Pre-stretched hydrogel nerve conduit for promoting directional growth of nerves and preparation method of pre-stretched hydrogel nerve conduit

Abstract

The invention discloses a pre-stretched hydrogel nerve conduit for promoting directional growth of nerves and a preparation method of the pre-stretched hydrogel nerve conduit, and belongs to the field of preparation of multifunctional hydrogel biomedical materials. The preparation method of the nerve conduit comprises the following preparation steps: preparing a polyvinyl alcohol solution, adding gelatin into the polyvinyl alcohol solution, stirring to enable the gelatin to be completely dissolved in the polyvinyl alcohol solution to prepare a mixed solution, putting the mixed solution into a mold, gelatinizing for a certain time, and performing circulating freezing-melting cross-linking to obtain the nerve conduit. And soaking in a sodium chloride / borax solution, and circularly stretching the hydrogel to obtain the pre-stretched hydrogel nerve conduit. The inside of the pre-stretched hydrogel nerve conduit has a three-dimensional network porous structure, and the inside of the pre-stretched hydrogel nerve conduit is of a porous structure, so that nutrient substance transfer and metabolite discharge in the nerve repair process are facilitated. The surface of the pre-stretched hydrogel nerve conduit is provided with a topological structure for promoting growth of nerve cells, and the nerve cells are guided to grow directionally in the repairing process. Besides, the pre-stretched hydrogel nerve conduit has excellent biocompatibility and adjustable mechanical properties, can be used for bridging operations after peripheral nerve injury, and is gradually degraded and absorbed by organisms in the nerve regeneration process. The nerve conduit provided by the invention shows wide application potential in the field of biomedicine, and can efficiently promote repair and regeneration of human tissues such as nerves and blood vessels.

Description

Technical Field

[0001] The present invention relates to the field of preparation of multifunctional hydrogel biomaterials, and particularly relates to a pre-stretched hydrogel nerve conduit for promoting nerve directional growth and a preparation method thereof. Background Art

[0002] Peripheral nerve injury (PNI) refers to the destruction of the structure and function of peripheral nerves caused by trauma, disease or surgery. Its clinical manifestations include paresthesia, muscle weakness or even paralysis, seriously affecting the quality of life of patients. PNI is a common and frequently-occurring disease, especially common in traffic accidents, industrial injuries and sports injuries. Although peripheral nerves have a certain regenerative ability, the repair after injury still faces many obstacles, such as slow regeneration speed of nerve fibers, poor regeneration directionality and atrophy of nerve target areas.

[0003] Although autologous nerve transplantation is the current "gold standard" treatment method, it is limited by problems such as insufficient donor nerves and loss of donor site function. Alternative technologies such as artificial nerve conduits can play the role of a bridging chamber between the two severed nerve ends. However, its current clinical effect has not yet reached the expectation. Therefore, the development of a new type of nerve repair material with strong biomimicry, good biocompatibility and precise axon guidance function has become a hot spot and a difficult point in the research of peripheral nerve regeneration. Existing nerve guidance conduits have problems in mechanical properties, biocompatibility and lack of biomimetic structures.

[0004] When preparing nerve conduits, in order to prepare a biomimetic structure for the directional growth of nerve fibers, the surface topology can be designed by 3D printing, electrospinning technology or template method, etc. However, this method is relatively complex in large-scale preparation, has a high cost, and requires high equipment and operators. In clinical applications, a synthetic material that is simple to manufacture, easy to operate, has good safety, mechanical properties, biodegradability, and at the same time has the characteristics of a nerve fiber biomimetic structure is expected. Summary of the Invention

[0005] Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a pre-stretched hydrogel nerve conduit for promoting the directional growth of nerve cells and a preparation method thereof, which can enable neuron cells to grow directionally along the hydrogel topology structure, and at the same time has excellent biocompatibility and adjustable mechanical properties. A hydrogel conduit prepared by the present invention has appropriate mechanical strength, good biocompatibility and a biomimetic oriented topological structure. The oriented fiber structure is an inherent natural feature of nerve tissue. Therefore, the hydrogel of the present invention effectively guides the directional growth of neuron cells on the surface of the hydrogel nerve conduit. Its adjustable mechanical properties solve the strain problem caused by impact during the nerve repair process, and at the same time, conductive polymers such as polypyrrole and graphene can be added to increase conductivity and promote nerve repair.

[0006] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions.

[0007] The present invention discloses a preparation method of a pre-stretched hydrogel nerve conduit for promoting nerve directional growth, which is characterized by comprising the following steps: Prepare a polyvinyl alcohol solution: Add polyvinyl alcohol to deionized water according to the mass ratio, stir for 2 - 4 h under a water bath condition of 90 °C, and the stirring speed is 250 rpm / min to completely dissolve the polyvinyl alcohol, obtaining a polyvinyl alcohol solution; Prepare a mixture: Add gelatin powder to the above polyvinyl alcohol solution, stir for 1 - 3 h under a water bath condition of 50 - 60 °C according to the mass ratio, and the stirring speed is 200 - 250 rpm / min to completely dissolve the gelatin powder, obtaining a mixture; Hydrogel preparation: Pour the mixture into a mold, gelate for 1.5 h, then perform 3 freeze-thaw cycles, the freezing temperature is -80 °C, the freezing time is 8 h, the melting temperature is 20 °C, the melting time is 2 h, and then soak it in a mixed solution of 25 wt% sodium chloride and 0.8 wt% borax for 6 h; Pre-stretched hydrogel preparation: Perform cyclic stretching on the hydrogel, and the number of cyclic stretching times is 200 - 500 times to obtain a pre-stretched hydrogel nerve conduit.

[0008] Furthermore, the mass ratio of the polyvinyl alcohol to the deionized water is 10 parts : 90 parts, and the mass ratio of the gelatin powder to the polyvinyl alcohol solution is 5 parts : 95 parts.

[0009] Furthermore, in the cyclic stretching step, the size of the hydrogel is 10 × 2 × 0.1 cm in length, width, and height.

[0010] The present invention also discloses a pre-stretched hydrogel nerve conduit for promoting nerve directional growth, which is characterized in that the nerve conduit is prepared by the preparation method described in any one of the above.

[0011] Furthermore, the surface of the nerve conduit has a topological structure for promoting nerve cell growth, and the diameter of the topological structure is 5 μm - 30 μm.

[0012] Furthermore, the inside of the nerve conduit has a three-dimensional network porous structure, and the pore diameter of the porous structure is 2 μm - 20 μm.

[0013] Furthermore, the pre-stretched hydrogel nerve conduit has no obvious residual strain during 500 cyclic stretching times.

[0014] Furthermore, the application of the pre-stretched hydrogel nerve conduit for promoting nerve directional growth described in any one of the above in the preparation of products for repairing peripheral nerve injuries.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0016] First of all, the present invention discloses a pre-stretched hydrogel nerve conduit for promoting nerve directional growth, which is prepared by using specific raw materials and preparation processes. Due to the mechanical action of pre-stretching on the hydrogel surface, a unique topological structure is formed. The diameter of the topological structure is 5 μm to 30 μm, which conforms to the bionic characteristics of nerve fibers and helps the compliant growth of nerve cells.

[0017] The hydrogel has a three-dimensional network porous structure inside, and the pore diameter of the porous structure is 2 μm to 20 μm, which promotes the transport and exchange of nutrients and metabolites.

[0018] The pre-stretched hydrogel has excellent mechanical properties, especially high tensile strength and tensile strain, and can be better stretched and deformed to adapt to the strain stress at the nerve repair site. When subjected to mechanical forces (such as when a mouse is pressed or collided), the hydrogel will not break and can still maintain its original mechanical properties.

[0019] Moreover, due to the addition of borax, [B(OH)4]− ions react with the hydroxyl groups on the PVA chain to form borate ester groups, forming a dynamic reversible ionic crosslinking, which has excellent anti-fatigue performance and no obvious residual strain in 500 cyclic stretches.

[0020] While maintaining the good mechanical properties of the hydrogel, the hydrogel has excellent ionic conductivity of 4.6 mS / cm, which can conduct electrical signals during the sciatic nerve repair process and further promote its repair efficiency.

[0021] The pre-stretched hydrogel nerve conduit for promoting nerve directional growth provided by the present invention has the advantages of simple operation, strong controllability, easy regulation, large-scale preparation and low cost, and can be widely promoted. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a scanning electron microscope photograph of the surface topological structure of the pre-stretched hydrogel prepared in Example 1 after freeze-drying.

[0023] Figure 2 is a scanning electron microscope photograph of the cross-section of the pre-stretched hydrogel prepared in Example 1 after freeze-drying.

[0024] Figure 3 is a comparison of the mechanical properties of the pre-stretched hydrogels prepared in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0025] Figure 4Comparison of the ionic conductivity of the pre-stretched hydrogels prepared in Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0026] Figure 5 It is the identification of live / dead cells after culturing the RSC96 cell line in the leaching solution of the pre-stretched hydrogel prepared in Example 1.

[0027] Figure 6 Immunofluorescence image of the cytoskeleton of PC-12 cells cultured on the surface of the pre-stretched hydrogel with a directional structure prepared in Example 1.

[0028] Figure 7 Scanning electron microscope photograph of the surface topological structure of the pre-stretched hydrogel prepared in Example 2 after freeze-drying.

[0029] Figure 8 Scanning electron microscope photograph of the surface topological structure of the pre-stretched hydrogel prepared in Example 3 after freeze-drying. Detailed implementation manners

[0030] In order to make the technical solutions, objectives, and advantages of the present invention clearer, the present invention will be described in detail below in conjunction with specific embodiments. It should be noted that the described embodiments are only exemplary contents of the present invention and not restrictive descriptions. Any other implementation manners that those skilled in the art can obtain on the basis of the embodiments of the present invention without creative labor shall be regarded as the protection scope of the present invention.

[0031] Example 1.

[0032] This example provides the preparation of a pre-stretched hydrogel, including the following steps.

[0033] S1. Stock solution preparation: Weigh polyvinyl alcohol (G108396 polyvinyl alcohol, pharmaceutical grade, gel strength ~240 g Bloom, Shanghai Aladdin Biochemical Technology Co., Ltd.) and deionized water according to the mass ratio. The mass ratio of polyvinyl alcohol to deionized water is 10 parts: 90 parts. Add polyvinyl alcohol to deionized water and stir at 90 °C in a water bath for 2 h to completely dissolve polyvinyl alcohol in deionized water. The stirring speed is 250 rpm / min to prepare the stock solution.

[0034] S2. Mixed solution preparation: Weigh gelatin powder according to the mass ratio. The gelatin powder in this example (G776063, gel strength ~240 g Bloom, Shanghai Aladdin Biochemical Technology Co., Ltd.) and the above-mentioned stock solution are in a mass ratio of 5 parts: 95 parts. Add the gelatin powder to the above-mentioned stock solution and stir for 1 h under the condition of a 60°C water bath until the gelatin powder is completely dissolved in the stock solution. The stirring speed is 200 rpm / min to obtain a mixed solution.

[0035] S3. Hydrogel preparation: After pouring the mixed solution into a mold and gelating for 1.5 h, perform 3 freeze-thaw cycles. The freezing temperature is -80°C (freezing time is 8 h), and the melting temperature is 20°C (melting time is 2 h). Then soak it in the sodium chloride / borax solution for 6 h. The concentration of the sodium chloride solution is 25 wt%, and the concentration of the borax solution is 0.8 wt%.

[0036] S4. Preparation of pre-stretched hydrogel: Perform cyclic stretching on the hydrogel prepared in S3. The size is 10*2*0.1 cm in length, width, and height, and perform cyclic stretching 500 times.

[0037] Microstructure test: Characterize the microstructure of the pre-stretched hydrogel using a scanning electron microscope (SEM). Figure 1 This is the scanning electron microscope of the surface of the pre-stretched hydrogel finally prepared in step S4 of Example 1 after freeze-drying. It can be seen from Figure 1 that the surface of the pre-stretched hydrogel has a topological structure that promotes the growth of nerve cells. The diameter of the topological structure is 5 μm to 30 μm. As Figure 2 shown, the hydrogel has a three-dimensional network porous structure inside. The pore size of the porous structure is 2 μm to 20 μm.

[0038] Mechanical property test: Perform a tensile test on the pre-stretched hydrogel using a universal mechanical testing machine. Cut the pre-stretched hydrogel finally prepared in step S4 of Example 1 to prepare a tensile sample (length and width are 12*4 mm). The thickness before freeze-drying is 1 - 10 mm, and the thickness after freeze-drying is 0.1 - 1 mm. The tensile rate is 100 mm / min. Calculate the tensile stress and strain according to the initial cross-sectional area and initial length of the test sample. The tensile stress-strain curve of the pre-stretched hydrogel of Example 1 is as Figure 3 shown. The tensile strength of the pre-stretched hydrogel of Example 1 is measured to be 3.3 MPa, and the tensile strain is 654.8%. It can be seen that the pre-stretched hydrogel prepared in Example 1 has excellent mechanical properties.

[0039] Ionic conductivity test: The conductivity of the pre-stretched hydrogel was tested using an electrochemical workstation. The pre-stretched hydrogel finally prepared in step S3 of Example 1 was cut to prepare a conductivity test sample (the sample size was 12 * 4 * 2 mm in length, width and height). The test sample was connected to the test port of the electrochemical workstation, and the ionic conductivity of the test sample could be calculated from the transmitted electrical signal. The calculation formula was: 𝛿 = 𝐿 / RS, where 𝛿 was the ionic conductivity; L was the length of the test sample, S was the cross-sectional area of the test sample; and R was the resistance. As Figure 4 shown, the ionic conductivity of the pre-stretched hydrogel of Example 1 was measured to be 4.6 mS / cm. It can be seen that the pre-stretched hydrogel prepared in Example 1 had excellent conductive properties.

[0040] Biocompatibility test: Cell viability was evaluated using a live / dead cell staining dye (LIVE / DEAD ® Viability / Cytotoxicity Kit, L3224, Invitrogen). Live cells converted calcein AM into strongly green fluorescent calcein (excitation / emission approximately 495 nm / 515 nm) through esterase activity. EthD-1 only entered cells with damaged membranes and emitted red fluorescence after binding to nucleic acids (excitation / emission approximately 495 nm / 635 nm). This method evaluated the cell life and death status based on these characteristics. The hydrogel prepared in Example 1 was soaked in 75% ethanol solution for 2 hours, then washed clean with sterile PBS, and sterilized by irradiation under ultraviolet light for 2 hours. The sterilized hydrogel was added to the complete DMEM medium at a concentration of 0.1 g / mL and incubated in an incubator at 37 °C, 100% relative humidity, and 5% carbon dioxide for 24 hours. After culturing RSC96 cells in the hydrogel extract for 24 hours, live / dead cell staining was performed. As Figure 5 shown, the cells cultured in the hydrogel extract adhered well to the surface of the hydrogel, most of them were stained green, and the proportion of dead cells was low.

[0041] Cytoskeleton immunofluorescence detection: PC12 cells were cultured on the surface of the hydrogel obtained in Example 1 above and cultured for 24 hours under the above cell culture conditions. The cells were fixed and stained with a Tuj1 marker (green), and the cell nuclei were stained with DAPI (blue). The distribution of the cells was observed under a confocal microscope. As Figure 6 shown, PC12 grew orderly along the ultrastructural topology.

[0042] The effect of different stretching times in Example 2 on the material.

[0043] This example provides a preparation of a pre-stretched hydrogel, including the following steps.

[0044] S1. Stock solution preparation: Weigh polyvinyl alcohol (G108396 polyvinyl alcohol, pharmaceutical grade, gel strength ~240 g Bloom, Shanghai Aladdin Biochemical Technology Co., Ltd.) and deionized water according to the mass ratio. The mass ratio of polyvinyl alcohol to deionized water is 10 parts: 90 parts. Add polyvinyl alcohol to deionized water and stir for 4 h under the condition of a 90 °C water bath until the polyvinyl alcohol is completely dissolved in the deionized water. The stirring speed is 250 rpm / min to obtain the stock solution.

[0045] S2. Mixture preparation: Weigh gelatin powder. The gelatin powder in this example (G776063, gel strength ~240 G BLOOM, Shanghai Aladdin Biochemical Technology Co., Ltd.) and the above-mentioned stock solution are in a mass ratio of 5 parts: 90 parts. Add the gelatin powder to the above-mentioned stock solution and stir for 3 h under the condition of a 50 °C water bath until the gelatin powder is completely dissolved in the stock solution. The stirring speed is 250 rpm / min to obtain the mixture.

[0046] S3. Hydrogel preparation: After pouring the mixture into a mold and gelating for 1.5 h, perform 3 freeze-thaw cycles. The freezing temperature is -80 °C (freezing time is 8 h), and the melting temperature is 20 °C (melting time is 2 h). Then soak it in the sodium chloride / borax solution for 6 h. The concentration of the sodium chloride solution is 25 wt%, and the concentration of the borax solution is 0.8 wt%.

[0047] S4. Preparation of pre-stretched hydrogel: Perform cyclic stretching on the hydrogel prepared in S3. The size is 10 * 2 * 0.1 cm in length, width, and height, and perform cyclic stretching 200 times.

[0048] Use the same test method as in Example 1 to test the pre-stretched hydrogel finally prepared in step S4 of Example 2. As Figure 7 shown, it is measured that the surface of the pre-stretched hydrogel finally prepared in step S4 of Example 2 is a topological structure. However, due to insufficient stretching, only a preliminary topological structure is shown. The measured tensile strength of the pre-stretched hydrogel in Example 2 is 3.21 MPa, the tensile strain is 651.4%, and the ion transport efficiency is 4.3 mS / cm. It can be seen that the pre-stretched hydrogel prepared in Example 2 has adjustable mechanical properties and ion conductive properties. However, due to the limitation of the number of stretching cycles, it is difficult to form a topologically parallel structure on the surface.

[0049] The influence of different stretching times on the material in Example 3.

[0050] This example provides a preparation method of a pre-stretched hydrogel, including the following steps.

[0051] S1. Stock solution preparation: Weigh polyvinyl alcohol (G108396 polyvinyl alcohol, pharmaceutical grade, gel strength ~240 g Bloom, Shanghai Aladdin Biochemical Technology Co., Ltd.) and deionized water according to the mass ratio. The mass fraction ratio of polyvinyl alcohol to deionized water is 10 parts: 90 parts. Add polyvinyl alcohol to deionized water and stir at 90 °C in a water bath for 4 h until the polyvinyl alcohol is completely dissolved in the deionized water. The stirring speed is 250 rpm / min to prepare the stock solution.

[0052] S2. Mixed solution preparation: Weigh gelatin powder according to the mass ratio. The gelatin powder in this example (G776063, gel strength ~240 G BLOOM, Shanghai Aladdin Biochemical Technology Co., Ltd.) and the above-mentioned stock solution have a mass fraction ratio of 5 parts: 90 parts. Add the gelatin powder to the above-mentioned stock solution and stir at 50 °C in a water bath for 3 h until the gelatin powder is completely dissolved in the stock solution. The stirring speed is 250 rpm / min to prepare the mixed solution.

[0053] S3. Hydrogel preparation: After pouring the mixed solution into the mold and gelating for 1.5 h, it undergoes 3 freeze-thaw cycles. The freezing temperature is -80 °C (freezing time is 8 h), and the melting temperature is 20 °C (melting time is 2 h). Then, it is soaked in the sodium chloride / borax solution for 6 h. The concentration of the sodium chloride solution is 25 wt%, and the concentration of the borax solution is 0.8 wt%.

[0054] S4. Preparation of pre-stretched hydrogel: The hydrogel prepared in S3 is subjected to cyclic stretching. The size is 10*2*0.1 cm in length, width, and height, and the number of cyclic stretching is 0 times.

[0055] The pre-stretched hydrogel finally prepared in step S4 of Example 3 is tested using the same test method as in Example 1. As Figure 8 It is measured that the surface of the hydrogel finally prepared in step S4 of Example 3 is basically smooth. The tensile strength of the pre-stretched hydrogel in Example 3 is 3.33 MPa, the tensile strain is 654.9%, and the ion transport efficiency is 4.1 mS / cm. It can be seen that the pre-stretched hydrogel prepared in Example 3 has adjustable mechanical properties and ion conductive properties. Also, due to the limitation of the number of stretching cycles, it is difficult to form a topologically parallel arrangement on the surface.

[0056] Comparative Example 1.

[0057] S1. Stock solution preparation: Weigh polyvinyl alcohol (G108396 polyvinyl alcohol, pharmaceutical grade, gel strength ~240 g Bloom, Shanghai Aladdin Biochemical Technology Co., Ltd.) and deionized water according to the mass ratio. The mass ratio of polyvinyl alcohol to deionized water is 10 parts: 90 parts. Add polyvinyl alcohol to deionized water and stir at 90 °C in a water bath for 4 h until the polyvinyl alcohol is completely dissolved in the deionized water. The stirring speed is 250 rpm / min to obtain the stock solution.

[0058] S2. Mixture preparation: Weigh collagen powder according to the mass ratio. The collagen powder (C835547, Shanghai Aladdin Biochemical Technology Co., Ltd.) and the above-mentioned stock solution in this comparative example have a mass ratio of 5 parts: 90 parts. Add the collagen powder to the above-mentioned stock solution and stir at 50 °C in a water bath for 3 h until the collagen powder is completely dissolved in the stock solution. The stirring speed is 250 rpm / min to obtain the mixture.

[0059] S3. Hydrogel preparation: After pouring the mixture into a mold and gelating for 1.5 h, perform 3 freeze-thaw cycles. The freezing temperature is -80 °C (freezing time is 8 h), and the melting temperature is 20 °C (melting time is 2 h). Then soak it in the sodium chloride / borax solution for 6 h. The concentration of the sodium chloride solution is 10 wt%, and the concentration of the borax solution is 0.8 wt%.

[0060] S4. Preparation of pre-stretched hydrogel: Perform cyclic stretching on the hydrogel prepared in S3 with dimensions of length × width × height of 10 * 2 * 0.1 cm and stretch it cyclically 5 times.

[0061] Use the same test method as in Example 1 to test the pre-stretched hydrogel finally prepared in step S4 of Comparative Example 1. It is measured that the surface of the pre-stretched hydrogel finally prepared in step S4 of Comparative Example 1 is basically smooth. The tensile strength of the pre-stretched hydrogel of Comparative Example 1 is 1.49 MPa, the tensile strain is 818.9%, the ion transport efficiency is 2.5 mS / cm, and no obvious topological structure is observed on the surface.

[0062] Comparative Example 2.

[0063] S1. Stock solution preparation: Weigh polyvinyl alcohol (G108396 polyvinyl alcohol, pharmaceutical grade, gel strength ~240 g Bloom, Shanghai Aladdin Biochemical Technology Co., Ltd.) and deionized water according to the mass ratio. The mass ratio of polyvinyl alcohol to deionized water is 10 parts: 90 parts. Add polyvinyl alcohol to deionized water and stir at 90 °C in a water bath for 4 h until the polyvinyl alcohol is completely dissolved in the deionized water. The stirring speed is 250 rpm / min to obtain the stock solution; S2. Mixed solution preparation: Weigh gelatin powder, the sodium alginate powder (S817374, Shanghai Aladdin Biochemical Technology Co., Ltd.) of this comparative example, and the above-mentioned stock solution according to the mass ratio. The mass fraction ratio is 5 parts: 90 parts. Add the sodium alginate powder to the above-mentioned stock solution, and stir at 50 °C in a water bath for 3 h to completely dissolve the sodium alginate powder in the stock solution. The stirring speed is 250 rpm / min to prepare a mixed solution; S3. Hydrogel preparation: After pouring the mixed solution into a mold and gelifying for 1.5 h, perform 3 freeze-thaw cycles. The freezing temperature is -80 °C (freezing time is 8 h), the melting temperature is 20 °C (melting time is 2 h), and then soak and treat in the sodium chloride / borax solution for 6 h. The concentration of the sodium chloride solution is 8 wt%, and the concentration of the borax solution is 0.6 wt%.

[0064] S4. Preparation of pre-stretched hydrogel: Perform cyclic stretching on the hydrogel prepared in S3. The size is 10 * 2 * 0.1 cm in length, width, and height, and perform cyclic stretching 500 times.

[0065] Use the same test method as in Example 1 to test the pre-stretched hydrogel finally prepared in step S4 of Comparative Example 2. It is measured that the surface of the pre-stretched hydrogel finally prepared in step S4 of Comparative Example 2 is a topological structure with a diameter of 5 μm to 30 μm. The tensile strength of the pre-stretched hydrogel of Comparative Example 2 is measured to be 1.17 MPa, the tensile strain is 856.4%, and the ion transport efficiency is 2.3 mS / cm. Under sufficient stretching conditions, Comparative Example 2 forms a topological structure with a unique surface morphology that is beneficial to the oriented growth of cells.

[0066] Comparative Example 3.

[0067] S1. Stock solution preparation: Weigh polyvinyl alcohol (G108396 polyvinyl alcohol, pharmaceutical grade, gel strength ~240 g Bloom, Shanghai Aladdin Biochemical Technology Co., Ltd.) and deionized water according to the mass ratio. The mass fraction ratio of polyvinyl alcohol and deionized water is 10 parts: 90 parts. Add polyvinyl alcohol to deionized water, and stir at 90 °C in a water bath for 4 h to completely dissolve polyvinyl alcohol in deionized water. The stirring speed is 250 rpm / min to prepare a stock solution; S2. Mixed solution preparation: Weigh xanthan gum powder, the xanthan gum powder (G810381, Shanghai Aladdin Biochemical Technology Co., Ltd.) of this comparative example, and the above-mentioned stock solution according to the mass ratio. The mass fraction ratio is 1 part: 100 parts. Add the xanthan gum powder to the above-mentioned stock solution, and stir at 60 °C in a water bath for 4 h to completely dissolve the xanthan gum powder in the stock solution. The stirring speed is 250 rpm / min to prepare a mixed solution; S3. Hydrogel preparation: After the mixed solution is poured into a mold and gelated for 1.5 h, it undergoes 3 freeze-thaw cycles. The freezing temperature is -60 °C (freezing time is 8 h), and the melting temperature is 30 °C (melting time is 1.5 h). Then it is soaked in the sodium chloride / borax solution for 2.5 h. The concentration of the sodium chloride solution is 6 wt%, and the concentration of the borax solution is 0.4 wt%.

[0068] S4. Preparation of pre-stretched hydrogel: The hydrogel prepared in S3 is subjected to cyclic stretching with dimensions of length×width×height of 10*2*0.1 cm and cyclic stretching is performed 400 times.

[0069] The pre-stretched hydrogel finally prepared in step S4 of Comparative Example 3 is tested using the same test method as in Example 1. It is measured that the surface of the pre-stretched hydrogel finally prepared in step S4 of Comparative Example 3 is a topological structure with a diameter of 2 μm to 30 μm. The tensile strength of the pre-stretched hydrogel of Comparative Example 3 is measured to be 0.91 MPa, the tensile strain is 528.2%, and the ion transport efficiency is 1.6 mS / cm. Under sufficient stretching conditions, Comparative Example 3 forms a topological structure with a unique surface morphology that is beneficial to the oriented growth of cells.

[0070] It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, improvements or transformations can be made according to the above description. All such improvements and transformations should fall within the protection scope of the appended claims of this application.

[0071] The above are only the preferred embodiments of the present invention and are not used to limit the patent scope of the present invention. For those skilled in the art, the present invention can have various changes and modifications. 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 method for preparing a pre-stretched hydrogel nerve conduit for promoting directional nerve growth, characterized in that: The following steps are involved: Prepare a polyvinyl alcohol solution: add polyvinyl alcohol into deionized water according to the mass ratio, stir for 2-4 hours in a 90°C water bath at a stirring speed of 250 rpm / min, so that the polyvinyl alcohol is completely dissolved to obtain a polyvinyl alcohol solution; Prepare a mixed solution: add gelatin powder to the above polyvinyl alcohol solution, according to the mass ratio, stir in a water bath at 50-60°C for 1-3 hours at a stirring speed of 200-250 rpm / min, so that the gelatin powder is completely dissolved to obtain a mixed solution; Preparation of hydrogel: the mixed solution was poured into a mold and gelled for 1.5 hours, and then subjected to 3 freeze-thaw cycles, with a freezing temperature of -80°C and a freezing time of 8 hours, a melting temperature of 20°C and a melting time of 2 hours, and then immersed in a mixed solution of 25wt% sodium chloride and 0.8wt% borax for 6 hours; Preparation of pre-stretched hydrogel: The hydrogel is subjected to cyclic stretching for 200-500 times to obtain a pre-stretched hydrogel nerve conduit.

2. The method for preparing a pre-stretched hydrogel nerve conduit for promoting directional nerve growth according to claim 1, characterized in that: The mass ratio of the polyvinyl alcohol to deionized water is 10 parts:90 parts, and the mass ratio of the gelatin powder to the polyvinyl alcohol solution is 5 parts:95 parts.

3. The method for preparing a pre-stretched hydrogel nerve conduit for promoting directional nerve growth according to claim 1, characterized in that: In the cyclic stretching step, the size of the hydrogel is 10×2×0.1 cm in length, width and height.

4. A pre-stretched hydrogel nerve conduit for promoting directional nerve growth, characterized in that: The nerve conduit is prepared by the preparation method according to any one of claims 1 to 3.

5. The pre-stretched hydrogel nerve conduit for promoting directional nerve growth according to claim 4, characterized in that: The surface of the nerve conduit has a topological structure that promotes the growth of nerve cells, and the diameter of the topological structure is 5 μm-30 μm.

6. The pre-stretched hydrogel nerve conduit for promoting directional nerve growth according to claim 4, characterized in that: The nerve conduit has a three-dimensional network porous structure inside, and the pore size of the porous structure is 2 μm-20 μm.

7. The pre-stretched hydrogel nerve conduit for promoting directional nerve growth according to claim 4, characterized in that: The pre-stretched hydrogel nerve conduit has no obvious residual strain after 500 cycles of stretching.

8. Use of the pre-stretched hydrogel nerve conduit for promoting directional nerve growth according to any one of claims 4 to 7 in the preparation of a product for repairing peripheral nerve damage.

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