CN110101918B - Hierarchical pore functional scaffold material for mobilizing endogenous neural stem cells to repair spinal cord injury and preparation method and application thereof - Google Patents
Hierarchical pore functional scaffold material for mobilizing endogenous neural stem cells to repair spinal cord injury and preparation method and application thereof Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
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Abstract
The invention relates to a hierarchical pore functional scaffold material for mobilizing endogenous neural stem cells to repair spinal cord injury, a preparation method and application thereof, wherein the material is prepared by the following method: mixing double bond-modified chitosan with cross-linking agent BIS, 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone and distilled water to obtain precursor solution, optionally adding growth factor to obtain gel precursor solution, placing in liquid nitrogen, guiding by ice crystal, and ultraviolet-irradiating at-20 deg.C to initiate gelling. The material provided by the invention is provided with guide holes with different inner diameters along the long axis of the material, provides a space structure required by nerve loop reconstruction, facilitates directional migration of endogenous neural stem cells to a material transplanting area and formation of new neurons, and enables nerve synapses to grow along a designed multi-stage structure to form a nerve network, reconstruct the nerve loop of an injured area and promote motor function recovery after spinal cord injury.
Description
Technical Field
The invention relates to the technical field of nerve regeneration repair, in particular to a hierarchical pore functional scaffold material for mobilizing endogenous neural stem cells to repair spinal cord injury and a preparation method and application thereof.
Background
Spinal Cord Injury (SCI) generally refers to direct spinal cord injury caused by trauma or other various causes, resulting in loss of motor and sensory functions to varying degrees below the level of the injury, and even complete loss of motor and sensory functions resulting in paraplegia. SCI has a high incidence, and may cause other secondary diseases besides quadriplegia and hemiparalysis of the body, which seriously affect the quality of life of the patient. The treatment and rehabilitation of SCI has been one of the major problems in the field of basic and clinical medicine.
Spinal cord injury initially often leads to constant loss of cells and tissues, and tissue engineering scaffolds can mimic the physiological state of extracellular matrix, thereby facilitating cell adhesion, migration, expansion and differentiation. Tissue engineering to repair spinal cord injury focuses on three aspects: seed cells, tissue engineering scaffolds, materials and cytokines. Materials for tissue engineering scaffolds can be classified into natural materials, which are generally polymers extracted from proteins or carbohydrates, and synthetic materials, which have been used as tissue scaffolds. Common tissue engineering scaffold materials are collagen, chitosan, agarose/alginate, fibronectin, synthetic polymers, polylactic acid, polyglycolic acid/polylactic acid, poly beta hydroxybutyric acid, magnetic nanoparticles, etc. However, most of the prior art is physical mixing, and related spinal cord repair materials are constructed by using materials through a physical method, so that an internal microscopic structure is not available, and a nerve loop reconstruction channel cannot be constructed. In addition, the students use a spinning structure or a multi-bundle fiber structure to construct a spinal cord repair material, but the spatial requirements of nerve regeneration and nerve loop reconstruction cannot be met. In addition, in the prior art, a system for realizing the controllable release of the correlation factor through a spatial structure is not provided. For example, patent document CN109106981A, published japanese 2019.01.01 discloses a double-modified collagen scaffold, which comprises a collagen scaffold material, and EGFR antibodies and microtubule stabilizing molecules modified on the collagen scaffold material, wherein the EGFR antibodies and microtubule stabilizing molecules are uniformly distributed in the interior and on the surface of the collagen scaffold material by physical adsorption.
In addition, patent document CN102727936A, published japanese patent No. 2012.10.17 discloses a slow-release NT-3 gelatin sponge cylinder stent material containing stem cells for repairing transectional spinal cord injury, which is characterized by the following morphological features: the surface of the cylindrical stent is wrapped by a thin wall formed by a polylactic-co-glycolic acid (PLGA) film, and the center of the cylinder is filled with gelatin sponge loaded with NT-3/silk fibroin; the NT-3/silk fibroin-loaded gelatin sponge is used for adsorbing the planted stem cells and the differentiated cells thereof to construct the sustained-release NT-3 gelatin sponge cylindrical stent which is beneficial to the regeneration of injured spinal nerves and the functional repair thereof. The cylindrical stent is composed of a PLGA tube wall with a thin outer layer and NT-3/silk fibroin-loaded gelatin sponge inside, and the NT-3/silk fibroin-loaded gelatin sponge is of a porous structure. But the porosity of the scaffold material is disordered.
At present, chitosan-based scaffold materials which comprise guide holes along the long axis direction of the materials and provide a space structure required by nerve loop reconstruction so as to facilitate the directional migration of endogenous neural stem cells are not seen.
Disclosure of Invention
The invention aims to provide a hierarchical porous functional scaffold material for mobilizing endogenous neural stem cells to repair spinal cord injury, aiming at the defects in the prior art.
The invention further aims to provide a preparation method of the hierarchical porous functional scaffold material for mobilizing endogenous neural stem cells to repair spinal cord injury.
The invention also aims to provide application of the hierarchical pore functional scaffold material.
In order to achieve the first purpose, the invention adopts the technical scheme that:
a hierarchical porous functional scaffold material for mobilizing endogenous neural stem cells to repair spinal cord injury is prepared by the following steps:
a) mixing glycidyl methacrylate crosslinked chitosan, a crosslinking agent N, N '-methylene bisacrylamide, a photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone and distilled water according to a certain proportion to obtain a precursor solution, wherein the mass ratio of the glycidyl methacrylate crosslinked chitosan to the crosslinking agent N, N '-methylene bisacrylamide, the photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone to the distilled water is 1 (0.5% -2%): 2% -4%): 35-45;
b) adding growth factors and preparing the growth factors and the precursor solution into colloidal precursor solution; or adding no growth factor, the precursor solution is the gel-forming precursor solution;
c) placing the gel-forming precursor solution in liquid nitrogen for ice crystal guiding;
d) and then ultraviolet irradiation is carried out at the temperature of minus 20 ℃ to initiate gelling, thus obtaining the product.
As a preferred example, between step b) and step c), the method further comprises the steps of: and centrifuging the gel-forming precursor solution to remove bubbles.
More preferably, the centrifugation conditions are: 4500-5500r/min, 2.5-4 min.
As another preferable example, the gel-forming precursor solution is injected into a glass tube, and then the glass tube is placed in liquid nitrogen at a constant speed for freezing guidance.
More preferably, the rate of exposure to liquid nitrogen is 2.5-3.5 mm/s.
As another preferred example, in the step d), the ultraviolet irradiation time is 3-18 min.
As another preferred example, the growth factor is selected from one or more of brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT-3), neurotrophin-4 (NT-4), Nerve Growth Factor (NGF), basic fibroblast growth factor (bFGF), Epidermal Growth Factor (EGF), insulin-like growth factor (IGF), Transforming Growth Factor (TGF), Vascular Endothelial Growth Factor (VEGF), Hepatocyte Growth Factor (HGF), glial neurotrophic growth factor (GDNF), Skeletal Growth Factor (SGF), platelet-derived growth factor (PDGF), and Stem Cell Factor (SCF), but is not limited thereto.
As another preferred example, the multi-level pore functional scaffold material is also planted with seed cells.
As another preferred example, the seed cells are neural stem cells, embryonic stem cells, Schwann cells, olfactory ensheathing cells, bone marrow stromal cells, oligodendrocytes, and the like.
In order to achieve the second object, the invention adopts the technical scheme that:
a preparation method of a hierarchical porous functional scaffold material for mobilizing endogenous neural stem cells to repair spinal cord injury comprises the following steps:
a) mixing glycidyl methacrylate crosslinked chitosan, a crosslinking agent N, N '-methylene bisacrylamide, a photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone and distilled water according to a certain proportion to obtain a precursor solution, wherein the mass ratio of the glycidyl methacrylate crosslinked chitosan to the crosslinking agent N, N '-methylene bisacrylamide, the photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone to the distilled water is 1 (0.5% -2%): 2% -4%): 35-45;
b) adding growth factors and preparing the growth factors and the precursor solution into colloidal precursor solution; or adding no growth factor, the precursor solution is the gel-forming precursor solution;
c) placing the gel-forming precursor solution in liquid nitrogen for ice crystal guiding;
d) and then ultraviolet irradiation is carried out at the temperature of minus 20 ℃ to initiate gelling, thus obtaining the product.
As a preferred example, between step b) and step c), the method further comprises the steps of: and centrifuging the gel-forming precursor solution to remove bubbles.
More preferably, the centrifugation conditions are: 4500-5500r/min, 2.5-4 min.
As another preferable example, in the step c), the gel-forming precursor solution is injected into a glass tube, and then the glass tube is placed in liquid nitrogen at a constant speed for freezing and guiding.
More preferably, the rate of exposure to liquid nitrogen is 2.5-3.5 mm/s.
As another preferred example, in the step d), the ultraviolet irradiation time is 3-18 min.
In order to achieve the third object, the invention adopts the technical scheme that:
the application of the hierarchical porous functional scaffold material in preparing a product for repairing spinal cord injury.
The invention has the advantages that:
1. the invention provides a multi-level hole material according to the multi-level characteristics of neuron axon growth, which is prepared by a special method, wherein guide holes with different sizes along the long axis and the inner diameter of the material are formed inside the material, so that a space structure required by nerve loop reconstruction is provided, endogenous nerve stem cells can be conveniently and directionally migrated to a material transplanting area to form new neurons, nerve synapses grow along the designed multi-level structure to form effective synapse connection to form a nerve network, the nerve loop of a damaged area is reconstructed, and the motor function recovery after spinal cord injury is promoted.
2. The hierarchical porous material has good mechanical strength and proper aperture of the guide hole, and animal experiments show that the transplantation treatment of the spinal cord injury of the mouse can obviously promote the recovery of the lower limb movement function of the mouse.
3. The material of the invention can load related factors in the construction process to construct a controlled release system, thereby better promoting the regeneration and repair.
4. The material of the invention can also be loaded with exogenous neural stem cells.
5. The material disclosed by the invention has the advantages of adjustable pore diameter (adaptive to cell culture), controllable strength (capable of tolerating the loading of acoustomagnetic and electronic physical factors), capability of 3D printing (integrating a nano controlled release system) and biocompatibility.
Drawings
FIG. 1: infrared pattern of chitosan with and without modified double bond.
FIG. 2 is a drawing: compression test plots of colloidal materials under different illumination times.
FIG. 3: mercury intrusion test pore size distribution profiles with and without freeze-directed colloidal materials.
FIG. 4 is a drawing: gel forming test chart of the colloidal material.
FIG. 5: SEM test pattern of colloidal material.
FIG. 6: the invention discloses a schematic diagram of repairing spinal cord injury by using a hierarchical porous functional scaffold material.
FIG. 7: the constructed multi-level hole functional stent is transplanted to treat spinal cord injury. A is a transplantation schematic diagram of a multi-level hole functional scaffold, an in vivo experiment is carried out by utilizing a spinal cord injury full-transection model constructed in an earlier stage, and the constructed multi-level hole functional scaffold is placed in an injury area; the upper part of the graph B is a general specimen during transplantation, the damaged area is completely filled with the multi-level pore functional scaffold after transplantation, the damaged group is compared with the general specimen of the control group after 8 weeks in the lower part, the tissue necrosis of the control group is seen, and the new tissue exists in the transplantation treatment group; the C picture is the ethological score of the mouse, and the ethological recovery of the transplantation treatment group is obviously statistically different from that of the control group; the D picture is the electrophysiological detection of the spinal cord injured mice, and can be seen that the control group has no obvious electrophysiological waveform, while the transplantation treatment group has obvious amplitude; it was revealed that the multi-stage pore functional scaffold promotes the recovery of nerve function after spinal cord injury.
FIG. 8: staining results of spinal cord sections after 8 weeks of multi-level pore functional stent transplantation treatment. The new neurons are found in the transplantation treatment area, more neurons are near the injury boundary, fewer neurons are in the injury central area, a certain trend gradient effect is presented, the distribution effect is not existed in normal tissues, and the multi-level pore functional scaffold is presumed to possibly induce the directional migration of endogenous neural stem cells (the white boundary is the boundary between the injury area and the normal tissues, and the lower half part is a local amplification image).
Detailed Description
The following detailed description of the present invention will be made with reference to the accompanying drawings.
EXAMPLE 1 preparation of the hierarchical pore functional scaffold Material of the invention
1. Chitosan grafting double bond:
2.0g of chitosan was weighed into a three-necked flask, 150ml of 2% aqueous acetic acid was added thereto, and a small amount of aqueous KOH solution was added dropwise to adjust the pH to 3.8. Then, 3.51g of Glycidyl Methacrylate (GMA) was slowly added dropwise thereto, and the mixture was reacted at 60 ℃ for 6 hours with continuous stirring. And then pouring the reaction mixed solution into acetone to obtain a large amount of flocculent precipitate, pouring the flocculent into acetonitrile to obtain powdery precipitate, finally washing the powdery precipitate by acetone for a plurality of times, dissolving the powdery precipitate by water, putting the powdery precipitate into a dialysis bag with the molecular weight of 3500 for dialysis for 3 days, and freeze-drying to obtain a raw material Chitosan-GMA for later use. And (3) characterizing the chitosan grafted with the double bonds by utilizing infrared spectrum and H-NMR.
As a result: FIG. 1 is an infrared image of chitosan with and without modified double bonds, showing that the double bond transmission peak appears at 1710.39 for the double bond modified chitosan versus the chitosan without modified double bonds, indicating that the double bond was successfully modified on the double bond chitosan.
2. Preparation of the material:
50mg (2.5%) of Chitosan-GMA was weighed into a glass vial, 100. mu.L of BIS (N, N '-methylenebisacrylamide, 10mg/mL) crosslinker (2% of monomer) was added, 1.5mg of photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone (3% of monomer) was added, and 1900. mu. L H was added2And O, stirring for 2 hours at 37 ℃ to dissolve the precursor to form precursor liquid, namely the gel-forming precursor liquid.
And after the solution is completely dissolved, centrifuging the gel-forming precursor solution for 3min at 5000r/min to remove bubbles. The solution was then injected into the capillary using an ear-washing bulb.
The capillary tube into which the solution was injected was placed in liquid nitrogen at a constant speed (3.3mm/s) using an elevator for freeze guiding. Immediately after the guiding was completed, the capillary was placed in a-20 ℃ refrigerator and frozen UV-gelled using a freezing UV lamp (UV Led Curing System, model UP3-304, available from Mean Well International co.
The mechanical strength of the material is adjusted by adjusting the dosage of the chitosan and the cross-linking agent and the ultraviolet illumination time. And (3) testing mechanical properties: the diameter and height of the cylindrical colloidal material were measured first, and the parameters were input into a universal mechanical tester (mitsung crossbar 2505), and then compression test was performed using the universal mechanical tester at a compression rate of 5 mm/min.
The size of the aperture is adjusted by adjusting the rate of descent of the elevator.
And (3) characterizing the pore diameter and pore diameter distribution of the material by a mercury intrusion method.
And testing the gelling time by using a rheometer.
After removing ice crystals from the gel by freeze drying, the guidance structure and pore structure of the gel are observed by using a scanning electron microscope.
As a result:
FIG. 2 is a compression test chart of the colloid material under different illumination time, the compression strength of the colloid first rises and then falls, and the colloid material has the maximum compression strength when being illuminated for 12 min. Fig. 3 is a plot of mercury intrusion pore size distribution for colloidal materials with and without freeze steering. Fig. 4 is a gelling test chart of the colloidal material. Fig. 5 is an SEM test chart of the colloidal material.
Example 2 preparation of the hierarchical pore functional scaffold Material of the invention
1. Chitosan grafting double bond:
the same as in example 1.
2. Preparation of the material:
50mg of Chitosan-GMA was weighed into a glass vial, then 25. mu.L of BIS (N, N '-methylenebisacrylamide, 10mg/mL) crosslinker (0.5% of monomer) was added, 2mg of photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone (4% of monomer) was added, and 1700. mu. L H was added2And O, stirring for 2 hours at 37 ℃ to dissolve the precursor to form precursor liquid, namely the gel-forming precursor liquid.
After the solution is completely dissolved, the gel-forming precursor solution is centrifuged at 4500r/min for 4min to remove bubbles. The solution was then injected into the capillary using an ear-washing bulb.
The capillary tube into which the solution was injected was placed in liquid nitrogen at a constant speed (2.5mm/s) using an elevator for freeze guiding. Immediately after the guiding was completed, the capillary was placed in a refrigerator at-20 ℃ to be frozen and UV-gelled using a freezing UV lamp (UV Led Curing System, model UP3-304, available from Mean Well International co.ltd), with a UV-irradiation time of 15 min.
Example 3 preparation of the hierarchical pore functional scaffold material of the invention
1. Chitosan grafting double bond:
the same as in example 1.
2. Preparation of the material:
50mg of Chitosan-GMA was weighed into a glass vial, 50. mu.L of BIS (N, N '-methylenebisacrylamide, 10mg/mL) crosslinker (1% of monomer) was added, 1mg of photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone (2% of monomer) was added, and 2200. mu. L H% of the mixture was added2And O, stirring for 2 hours at 37 ℃ to dissolve the precursor to form precursor liquid, namely the gel-forming precursor liquid.
After the solution is completely dissolved, the gel-forming precursor solution is centrifuged at 4500r/min for 4min to remove bubbles. The solution was then injected into the capillary using an ear-washing bulb.
The capillary tube into which the solution was injected was placed in liquid nitrogen at a constant speed (3.5mm/s) using an elevator for freeze guiding. Immediately after the guiding was completed, the capillary was placed in a refrigerator at-20 ℃ to be frozen and UV-gelled using a freezing UV lamp (UV Led Curing System, model UP3-304, available from Mean Well International co.ltd) for 3 min.
EXAMPLE 4 preparation of the hierarchical pore functional scaffold Material of the Invention (IV)
1. Chitosan grafting double bond:
the same as in example 1.
2. Preparation of the material:
50mg of Chitosan-GMA was weighed into a glass vial, then 25. mu.L of BIS (N, N '-methylenebisacrylamide, 10mg/mL) crosslinker (0.5% of monomer) was added, 1.5mg of photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone (3% of monomer) was added, and 1950. mu. L H was added2O, dissolved by stirring at 37 ℃ for 2h to form a precursor solution. Adding related factors and the precursor liquid into the precursor liquid to prepare the precursor liquid, wherein the added related factors are one or more of BDNF, NT-3, NT-4, NGF, bFGF, EGF, IGF, TGF, VEGF, HGF, GDNF, SGF, PDGF and SCF.
After the solution is completely dissolved, the gel-forming precursor solution is centrifuged at 5500r/min for 2.5min to remove bubbles. The solution was then injected into the capillary using an ear-washing bulb.
The capillary tube into which the solution was injected was placed in liquid nitrogen at a constant speed (3.0mm/s) using an elevator for freeze guiding. Immediately after the guiding was completed, the capillary was placed in a refrigerator at-20 ℃ to be frozen and UV-gelled using a freezing UV lamp (UV Led Curing System, model UP3-304, available from Mean Well International co.ltd) for 3 min.
EXAMPLE 5 preparation of the hierarchical pore functional scaffold material of the present invention (V)
1. Chitosan grafting double bond:
the same as in example 1.
2. Preparation of the material:
50mg of Chitosan-GMA was weighed into a glass vial, 100. mu.L of BIS (N, N '-methylenebisacrylamide, 10mg/mL) crosslinker (2% of monomer) was added, 2mg of photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone (4% of monomer) was added, and 1950. mu. L H was added2O, dissolved by stirring at 37 ℃ for 2h to form a precursor solution. Adding related factors and the precursor liquid into the precursor liquid to prepare the precursor liquid, wherein the added related factors are one or more of BDNF, NT-3, NT-4, NGF, bFGF, EGF, IGF, TGF, VEGF, HGF, GDNF, SGF, PDGF and SCF.
And after the solution is completely dissolved, centrifuging the gel-forming precursor solution for 4min at 5000r/min to remove bubbles. The solution was then injected into the capillary using an ear-washing bulb.
The capillary tube into which the solution was injected was placed in liquid nitrogen at a constant speed (3.0mm/s) using an elevator for freeze guiding. Immediately after the guiding was completed, the capillary was placed in a refrigerator at-20 ℃ to be frozen and UV-gelled using a freezing UV lamp (UV Led Curing System, model UP3-304, available from Mean Well International co.ltd), with a UV-irradiation time of 18 min.
Example 6
Referring to fig. 6, fig. 6 is a schematic view of repairing spinal cord injury with the multi-level porous functional scaffold material of the present invention, the multi-level porous functional scaffold material of the present invention is transplanted at the damaged spinal cord defect, because the material has guiding holes with a size not equal to the inner diameter of the long axis of the material, the material can facilitate the directional migration of endogenous neural stem cells to the material transplantation area and form new neurons, and the neural synapses grow along the designed multi-level structure to form a neural network to reconstruct the neural loop of the damaged area, and promote the motor function recovery after spinal cord injury. Related factors can be loaded in the material construction process to construct a controlled release system, so that the regeneration and repair are better promoted.
Animal experiments prove that the multi-level pore functional scaffold material has the effects of promoting endogenous neural stem cells to reconstruct neural loops of a spinal cord injury area and repairing spinal cord injury.
The multi-level pore functional scaffold material constructed in the embodiment 1 is used for transplantation treatment of spinal cord injury of mice, and compared with a blank control group, the multi-level pore functional scaffold material transplantation can effectively promote recovery of lower limb motor functions of the mice, the ethological score is obviously improved compared with the control group, and electrophysiological detection shows that the multi-level pore functional scaffold material transplantation treatment group has obvious electrophysiological activities (fig. 7). Through technical means such as immunohistochemistry and the like, new neurons are found in the transplantation treatment area, more neurons are close to the damaged boundary, fewer neurons are in the damaged central area, and a certain trend gradient effect is presented (fig. 8).
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.
Claims (10)
1. A hierarchical porous functional scaffold material for mobilizing endogenous neural stem cells to repair spinal cord injury is prepared by the following steps:
a) mixing glycidyl methacrylate crosslinked chitosan, a crosslinking agent N, N '-methylene bisacrylamide, a photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone and distilled water according to a certain proportion to obtain a precursor solution, wherein the mass ratio of the glycidyl methacrylate crosslinked chitosan to the crosslinking agent N, N '-methylene bisacrylamide, the photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone to the distilled water is 1 (0.5% -2%): 2% -4%): 35-45;
b) adding growth factors, and preparing a gel precursor solution with the precursor solution; or adding no growth factor, the precursor solution is the gel-forming precursor solution;
c) the gel-forming precursor liquid is placed in liquid nitrogen for ice crystal guiding, and the speed of placing the gel-forming precursor liquid in the liquid nitrogen is 2.5-3.5 mm/s;
d) and then ultraviolet irradiation is carried out at the temperature of minus 20 ℃ to initiate gelling, thus obtaining the product.
2. The multi-stage pore functional scaffold material according to claim 1, further comprising, between step b) and step c), the steps of: and centrifuging the gel-forming precursor solution to remove bubbles.
3. The multi-stage hole functional scaffold material according to claim 1, wherein in step c), the gel-forming precursor solution is injected into a glass tube, and then the glass tube is placed in liquid nitrogen at a constant speed for freezing guidance.
4. The multi-stage pore functional scaffold material according to claim 1, wherein in step d), the ultraviolet light irradiation time is 3-18 min.
5. The multi-stage pore functional scaffold material according to claim 1, wherein the growth factor is selected from one or more of brain-derived neurotrophic factor, neurotrophic factor-3, neurotrophin-4, nerve growth factor, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor, transforming growth factor, vascular endothelial growth factor, hepatocyte growth factor, glial neurotrophic growth factor, bone growth factor, platelet-derived growth factor, and stem cell factor.
6. A preparation method of a hierarchical porous functional scaffold material for mobilizing endogenous neural stem cells to repair spinal cord injury is characterized by comprising the following steps:
a) mixing glycidyl methacrylate crosslinked chitosan, a crosslinking agent N, N '-methylene bisacrylamide, a photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone and distilled water according to a certain proportion to obtain a precursor solution, wherein the mass ratio of the glycidyl methacrylate crosslinked chitosan to the crosslinking agent N, N '-methylene bisacrylamide, the photoinitiator 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone to the distilled water is 1 (0.5% -2%): 2% -4%): 35-45;
b) adding growth factors, and preparing a gel precursor solution with the precursor solution; or adding no growth factor, the precursor solution is the gel-forming precursor solution;
c) the gel-forming precursor liquid is placed in liquid nitrogen for ice crystal guiding, and the speed of placing the gel-forming precursor liquid in the liquid nitrogen is 2.5-3.5 mm/s;
d) and then ultraviolet irradiation is carried out at the temperature of minus 20 ℃ to initiate gelling, thus obtaining the product.
7. The method of claim 6, further comprising, between step b) and step c), the steps of: and centrifuging the gel-forming precursor solution to remove bubbles.
8. The preparation method according to claim 6, wherein in step c), the gel-forming precursor solution is injected into a glass tube, and the glass tube is placed in liquid nitrogen at a constant speed for freezing guidance.
9. The method according to claim 6, wherein the UV irradiation time in step d) is 3-18 min.
10. Use of the hierarchical porous functional scaffold material of any one of claims 1 to 5 in the preparation of a product for repairing spinal cord injury.
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Inventor after: Cheng Liming Inventor after: Wang Qigang Inventor after: Cheng Heli Inventor after: Xu Wei Inventor before: Cheng Liming Inventor before: Wang Qigang Inventor before: Cheng Heli Inventor before: Xu Wei |