, the chemical cross-linking temperature is -20 to -80°C, and the time is 12 to 24 hours.

[0014] Thirdly, the present invention provides an application of the above-mentioned multi-level structured bioactive three-dimensional scaffold in the preparation of multi-cell interaction and bone repair materials.

[0015] Beneficial effects (1) This invention combines a bioceramic scaffold with a macroscopic channel structure and a hydrogel scaffold with a directional microchannel structure to prepare a multi-level scaffold with a macroscopic and microscopic directional channel structure. This bioactive scaffold can provide a three-dimensional biomimetic platform for exploring the interaction between nerve cells and osteocytes. In vitro experiments show that under the physical stimulation of the directional microchannel structure, the chemical stimulation of the release of bioactive ions, and the co-culture environment of osteoblasts and nerve cells, the two types of cells can communicate with each other by secreting extracellular vesicles and mitochondrial transfer, thereby enhancing their respective migration ability, osteogenic differentiation and neural differentiation ability, and the energy metabolism level is also significantly improved. (2) The bioactive scaffold with a multi-level channel structure constructed in this invention exhibits excellent neurogenic bone regeneration effect in animals. Nerve cells can provide a neurally activated regenerative microenvironment. By recruiting endogenous stem cells to migrate to the injury site along the directional microchannel, the interaction between the two types of cells is realized, thereby promoting bone regeneration and nerve innervation at the defect site. In addition, in vivo critical bone defect experiments further verified that the bioactive scaffold with a multi-level channel structure can effectively promote neurogenic bone regeneration. (3) The bioactive scaffold prepared by the present invention with a multi-level channel structure is a novel bone repair biomaterial that combines multi-level directional structure and bioactivity, providing a new strategy for complex tissue regeneration. Attached Figure Description

[0016] Figure 1The physicochemical properties of the magnesium feldspar bioceramic scaffold are characterized as follows: a is the SEM image of magnesium feldspar powder; b is the SEM image of the magnesium feldspar bioceramic scaffold with macroscopic channel structure; c is the XRD pattern of the 3D printed magnesium feldspar bioceramic scaffold; d is the optical image of magnesium feldspar bioceramic scaffolds with different channel pore sizes; e is the elemental distribution diagram of the magnesium feldspar bioceramic scaffold. Figure 2 Physicochemical characterization of bioactive scaffolds with multi-level channel structures: a) SEM images of multi-level channel bioactive scaffolds with different pore sizes, composed of 1% methacrylated gelatin (AKT-1GM), 3% methacrylated gelatin (AKT-3GM), and 5% methacrylated gelatin (AKT-5GM); b) directional microchannels of the three scaffolds (AKT-1GM, AKT-3GM, and AKT-5GM). c represents the diameter of the directional microchannels of the three AKT-1GM, AKT-3GM, and AKT-5GM stents, calculated from a top view; d represents the diameter of the directional microchannels of the three AKT-1GM, AKT-3GM, and AKT-5GM stents, calculated from a side view; e represents the stress-strain curves of the three AKT-1GM, AKT-3GM, and AKT-5GM stents; f represents the compressive strength of the three AKT-1GM, AKT-3GM, and AKT-5GM stents. Figure 3 The versatility of 3D printing strategies combined with ice template method for preparing multi-level structures: a) SEM images of AKT bioceramic scaffolds with different external macroscopic structures and bioactive scaffolds with multi-level channel structures composed of directional microchannels filled with methacryloyl gelatin; b) SEM images of AKT bioceramic scaffolds with macroscopic channel structures and scaffolds filled with different types of polymer materials (polyvinyl alcohol and chitosan). Figure 4 Characterization of bone marrow mesenchymal stem cell migration within a bioactive scaffold with a multilevel channel structure: a) Fluorescent images of cell migration after 1 and 3 days of culture in AKT-1GM, AKT-3GM, and AKT-5GM; b) Quantitative analysis results of cell migration distance. Figure 5Characterization of the migration, morphology, and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) within a multi-level channel bioactive scaffold: a) Confocal three-dimensional reconstructed fluorescence images of BMSC migration and distribution within each scaffold group after 1 and 3 days of culture. BMSCs were labeled with red fluorescent dye before seeding onto the scaffold surface; b) Quantitative statistics of the migration distance of BMSCs within the scaffold; c) Cell morphology of BMSCs in different scaffolds after 3 days of culture; d) Cytoskeleton orientation analysis; e) Representative immunofluorescence staining images of the osteogenic marker OPN in BMSCs after 5 days of culture; f) Expression of osteogenic differentiation-related genes RunX2, OPN, and ALP in BMSCs after 5 days of culture; g) Semi-quantitative analysis of the average fluorescence intensity of the osteogenic marker OPN. Figure 6 Characterization of Schwann cell migration, morphology, and neurogenic differentiation within a multi-level channel bioactive scaffold: a) Confocal 3D reconstructed fluorescence images of Schwann cell migration and distribution within each scaffold group after 1 and 3 days of culture; Schwann cells were labeled with red fluorescent dye before seeding onto the scaffold surface; b) Quantitative statistics of Schwann cell migration distance within the scaffold; c) Cell morphology of Schwann cells in different scaffolds after 3 days of culture; d) Cytoskeleton orientation analysis; e) Representative immunofluorescence staining images of GDNF, a neurogenic differentiation marker of Schwann cells, after 5 days of culture; f) Expression of osteogenic differentiation-related genes NGF, GDNF, and NCAM in Schwann cells after 5 days of culture; g) Semi-quantitative analysis of the average fluorescence intensity of the neurogenic differentiation marker GDNF. Figure 7 Characterization of cell migration of bone marrow mesenchymal stem cells and Schwann cells in single co-culture in bioactive scaffolds with different multilevel structures: a) Distribution of bone marrow mesenchymal stem cells and Schwann cells in the scaffold after 1, 3 and 5 days of vertical co-culture, with bone marrow mesenchymal stem cells labeled with green dye and Schwann cells labeled with red dye; b) Quantitative statistics of migration distance of bone marrow mesenchymal stem cells and Schwann cells in different groups. Figure 8 Characterization of cell differentiation experiments of bone marrow mesenchymal stem cells and Schwann cells in monoculture and coculture in bioactive scaffolds with different multilevel structures: a) Expression levels of osteogenic-related genes in bone marrow mesenchymal stem cells in different groups; b) Expression levels of neural differentiation-related genes in Schwann cells in different groups; c and d) Representative immunofluorescence staining images and semi-quantitative statistical analysis of osteogenic marker OCN in bone marrow mesenchymal stem cells in different groups; e and f) Representative immunofluorescence staining images and semi-quantitative statistical analysis of neural marker GDNF in Schwann cells in different groups. Figure 9Characterization of energy metabolism levels of bone marrow mesenchymal stem cells and Schwann cells in monoculture and coculture in a multilevel bioactive scaffold: a) Quantitative statistics of ATP production and energy metabolism-related gene expression levels of bone marrow mesenchymal stem cells in monoculture and coculture with Schwann cells in a scaffold with a multilevel channel structure; b) Quantitative statistics of ATP production and energy metabolism-related gene expression levels of Schwann cells in monoculture and coculture with bone marrow mesenchymal stem cells in a scaffold with a multilevel channel structure. Figure 10 In vivo evaluation of nerve-innervated bone regeneration 12 weeks after implantation of a Schwann cell-loaded multilevel channel scaffold into a rabbit femoral condyle defect: a) Micro-CT reconstructed images of the bone defect area after scaffold implantation in different groups, with red indicating the implanted scaffold and green indicating newly formed bone; b) Statistical analysis of bone volume fraction (BV / TV) and trabecular bone number (Tb.N) of newly formed bone in different groups; c) Schematic diagram of dividing the defect into upper, middle, and lower regions for quantitative analysis; d) Statistical analysis of bone volume fraction (BV / TV) and trabecular bone number (Tb.N) of newly formed bone in the upper, middle, and lower layers, respectively; e) Representative H&E staining images of rabbit femoral defects in different groups, with the black dashed line representing the middle layer of the defect and NB representing newly formed bone tissue; Figure 11 Immunofluorescence staining images of the middle part of the defect area in different groups: a is the immunofluorescence image of osteogenic marker OCN; b is the immunofluorescence image of Schwann cell-specific marker S100β; c is the immunofluorescence image of neurogenic marker NF; d is the immunofluorescence image of vascularization-specific marker CD31; eh is the statistical analysis of positive areas of OCN(e), S100β(f), NF(j), and CD31(h) in each group. Detailed Implementation

[0017] The present invention will be further illustrated by the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0018] First, this invention provides a multi-level bioactive three-dimensional scaffold for multi-cell interaction and neuralized bone regeneration. The multi-level bioactive three-dimensional scaffold may include: a bioactive scaffold matrix with macroscopic channel structures, and microscopic oriented topological structures filling the interior of the bioactive scaffold matrix; the microscopic oriented topological structures may include ordered, oriented micro / nanoscale channels.

[0019] In some embodiments, the material of the bioactive scaffold matrix may be selected from at least one of bioceramics, bioactive glass, biometals, metal oxides, biopolymers, and carbon-based nanomaterials, preferably bioceramics; more preferably, the bioceramic material may include β-tricalcium phosphate or magnesium feldspar.

[0020] In some embodiments, the macroscopic channel structure of the bioactive scaffold matrix may include at least one of 3D-printed directional channel structures, branched channels, and cross-stacked structures.

[0021] In some embodiments, the diameter of the bioactive scaffold substrate can be 0.2–5 mm, preferably 0.6–2 mm, and the height can be 1–10 mm, preferably 1–6 mm. The scaffold size must match the size of the defect in the animal model; if the scaffold size is too large, it cannot be implanted into the defect, and if it is too small, it is easy to fall off from the defect.

[0022] In some embodiments, the diameter of the micro / nano-scale channels can be 5–200 μm, preferably 10–50 μm. The microchannel size needs to match the cell size to achieve the effect of reshaping cell morphology and guiding cell migration. If the microchannel size is too large, the responsiveness of cells to micro / nano structures cannot be reflected; if the microchannel size is too small, cells cannot penetrate into the scaffold.

[0023] The following exemplifies the method for fabricating a multi-level bioactive three-dimensional scaffold provided by the present invention. The fabrication method may include the following steps: using a precursor solution for electrospinning, electric / magnetic field-induced self-assembly, or directional freezing ice template method to form ordered, oriented micro / nano-scale channels within a 3D-printed bioactive scaffold matrix with a macroscopic channel structure, thereby obtaining the multi-level bioactive three-dimensional scaffold.

[0024] In some embodiments, the preparation method of the bioactive scaffold matrix with macroscopic channel structure may include the following steps: forming a scaffold green body by photopolymerization 3D printing, followed by crosslinking and high-temperature calcination to obtain the final product.

[0025] In the photopolymerization 3D printing process, the mass ratio of raw material powder to photosensitive resin in the printing slurry can be 1:(1.22~2.2). The raw material powder can include bioceramics, biocompatible metals, and polymers, preferably β-tricalcium phosphate (β-TCP) and arsenic tricalcium phosphate (AKT). The photosensitive resin can include water-washed photosensitive resin, standard resin, and flexible resin, preferably water-washed photosensitive resin. The printing process can include: pouring the printing slurry into the printing tank, printing the substrate according to the pre-set printing file, setting the printing parameters of the photopolymerization printer as needed (e.g., model layer thickness 50μm, exposure time per layer 5s, light power 10%), and cleaning to remove any uncured photosensitive resin remaining on the support modules after the printing process is completed. Further, all support modules can be collected and soaked in deionized water and alcohol respectively, and then cleaned in an ultrasonic cleaner for at least 5 minutes to remove any remaining uncured photosensitive resin.

[0026] The cross-linking process of the stent blank may include: placing the stent blank under 450nm blue light to achieve deep cross-linking for more than 2 hours.

[0027] The parameters for the high-temperature calcination may include: heating rate of 2℃ / min, holding temperature of 1150-1350℃, holding time of 3-5 hours, furnace cooling, and calcination in an air atmosphere.

[0028] In some embodiments, the preparation process of the precursor solution may include: adding 200 μL of a 2-6% (preferably 5%) initiator solution and 2-10 μL (preferably 4 μL) of catalyst to 20 mL of a 0.1-20% (preferably 1-6%, more preferably 1-3%) hydrogel solution to form the precursor solution. The ratio of initiator to catalyst is one of the factors determining the size of the microchannels. In this invention, the microchannel size of the multi-level scaffold needs to match the cell size to reshape cell morphology, guide and promote cell migration, and thus enable osteoblasts and nerve cells to interact better. The higher the catalyst concentration, the smaller the corresponding microchannel size and the higher the biotoxicity.

[0029] The initiator may include at least one of glutaraldehyde, genipin, ammonium persulfate (APS), and lithium phenyl (2,4,6-trimethylbenzoyl)phosphate; preferably, 0.2 to 0.6 g of ammonium persulfate is dissolved in 10 mL of deionized water to prepare a 2 to 6% ammonium persulfate initiator solution.

[0030] The catalyst may include tetramethylethylenediamine (TEMED).

[0031] The hydrogel may include at least one of gelatin, methacrylamide gelatin, chitosan, sodium alginate, polyvinyl alcohol, silk fibroin, and polyethylene glycol diacrylate; preferably, 0.2-0.6g of methacrylamide gelatin is dissolved in 20mL of water and heated in a 50℃ water bath for 3-4 hours to dissolve, thereby preparing a 1-3% hydrogel methacrylamide gelatin solution.

[0032] In some embodiments, the process of the directional freezing ice template method may include: in a directional freezing mold, injecting a precursor solution into the interior of a bioceramic scaffold until it is submerged, then placing the entire system in an ultra-low temperature environment to induce the directional growth of ice crystals to form directionally arranged micro-nano channels, then transferring the entire scaffold system to a low temperature environment for chemical cross-linking, and freeze-drying to remove the ice crystals to obtain the multi-level structured bioactive three-dimensional scaffold.

[0033] The material of the mold part in the directional freezing mold where the bioceramic support is placed can be polytetrafluoroethylene; the heat-conducting plate in the directional freezing mold can be a copper plate; preferably, liquid nitrogen is injected into the liquid storage chamber of the directional freezing mold for pre-cooling for 10 minutes.

[0034] The induced directional growth of ice crystals can be carried out in a liquid nitrogen environment at a temperature of -80 to -196°C for at least 20 minutes, preferably 20 to 40 minutes. Sufficient temperature difference between the upper and lower ends of the support is necessary for the formation of directional ice crystals.

[0035] The chemical cross-linking temperature can be -20 to -80°C, preferably -80°C, and the time can be 12 to 24 hours. Low-temperature cross-linking prevents the structure from melting and makes the structure more stable.

[0036] It should be noted that when the matrix is ​​bioceramic, the diameter of the internal oriented microchannels depends on the type, concentration, and temperature of the precursor solution. Specifically, when the concentration of methacrylamide gelatin is 1% (AKT-1GM), the size of the internal oriented microchannels is 50–60 μm; when the concentration of methacrylamide gelatin is 3% (AKT-3GM), the size of the internal oriented microchannels is 30–40 μm; and when the concentration of methacrylamide gelatin is 5% (AKT-5GM), the size of the internal oriented microchannels is approximately 20 μm. Furthermore, oriented channel structures can be fabricated within matrices of various external shapes (e.g., branched structures, cross-stacking structures).

[0037] In summary, the technical solution provided by this invention uses photopolymerization 3D printing technology to prepare a bioceramic scaffold with a macroscopic channel structure, and then uses different concentrations of methacrylamide gelatin to form a dimensionally controllable directional microchannel structure in the macroscopic channel through the ice template method.

[0038] By combining photopolymerization 3D printing with ice template technology, directional microchannel structures were successfully filled into hollow bioceramic scaffolds. These microchannels not only provide space for cell loading and attachment but also offer topographical cues for cell penetration and differentiation. Furthermore, the release of bioactive calcium, magnesium, and silicon ions from AKT and other sources enhanced cell differentiation activity, outperforming traditional TCP materials. On the other hand, the introduction of exogenous neural cells acted as a "magnet" attracting and recruiting endogenous stem cells, further guiding osteogenic differentiation of bone marrow mesenchymal stem cells. This scaffold achieves a dual-driven repair mode of "topographically guided cell penetration" and "functionally enhanced intercellular communication," effectively coordinating neurogenesis / osteogenesis coupling.

[0039] Furthermore, the spatiotemporal distribution of multiple cells and their interactions in three-dimensional space are crucial for regulating cell behavior and influencing tissue growth and development. Therefore, this invention designs a bioactive scaffold with a multi-level macro- and micro-channel structure to mimic the structure of natural bone tissue, thereby accelerating neurogenic bone regeneration by regulating intercellular interactions. The spatiotemporal interactions between nerve cells and osteoblasts within the multi-level channels are used to further explore the effects of this multi-level macro- and micro-channel structure on the migration, maturation, differentiation, and induction of innervation and bone regeneration of both cell types.

[0040] The bioactive scaffold with a multi-level directional channel structure provided by this invention can serve as a "spatial bridge" coordinating spatiotemporal interactions among multiple cells, effectively promoting the migration, energy metabolism, and specific differentiation capabilities between mesenchymal stem cells (MSCs) and Schwann cells (SCs). It has a beneficial promoting effect on the migration, energy metabolism levels, and specific differentiation behavior of various cells in three-dimensional space. The three-dimensional bioactive scaffold constructed by this invention is a novel bone repair biomaterial possessing both multi-level structure and bioactivity. The synergistic effect of the ordered multi-level structure and biochemical signals can achieve neurally innervated bone regeneration, especially promoting bone formation in the core area of ​​bone defects. This provides a new strategy for large-segment bone repair and other complex tissue reconstruction, and can be applied to the preparation of bone repair materials.

[0041] It is also worth noting that the bioactive scaffold prepared in this invention possesses a multi-level macro- and micro-level channel structure, aiming to provide a "bridge" for guiding and promoting intercellular interactions. This amplifies the effects of osteoblasts and nerve cells when used alone, thus enabling its application in the repair of neurogenic bone defects. The outer scaffold material with the macro-channel structure is made of magnesium feldspar bioceramics, which can release calcium, magnesium, and silicon ions to act on nerve cells and osteoblasts, thereby creating a neuro-metabolic activated bone regeneration microenvironment. Furthermore, magnesium feldspar has high biomechanical strength, providing mechanical support in the repair of load-bearing bone defects. The inner micro-channel structure is made of hydrogel material, which firstly possesses excellent biocompatibility, and secondly, through directional freezing, allows for microchannel sizes closely matching the cell size. This facilitates the penetration of osteoblasts and nerve cells into the scaffold, while also reshaping cell morphology, activating intracellular protein expression, and guiding and promoting the migration and interaction of the two cell types.

[0042] Unlike the macroscopic polymer materials used in conventional technical solutions, this material cannot release bioactive ions, has low mechanical strength, and is not suitable for repairing load-bearing bone defects. Furthermore, the size of its microchannels is generally 0.2–10 μm or 1–200 nm, which is outside the size range of cells. The gaps are too small, and cells cannot penetrate well into the scaffold. Therefore, it cannot realize the scaffold as a "bridge" to guide the interaction between the two types of cells mentioned in this invention, and it does not involve the interaction between nerve cells and osteoblasts, which is inconsistent with the scientific problem solved by this invention.

[0043] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0044] Example 1 The method for preparing the magnesium feldspar composite methacrylamide gelatin multi-level directional channel structure bioactive three-dimensional scaffold provided in this embodiment includes the following steps: (1) Preparation of magnesium feldspar bioactive scaffold matrix with macroscopic directional channel structure: The mass ratio of raw material powder to photosensitive resin in the photocuring printing slurry can be 1:1.22. Pour the printing slurry into the printing tank and print the blank according to the printing file built in 3Dmax. The printing parameters of the photocuring printer are set as follows: the model layer thickness is 50μm, the exposure time of each layer is 5s, and the light power is 10%. After the printing program is completed, all the scaffold modules are collected and soaked in deionized water. Then, the scaffold is cleaned with water and anhydrous ethanol in an ultrasonic cleaner for at least 5 minutes to remove the uncured photosensitive resin remaining on the scaffold module. Next, the blank of the scaffold module is placed under 450nm blue light to make it deeply crosslinked for more than 3 hours. The blanks of all the scaffold modules are calcined at high temperature to obtain bioactive scaffolds. The calcination parameters of the AKT scaffold blank are: heating rate 2℃ / min, holding temperature 1350℃, holding time 3 hours, and cooling method is furnace cooling. (2) Preparation of precursor solution: Take 200 μL of 5% ammonium persulfate aqueous solution as initiator and 2 μL of tetramethylethylenediamine as catalyst, and add them to 20 mL of 1%, 3% and 5% methacrylamide aqueous solution respectively to form precursor solution; (3) Preparation of multi-level structured bioactive three-dimensional scaffolds: Micron-level directional microchannels were constructed inside a bioceramic scaffold with a macro-channel structure using the ice template method. In the specific operation, liquid nitrogen was injected into the storage chamber of the directional freezing mold and pre-cooled for 10 min. The bioceramic scaffold was placed in the polytetrafluoroethylene mold, and the prepared precursor solution was injected into the bioceramic scaffold until it was completely submerged. During the process, air bubbles were avoided. The entire polytetrafluoroethylene mold was then placed on the copper plate of the directional freezing mold for at least 1 h until the solution was completely frozen. Subsequently, the mold was transferred to a -80℃ environment for 24 h for chemical cross-linking and structural stabilization. The entire mold was then placed in a freeze dryer to remove ice crystals, and finally a bioactive scaffold with a multi-level directional channel structure was formed. According to the concentration of the precursor solution, the scaffolds were named AKT-1GM, AKT-3GM, and AKT-5GM, respectively.

[0045] Example 2 The preparation method of the magnesium feldspar composite methacrylamide gelatin branched multichannel and cross-stacked multichannel bioactive three-dimensional scaffold provided in this embodiment is the same as in Example 1, with the main difference being: In step (1), a model is created based on 3Dmax, and an AKT bioactive ceramic scaffold with branched and cross-stacked structures is printed using photopolymerization. In step (2), a 3% methacrylamide gelatin precursor solution is prepared; The resulting bioactive three-dimensional scaffold with a multi-level structure is designated AKT-3GM.

[0046] Example 3 The preparation method of the magnesium feldspar composite polyvinyl alcohol multi-level directional channel structure bioactive three-dimensional scaffold provided in this embodiment is the same as in Example 1, with the main difference being: In step (2), a 2% polyvinyl alcohol precursor solution is prepared; The resulting bioactive scaffold with a multi-level directional channel structure is designated as the AKT-PVA scaffold.

[0047] Example 4 The preparation method of the magnesium feldspar composite chitosan multi-level oriented channel structure bioactive three-dimensional scaffold provided in this embodiment is the same as in Example 1, with the main difference being: In step (2), a 2% chitosan precursor solution is prepared; The resulting bioactive scaffold with a multi-level directional channel structure is designated as the AKT-CTS scaffold.

[0048] Example 5 The preparation method of the tricalcium phosphate composite methacrylamide gelatin multilevel directional channel structure bioactive three-dimensional scaffold provided in this embodiment is the same as in Example 1, with the main difference being: In step (1), the parameters for calcining the β-TCP scaffold blank are: heating rate 2℃ / min, holding temperature 1150℃, holding time 3 hours, and cooling method is furnace cooling. In step (2), a 3% methacrylamide gelatin precursor solution is prepared; The resulting bioactive scaffold with a multi-level directional channel structure is designated as the TCP-3GM scaffold.

[0049] Example 6 A neural-bone co-culture model was constructed using the AKT bioactive scaffold (AKT-A) with a multi-level directional channel structure. The AKT-A scaffold was placed into a 48-well plate, and 80 μL of bone marrow mesenchymal stem cell suspension (2 × 10⁻⁶) was added first. 6 Schwann cell suspension (2 × 10⁻⁶ cells / mL) was seeded on the top surface of the scaffold and cultured for 2 hours; after bone marrow mesenchymal stem cells attached, Schwann cell suspension (2 × 10⁻⁶ cells / mL) was added. 6 (1 cell / mL) was seeded on the other side of the scaffold and cultured for 2 hours to promote cell attachment; then, the scaffold carrying bone marrow mesenchymal stem cells / Schwan cells was cultured horizontally while supplementing with mixed culture medium (MEM-α:DMEM=1:1).

[0050] Comparative Example 1 The AKT bioactive scaffold provided in this comparative example is prepared using the same process as the sintered AKT bioceramic scaffold in Example 1.

[0051] Comparative Example 2 The comparative example provided is a TCP bioactive scaffold, and the preparation process is the same as that of the sintered TCP bioceramic scaffold in Example 5.

[0052] Comparative Example 3 The preparation method of the magnesium feldspar composite methacrylamide gelatin multi-level disordered structure scaffold provided in this comparative example is the same as that in Example 1, with the main difference being: In step (3), the precursor solution is directly injected into the AKT scaffold and placed in a -80℃ refrigerator for crosslinking, denoted as AKT-R.

[0053] Comparative Example 4 The preparation method of the tricalcium phosphate composite methacryloyl gelatin multilevel disordered structure scaffold provided in this comparative example is the same as that in Example 5, with the main difference being: In step (3), the precursor solution is directly injected into the TCP scaffold and placed in a -80℃ refrigerator for cross-linking, denoted as TCP-R.

[0054] Comparative Example 5 A neural-bone co-culture model was constructed using a multi-level disordered scaffold composed of magnesium feldspar and methacrylamide gelatin. The AKT-R scaffold was placed into a 48-well plate, and 80 μL of bone marrow mesenchymal stem cell suspension (2 × 10⁻⁶) was added first. 6 Schwann cell suspension (2 × 10⁻⁶ cells / mL) was seeded on the top surface of the scaffold and cultured for 2 hours; after bone marrow mesenchymal stem cells attached, Schwann cell suspension (2 × 10⁻⁶ cells / mL) was added. 6 (1 cell / mL) was seeded on the other side of the scaffold and cultured for 2 hours to promote cell attachment; then, the scaffold carrying bone marrow mesenchymal stem cells / Schwan cells was cultured horizontally while supplementing with mixed culture medium (MEM-α:DMEM=1:1).

[0055] Morphological characterization of AKT bioceramic scaffolds.

[0056] Figure 1The physicochemical properties of the MgO feldspar bioceramic scaffold are characterized as follows: a) is the SEM image of the MgO feldspar powder; b) is the SEM image of the MgO feldspar bioceramic scaffold with a macroscopic channel structure; c) is the XRD pattern of the 3D-printed MgO feldspar bioceramic scaffold; d) is the optical pattern of the MgO feldspar bioceramic scaffold with different channel pore sizes; e) is the elemental distribution map of the MgO feldspar bioceramic scaffold. As shown in the figures, the 3D-printed AKT bioceramic scaffold has a macroscopic directional channel structure, enabling printing in the diameter range of 0.6–1.5 mm; the scaffold after sintering still retains the MgO feldspar phase; and the elemental distribution map shows the uniform distribution of oxygen, magnesium, silicon, and calcium in the AKT scaffold.

[0057] Morphological characterization of bioactive scaffolds with multi-level directional channel structures.

[0058] Since the concentration of the precursor solution affects the size of ice crystals, which in turn affects the pore size of microchannels, multi-level channel bioactive scaffolds with methacrylamide gelatin concentrations of 1%, 3%, and 5% were prepared and named AKT-1GM, AKT-3GM, and AKT-5GM, respectively. Scanning electron microscopy images show top and side views of these scaffolds, revealing that the hollow tubes of the bioceramic scaffolds are completely filled with neatly arranged methacrylamide gelatin. Furthermore, higher concentrations resulted in a significant reduction in the diameter of these vertically aligned microchannels. False-color images and orientation analysis further demonstrated that all methacrylamide gelatin maintained a well-oriented microchannel structure with angles close to 90°. Subsequently, by measuring the pore size of the methacrylamide gelatin in the transverse section and the microchannel size in the longitudinal section, the average microchannel sizes of AKT-1GM, AKT-3GM, and AKT-5GM in the side view were found to be 50.28±10.44, 29.71±8.50, and 19.01±6.30 μm, respectively. Furthermore, the mechanical properties of these scaffolds were measured using a universal testing machine. The stress-strain curves and corresponding compressive strength results showed that the addition of methacrylamide gelatin slightly increased the mechanical strength, but the difference was not significant. The mechanical strength of the multi-level channel scaffolds was primarily attributed to the bioceramic framework, while the effect of methacrylamide gelatin was negligible (see [link to relevant documentation]). Figure 2 ).

[0059] To further verify that combining 3D printing with the ice template method is a universal approach for fabricating hierarchical multilevel scaffolds, cross-stacked and branched bioceramic scaffolds filled with methacrylamide gelatin solution also exhibited good microchannel structures. Furthermore, AKT scaffolds filled with other polymer hydrogels (such as polyvinyl alcohol and chitosan hydrogels) were also successfully fabricated, demonstrating the versatility of this strategy of combining 3D printing technology with the ice template method (see [link to documentation]). Figure 3 ).

[0060] By combining the scaffold pore size with the distance cells migrate within directional microchannels of different diameters, the AKT-3GM bioactive scaffold is considered to have a relatively stable structure and is more suitable for cell culture (see [link]). Figure 4 ).

[0061] Construction of in vitro single-culture models of bone marrow mesenchymal stem cells and Schwann cells.

[0062] To investigate the effects of the topological structure and chemical composition of multi-level structures on cell behavior, six groups were established: AKT (pure AKT bioceramic scaffold), AKT-R (AKT bioceramic scaffold filled with 3% methacrylamide gelatin with random structures), AKT-A (AKT bioceramic scaffold filled with 3% methacrylamide gelatin with directional microchannel structures), TCP (pure TCP bioceramic scaffold), TCP-R (TCP bioceramic scaffold filled with 3% methacrylamide gelatin with random structures), and TCP-A (TCP bioceramic scaffold filled with 3% methacrylamide gelatin with directional microchannel structures). Cell migration distance was observed, revealing that scaffolds with ordered microchannels significantly enhanced the permeability of BMSCs and SCs, showing better results compared to random microstructures, while no significant differences were found among the different bioceramic matrix material groups. Cell morphology images showed that the cytoskeleton of BMSCs and SCs was elongated in the AKT-A and TCP-A scaffolds, while the cytoskeleton was randomly distributed in the other groups. Therefore, it can be concluded that ordered microchannel structures can provide topological cues to guide cell alignment and permeation. Subsequently, the effects of multilevel scaffolds on osteogenic differentiation of bone mesenchymal stem cells (BMSCs) and neural differentiation of scaffolds were evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence staining. The results showed that, under the combined effects of directional channels and AKT ion release, the expression levels of both osteogenic and neural differentiation proteins were higher in the AKT-A group, indicating that AKT-A enhanced the specific differentiation of BMSCs and scaffolds (see [link to study]). Figure 5 and Figure 6 ).

[0063] Construction of an in vitro co-culture model of bone marrow mesenchymal stem cells and Schwann cells.

[0064] This invention seeded BMSCs and SCs at opposite ends of a multi-level scaffold and co-cultured them horizontally to investigate their cell interactions in three-dimensional space. To explore the effects of monoculture / co-culture and material structural characteristics on cell behavior, four groups were established: R-Mono (cell monoculture on an AKT bioceramic scaffold filled with 3% methacrylamide gelatin with a random structure), R-Co (cell co-culture on an AKT bioceramic scaffold filled with 3% methacrylamide gelatin with a random structure), A-Mono (cell monoculture on an AKT bioceramic scaffold filled with 3% methacrylamide gelatin with a directional microchannel structure), and A-Co (cell co-culture on an AKT bioceramic scaffold filled with 3% methacrylamide gelatin with a directional microchannel structure). BMSCs were labeled with green fluorescence, and SCs with red fluorescence to monitor their migration behavior. The results showed that co-culture on a multi-level oriented scaffold (A-Co group) significantly promoted the migration of BMSCs and SCs compared to monoculture on a multi-level oriented scaffold (A-Mono group). This confirmed that when BMSCs and SCs are co-cultured, one cell type attracts the other, thereby promoting their mutual migration. Co-localization of red and green fluorescence was observed on day 5, indicating that BMSCs and SCs actually reached direct contact. Furthermore, to further demonstrate the guiding advantage of well-aligned microchannel structures for cell migration in BMSCs / SCs co-culture experiments, cell migration distances were also measured in co-culture on a multi-level disordered scaffold (R-Co group) and monoculture on a multi-level disordered scaffold (R-Mono group). Quantitative analysis showed that in both BMSCs and SCs, cells with ordered microchannel structures migrated farther than those with randomly arranged scaffolds (reference...). Figure 7 ).

[0065] The osteogenic differentiation activity of bone marrow mesenchymal stem cells (BMSCs) and the neural differentiation activity of stem cells (SCs) were further assessed using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence staining. Results showed that the expression of osteogenic-related genes (including ALP, OPN, and RunX2) in BMSCs from the co-culture group was significantly higher than that from the monoculture group, both in ordered and disordered microchannel structures, with the highest expression in the A-Co group. Immunofluorescence analysis confirmed that the expression level of OPN in the A-Co group was 1.48 times that of the A-Mono group, and under the same cell culture modulus, it was higher than that in the random structure group. Similarly, compared to A-Mono and R-Co, the expression of NGF and GDNF in SCs was significantly upregulated in the A-Co group, while NCAM expression was lower. The expression intensity of GDNF protein in SCs from the A-Co group was 1.49 times that of the A-Mono group and 1.98 times that of the R-Co group. Therefore, these results confirm that layered multichannel scaffolds containing BMSCs / SCs co-culture can promote osteogenic differentiation of BMSCs and neural differentiation of SCs, indicating that this close interaction helps enhance their specific differentiation behaviors (see reference). Figure 8 ).

[0066] Previous studies have found that cells can significantly enhance each other's energy metabolism activity through pathways such as mitochondrial transfer, thereby promoting specific differentiation. Further analysis of ATP content and energy metabolism-related genes revealed that co-culturing the two cell types increased ATP expression and energy metabolism gene expression compared to monoculture. This further validated the close interaction between BMSCs and SCs co-cultured in the AKT-A multilevel channel scaffold, accelerating the migration and differentiation of both cell types by enhancing their respective energy metabolism activities (see reference). Figure 9 ).

[0067] Bioactive scaffolds with multi-level channel structures exhibit in vivo neurogenic osteogenic bioactivity.

[0068] AKT, AKT-R, AKT-A, AKT-R@SCs (pre-loaded Schwann cells on a multilevel disordered scaffold), and AKT-A@SCs (pre-loaded Schwann cells on a multilevel directional channel scaffold) were implanted into lateral femoral condyle defects in rabbits, and samples were harvested after 12 weeks. Micro-CT results showed that, compared with other groups, the amount of new bone formation in the AKT-R@SCs (G5) and AKT-A@SCs (G6) groups showed a progressively increasing trend, with the AKT-A@SCs group (G6) achieving highly penetrating bone formation within these macroscopic channels. Further, the defect area was divided into upper, middle, and lower layers for quantitative analysis. It was found that there was almost no difference in BV / TV and Tb.N values ​​between the ordered microchannel structure groups (G4 and G6) and the random structure groups (G3 and G5) in the upper and lower layers. However, in the middle layer, the BV / TV and Tb.N values ​​of the ordered microchannel structures (G4 and G6) were approximately 1.5 times higher than those of the random structures (G3 and G5), indicating significant new bone formation in the core region of the bone defect. H&E staining also showed the same results (see reference). Figure 10 ).

[0069] Immunofluorescence staining of the tissue in the middle of the defect showed that the AKT-A@SCs (G6) group exhibited the highest expression levels in bone matrix expression, nerve innervation, and angiogenesis. These results indicate that the AKT-A@SCs scaffold, with its ordered microchannel structure and Schwann cell-loaded structure, can create a microenvironment conducive to neural activation, thereby achieving bone regeneration under neural innervation (see reference). Figure 11 ).

[0070] In summary, this invention combines 3D printing technology with the ice template method to prepare a multi-level bioactive three-dimensional scaffold for multi-cell interaction and regeneration of neurogenic bone. It can serve as a bridge and a multifunctional platform to coordinate the spatiotemporal interactions of multiple cells, thereby achieving complex regeneration of neurogenic bone tissue. It also provides a new approach for achieving the regeneration of other complex tissues.

[0071] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A multi-level structure bioactive three-dimensional scaffold for multi-cellular interactions and neuralized bone regeneration, characterized in that, The multi-level structure bioactive three-dimensional scaffold comprises a bioactive scaffold matrix with macroscopic channel structure and a microcosmic directional topological structure filled inside the matrix; the microcosmic directional topological structure comprises micro-nano level channels arranged in order. The multi-level structure bioactive three-dimensional scaffold can serve as a "spatial bridge" for coordinating the spatiotemporal interaction of multiple cells, and has beneficial promoting effect on the mutual migration, energy metabolism level and specific differentiation behavior of multiple cells in three-dimensional space.

2. The multi-level structured bioactive three-dimensional scaffold of claim 1, wherein, The material of the bioactive scaffold matrix is selected from at least one of bioceramics, bioactive glass, biological metal, metal oxide, biological polymer and carbon-based nanomaterials, and is preferably bioceramics; more preferably, the material of the bioceramics comprises β-tricalcium phosphate or hardystonite.

3. The multi-level structured bioactive three-dimensional scaffold according to claim 1 or 2, wherein, The macroscopic channel structure of the bioactive scaffold matrix comprises at least one of directional channel structure, branch channel and cross-stacked structure formed by 3D printing. Preferably, the diameter of the bioactive scaffold matrix is 0.2-5 mm, and the height is 1-10 mm. Preferably, the diameter of the micro-nano level channel is 5-200 μm.

4. A method of producing a multi-level structured bioactive three-dimensional scaffold according to any one of claims 1 to 3, characterized in that, The preparation method comprises the following steps: electrospinning, electric field / magnetic field induced self-assembly or directional freeze ice template method are adopted to form micro-nano level channels arranged in order inside the bioactive scaffold matrix with macroscopic channel structure formed by 3D printing, so as to obtain the multi-level structure bioactive three-dimensional scaffold.

5. The production method according to claim 4, characterized by, The preparation process of the precursor solution comprises: 200 μL of 2-6 % initiator solution and 2-10 μL of catalyst are added into 20 mL of 0.1-20 % hydrogel solution to form a precursor solution.

6. The production method according to claim 4 or 5, characterized by, The initiator comprises at least one of glutaraldehyde, genipin, ammonium persulfate and lithium phenyl (2,4,6-trimethylbenzoyl) phosphate. The catalyst comprises tetramethyl ethylenediamine. The hydrogel comprises at least one of gelatin, methacrylated gelatin, chitosan, sodium alginate, polyvinyl alcohol, silk fibroin and polyethylene glycol diacrylate.

7. The production method according to any one of claims 4 to 6, characterized by, The process of the directional freeze ice template method comprises: in a directional freeze mold, the precursor solution is filled into the inside of the bioceramic scaffold to be immersed, then the whole system is placed in an ultralow temperature environment to induce directional growth of ice crystals to form micro-nano level channels arranged in order, then the whole scaffold system is transferred to a low temperature environment for chemical crosslinking, and the ice crystals are removed by freeze drying to obtain the multi-level structure bioactive three-dimensional scaffold.

8. The production method according to any one of claims 4 to 7, characterized by, The directional growth of ice crystals is induced in a liquid nitrogen environment with a temperature of-80--196 ℃ for more than 20 min, preferably 20-40 min.

9. The production method according to any one of claims 4 to 8, characterized by, The temperature of the chemical crosslinking is-20--80 ℃ for 12-24 h.

10. Application of the multi-level structure bioactive three-dimensional scaffold of any one of claims 1-3 in the preparation of materials for multiple cell interaction and bone repair.

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