Resveratrol and nerve growth factor loaded chitosan-polydopamine modified 3D printing biological material as well as preparation method and application thereof in spinal cord injury repair

Abstract

The invention discloses a resveratrol and nerve growth factor loaded chitosan-polydopamine modified 3D printing biological material as well as a preparation method and application thereof in spinal cord injury repair, the structure and function of the biological material simulate a natural extracellular matrix (ECM) tissue structure to fill cavities in a spinal cord injury area, and tissue regeneration can be guided; meanwhile, the compound can be used as a carrier of medicines, cells or bioactive factors. According to the material, a three-dimensional porous PLA support with an accurate structure is printed through a fused deposition modeling (FDM) technology to serve as a carrier for cell compatibility and physical supporting, CS is loaded in the three-dimensional porous PLA support through a freeze drying technology, PDA carrying NGF is loaded on the surface of the three-dimensional porous PLA support, Res is loaded through the physical adsorption effect, and the multifunctional 3D biological composite material for spinal cord injury repair is prepared. Through the synergistic effect of anti-inflammatory, anti-oxidation, anti-apoptosis and other ways, a microenvironment beneficial to nerve regeneration is created together, and functional recovery after spinal cord injury is significantly promoted.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and neural regeneration materials, and in particular to a chitosan-polydopamine modified 3D printed biomaterial loaded with resveratrol and nerve growth factor, its preparation method, and its application in spinal cord injury repair. Background Technology

[0002] Spinal cord injury (SCI) is a highly disabling central nervous system trauma with a complex pathological mechanism, primarily involving primary mechanical injury and a secondary biochemical cascade. The secondary injury stage involves multiple interconnected malignant processes, including uncontrolled inflammatory responses, oxidative stress bursts, glutamate excitotoxicity, apoptosis, and inhibitory glial scar formation, ultimately leading to expanded injury extent and permanent loss of neurological function. Current clinical treatment strategies, such as surgical decompression and high-dose methylprednisolone pulse therapy, have limited efficacy and are accompanied by significant side effects, failing to effectively reverse neurological deficits. Therefore, developing novel comprehensive treatment strategies that can intervene in secondary injury and promote nerve regeneration has become a major challenge and urgent need in this field.

[0003] Tissue engineering, particularly neural scaffold technology, offers a promising direction for SCI repair. An ideal neural scaffold should possess the following functions: (1) bridging the injury defect, providing physical support and guidance for cell migration and axonal regeneration; (2) loading and controlling the release of therapeutic drugs / factors to regulate the harmful injury microenvironment; and (3) possessing good biocompatibility and biodegradability, ultimately being replaced by new tissue. Among numerous fabrication technologies, fused deposition modeling (FDM) 3D printing technology is widely used to prepare synthetic polymer scaffolds, represented by polylactic acid (PLA), because it can precisely customize the macroscopic structure of the scaffold (such as pore size and porosity) and perfectly match the injury area. PLA has good printability, mechanical strength, and biodegradability, making it an ideal material for constructing scaffold structures.

[0004] However, the clinical application of PLA scaffolds faces several key bottlenecks: (1) Bioinertness and hydrophobicity: PLA material itself lacks cell recognition sites and has a hydrophobic surface, which is not conducive to the adhesion, spread and function of nerve cells, and may even cause mild foreign body reactions; (2) Single function limitation: Simple PLA scaffolds only provide physical support and cannot actively regulate the pathological microenvironment or promote nerve regeneration. In order to endow the scaffold with bioactivity, surface modification and functional loading strategies are often adopted. CS is a natural cationic polysaccharide with good biocompatibility, antibacterial and nerve regeneration capabilities. PDA is modeled after mussel adhesive protein and has extremely strong adhesion. It can form a coating on the surface of various materials, effectively improve the hydrophilicity of the materials, and can serve as a platform for secondary reactions to further fix functional molecules (chitosan). At the same time, PDA itself also has certain antioxidant properties. In terms of drug selection, Res, as a natural polyphenol, has strong anti-inflammatory (such as inhibiting the NF-κB pathway), antioxidant (such as activating the Nrf2 pathway), anti-apoptotic and neuroprotective effects, and is an ideal candidate drug for intervening in early secondary injury. NGF is a core member of the neurotrophic factor family. It can effectively support the survival of sensory neurons and cholinergic neurons, and strongly induce PC-12 cells to differentiate into neuron-like structures, promoting axonal growth. It is crucial for neurogenesis in the middle and late stages.

[0005] While each of the aforementioned individual strategies has its advantages, existing technologies face profound technical contradictions and limitations when integrating multiple functions: Res is a hydrophobic small molecule, while NGF is a hydrophilic macromolecular protein, resulting in fundamental conflicts in solubility, loading mechanisms, and release kinetics; SCI repair is a multi-stage dynamic process, and the ideal treatment strategy should rapidly release anti-inflammatory and antioxidant drugs (such as Res) in the early stages (hours to days after injury) to curb the expansion of secondary damage, while continuously releasing neurotrophic factors (such as NGF) in the mid-to-late stages (days to weeks) to promote regeneration. Most existing co-loading systems cannot achieve this spatiotemporally sequential intelligent release, and the release behaviors of different molecules interfere with each other, leading to reduced efficacy; NGF and other protein factors are extremely sensitive to the microenvironment (pH, temperature, organic solvents) and are prone to conformational changes and inactivation during scaffold preparation and loading. Many preparation processes involving organic solvents or harsh reaction conditions are not suitable for NGF loading. However, this invention systematically and comprehensively integrates the structural advantages of 3D printing, the surface modification and loading platform advantages of PDA, the bioactivity of CS, and the synergistic therapeutic advantages of Res and NGF, which is of great significance for the repair of spinal cord injury. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a chitosan-polydopamine modified 3D printing biomaterial loaded with resveratrol and nerve growth factor, its preparation method, and its application in spinal cord injury repair.

[0007] This invention utilizes a 3D-printed porous polylactic acid (PLA) scaffold. Chitosan (CS) is loaded internally, and polydopamine (PDA) containing nerve growth factor (NGF) is loaded onto the surface. Finally, resveratrol (Res) is physically adsorbed onto the surface to create a chitosan-polydopamine-modified 3D-printed PLA scaffold loaded with resveratrol and NGF. Each component and its assigned function is distinct. Using 3D printing technology, the macroscopic structure of the scaffold (such as pore size and porosity) can be precisely controlled, allowing it to perfectly match the spinal cord injury area and provide an optimal topology for cell migration and tissue ingrowth. PLA is a biocompatible and biodegradable plastic polymer material, easily printed into precisely structured three-dimensional porous scaffolds using fused deposition modeling (FDM) technology. However, the hydrophobic surface and lack of bioactivity of PLA limit its integration with neural tissue and its functional applications. The introduction of CS further enhances the biocompatibility and hydrophilicity of the scaffold, and its inherent antibacterial and nerve growth-promoting properties complement the pharmacological effects of Res. The PDA coating perfectly solves the problems of PLA surface inertness and hydrophobicity, significantly improving hydrophilicity and cell adhesion, while providing an active platform for subsequent Res loading and facilitating the sustained release of NGF. The dual action of PDA and CS achieves efficient loading and controlled sustained release of hydrophobic Res, specifically inhibiting inflammatory responses and oxidative stress, and protecting neurons and oligodendrocytes. NGF can promote neuronal survival and the differentiation of neural stem cells into mature neuronal phenotypes, thereby achieving nerve regeneration and the effect of spinal cord injury repair. The scaffold prepared in this invention integrates physical support (PLA), interface modification (PDA), bioactivity enhancement (CS), drug therapy (Res), and bioactive factor delivery (NGF) into one unit. Through the synergistic effects of anti-inflammatory, antioxidant, and anti-apoptotic pathways, it jointly creates a microenvironment conducive to nerve regeneration, significantly promoting functional recovery after spinal cord injury.

[0008] This invention is achieved through the following technical solution: A chitosan-polydopamine modified 3D-printed biomaterial loaded with resveratrol and nerve growth factor mimics the structure and function of natural extracellular matrix (ECM) tissue to fill cavities in spinal cord injury areas and guide tissue regeneration. It can also serve as a carrier for drugs, cells, or bioactive factors. A three-dimensional porous PLA scaffold was printed using fused deposition modeling (FDM) to serve as a carrier for cell compatibility and physical support. Chitosan was loaded inside the carrier using freeze-drying technology, and polydopamine carrying nerve growth factor was loaded on the carrier surface. Finally, resveratrol was loaded onto the carrier surface through physical adsorption to prepare a multifunctional 3D biocomposite material for spinal cord injury repair. Through synergistic effects of anti-inflammatory, antioxidant, and anti-apoptotic pathways, it creates a microenvironment conducive to nerve regeneration, significantly promoting functional recovery after spinal cord injury.

[0009] A method for preparing chitosan-polydopamine modified 3D printing biomaterial loaded with resveratrol and nerve growth factor includes the following steps: a. Design a three-dimensional porous scaffold model and print the PLA scaffold using fused deposition modeling technology; after printing, clean the PLA scaffold, dry it, and set it aside for use. b. Weigh a certain amount of chitosan and add it to an acetic acid aqueous solution. After it is fully dissolved, add the PLA scaffold from step a to this solution and stir. Then freeze-dry it and record it as a PC scaffold. c. Synthesis of polydopamine solution: Add dopamine hydrochloride to tris(hydroxymethyl)aminomethane distilled water buffer to synthesize polydopamine solution, and then stir the polydopamine solution in the dark for 6-24 hours before use; d. Prepare nerve growth factor solution: Add nerve growth factor powder to phosphate buffer solution for later use; e. Dilute the nerve growth factor solution in step d, add it to the polydopamine solution in step c, stir, let stand, and protect from light for later use. f. Weigh an appropriate amount of resveratrol (Res) and add it to a methanol solution. Dissolve the resveratrol in methanol by ultrasonication until homogeneous. Protect the solution from light and set aside for later use. g. The PC scaffold obtained in step b is immersed in the polydopamine solution obtained in step c, and the scaffold obtained by freeze-drying is denoted as PCP scaffold; the PC scaffold obtained in step b is added to the resveratrol methanol solution obtained in step f, and the scaffold obtained by freeze-drying is denoted as PCP-Res scaffold; the PC scaffold obtained in step b is added to the polydopamine solution containing nerve growth factor obtained in step e, freeze-dried, and then immersed in the resveratrol methanol solution obtained in step f, and the scaffold obtained by freeze-drying is denoted as PCP-Res-NGF scaffold.

[0010] In step a, a three-dimensional porous support model is designed using computer-aided design (CAD). The resulting PLA support is a cuboid with dimensions of 10×10×2mm. After the PLA support is printed, it is cleaned with an ultrasonic cleaner for 30~60 minutes at a temperature controlled at 37℃. After cleaning, it is dried and ready for use.

[0011] In step b, the relative concentration of chitosan to acetic acid aqueous solution is 1~2 wt / v; in the acetic acid aqueous solution, the relative concentration of acetic acid to deionized water is 1~2 v / v.

[0012] In step c, the concentration of dopamine hydrochloride in the tris(hydroxymethyl)aminomethane distilled water buffer is 10 mg / mL to 40 mg / mL; the amount of tris(hydroxymethyl)aminomethane distilled water buffer is 10 mmol to 20 mmol; the volume used is 50 to 100 mL; and the pH of the tris(hydroxymethyl)aminomethane distilled water buffer is 7.4 to 8.5.

[0013] In step d, the nerve growth factor solution is prepared by adding 1 mg of nerve growth factor powder to 1 mL of phosphate buffer solution to obtain a 1 mg / mL nerve growth factor solution, which is then sealed and stored at -20°C for later use.

[0014] In step e, the nerve growth factor solution is diluted to 0.25-0.5 mg / mL and then added to the polydopamine solution in step c. The nerve growth factor solution accounts for 1-2.5 wt / v of the total polydopamine and nerve growth factor solution.

[0015] In step f, a 2 mg / mL resveratrol methanol solution is prepared.

[0016] The freeze-drying process involves placing the container in the dark for 12-24 hours, freezing it at -80°C for 24-48 hours, then freeze-drying it at -50°C for 12-24 hours using a freeze dryer. After drying, the container is removed and sealed for storage at -20°C.

[0017] The application of a chitosan-polydopamine modified 3D-printed biomaterial loaded with resveratrol and nerve growth factor as described in claim 1 as a medical scaffold material for treating spinal cord injury.

[0018] The advantages of the present invention are: (1) Structural advantages: The present invention uses 3D printing technology, which can precisely control the macroscopic structure of the scaffold (such as pore size, porosity, etc.), and internally loads CS to increase porosity, so that it can perfectly match the spinal cord injury area and provide the optimal topological structure for cell migration and tissue ingrowth.

[0019] (2) Functional Synergy: The three-dimensional porous PLA scaffold of this invention serves as a carrier for cell compatibility and physical support; the introduction of CS further enhances the biocompatibility of the scaffold, and its inherent antibacterial and nerve growth-promoting properties complement the pharmacological effects of Res, while also increasing the hydrophilicity of the 3D printed PLA scaffold; the PDA coating perfectly solves the problems of PLA surface inertness and hydrophobicity, significantly improving hydrophilicity and cell adhesion, while providing an active platform for subsequent continuous release of NGF and loading of Res. Res is rapidly released in the early stage to inhibit the expansion of secondary damage, while NGF is continuously released in the middle and late stages (several days to several weeks) to promote nerve regeneration.

[0020] (3) Synergistic treatment mechanism: The scaffold of the present invention integrates physical support (PLA), interface modification (PDA), bioactivity enhancement (CS) and drug therapy (Res). Through the synergistic effects of anti-inflammatory, antioxidant and anti-apoptotic pathways, it jointly creates a microenvironment conducive to nerve regeneration and significantly promotes functional recovery after spinal cord injury.

[0021] (4) Simple and controllable process: The preparation method of this invention is simple, the conditions are mild, it is easy to scale up production, and it has a good prospect for clinical translation. Attached Figure Description

[0022] Figure 1 A 3D model of a 3D-printed PLA (using a=0.8cm, b=0.52cm as an example); Figure 2 These are SEM images of Example 1, where (a), (b), (c), and (d) are SEM images of PLA, PC, PCP, and PCP-Res, respectively. Figure 3 The above are in vitro free radical capture maps of Example 1, where (a) is the UV-Vis spectrum of DPPH, PLA, PC, PCP, and PCP-Res, (b) is the UV-Vis spectrum of H2O2, PLA, PC, PCP, and PCP-Res, (c) is the UV-Vis spectrum of ·OH, PLA, PC, PCP, and PCP-Res, and (d) is the DPPH, H2O2, and ·OH scavenging rates of PLA, PC, PCP, and PCP-Res. Figure 4 Cell compatibility of PLA, PC, PCP, PCP-Res, and PCP-Res-NGF in Example 1; Figure 5The diagram shows the ROS scavenging ability of PC-12 cells in Example 1, where (a) is the ROS fluorescence diagram of PC-12 cells co-cultured with PLA, PC, PCP, PCP-Res, and PCP-Res-NGF for 6 and 12 h, and (b) is the relative fluorescence intensity of ROS of PC-12 cells co-cultured with PLA, PC, PCP, PCP-Res, and PCP-Res-NGF for 6 and 12 h. Figure 6 This is a cell differentiation diagram of Example 1, showing the fluorescence of PC-12 cells and cells co-cultured with LPS, PCP-Res-NGF (LPS), PCP-Res-NGF (LPS-PR), and PCP-Res-NGF for 3 days. PCP-Res-NGF (LPS-PR) is a PCP-Res-NGF scaffold co-cultured with PC-12 and Raw246.7 cells under LPS stimulation. Detailed Implementation

[0023] A chitosan-polydopamine modified 3D-printed biomaterial loaded with resveratrol and nerve growth factor (NGF) has been developed. The material's structure and function mimic the natural extracellular matrix (ECM) tissue structure to fill cavities in spinal cord injury areas and guide tissue regeneration. It can also serve as a carrier for drugs, cells, or bioactive factors. The material is printed into a precisely structured three-dimensional porous PLA scaffold using fused deposition modeling (FDM) technology, serving as a carrier for cell compatibility and physical support. A multifunctional 3D biocomposite material for spinal cord injury repair is prepared by loading resveratrol (CS) internally, NGF-carrying PDA on the surface, and Res through physical adsorption. Through synergistic effects of anti-inflammatory, antioxidant, and anti-apoptotic pathways, it creates a microenvironment conducive to nerve regeneration, significantly promoting functional recovery after spinal cord injury.

[0024] A method for preparing chitosan-polydopamine modified 3D printing biomaterial loaded with resveratrol and nerve growth factor includes the following steps: a. A three-dimensional porous scaffold model was designed using computer-aided design (CAD), and PLA scaffolds were printed using fused deposition modeling (FDM) technology. The scaffolds were 10×10×2mm cuboids. Different sizes were designed, while maintaining a constant porosity of 40% and a constant pore diameter of a=0.8cm. Specific dimensions are shown in Tables 1 and 2. After printing, the PLA scaffolds were cleaned using an ultrasonic cleaner for 30-60 minutes at 37℃, and then dried for later use. (PLA scaffolds with a=0.8cm and b=0.52cm were selected for subsequent experiments; these are referred to as PLA scaffolds). Table 1. 3D printed PLA scaffold (porosity 40%) a / cm b / cm 1 0.7 0.4 2 0.8 0.52 3 0.8 0.6 4 1 0.62 Table 2 3D printed PLA bracket (aperture a=0.8cm) a / cm b / cm 1 0.8 0.5 2 0.8 0.6 3 0.8 0.7 4 0.8 0.8 b. Weigh a certain amount of chitosan and add it to an acetic acid aqueous solution. After it is fully dissolved, add the PLA scaffold from step a to this solution and stir. Then freeze-dry it and record it as a PC scaffold. c. Synthesis of polydopamine solution: Add dopamine hydrochloride to tris(hydroxymethyl)aminomethane distilled water buffer to synthesize polydopamine solution, and then stir the polydopamine solution in the dark for 6-24 hours before use; d. Prepare nerve growth factor solution: Add nerve growth factor powder to phosphate buffer solution for later use; e. Dilute the nerve growth factor solution in step d, add it to the polydopamine solution in step c, stir, let stand, and protect from light for later use. f. Weigh an appropriate amount of resveratrol (Res) and add it to a methanol solution. Dissolve the resveratrol in methanol by ultrasonication until homogeneous. Protect the solution from light and set aside for later use. g. The PC scaffold obtained in step b is immersed in the polydopamine solution obtained in step c, and the scaffold obtained by freeze-drying is denoted as PCP scaffold; the PC scaffold obtained in step b is added to the resveratrol methanol solution obtained in step f, and the scaffold obtained by freeze-drying is denoted as PCP-Res scaffold; the PC scaffold obtained in step b is added to the polydopamine solution containing nerve growth factor obtained in step e, freeze-dried, and then immersed in the resveratrol methanol solution obtained in step f, and the scaffold obtained by freeze-drying is denoted as PCP-Res-NGF scaffold.

[0025] In step b, the relative concentration of chitosan to acetic acid aqueous solution is 1~2 wt / v; in the acetic acid aqueous solution, the relative concentration of acetic acid to deionized water is 1~2 v / v.

[0026] In step c, the concentration of dopamine hydrochloride in the tris(hydroxymethyl)aminomethane distilled water buffer is 10 mg / mL to 40 mg / mL; the amount of tris(hydroxymethyl)aminomethane distilled water buffer is 10 mmol to 20 mmol; the volume used is 50 to 100 mL; and the pH of the tris(hydroxymethyl)aminomethane distilled water buffer is 7.4 to 8.5.

[0027] In step d, the nerve growth factor solution is prepared by adding 1 mg of nerve growth factor powder to 1 mL of phosphate buffer solution to obtain a 1 mg / mL nerve growth factor solution, which is then sealed and stored at -20°C for later use.

[0028] In step e, the nerve growth factor solution is diluted to 0.25-0.5 mg / mL and then added to the polydopamine solution in step c. The nerve growth factor solution accounts for 1-2.5 wt / v of the total polydopamine and nerve growth factor solution.

[0029] In step f, a 2 mg / mL resveratrol methanol solution is prepared.

[0030] The freeze-drying process involves placing the container in the dark for 12-24 hours, freezing it at -80°C for 24-48 hours, then freeze-drying it at -50°C for 12-24 hours using a freeze dryer. After drying, the container is removed and sealed for storage at -20°C.

[0031] Application of a chitosan-polydopamine modified 3D-printed biomaterial loaded with resveratrol and nerve growth factor as a medical scaffold material for treating spinal cord injury.

[0032] The present invention will be further described in detail below with reference to specific embodiments.

[0033] Example 1: This example is carried out according to the following steps, and the specific data is shown in the attached diagram. Figures 2-6 As shown: 1. A three-dimensional porous scaffold model was designed using computer-aided design (CAD), and the PLA scaffold was printed using fused deposition modeling (FDM) technology. For example... Figure 1 As shown. A cuboid with dimensions of 10×10×2mm, a=0.8cm, b=0.52cm. After printing, the PLA bracket is cleaned with an ultrasonic cleaner for 30~60 minutes at a temperature controlled at 37℃. After cleaning, it is dried and ready for use. 2. Weigh 0.1g of chitosan and add it to 50mL of acetic acid aqueous solution (add 49mL of deionized water to 1mL of acetic acid). After it is fully dissolved, add the PLA scaffold from step 1 to this solution and stir for a period of time. Then freeze it in a -80℃ freezer for more than 48 hours. Then transfer it to a freeze dryer and freeze dry it at -50℃ for 48 hours. The freeze-dried scaffold is denoted as PC. 3. Add 1.2113 g of tris(hydroxymethyl)aminomethane to deionized water, stir to dissolve, and adjust the pH to 8.5 using a pH meter to prepare a 10 mM tris(hydroxymethyl)aminomethane buffer solution. The amount of the prepared tris(hydroxymethyl)aminomethane buffer solution is 10 mmol of polydopamine solution. Then, stir the polydopamine solution in the dark for 24 hours before use. 4. Prepare nerve growth factor solution: Take 1 mg of nerve growth factor powder and add it to 1 mL of phosphate buffer solution for later use. This diluted solution will be used subsequently. 5. Dilute the nerve growth factor solution from step 4 to 0.5 mg / mL. Add 0.2 mL of diluted nerve growth factor (the concentration of nerve growth factor relative to phosphate buffer is 0.5 mg / mL) to the prepared polydopamine solution, so that the added nerve growth factor content is 2% of the polydopamine solution. Keep the materials in the dark during the experiment. 6. Weigh 0.05g of resveratrol and add it to 1mL of methanol. Dissolve the mixture evenly by ultrasonication and then protect it from light for later use. 7. The PC scaffold obtained in step 2 is immersed in polydopamine and then freeze-dried to obtain a scaffold denoted as PCP. The PC obtained in step 2 is added to a resveratrol solution and then freeze-dried to obtain a scaffold denoted as PCP-Res. The PC obtained in step 2 is added to a polydopamine solution containing nerve growth factor, freeze-dried, and then immersed in a resveratrol solution and freeze-dried to obtain a scaffold denoted as PCP-Res-NGF. In all freeze-drying processes, the material is taken out of the solution and placed in a freezer at -80℃ for more than 48 hours, and then transferred to a freeze dryer at -50℃ for 48 hours.

[0034] Example 2, this example is carried out according to the following steps: 1. A three-dimensional porous scaffold model was designed using computer-aided design (CAD), and the PLA scaffold was printed using fused deposition modeling (FDM) technology. The scaffold was a cuboid with dimensions of 10×10×2mm, a=0.8cm, b=0.52cm. After printing, the PLA scaffold was cleaned with an ultrasonic cleaner for 30~60 minutes at a temperature controlled at 37℃. After cleaning, it was dried and ready for use. 2. Weigh 0.1g of chitosan and add it to 50mL of acetic acid aqueous solution (add 49mL of deionized water to 1mL of acetic acid). After it is fully dissolved, add the PLA scaffold from step 1 to this solution and stir for a period of time. Then freeze it in a -80℃ freezer for more than 48 hours. Then transfer it to a freeze dryer and freeze dry it at -50℃ for 48 hours. The freeze-dried scaffold is denoted as PC. 3. Add 2.4226 g of tris(hydroxymethyl)aminomethane to deionized water, stir to dissolve, and adjust the pH to 8.5 using a pH meter to prepare a 10 mM tris(hydroxymethyl)aminomethane buffer solution. The prepared tris(hydroxymethyl)aminomethane buffer solution is used to synthesize a polydopamine solution of 20 mmol. Then, the polydopamine solution is stirred in the dark for 24 hours before use. 4. Prepare nerve growth factor solution: Take 1 mg of nerve growth factor powder and add it to 1 mL of phosphate buffer solution for later use. This diluted solution will be used subsequently. 5. Dilute the nerve growth factor solution from step 4 to 0.5 mg / mL. Add 0.2 mL of diluted nerve growth factor (the concentration of nerve growth factor relative to phosphate buffer is 0.5 mg / mL) to the prepared polydopamine solution, so that the added nerve growth factor content is 2% of the polydopamine solution. Keep the materials in the dark during the experiment. 6. Weigh 0.05g of resveratrol and add it to 1mL of methanol. Dissolve the mixture evenly by ultrasonication and then protect it from light for later use. 7. The PC scaffold obtained in step 2 is immersed in polydopamine and then freeze-dried to obtain a scaffold denoted as PCP. The PC obtained in step 2 is added to a resveratrol solution and then freeze-dried to obtain a scaffold denoted as PCP-Res. The PC obtained in step 2 is added to a polydopamine solution containing nerve growth factor, freeze-dried, and then immersed in a resveratrol solution and freeze-dried to obtain a scaffold denoted as PCP-Res-NGF. In all freeze-drying processes, the material is taken out of the solution and placed in a freezer at -80℃ for more than 48 hours, and then transferred to a freeze dryer at -50℃ for 48 hours.

[0035] Example 3, this example is carried out according to the following steps: 1. A three-dimensional porous scaffold model was designed using computer-aided design (CAD), and the PLA scaffold was printed using fused deposition modeling (FDM) technology. The scaffold was a cuboid with dimensions of 10×10×2mm, a=0.8cm, b=0.52cm. After printing, the PLA scaffold was cleaned with an ultrasonic cleaner for 30~60 minutes at a temperature controlled at 37℃. After cleaning, it was dried and ready for use. 2. Weigh 0.1g of chitosan and add it to 50mL of acetic acid aqueous solution (add 49mL of deionized water to 1mL of acetic acid). After it is fully dissolved, add the PLA scaffold from step 1 to this solution and stir for a period of time. Then freeze it in a -80℃ freezer for more than 48 hours. Then transfer it to a freeze dryer and freeze dry it at -50℃ for 48 hours. The freeze-dried scaffold is denoted as PC. 3. Add 1.2113 g of tris(hydroxymethyl)aminomethane to deionized water, stir to dissolve, and adjust the pH to 8.5 using a pH meter to prepare a 10 mM tris(hydroxymethyl)aminomethane buffer solution. The amount of the prepared tris(hydroxymethyl)aminomethane buffer solution is 10 mmol of polydopamine solution. Then, stir the polydopamine solution in the dark for 24 hours before use. 4. Prepare nerve growth factor solution: Take 1 mg of nerve growth factor powder and add it to 1 mL of phosphate buffer solution for later use. This diluted solution will be used subsequently. 5. Dilute the nerve growth factor solution from step 4 to 0.25 mg / mL. Add 0.2 mL of diluted nerve growth factor (0.5 mg / mL relative to phosphate buffer) to the prepared polydopamine solution, so that the added nerve growth factor content is 2% of the polydopamine solution. Keep the materials in the dark during the experiment. 6. Weigh 0.05g of resveratrol and add it to 1mL of methanol. Dissolve the mixture evenly by ultrasonication and then protect it from light for later use. 7. The PC scaffold obtained in step 2 is immersed in polydopamine and then freeze-dried to obtain a scaffold denoted as PCP. The PC obtained in step 2 is added to a resveratrol solution and then freeze-dried to obtain a scaffold denoted as PCP-Res. The PC obtained in step 2 is added to a polydopamine solution containing nerve growth factor, freeze-dried, and then immersed in a resveratrol solution and freeze-dried to obtain a scaffold denoted as PCP-Res-NGF. In all freeze-drying processes, the material is taken out of the solution and placed in a freezer at -80℃ for more than 48 hours, and then transferred to a freeze dryer at -50℃ for 48 hours.

Claims

1. A chitosan-polydopamine modified 3D printing biomaterial loaded with resveratrol and nerve growth factor, characterized in that: A three-dimensional porous PLA scaffold was printed using fused deposition modeling technology to serve as a carrier for cell compatibility and physical support. Chitosan was loaded inside the carrier using freeze-drying technology, and polydopamine carrying nerve growth factor was loaded on the surface of the carrier. Finally, resveratrol was loaded on the surface of the carrier through physical adsorption to prepare a multifunctional 3D biocomposite material for spinal cord injury repair.

2. A method for preparing chitosan-polydopamine modified 3D printing biomaterial loaded with resveratrol and nerve growth factor, characterized in that: Includes the following steps: a. Design a three-dimensional porous scaffold model and print the PLA scaffold using fused deposition modeling technology; after printing, clean the PLA scaffold, dry it, and set it aside for use. b. Weigh a certain amount of chitosan and add it to an acetic acid aqueous solution. After it is fully dissolved, add the PLA scaffold from step a to this solution and stir. Then freeze-dry it and record it as a PC scaffold. c. Synthesis of polydopamine solution: Add dopamine hydrochloride to tris(hydroxymethyl)aminomethane distilled water buffer to synthesize polydopamine solution, and then stir the polydopamine solution in the dark for 6-24 hours before use; d. Prepare nerve growth factor solution: Add nerve growth factor powder to phosphate buffer solution for later use; e. Dilute the nerve growth factor solution in step d, add it to the polydopamine solution in step c, stir, let stand, and protect from light for later use. f. Weigh an appropriate amount of resveratrol (Res) and add it to a methanol solution. Dissolve the resveratrol in methanol by ultrasonication until homogeneous. Protect the solution from light and set aside for later use. g. Immerse the PC scaffold obtained in step b into the polydopamine solution obtained in step c, and freeze-dry the resulting scaffold, which is denoted as PCP scaffold; add the PC scaffold obtained in step b to the resveratrol methanol solution obtained in step f, and freeze-dry the resulting scaffold, which is denoted as PCP-Res scaffold. The PC scaffold obtained in step b was added to the polydopamine solution containing nerve growth factor obtained in step e, and then freeze-dried before being immersed in the resveratrol methanol solution obtained in step f. The scaffold obtained by freeze-drying is denoted as PCP-Res-NGF scaffold.

3. The method for preparing chitosan-polydopamine modified 3D printing biomaterial loaded with resveratrol and nerve growth factor according to claim 2, characterized in that: In step a, a three-dimensional porous support model is designed using computer-aided design (CAD). The resulting PLA support is a cuboid with dimensions of 10×10×2mm. After the PLA support is printed, it is cleaned with an ultrasonic cleaner for 30~60 minutes at a temperature controlled at 37℃. After cleaning, it is dried and ready for use.

4. The method for preparing chitosan-polydopamine modified 3D printing biomaterial loaded with resveratrol and nerve growth factor according to claim 2, characterized in that: In step b, the relative concentration of chitosan to acetic acid aqueous solution is 1~2 wt / v; in the acetic acid aqueous solution, the relative concentration of acetic acid to deionized water is 1~2 v / v.

5. The method for preparing chitosan-polydopamine modified 3D printing biomaterial loaded with resveratrol and nerve growth factor according to claim 2, characterized in that: In step c, the concentration of dopamine hydrochloride in the tris(hydroxymethyl)aminomethane distilled water buffer is 10 mg / mL to 40 mg / mL; the amount of tris(hydroxymethyl)aminomethane distilled water buffer is 10 mmol to 20 mmol; the volume used is 50 to 100 mL; and the pH of the tris(hydroxymethyl)aminomethane distilled water buffer is 7.4 to 8.

5.

6. The method for preparing a chitosan-polydopamine modified 3D printing biomaterial loaded with resveratrol and nerve growth factor according to claim 2, characterized in that: In step d, the nerve growth factor solution is prepared by adding 1 mg of nerve growth factor powder to 1 mL of phosphate buffer solution to obtain a 1 mg / mL nerve growth factor solution, which is then sealed and stored at -20°C for later use.

7. The method for preparing chitosan-polydopamine modified 3D printing biomaterial loaded with resveratrol and nerve growth factor according to claim 2, characterized in that: In step e, the nerve growth factor solution is diluted to 0.25-0.5 mg / mL and then added to the polydopamine solution in step c. The nerve growth factor solution accounts for 1-2.5 wt / v of the total polydopamine and nerve growth factor solution.

8. The method for preparing chitosan-polydopamine modified 3D printing biomaterial loaded with resveratrol and nerve growth factor according to claim 2, characterized in that: In step f, a 2 mg / mL resveratrol methanol solution is prepared.

9. A method for preparing a chitosan-polydopamine modified 3D printing biomaterial loaded with resveratrol and nerve growth factor according to any one of claims 2-8, characterized in that: The freeze-drying process involves placing the container in the dark for 12-24 hours, freezing it at -80°C for 24-48 hours, then freeze-drying it at -50°C for 12-24 hours using a freeze dryer. After drying, the container is removed and sealed for storage at -20°C.

10. The application of the chitosan-polydopamine modified 3D-printed biomaterial loaded with resveratrol and nerve growth factor as described in claim 1 as a medical scaffold material for treating spinal cord injury.

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