Construction method of conductive material-stem cell-hydrogel living material bio-ink and its application in 4D printing neural tissue

Abstract

This invention provides a method for constructing a bio-ink based on a conductive material, stem cells, and hydrogel, and its application in 4D-printed neural tissue-like structures. By anchoring uniformly sized conductive graphene onto the surface of neural stem cells and then binding it to a hydrogel, a bio-ink based on a conductive material, stem cells, and hydrogel can be efficiently constructed, ensuring stable binding of the conductive material and neural stem cells within the hydrogel. 3D printing allows for the design of desired structures and shapes through adjustments to printing parameters, achieving controllable spatial distribution. Conversely, the electrical signals mediated by the conductive material can precisely stimulate stem cells, regulating their efficient and rapid differentiation into functional neurons at the neural level, enabling spatiotemporally controllable differentiation of NSCs, thereby constructing 4D-printed neural tissue-like structures. This provides an efficient and precise pathway for in vivo neural injury repair using 4D-printed neural tissue-like structures, promoting the development of regenerative medicine and intelligent tissue engineering.

Description

Technical Field

[0001] This invention relates to a method for constructing a conductive material-stem cell-hydrogel bio-ink and its application in 4D printing of neural tissue-like materials, belonging to the biomedical field. Background Technology

[0002] Neural-like tissues are a type of biomimetic functional neural tissue constructed using artificial techniques, possessing the core essential attributes of natural neural tissue. Their key characteristic lies not only in their ability to structurally mimic the cellular composition and spatial arrangement of natural neural tissue, but also in their ability to functionally replicate the signal reception, conduction, integration, and synergistic effects of neural tissue. The application prospects of neural-like tissues extend from the core field of nerve injury repair to multiple key areas such as medical research, drug development, and brain science exploration, demonstrating significant clinical and scientific research value. Customized neural-like tissues can guide the regeneration of spinal cord nerve axons, reconstructing severed neural conduction pathways and replacing damaged neural tissue. Furthermore, neural-like tissues can be used to construct in vitro disease models, providing "living in vitro models" for elucidating disease pathogenesis and replacing traditional animal models for neuropharmaceutical screening.

[0003] 3D printing of living materials composed of stem cells and hydrogels offers tremendous potential for creating complex neural-like tissues. 3D printing allows for precise control over the arrangement of stem cells and the morphology of the tissue, enabling it to exhibit structures similar to the natural nervous system in vitro, providing higher spatial resolution and design freedom for neural-like tissue construction. However, the development of neural-like tissues from living materials composed of stem cells and hydrogels is a highly complex and dynamic process. Efficient construction and precise control of the structure and function of neural-like tissues cannot be achieved solely through static 3D printing and natural differentiation of stem cells. This is because regulating the directed differentiation of stem cells into functional nerve cells is difficult, and the entire differentiation cycle is long and inefficient. While growth factors such as neurotrophic factors can regulate the direction and speed of stem cell differentiation, these factors are not only expensive but also suffer from drawbacks such as easy inactivation, rapid degradation, short half-life, and uncontrollable distribution in three-dimensional space.

[0004] By combining time-dimensional extensions in 3D printing technology, programmable morphological and functional changes in printed scaffolds can be achieved in response to external stimuli. Utilizing "material responsiveness" and "external field modulation," static 3D-printed scaffolds can undergo programmable morphological changes and functional maturation over time, enabling the regulation of stem cell differentiation and neural development processes. This satisfies the multi-stage needs of neural development and repair, thus constructing 4D-printed neural tissue-like structures. However, the efficiency of inducing stem cell neural differentiation using controlled deformation and stress is low, resulting in low efficiency and poor controllability in constructing neural tissue-like structures using 4D-printed responsive deformation materials.

[0005] Nerve tissue is electrically active, and electrical stimulation is widely used to promote neural differentiation of stem cells and repair of nerve tissue. Stem cell differentiation can be regulated by modifying the function of transmembrane proteins, altering ion channels on the cell membrane, and changing the distribution of membrane receptors. Compared to deformable materials and growth factors, conductive materials can generate controllable radio stimulation under the drive of a rotating magnetic field, offering advantages such as adjustable parameters, controllable stimulation area, controllable stimulation time, long propagation distance, and non-invasiveness. The key to constructing 4D-printed neural tissue using electrical stimulation lies in ensuring direct contact between the conductive material and the stem cells. This allows the electrical stimulation generated under the drive of the magnetic field to effectively and precisely target specific stem cells, enabling them to differentiate into neurons or glial cells at the correct time and location, thus forming functional neural tissue.

[0006] Therefore, how to develop a bio-ink that combines conductive materials, stem cells, and hydrogels for 4D printing, precisely assembles conductive materials and stem cells within a hydrogel, achieves precise spatial distribution control through 3D printing, and utilizes the radio stimulation generated by the conductive materials under magnetic field drive to precisely act on stem cells, inducing spatiotemporally controllable differentiation of stem cells, is a key problem that urgently needs to be solved in constructing 4D-printed neural tissue with controllable structure and function. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a method for constructing a bio-ink using a conductive material-stem cell-hydrogel living material, as well as a bio-ink using the above method.

[0008] This invention also provides the application of the above-mentioned living material bio-ink. Using this living material bio-ink, the spatial distribution can be precisely controlled through 3D printing, and the radio stimulation generated by the conductive material under the drive of a magnetic field can be precisely applied to stem cells to induce spatiotemporally controllable differentiation of stem cells, thereby realizing 4D printed neural tissue with controllable structure and function.

[0009] This invention is achieved through the following technical solution: A highly efficient mass assembly method for conductive materials and stem cells includes the following steps: 1) The conductive material is sterilized and dispersed in deionized water, and then incubated with a laminin solution to obtain a protein-modified conductive material; the conductive material is graphene nanosheets. 2) After centrifuging the stem cells, they were re-dispersed by pipetting with differentiation medium to obtain a cell suspension; the stem cells were neural stem cells; 3) Add a conductive material suspension to the neural stem cell suspension to obtain a mixture, and then incubate it; 4) Centrifuge the stem cells anchored to the conductive material, remove the supernatant, and obtain the conductive material-stem cell assembly.

[0010] According to a preferred embodiment of the present invention, in step 1), the size of the conductive material is 1-10 μm.

[0011] According to a preferred embodiment of the present invention, in step 1), the sterilization treatment is a soaking in 75% alcohol for 0.5-3 hours.

[0012] According to a preferred embodiment of the present invention, in step 1), after sterilization, the concentration of conductive material in the conductive material dispersion is 1-10 mg / mL.

[0013] According to a preferred embodiment of the present invention, in step 1), the concentration of the laminin solution is 0.5-5 μg / mL.

[0014] According to a preferred embodiment of the present invention, in step 1), the incubation temperature is 37°C. o C, incubation time is 1-8 hours.

[0015] According to a preferred embodiment of the present invention, in step 2), the neural differentiation medium is an F12 medium containing 2% B27 neural culture additive, 1% N2 neural culture additive, 1% fetal bovine serum (FBS), 1% GlutaMax, and 1% double antibiotics.

[0016] F12 culture medium is a current technology.

[0017] According to a preferred embodiment of the present invention, in step 3), the concentration of the conductive material in the mixture is 5-100 μg / mL, and the concentration of cells is (0.5-10) × 10⁻⁶. 6 per mL.

[0018] According to a preferred embodiment of the present invention, in step 3), the incubation environment is a constant-temperature shaking incubator, and the incubation temperature is 37°C. o C, the shaking frequency of the shaker is 50-200 rpm, and the incubation time is 0.5-12 h.

[0019] According to a preferred embodiment of the present invention, in step 4), the centrifugation speed is 600-1300 rpm and the centrifugation time is 3-5 min.

[0020] According to a preferred embodiment of the present invention, in step 4), the in vitro culture is carried out for 3-10 days in a saturated humidity environment containing 5% CO2 at 37 °C.

[0021] The present invention also discloses a conductive material-stem cell assembly, which is prepared by the above method.

[0022] The conductive materials used in this invention range in size from 1 to 10 micrometers, exhibiting uniform size distribution, intact structure, and few defects, thus demonstrating excellent conductivity. The large size of the materials effectively prevents endocytosis by cells, and through protein modification, the patches can bind efficiently, stably, and persistently to the cell membrane surface, exhibiting good biocompatibility and safety. The assembly method is simple, efficient, and inexpensive, showing broad application prospects in the biomedical field, especially in cell interface regulation.

[0023] This invention also discloses a method for the mass production of a conductive material-stem cell-hydrogel bio-ink, comprising the following steps: 1) Place the hydrogel at 37°C o Preheating in a water bath in the dark; the hydrogel is methacrylamide gelatin (GelMA) hydrogel; 2) After centrifuging the stem cells anchored to the conductive material, use 37... o C. Resuspend the preheated solution; According to a preferred embodiment of the present invention, in step 1), the concentration of the hydrogel is 2%-15%.

[0024] According to a preferred embodiment of the present invention, in step 1), the hydrogel is at 37°C. o The preheating time for C is 5-30 minutes.

[0025] According to a preferred embodiment of the present invention, in step 2), the concentration of cells in the suspension is (0.5-10) × 10⁻⁶. 7 per mL.

[0026] The conductive material-stem cell-hydrogel bio-ink prepared in this invention can be 3D printed into different shapes and structures, thereby constructing tissue-like structures of various shapes and structures. Different needs can be met by controlling the rheological, mechanical, and degradation properties of the hydrogel, as well as the distribution of stem cells within the hydrogel. More importantly, it can ensure the stable binding and synergistic function of the conductive material and stem cells before and after printing.

[0027] The application of the aforementioned conductive material-stem cell-hydrogel bio-ink utilizes the conductive material to mediate electrical stimulation under magnetic field drive to regulate the rapid differentiation of stem cells and construct a 4D printed neural tissue-like method.

[0028] According to a preferred embodiment of the present invention, the method for constructing 4D-printed neural tissue is as follows: 1) Place the conductive material-stem cell-hydrogel bio-ink in a 4-layer container. o C pre-cooling; 2) 3D printing using conductive material-stem cell-hydrogel bio-ink.

[0029] 3) Add neural differentiation culture medium to the 3D-printed conductive material-stem cell-hydrogel living material and place it in a 37°C incubator; 4) After cleaning the sample, replace it with a new culture medium; 5) Stimulate with a rotating magnetic field to construct 4D-printed neural tissue.

[0030] According to a preferred embodiment of the present invention, in step 1), the conductive material-stem cell-hydrogel bio-ink is in a 4 o The pre-cooling time for C is 5-20 minutes.

[0031] According to a preferred embodiment of the present invention, in step 2), the air pressure for 3D printing is 0.1-0.5 MPa, and the platform temperature is -5-15°C. o C, needle temperature is 4-20°C o C, printing speed is 3-10 mm / s.

[0032] According to a preferred embodiment of the present invention, in step 2), the intensity of photocuring is 30-80 mW / cm. 2 The photocuring time is 10-60 seconds.

[0033] According to a preferred embodiment of the present invention, in step 3), the incubation time in an incubator at 37°C is 15-30 min; According to a preferred embodiment of the present invention, in step 4), the washing is performed 1-3 times, and a neural differentiation culture medium is used; According to a preferred embodiment of the present invention, in step 5), the culture time is 5-15 days, the distance from the cell culture plate to the magnet is 0.5-1 cm, and the rotational magnetic field stimulation conditions are: rotation speed of 100-1000 rpm, magnetic field strength of 50-200 mT, stimulation time of 5-30 min each time, and stimulation 1-5 times a day.

[0034] Technical features and advantages of the present invention: 1. The conductive material-stem cell assembly method proposed in this invention fully utilizes the advantages of conductive nanomaterials, such as uniform size and good conductivity, to efficiently, stably, and for a long time bind conductive nanomaterials to the cell membrane surface, avoiding endocytosis by the cell and achieving efficient batch assembly.

[0035] 2. The conductive material-stem cell-hydrogel bio-ink proposed in this invention can be constructed with the required structure and shape through 3D printing, while ensuring the precise assembly of the conductive material and stem cells in the hydrogel. This ensures the precise regulation of stem cells by the electrical stimulation mediated by the conductive material. The preparation method is simple, efficient, low-cost, and highly adaptable to various technologies.

[0036] 3. The method for constructing 4D-printed neural tissue proposed in this invention utilizes conductive materials, stem cells, hydrogel bio-inks, and 3D printing technology. This method can achieve spatial structural regulation and, through radio stimulation mediated by conductive materials, controllable differentiation of stem cells within the hydrogel over time, thus constructing neural tissue with controllable structure and function. This method has broad application prospects in the biomedical field, especially in neural tissue regeneration, disease model construction, and drug screening. Attached Figure Description

[0037] Figure 1 Fluorescence co-localization image of graphene nanopatch assembled with stem cells; Figure 2 Scanning electron microscope image of graphene nanopatterns assembled with stem cells; Figure 3 Dynamic modulus analysis of GelMA hydrogel; Figure 4 Swelling rate analysis of GelMA hydrogel; Figure 5 Fluorescent colocalization image of graphene nanopatch-stem cell-hydrogel assembly; Figure 6 This is a test diagram of the electrical signals of the graphene nanopatch. Figure 7 An experiment to activate cell membrane calcium ion channels through radio stimulation mediated by graphene nanopatch; Image a shows the activation of the calcium ion pathway in the cell membrane of the hydrogel by radio stimulation generated by the nanopatch under a rotating magnetic field; image b shows... Figure 7 A diagram illustrating the changes in cellular fluorescence in cell a. Figure 8 PCR analysis of Tuj1, MAP2, and GFAP genes after five days of culture for each treatment in Experiment Example 4; Figure 9 For the neural immunofluorescence assay in each treatment group; In this example, a represents the immunofluorescence staining of Tuj1 protein after five days of culture for each treatment in Experiment 4, and b represents the immunofluorescence staining of MAP2 protein after five days of culture for each treatment in Experiment 4. Figure 10 For neural Western blot testing in each treatment group Where a represents the Western blotting bands of Tuj1 and MAP2 proteins after five days of culture for each treatment in Experiment Example 4, and b represents... Figure 10 Gray-scale analysis of the band 'a' in the middle; Figure 11 Top and side views of 3D printed hydrogel; Figure 12Images showing stem cell proliferation after 3 and 10 days of culture in a 3D-printed hydrogel; Figure 13 Experiments to construct 4D-printed hydrogels; In this figure, a is a schematic diagram of 4D printed hydrogel, and b is a fluorescence image of 4D printed neural tissue constructed in Experiment 6; Figure 14 Electrical signal detection for the 4D-printed neural tissue constructed in Experiment Example 7; Figure 15 This shows the performance of Experiment 8 and the slope after different treatments in the slope experiment. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0039] Furthermore, unless otherwise specified, the experimental methods described in the following embodiments are all conventional methods; and unless otherwise specified, the reagents, materials and equipment are all commercially available.

[0040] F12 complete medium is F12 medium supplemented with 2% B27 neural culture additive, 1% N2 neural culture additive, 1% fetal bovine serum (FBS), 1% GlutaMax and 1% antibiotics.

[0041] In this example, neural stem cells (NSCs) were obtained from Wistar rat embryos aged 12-14 days under aseptic conditions and cultured for three generations in proliferation medium. The proliferation medium was F12 medium containing 2% B27 neural culture additive, 1% N2 neural culture additive, 1% fetal bovine serum (FBS), 1% GlutaMax, 1% penicillin-dextrose antibody, 20 ng / mL mEGF, and 20 ng / mL mbFGF.

[0042] Example 1 The assembly method of graphene nanopatch-neural stem cells is as follows: 1) After centrifuging and re-dispersing, neural stem cells were redispersed in neural differentiation medium at 700 rpm for 5 min to obtain a neural stem cell suspension with a cell concentration of 1×10⁻⁶ cells / mL. 6 cells / mL; 2) Add graphene nanopatch dispersion to neural stem cell suspension to obtain a mixture, and heat at 37°C. o Incubate on a shaker at C for 1 h, with a cell concentration of 1×10⁻⁶ cells in the mixture. 6 The concentration of graphene nanoparticles was 60 μg / mL, and the shaking speed was 100 rpm. 3) Centrifuge the above suspension at 750 rpm for 5 min, remove the supernatant, and obtain neural stem cells anchored by graphene nanopatch.

[0043] Example 2 The assembly method of graphene nanopatch-neural stem cells-GelMA hydrogel bio-ink is as follows: 1) Place the GelMA hydrogel at 37°C. o Preheat a water bath (C) with a GelMA hydrogel concentration of 5% for 20 minutes. 2) The neural stem cells anchored in the graphene nanopatch obtained in Example 1 were centrifuged and then redispersed in preheated GelMA solution. The centrifugation speed was 700 rpm and the centrifugation time was 5 min to obtain a graphene nanopatch-neural stem cell-GelMA hydrogel bio-ink. The cell concentration in the bio-ink was 1×10⁻⁶. 7 per mL.

[0044] Example 3 Example 2 describes a method for rapid neural differentiation of stem cells in a graphene nanopatch-neural stem cell-GelMA hydrogel bio-ink. The specific method is as follows: 1) Place the hydrogel in a neural differentiation culture medium at 37°C. o Incubate in a C2 incubator for 15 min; 2) Replace with a completely new differentiation medium; 3) Apply a rotating magnetic field with a strength of 160 mT, a rotation speed of 600 rpm, and a stimulation time of 30 min. Stimulate once a day for 5 days.

[0045] Example 4 Example 2 describes a method for 3D printing using the graphene nanopatch-neural stem cell-GelMA hydrogel bio-ink prepared in this study. The specific method is as follows: 1) Graphene nanopatch-stem cell-hydrogel living biomaterial was used as bio-ink in a 4-layer substrate. o Pre-cool at C for 15 minutes; 2) Set the 3D printer platform temperature to 4. o C, needle temperature set to 4 o C. Printing speed is 10 mm / s, printing air pressure is 0.5 MPa, and printing is performed according to the design pattern; 3) The printed gel was then photocured to a strength of 35 mW / cm². 2 The photocuring time is 30 seconds.

[0046] Example 5 Example 4 describes a method for constructing 4D-printed neural tissue-like materials from 3D-printed graphene nanopatch-neural stem cells-GelMA hydrogel under a rotating magnetic field. The specific method is as follows: 1) Place the printed hydrogel in a neural differentiation culture medium at 37°C. o Incubate in a C2 incubator for 15 min; 2) Replace with a completely new differentiation medium; 3) Apply a rotating magnetic field with a strength of 160 mT, a rotation speed of 600 rpm, and a stimulation time of 30 min. Stimulate once a day for 10 days.

[0047] Experimental Example 1 1. The graphene nanopatch and cell membrane were fluorescently labeled before incubation in step 2) of Example 1. The graphene nanopatch and cells were co-incubated for 3 hours. Fluorescent co-localization was then performed on the stem cells anchored by the graphene nanopatch. Figure 1 As shown, red fluorescence is consistent with green fluorescence, and red fluorescence is present in almost all cells, indicating that the graphene nanopatch of the present invention can bind tightly to stem cells.

[0048] 2. After incubating graphene nanopatch with stem cells for 3 hours, the cell scanning electron microscope results were as follows: Figure 2 As shown, obvious graphene nanoparticles are observed adhering to the cell surface, indicating that the graphene nanoparticles are successfully anchored on the surface of stem cells.

[0049] Experimental Example 2 Rheological tests were performed on the GelMA hydrogels of different amino substitution degrees (30, 60) and different concentrations (5%, 10%) in Example 2, such as... Figure 3 As shown, the storage modulus G' of all types of hydrogels is greater than the loss modulus G'', indicating that a stable gel has been formed. Meanwhile, with the increase of the degree of substitution of the GelMA hydrogel, the storage modulus G' of the hydrogel increases significantly, indicating an increase in gel hardness. Similarly, with the increase of the GelMA hydrogel concentration, the storage modulus G' of the hydrogel also increases significantly, again indicating an increase in gel hardness. Figure 4 As shown, the swelling rate of GelMA hydrogel increases rapidly in the first two hours and reaches swelling equilibrium after 24 hours, with a swelling rate of about 40%.

[0050] Experimental Example 3 The graphene nanopatch within the hydrogel in Example 2 was subjected to fluorescence co-localization with stem cells, such as... Figure 5 As shown, the red and green fluorescence are consistent, indicating that the graphene nanopatch and stem cells can maintain a tight bond within the hydrogel. Electrical signal detection is as follows... Figure 6As shown, the voltage intensity generated by the biodegradable electrode patch increases with the increase of the magnetic field rotation speed, reaching its highest value at 600 rpm. This also verifies the activation effect of radio stimulation on cell membrane potential, such as... Figure 7 As shown in a, a significant change in fluorescence was observed after the application of radio stimulation, and the fluorescence changes were analyzed (e.g., Figure 7 (b) in the diagram demonstrates that the radio stimulation generated by the nanopatch under a rotating magnetic field can activate the voltage-gated calcium ion channels on the surface of stem cell membranes within the gel, promoting calcium ion influx.

[0051] Test Example 4 After culturing the graphene nanopatch-neural stem cells-GelMA hydrogel living material from Example 3 with a magnetic field applied (GelMA+Graphene@NSCs+MF) for five days, the genes and proteins of neurons were detected. At the same time, the magnetic field group (Ctrl+MF), the mixed graphene nanopatch group (GelMA+Graphene+NSCs), the graphene-stem cell-nanopatch group without a magnetic field applied (GelMA+Graphene@NSCs), and the mixed graphene nanopatch with a magnetic field applied (GelMA+Graphene+NSC+MF) group were used as controls. The magnetic field group is the method of Example 3, in which only neural stem cells are cultured in the gel and a rotating magnetic field is applied; The mixed graphene nanopatch group was cultured in an in-gel mixture of graphene nanopatch and neural stem cells without the application of a rotating magnetic field, as described in Example 3. The graphene-stem cell-nanopatch group without a magnetic field applied is the method of Example 3, where no rotating magnetic field is applied.

[0052] The method used in Example 3 involved mixing graphene nanopatches and neural stem cells within a gel and culturing them under a rotating magnetic field. After five days of culture, the expression of neuronal markers β-tubulin (Tuj1) and microtubule-binding protein (MAP2) in each treatment group was analyzed. Figure 8 As shown, at a magnetic field rotation speed of 600 rpm, radio stimulation mediated by graphene nanopatch significantly promoted rapid neuronal differentiation of stem cells within the gel. The expression levels of neuronal markers Tuj1 and MAP2 were increased compared to other groups, while the expression of glial GFAP showed no significant change. Figure 9 Immunofluorescence staining of samples a and b also showed that in the electrically stimulated group, the neuronal morphology was more pronounced, and the fluorescence of Tuj1 and MAP2 was also more obvious, indicating a significant promotion of neural differentiation. Further Western blot experiments were performed, such as... Figure 10The bands and grayscale analysis of a and b in the data show that the expression level of Tuj1 increased by 4.5 times and the expression level of MAP2 increased by 5.5 times in the electrical stimulation group.

[0053] Experimental Example 5 In Example 4, Mingchang photographed and observed the 3D-printed hydrogel obtained in Example 2. By optimizing the printing process and parameters, gel printing of different structural sizes can be achieved. Figure 11 As shown, it can print sizes larger than 1×1×1 cm. 3 Nerve-like tissues. The activity of NSCs in the 3D-printed hydrogel was analyzed using live / dead staining. Figure 12 As shown, after 3 days of cultivation following printing, there was no significant difference in the activity of NSCs in the hydrogels of the two groups of experiments. However, after 10 days of cultivation, it was evident that NSCs showed significant proliferation in the hydrogels, indicating that 3D printing did not affect cell activity.

[0054] Experimental Example 6 In Example 5, 3D-printed hydrogels with different structures were designed to further explore the ability to construct 4D-printed in vitro neural tissue-like structures. Different bio-inks (GelMA+NSCs, GelMA@Nanopatch+NSCs) were combined with 3D printing to construct 4D-printed neural tissue-like structures (e.g.,...). Figure 13 (a) Under magnetic field excitation, the differentiation of NSCs in hydrogels of various structures is regulated (e.g., ... Figure 13 (b) can construct neural-like tissues of different shapes, realizing spatiotemporally controllable differentiation of NSCs.

[0055] Experimental Example 7 Functional verification was performed on the 4D-printed in vitro neural tissue constructed in Example 5, such as... Figure 14 As shown, neural-like tissue can respond to electrical stimulation and conduct electrical signals, exhibiting the function of a regionalized neural network.

[0056] Experimental Example 8 The 4D-printed neural tissue obtained in step 3) of Example 5 was implanted into the complete spinal cord resection site of mice; at the same time, blank group, disease group, cell group, and mixed graphene nanopatch with magnetic field group were used as controls.

[0057] The blank group consisted of normal spinal cord tissue (Sham); The spinal cord of mice was removed to form the total spinal cord resection group (SCI). The cell group consisted of neural stem cells cultured in GelMA gel for ten days and then implanted into the spinal cord resection site of mice (SCI+NSCs). The hybrid graphene nanopatch with magnetic field group: GelMA with hybrid graphene nanopatch and NSCs and cultured under a rotating magnetic field for 10 days was implanted into the spinal cord resection site of mice (SCI+NSCs+Graphene+MF). The performance of each group of experimental mice in the inclined plane test after treatment was as follows: Figure 15 It can be seen that the climbing ability of mice in the experimental group was significantly improved, which indicates that the constructed 4D printed neural tissue can significantly improve the behavioral ability of mice with total spinal cord resection and achieve the repair of damaged tissue.

Claims

1. A method for batch assembly of a conductive material-stem cell-hydrogel bio-ink, characterized in that... Includes the following steps: 1) The conductive material is sterilized and dispersed in deionized water, then incubated with a laminin solution to obtain a protein-modified conductive material. After redispersing, a conductive material dispersion is obtained; the conductive material is graphene nanosheets. 2) After centrifuging the stem cells, they were re-dispersed by pipetting with differentiation medium to obtain a cell suspension; the stem cells were neural stem cells; 3) Add a conductive material suspension to the neural stem cell suspension to obtain a mixture, and then incubate it; 4) Centrifuge the stem cells anchored with the conductive material, remove the supernatant, and obtain the conductive material-stem cell assembly; 5) Place the hydrogel at 37°C o Preheating in a water bath in the dark; the hydrogel is methacrylamide gelatin hydrogel. 6) Use 37 o The conductive material-stem cell assembly is suspended in a preheated hydrogel solution to obtain a conductive material-stem cell-hydrogel bio-ink.

2. The method for batch assembly of conductive material-stem cell-hydrogel bio-ink according to claim 1, characterized in that, In step 1), the size of the conductive material is 1-10 μm, and the sterilization method is to soak it in 75% alcohol for 0.5-3 hours. In step 1), the concentration of the conductive material is 1-10 mg / mL, the concentration of the laminin solution is 0.5-5 μg / mL, and the incubation temperature is 37°C. o C, incubation time is 1-8 h, and the concentration of conductive material dispersion is 10-1000 μg / mL.

3. The method for batch assembly of conductive material-stem cell-hydrogel bio-ink according to claim 1, characterized in that, In step 2), the neural differentiation medium is F12 medium containing 2% B27 neural culture additive, 1% N2 neural culture additive, 1% fetal bovine serum, 1% GlutaMax, and 1% penicillin-dextrose antibody; the cell concentration in the stem cell suspension is (0.5-10)×10⁻¹⁰. 6 cells / mL; In step 3), the ratio of stem cell suspension to conductive material suspension is 1:1, and the cell concentration is (0.25-5)×10⁻⁶. 6 The concentration of conductive material was 5-500 μg / mL, the oscillation frequency was 50-200 rpm, and the incubation temperature was 37°C. o C, incubation time is 0.5-12 h; In step 4), the centrifugation speed is 600-1300 rpm and the centrifugation time is 3-5 min.

4. The method for mass assembly of conductive material-stem cell-hydrogel bio-ink according to claim 1, characterized in that: In step 5), the concentration of the hydrogel is 2%-15%, and the hydrogel is kept at 37°C. o The preheating time for C is 5-30 minutes; In step 6), the concentration of stem cells in the conductive material-stem cell-hydrogel bio-ink is (0.5-10)×10⁻⁶. 7 per mL.

5. A conductive material-stem cell-hydrogel bio-ink, characterized in that, It is prepared by the method described in claim 1.

6. A method for constructing 4D-printed neural tissue, characterized in that... Includes the following steps: 1) The conductive material-stem cell-hydrogel bio-ink prepared according to claim 1 is placed in 4... o C pre-cooling; 2) 3D printing using pre-cooled conductive material-stem cell-hydrogel bio-ink; 3) Add neural differentiation culture medium to the 3D-printed living biological material and place it in an incubator at 37°C; 4) After cleaning the sample, replace it with a new culture medium; 5) Stimulate with a rotating magnetic field to construct 4D-printed neural tissue.

7. The method according to claim 6, characterized in that, In step 1), the conductive material-stem cell-hydrogel bio-ink is in 4 o The pre-cooling time for C is 5-20 minutes.

8. The method according to claim 6, characterized in that, In step 2), the air pressure for 3D printing is 0.1-0.5 MPa, and the platform temperature is -5 to 15 degrees Celsius. o C, needle temperature is 4-20°C o C, printing speed is 3-10 mm / s, and photocuring strength is 30-80 mW / cm². 2 The photocuring time is 10-60 seconds.

9. The method according to claim 6, characterized in that, In step 3), the incubation time in a 37℃ incubator is 15-30 min; In step 4), the washing is performed 1-3 times using neural differentiation culture medium; In step 5), the culture time is 5-15 days, the distance between the cell culture plate and the magnet is 0.5-1 cm, and the rotational magnetic field stimulation conditions are: rotation speed of 100-1000 rpm, magnetic field strength of 50-200 mT, stimulation time of 5-30 min each time, and stimulation 1-5 times a day.

10. A 4D-printed neural tissue, characterized in that: It is prepared by the method described in claims 6-9.

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