Method for preparing a micro bionic intervertebral disc scaffold by using near-field direct writing 3D printing technology

Abstract

The present invention discloses a method for preparing a micro bionic intervertebral disc scaffold by using near-field direct writing 3D printing technology. The present invention uses near-field direct writing 3D printing technology to print fiber ring laminated scaffolds with a reticular laminated intersecting structure of various specifications, arranges the fiber ring laminated scaffolds in the order of increasing pore size, heats the fiber ring laminated scaffolds, and manually curls the fiber ring laminated scaffolds at an angle of 45° obliquely to imitate the structure of the rat fiber ring, obtaining a simulated fiber ring laminated scaffold with a gradient change in pore size from inside to outside; methyl acrylated gelatin GelMA is perfused into the center of the fiber ring laminated scaffold as the nucleus pulposus to obtain a highly bionic intervertebral disc scaffold. The intervertebral disc scaffold prepared by the present invention has the characteristics of small size, ultra-high precision, high bionics, and good repair effect, and has been successfully used for micro spinal repair of small vertebrates, and is expected to be applied to intervertebral disc replacement and repair of vertebrates.

Description

Technical Field

[0001] The present invention relates to the field of bionic technology, and specifically to a method for preparing a micro bionic intervertebral disc scaffold by using near-field direct writing 3D printing, which is used for bionics of intervertebral disc scaffolds of small animals such as rats. Background Art

[0002] Intervertebral disc degeneration is a common lumbar disease with a high disability rate. With the development of electronic products and the change of people's work and lifestyle, more and more people have this disease due to incorrect sitting postures for a long time.

[0003] Currently in clinical practice, the treatment methods for intervertebral disc degeneration are very limited and can only relieve symptoms, unable to be fundamentally cured. Theoretically, intervertebral disc replacement is the most ideal method for treating intervertebral disc degeneration. With the development of intervertebral disc replacement technology and the continuous progress of material preparation technology, the construction of a whole tissue-engineered intervertebral disc (TE-IVD) better meets the treatment needs.

[0004] As the largest load-bearing organ in the human body, the intervertebral disc has a complex structure and mechanical environment. Among them, the macroscopic structure with a gradient change in mechanical properties in different regions inside and outside the intervertebral disc, and the microscopic structure with an intersecting and overlapping adjacent layer between the annulus fibrosus in the intervertebral disc are two very important structural characteristics of the intervertebral disc. In the precise construction of a whole tissue-engineered intervertebral disc, how to accurately bionically simulate the macroscopic and microscopic complex structures of the intervertebral disc has become the main problem.

[0005] In the research on intervertebral disc tissue engineering, some researchers have prepared annulus fibrosus-like laminated scaffolds with different shapes and internal pore structures by using traditional technologies such as freeze-drying, electrospinning, and gas foaming. The cell orientation growth can be regulated by controlling the macroscopic and microscopic structures of the materials, thereby promoting the expression of annulus fibrosus-related genes and realizing tissue repair. However, due to the non-uniformity of the intervertebral disc tissue, the structure and mechanical microenvironment are extremely complex, and traditional material preparation processes cannot achieve precise and complex forming, which greatly limits the construction of tissue-engineered intervertebral discs with a multi-level gradient distribution structure.

[0006] Some researchers prepared a TE-IVD scaffold using the reverse reconstruction method. The annulus fibrosus part of this scaffold contains a concentrically arranged layered structure, and there is a + / −30°-45° overlapping structure between adjacent layers. This scaffold is prepared from non-degradable materials such as bacterial cellulose and lacks necessary biological activity. Moreover, this scaffold only bionics the microscopic overlapping structure between adjacent layers of the annulus fibrosus, and does not effectively bionics the gradually changing structure with gradient changes in the inner, middle, and outer regions of the entire intervertebral disc. In addition, the mechanical properties of this bionic scaffold are insufficient, and the ideal mechanical support effect is not achieved after implantation into the rat caudal vertebra. And during the operation, it is often necessary to nail the upper and lower vertebrae of the replaced area into the external scaffold for fixation, which not only increases the surgical difficulty and causes a large amount of additional trauma, but also there is a possibility of deformation and slippage after the annulus fibrosus layered scaffold is implanted.

[0007] Since the currently prepared tissue-engineered intervertebral discs cannot effectively simulate the complex structure of the in-situ intervertebral discs, the effect of the prepared intervertebral disc scaffolds in animal in-vivo experiments is not ideal. In addition, since the animal models selected for in-vivo experiments are often animals such as rats or white rabbits, the intervertebral disc sizes of these animals are extremely small, often less than one-tenth of the normal human intervertebral disc. For example, the size of the rat caudal vertebra intervertebral disc is: Φ3mm×0.5mm, height h = 1.5mm, which further increases the preparation difficulty of the tissue-engineered intervertebral disc scaffold and also makes it difficult to implement the in-vivo verification effect of the tissue-engineered intervertebral disc. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for preparing a micro bionic intervertebral disc scaffold using near-field direct writing 3D printing, which can highly bionic the macroscopic and microscopic complex structures of the intervertebral disc and further reduce the structure to a size suitable for in-vivo experiments on rats, providing new ideas for the development of tissue-engineered intervertebral discs.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] A method for preparing a micro bionic intervertebral disc scaffold using near-field direct writing 3D printing, comprising the following steps:

[0011] Step 1. Modeling and bionic optimization: Taking the size of the annulus fibrosus of the rat caudal vertebra as a reference, use 3D software to digitally model the intervertebral disc scaffold to obtain a 3D model of the intervertebral disc scaffold. Then import the 3D model into the COMSOL Multiphysics simulation software to perform finite element analysis on the structure of the scaffold and the deformation amount under pressure. Set the material, the constrained object, and the boundary load of the 3D model of the intervertebral disc scaffold. Cut the annulus fibrosus in the 3D model of the intervertebral disc scaffold into several laminated scaffolds, simulate the rat's upright, twisting, and forward flexion movements, detect the longitudinal pressure and transverse pressure parameters borne by each laminated scaffold of the annulus fibrosus, and optimize and improve the 3D model of the intervertebral disc scaffold according to the obtained finite element analysis results to determine the appropriate number, pore size, and fiber diameter of the laminated scaffolds of the annulus fibrosus;

[0012] Step 2. Print the laminated scaffolds of the annulus fibrosus using 3D printing technology:

[0013] Mix polycaprolactone PLC with a mass ratio of 3:4.5 - 5 and glacial acetic acid evenly, and ultrasonicate for 2 - 3 hours until the polycaprolactone PLC is fully dissolved to obtain printing ink; add the printing ink to a near-field direct writing 3D printing device, set the printing parameters according to the optimized model of the laminated scaffolds of the annulus fibrosus in Step 1, and print out several laminated scaffolds of the annulus fibrosus with a reticular laminated intersecting structure with a pore size of 50 - 500 μm and a fiber diameter of 20 - 50 μm;

[0014] Step 3. Prepare the laminated scaffolds of the annulus fibrosus

[0015] Arrange the several laminated scaffolds of the annulus fibrosus with different pore sizes printed in Step 2 in ascending order of pore size, heat the laminated scaffolds of the annulus fibrosus to a temperature of 50 - 60 °C, and manually curl the laminated scaffolds of the annulus fibrosus at an angle of 45° to imitate the structure of the rat annulus fibrosus to obtain a simulated laminated scaffold of the annulus fibrosus with a gradient change in pore size from the inside to the outside;

[0016] Step 4. Assembly of the whole tissue engineering intervertebral disc

[0017] Then disinfect the laminated scaffolds of the annulus fibrosus prepared in Step 3, perfuse sterile methacrylated gelatin GelMA into the center of the laminated scaffolds of the annulus fibrosus as the nucleus pulposus, and irradiate it with ultraviolet light to make it photocured and crosslinked into a shape, and assemble it into a complete bio - simulated whole tissue engineering intervertebral disc scaffold. The size of the bionic intervertebral disc scaffold is adapted to the size of the intervertebral disc to be transplanted in the rat caudal vertebra and implant it into the rat caudal vertebra.

[0018] Preferably, the molecular weight of the polycaprolactone PLC in Step 2 is 8×10 4 -8.5×10 4 , and the mass percentage concentration of glacial acetic acid ≥ 99.8%.

[0019] Preferably, the pore sizes of the annular fiber stacked scaffold printed in step 2 are 50 μm, 100 μm, 300 μm or 500 μm respectively.

[0020] Preferably, the size of the annulus fibrosus stacked scaffold prepared in step 3 is compatible with the size of the intervertebral disc to be transplanted in the rat coccygeal vertebra, and the pore sizes of the side walls of the annulus fibrosus stacked scaffold are 50×50 μm from inside to outside. 2 , 100×100μm 2 、300×300μm 2 , 500×500μm 2 .

[0021] The present invention uses near-field direct writing 3D printing technology to print out a variety of specifications of mesh-like stacked intersecting structures of annulus fiber stacking scaffolds, and arranges the annulus fiber stacking scaffolds in order of pore size from small to large. After heating the annulus fiber stacking scaffold, the annulus fiber stacking scaffold is manually curled at an angle of 45° to imitate the structure of the rat annulus fiber, and a simulated annulus fiber stacking scaffold with a gradient pore size change from the inside to the outside is obtained; methacrylic anhydride gelatin GelMA is infused into the center of the annulus fiber stacking scaffold as the nucleus pulposus to obtain a highly bionic intervertebral disc scaffold. Compared with the prior art, the present invention replicates the stacked and staggered structure of the original annulus fiber and the gradient distribution of the inner, middle and outer regions, and also reduces the size of the scaffold to a barrel type with an outer diameter of 3mm, which is completely matched with the micro-vertebral structure and size of small animals; the constructed gradient aperture precision structure can gradiently regulate cell behavior, guide cell directional growth, secrete extracellular matrix, and finally form a disc-like tissue with a gradient distribution of cell types to play its physiological role; it avoids the trauma caused by the need for additional mechanical scaffold fixation in traditional intervertebral disc replacement surgery,

[0022] The scaffold prepared by the present invention has good mechanical properties. The pressure it withstands is ≥3Mp after testing. It provides necessary mechanical support to the replaced area at the initial stage of implantation and after implantation, ensuring the long-term stable recovery environment required for the intervertebral disc regeneration process.

[0023] The present invention uses polycaprolactone PLC and glacial acetic acid as printing inks, and injects sterile methacrylic anhydride gelatin GelMA into the center of the stent as the nucleus pulposus. The prepared intervertebral disc stent can be slowly absorbed in the animal body, and the animal itself eventually generates a new intervertebral disc.

[0024] COMSOL Multiphysics is a large-scale advanced numerical simulation software. Based on the finite element method, it realizes the simulation of real physical phenomena by solving partial differential equations (single-field) or systems of partial differential equations (multi-field), and is called "the first truly arbitrary multi-physics direct coupling analysis software" by scientists around the world today. Solving real-world physical phenomena by mathematical methods, COMSOL Multiphysics achieves highly accurate numerical simulations with high computational performance and outstanding multi-field two-way direct coupling analysis capabilities. It has been widely used in the fields of acoustics, bioscience, chemical reactions, dispersion, electromagnetics, fluid dynamics, fuel cells, earth science, heat conduction, microsystems, microwave engineering, optics, photonics, porous media, quantum mechanics, radio frequency, semiconductors, structural mechanics, transport phenomena, wave propagation, etc.

[0025] The near-field direct writing 3D printing technology is widely used in scientific experimental fields such as biomedicine, animals, pharmaceuticals, chemistry, and physics. It also has injection and withdrawal functions, can control the ink flow rate, and can control the injection flow through a program. Brief Description of the Drawings

[0026] Figure 1 It is a schematic diagram of the structure of the annulus fibrosus of the rat caudal vertebra;

[0027] Figure 2 It is a schematic diagram of the structure of the intervertebral disc scaffold prepared by the present invention;

[0028] Figure 3 is Figure 2 The microscopic structure diagram of the intervertebral disc scaffold in

[0029] Figure 4 is Figure 2 The schematic diagram of the microscopic structure of the interlaced annulus fibrosus tissue layer in

[0030] Figure 5 is Figure 2 The top view of the microscopic structure of the intervertebral disc scaffold in

[0031] Figure 6 It is the growth of cells on the intervertebral disc scaffold after the present invention is implanted into the rat body;

[0032] Figure 7 It is a photo of the rat caudal vertebra intervertebral disc replacement test;

[0033] Figure 8 It is a photo of the MRI image observation in the rat in vivo experiment. Detailed Description of the Invention

[0034] Taking the bionic intervertebral disc scaffold with an outer diameter of 3 mm, an inner diameter of 2.5 mm, and a height h = 1.5 mm as an example, the present invention will be further described in detail below with reference to the accompanying drawings.

[0035] A method for preparing a high-precision micro bionic intervertebral disc scaffold using 3D printing technology, comprising the following steps:

[0036] Step 1. Modeling and bionic optimization:

[0037] Taking the size of the annulus fibrosus of the rat caudal vertebra as a reference, digitally modeling the intervertebral disc scaffold using 3D software to obtain a 3D model of the intervertebral disc scaffold, and then importing the 3D model into COMSOL Multiphysics simulation software to perform finite element analysis on the structure of the scaffold and the deformation amount under pressure. Set the material, constraint object, and boundary load of the 3D model of the intervertebral disc scaffold. Cut the annulus fibrosus in the 3D model of the intervertebral disc scaffold into several laminated scaffolds, simulate the rat's upright, rotation, and forward flexion movements, detect the longitudinal pressure and transverse pressure parameters borne by each laminated scaffold of the annulus fibrosus, and optimize and improve the 3D model of the intervertebral disc scaffold according to the obtained finite element analysis results to determine the appropriate number, pore size, and fiber diameter of the laminated scaffolds of the annulus fibrosus;

[0038] Step 2. Printing laminated scaffolds of the annulus fibrosus with multiple specifications using 3D printing technology:

[0039] (1) Preparation of a laminated scaffold of the annulus fibrosus with a pore size of 100×100μm 2 , and a fiber diameter of 30μm:

[0040] Mix polycaprolactone PCL particles (Sigma-Aldrich, 440744) and glacial acetic acid (MACKLIN, A801295) with a mass ratio of 3:4.5 evenly, and perform ultrasonic treatment (50°C, power ratio 90%) for 120 min until the polycaprolactone PCL particles are fully dissolved to obtain printing ink; load the prepared printing ink into a special ink cartridge and let it stand at 30°C for 24 hours;

[0041] Load the special ink cartridge into an EHDP 3D printer, set the printing path through Emsemble software, set the long-axis movement distance to 40 mm, the short-axis movement distance to 0.2 mm, the acceleration to 500, the single printing cycle to 100, and repeat the cycle 10 times; set the printer load voltage to 2.5 kV, the extrusion rate to 2 μl, and the printing head height to 250 μm, start the printing program and perform printing, and keep the indoor temperature at 24°C and the humidity at 60%;

[0042] After printing, turn off the power and let it stand for 10 min until the printed scaffold is completely dry to obtain a pore size of 100×100μm 2, an annulus fibrosus laminated scaffold with a reticular laminated intersecting structure having a fiber diameter of 30 μm and 6 layers; after testing, the tensile strength of the annulus fibrosus laminated scaffold is 2.5 MPa and the elongation at break is 568.5%;

[0043] (2) The pore size is 50×50 μm 2 , Preparation of an annulus fibrosus laminated scaffold with a fiber diameter of 20 μm:

[0044] Mix polycaprolactone PCL particles (Sigma-Aldrich, 440744) and glacial acetic acid (MACKLIN, A801295) with a mass ratio of 3:5 evenly, and perform ultrasonic treatment (60 °C, power ratio 90%) for 180 min until the polycaprolactone PCL particles are fully dissolved to obtain printing ink; Load the prepared printing ink into a special ink cartridge and let it stand at 30 °C for 24 hours;

[0045] Load the special ink into an EHDP 3D printer, set the printing path through Emsemble software, set the long-axis moving distance to 40 mm, the short-axis moving distance to 0.1 mm, the acceleration to 500, the single printing cycle to 200, and repeat the cycle 10 times; Set the printer load voltage to 3 kV, the extrusion rate to 2 μl, and the print head height to 250 μm, start the printing program and perform printing, keeping the indoor temperature at 24 °C and the humidity at 60%;

[0046] After printing, turn off the power and let it stand for 10 min until the printed scaffold is completely dry to obtain a pore size of 50×50 μm 2 , an annulus fibrosus laminated scaffold with a reticular laminated intersecting structure having a fiber diameter of 20 μm and 6 layers; after testing, the tensile strength of the annulus fibrosus laminated scaffold is 3.5 MPa and the elongation at break is 325.5%;

[0047] (3) The pore size is 300×300 μm 2 , Preparation of an annulus fibrosus laminated scaffold with a fiber diameter of 40 μm:

[0048] Mix polycaprolactone PCL particles (Sigma-Aldrich, 440744) and glacial acetic acid (MACKLIN, A801295) with a mass ratio of 3:4.7 evenly, perform ultrasonic treatment (60 °C, power ratio 90%) for 170 min until the polycaprolactone PCL particles are fully dissolved to obtain printing ink, load the prepared printing ink into a special ink cartridge and let it stand at 30 °C for 24 hours;

[0049] Load a dedicated ink cartridge into the EHDP 3D printer. Set the printing path through the Emsemble software. Set the long-axis movement distance to 40 mm, the short-axis movement distance to 0.5 mm, the acceleration to 100, the single printing cycle to 40, and repeat the cycle 10 times. Set the printer load voltage to 2.5 kV, the extrusion rate to 2 μl, and the print head height to 250 μm. Start the printing program and perform printing, keeping the indoor temperature at 24 °C and the humidity at 60%.

[0050] After printing, turn off the power and let it stand for 10 min until the printed scaffold is completely dry, obtaining a fiber annulus laminated scaffold with a pore size of 300×300 μm 2 、a fiber diameter of 40 μm, and a six-layer reticular laminated and intersecting structure; After testing, the tensile strength of this fiber annulus laminated scaffold is 1.7 MPa, and the elongation at break is 215%;

[0051] (4)Preparation of a fiber annulus laminated scaffold with a pore size of 500×500 μm 2 and a fiber diameter of 50 μm:

[0052] Mix polycaprolactone PCL particles (Sigma-Aldrich, 440744) and glacial acetic acid (MACKLIN, A801295) with a mass ratio of 3:4.8 evenly, and perform ultrasonic treatment (60 °C, power ratio 90%) for 150 min until the polycaprolactone PCL particles are fully dissolved to obtain printing ink. Load the prepared printing ink into a dedicated ink cartridge and let it stand at 30 °C for 24 hours;

[0053] Load a dedicated ink cartridge into the EHDP 3D printer. Set the printing path through the Emsemble software. Set the long-axis movement distance to 40 mm, the short-axis movement distance to 0.5 mm, the acceleration to 100, the single printing cycle to 40, and repeat the cycle 10 times. Set the printer load voltage to 2.5 kV, the extrusion rate to 2 μl, and the print head height to 250 μm. Start the printing program and perform printing, keeping the indoor temperature at 24 °C and the humidity at 60%.

[0054] After printing, turn off the power and let it stand for 10 min until the printed scaffold is completely dry, obtaining a fiber annulus laminated scaffold with a pore size of 500×500 μm 2 、a fiber diameter of 50 μm, and a six-layer reticular laminated and intersecting structure; After testing, the tensile strength of this fiber annulus laminated scaffold is 0.6 MPa, and the elongation at break is 168%;

[0055] Step 3: Prepare the fiber annulus laminated scaffold

[0056] Arrange the fiber ring laminated scaffolds printed in Step 2 in ascending order of pore size, heat the fiber ring laminated scaffolds for 1 - 3 min until the temperature reaches 50 - 60 °C, and manually curl the fiber ring laminated scaffolds at an angle of 45° to imitate the structure of the rat annulus fibrosus to obtain a simulated fiber ring laminated scaffold with a gradient change in pore size from inside to outside;

[0057] Step 4. Assembly of the whole tissue-engineered intervertebral disc

[0058] Then disinfect the fiber ring laminated scaffolds prepared in Step 3, pour sterile methacrylated gelatin GelMA into the center of the fiber ring laminated scaffolds as the nucleus pulposus, and irradiate it with ultraviolet light to make it photocured and crosslinked into a complete bio-simulated whole tissue-engineered intervertebral disc scaffold. The size of the bionic intervertebral disc scaffold is 3 mm in outer diameter, 2.5 mm in inner diameter, and the height h = 1.5 mm, and implant it into the rat caudal vertebra.

[0059] For the intervertebral disc scaffold prepared according to the above method, observe its microstructure by scanning electron microscope SEM. The pore size of the bionic intervertebral disc scaffold is about 300×300 μm 2 , the fiber diameter is about 30 μm, and it has a microscopic structure of simulated fiber ring tissue laminated and staggered as shown in Figure 4 ; The top view of the microstructure of the prepared intervertebral disc scaffold is as shown in Figure 5 . The intervertebral disc scaffold is composed of fiber ring laminated scaffolds, and each fiber ring laminated scaffold is formed by the intersection of multiple layers of fibers.

[0060] The intervertebral disc scaffold prepared by the present invention highly imitates the complex microstructure of the annulus fibrosus in the rat intervertebral disc, and can prepare various specifications of intervertebral discs according to the size of the rat caudal vertebra intervertebral disc to be transplanted, providing an effective technical guarantee for further experiments.

[0061] The operator planted annulus fibrosus cells (AFCs) on the prepared intervertebral disc scaffold and observed the growth of cells on the intervertebral disc scaffold through a fluorescence microscope, as shown in Figure 6 . The results show that AFCs can grow and proliferate well on the scaffold, indicating that the scaffold has good biocompatibility.

[0062] Intervertebral disc replacement experiment in rats

[0063] In the experiment, the fiber ring laminated scaffolds prepared in this example were assembled with methacrylated gelatin GelMA as the nucleus pulposus to prepare a tissue-engineered intervertebral disc (TE-IVD) with a laminated and staggered structure and a single pore size, and a rat caudal vertebra intervertebral disc replacement experiment was carried out (as shown inFigure 7 ). Due to the good mechanical properties of the stent material, no external stent fixation is required during the experiment, and the implanted stent can remain in the original intervertebral disc position without slippage. Compared with the traditional intervertebral disc replacement surgery, this method simplifies the surgical procedure and reduces surgical trauma while ensuring the implantation effect.

[0064] The implantation effect was evaluated by MRI imaging after implantation, as Figure 8 shown. It can be concluded from the imaging that the implanted TE-IVD can not only provide certain mechanical support to the intervertebral disc area after implantation, but also has a more obvious nucleus pulposus signal compared with the control group. It is preliminarily judged that this tissue has a good implantation effect.

Claims

1. A method for preparing a micro bionic intervertebral disc scaffold by near-field direct writing 3D printing, characterized in that: It includes the following steps: Step 1, Modeling and bionic optimization: Taking the size of the annulus fibrosus of the rat caudal vertebra as a reference, using 3D software to digitally model the intervertebral disc scaffold to obtain a 3D model of the intervertebral disc scaffold, and then importing the 3D model into COMSOL Multiphysics simulation software to perform finite element analysis on the structure of the scaffold and the deformation amount under pressure. Set the material, constraint object and boundary load of the 3D model of the intervertebral disc scaffold, cut the annulus fibrosus in the 3D model of the intervertebral disc scaffold into several laminated scaffolds, simulate the upright, rotation and forward flexion movements of the rat, detect the longitudinal pressure and transverse pressure parameters borne by each laminated scaffold of the annulus fibrosus, and optimize and improve the 3D model of the intervertebral disc scaffold according to the obtained finite element analysis results to determine the appropriate number, pore size and fiber diameter of the laminated scaffolds of the annulus fibrosus; Step 2, Printing the laminated scaffolds of the annulus fibrosus using 3D printing technology: Mix polycaprolactone PLC and glacial acetic acid with a mass ratio of 3:4.5 - 5 evenly, and ultrasonicate for 2 - 3 hours until the polycaprolactone PLC is fully dissolved to obtain printing ink; Add the printing ink to the near-field direct writing 3D printing device, set the printing parameters according to the optimized model of the laminated scaffolds of the annulus fibrosus in Step 1, and print out several laminated scaffolds of the annulus fibrosus with a reticular laminated intersection structure with a pore size of 50 - 500 μm and a fiber diameter of 20 - 50 μm; Step 3, Preparing the laminated scaffolds of the annulus fibrosus Arrange the several laminated scaffolds of the annulus fibrosus with different pore sizes printed in Step 2 in the order from small to large pore size from the inside to the outside, heat the laminated scaffolds of the annulus fibrosus to a temperature of 50 - 60 °C, and manually curl the laminated scaffolds of the annulus fibrosus at an angle of 45° to imitate the structure of the rat annulus fibrosus to obtain a simulated laminated scaffold of the annulus fibrosus with a gradient change in pore size from the inside to the outside; Step 4, Assembly of the whole tissue engineering intervertebral disc Then disinfect the laminated scaffolds of the annulus fibrosus prepared in Step 3, perfuse sterile methacrylated gelatin GelMA into the center of the laminated scaffolds of the annulus fibrosus as the nucleus pulposus, and irradiate it with ultraviolet light to make it photocured and crosslinked into a shape, and assemble it into a complete biological simulation whole tissue engineering intervertebral disc scaffold. The size of the bionic intervertebral disc scaffold is adapted to the size of the intervertebral disc to be transplanted in the rat caudal vertebra, and it is implanted into the rat caudal vertebra.

2. The method for preparing a micro bionic intervertebral disc scaffold by near-field direct writing 3D printing according to claim 1, characterized in that: In the second step, the molecular weight of polycaprolactone (PLC) is 8×10 4 - 8.5×10 4 , and the mass percentage concentration of glacial acetic acid is ≥99.8%.

3. The method for preparing a micro bionic intervertebral disc scaffold by near-field direct writing 3D printing according to claim 1 or 2, characterized in that: The pore sizes of the laminated scaffolds of the annulus fibrosus printed in Step 2 are 50 μm, 100 μm, 300 μm or 500 μm respectively.

4. The method for preparing a micro bionic intervertebral disc scaffold by near-field direct writing 3D printing according to claim 3, characterized in that: The size of the annulus fibrosus laminated scaffold prepared in the third step is adapted to the size of the intervertebral disc to be transplanted in the rat caudal vertebra, and the side wall pore diameters of the annulus fibrosus laminated scaffold are 50×50μm, 2 100×100μm, 2 300×300μm, 2 500×500μm, 2 .

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