Gradient cross-linking self-curling neural restoration hydrogel film as well as preparation method and application of gradient cross-linking self-curling neural restoration hydrogel film

Abstract

The invention discloses a gradient cross-linking self-curling neural restoration hydrogel film and a preparation method and application thereof.The method comprises the steps that 1, a macromolecular solution is prepared, specifically, a macromolecular polymer is dissolved in deionized water and stirred to be uniform, macromolecular solutions with the same or different concentrations are prepared, adhesive molecules with adhesive groups are added, stirring and dispersing are conducted to be uniform, and a hydrogel solution is obtained; high-molecular adhesive liquids with the same or different concentrations are obtained; 2) high-molecular film pasting: pouring the high-molecular adhesive liquid into a mold, and drying to form a high-molecular film; or sequentially pouring the high-molecular adhesive liquids with different concentrations into the mold, and drying to form high-molecular films with different gradients; and 3) crosslinking: pouring the polymer crosslinking agent solution onto the polymer film to crosslink the polymer film from top to bottom, and then drying to form the gradient crosslinking self-curling neural restoration hydrogel film with different pore diameters. Different swelling gradient characteristics are formed by regulating and controlling the crosslinking density of macromolecular chains in different areas of the film, and the film can be applied to neural restoration.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and specifically relates to a gradient cross-linked self-curling nerve repair hydrogel film, a preparation method thereof, and an application thereof. Background Art

[0002] The repair of peripheral nerve injuries is a major problem in clinical medicine. Especially when dealing with long nerve gaps or nerve defects (such as nerve defects or brachial plexus root avulsion injuries), traditional direct suture techniques often face the risk of repair failure due to complex operations and poor material adaptability. The main problems are manifested in excessive tension during suture, epineurium exfoliation, neuroma formation, etc. In addition, the repair effect highly depends on the surgical skills of senior physicians, making the treatment process extremely demanding on the skills of physicians and having low popularity.

[0003] Although autologous nerve transplantation is regarded as the "gold standard", its clinical application is limited by many factors such as donor site injury, limited donor source, size mismatch, and the need for secondary surgery. New nerve conduit materials such as chitosan sleeves can assist nerve repair, but still require fine suture in clinical operations, which not only has extremely high requirements for the skills of physicians, but also has a cumbersome process, is prone to secondary damage to the sleeve, exacerbates the difficulty of nerve repair and prolongs the recovery time. Therefore, it is particularly important to develop an artificial nerve conduit stent with "rapid closure, seamless suture, excellent adhesiveness and adaptability". In particular, a conduit with good curling property and automatic closure function can efficiently adapt to nerve stumps without complex suture operations, reduce the operation difficulty of physicians, reduce surgical trauma, and improve the clinical effect of nerve repair. The breakthrough of this technology has important clinical significance and broad application prospects for solving the problem of peripheral nerve injury repair. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a gradient cross-linked self-curling nerve repair hydrogel film, a preparation method thereof, and an application thereof. The hydrogel film has a pore size gradient from top to bottom. By regulating the cross-linking density of polymer chains in different regions of the film, different swelling gradient characteristics are formed. As a nerve repair material, it has the advantages of adjustable structure, rapid response, high efficiency and controllability, and can be widely applied in the biomedical field.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A preparation method of a gradient cross-linked self-curling nerve repair hydrogel film, comprising the following steps:

[0007] 1) Preparation of polymer solution: Dissolve the polymer in deionized water, stir evenly to prepare polymer solutions with the same or different concentrations, and add adhesion molecules with adhesion groups, then stir and disperse evenly to obtain polymer adhesion liquids with the same or different concentrations and adhesion functions;

[0008] 2) Polymer film formation: Pour the polymer adhesion liquid into a mold and dry it at a certain temperature to form a polymer film; or

[0009] Pour polymer adhesion liquids with different concentrations into a fixed mold in sequence and dry it at a certain temperature to form polymer films with different gradients;

[0010] 3) Crosslinking: Dissolve the polymer crosslinking agent in deionized water to prepare a polymer crosslinking agent solution, and then pour it onto the polymer film prepared in step 2) to cause crosslinking from top to bottom, and then dry it to form a gradient crosslinked self - curling nerve repair hydrogel film with different pore sizes.

[0011] Among them, by controlling the crosslinking density of the polymer chain from top to bottom, different swelling gradient differences are realized. Under humidity stimulation, swelling differences on the upper and lower surfaces are induced to form a self - curling hydrogel film. That is, by controlling the concentration of the polymer chain or the concentration of the added crosslinking agent to be different, the crosslinking density gradient is achieved, thereby realizing the swelling gradient difference. When the concentration of the polymer solution is the same, the polymer solution cannot form a swelling gradient, but a crosslinking gradient will be formed after crosslinking; for polymer solutions with different concentrations, a swelling gradient can be directly formed, and then a crosslinking gradient is formed.

[0012] The preparation process of this application is simple. By constructing a hydrogel film with a swelling rate gradient from top to bottom, precise response under humidity stimulation is achieved. By regulating the crosslinking density of the polymer chain in different regions of the film, significant swelling gradient characteristics are formed. The film can exhibit regional swelling differences under humidity stimulation, enabling it to wrap the nerve stump and conformally contact the nerve, thus effectively promoting the repair of nerve injury. Since polymer solutions with different concentrations can form a gradient crosslinking density, and polymer solutions with the same concentration can be regulated by crosslinking agents to form a gradient crosslinking density, the gradient crosslinking density of the polymer film can be controlled by adjusting the concentration of the polymer chain or introducing crosslinking agents with different concentrations, thereby realizing the design and optimization of the swelling gradient. As a new type of nerve repair material, this hydrogel film shows important research value and broad clinical application prospects.

[0013] In an embodiment of the present invention, in step 2), pour the polymer adhesion liquid into a mold, place it in an oven, and dry it at 35 - 38 °C for 12 - 24 hours to form a polymer film;

[0014] For example, dry at 37°C for 14 hours.

[0015] In one embodiment of the present invention, the polymer is selected from one or more of polyvinyl alcohol, chitosan, gelatin, carboxymethyl cellulose, sodium alginate, polyacrylamide, and polyacrylic acid.

[0016] In one embodiment of the present invention, the mass concentration of the polymer solution is 5% to 50%, preferably 10% to 30%.

[0017] For example, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%.

[0018] In one embodiment of the present invention, the molecule with adhesion function is selected from one or more of dopamine nanoparticles, tannic acid, gallic acid, anthocyanin, giant salamander secretion, and acryloyl-N-hydroxysuccinimide.

[0019] In one embodiment of the present invention, the mass concentration of the polymer adhesion liquid is 5% to 30%, preferably 10% to 20%.

[0020] For example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%.

[0021] In one embodiment of the present invention, the crosslinking agent is selected from one or more of glutaraldehyde, genipin, phosphate triester, propylene oxide, and hexamethylene diisocyanate.

[0022] In one embodiment of the present invention, the mass concentration of the crosslinking agent solution is 0.1% to 10%, preferably 0.5% to 5%.

[0023] For example, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%.

[0024] By controlling the concentration of the crosslinking agent solution, the crosslinking degree of the polymer can be controlled. If the concentration of the crosslinking agent solution is too high, it is easy to over-crosslink, which is not conducive to the formation of the crosslinking gradient density; on the contrary, if the concentration of the crosslinking agent solution is too low, the crosslinking density may be insufficient, which is not conducive to the gel stability and the matching of mechanical properties. After comprehensive consideration, the concentration of the crosslinking agent solution in this application is preferably controlled within the above range.

[0025] In one embodiment of the present invention, polymer adhesion liquids with different concentrations are poured into a fixed mold in sequence, and the mass concentrations of the polymer adhesion liquids in different layers differ by 4%.

[0026] In one embodiment of the present invention, the preparation method further includes a step of film laminating and forming. The prepared hydrogel film is cut into a specific size for nerve repair.

[0027] The present invention also provides a gradient cross-linked self-curling nerve repair hydrogel film with a gradient cross-linking density. The hydrogel film is a gradient-regulated self-deforming nerve repair hydrogel film, which can quickly self-curl into a tubular structure under humidity stimulation and aqueous medium conditions for nerve injury repair.

[0028] In one embodiment of the present invention, the self-curling nerve repair hydrogel film has 1 to 6 film layers with a swelling rate gradient from top to bottom; preferably 1 to 3 layers.

[0029] Preferably, the pore size of the hydrogel film is 20 μm to 500 μm, preferably 50 μm to 200 μm; the pore sizes of the top layer and the bottom layer differ by 20% to 50%.

[0030] In one embodiment of the present invention, the porosity of the hydrogel film is 30% to 80%, and the porosity difference between the top layer and the bottom layer is 20% to 50%.

[0031] Preferably, the thickness of the hydrogel film is 1 mm to 5 mm, preferably 1 mm to 3 mm.

[0032] The present invention also provides an application of the gradient-regulated self-curling nerve repair hydrogel film in nerve injury, preferably in the repair of sciatic nerve injury, traumatic peripheral nerve injury, radial nerve injury, and peroneal nerve injury.

[0033] Advantages of the present invention:

[0034] The present invention provides a gradient-regulated self-curling nerve repair hydrogel film. The present invention forms a hydrogel film patch with a gradient pore size through gradient cross-linking or gradient concentration change of a polymer solution. The hydrogel film has a swelling rate gradient from top to bottom, and a swelling gradient characteristic is formed by regulating the cross-linking density of polymer chains in different regions of the film. Specifically, it has the following advantages:

[0035] 1) Gradient swelling characteristic design: By regulating the cross-linking density of polymer chains, a hydrogel film with a swelling rate gradient from top to bottom is constructed. This gradient characteristic is the key to material design, endowing the film with the ability to exhibit regional swelling differences under humidity stimulation conditions. The gradient characteristic optimizes the function of the hydrogel film in nerve repair and has unexpected advantages and effects.

[0036] 2) Humidity-responsive wrapping ability: Under humidity stimulation, the hydrogel film can exhibit regional swelling differences. The hydrogel film can quickly adjust its own morphology to form a wrap around the nerve stump and conformal contact with the nerve, thus achieving efficient nerve protection and repair. This humidity-responsive dynamic behavior provides higher adaptability and efficiency for nerve repair, and thus is applicable to the field of nerve injury repair.

[0037] 3) Simplify the repair process: The hydrogel film can quickly form effective contact with the nerve stump, reduce secondary injuries caused by external stimuli, and shorten the operation time. This rapid adaptation and functional response are difficult to achieve with existing technologies, significantly improving the efficiency of clinical operations.

[0038] 4) The hydrogel film has a top-down swelling rate gradient and can exhibit regional swelling differences under humidity stimulation in an aqueous medium condition, enabling it to quickly form a functional film structure for nerve injury repair. Therefore, it is a novel nerve repair material with adjustable structure, rapid response, high efficiency and controllability, and can be widely applied in the biomedical field. Brief Description of the Drawings

[0039] Figure 1 is a schematic diagram of the cross-linking process of the gradient cross-linked self-curling nerve repair hydrogel film;

[0040] Figure 2 is a physical picture of the gradient cross-linked self-curling nerve repair hydrogel film prepared in Example 1;

[0041] Figure 3 is the SEM image of the gradient cross-linked self-curling nerve repair hydrogel film prepared in Example 1;

[0042] Figure 4 is the automatic curling process diagram of the gradient cross-linked self-curling nerve repair hydrogel film prepared in Example 1 under humidity conditions;

[0043] Figure 5 is the stress-strain curve diagram of the nerve repair hydrogel film prepared in Example 1;

[0044] Figure 6 is the adhesion curve diagram of the nerve repair hydrogel film prepared in Example 1 adhered to nerve tissue;

[0045] Figure 7 is the cell viability diagram of the nerve repair hydrogel film prepared in Example 1;

[0046] Figure 8Neural characterization diagrams of the self - curling nerve repair hydrogel film prepared in Example 1 for repairing the sciatic nerve of SD rats; (A) is the NF200 staining of the rat sciatic nerve, indicating good axon growth during the treatment process; (B) is the S100 staining of the rat sciatic nerve, indicating good axon growth during the treatment process; (C) is the toluidine blue staining of the rat sciatic nerve, showing good nerve recovery; (D) is the scanning electron micrograph of the rat sciatic nerve repair, and it can be seen that its axons are well - recovered.

[0047] Figure 9 Functional characterization diagrams of the nerve repair hydrogel film prepared in Example 1 for repairing the sciatic nerve of SD rats; (A) is the Masson staining of the muscle; (B) is the muscle comparison diagram, indicating good innervation of the distal gastrocnemius and soleus muscles; (C) is the result of nerve electrophysiological testing, and it can be seen that the signal conduction function is good; (D) is the Carwalk paw print diagram, showing good recovery of SD rats after nerve regeneration;

[0048] Figure 10 Scanning electron micrograph of the nerve repair hydrogel film prepared in Example 2;

[0049] Figure 11 Scanning electron micrograph of the nerve repair hydrogel film prepared in Example 3;

[0050] Figure 12 Schematic diagram of the process of wrapping the nerve repair with the nerve repair hydrogel film prepared in Example 3. Detailed implementation manners

[0051] The following will further elaborate on the present invention in conjunction with specific embodiments. It should be understood that the following embodiments are only for illustrative and explanatory purposes of the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0052] Example 1

[0053] 1) Prepare a 5% chitosan solution: Add 5 g of chitosan powder to 100 mL of 1% acetic acid solution by mass, and stir well to completely dissolve it. Add 1% (percentage by mass of chitosan) of dopamine nanoparticles with adhesion function to the solution to make it evenly dispersed, obtaining a chitosan solution with adhesion function. The concentration of the chitosan solution is 5%.

[0054] 2) High - molecular film - making: Pour the chitosan solution into a mold, place it in an oven, and dry it at 38 °C for 24 hours to form a chitosan film.

[0055] 3) Crosslinking: Pour the genipin solution with a mass concentration of 0.5% onto the upper layer of the chitosan film prepared above, allow it to penetrate from top to bottom, and let it stand for crosslinking at room temperature for 12 h to form a hydrogel film with gradient crosslinking, as Figure 2 shown.

[0056] 4) Film laminating and forming: Cut the prepared gradient hydrogel film into small pieces with a size of 10 mm × 10 mm for nerve injury repair experiments.

[0057] Figure 3 Figure Figure 2 is the SEM image of the repair hydrogel film prepared in Example 1. It can be seen that the hydrogel film has an obvious pore structure gradient from top to bottom. The crosslinking density pore size of the upper layer is smaller, and that of the lower layer is larger. This is because after genipin is added to the polymer solution, it starts to penetrate from top to bottom, resulting in a large crosslinking density in the upper layer and a relatively small pore size. As genipin penetrates, the concentration of genipin reaching the bottom decreases, so its crosslinking density becomes smaller, thus forming a small pore size in the upper layer and a large pore size in the lower layer.

[0058] The pore size and porosity of the hydrogel film prepared in Example 1 were calculated and statistically analyzed by ImageJ. The thickness of the hydrogel film is 1.2 mm. The pore size of the top layer of the hydrogel film is 50 μm, and that of the bottom layer is 200 μm. The porosity of the top layer of the hydrogel film is 70%, and the porosity of the bottom layer is 50%. The difference in porosity is 40%.

[0059] As Figure 4 Figure Figure 4 is the automatic curling process diagram of the nerve repair hydrogel film prepared in Example 1 under humid conditions. It can be seen that the hydrogel film curls from left to right, curling from a rectangle to a circle. This is because the hydrogel has a certain difference in porosity, and its water absorption rate is different after entering the water, resulting in bending.

[0060] According to the international standard: the test method of ASTM D3039 (Standard Test Method for Tensile Properties of Polymer Matrix Composites), the mechanical properties of the gel film prepared in Example 1 were tested by a universal tensile machine, and the stress-strain curve was obtained by calculating with the following formula.

[0061] Stress = F (force) / A (area of force application)

[0062] Strain = ΔL (deformation distance) / L (initial length)

[0063] The stress-strain curve is as Figure 5 shown. From the figure, its maximum tensile strength can be obtained as 4.8 Mpa, which fully meets the mechanical property requirements for peripheral nerve repair.

[0064] Adhesion force test:

[0065] Reference: Dry double-sided tape for adhesion of wet tissues and devices. Nature 2019, 575(7781): 169-174. The adhesion curve of the hydrogel film prepared in Example 1 adhered to nerve tissue was measured by peeling using a universal tensile machine. As Figure 6 shown, during the peeling process, an adhesion force of 160 J·m -2 is required, proving that its adhesion force is strong, it can adhere well to the nerve, prevent it from falling off and failing, and will not damage the nerve, and can effectively treat damaged nerves.

[0066] Cytotoxicity test of the material:

[0067] The hydrogel film was immersed in DMEM / FBS medium and soaked at 37 °C for 24 h, and the leachate of the hydrogel film was extracted and co-incubated with RSC96 cells for 24 h. After digestion, the cells were centrifuged and resuspended. The cell suspension was inoculated into a 96-well plate at 100 μl / well, and 10 μl / well of CCK-8 solution was added to each well. After incubation for 4 h, the absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and its cell viability was calculated by the following formula:

[0068] Cell viability (%) = OD value of the sample / OD value of the blank group.

[0069] As Figure 7 shown, it can be found that when the cells are at 200 mg / ml, the cell survival rate reaches 65%, and when the cells are at 100 mg / ml, the cell survival rate reaches over 90%, proving that the hydrogel film has good cell viability.

[0070] An animal model of 10 mm injury repair of the sciatic nerve of SD rats was constructed. The specific surgical methods and steps are as follows: The rats were placed in a small animal anesthetic induction box, and anesthetized by induction with 5% isoflurane (flow rate 600 mL / min). After complete anesthesia, it was changed to 2% isoflurane (500 mL / min) to maintain anesthesia. The rats were fixed prone on the operating table, and the left lower limb was shaved and disinfected 3 times with 75% alcohol. At 2 mm below the femur of the rat, a diagonal incision about 1 cm long was made from the knee joint to the ischial tuberosity. The gluteal muscle space was bluntly dissected, and the surgical wound was enlarged using a retractor to expose the left sciatic nerve. The sciatic nerve was dissected free, and the sciatic nerve was transected 1 mm proximal to the branch of the sural nerve from the lower edge of the piriformis muscle, causing a 10 mm injury to the sciatic nerve.

[0071] Under a surgical microscope, the self-curling nerve repair hydrogel film prepared in Example 1 was used to repair the sciatic nerve of SD rats. The 2 mm at both ends of the sleeve tube were the nerve stumps, and the 2 mm in the middle was the nerve injury area. After the implantation was completed, the surgical wound was rinsed with normal saline, the gluteal muscle space was sutured with 4-0 absorbable braided sutures, and the skin was sutured with 4-0 non-absorbable braided sutures. The surgical wound was disinfected by wiping with iodophor and the rats were prevented from licking or biting the surgical wound. After disinfecting the left ear with iodophor, an ear tag was used for numbering and marking. After the animals recovered from anesthesia, they were placed back in the cage, given pain-relieving jelly for analgesia, allowed to drink water and eat normally, and raised for 12 weeks. After the operation, the surgical side and the condition of the paw of the rats were closely observed: the healing condition of the surgical wound, the movement of the toes, and whether there were ulcers or autophagy of the toes.

[0072] Figure 8 The repair effect of the sciatic nerve of the rats in the treatment group is shown. The nerve characterization is as follows: (A) is the NF200 staining of the sciatic nerve of the rat, indicating that axons grow well during the treatment; (B) is the S100 staining of the sciatic nerve of the rat, showing that axons grow well during the treatment; (C) is the toluidine blue staining of the sciatic nerve of the rat, showing good nerve recovery; (D) is the scanning electron micrograph of the repair of the sciatic nerve of the rat, and it can be seen that its axons recover well.

[0073] Figure 9 The functional effect after the repair of the sciatic nerve of the treated rats is shown: (A) Masson staining of the muscle; (B) is a muscle comparison diagram, indicating good innervation of the distal gastrocnemius and soleus muscles; (C) is the result of nerve electrophysiological testing, showing good signal conduction function; (D) is the Carwalk paw print diagram, showing good recovery of SD rats after nerve regeneration. Figure 9 It is proved that the nerve repair hydrogel film has good curative effect on nerve injury.

[0074] Example 2

[0075] 1) Preparation of polymer solution: 20 g, 10 g, and 5 g of silk fibroin powder were respectively added to 100 mL of deionized water and stirred well to completely dissolve them, preparing silk fibroin solutions with mass concentrations of 20%, 10%, and 5% respectively. Dopamine nanoparticles with a mass ratio of 2% (the mass of 2% silk fibroin) were added to the 20%, 10%, and 5% silk fibroin solutions respectively to make them evenly dispersed, obtaining silk fibroin solutions with adhesion functions.

[0076] 2) Polymer film application: The above-prepared chitosan solutions were poured into the mold in order of decreasing concentration. The mass concentrations of different layers of polymer adhesion solutions differed by 4%. Then, they were dried at 25 °C for 10 hours. After each layer of solution was dried into a film, the next solution was poured on top to form a three-layer silk fibroin film with a swelling gradient characteristic.

[0077] 3) Crosslinking: Immerse the above-prepared three-layer silk fibroin film in a 0.5 wt% genipin crosslinking agent solution, and let it stand at room temperature for 12 hours to allow the genipin solution to crosslink gradually from top to bottom, forming a gradient hydrogel film. After 12 hours of crosslinking, wash away the unreacted genipin with deionized water. The silk fibroin solution concentration in the upper layer is relatively high, so it can be crosslinked by more crosslinking agents, resulting in a relatively dense void structure; the silk fibroin solution concentration in the middle layer is moderate, and thus its crosslinking density is also moderate; while the silk fibroin solution concentration in the bottom layer is the lowest, and it can only be crosslinked by a small amount of genipin, thereby forming a hydrogel film with a relatively low crosslinking density and a larger void structure. The scanning electron micrograph is as shown in Figure 10 shown, and the thickness of the hydrogel film is 1.5 mm.

[0078] 4) Film forming: Cut the above-prepared gradient hydrogel film into small pieces of 10 mm × 10 mm in size for nerve injury repair experiments. The differences in pore size and porosity of the hydrogel film prepared in Example 2 are shown in Table 1.

[0079] Table 1

[0080] Level Aperture (nm) Porosity (%) Bottom layer 200μm 30 Middle layer 100μm 50 Top layer 20μm 70

[0081] Example 3

[0082] 1) Preparation of polymer solution: Dissolve 5 g of alginic acid and 5 g of gelatin in 100 mL of deionized water, stir well to dissolve, and prepare a composite polymer solution with a mass concentration of 10%. Then add the adhesion molecule tannic acid with an adhesion group, so that its mass fraction in the composite solution is 0.5%, and stir well to obtain a composite polymer adhesion liquid with adhesion function.

[0083] 2) Polymer film formation: Pour the above composite polymer adhesion liquid evenly into a mold, and let it stand and dry at 35 °C for 12 hours to form a uniform single-layer film. Continue to pour the composite polymer solution on the single-layer film, and repeat this step twice. Finally, obtain a three-layer composite film with a total thickness of about 1.5 mm.

[0084] 3) Crosslinking: Crosslink the above-prepared three-layer composite film with a 0.2 wt% glutaraldehyde solution. The crosslinking temperature is 37 °C, and the crosslinking time is controlled for 6 hours to crosslink gradually from top to bottom to form a gradient hydrogel film. The top layer of the hydrogel film forms a relatively dense structure due to the longer contact time with the crosslinking agent, while the bottom layer maintains a larger pore size. The SEM image is as shown in Figure 11 shown.

[0085] 4) Film forming: Cut the prepared gradient hydrogel film into circular pieces with a diameter of 10 mm for nerve injury repair experiments. Figure 12 The process diagram of the hydrogel film wrapping nerve repair is shown.

[0086] The pore size and porosity of the hydrogel film prepared in Example 3 are shown in Table 2.

[0087] Table 2

[0088] Level Aperture (nm) Porosity (%) Lower layer 230μm 28 Top layer 30μm 53

[0089] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a gradient cross-linked self-curling nerve repair hydrogel film, characterized in that, It includes the following steps: 1) Prepare a polymer solution: Dissolve the polymer in deionized water, stir evenly to prepare polymer solutions with the same or different concentrations, and add adhesion molecules with adhesion groups, then stir and disperse evenly to obtain polymer adhesion liquids with the same or different concentrations and with adhesion functions; 2) Polymer film forming: Pour the polymer adhesion liquid into a mold and dry it at a certain temperature to form a polymer film; or Pour polymer adhesion liquids with different concentrations into a fixed mold in sequence and dry it at a certain temperature to form a polymer film with different concentration gradients; 3) Crosslinking: Dissolve the polymer crosslinking agent in deionized water to prepare a polymer crosslinking agent solution, then pour it onto the polymer film prepared in step 2) so that crosslinking occurs from top to bottom, and then dry it to form a gradient crosslinked self - curling nerve repair hydrogel film with different pore sizes.

2. The preparation method according to claim 1, wherein, The polymer is selected from one or more of polyvinyl alcohol, chitosan, gelatin, carboxymethyl cellulose, sodium alginate, polyacrylamide, and polyacrylic acid.

3. The preparation method according to claim 1, characterized in that, The mass concentration of the polymer solution is 5% - 50%, preferably 10% - 30%; For example, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%.

4. The preparation method according to claim 1, characterized in that, The molecules with adhesion functions are selected from one or more of dopamine nanoparticles, tannic acid, gallic acid, anthocyanin, giant salamander secretion, and acrylic acid - N - hydroxysuccinimide; Preferably, the mass concentration of the polymer adhesion liquid is 5% - 30%, preferably 10% - 20%; For example, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%; Preferably, in step 2), it is dried at 35 - 38 °C for 12 - 24 hours.

5. The preparation method according to claim 1, characterized in that, The crosslinking agent is selected from one or more of glutaraldehyde, genipin, phosphate triester, propylene oxide, and hexamethylene diisocyanate; Preferably, the mass concentration of the crosslinking agent solution is 0.1% - 10%, preferably 0.5% - 5%.

6. The preparation method according to claim 1, characterized in that, It also includes cutting the hydrogel film into a specific size, and then sterilizing and packaging it.

7. A gradient crosslinked self - curling nerve repair hydrogel film, characterized in that, It includes a film layer with a porosity gradient from top to bottom. The hydrogel film can quickly self - curl into a tubular structure under humidity stimulation and aqueous medium conditions for nerve injury repair.

8. The gradient cross-linked self-curling nerve repair hydrogel film according to claim 7, wherein The pore size of the hydrogel film is 20 μm - 500 μm, preferably 50 μm - 200 μm; the pore sizes of the top layer and the bottom layer differ by 20% - 50%.

9. The gradient cross-linked self-curling nerve repair hydrogel film according to claim 7, characterized in that, The porosity of the hydrogel film is 30% - 80%, and the porosity differences between the top layer and the bottom layer are 20% - 50%; Preferably, the thickness of the hydrogel film is 1 mm - 5 mm, preferably 1 mm - 3 mm.

10. Application of the gradient crosslinked self - curling nerve repair hydrogel film according to any one of claims 7 to 9 in nerve injury, preferably in the application of sciatic nerve injury, traumatic peripheral nerve injury, radial nerve injury repair, and peroneal nerve injury repair.

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