Hydrogel composite scaffold loaded with human spinal cord V3 precursor cells for spinal cord injury repair and preparation method of hydrogel composite scaffold

Abstract

The invention belongs to the field of biomedical materials, and particularly relates to a hydrogel composite scaffold loaded with human spinal cord V3 precursor cells for spinal cord injury repair and a preparation method of the hydrogel composite scaffold. The hydrogel composite scaffold comprises hydrogel and human spinal cord V3 neural precursor cells, hydrogel is used as a carrier, the human spinal cord V3 neural precursor cells are loaded on the hydrogel, and the hydrogel is formed by oxidized hyaluronic acid and carboxymethyl chitosan through a Schiff base cross-linking reaction. The specific preparation method comprises the following steps: mixing the human spinal cord V3 neural precursor cells with hydrogel to obtain the hydrogel composite scaffold loaded with the human spinal cord V3 neural precursor cells. Therefore, the technical problems of low cell survival rate, non-uniform cell distribution and difficulty in controlling the cell differentiation direction in spinal cord injury repair in the prior art are solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical materials, and more particularly relates to a hydrogel composite scaffold loaded with human spinal cord V3 precursor cells for spinal cord injury repair and a preparation method thereof. BACKGROUND

[0002] Spinal cord injury (SCI) is a severe central nervous system disease, which can cause severe loss of sensation and motor function below the injury plane, even permanent paralysis. The neurological dysfunction caused by SCI is a major clinical challenge, and its repair faces multiple obstacles such as inhibitory microenvironment, poor nerve regeneration ability, and easy secondary infection.

[0003] In recent years, cell replacement therapy has brought new hope for spinal cord injury repair. Among them, neural stem cell / progenitor cell transplantation is one of the most widely studied strategies. However, this therapy faces severe challenges: first, the survival rate of transplanted cells in the harsh microenvironment of the injury area (such as inflammation, ischemia, and excitotoxicity) is extremely low; second, transplanted cells are difficult to effectively migrate and integrate with the host neural network; third, the differentiation direction of transplanted cells is uncontrollable, and they are extremely likely to differentiate into astrocytes, which may exacerbate the formation of glial scar and even induce neuropathic pain, affecting the treatment effect.

[0004] In terms of cell selection, the key to achieving functional recovery lies in precisely replacing the specific neuron types lost in the injury. Studies have shown that spinal cord V3 interneurons are the core components of central pattern generators (CPGs) and are crucial for coordinating rhythmic movements. Therefore, transplanting V3 precursor cells is considered a promising strategy for restoring motor function. Human pluripotent stem cell technology has made it possible to scale the in vitro production of V3 precursor cells. However, how to safely and efficiently deliver these functionally specialized cells to the injury site and provide a suitable microenvironment for their survival, differentiation, and functional integration remains a bottleneck for current technology. SUMMARY

[0005] In view of the above defects or improvement needs of the prior art, the present application provides a hydrogel composite scaffold loaded with human spinal cord V3 precursor cells for spinal cord injury repair and a preparation method thereof, thereby solving the technical problems of low cell survival rate, uneven cell distribution, and difficult control of cell differentiation direction in spinal cord injury repair, and providing a functionalized scaffold that not only physically supports cells but also actively regulates the injury microenvironment, providing suitable three-dimensional support for cell survival, differentiation, and functional integration.

[0006] To achieve the above object, according to one aspect of the present application, a hydrogel composite scaffold loaded with human spinal cord V3 nerve precursor cells is provided, comprising a hydrogel and human spinal cord V3 nerve precursor cells; the hydrogel is used as a carrier, and the human spinal cord V3 nerve precursor cells are loaded on the hydrogel; the hydrogel is formed by Schiff base cross-linking reaction of oxidized hyaluronic acid and carboxymethyl chitosan.

[0007] According to another aspect of the present application, a preparation method of a hydrogel composite scaffold loaded with human spinal cord V3 nerve precursor cells is provided, which comprises mixing human spinal cord V3 nerve precursor cells with a hydrogel, and then obtaining a hydrogel composite scaffold loaded with human spinal cord V3 nerve precursor cells, wherein the hydrogel is formed by Schiff base cross-linking reaction of oxidized hyaluronic acid and carboxymethyl chitosan.

[0008] Preferably, the method specifically comprises the following steps: (1) oxidizing hyaluronic acid by using an oxidizing agent, adding ethylene glycol to terminate the reaction after the reaction is completed, and freeze-drying after dialysis to obtain oxidized hyaluronic acid; (2) alkalinizing chitosan in a strong alkali solution, then adding potassium iodide and chloroacetic acid, and reacting at 55-65°C for 1-3 hours, and freeze-drying after dialysis to obtain carboxymethyl chitosan; (3) dissolving the oxidized hyaluronic acid and the carboxymethyl chitosan in a phosphate buffer solution respectively to obtain an oxidized hyaluronic acid solution and a carboxymethyl chitosan solution respectively; (4) mixing the carboxymethyl chitosan solution with human spinal cord V3 nerve precursor cells first, then adding the oxidized hyaluronic acid solution, and obtaining the hydrogel composite scaffold loaded with human spinal cord V3 nerve precursor cells after mixing.

[0009] Preferably, the oxidizing agent is selected from one or more of sodium periodate, potassium periodate, and tetramethylpiperidine oxide; Preferably, the strong alkali solution is selected from one or more of sodium hydroxide or potassium hydroxide.

[0010] Preferably, the mass fraction of the solute in the strong alkali solution is 30-50%; and the alkalinization time is 5-7 hours.

[0011] Preferably, in step (1), the dialysis adopts a dialysis bag with a molecular weight cut-off of 3.5 kDa, and the dialysis is performed in deionized water for 48-96 hours to ensure complete removal of reaction byproducts.

[0012] Preferably, in step (3), the mass of the oxidized hyaluronic acid to the volume of the phosphate buffer solution is 2%-4%; and the mass of the carboxymethyl chitosan to the volume of the phosphate buffer solution is 2%-4%.

[0013] Preferably, the volume ratio of the hyaluronic acid oxidized solution to the carboxymethyl chitosan solution is 1: (1~3).

[0014] Preferably, in step (2), the chloroacetic acid is added in three times with an interval of 10~15 minutes; the mass of the potassium iodide is 2.5~3.5% of the mass of the chitosan.

[0015] According to another aspect of the present application, the use of the hydrogel composite scaffold loaded with human spinal cord V3 neural precursor cells for preparing a spinal cord injury repair preparation is provided.

[0016] Overall, compared with the prior art, the above technical solutions conceived by the present application can achieve the following beneficial effects: (1) The present application innovatively crosslinks hyaluronic acid oxidized and carboxymethyl chitosan to obtain a hydrogel, which is combined with human spinal cord V3 neural precursor cells to construct a new cell-scaffold composite treatment system. The hydrogel can safely and efficiently load and deliver human spinal cord V3 precursor cells, and also provides a long-term three-dimensional survival space for the cells after implantation. Meanwhile, the mechanical properties, degradation rate and biological activity of the hydrogel can actively regulate cell behavior and effectively resist the inhibitory microenvironment, ultimately achieving the goal of promoting axon regeneration, bridging the broken ends of the injury and reconstructing the nerve function. This system not only solves the problem of low cell survival rate and easy loss in spinal cord injury repair, but also inhibits the formation of glial scar, significantly improves the integration and functional reconstruction of cells in the injury area, and provides a comprehensive solution for spinal cord injury repair.

[0017] (2) The hydrogel formed by Schiff base crosslinking reaction of hyaluronic acid oxidized and carboxymethyl chitosan in the present application not only promotes the differentiation of V3 neural precursor cells to mature neurons, but also perfectly maintains the normal development of cytoskeleton and the specificity of neurotransmitter phenotype, which is crucial for generating functional neurons and is the key mechanism and significant advantage of the system for successful application in nerve regeneration.

[0018] (3) The self-healing hydrogel of hyaluronic acid oxidized-carboxymethyl chitosan prepared in the present application has good injectability and self-healing ability, can be smoothly extruded through a syringe, and the elastic modulus can quickly recover to close to the initial modulus after strain elimination; at the same time, the hydrogel has a three-dimensional porous network structure with interconnected pairs, and the pore size can reach more than 100 μm, meeting the demand of loading cells; the elastic modulus of the hydrogel is about 100 Pa, which matches the mechanical properties of central nervous tissue.

[0019] (4) The preparation process of the present application is mild and free of toxic reagents throughout, which greatly guarantees the cell activity, has good biocompatibility, and has broad clinical transformation prospects. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 A flow chart of the present application.

[0021] Figure 2 A scanning electron microscope image of the self-healing hydrogel of the present application, wherein A is an electron microscope image of the hydrogel with a volume ratio of OHA to CMCS of 1:2; B is an electron microscope image of the hydrogel with a volume ratio of OHA to CMCS of 1:3.

[0022] Figure 3 A display of the injectability of the self-healing hydrogel of the present application, Figure 3 A "HUST" logo injected by the self-healing hydrogel through a 1 mL syringe in A; Figure 3 B is the shear-thinning behavior test result of the OHA-CMCS hydrogel in B.

[0023] Figure 4 A display of the self-healing performance of the self-healing hydrogel of the present application, Figure 4 A macroscopic observation graph in A; Figure 4 B is the rheological self-healing performance test of the hydrogel in B.

[0024] Figure 5 The evaluation result of the self-healing hydrogel of the present application on the cytotoxic effect of SH-SY5Y cells. Among them, Figure 5 A statistical graph of cell activity detected by CCK-8 method in A; Figure 5 B is a live and dead cell staining fluorescence graph, wherein green fluorescence represents live cells and red fluorescence represents dead cells.

[0025] Figure 6 The evaluation result of the self-healing hydrogel of the present application on the growth of human spinal cord V3 neural precursor cells. Among them, Figure 6 A normal optical microscope observation graph in A; Figure 6 B is an immunofluorescence staining graph.

[0026] Figure 7 The evaluation result of the self-healing hydrogel of the present application loaded with V3 neural precursor cells on the recovery of motor function and tissue repair of spinal cord injury rats after transplantation. Among them, Figure 7 A statistical graph of the motor function of the hind limbs of rats in A; Figure 7 B is an HE staining section graph of the spinal cord tissue in the injury area. DETAILED DESCRIPTION

[0027] In order to make the objectives, technical solutions, and advantages of the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific examples described herein are merely intended to explain the present application and are not intended to limit the present application. Furthermore, the technical features involved in the various embodiments of the present application described below can be combined with each other as long as they do not conflict with each other.

[0028] Example 1 The present example provides a preparation method of a hydrogel composite scaffold loaded with human spinal cord V3 neural precursor cells, and the specific steps are as follows: (1) Preparation of oxidized hyaluronic acid (OHA), and the specific preparation steps are as follows: 2 g of hyaluronic acid was added to 200 ml of PBS buffer, and after complete dissolution by stirring, 1 g of sodium periodate was added to the solution. After 6 hours of reaction in the dark, 2 ml of ethylene glycol was added to terminate the reaction. After dialysis for three days, the oxidized hyaluronic acid was obtained by freeze-drying and was ready for use.

[0029] (2) Preparation of carboxymethyl chitosan (CMCS), and the specific preparation steps are as follows: 2 g of chitosan was dissolved in 12.5 g of 40% sodium hydroxide solution, and alkalization was performed for 6 hours. After alkalization, 3% potassium iodide was added as a catalyst, and then 6 g of chloroacetic acid was added in three portions with an interval of 10 minutes. The reaction was performed at 60°C for 2 hours. Finally, the carboxymethyl chitosan was obtained by dialysis and freeze-drying and was ready for use.

[0030] (3) Preparation of self-healing hydrogel, and the self-healing hydrogel was prepared by Schiff base reaction, and the specific steps are as follows: Oxidized hyaluronic acid and carboxymethyl chitosan were taken respectively, and then were dissolved in PBS buffer according to 3 (w / v)%. Then, the oxidized hyaluronic acid and the carboxymethyl chitosan were mixed according to a volume ratio of 1:3, and Schiff base crosslinking was performed, so as to obtain OHA-CMCS self-healing hydrogel.

[0031] (4) Human spinal cord V3 neural precursor cells were added to the OHA-CMCS self-healing hydrogel, so as to obtain a hydrogel composite scaffold loaded with human spinal cord V3 neural precursor cells.

[0032] The self-healing hydrogel prepared in the present example was freeze-dried, and its micro-morphology was observed by high-magnification scanning electron microscopy (SEM), and the results are shown in Figure 2 As can be clearly seen from the figure, the hydrogel has a continuous and uniform three-dimensional porous network structure, the pore size can reach more than 100 μm, the pore size distribution is appropriate, and the pores are interconnected. This structure is conducive to the transmission of nutrients and metabolic waste, and provides sufficient physical space for cell migration, proliferation, and extracellular matrix secretion, indicating that it is very suitable for cell loading and three-dimensional culture.

[0033] Comparative Example 1 Except that no sodium periodate was added to perform the oxidation reaction, the remaining steps were the same as Example 1. The obtained hyaluronic acid solution could not form a stable hydrogel after being mixed with the CMCS solution, and could not be used for cell loading.

[0034] Example 2 This example provides a hydrogel composite scaffold loaded with human spinal cord V3 neural precursor cells, and the specific steps are as follows: (1) Preparation of oxidized hyaluronic acid, and the specific preparation steps are as follows: 2g of hyaluronic acid was added to 200ml of PBS buffer, and after complete dissolution by stirring, 1g of sodium periodate was added to the solution. After 6 hours of reaction in the dark, 2ml of ethylene glycol was added to terminate the reaction. After dialysis for three days, the oxidized hyaluronic acid was obtained by freeze-drying and was ready for use.

[0035] (2) Preparation of carboxymethyl chitosan, and the specific preparation steps are as follows: 2g of chitosan was dissolved in 12.5g of 40% sodium hydroxide solution, and alkalization was performed for 6 hours. After alkalization, 3% potassium iodide was added as a catalyst, and then 6g of chloroacetic acid was added in three portions with an interval of 10 minutes. The reaction was performed at 60°C for 2 hours. Finally, carboxymethyl chitosan was obtained by dialysis and freeze-drying and was ready for use.

[0036] (3) Preparation of self-healing hydrogel, and the self-healing hydrogel was prepared by Schiff base reaction, which specifically included the following steps: Oxidized hyaluronic acid and carboxymethyl chitosan were taken respectively, and then dissolved in PBS buffer according to 3(w / v)%. Then, the oxidized hyaluronic acid and the carboxymethyl chitosan were mixed according to 1:3, and Schiff base crosslinking was performed, so that the OHA-CMCS self-healing hydrogel was obtained.

[0037] (4) Human spinal cord V3 neural precursor cells were added to the OHA-CMCS self-healing hydrogel to obtain a hydrogel composite scaffold loaded with human spinal cord V3 neural precursor cells.

[0038] Performance test of the self-healing hydrogel, including the following steps: The self-healing performance of the hydrogel was evaluated by two methods, namely macroscopic direct observation and rotational rheometer. The injectability of the hydrogel was evaluated by syringe extrusion method.

[0039] The self-healing hydrogel prepared in this example could also be extruded through a 1ml syringe, and the hydrogel could form a continuous and uniform extrusion flow and be accurately shaped into the "HUST" character. No obvious flow blockage or fracture phenomenon occurred, which directly proved that the hydrogel had excellent injectability and could meet the demand for precise filling of irregular defects in minimally invasive surgery applications (such as shown in Fig. A). Figure 3 Figure 3 ​The shear-thinning behavior of the OHA-CMCS hydrogel was tested, and the results showed that the viscosity of the hydrogel decreased significantly with the increase of the shear rate, exhibiting typical non-Newtonian fluid shear-thinning characteristics. The rheological behavior ensured that the hydrogel could be smoothly extruded by reducing the viscosity under the action of high shear force during injection, and quickly restored the viscosity to maintain the integrity of the three-dimensional structure after the injection was completed.

[0040] The macroscopic self-healing of the self-healing hydrogel obtained in this example is shown in Figure 4 As shown in FIG. 1A, the hydrogel was dyed using rhodamine B and methylene blue, and the hydrogel was cut into two pieces. Then, the two pieces of different colored hydrogels were placed together at room temperature for 2 min without any external stimulus. Subsequently, the hydrogel was completely healed into a whole, and no rupture occurred under the action of gravity, indicating that it has a fast and significant self-healing ability. The results of rheology are shown in Figure 4 As shown in FIG. 1B, it can be seen that the storage modulus of the hydrogel is about 100 Pa; then through the continuous step strain test, it can be seen that under the condition of alternating 1% (small strain, gel state) and 600% (large strain, sol state) strain, the storage modulus (G') and the loss modulus (G'') change accordingly; when the strain value increases from 1% to 600%, the storage modulus of the hydrogel decreases sharply and is lower than the loss modulus, at this time it indicates that the structure of the hydrogel is damaged. When the strain recovers from 600% to 1%, G' quickly recovers to the initial value, indicating that the hydrogel has experienced a rapid sol-gel transition, and the gel network structure is reversible and quickly rebuilt. This result shows that the prepared hydrogel has self-healing ability, can form a hydrogel in situ, and meets the application requirements of filling irregular wounds.

[0041] Biocompatibility test of the self-healing hydrogel: including the following steps: (1) The prepared hydrogel was placed in the complete culture medium of SH-SY5Y cells, and was immersed at 37°C, 5% CO2 for 24 hours. The supernatant was collected, filtered through a 0.22 μm filter membrane to remove bacteria, and the hydrogel extract was obtained.

[0042] (2) Human neuroblastoma cells (SH-SY5Y) were cultured in complete culture medium.

[0043] (3) The SH-SY5Y cells were inoculated into a 96-well plate at a density of 3×10 3 cells / well, the experimental group was replaced with the hydrogel extract, and the control group was cultured with complete culture medium. After 1-3 days of culture, the cell activity was detected by CCK-8 method.

[0044] (4) Similarly, the SH-SY5Y cells were inoculated onto a confocal culture dish, and the live and dead cells were observed by using live and dead cell staining, and the fluorescence image of live and dead cell staining was taken.

[0045] The biocompatibility of the obtained self-healing hydrogel is shown in Figure 5 Compared with the blank control group, the cell viability of the hydrogel extract group remained above 90% after 24, 48 and 72 hours of culture, and there was no statistical difference, indicating that the material extract had no cytotoxicity and had good biocompatibility. Figure 5 The live and dead staining results of the hydrogel group in Example B show that almost no dead cells are observed, and there is no obvious difference from the control group, further proving that the self-healing hydrogel has excellent cell compatibility and can provide a safe growth microenvironment for nerve cells. No obvious cell death is observed in the self-healing hydrogel of the present application, indicating that the hydrogel of the present application has good biocompatibility.

[0046] Characterization of the effect of the self-healing hydrogel on the survival and differentiation of human spinal cord V3 neural precursor cells: The specific steps are as follows: (1) The prepared hydrogel is placed in the culture medium of human spinal cord V3 neural precursor cells and extracted for 24 hours at 37°C and 5% CO2. The supernatant is collected, filtered through a 0.22 μm filter membrane to remove bacteria, and the hydrogel extract is obtained for use.

[0047] (2) Human pluripotent stem cells are induced to differentiate into human spinal cord V3 neural precursor cells under the action of various small molecules, and form neural spheres by aggregation.

[0048] (3) Human spinal cord V3 neural precursor cells are inoculated into a 24-well plate, supplemented with nutritional factors, further dissociated into neural progenitor cells, and differentiated into post-mitotic neurons.

[0049] (4) The culture medium of the experimental group is replaced with the differentiation culture medium of the hydrogel extract, and the control group uses the standard differentiation culture medium. Fresh culture medium or hydrogel extract is replaced every 2-3 days, and the results are observed after 7 days of culture.

[0050] (5) The growth and differentiation of cells are observed using an ordinary light microscope and immunofluorescence staining.

[0051] The results of the effect of the prepared self-healing hydrogel on the survival and differentiation of human spinal cord V3 neural precursor cells are as follows, as shown in Figure 6 The V3 neural precursor cells in the experimental group form regular neural spheres with clear boundaries, and the human spinal cord V3 neural progenitor cells enriched in the neural spheres cultured by the hydrogel can be well dissociated and differentiated into post-mitotic neurons, and the cell state is comparable to that of the control group, indicating that the hydrogel can effectively support the survival and growth of V3 cells. After 7 days of culture, immunofluorescence staining can be seen (as shown in Figure 6As shown in FIG. 12B, a large number of cells in the experimental group highly expressed the mature neuron marker MAP (red) and the V3 neuron-specific glutamatergic marker VGLUT2 (green), indicating that the self-healing hydrogel prepared by the application promotes the differentiation of V3 neural precursor cells into mature functional neurons without affecting the normal establishment of cytoskeleton structure or the specific maintenance of neurotransmitter phenotype. At the same time, the normal establishment of cytoskeleton structure and the formation of synaptic connections are maintained, providing an ideal cell carrier platform for spinal cord injury repair.

[0052] Comparative Example 2 Except that the volume ratio of OHA to CMCS is changed to 3:1, the remaining steps are the same as those in Example 2. The resulting hydrogel has a crosslinking time greater than 20 minutes, and the mechanical strength of the hydrogel is insufficient and does not meet the subsequent experimental requirements.

[0053] Example 3 A method for preparing a hydrogel composite scaffold loaded with human spinal cord V3 neural precursor cells, the specific steps being as follows: (1) A T9 spinal cord segment of a nude rat is exposed by laminectomy, and a 2mm full transection spinal cord injury model is constructed using a microscissors.

[0054] (2) The CMCS solution obtained in Example 2 is mixed with human spinal cord V3 neural precursor cells (10 6 cells), and then mixed with the oxidized hyaluronic acid solution to form a hydrogel composite scaffold loaded with human spinal cord V3 neural precursor cells.

[0055] (3) Immediately after modeling, transplantation is performed. The cell-loaded hydrogel is injected into the center of the injury area by a microsyringe, and the transplantation surgery is completed. The muscle, fascia and skin are sutured layer by layer.

[0056] (4) At 16 weeks after the operation, behavioral assessment is performed, and at 16 weeks, the spinal cord tissue is removed under anesthesia, fixed in 4% paraformaldehyde for 24 hours, and finally dehydrated in a 30% sucrose solution until the tissue sinks. Subsequently, the frozen section is stained, and the distribution of neurons and the condition of glial scar are observed and photographed under a confocal microscope.

[0057] The present embodiment establishes a rat T9 segment spinal cord full transection injury model, and systematically evaluates the repair effect of the self-healing hydrogel loaded with human spinal cord V3 neural precursor cells in vivo. First, the Basso, Beattie, Bresnahan (BBB) motor function score scale is used to detect the recovery of hind limb motor function. The results show that the hydrogel combined with V3 cell treatment group of rats has the most significant recovery of hind limb motor function, and the score reaches (7.8±0.48) points at 16 weeks after operation, which is significantly higher than that of the control SCI group, indicating that the hind limb function of the rats has been significantly improved, and this combined treatment method can effectively promote the reconstruction of motor function. At 16 weeks after operation, the tissue morphology of the injury area is analyzed by hematoxylin-eosin (HE) staining. Compared with the blank control group (only injury without treatment), the simple hydrogel group and the simple V3 cell transplantation group, the tissue structure of the injury area of the hydrogel combined with V3 cell treatment group is the most complete, and the cavity area is significantly smaller than that of the other groups, indicating that this combined treatment method has good tissue bridging and repair support ability. The above results show that the self-healing hydrogel loaded with V3 neural precursor cells can significantly promote the structural reconstruction and functional recovery after spinal cord injury through multiple synergies such as reducing the cavity area, inhibiting the formation of glial scar and supporting the differentiation of functional neurons.

[0058] The above embodiments show that the OHA / CMCS self-healing hydrogel prepared by the present application can effectively load human spinal cord V3 neural precursor cells, promote their differentiation into functional neurons, and show good therapeutic effect in the repair of spinal cord injury.

[0059] It is easily understood by those skilled in the art that the above description is only the preferred embodiment of the present application, and is not used to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A hydrogel composite scaffold loaded with human spinal cord V3 neural progenitor cells, characterized in that, It includes a hydrogel and human spinal cord V3 neural progenitor cells; the human spinal cord V3 neural progenitor cells are loaded on the hydrogel as a carrier; the hydrogel is formed by oxidized hyaluronic acid and carboxymethyl chitosan through a Schiff base crosslinking reaction.

2. The method for preparing a hydrogel composite scaffold loaded with human spinal cord V3 neural progenitor cells as described in claim 1, characterized in that, Human spinal cord V3 neural progenitor cells were mixed with hydrogel to obtain a hydrogel composite scaffold loaded with human spinal cord V3 neural progenitor cells. The hydrogel was formed by a Schiff base crosslinking reaction of oxidized hyaluronic acid and carboxymethyl chitosan.

3. The method for preparing a hydrogel composite scaffold loaded with human spinal cord V3 neural progenitor cells as described in claim 2, characterized in that, Specifically, the steps include the following: (1) Oxidize hyaluronic acid with an oxidizing agent. After the reaction is complete, add ethylene glycol to terminate the reaction. After dialysis, freeze dry to obtain oxidized hyaluronic acid. (2) Alkalize chitosan in an alkaline solution, then add potassium iodide and chloroacetic acid, react at 55~65℃ for 1~3 hours, dialyze and freeze dry to obtain carboxymethyl chitosan; (3) Dissolve the oxidized hyaluronic acid and the carboxymethyl chitosan in phosphate buffer solution respectively to obtain oxidized hyaluronic acid solution and carboxymethyl chitosan solution respectively; (4) The carboxymethyl chitosan solution is first mixed with human spinal cord V3 neural progenitor cells, and then the oxidized hyaluronic acid solution is added. After mixing, the hydrogel composite scaffold loaded with human spinal cord V3 neural progenitor cells is obtained.

4. The method for preparing a hydrogel composite scaffold loaded with human spinal cord V3 neural progenitor cells as described in claim 3, characterized in that, The oxidant is selected from one or more of sodium periodate, potassium periodate, and tetramethylpiperidine oxide; The alkaline solution is selected from one or more of sodium hydroxide or potassium hydroxide.

5. The method for preparing a hydrogel composite scaffold loaded with human spinal cord V3 neural progenitor cells as described in claim 3, characterized in that, The mass fraction of the solute in the alkaline solution is 30-50%; the alkalization time is 5-7 hours.

6. The method for preparing a hydrogel composite scaffold loaded with human spinal cord V3 neural progenitor cells as described in claim 3, characterized in that, In step (3), the mass ratio of the oxidized hyaluronic acid to the volume ratio of the phosphate buffer is 2% to 4%; the mass ratio of the carboxymethyl chitosan to the volume ratio of the phosphate buffer is 2% to 4%.

7. The method for preparing a hydrogel composite scaffold loaded with human spinal cord V3 neural progenitor cells as described in claim 6, characterized in that, The volume ratio of the oxidized hyaluronic acid solution to the carboxymethyl chitosan solution is 1:(1~3).

8. The preparation method according to claim 3, characterized in that, In step (2), the chloroacetic acid is added in three portions, with an interval of 10 to 15 minutes between each addition; the mass of the potassium iodide is 2.5 to 3.5% of the mass of the chitosan.

9. The application of the hydrogel composite scaffold loaded with human spinal cord V3 neural progenitor cells according to claim 1 in the preparation of spinal cord injury repair agents.

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