Stem cell-loaded nerve scaffolds, methods of making and using the same

Abstract

The present application relates to the biomedical technology field, specifically relates to a kind of stem cell loaded nerve scaffold and its preparation method and application.The preparation method of a kind of stem cell loaded nerve scaffold provided by the present application includes seeding stem cell on the surface of two-dimensional material of fiber directional arrangement nanofiber membrane, biocompatible hydrogel is applied to the surface of seeded stem cell and is solidified, stem cell loaded nanofiber membrane is used to construct three-dimensional structure stem cell loaded nerve scaffold.The method can control the directional arrangement distribution of stem cell, after cell growth on two-dimensional plane, through the way of reassembling into three-dimensional stem cell loaded nerve scaffold by applying hydrogel layer, then the activity of cell is maintained, so the method can maintain the activity of stem cell and regulate stem cell arrangement in nerve scaffold.

Description

TECHNICAL FIELD

[0001] The present application relates to the biomedical technology field, in particular to a nerve scaffold loaded with stem cells and a preparation method and application thereof. BACKGROUND

[0002] Peripheral nerve injury can cause severe disability, bring great physical and mental pain to patients, and also bring heavy economic burden to the patient's family and the whole society. In recent years, people have developed various nerve tissue engineering scaffolds to promote the repair after nerve injury, among which the nerve scaffold combined with stem cells can differentiate into damaged nerve cells or supportive Schwann cells to promote the reconstruction of nerve network, and secrete beneficial nutritional factors to promote nerve axon regeneration and new blood vessel regeneration.

[0003] A key challenge of the tissue engineering scaffold loaded with stem cells is to construct a three-dimensional nerve scaffold that can allow stem cells to grow and function well at the implant site. There are many related researches on integrating stem cells into tissue engineering scaffolds at present, mostly using direct seeding or 3D printing methods. The method of directly seeding stem cells on the already formed three-dimensional scaffold material or the method of injecting cell suspension into the three-dimensional scaffold cavity cannot control the distribution of cells in the scaffold. Although the 3D printing method can arrange cells in a predetermined manner, it cannot guarantee the viability of the loaded stem cells due to the complexity of the operation, so these methods will affect the good function of stem cells after implantation, and thus it is difficult to achieve the effect of promoting repair.

[0004] Based on the above defects of the prior art, it is very important to provide a method that can maintain the activity of stem cells and realize the arrangement and regulation of stem cells in the nerve scaffold. SUMMARY

[0005] In view of the deficiencies of the prior art, the first purpose of the present application is to provide a preparation method of a nerve scaffold loaded with stem cells, which can maintain the activity of stem cells and regulate the arrangement of stem cells in the nerve scaffold.

[0006] The second purpose of the present application is to provide a nerve scaffold loaded with stem cells, which maintains good activity of stem cells and the stem cells grow according to the material morphology.

[0007] The third purpose of the present application is to provide the application of a nerve scaffold loaded with stem cells in the preparation of a drug or medical device for treating peripheral nerve injury, which shows good therapeutic effect.

[0008] In order to achieve the above purposes, the technical solution provided by the present application is:

[0009] According to the first aspect of the present application, the present application provides a method for preparing a stem cell-loaded nerve scaffold, comprising seeding stem cells on a two-dimensional material surface of a nanofiber membrane with fiber orientation arrangement, applying a biocompatible hydrogel to the seeded stem cell surface and curing, and constructing a three-dimensional stem cell-loaded nerve scaffold using the stem cell-loaded nanofiber membrane.

[0010] Further, the method for constructing a three-dimensional stem cell-loaded nerve scaffold using the stem cell-loaded nanofiber membrane comprises winding the stem cell-attached nanofiber membrane into a reel along the direction of fiber orientation arrangement, and then integrating the reel into a tubular porous shell to obtain the three-dimensional stem cell-loaded nerve scaffold.

[0011] Further, the reel has a multi-layer spiral structure or a single-layer structure; preferably, the number of layers of the multi-layer spiral structure is 2-5 layers.

[0012] Further, the method for preparing the tubular porous shell comprises dissolving poly-L-lactic acid in a low-boiling-point solvent, adding a porogen, removing the solvent to prepare a PLLA porous film, and then using high temperature to prepare the tubular porous shell into a hollow cylindrical shape.

[0013] Preferably, the inner diameter of the tubular porous shell is 1-3 mm.

[0014] Further, it further comprises adding a culture medium to the cured hydrogel for culture.

[0015] Preferably, the culture medium is an α-MEM culture medium containing 1% penicillin-streptomycin and 10% fetal bovine serum.

[0016] Further, the seeding is performed on one surface of the nanofiber membrane with fiber orientation arrangement, or the seeding is performed on both surfaces of the nanofiber membrane with fiber orientation arrangement.

[0017] Further, the method for seeding comprises adding a culture solution containing stem cells dropwise on the two-dimensional material surface of the nanofiber membrane with fiber orientation arrangement, spreading the culture solution evenly on the nanofiber membrane, and standing to allow the stem cells to adhere; specifically, the culture solution is spread evenly on the nanofiber membrane by shaking.

[0018] Preferably, the seeding density of the stem cells is 1×10 5 / mL-9×10 6 / mL; preferably, the seeding density of the stem cells is 1×10 6 / mL.

[0019] Preferably, the stem cells are selected from at least one of embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, neural stem cells.

[0020] Further, the biocompatible hydrogel is a biocompatible hydrogel with photo-curing characteristics.

[0021] Preferably, the biocompatible hydrogel with photo-curing characteristics is selected from at least one of methacrylated hyaluronic acid, methacrylated sodium alginate, methacrylated gelatin.

[0022] Further, the preparation raw material of the fiber-aligned nanofiber membrane is selected from at least one of poly-L-lactic acid, polyvinyl alcohol and polycaprolactone.

[0023] Preferably, the preparation raw material of the fiber-aligned nanofiber membrane is poly-L-lactic acid with a molecular weight of 200000-300000; preferably, the molecular weight of the poly-L-lactic acid is 260000.

[0024] Preferably, the preparation method of the fiber-aligned nanofiber membrane is selected from at least one of electrospinning, self-assembly, stretching method and template synthesis method; preferably, the nanofiber membrane is prepared by electrospinning.

[0025] According to the second aspect of the present application, the present application provides a stem cell-loaded nerve scaffold, which is prepared according to the above method.

[0026] According to the third aspect of the present application, the present application provides use of the stem cell-loaded nerve scaffold as described above in the preparation of a medicament or medical device for treating peripheral nerve injury.

[0027] Compared with the prior art, the present application has the following beneficial effects:

[0028] 1. The preparation method of the stem cell-loaded nerve scaffold provided by the present application directly seeds stem cells on the surface of the fiber-aligned nanofiber membrane two-dimensional material, so that the stem cells adhere well to the material surface and grow according to the material topography, the directional arrangement and distribution of the stem cells are controlled, and the subsequent function of the stem cells is better promoted. After the cells grow on the two-dimensional plane, the stem cell-loaded nerve scaffold is reassembled into a three-dimensional form by applying a hydrogel, the biocompatible hydrogel can provide support for the growth of stem cells, and thus maintain the activity of the cells, so that the method can maintain the activity of the stem cells and regulate the arrangement of the stem cells in the nerve scaffold.

[0029] 2. The stem cell loaded nerve scaffold provided by the application shows good therapeutic effect in cell and rat sciatic nerve injury model. The cell experiment result shows that the stem cells loaded in the scaffold can grow well on the scaffold material and differentiate into Schwann cells. The animal experiment result shows that the stem cell loaded spiral structure scaffold can better promote the nerve regeneration and functional recovery of the rat sciatic nerve after injury, which provides a new idea for the treatment of related nerve injury diseases.

[0030] Drawings of the specification

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed to be used in the embodiment or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings;

[0032] Figure 1 Preparation flow chart of the nerve scaffold in Example 1;

[0033] Figure 2 Surface morphology characterization of the PLLA nanofiber membrane in Example 1;

[0034] Figure 3 Morphology diagram of the cylindrical porous shell in Example 1;

[0035] Figure 4 Structure schematic diagram of the nerve scaffold prepared in Example 1;

[0036] Figure 5 SEM diagram of the nerve scaffold prepared in Example 1;

[0037] Figure 6 Biocompatibility evaluation result in Example 2;

[0038] Figure 7 Immunofluorescence diagram of the cell distribution on the nerve scaffold in Example 2;

[0039] Figure 8 Stem cell differentiation characterization result on the nerve scaffold in Example 2;

[0040] Figure 9 Stem cell secretion characterization result on the nerve scaffold in Example 2;

[0041] Figure 10 Surgical implantation photo of the nerve scaffold in Example 2;

[0042] Figure 11 Morphological characterization result of nerve repair in Example 2;

[0043] Figure 12 Results of characterization of the recovery of the gastrocnemius muscle innervated in Example 2. DETAILED DESCRIPTION

[0044] The technical solutions of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. The described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0045] The embodiment of the present application provides a preparation method of a stem cell loaded nerve scaffold, which comprises seeding stem cells on a two-dimensional material surface of a fiber directionally arranged nanofiber membrane, applying a biocompatible hydrogel to the seeded stem cell surface and curing, and constructing a three-dimensional structure stem cell loaded nerve scaffold by using the stem cell loaded nanofiber membrane.

[0046] In the above technical solution, the stem cells are seeded directly on the two-dimensional material surface of the fiber directionally arranged nanofiber membrane first, so that the stem cells adhere well to the material surface and grow according to the material topography, which can control the directional arrangement and distribution of the stem cells, avoid the random arrangement of the cells caused by seeding the cells directly in the already formed three-dimensional scaffold, and further promote the subsequent function of the stem cells. After the cells grow on the two-dimensional plane, the three-dimensional stem cell loaded nerve scaffold is reassembled by applying a biocompatible hydrogel layer, the water content of the hydrogel is high, the modulus of the hydrogel is close to that of the tissue, and the hydrogel has high biological tissue adaptability with the tissue, which can provide support for the growth of the stem cells and further maintain the activity of the cells. In addition, a three-dimensional structure with well-distributed stem cells is formed, which constructs a three-dimensional nerve scaffold that can allow the stem cells to grow and function well in the implantation site. Although the 3D printing method in the prior art can directly form a three-dimensional scaffold structure by loading a cell solution, the activity of the stem cells is affected after long-term compression and extrusion molding.

[0047] It should be noted that the method of constructing the stem cell loaded nerve scaffold by using the stem cell loaded nanofiber membrane can be performed by a conventional method. Since the stem cell loaded nanofiber membrane is constructed into a nerve scaffold, attention should be paid to the influence of the construction method on the activity of the stem cells during the construction process.

[0048] As a preferred embodiment, the present application provides a method of constructing a three-dimensional structure stem cell loaded nerve scaffold by using a stem cell loaded nanofiber membrane, which comprises winding the stem cell attached nanofiber membrane along the direction of fiber directional arrangement into a reel, and then integrating the reel into a tubular porous shell to obtain a three-dimensional structure stem cell loaded nerve scaffold.

[0049] It should be noted that by the above construction method, a spool with single-layer stem cells can be obtained, and a spool with multi-layer stem cells can also be obtained.

[0050] As a preferred embodiment, the spool has a multi-layer spiral structure, which is designed to improve the stem cell load in the nerve scaffold. After loading the stem cells, the superimposed biocompatible hydrogel material can provide a better environment for the growth and differentiation of stem cells in the scaffold, maintain the viability of the cells, and avoid interference between the multi-layer cells. Through the design of multi-layer stem cells and the support of the material itself, a better effect of promoting nerve repair is achieved.

[0051] It should be noted that the number of layers in the multi-layer spiral structure in the present application refers to more than 1 layer (not including the number of one layer), which can be any value more than 1, such as any natural number more than 1, and non-natural numbers such as 1.1 and 1.5.

[0052] As a preferred embodiment, the number of layers of the multi-layer spiral structure is 2-5 layers; as an exemplary embodiment, the number of layers of the multi-layer spiral spool is, for example, 1.5 layers, 2 layers, 2.5 layers, 3 layers, 4 layers, 5 layers, etc.

[0053] The tubular porous shell in the present application can provide structural support for the spiral spool. The shell is perforated to achieve material exchange between the internal and external environments of the nerve scaffold during use. It should be noted that the tubular porous shell can be prepared by conventional methods for preparing a nerve scaffold shell; as a preferred embodiment, the preparation method of the tubular porous shell is to dissolve poly-L-lactic acid (PLLA) in a low-boiling point solvent, add a porogen, remove the solvent to form a PLLA porous film, and then use high temperature to form a hollow cylindrical tubular porous shell.

[0054] In the above technical solution, the low-boiling point solvent includes but is not limited to dichloromethane, and the porogen includes but is not limited to PEG200. The diameter of the tubular porous shell and the PLLA porous film can be selected as needed during actual operation, and as a preferred embodiment, the inner diameter of the tubular porous shell is 1-3 mm.

[0055] The method of seeding stem cells on the two-dimensional surface of the nanofiber membrane is not particularly limited. The seeding can be performed on one surface of the nanofiber membrane, or on both surfaces of the nanofiber membrane. Considering the preparation difficulty and actual requirements, the seeding method is preferably performed on one surface of the nanofiber membrane. This preferred embodiment does not constitute a limitation on the present application.

[0056] As an example, the seeding method provided by the present application is that a culture solution containing stem cells is added dropwise on the surface of the nanofiber membrane two-dimensional material, the nanofiber membrane is slightly shaken to evenly spread the added culture solution on the nanofiber membrane, and the stem cells are adhered by standing treatment; the seeding density of the stem cells is not particularly limited in the present application, and the stem cells can be well grown, differentiated and distributed on the nanofiber membrane; as a preferred embodiment, the seeding density of the stem cells is 1×10 5 / mL to 9×10 6 / mL; as an example, the seeding density of the stem cells is preferably 1×10 6 / mL.

[0057] The preparation method of the stem cell-loaded nerve scaffold of the present application can be applied to the loading of various stem cells, and thus the type of the stem cells is not particularly limited, and the type of the stem cells includes but is not limited to at least one of embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells and neural stem cells; as an example, the stem cells are mesenchymal stem cells.

[0058] The selection of the biocompatible hydrogel in the present application is not particularly limited, and the material can not affect the normal activity of cells and tissues after being in contact with the biological tissues and body fluids. The biocompatible hydrogel of the present application can be cured by conventional curing methods such as photocuring and solvent evaporation curing; as a preferred embodiment, the biocompatible hydrogel is a biocompatible hydrogel with photocuring characteristics; as an example, the biocompatible hydrogel with photocuring characteristics is selected from at least one of methacrylated hyaluronic acid, methacrylated sodium alginate and methacrylated gelatin.

[0059] The preparation method and material of the fiber-aligned nanofiber membrane in the present application are not particularly limited, as a preferred embodiment, the preparation raw material of the fiber-aligned nanofiber membrane is selected from at least one of poly-L-lactic acid, polyvinyl alcohol and polycaprolactone; wherein, the poly-L-lactic acid is non-toxic, non-irritating, biodegradable and absorbable, has good biocompatibility, and has high strength and plasticity, and is easy to process; as a preferred embodiment, the preparation raw material of the fiber-aligned nanofiber membrane of the present application is poly-L-lactic acid with a molecular weight of 200000-300000, and the specific selected molecular weight is, for example, 200,000, 260,000 or 300,000, etc.; the preparation method of the fiber-aligned nanofiber membrane using the above raw material is not particularly limited, and includes but is not limited to at least one of electrospinning, self-assembly, stretching method and template synthesis method; as a preferred embodiment, the nanofiber membrane is prepared by electrospinning; as an example, the fiber-aligned nanofiber membrane is prepared by electrospinning using poly-L-lactic acid with a molecular weight of 260,000, and the thickness of the above fiber-aligned nanofiber membrane is 50-70 μm.

[0060] The three-dimensional structure can be constructed after the culture medium is added to the cured hydrogel for a period of time, so that the stem cells can stably grow on the nanofiber membrane, and the cell viability can be maintained. As a preferred embodiment, the application further comprises adding the culture medium to the cured hydrogel for culture. The type of culture medium can be a conventional stem cell culture medium for maintaining or promoting the growth of stem cells. As an example, the culture medium is an α-MEM culture medium containing 1% penicillin-streptomycin and 10% fetal bovine serum.

[0061] The application further provides a nerve scaffold loaded with stem cells, which is prepared according to the above method. The nerve scaffold loaded with stem cells provided by the application can directly seed stem cells on the two-dimensional surface of the nanofiber membrane with aligned fibers, can regulate the arrangement of stem cells in the scaffold, is conducive to the uniform distribution of stem cells on the scaffold, and promotes the neural differentiation of stem cells. After seeding, the hydrogel material with good biocompatibility is superimposed, and the culture medium is added dropwise for culture, so that a better environment can be provided for the growth and differentiation of stem cells in the nanofiber membrane, and the cell viability can be maintained. Therefore, the nerve scaffold provided by the application can maintain the activity of stem cells and regulate the arrangement of stem cells in the nerve scaffold.

[0062] The application further provides the use of the nerve scaffold loaded with stem cells as described above in the preparation of a drug or medical device for treating peripheral nerve injury. The application provides experimental verification related to cells and rat sciatic nerve injury models. The cell results show that the stem cells loaded in the scaffold can grow well on the scaffold material and differentiate into Schwann cells. In animal experiments, the spiral structure scaffold loaded with stem cells can better promote the nerve regeneration and functional recovery of rats after sciatic nerve injury.

[0063] To make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below. The specific conditions are not indicated in the embodiments, and the conventional conditions or the conditions recommended by the manufacturer are used. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be purchased on the market.

[0064] Example 1: Preparation of a nerve scaffold

[0065] Please refer to Figure 1 The preparation method of the nerve scaffold provided in the embodiment specifically comprises the following steps:

[0066] (1) Preparation of PLLA directional fiber membrane

[0067] A sample bottle was filled with 0.5 g of PLLA powder and 6.2 g of hexafluoroisopropanol, and mixed on a magnetic stirring platform for 3 hours to form a solution with a mass fraction ratio of 7.5% (w / w). The solution was then poured into a syringe with a 20-gauge needle, and a voltage of 12 kV was added to the needle, and a negative voltage of -3 kV was added to the receiver with aluminum foil. The distance between the needle tip and the nanofiber receiver was 20 cm. After electrospinning, the aluminum foil was removed from the receiver and dried in a vacuum oven at 40°C for 8 hours until the residual solvent in the nanofiber film was completely removed. The film was then annealed in a constant temperature oven at 155°C for 4 h. After cooling to room temperature, the PLLA oriented fiber film was removed from the aluminum foil, and its surface morphology was characterized as shown in Figure 2 Figure 2 It can be seen from the PLLA nanofiber film SEM photos and diameter statistics that the fiber diameter distribution of the obtained PLLA nanofiber film is uniform and has good orientation, with fiber diameters distributed between 0.4 μm and 1.6 μm, and a fiber diameter of about 1 μm.

[0068] (2) Preparation of porous shell

[0069] PLLA 1 g was dissolved in 10 mL of dichloromethane, and after the PLLA was completely dissolved, 2 mL of PEG200 solution was added and stirred uniformly. The above mixed solution was poured into a 10 cm x 10 cm stainless steel tank, and after the solvent was naturally volatilized at room temperature for 24 hours, it was taken out and washed with deionized water and alcohol alternately for 3 times to obtain a PLLA porous film. The PLLA porous film was cut into a rectangular strip with a width of 5 mm, then wrapped around a stainless steel cylinder with a diameter of 1.3 mm, and the outside was wrapped with a PTFE film to fix it. After being kept at 70°C for 20 min, it was taken out, the PTFE film was removed, and the stainless steel cylinder was extracted to obtain a PLLA cylindrical porous film shell with an inner diameter of about 1.3 mm. The morphology of the obtained cylindrical porous shell is shown in Figure 3

[0070] (3) Extraction of mesenchymal stem cells

[0071] Rat mesenchymal stem cells were isolated from the femur and tibia of a newborn rat (3 days old) and cultured in an α-MEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS). The cells were maintained in a humidified atmosphere at 37°C, 5% CO2, and the culture medium was replaced every two days. Passage 2 to 4 was used for the following experiments.

[0072] (4) Seeding of stem cells

[0073] The mesenchymal stem cells extracted from the rat suckling mouse were seeded at a concentration of 1 x 10 6 ​​ / mL density seeding on the fiber membrane, gently shaking to make the cells evenly spread, after the cells adhere, 2.5% light-cured hydrogel solution is gently added on the surface of the cells, the composition of the light initiator is lithium phenyl (2, 4, 6-trimethylbenzoyl) phosphate, and the composition of the hydrogel solution is methacrylated hyaluronic acid. After irradiation with ultraviolet light for 10s, 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) α-MEM medium is added on the hydrogel, and the culture box is placed in a humidified atmosphere at 37℃, 5% CO2 for 2 days.

[0074] (5) Assembly of stem cell-loaded nerve scaffolds

[0075] Please refer to Figure 4 The structure diagram of the stem cell-loaded nerve scaffold provided is shown, wherein A is a front structure diagram, and B is a top structure diagram. The PLLA oriented fiber membrane covered with the hydrogel is rolled into a spiral-shaped concentric spool along the direction of the fiber orientation arrangement, and is inserted into a preformed PLLA cylindrical porous shell to obtain an assembled stem cell-loaded nerve scaffold.

[0076] After the assembled nerve scaffold is rapidly frozen in liquid nitrogen, it is embedded in a frozen section embedding agent (OCT embedding agent), and then a 30μm section is cut out with a freezing microtome and attached to a clean silicon wafer for SEM observation. Before observation, gold spraying equipment is used to spray gold for 60s, and then SEM observation is performed, and the SEM morphology diagram is shown in Figure 5 As can be seen from Figure 5 It can be seen that the cross-sectional structure of the entire scaffold can be seen at a small magnification, the outer layer is a porous PLLA shell, and the inner layer is a multilayer PLLA fiber.

[0077] Example 2: Effect evaluation

[0078] (1) Biocompatibility test

[0079] The prepared nerve scaffold is subjected to biocompatibility test, and three comparisons are set, wherein the control group is to culture the extracted mesenchymal stem cells directly on the cell plate; the fiber membrane group is to culture the extracted mesenchymal stem cells on the prepared PLLA nanofiber membrane; and the hydrogel group is to culture the extracted mesenchymal stem cells on the hydrogel material; the culture conditions are as in step (3) of Example 1, and the effects of the scaffold material on the cell activity are evaluated by live and dead cell staining and CCK8 at 1st, 3rd, 5th and 7th days of cell culture, and the biocompatibility test results of Example 1 and the three comparisons are shown in Figure 6 , wherein A is the live and dead staining test result of the cells; B is the CCK8 test result at 1st, 3rd, 5th and 7th days.

[0080] From Figure 6It can be seen from the results that the nerve scaffold material of the embodiment 1 of the present application has good compatibility with stem cells, and the scaffold material does not affect the normal activity of the cells.

[0081] (2) Cell distribution test results

[0082] According to the culture method in the biocompatibility test, the cells are cultured on the nerve scaffold of the present application for 14 days, and then fixed with 4% paraformaldehyde (PFA) for 10 minutes, permeabilized with 0.5% Triton X-100 for 10 minutes, and blocked with 5% goat serum for 40 minutes at room temperature. Then, the antibodies specific to the proteins GFAP and S100 expressed by Schwann cells are used for staining overnight at 4°C. After incubation, the cells are washed with PBS three times, and then stained with secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgG) for 2 hours at room temperature. Finally, the cells are stained with DAPI for 10 minutes, and observed by laser scanning confocal microscopy. The immunofluorescence images of the distribution of stem cells in the nerve scaffold are shown in Figure 7 .

[0083] It can be seen from Figure 7 that the cells have good directional arrangement on the fibers, and the positive areas of S100 and GFAP indicate that the mesenchymal stem cells loaded on the material have been mostly differentiated into Schwann cells.

[0084] (3) Evaluation of stem cell differentiation on the surface of the material loaded with stem cells

[0085] According to the culture method in the biocompatibility test, the cells are cultured on the nerve scaffold material of the present application (i.e., the Scaffold group) and on ordinary cell culture plates (i.e., the TCP group), respectively, and then the cells after 21 days of culture are subjected to immunofluorescence staining, in which GFAP and S100 are both specific indicators of Schwann cells. The test results are shown in Figure 8 , in which A is the image of the immunofluorescence staining of the cells, B and C are the proportions of the relative GFAP and S100 positive areas, respectively, which are counted according to the results of the immunofluorescence, and Merge refers to the superposition of the three immunofluorescences of GFAP, S100 and DAPI.

[0086] The results show that the cells cultured on the nerve scaffold material of the present application are more inclined to differentiate into Schwann cells.

[0087] (4) Evaluation of stem cell secretion on the surface of the material loaded with stem cells

[0088] According to the culture method in the biocompatibility test, the cells were cultured on the general cell culture plate (i.e. TCP group) and the nerve scaffold material of the application (i.e. Scaffold group) respectively. After 14 days of cell culture, the supernatant of the cells in the two groups was taken, and the level of the nutrient factors (brain-derived neurotrophic factor (BDNF), beta neurotrophic factor (beta-NGF) and vascular endothelial growth factor (VEGF)) secreted by the cells in the culture supernatant was evaluated by enzyme-linked immunosorbent method (Elisa), and the test results are shown in Figure 9 , wherein A, B and C are respectively the concentration of BDNF, beta-NGF and VEGF in the supernatant of the cells tested by elisa.

[0089] The results show that the level of the three kinds of nutrient factors in the Scaffold group of the scaffold material loaded with stem cells of the application is much higher than that in the TCP group.

[0090] (5) Repair evaluation of rat sciatic nerve injury animal experiment

[0091] After the sciatic nerve injury model of the rat was constructed (referring to CN113100994A), the nerve scaffold loaded with stem cells prepared by the application was implanted, and the implantation photo is shown in Figure 10 . 12 weeks after the operation, the myelinated axons of the regenerated nerves were morphologically evaluated by transmission electron microscopy, and the results are shown in Figure 11 , wherein A and C are respectively the low-magnification and high-magnification micrographs of the myelinated axons of the regenerated nerves in the autograft group; B and D are respectively the low-magnification and high-magnification micrographs of the myelinated axons of the regenerated nerves in the nerve scaffold group of the application; Figure 11 The results in Figure 12 show that after treatment with the nerve scaffold loaded with stem cells, the regenerated nerves are remyelinated, and have a dense myelin sheath comparable to the level of the positive control autograft group. In addition, sciatic nerve injury also causes atrophy of the innervated muscles. The recovery of the muscles after treatment was evaluated 12 weeks after the operation, and the morphology and Masson staining results of the gastrocnemius muscle are shown in Figure 12 , wherein A and B are respectively the ex vivo photos of the left and right leg gastrocnemius muscles innervated by the sciatic nerve of the rat in the autograft group and the stem cell scaffold group, and C and D are respectively the Masson staining results of the tissue sections of the muscle tissues in the autograft group and the stem cell scaffold group; it can be seen that the anatomical morphology of the left and right gastrocnemius muscles of the rat in the treatment group with the nerve scaffold loaded with stem cells is less different and close to the autograft group, and the proportion of collagen in the Masson staining is also close to the autograft group.

[0092] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not to limit the present application; although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the specific implementation of the present application can be modified or some technical features can be replaced by equivalent ones, and all of these should be included in the technical solution range of the present application claimed.

Claims

1. A method for preparing a neural scaffold loaded with stem cells, characterized in that, The process includes seeding stem cells on the surface of a two-dimensional material, a nanofiber membrane with oriented fiber arrangement, applying a biocompatible hydrogel to the seeded stem cell surface and solidifying it, and using the stem cell-loaded nanofiber membrane to construct a three-dimensional neural scaffold loaded with stem cells. The seeding method involves dripping a culture medium containing stem cells onto the surface of a two-dimensional material, a nanofiber membrane with oriented fibers, spreading the culture medium evenly on the nanofiber membrane, and allowing it to stand to allow the stem cells to adhere to the membrane. The method of constructing a three-dimensional stem cell-loaded neural scaffold using a nanofiber membrane loaded with stem cells involves rolling the nanofiber membrane with attached stem cells along the direction of fiber orientation, and then integrating the roll into a tubular porous shell to obtain a three-dimensional stem cell-loaded neural scaffold. The biocompatible hydrogel is a biocompatible hydrogel with photocuring properties.

2. The method for preparing a neural scaffold loaded with stem cells according to claim 1, characterized in that, The reel has a multi-layer spiral structure or a single-layer structure.

3. The method for preparing a neural scaffold loaded with stem cells according to claim 2, characterized in that, The number of layers in the multi-layered spiral structure is 2 to 5.

4. The method for preparing a neural scaffold loaded with stem cells according to claim 1, characterized in that, The method for preparing the tubular porous shell is as follows: poly-L-lactic acid is dissolved in a low-boiling-point solvent, a pore-forming agent is added, the solvent is removed to form a PLLA porous film, and then the PLLA porous film is formed into a hollow cylindrical tubular porous shell using high temperature.

5. The method for preparing a neural scaffold loaded with stem cells according to claim 4, characterized in that, The inner diameter of the tubular porous shell is 1mm to 3mm.

6. A method for preparing a neural scaffold loaded with stem cells according to any one of claims 1 to 5, characterized in that, It also includes adding culture medium to the solidified hydrogel for cultivation.

7. The method for preparing a neural scaffold loaded with stem cells according to claim 6, characterized in that, The culture medium was α-MEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum.

8. The method for preparing a neural scaffold loaded with stem cells according to claim 1, characterized in that, The seeding is performed by placing the stem cells on one surface of the fiber-defined nanofiber membrane, or the seeding is performed on both surfaces of the fiber-defined nanofiber membrane.

9. A method for preparing a neural scaffold loaded with stem cells according to claim 1 or 8, characterized in that: The seeding density of the stem cells is 1×10⁻⁶. 5 / mL~9×10 6 / mL.

10. The method for preparing a neural scaffold loaded with stem cells according to claim 9, characterized in that, The seeding density of the stem cells is 1×10⁻⁶. 6 / mL.

11. A method for preparing a neural scaffold loaded with stem cells according to claim 1 or 8, characterized in that, The stem cells are selected from at least one of embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, and neural stem cells.

12. The method for preparing a neural scaffold loaded with stem cells according to claim 1, characterized in that, The biocompatible hydrogel with photocurable properties is selected from at least one of methacrylamide hyaluronic acid, methacrylamide sodium alginate, and methacrylamide gelatin.

13. The method for preparing a neural scaffold loaded with stem cells according to claim 1, characterized in that, The raw materials for preparing the nanofiber membrane with oriented fiber arrangement are selected from at least one of poly-L-lactic acid, polyvinyl alcohol, and polycaprolactone.

14. The method for preparing a neural scaffold loaded with stem cells according to claim 13, characterized in that, The molecular weight of the poly-L-lactic acid is 200,000 to 300,000.

15. The method for preparing a neural scaffold loaded with stem cells according to claim 13, characterized in that, The molecular weight of the poly-L-lactic acid is 260,000.

16. The method for preparing a neural scaffold loaded with stem cells according to claim 1, characterized in that, The method for preparing the nanofiber membrane with oriented fiber arrangement is selected from at least one of electrospinning, self-assembly, stretching, and template synthesis.

17. The method for preparing a neural scaffold loaded with stem cells according to claim 16, characterized in that, The nanofiber membrane was prepared by electrospinning.

18. A neural scaffold loaded with stem cells, characterized in that, The neural scaffold is prepared according to any one of claims 1 to 17.

19. The use of the stem cell-loaded neural scaffold as described in claim 18 in the preparation of drugs or medical devices for treating peripheral nerve injury.

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