CN114606189A

CN114606189A - Acellular spinal cord-GelMA hydrogel composite material bracket for promoting proliferation and differentiation of neural stem cells

Info

Publication number: CN114606189A
Application number: CN202210268899.XA
Authority: CN
Inventors: 居丁玥; 董传明; 王庆华; 杨丹; 陈世园; 何文华
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2022-06-10

Abstract

The invention provides an acellular spinal cord-GelMA hydrogel composite scaffold for promoting proliferation and differentiation of neural stem cells, and relates to the technical field of regenerative medicine and tissue engineering. The application provides a good microenvironment for NSCs by constructing the acellular spinal cord/GelMA composite scaffold, promotes the survival and migration of NSCs, and regulates and controls the proliferation and differentiation behaviors of NSCs. The acellular spinal cord retains hyaluronic acid, laminin and growth factors required by neuron growth, provides nutrients for neural stem cells, and the GelMA hydrogel makes up for the defect that the acellular spinal cord cannot be stable for a long time, and provides a long-term stable growth environment for the neural stem cells.

Description

Acellular spinal cord-GelMA hydrogel composite material bracket for promoting proliferation and differentiation of neural stem cells

Technical Field

The invention relates to the technical field of regenerative medicine and tissue engineering, in particular to a decellularized spinal cord-GelMA hydrogel composite material bracket for promoting proliferation and differentiation of neural stem cells.

Background

The incidence of Spinal Cord Injury (SCI) is increasing worldwide. SCI leads to massive neuronal cell death, leading to motor and sensory dysfunction. Since the spinal cord has little ability to regenerate, mature nerve cells cannot divide and proliferate to replace lost cells after spinal cord injury, and glial cells at the site of injury are activated, proliferate extensively to form glial scars, and the myelin sheaths of damaged cells produce large amounts of factors that inhibit axonal growth, such as: nogo, myelin associated protein (MAG), oligodendrocyte-myelin glycoprotein (OMgp), and the like. These internal and external factors form a microenvironment that inhibits nerve regeneration.

With the development of stem cell research, stem cell transplantation is expected to promote anatomical repair and functional recovery after spinal cord injury. Neural Stem Cells (NSCs) are the most promising therapeutic strategy for injuries to the nervous system and neurodegenerative diseases. NSCs have certain capacity of differentiating into neurons, astrocytes and oligodendrocytes, can replace necrotic cells, and promote repair of spinal cord structure and function. However, transplanted neural stem cells face a series of problems: the damaged area can lead to extensive cell death, poor neuronal differentiation and leakage of neural stem cells from the site of injury.

Exogenously engineered scaffolds have great potential for nerve regeneration and functional recovery in vivo, and can also overcome many of the problems faced in transplanting stem cells in the damaged spinal cord. The tissue engineering scaffold can fill a diseased cavity, improve the microenvironment of a damaged area, and promote the proliferation, migration and differentiation of endogenous and exogenous cells. Ideally, the tissue engineering scaffold should have good biocompatibility, be non-toxic, and promote nerve regeneration. Here we have selected a natural biomaterial acellular spinal cord extracellular matrix. Compared with synthetic materials, the decellularized spinal cord has a natural three-dimensional network structure, and contains laminin and proteoglycan necessary for survival and growth of motor neurons. The cell components are removed, and the immunogenicity is greatly reduced. The acellular extracellular matrix (dECM) can also be used as a carrier to carry medicaments, stem cells and nutritional factors and promote nerve regeneration at the damaged part. Therefore, the method has wide application in tissue engineering and regenerative medicine. However, decellularized spinal cords often do not perfectly match the site of injury. Researchers have found that decellularized spinal cords can be digested with enzymes to yield a decellularized spinal cord solution, a thermal gel hydrogel, that can be injected into the injury site, and that fits completely with the injury site. In vitro cell experiment results show that the acellular spinal cord scaffold can promote the adhesion, proliferation and differentiation of NSCs. However, the acellular spinal cord scaffold has poor structural stability and mechanical property and high degradation speed.

Disclosure of Invention

The invention aims to solve the technical problems of crossed structural stability and mechanical property and high degradation speed of a decellularized spinal cord scaffold in the prior art, and methacrylic anhydride gelatin (GelMA) prepared from methacrylic anhydride and gelatin is introduced. It is a photosensitive biological hydrogel material. The double bond structure of GelMA has been modified to be crosslinkable and prone to change its mechanical properties. GelMA hydrogels have been demonstrated to have low cytotoxicity and high cell viability. GelMA hydrogels may lack sufficient bioactivity and have limited healing efficacy compared to natural biomaterials. Therefore, we mixed GelMA hydrogel with decellularized spinal cord solution as a new composite scaffold for research. The combination of the two prolongs the molecular chain of the photosensitive molecule, can obtain the characteristics of high water content and soft elasticity, and is more suitable for cell growth. In vitro experiments show that the composite scaffold has good biocompatibility and supports cell proliferation and differentiation. This provides an opportunity for the formulation of effective transplantation strategies and a new idea for promoting the formation of new neuronal networks and functional connections.

In order to achieve the purpose, the invention adopts the following technical scheme:

a acellular spinal cord-GelMA hydrogel composite scaffold for promoting proliferation and differentiation of neural stem cells consists of acellular matrix liquid prepared from adult pig acellular spinal cords and GelMA hydrogel.

Preferably, the mixing ratio of the acellular matrix liquid to the GelMA hydrogel is 1:9 or 3: 7.

Preferably, the GelMA concentration is 5%.

The application also provides a preparation method of the acellular spinal cord-GelMA hydrogel composite scaffold for promoting proliferation and differentiation of neural stem cells, which comprises the following steps:

s1: preparing adult pig acellular spinal cords;

s2: preparing GelMA hydrogel;

s3: and (3) preparing a mixed scaffold of the decellularized spinal cord and GelMA.

Preferably, the specific step of S3 is: mixing the dissolved acellular solution with 5% GelMA hydrogel according to a certain proportion, and carrying out photocrosslinking under ultraviolet illumination to obtain the acellular scaffold solution-GelMA hydrogel composite material.

Preferably, the GelMA concentration in S3 is 5%, and the mixing ratio of the acellular matrix solution to the GelMA hydrogel is 1:9 or 3: 7.

The method for preparing the acellular spinal cord-GelMA hydrogel composite scaffold for promoting the proliferation and differentiation of neural stem cells according to claim 5, wherein the acellular spinal cord-GelMA hydrogel composite scaffold comprises the following steps: the wavelength of ultraviolet light in the S3 is 405nm, and the illumination time is 18 seconds.

The application also provides a verification method of the acellular spinal cord-GelMA hydrogel composite material bracket for promoting the proliferation and differentiation of the neural stem cells, which comprises the following steps: comprises the following steps:

a1: separating and culturing neural stem cells;

a2: staining live and dead cells;

a3: staining with phalloidin;

a4: EdU proliferation assay;

a5: NSCs differentiation assay.

The application provides a decellularized spinal cord-GelMA hydrogel composite material support for promoting proliferation and differentiation of neural stem cells, which overcomes the defects of the prior technology and method that NSCs are difficult to transplant after SCI and cell death is caused by an inhibitory microenvironment at a damage part, the problem can be effectively avoided by applying the composite support constructed by the inventor, and the microenvironment formed by the composite support is beneficial to regulating and controlling the behaviors of NSCs. The application provides a good microenvironment for NSCs by constructing the acellular spinal cord-GelMA composite scaffold, promotes the survival and migration of NSCs, and regulates and controls the proliferation and differentiation behaviors of NSCs. The acellular spinal cord retains hyaluronic acid, laminin and some growth factors required by neuron growth, provides nutrients for the neural stem cells, and the GelMA hydrogel makes up for the defect that the acellular spinal cord cannot be stable for a long time, and provides a long-term stable growth environment for the neural stem cells.

Drawings

FIG. 1 is a schematic diagram of the physical properties of GelMA hydrogel, such as its polymerizability, stability and adhesion, according to an embodiment of the present invention;

FIG. 2 is a sample and analysis diagram during the preparation of a mixed spinal cord and GelMA scaffold according to an embodiment of the present invention;

FIG. 3 is a graph of the identification of neural stem cells and the assessment of the biocompatibility of the sample by a live and dead cell staining test;

FIG. 4 is a schematic representation of a sample after phalloidin staining;

FIG. 5 is a sample and analytical chart during an EdU proliferation experiment;

FIG. 6 is a sample and analysis chart of neural stem cell differentiation experiment process.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments.

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.

The application provides an acellular spinal cord-GelMA hydrogel composite material bracket for promoting proliferation and differentiation of neural stem cells, which consists of acellular matrix liquid prepared from adult pig acellular spinal cords and GelMA hydrogel.

In one embodiment, the mixing ratio of the acellular matrix solution to the GelMA hydrogel is 1:9 or 3: 7. The GelMA concentration is 5%.

Referring to fig. 1, the present application also provides a method for preparing a decellularized spinal cord-GelMA hydrogel composite scaffold for promoting proliferation and differentiation of neural stem cells, which is used for preparing the above decellularized spinal cord-GelMA hydrogel composite scaffold, and comprises the following steps:

s1: adult pig decellularized spinal cord preparation:

specifically, in one embodiment, the method comprises the following steps:

1. taking the adult pig thoracic spinal cord, removing the dura mater, and longitudinally cutting into 4cm sections. And washing with PBS buffer solution to remove surface blood stains.

2. The treated spinal cords were stored frozen at-80 ℃ for 2 hours and thawed at room temperature.

3. The spinal cord is soaked in distilled water for 6 hours at room temperature, and the liquid is changed every 1 hour.

4. The spinal cord was placed in 3% TritonX-100/0.01MPBS solution and shaken continuously for 12 hours (25 ℃, 120 rpm).

5. Shaking and soaking with distilled water for 3 hours, and changing the solution for 1 time in 1 hour.

6. Shaking 4% sodium deoxycholate/0.01 MPBS solution was continued for 12 hours (25 ℃, 120 rpm).

7. Soaking in distilled water for 3 hr, and changing the solution 1 time after 1 hr.

8. And (4) repeating the steps (4) to (7) once.

9. Defatting with 4% ethanol for 12 hr.

10. The spinal cord decellularized scaffold was lyophilized in a lyophilizer for at least 24 h.

11. The freeze-dried decellularized scaffold was minced with sterile surgical scissors.

12. The minced scaffolds were placed on a constant temperature shaker (37 ℃ 350 rpm) and dissolved for 12h with pepsin solution (1 mg pepsin in 1ml 0.01 MHCl). 5mg of the scaffold was dissolved in 1mL of pepsin solution.

13. Undigested particles were ultracentrifuged at 30000 rpm for 30 minutes. The pH of the solution was adjusted to around 7.4 with 0.1N NaOH, the ionic strength was adjusted using 10 XPBS, and the pre-gel solution was aliquoted and stored at-80 ℃ until use.

S2: preparation of GelMA hydrogel:

specifically, the method comprises the following steps:

1. preparing 0.25% (w/v) photoinitiator solution;

(1) 20ml of sterile 1 XPBS were added to a brown bottle (0.05 g in) containing the photoinitiator;

(2) dissolving in 40-50 deg.C water bath for 15 min while shaking for several times.

2. Preparing GelMA solution (the concentration of GelMA is 5%)

(1) Putting GelMA with required mass into a centrifuge tube;

(2) adding a photoinitiator standard solution into the centrifugal tube, and oscillating to fully soak GelMA;

(3) heating in 60-70 deg.C water bath in dark place for 15 min, and oscillating for several times;

(4) the GelMA solution was immediately sterilized (to prevent low temperature gelation) with a 0.22 μm sterile needle filter.

Referring to fig. 1, fig. 1 shows the polymerization, stability and physical properties of GelMA hydrogel. Wherein (A, B, C) polymerization properties: the GelMA polymerization capacities were 2.5%, 5% and 7.5% (wt/vol) at different LAP concentrations and UV irradiation times, respectively. "+ stable" means that the hydrogel can polymerize into a gel, and "-unstable" means that the hydrogel cannot polymerize into a gel. In FIG. 1 (D, E) the polymerized hydrogel was immersed in 1 XPBS for 24 hours at 37 ℃ to ensure structural integrity. Fig. 1 (F) porosity of GelMA hydrogel. The GelMA hydrogel of fig. 1 (G) can be adhered to a smooth glass plate. Significant level isP < 0.05，**P < 0.01。

S3: preparing a mixed scaffold of the decellularized spinal cord and GelMA:

specifically, in one embodiment, the dissolved acellular solution and 5% GelMA hydrogel are mixed according to a certain proportion, and are subjected to photocrosslinking under ultraviolet illumination, so that the acellular scaffold solution-GelMA hydrogel composite material is obtained.

Specifically, the method comprises the following specific steps:

1. the preparation method of GelMA comprises the following steps: 0.1g GelMA was dissolved in 2mL of the photoinitiator solution. Namely 5 percent of mass ratio;

2. mixing with the acellular matrix solution according to different volume ratios, wherein in one embodiment, the mixing ratio of the acellular matrix solution to GelMA is as follows: acellular matrix liquid: GelMA =1:9 (designated 1E 9G), and in another embodiment, the acellular matrix fluid: GelMA =3:7 (named 3E 7G);

3. the ultraviolet light wavelength is 405nm, and the illumination time is 18 seconds.

Referring to fig. 2, we first obtain decellularized spinal cord ECM by physical and chemical methods using porcine spinal cord as raw material (fig. 2A). We observed no nuclear signal in histological hematoxylin-eosin (H & E) staining after decellularization (fig. 2B). The DNA content in the decellularized spinal cord was significantly reduced: 62.7. + -. 5.4 ng DNA/mg wet tissue in decellularized spinal cord was compared to 2184.14. + -. 149.27 ng DNA/mg wet tissue in native spinal cord (FIG. 2C). It indicates that the cells were almost completely removed. The dECM was lyophilized and digested to dECM-gel, which was mixed with varying proportions of 5% GelMA. The compressive moduli of the GelMA, 1E9G, and 3E7G stents were 8.14. + -. 0.76 kPa, 6.04. + -. 0.68 kPa, and 4.02. + -. 0.32 kPa, respectively (FIG. 2D). The results show that the stiffness of the 3E7G scaffold was the lowest among the three different concentrations of GelMA. The high water content and wettability of the scaffold facilitates the transport of nutrients to the cells. A smaller contact angle results in a stronger wettability. In our experiments, the porosity of GelMA, 1E9G, and 3E7G scaffolds were 62.05 ± 2.1%, 73.81 ± 2.48%, 84.15 ± 2.82%, respectively; the contact angles were 27.2 + -0.64 deg., 23.3 + -1.02 deg., and 17.37 + -0.45 deg., respectively (FIG. 2E-F). The water retention capacity of the 3E7G scaffold was higher than that of the GelMA and 1E9G scaffolds at different time points. This is probably due to the lower crosslink density, larger pores and better water retention of the 3E7G hydrogel (fig. 2G). Therefore, the 3E7G hydrogel has the highest wettability and hydrophilicity, and is more favorable for cell growth than GelMA and 1E9G scaffolds. This offers potential for future in vivo applications. There were no significant differences in degradation rates for these three scaffolds (FIG. 2H).

The application also provides a verification method of the acellular spinal cord-GelMA hydrogel composite material bracket for promoting the proliferation and differentiation of the neural stem cells, which comprises the following steps:

a1: isolation and culture of neural stem cells

NSCs were isolated from embryonic hippocampus of day 15 pregnant SD rats and placed in fresh ice-cold Dulbecco's modified Eagle Medium/F12 (DMEM/F12). Hippocampus tissues were separated into approximately 1mm volumes using sterile microtubes³The separated tissue pieces were transferred to a 15ml centrifuge tube, pipetted to a chyliform state, passed through a cell net (40 μm), centrifuged (1000 r/min, 3min), the supernatant was discarded, added to a petri dish, and placed in an incubator (37 ℃, 5% CO)₂) And (4) medium culture. The cell suspension was cultured in the proliferative NSCs medium DMEM/F12 containing 2% B27, 20 ng/mL EGF, 20 ng/mL FGF, 1% penicillin-streptomycin. After 4-5 days, primary neurospheres were harvested and dissociated into single cells using Accutase, and cultured in fresh proliferation medium. The NSCs used in this study were between generations 3-4.

Referring to FIG. 3, NSCs were isolated from hippocampus pregnant with 15 embryos in the presence of Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF). During passage, NSCs retained their stem cell characteristics and stained positive for SOX2 and Nestin (FIG. A). The biocompatibility of the composite scaffold was assessed by live/dead cell assay. After 3 days of co-culture with the composite scaffolds, different fields of view were randomly selected and the number of live and dead cells counted. Cell viability for GelMA, 1E9G, and 3E7G scaffolds were 78.91% ± 1.74%, 85.75% ± 1.47%, and 94.03% ± 2.63%, respectively (fig. B-C). The results show that the cell viability of the 3E7G scaffold is much higher than that of the other two scaffolds (II) ((III))p < 0.01）。

A2: staining of live and dead cells

Separating the NSCs at 5 × 10⁴The concentration of individual cells/well was seeded on poly-L-lysine pre-coated 24-well plates and co-cultured with the composite scaffold. Cell behavior was examined in two-dimensional culture environments with different concentrations of composite scaffolds. After 3 days of culture, the effect of different scaffolds on the survival of NSCs was evaluated. The Calcein-AM/PI (YEASEN, China) kit was used to inoculate the viability of NSCs. The reaction solution was added to the plate and incubated at 37 ℃ for 30 minutes according to the manufacturer's instructions. After washing several times with 1 × PBS, the samples were observed with an inverted fluorescence microscope (Ts 2R-FL, Nikon).

A3: coprinus cinereus cyclopeptide staining

Injecting the composite scaffold into 48-well plate, irradiating with ultraviolet light (450 nm), and inoculating 2 × 10⁴Individual cells/well. Cells were cultured for 3 days, the medium was aspirated, cells were washed 2 times with 1 × PBS pre-warmed at 37 ℃, and fixed with 4% paraformaldehyde at room temperature for 10 minutes. The cells were then infiltrated with 0.5% Triton X-100 (Sigma, USA) for 5 minutes and TRITC-phalloidin (YEASEN, China) was incubated for 30 minutes at room temperature in the dark. After washing with 1 × PBS, nuclei were stained with DAPI. The samples were observed using an inverted fluorescence microscope (Ts 2R-FL, Nikon).

Referring to fig. 4, adhesion is critical for cell survival and growth. Actin filaments were stained with phalloidin to assess the spreading of the cytoskeleton on different scaffolds. According to the figure, it can be seen that the expansion and migration of NSCs on the surface of the scaffolds of 1E9G and 3E7G are greater, the fusion of adherent cells is also higher, while the morphological extension of GelMA surface cells is shorter, and the number of adherent cells is also less.

A4: EdU proliferation assay

For the EdU assay, NSCs cells co-cultured with the composite scaffold were incubated with 50 μ M proliferation diffusion assay kit (RiboBio, china) at 37 ℃ for 2 hours and then fixed with 4% paraformaldehyde for 30 minutes at room temperature. The fixative was discarded and incubated for 5 minutes with 2mg/ml glycine. After washing twice with 1 XPBS, 0.5% Triton X-100 was added to the osmotic shaker and incubated for 10 minutes. Next, the cells were incubated for 30 minutes with 200. mu.l of 1X Apollo ® dyeing reaction solution. After washing with 1 × PBS, Nestin (Abcam, usa) was incubated at room temperature for 2 hours, and finally nuclei were stained with DAPI for 10 minutes. The samples were observed using an inverted fluorescence microscope (Ts 2R-FL, Nikon).

Referring to FIG. 5, the proliferative capacity of NSCs co-cultured with scaffolds is indicated by EdU. After 3 days of culture, a large amount of EdU was observed in the 3E7G group⁺A cell. Statistical analysis showed that the proliferation rate of cells in group 3E7G was higher than that in GelMA and 1E 9G. (3E7G: 24.81% + -1.06%, 1E9G: 15.09% + -0.96%, GelMA: 11.69% + -1.7%,p <0.01) (FIGS. A-B). The CCK8 assay can be used to assess cell viability. After 1, 3 and 7 days of culture, the cell viability of the 3E7G group was significantly higher than that of the GelMA and 1E9G groups (p<0.05) (fig. C). These results indicate that group 3E7G has good biocompatibility and can provide better support for cell proliferation.

A5: NSCs differentiation assay

The neurospheres were grown logarithmically and digested into single cells, seeded on poly-L-lysine precoated 24-well plates, approximately 10%⁵Individual cells/well, co-cultured with composite scaffolds, placed in 2% FBS, 1% PS in DMEM/F12 medium. At 37 ℃ in 5% CO₂And (4) carrying out incubation. Then, immunofluorescent staining was performed on

days

7 and 14, respectively.

Referring to fig. 6, to explore whether the composite scaffolds modulate the differentiation of NSCs, we examined the expression levels of Tuj1 (early neuronal marker) and GFAP (astrocytic marker) by immunofluorescence and western blot. NSCs were co-cultured with composite scaffolds and cultured in NSC differentiation medium for 7 days. The results showed a significant increase in the number of Tuj 1-positive cells and a significant decrease in the number of GFAP-positive cells in group 3E7G compared to GelMA and group 1E9G (FIGS. A-C). The protein expression level was consistent with the immunofluorescence results (fig. D), indicating that 3E7G group cultured NSCs promoted neuronal differentiation and inhibited astrocyte differentiation. In addition, the composite scaffold promotes neurite outgrowth of neurospheres. The bright field photograph results showed that the average neurite length (31.03. + -. 3.55 mm) on the 3E7G scaffold was longer than that of the GelMA (10.21. + -. 0.99 mm) and 1E9G scaffolds (16.81. + -. 1.47 mm). The longest length on the 3E7G scaffold exceeded 150 μm (FIGS. E-F).

The application provides a decellularized spinal cord-GelMA hydrogel composite material support for promoting proliferation and differentiation of neural stem cells, which overcomes the defects of the prior technology and method that NSCs are difficult to transplant after SCI and cell death is caused by an inhibitory microenvironment at a damage part, the problem can be effectively avoided by applying the composite support constructed by the inventor, and the microenvironment formed by the composite support is beneficial to regulating and controlling the behaviors of NSCs. The application provides a good microenvironment for NSCs by constructing the acellular spinal cord-GelMA composite scaffold, promotes the survival and migration of NSCs, and regulates and controls the proliferation and differentiation behaviors of NSCs. The acellular spinal cord retains hyaluronic acid, laminin and growth factors required by neuron growth, provides nutrients for neural stem cells, and the GelMA hydrogel makes up for the defect that the acellular spinal cord cannot be stable for a long time, and provides a long-term stable growth environment for the neural stem cells.

The above description is only an example of the present invention, and is not intended to limit the present invention, and it is obvious to those skilled in the art that various modifications and variations can be made in the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A acellular spinal cord-GelMA hydrogel composite scaffold for promoting proliferation and differentiation of neural stem cells is characterized in that: consists of acellular matrix liquid prepared from adult pig acellular spinal cords and GelMA hydrogel.

2. The acellular spinal cord-GelMA hydrogel composite scaffold for promoting proliferation and differentiation of neural stem cells according to claim 1, wherein: the mixing ratio of the acellular matrix liquid to the GelMA hydrogel is 1:9 or 3: 7.

3. The acellular spinal cord-GelMA hydrogel composite scaffold for promoting proliferation and differentiation of neural stem cells according to claim 1, wherein: the GelMA concentration is 5%.

4. A preparation method of a decellularized spinal cord-GelMA hydrogel composite material bracket for promoting proliferation and differentiation of neural stem cells is characterized in that: comprises the following steps:

s1: preparing adult pig acellular spinal cords;

s2: preparing GelMA hydrogel;

5. The method for preparing the acellular spinal cord-GelMA hydrogel composite scaffold for promoting the proliferation and differentiation of neural stem cells according to claim 4, wherein the acellular spinal cord-GelMA hydrogel composite scaffold comprises the following steps: the specific steps of S3 are as follows: mixing the dissolved acellular solution with 5% GelMA hydrogel according to a certain proportion, and carrying out photocrosslinking under ultraviolet illumination to obtain the acellular scaffold solution-GelMA hydrogel composite material.

6. The method for preparing the acellular spinal cord-GelMA hydrogel composite scaffold for promoting the proliferation and differentiation of neural stem cells according to claim 5, wherein the acellular spinal cord-GelMA hydrogel composite scaffold comprises the following steps: the GelMA concentration in the S3 is 5%, and the mixing ratio of the acellular matrix liquid to the GelMA hydrogel is 1:9 or 3: 7.

7. The method for preparing the acellular spinal cord-GelMA hydrogel composite scaffold for promoting the proliferation and differentiation of neural stem cells according to claim 5, wherein the acellular spinal cord-GelMA hydrogel composite scaffold comprises the following steps: the wavelength of ultraviolet light in the S3 is 405nm, and the illumination time is 18 seconds.

8. A verification method of a decellularized spinal cord-GelMA hydrogel composite material bracket for promoting proliferation and differentiation of neural stem cells comprises the following steps: comprises the following steps:

a1: separating and culturing neural stem cells;

a2: staining live and dead cells;

a3: staining with phalloidin;

a4: EdU proliferation assay;

a5: NSCs differentiation assay.