Nerve radiation field 3D printing-based spinal cord-like tissue as well as preparation method and application thereof

Abstract

The invention discloses a spinal cord-like tissue based on neural radiation field 3D printing as well as a preparation method and application of the spinal cord-like tissue. The preparation method of the spinal cord-like tissue comprises the following steps: S1, constructing a three-dimensional model of the spinal cord tissue by utilizing a nerve radiation field; s2, performing 3D printing of an entity based on the three-dimensional model in the S1 to obtain a microporous structure scaffold; and S3, implanting seed cells and bioactive factors into the microporous structure scaffold in the S2 to obtain the spinal cord-like tissue. According to the method, the spinal cord models of rats with different genders and weights are generated through NeRF, the spinal cord model database is constructed, the NeRF and the 3D printing technology are combined, a personalized spinal cord three-dimensional model can be generated according to the specific SCI condition of a patient, and the SCI treatment effect is improved.

Description

Technical Field

[0001] The present invention relates to the field of regenerative medicine and tissue engineering technology, and in particular to a spinal cord-like tissue based on 3D printing of a neural radiation field, and a preparation method and application thereof. Background Art

[0002] Spinal cord injury (SCI) is a highly disabling and destructive central nervous system injury that destroys the nerve contact between other parts of the body and the brain, leading to varying degrees of sensory and motor disorders, seriously hindering the patient's daily life. According to statistics, 250,000 to 500,000 people suffer from SCI each year worldwide, and the average treatment cost of patients with traumatic SCI exceeds US$500,000, which brings a huge economic burden to the patient's family and society. The massive death of neurons in the SCI site, the lack of neurotrophic factors, and the presence of inhibitory molecules, free radicals, excitotoxins and inflammatory mediators in the microenvironment lead to limited nerve regeneration at the injury site, making the repair of SCI one of the most challenging medical problems in the field of neuroscience. SCI destroys the structural and functional connection between the higher centers and the spinal cord, and is divided into primary injury and secondary injury, which generally manifests as limb sensory, motor and autonomic dysfunction below the injury plane. The key to repairing SCI is to achieve neural circuit remodeling through the regeneration and functional recovery of neurons and axons. At present, surgical treatment and drug therapy are the main clinical treatments for SCI, but they can only reduce secondary injuries and cannot solve the problem of poor nerve regeneration. To solve this problem, researchers have developed tissue engineering therapy, using functional nerve regeneration scaffolds to fill and repair the damaged area. The scaffold provides properties similar to the extracellular microenvironment, in which the filled biomaterials provide structural support for nerve regeneration, stem cells provide cellular support, and biological factors provide nutritional support, effectively promoting nerve regeneration at SCI. However, the conditions of SCI in different individuals are different, and it is difficult to perfectly bridge the damaged area and achieve the ideal treatment effect using a uniform tissue engineering scaffold.

[0003] With the continuous development of tissue engineering technology in the field of regenerative medicine, traditional methods for preparing 3D scaffolds, such as electrospinning, freeze-drying, and model making, cannot spatially adjust the porosity, mechanical anisotropy, and cell distribution of 3D structures, and cannot accurately simulate the complex three-dimensional structure of human tissues / organs, which can easily lead to poor prognosis of functional recovery in patients, affect the biosafety of implants, and cause physiological pain and psychological trauma to patients. Compared with traditional 3D scaffold construction methods, 3D printing technology has its unique advantages. It is a technology that uses digital models to print various materials layer by layer to construct objects with precise and complex structures. In addition, 3D printing can achieve customized design and on-demand manufacturing, with stable high repeatability, and is suitable for patients with different degrees of spinal cord defects. 3D printing technology provides a platform for rapid and accurate construction of scaffolds for tissue engineering repair of the spinal cord, simulating spinal cord tissue in terms of biological functions, structural construction, and mechanical properties, thereby providing new treatment strategies for the repair of SCI.

[0004] However, current 3D printing technology is still based on traditional three-dimensional modeling solutions, which cannot meet the accuracy requirements of tissue engineering scaffolds and has the following limitations: (1) Depth estimation based on monocular images often loses scale information and has large errors; (2) Existing pose solution methods are usually relative pose estimation, which has the problem of error accumulation; (3) The fusion, splicing and drawing methods of three-dimensional point clouds are computationally complex and require a lot of time.

[0005] Therefore, there is an urgent need to provide a method for constructing spinal cord-like tissue with high precision and personalized customization to provide new ideas and therapeutic effects for SCI treatment. Summary of the invention

[0006] In view of the defects in the prior art, the present invention proposes a spinal cord-like tissue based on neural radiation field 3D printing and its preparation method and application. The present invention focuses on the personalization of tissue engineering scaffolds in the SCI repair process, combines neural radiation field (NeRF) and 3D printing technology, constructs a customized methacrylated gelatin (GelMA) hydrogel scaffold and loads neural stem cells (NSCs) and neurotrophic factor 3 (NT-3) to form a spinal cord-like tissue with a spinal cord-like structure. NeRF is a new viewpoint generation method based on microscopic rendering and three-dimensional representation of neural fields, which can generate a three-dimensional model of the spinal cord according to the magnetic resonance imaging of the spinal cord. After data processing, personalized tissue engineering scaffold modeling can be completed, and then the model data can be imported into a 3D printer to construct a customized spinal cord-like tissue, realizing the precision and customization of spinal cord tissue engineering therapy.

[0007] The present invention provides a method for preparing spinal cord-like tissue based on 3D printing of neural radiation field, comprising the following steps:

[0008] S1: Construction of a three-dimensional model of spinal cord tissue using neural radiation fields;

[0009] S2: Performing 3D printing of a solid body based on the three-dimensional model described in S1 to obtain a microporous structure scaffold;

[0010] S3: Implanting seed cells and bioactive factors into the microporous structure scaffold described in S2 to obtain the spinal cord-like tissue.

[0011] In some embodiments, S1 specifically comprises the following steps:

[0012] S1-1: Acquire a two-dimensional image of the spinal cord tissue collected at each preset posture and posture information of the two-dimensional image to obtain a data set;

[0013] S1-2: Input the data set described in S1-1 into the deep neural network to complete the neural radiation field model training;

[0014] S1-3: Using the neural radiation field model obtained through training in S1-2, a three-dimensional model corresponding to the spinal cord tissue is generated through volume rendering technology.

[0015] In some embodiments, S1 further comprises the following steps:

[0016] S1-4: Observe the cross-sectional shape and diameter of the spinal cord tissue, and verify and optimize the accuracy and authenticity of the three-dimensional model described in S1-3.

[0017] In some embodiments, the porosity of the microporous structure scaffold described in S2 is 30-60%, preferably 36-54%, and more preferably 49.93%. High porosity helps cell migration, proliferation, and nutrient exchange, while accelerating drug release and promoting tissue regeneration, but too high porosity may reduce mechanical strength and scaffold stability. Low porosity can enhance mechanical properties, but too low porosity may limit cell growth and tissue repair.

[0018] In some embodiments, the seed cells described in S3 are any one of neural stem cells, umbilical cord mesenchymal stem cells and bone marrow mesenchymal stem cells.

[0019] In some embodiments, the bio-ink used in the 3D printing described in S2 is methacrylated gelatin;

[0020] The seed cells described in S3 are neural stem cells, and the bioactive factor described is neurotrophic factor 3.

[0021] The combination of NSCs, NT-3 and GelMA can achieve significant nerve regeneration effects. GelMA provides a highly biocompatible scaffold to promote the attachment and proliferation of NSCs, while NT-3 enhances the differentiation and survival of neurons. Under this synergistic effect, compared with traditional technologies, this combination not only improves nerve growth and functional recovery, but also achieves sustained release of NT-3 by regulating the degree of cross-linking of GelMA, further promoting the repair effect. This makes it show good application prospects in the treatment of nerve injuries.

[0022] In some embodiments, the concentration of the methacrylated gelatin is 2-8% (m / v), preferably 2.5-7.5% (m / v), and more preferably 5% (w / v); the grafting rate of the methacrylated gelatin is 30-60%, preferably 30%. The grafting rate of GelMA is crucial to the performance of the scaffold and cell behavior. Too high a grafting rate will enhance the mechanical properties, but may cause the scaffold to be too rigid, reduce biocompatibility and porosity, and thus affect cell growth; while too low a grafting rate may cause the scaffold to be soft, insufficiently supported, and degrade faster, and it will not be able to effectively retain cells and nutrients.

[0023] In some embodiments, the concentration of the neurotrophic factor 3 is 10-100 ng / mL, preferably 10 ng / mL.

[0024] In some embodiments, the parameters of 3D printing include:

[0025] The concentration of the hydrogel is 2.5-7.5% (w / v); the ratio of the photoinitiator is 0.10-1% (w / v); the UV curing time is 10-60s; the pre-cooling temperature is 4--20°C; the pre-cooling time is 5-10min; the printing speed is 600-2000mm / min; and the printing wire diameter is 0.2-0.8mm.

[0026] The present invention also provides spinal cord-like tissue obtained by the preparation method.

[0027] The present invention also provides the use of the spinal cord-like tissue in preparing a tissue engineering scaffold for repairing spinal cord injury.

[0028] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0029] 1. This application uses the NeRF strategy to reconstruct the three-dimensional model of the spinal cord, which can capture the complex structure of the spinal cord injury site in a more detailed manner, making it less likely to lose details or accumulate errors. It overcomes the limitation that traditional three-dimensional spinal cord models need to be constructed through X-rays or long-term CT scans, MRI and other images, and improves the high precision and continuity in the process of 3D printing spinal cord tissue engineering scaffolds.

[0030] 2. This application uses NeRF to generate rat spinal cord models of different genders and weights, constructs a spinal cord model database, and combines NeRF and 3D printing technology to generate personalized spinal cord three-dimensional models based on the patient's specific SCI condition, thereby improving the treatment effect of SCI.

[0031] 3. The present application uses methacryloyl gelatin as the biological ink, which has high compatibility with neural stem cells. Both two-dimensional and three-dimensional cultured spinal cord-like tissues can inhibit scar growth and promote nerve regeneration, and the three-dimensional cultured spinal cord-like tissue has good in vitro effectiveness. By controlling the position and interaction of the biomaterial methacryloyl gelatin, neural stem cells and the bioactive factor neurotrophic factor 3 in the spinal cord-like engineering scaffold, the physiological structure and biological function of the spinal cord can be better simulated, thereby achieving spinal cord regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0033] Figure 1 This is a flow chart of the method for 3D printing spinal cord tissue engineering scaffolds based on nerve radiation fields of the present invention;

[0034] Figure 2 The NeRF architecture and rat spinal cord model database flow chart are constructed for the present invention;

[0035] Figure 3 Schematic diagram of constructing NeRF architecture and printing spinal cord-like tissue in the present invention; Figure A is a multi-angle two-dimensional picture of the rat spinal cord; Figure B is a spinal cord model generated by NeRF; Figure C is a spinal cord-like tissue model for filling the defect; Figure D is the state of the hydrogel under different printing parameters; Figure E is the printed spinal cord-like tissue;

[0036] Figure 4 This is a flow chart of 3D printing of spinal cord tissue in the present invention;

[0037] Figure 5 The extraction and identification results of neural stem cells of the present invention; Figure A is the result of observing NSCs under a microscope, scale: 1000 μm (left), 200 μm (right); Figure B is the result of identifying NSCs by flow cytometry; Figure C is the result of identifying NSCs by immunofluorescence staining;

[0038] Figure 6 The cell compatibility and factor release curve results of the three GelMAs of the present invention;

[0039] Figure 7 Figure 1 is the material characterization of GelMA-30 of the present invention; Figure A is the infrared spectrum of GelMA; Figure B is the porosity and swelling rate of GelMA-30; Figure C is the degradation curve of GelMA-30;

[0040] Figure 8 This is a flow chart of the in vitro experiment of the present invention to explore the safety and effectiveness of spinal cord-like tissue;

[0041] Fig. 9 The results of in vitro effectiveness of two-dimensional culture and three-dimensional culture of spinal cord tissue; Figure A shows the protein expression in two-dimensional culture; Figure B shows the gene transcription in two-dimensional culture; Figure C shows the protein expression in three-dimensional culture; Figure D shows the gene transcription in three-dimensional culture;

[0042] Fig.10 This is a flow chart of the in vivo experiment of the present invention to explore the safety and effectiveness of spinal cord-like tissue;

[0043] Fig.11 The results of the effects of spinal cord-like tissues prepared with three GelMA materials on cell proliferation and apoptosis; Figure A shows cell proliferation; Figure B shows cell apoptosis; Figure C shows cell cycle;

[0044] Fig.12 This is a graph showing the results of the in vivo experiment to explore the safety and effectiveness of the spinal cord-like tissue of the present invention; Figure A is the BBB score of SCI rats; Figure B is the gait analysis of rats; and Figure C is the overall view of the spinal cord recovery of rats. DETAILED DESCRIPTION

[0045] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0046] Based on the individualization of SCI tissue engineering scaffolds, this invention combines NeRF and 3D printing technology to construct customized "spinal cord-like tissues", providing new ideas for SCI treatment. The specific methods are as follows: to screen 3D printing materials suitable for loading neural stem cells (NSCs) and perform characterization tests; to build and optimize the NeRF architecture, and to perform 3D reconstruction of the two-dimensional image of the SD rat spinal cord; to establish spinal cord-like tissues loaded with NSCs and neurotrophic factor 3 through 3D printing; and then to test the safety and effectiveness of spinal cord-like tissues in vitro and in vivo. The technical route is as follows Figure 1 shown.

[0047] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified.

[0048] Example 1 Construction of NeRF architecture and rat spinal cord model database

[0049] The NeRF architecture can learn and generate a continuous, panoramic 3D scene model from a series of 2D images, capable of handling transparency and complex lighting conditions, and can be represented with arbitrary accuracy and level of detail.

[0050] Building the NeRF architecture mainly includes the following steps:

[0051] 1) Data collection: NeRF uses a series of 2D pictures taken from different angles and positions as input. 2) Neural network training: NeRF uses a deep neural network to represent the 3D radiance field. This network receives a 3D coordinate and a direction as input, and outputs the color and volume density at that coordinate. This network is trained by minimizing the error between the color predicted by the model and the color of the actual image. 3) Volume rendering: After training the neural network, NeRF will generate a 3D image from the neural radiance field through volume rendering technology, specifically by calculating the weighted sum of the color and volume density along the direction of each pixel ray to generate the final 3D image.

[0052] Reference Figure 2 The spinal cord tissue of SD rats was collected according to the process. On this basis, the plane graphics of the spinal cord tissue at various angles were captured by a high-resolution camera, loaded into the constructed NeRF architecture, and trained to generate a three-dimensional model of the spinal cord structure. The reliability of the model generated by the NeRF architecture was verified by observing the different cross-sectional shapes and diameters of the spinal cord tissue. Finally, spinal cord models of rats of different genders and weights were generated through NeRF, and a spinal cord model database was constructed.

[0053] The multi-angle two-dimensional images of the rat spinal cord obtained in this embodiment are as follows: Figure 3 As shown in A, SD rats of different genders and ages were selected to obtain spinal cords and spinal cord images were taken from multiple angles. The shooting angles include at least the front, side, and top. The more different angles, the higher the clarity of the photos, and the better the subsequent modeling effect. The generated spinal cord model is as follows Figure 3 As shown in B, a 3D model of the rat spinal cord was reconstructed using NeRF based on multi-angle 2D photos of the rat spinal cord. Figure 3 As shown in C, the model data generated by NeRF is processed to establish a spinal cord tissue model that fills the defect site, which can be completed with the help of Blender software.

[0054] Example 2 3D printing to construct spinal cord-like tissue

[0055] The flowchart of 3D printing to construct spinal cord-like tissue is as follows Figure 4 The printer used in this embodiment is an extrusion-type biological 3D printer (model: BP6601), and methacrylated gelatin (GelMA) is used as printing ink.

[0056] 3D printing parameter optimization includes: hydrogel concentration, photoinitiator ratio, UV curing time, pre-cooling temperature and time, etc.; printer settings consider printing speed, printing filament diameter, etc.

[0057] In this embodiment, the concentration of the hydrogel is 5% (w / v), the photoinitiator ratio is 0.25% (w / v), the UV curing time is 15s, the pre-cooling temperature is -20°C, the pre-cooling time is 7min, the printing speed is 1000mm / min, and the printing wire diameter is 0.3mm.

[0058] The specific printing steps are as follows: (1) Material preparation: Weigh a certain amount of GelMA, add the photoinitiator diluted with PBS, and heat in a 60°C water bath to dissolve. Filter and sterilize, cool to about 37°C, add cells and factors, and precool in a refrigerator.

[0059] (2) Printing settings: Turn on the water circulation, set the printing platform temperature to 4°C, and the printing nozzle temperature to 20°C. Import the 3D model, set the 3D printer parameters, print filament diameter 0.3mm, print speed 1000mm / min, layer height 0.3mm, etc., and export the G-code file.

[0060] (3) Printing process: Load the pre-cooled bio-ink into the printer, print according to the G-code, and monitor the process.

[0061] (4) Curing step: Immediately after printing, irradiate with UV light for 15 seconds for curing.

[0062] (5) Post-treatment: The scaffolds were washed three times with PBS or saline and then cultured in complete culture medium.

[0063] The state of the hydrogel under different printing parameters is shown in Figure 2. Figure 3 As shown in D, when the air pump pressure is appropriate, the extruded hydrogel is uniform filaments and can be used to print scaffolds; when the air pump pressure is inappropriate, the extruded hydrogel is hanging or spindle-shaped, and the air pressure needs to be adjusted. Figure 3 As shown in E, it is approximately cylindrical and can maintain its three-dimensional structure without deformation after solidification.

[0064] Characterization tests of spinal cord-like tissue include: cell compatibility, scanning electron microscopy, factor release curve, infrared spectrum, porosity, swelling rate, and in vitro degradation curve.

[0065] (1) Cytocompatibility: The spinal cord-like tissue containing NSCs was cultured for a certain period of time and then washed with PBS. Finally, the cell survival rate was evaluated by the live-dead staining method, and the cell viability was detected by the CCK8 method to evaluate the cytocompatibility of the spinal cord-like tissue.

[0066] As shown in 6A and D, the live-dead staining results showed that in the three GelMA materials, most NSCs could survive, indicating that GelMA materials were non-toxic to NSCs cells. As shown in 6B and C, among the three GelMA materials, the relative activity of NSCs in the GelMA-30 and GelMA-PR-002 groups was higher than that in GelMA-60, and had better cell compatibility. Among several different concentrations of GelMA-30, the 5% GelMA-30 group had the highest relative activity of NSCs.

[0067] (2) Scanning electron microscopy (SEM): First, prepare the GelMA sample and perform appropriate metal coating treatment to improve conductivity. Then, place the sample in a scanning electron microscope, set the appropriate acceleration voltage and magnification, observe the surface morphology and microstructure of the material, and record the image for analysis.

[0068] like Figure 6 As shown in Figure E, several GelMA materials have a large number of pores on their surfaces, with pore sizes ranging from tens to two hundred microns, forming an open three-dimensional network. This structure not only gives the samples good porosity and biocompatibility, but also improves the adhesion ability of cells. In addition, the sample surface is relatively smooth and has moderate roughness, and the overall morphology is consistent, indicating good repeatability and stability during the preparation process.

[0069] (3) Factor release curve: In the in vitro experiment, spinal cord tissue samples loaded with NT-3 were immersed in PBS, samples were taken at different time points and the factor concentration in the release solution was measured, the factor release curve was drawn, and the controlled release characteristics were evaluated.

[0070] As shown in 6F and G, the three GelMAs all reached the maximum NT-3 release rate at the 6th hour; all of them could achieve sustained release of NT-3 for up to one week.

[0071] (4) Infrared spectroscopy: The infrared spectroscopy detection steps of GelMA include sample preparation, powder grinding, mixing the sample with KBr and pressing it into a pellet, and scanning on a FTIR instrument to obtain spectral data.

[0072] As shown in 7A, the test results show characteristic absorption peaks, NH stretching vibration at 3300-3500 cm-1, indicating the presence of amino functional groups; C=O stretching vibration at 1650 cm-1, reflecting amide groups; CH bending vibration near 1400 cm-1, reflecting polymer chain structure; and COC bond stretching vibration in the 1000-1200 cm-1 region. This result verifies the chemical structure of GelMA and its functional groups.

[0073] (5) Porosity: The spinal cord tissue sample was dried to a constant weight and then weighed. The pore volume was then determined using the liquid exclusion method, and the porosity was calculated to evaluate the internal pore structure of the material.

[0074] The porosity results are as follows Figure 7 As shown in Figure B, the porosity of GelMA with a concentration of 2.5-7.5% is in the range of 37.60-52.07%. As the concentration of GelMA increases, the porosity decreases. A higher concentration means that more polymer chains are cross-linked with each other, forming a denser network structure, which reduces the number and volume of pores.

[0075] (6) Swelling rate: The GelMA sample was immersed in PBS and a specific time interval was set. Each time the sample was taken out, the surface water was absorbed with filter paper, the mass change was weighed, and the swelling rate was calculated to understand the hydrophilicity of the material.

[0076] like Figure 7 As shown in Figure B, the swelling ratio of GelMA with a concentration of 2.5-7.5% is in the range of 498.47-610.17%. The increase in GelMA concentration leads to a decrease in the swelling ratio. The higher concentration of GelMA, due to its denser network structure, limits the ability of water molecules to enter, thereby inhibiting the degree of swelling of the material.

[0077] (7) In vitro degradation curve: The spinal cord tissue-like sample was placed in PBS, samples were taken regularly and their mass changes were measured. The mass loss during the degradation process was recorded and an in vitro degradation curve was drawn to evaluate the degradation rate of the material.

[0078] As shown in 7C, the degradation of GelMA-30 at three different concentrations was relatively slow and could be stably present in vitro for more than one month.

[0079] Example 3 In vitro experiments to investigate the safety and effectiveness of spinal cord tissue

[0080] The experimental process of this embodiment is as follows Figure 8 As shown, the biocompatibility of the 3D printed spinal cord tissue scaffold was first investigated by CCK8, Live / Dead and EDU staining. The specific steps are as follows:

[0081] 2D culture: Weigh a certain amount of GelMA, add 0.25% (w / v) photoinitiator diluted with PBS, and prepare a 5% (w / v) GelMA mixture. Heat in a 60°C water bath to dissolve for about 20 minutes, and oscillate several times during the period to mix. Filter and sterilize, cool to about 37°C, add NT-3 to a concentration of 10 ng / mL, mix well and use for 3D printing. Place the 3D printed spinal cord tissue engineering scaffold in a sterile 24-well plate. Make sure that the size of the scaffold matches the well plate and that the scaffold is evenly distributed in each well. Wash the scaffold three times with PBS or saline, and then culture in complete culture medium. The neural stem cells were cultured at 2×10 6 Resuspend in complete medium at a concentration of 100 μg / mL.

[0082] Among them, the neural stem cell culture medium is DMEM / F12 culture medium + 2% B27 + 1% double antibody + 20ng / mL EGF + 20ng / mL b-FGF; the differentiation medium, that is, the complete culture medium, is DMEM / F12 culture medium + 10% FBS + 1% double antibody.

[0083] NSCs are routinely cultured and subcultured using neural stem cell culture medium; mixed with materials and cultured after printing, or materials printed and then loaded with NSCs and cultured again, using complete culture medium. Cells are added evenly to the well plate. Ensure that neural stem cells are evenly distributed on each scaffold so that they can attach to the scaffold.

[0084] 3D culture: Use NSCs at a concentration of 2 × 10 6 Cell-containing spinal cord tissue was printed with GelMA-30 bio-ink at a concentration of 100 cells / mL. After washing with PBS, complete culture medium was added for culture.

[0085] The neural stem cells were used as the control group, and the detection work was carried out after adding complete culture medium for 5 days. Among them, CCK8 was incubated for 2 to 4 hours after adding CCK8 reagent, and the absorbance at 450nm was measured by microplate reader to reflect the relative viability of cells. After Live / Dead and EDU staining, the live cell imager was used to observe the live-dead cell ratio or the proliferating cell ratio. Cell apoptosis and cell cycle were detected by flow cytometry.

[0086] As shown in 6A and D, the live-dead staining results show that in the three GelMA materials, the vast majority of NSCs can survive, indicating that the materials are non-toxic to NSCs cells. As shown in 6B and C, among the three GelMA materials, the NSCs in the GelMA-30 and GelMA-PR-002 groups have higher relative activity, that is, they have better cell compatibility. Among them, GelMA-PR-002 is too thermosensitive and not suitable for 3D printing, so GelMA-30 is a better choice. Among several different concentrations of GelMA-30, the 5% GelMA-30 group has the highest relative activity of NSCs, so 5% GelMA-30 is preferred.

[0087] As shown in Figure 11A, the EDU staining results showed that all three material groups could effectively promote the proliferation of NSCs, among which the GelMA-30 group had the most significant promoting effect, that is, GelMA-30 was the preferred material in promoting cell proliferation.

[0088] Apoptosis experiments use staining methods (such as Annexin V / PI double staining) to analyze different cell death states. This method is based on changes in the cell membrane, especially the externalization of phosphatidylserine and changes in cell membrane integrity. Fig.11 In B, the four quadrants represent different cell states: lower left quadrant (Annexin V-, PI-): living cells, intact cell membrane, no apoptosis or necrosis; lower right quadrant (Annexin V+, PI-): early apoptotic cells, everted phosphatidylserine (PS) exposed, but the cell membrane has not yet ruptured, and has not entered the necrosis stage; upper left quadrant (Annexin V-, PI+): necrotic cells, cell membrane rupture, PI can enter the cell and stain; upper right quadrant (Annexin V+, PI+): late apoptotic or necrotic cells, cell membrane rupture, everted phosphatidylserine exposed, and incomplete cell membrane. The experimental results show that among the three materials, the GelMA-30 group has the largest proportion of normal cells and the smallest proportion of apoptotic cells, that is, the cell survival state is the best.

[0089] The cell cycle includes G1 phase, S phase, G2 phase and M phase, and the DNA content in each phase is different. The dye PI is used to bind to DNA, and the fluorescence intensity of the dye is proportional to the DNA content in the cell. After the cells are labeled with the dye, the flow cytometer analyzes the fluorescence intensity of each cell. G1 phase cells with low DNA content will show lower fluorescence intensity, the DNA content of S phase cells gradually increases, and the DNA content of G2 / M phase cells is higher. Finally, the distribution of cells in different cycle stages is quantitatively analyzed by fluorescence intensity distribution. Fig.11As shown in C, among the three materials, the GelMA-30 group had the lowest proportion of cells in the G1 phase and the highest proportion of cells in the S+G2 phase, that is, the cell division activity was the most active and the cell proliferation was the fastest.

[0090] After inducing NSCs differentiation and culture for 7 days, spinal cord-like tissues were collected, and the expression of glial fibrillary acid protein (GFAP) and β-tubulin (TUJ-1) was double-labeled by immunofluorescence (IF) and recorded by confocal fluorescence microscopy. Spinal cord-like tissue proteins were extracted, and the relative expression of marker proteins of neurons, oligodendrocytes, and astrocytes was detected by Western Blot; spinal cord-like tissues were extracted, cDNA was obtained using a reverse transcription kit, and the relative expression of marker proteins of neurons, oligodendrocytes, and astrocytes was determined by qPCR to evaluate the differentiation of NSCs in spinal cord-like tissues.

[0091] The expression of marker proteins and gene transcription of cells after 7 days of spinal cord culture Fig. 9 As shown, Fig. 9 A and B are two-dimensional cultures. Fig. 9 C and D are three-dimensional cultures. Western blot and qPCR experimental results showed that both two-dimensional and three-dimensional cultured spinal cord-like tissues could effectively inhibit the expression of GFAP and promote the expression of TUJ-1, that is, inhibit scar growth and promote nerve regeneration, and the three-dimensional cultured spinal cord-like tissue had better in vitro effectiveness.

[0092] Example 4 In vivo experiments to investigate the safety and effectiveness of spinal cord tissue

[0093] The experimental flow chart is as follows Fig.10 The specific experimental steps are as follows:

[0094] (1) Construction of spinal cord defect rat model:

[0095] Take 8-week-old SD rats, weigh the SD rats, and inject 1% pentobarbital (40 mg / kg) intraperitoneally for anesthesia. Fix the rats on the operating table in a prone position, shave the hair on the back to expose the skin, and wipe the skin with iodine to disinfect. At the 7th to 10th spine, cut the skin and muscles to expose the vertebrae. Use bone rongeurs to bite off the 8th to 9th vertebrae to expose the spinal cord, 2mm spinal cord respectively, and after hemostasis, perform different treatments according to the control group or treatment group. Then suture the muscles and skin layer by layer, wipe the wound with iodine to disinfect, and return to the cage after waking up, and inject penicillin continuously for 5 days after surgery.

[0096] (2) Animal grouping, treatment and sampling:

[0097] 1) Grouping: This experiment was divided into 3 groups, namely sham group (Sham), control group (Control), and treatment group (Implant). Among them, the sham group: only the spinal cord was exposed but not damaged; the control group: no treatment was given after spinal cord defect; the treatment group: after spinal cord injury, spinal cord tissue was transplanted in situ.

[0098] 2) Animal sampling: 8 weeks after surgery, 4 rats were randomly selected from each group for sampling and testing. Rats were injected with an overdose of 1% sodium pentobarbital (40 mg / kg) intraperitoneally. The operation was started after the rats entered deep anesthesia. The rats were placed in a supine position, the chest cavity was cut open and the heart was exposed. The catheter was inserted from the apex of the heart until it entered the aorta and was fixed, and finally the right atrial appendage was cut open. First, perfuse with normal saline until the outflowing liquid becomes clear, and then perfuse with 4% paraformaldehyde, and stop when the rat's whole body is stiff. The rat was placed in a prone position, and the injured part on the back of the rat was opened with scissors to remove the spinal cord tissue.

[0099] 3) Evaluation of SCI repair effect: The rats were scored with BBB (Basso, Beattie, Bresnahan) every week within 8 weeks after surgery, and gait analysis was performed 8 weeks later to evaluate the recovery of the rats' limb function. The spinal cord recovery of the rats was observed after sampling.

[0100] The BBB score is a scoring system commonly used to evaluate the recovery of motor function after spinal cord injury. This experiment is used to evaluate the motor ability of the lower limbs of rats. The scoring system ranges from 0 to 21 points, with 0 indicating complete loss of motor ability and 21 indicating normal motor ability. The BBB score is generally performed by observing the performance of rats in different motor tasks. Specific observation items include: gait: whether they can walk normally, whether there is lameness or gait incoordination; support ability: whether the rat can support the body with its limbs and whether it can stand; coordination: whether the gait is smooth and whether there is obvious motor incoordination; range of motion: the range of motion of the limbs, whether there is paralysis or limited activity. Specific steps for scoring: Place the rat on a smooth surface and observe its walking, standing and turning performance. Record the motor function performance of the rat at different time points and score it according to the scoring criteria (from 0 to 21 points). Such as Fig.12 As shown in A, the BBB scores of the transplanted spinal cord group were higher than those of the untreated group every week, indicating that the motor function recovery effect of the rats in this group was better than that of the untreated group.

[0101] Footprint analysis (or gait analysis) of rats after spinal cord injury is a method to evaluate the recovery of their motor function by analyzing the rat's gait and footprints. First, the rat's hind limbs are painted with non-toxic red ink; then the rat is placed in a long wooden box with paper pads and made to walk in a straight line in one direction, leaving clear footprints on the paper; finally, the footprints are observed to evaluate the rat's motor function and recovery.

[0102] like Fig.12 As shown in B, the rats in the sham-operated group showed normal gait, symmetrical stride, good coordination, reasonable support time, and no significant abnormalities. In contrast, the rats in the untreated group had obviously asymmetrical gait, less hindlimb force, unstable gait, poor coordination between the front and rear limbs, and less support from the hind limbs. The rats in the treated group restored gait symmetry and coordination to a certain extent, their stride increased, and gait asymmetry and shortened stride were alleviated, but overall they still did not fully recover to the normal level of the sham-operated group. In short, transplanted spinal cord tissue improved the behavioral function of SSCI rats.

[0103] like Fig.12 As shown in C, the spinal cord in the sham operation group had a normal appearance, with a white body and a smooth surface, and no signs of injury or glial reaction. The spinal cord injury area in the untreated group had obvious signs of injury, which was yellow, indicating hematoma, necrotic tissue or lipid deposition, and the stump of the spinal cord was not effectively repaired. There may be obvious inflammatory reactions and glial scar formation around the injury area. The spinal cord injury site in the treatment group showed signs of partial recovery, with a relatively flat local tissue morphology, a mild glial reaction, and a small scar tissue.

[0104] In summary, the rats survived after the transplantation of spinal cord tissue, and no tumor formation or other negative effects were found, indicating that the spinal cord tissue is safe in vivo. Compared with the untreated group, the behavioral ability of rats transplanted with spinal cord tissue was significantly improved, and the spinal cord recovered better in appearance, indicating that the transplantation of spinal cord tissue can promote spinal cord repair and has in vivo effectiveness.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing spinal cord-like tissue based on 3D printing of neural radiation field, characterized in that: The steps include: S1: Construction of a three-dimensional model of spinal cord tissue using neural radiation fields; S2: Performing 3D printing of a solid body based on the three-dimensional model described in S1 to obtain a microporous structure scaffold; S3: Implanting seed cells and bioactive factors into the microporous structure scaffold described in S2 to obtain the spinal cord-like tissue.

2. The preparation method according to claim 1, characterized in that: S1 specifically includes the following steps: S1-1: Acquire a two-dimensional image of the spinal cord tissue collected at each preset posture and posture information of the two-dimensional image to obtain a data set; S1-2: Input the data set described in S1-1 into the deep neural network to complete the neural radiation field model training; S1-3: Using the neural radiation field model obtained through training in S1-2, a three-dimensional model corresponding to the spinal cord tissue is generated through volume rendering technology.

3. The preparation method according to claim 2, characterized in that: S1 further comprises the following steps: S1-4: Observe the cross-sectional shape and diameter of the spinal cord tissue, and verify and optimize the accuracy and authenticity of the three-dimensional model described in S1-3.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The porosity of the microporous structure support described in S2 is 30-60%.

5. The preparation method according to any one of claims 1 to 3, characterized in that: The seed cells described in S3 are any one of neural stem cells, umbilical cord mesenchymal stem cells and bone marrow mesenchymal stem cells.

6. The preparation method according to claim 5, characterized in that: The bio-ink used in the 3D printing described in S2 is methacrylated gelatin; The seed cells described in S3 are neural stem cells, and the bioactive factor described is neurotrophic factor 3.

7. The preparation method according to claim 6, characterized in that: The concentration of the methacrylated gelatin is 2-8% (w / v).

8. The preparation method according to claim 6, characterized in that: The concentration of the neurotrophic factor 3 is 10-100 ng / mL.

9. The spinal cord-like tissue obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the spinal cord-like tissue according to claim 9 in preparing a tissue engineering scaffold for repairing spinal cord injury.

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