A conductive three-dimensional biomimetic oriented gel scaffold and its preparation method and application

Abstract

The present invention provides a conductive three-dimensional biomimetic oriented gel scaffold and its preparation method and application, comprising the following steps: adding a mass of a type B gelatin aqueous solution or a collagen aqueous solution dropwise to a KGM aqueous solution, heating to a gel temperature above the gel temperature of the type B gelatin or collagen, and mixing to obtain a first mixed solution, wherein the final concentration of the type B gelatin or collagen is 0.5%-3.5%, and the final concentration of the KGM is 0.3%; maintaining above the gel temperature of the type B gelatin or collagen, adding PCNT and a cross-linking agent to the first mixed solution to obtain a third mixed solution; after the third mixed solution is gelled at room temperature, unidirectional freezing and freeze-drying are performed to obtain a conductive three-dimensional biomimetic oriented gel scaffold. The conductive three-dimensional biomimetic oriented gel scaffold of the present invention has good biocompatibility and degradation performance, has an oriented arrangement of micropores and conductive particles, can better simulate the microchannels of the endoneurial tube, and has great potential in nerve tissue repair and regeneration.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical material preparation, and in particular relates to a conductive three-dimensional bionic oriented gel scaffold, a preparation method and an application thereof. Background Art

[0002] Peripheral nerve injury (PNI) caused by lifestyle, exercise, car accidents or surgery is a global clinical problem, with millions of cases occurring each year, significantly reducing patients' quality of life and creating a heavy economic burden. PNI is divided into three degrees: neuropraxia, axonal injury and nerve defect. Nerve defect is characterized by physiological severance of myelin, axons and other supporting tissues, and is the most severe type of PNI. For nerve injuries without defects at the rupture site, direct surgical anastomosis is currently the main method, relying on the body's own regenerative ability to repair the damaged nerve. For nerve injuries with defects at the rupture site, due to the lack of structural and mechanical support of the natural tissue basal layer, nerve cell function is impaired, and Waller axons cannot reach the original site, thus losing tissue regeneration function. Autologous transplantation is the "gold standard" for the clinical treatment of PNI because it retains the patient's own neural structure and the original microenvironmental support of nerve cells. However, there are problems such as limited donor resources and loss of function in the donor site. Therefore, in clinical applications, nerve conduits are often used as scaffolds, aiming to use synthetic or natural biomaterials to simulate the axial microenvironment of nerve regeneration, support nerve cell growth and axon differentiation and elongation, and bridge and repair severe nerve damage. The ideal scaffold should have good biocompatibility and an oriented topological microstructure to guide the directional alignment of nerve cells and the directional elongation of new axons along the long axis of the scaffold micropores, and contact each other. There are many existing nerve repair scaffolds made of natural materials such as chitosan and silk fibroin. They have good biocompatibility but poor mechanical strength and lack of oriented topological structure. Their performance in promoting axon elongation is limited, making the repair effect of long-distance nerve defects less than ideal. Although the introduction of synthetic materials into scaffold construction can improve its mechanical properties, it will inevitably affect the biocompatibility of the scaffold and cause problems such as uncontrollable subsequent degradation process. Therefore, the development of biomimetic scaffolds with good biocompatibility and mechanical properties, structural orientation, and degradability to replace autologous transplantation remains a huge challenge. Summary of the Invention

[0003] In order to solve the problems existing in the prior art, the present invention provides a conductive three-dimensional biomimetic oriented gel scaffold and its preparation method and application. The conductive three-dimensional biomimetic oriented gel scaffold is formed based on the assembly of natural macromolecules, has good biocompatibility and degradation performance, and can adjust the network structure of the natural scaffold based on the freezing structure principle to achieve the oriented arrangement of micropores and conductive particles, which can better simulate the microchannels of the endoneurial tube and has great potential in the repair and regeneration of neural tissue.

[0004] To achieve the above object, the present invention provides the following technical solution: a method for preparing a conductive three-dimensional biomimetic oriented gel scaffold, comprising the following steps:

[0005] S1: adding equal mass of type B gelatin aqueous solution or collagen aqueous solution to KGM aqueous solution, heating to above the gel temperature of type B gelatin or collagen, and mixing to obtain a first mixed solution, wherein the final concentration of type B gelatin or collagen is 0.5%-3.5%, and the final concentration of KGM is 0.3%.

[0006] S2 is maintained above the gelling temperature of type B gelatin or collagen, and PCNT and a cross-linking agent are added to the first mixed solution to obtain a third mixed solution;

[0007] S3 gels the third mixed solution at room temperature, performs unidirectional freezing, and freeze-dries to obtain a conductive three-dimensional biomimetic oriented gel scaffold.

[0008] Furthermore, in S2, the concentration of PCNT added is 0.01-0.1%.

[0009] Furthermore, in S2, the concentration of the cross-linking agent added is 0.05% to 0.1%.

[0010] Furthermore, in S2, the cross-linking agent is a genipin cross-linking agent.

[0011] Furthermore, in S2, the third mixed liquid is placed in a mold for gelation, and the mold is a cylindrical food-grade polytetrafluoroethylene mold.

[0012] Furthermore, in S3, the temperature of the one-way freezing is -20 to 80°C.

[0013] The present invention provides a conductive three-dimensional biomimetic oriented gel scaffold, which is prepared by the above-mentioned preparation method and has a directionally arranged microporous structure inside.

[0014] The present invention provides an application of a conductive three-dimensional biomimetic orientation gel scaffold, and nerve cell suspension is spread on the conductive three-dimensional biomimetic orientation gel scaffold to culture the nerve cells.

[0015] Furthermore, the proliferation state of nerve cells was regulated by adjusting the pore size and PCNT concentration of the conductive three-dimensional biomimetic oriented gel scaffold.

[0016] The present invention provides a stent for treating peripheral nerve damage diseases, comprising the stent or the stent prepared by the method and nerve cells cultured on the stent.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects:

[0018] The present invention provides a method for preparing a conductive three-dimensional biomimetic oriented gel scaffold. Type B gelatin or collagen and KGM (konjac glucomannan), both hydrophilic natural macromolecules, are mixed in an aqueous solution. PCNTs (polydopamine-coated carbon nanotubes) are then added to the fused KGM and type B gelatin or collagen to form a gel. KGM and type B gelatin or collagen are not conductive, while carbon nanotubes have high conductivity, a large specific surface area, and good biocompatibility. The polydopamine coating significantly enhances the carbon nanotubes' adhesion to cells. In the fused state, gelatin and KGM form a complementary, mutually penetrating network structure. The introduction of PCNT increases the conductivity of the scaffold while being more conducive to cell adhesion to the scaffold. At the same time, the addition of a cross-linker enhances the bonding between the two to form a stable hydrogel. The unidirectional freezing technology is used to induce ice crystal nucleation and directional growth. The PCNT is arranged in a directional manner. After further freeze-drying, an aerogel is obtained, which is a new type of conductive gel scaffold. The scaffold forms oriented micropores, which can better simulate the microchannels of the endoneurial tube, thereby guiding the directional growth of nerve cells.

[0019] In the present invention, the mass ratio of type B gelatin or collagen and KGM, the concentrations of genipin and PCNT, the temperature of the solution after solution mixing, and the temperature of the final directional frozen gel are strictly controlled during the preparation process, thereby obtaining a gel scaffold with an oriented structure that can simulate the extracellular matrix. The gel scaffold exhibits good proliferation ability and cell adhesion properties, and can induce the directional arrangement of nerve cells, which is beneficial to the spreading and mutual contact of new axons, providing a new idea for the preparation method of nerve tissue repair scaffolds.

[0020] The directional freezing technology used in the present invention converts hydrogels directly into aerogels by sublimating ice crystals without the need for solvent exchange, thereby achieving a more direct, cost-effective and sustainable process. The use of PCNT increases the solubility of carbon nanotubes while providing the physiological conductivity required for electrical signal transduction of nerve cells. Electrophysiological signals can promote the mutual communication between nerve cells in the matrix microenvironment. In cell signal transduction, the transmembrane transport process of ions is activated, causing the cell membrane to polarize, thereby generating an endogenous electric field in the intercellular space of the nervous tissue. The bioelectric signal caused by the change of cell membrane potential is one of the main ways of cell signal transduction, which is closely related to cytoskeleton rearrangement, cell membrane depolarization, Ca 2+ Influx affects physiological processes such as cell migration, proliferation, and differentiation. Therefore, conductivity is one of the important properties of nerve repair and is more conducive to the proliferation and function of nerve cells. The preparation method of the present invention is simple and can be completed at room temperature, which is convenient for practical application. The prepared gel scaffold has low density, high porosity, fast water absorption rate, good swelling capacity, and appropriate mechanical properties. In addition, the gel scaffold has oriented micropores, which can better simulate the parallel arrangement structure of nerve bundles and can be effectively used for in vitro culture of nerve cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The macroscopic phase diagram is a diagram of a final concentration of type B gelatin or collagen of 2.5-10% and a final concentration of KGM of 0.3%;

[0022] Figure 2 Microrheological diagrams of gels formed at different gelatin concentrations (2.5% and 3.5%);

[0023] Figure 3 Swelling ratio diagram of gel scaffolds formed with different gelatin concentrations (A is 2.5% gelatin concentration, B is 3.5% gelatin concentration);

[0024] Figure 4 Apparent density and porosity of gel scaffolds formed with different gelatin concentrations (A is 2.5% gelatin concentration, B is 3.5% gelatin concentration);

[0025] Figure 5 The graphs show the proliferation activity of cells cultured on gel scaffolds formed at different gelatin concentrations at a directional freezing temperature (-40°C) (A is 2.5% gelatin concentration, B is 3.5% gelatin concentration). DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0027] The present invention provides a method for preparing a conductive three-dimensional biomimetic oriented gel scaffold, comprising the following steps:

[0028] To explore the S1 phase fusion system, a 1-7% initial concentration of type B gelatin aqueous solution or collagen aqueous solution and a 0.6% KGM aqueous solution were prepared;

[0029] S2: Add equal mass of type B gelatin aqueous solution or collagen aqueous solution into KGM aqueous solution, heat above the gel temperature of type B gelatin or collagen and stir thoroughly to obtain a first mixed solution, in which the final concentration of type B gelatin or collagen is 0.5%-3.5%, and the final concentration of KGM is 0.3%.

[0030] S3, while maintaining the temperature above the gelation temperature of type B gelatin or collagen, adding PCNT with a concentration of 0.01-0.1% to the first mixed solution, mixing and stirring for 30 minutes to obtain a second mixed solution;

[0031] S4: adding 0.05% to 0.1% genipin crosslinker to the second mixed solution, mixing and stirring for 30 minutes to obtain a third mixed solution;

[0032] S4: placing the third mixed solution into a mold for cross-linking for 24 hours, and then unidirectionally freezing and freeze-drying to obtain a conductive three-dimensional biomimetic oriented gel scaffold.

[0033] Furthermore, in S4, the third mixed solution is placed at room temperature for cross-linking for 24 hours.

[0034] Furthermore, in S4, the one-way freezing temperature is -20 to 80°C.

[0035] Furthermore, in S4, the mold is a cylindrical food-grade polytetrafluoroethylene mold.

[0036] The conductive three-dimensional bionic oriented gel scaffold prepared by the invention has a cylindrical blue-black appearance, a thickness of 0.5 to 0.8 cm, and a directionally arranged microporous structure inside.

[0037] The conductive three-dimensional biomimetic oriented gel scaffold of the present invention can be used for the culture of nerve cells. The scaffold can be combined with the cultured nerve cells to treat peripheral nerve injury diseases. Specifically:

[0038] 1) The conductive three-dimensional biomimetic oriented gel scaffold was sterilized by ultraviolet light for 4 hours.

[0039] 2) The sterilized conductive three-dimensional biomimetic oriented gel scaffold was first equilibrated with PBS for 12 hours, and then with culture medium for 12 hours. The culture medium was aspirated, and a cell suspension of a certain density was plated on the conductive three-dimensional biomimetic oriented gel scaffold and cultured at 37°C for 24 hours.

[0040] Furthermore, the cell proliferation state was regulated by adjusting the pore size and PCNT concentration of the conductive three-dimensional biomimetic oriented gel scaffold.

[0041] Example 1

[0042] Type B gelatin was dispersed in distilled water and heated at 60°C for 1 hour to prepare a 1% (W / V) stock solution. A KGM stock solution with a concentration of 0.6% (W / V) was prepared by further heating at 60°C for 8 hours. Type B gelatin and KGM aqueous solution were then mixed in equal amounts, heated at 40°C with magnetic stirring for 30 minutes, and then 0.01% PCNT was added and mixed for 30 minutes. The final concentrations of type B gelatin and KGM were 0.5%, and 0.3%, respectively. After adding 0.05% genipin crosslinker and heating at 40°C for 30 minutes, the mixture was transferred into a food-grade polytetrafluoroethylene mold with an inner diameter of 40 mm and gelled at room temperature for 24 hours. The gel was placed in a directional freezing device and frozen at -20°C before being freeze-dried in a freeze dryer to produce a new gel scaffold.

[0043] Example 2

[0044] Type B gelatin was dispersed in distilled water and heated at 60°C for 1 hour to prepare a 2% (W / V) stock solution. A KGM stock solution with a concentration of 0.6% (W / V) was prepared by further heating at 60°C for 8 hours. Type B gelatin and KGM aqueous solution were then mixed in equal amounts, heated at 40°C with magnetic stirring for 30 minutes, and then 0.05% PCNT was added and mixed for 30 minutes. The final concentration of type B gelatin was 1%, and the final concentration of KGM was 0.3%. After adding 0.05% genipin crosslinker and heating at 40°C for 30 minutes, the mixture was transferred into a food-grade polytetrafluoroethylene mold with an inner diameter of 40 mm and gelled at room temperature for 24 hours. The gel was placed in a directional freezing device and frozen at -40°C, then placed in a freeze dryer for freeze drying to obtain a new gel scaffold.

[0045] Example 3

[0046] Type B gelatin was dispersed in distilled water and heated at 60°C for 1 hour to prepare a 5% (W / V) stock solution. A KGM stock solution with a concentration of 0.6% (W / V) was prepared by further heating at 60°C for 8 hours. Type B gelatin and KGM aqueous solution were then mixed in equal amounts, heated at 40°C with magnetic stirring for 30 minutes, and then 0.1% PCNT was added and mixed for 30 minutes. The final concentration of type B gelatin was 2.5%, and the final concentration of KGM was 0.3%. After adding a 0.05% genipin crosslinker and heating at 40°C for 30 minutes, the mixed system was transferred to a food-grade polytetrafluoroethylene mold with an inner diameter of 40 mm and gelled at room temperature for 24 hours. The gel was placed in a directional freezing device and frozen at -60°C, then placed in a freeze dryer for freeze drying to obtain a new gel scaffold.

[0047] Example 4

[0048] Type B gelatin was dispersed in distilled water and heated at 60°C for 1 hour to prepare a 6% (W / V) stock solution. A KGM stock solution with a concentration of 0.6% (W / V) was prepared by further heating at 60°C for 8 hours. Type B gelatin and KGM aqueous solution were then mixed in equal amounts, heated at 40°C with magnetic stirring for 30 minutes, and then 0.01% PCNT was added and mixed for 30 minutes. The final concentrations of type B gelatin were 3%, and those of KGM were 0.3%. After adding 0.05% genipin crosslinker and heating at 40°C for 30 minutes, the mixture was transferred into a food-grade polytetrafluoroethylene mold with an inner diameter of 40 mm and gelled at room temperature for 24 hours. The gel was placed in a directional freezing device and frozen at -40°C before being freeze-dried in a freeze dryer to produce a new gel scaffold.

[0049] Example 5

[0050] Type B gelatin was dispersed in distilled water and heated at 60°C for 1 hour to prepare a 7% (W / V) stock solution. A KGM stock solution with a concentration of 0.6% (W / V) was prepared by further heating at 60°C for 8 hours. Type B gelatin and KGM aqueous solution were then mixed in equal amounts, heated at 40°C with magnetic stirring for 30 minutes, and then 0.05% PCNT was added and mixed for 30 minutes. The final concentrations of type B gelatin were 3%, and those of KGM were 0.3%. After adding 0.05% genipin crosslinker and heating at 40°C for 30 minutes, the mixture was transferred into a food-grade polytetrafluoroethylene mold with an inner diameter of 40 mm and gelled at room temperature for 24 hours. The gel was placed in a directional freezing device and frozen at -40°C before being freeze-dried in a freeze dryer to produce a new gel scaffold.

[0051] Example 6

[0052] Type B gelatin was dispersed in distilled water and heated at 60°C for 1 hour to prepare a 7% (W / V) stock solution. A KGM stock solution with a concentration of 0.6% (W / V) was prepared by further heating at 60°C for 8 hours. Type B gelatin and KGM aqueous solution were then mixed in equal amounts, heated at 40°C with magnetic stirring for 30 minutes, and then 0.1% PCNT was added and mixed for 30 minutes. The final concentration of type B gelatin was 3.5%, and the final concentration of KGM was 0.3%. After adding 0.05% genipin crosslinker and heating at 40°C for 30 minutes, the mixture was transferred into a food-grade polytetrafluoroethylene mold with an inner diameter of 40 mm and gelled at room temperature for 24 hours. The gel was placed in a directional freezing device and frozen at -40°C, then placed in a freeze dryer for freeze drying to obtain a new gel scaffold.

[0053] Example 7

[0054] Collagen was dispersed in distilled water and heated at 60°C for 1 hour to prepare a 7% (W / V) stock solution. A KGM stock solution with a concentration of 0.6% (W / V) was prepared by further heating at 60°C for 8 hours. Then, equal masses of collagen and KGM aqueous solution were mixed, heated at 40°C with magnetic stirring for 30 minutes, and then 0.1% PCNT was added and mixed for 30 minutes. Among them, the final concentration of collagen was 3.5%, the final concentration of KGM was 0.3%, and after adding a crosslinker with a concentration of 0.05% genipin and heating at 40°C for 30 minutes, the above-mentioned mixed system was transferred to a food-grade polytetrafluoroethylene mold with an inner diameter of 40 mm and gelled at room temperature for 24 hours. The gel was placed in a directional freezing device and frozen at -40°C, and then placed in a freeze dryer for freeze drying to obtain a new gel scaffold.

[0055] Example 8

[0056] Collagen was dispersed in distilled water and heated at 60°C for 1 hour to prepare a 7% (W / V) stock solution. A KGM stock solution with a concentration of 0.6% (W / V) was prepared by further heating at 60°C for 8 hours. Then, equal amounts of collagen and KGM aqueous solution were mixed, heated at 40°C with magnetic stirring for 30 minutes, and then 0.05% PCNT was added and mixed for 30 minutes. The final concentration of collagen was 3%, the final concentration of KGM was 0.3%, and after adding a 0.05% genipin crosslinker and heating at 40°C for 30 minutes, the mixed system was transferred into a food-grade polytetrafluoroethylene mold with an inner diameter of 40 mm and gelled at room temperature for 24 hours. The gel was placed in a directional freezing device and frozen at -40°C, and then placed in a freeze dryer for freeze drying to obtain a new gel scaffold.

[0057] Example 9

[0058] Collagen was dispersed in distilled water and heated at 60°C for 1 hour to prepare a 5% (W / V) stock solution. A KGM stock solution with a concentration of 0.6% (W / V) was prepared by further heating at 60°C for 8 hours. Collagen and KGM aqueous solution were then mixed in equal amounts, heated at 40°C with magnetic stirring for 30 minutes, and then 0.1% PCNT was added and mixed for 30 minutes. The final concentration of collagen was 2.5%, the final concentration of KGM was 0.3%, and after adding a 0.05% genipin crosslinker and heating at 40°C for 30 minutes, the mixed system was transferred into a food-grade polytetrafluoroethylene mold with an inner diameter of 40 mm and gelled at room temperature for 24 hours. The gel was placed in a directional freezing device and frozen at -60°C, and then placed in a freeze dryer for freeze drying to obtain a new gel scaffold.

[0059] Comparative Example 1

[0060] Type B gelatin was dispersed in distilled water and heated at 60°C for 1 hour to prepare a 10% (W / V) stock solution. A 0.6% (W / V) KGM stock solution was further heated at 60°C for 8 hours. Equal amounts of type B gelatin and KGM aqueous solution were then mixed and heated at 40°C with magnetic stirring for 30 minutes. The final concentrations of type B gelatin and KGM were 5% and 0.3%, respectively. Phase separation was observed, with the upper layer consisting of transparent type B gelatin and the lower layer consisting of KGM, preventing the formation of a composite gel.

[0061] Comparative Example 2

[0062] Type B gelatin was dispersed in distilled water and heated at 60°C for 1 hour to prepare a 15% (W / V) stock solution. A 0.6% (W / V) KGM stock solution was further heated at 60°C for 8 hours. Equal amounts of type B gelatin and KGM aqueous solution were then mixed and heated at 40°C with magnetic stirring for 30 minutes. The final concentrations of type B gelatin and KGM were 7.5% and 0.3%, respectively. The system was observed to have a transparent upper layer of type B gelatin and a lower layer of KGM, indicating phase separation and failure to form a composite gel.

[0063] Comparative Example 3

[0064] Type B gelatin was dispersed in distilled water and heated at 60°C for 1 hour to prepare a 20% (W / V) stock solution. A 0.6% (W / V) KGM stock solution was further heated at 60°C for 8 hours. Equal amounts of type B gelatin and KGM aqueous solution were then mixed and heated at 40°C with magnetic stirring for 30 minutes. The final concentrations of type B gelatin and KGM were 10% and 0.3%, respectively. The system was observed to have a transparent upper layer of type B gelatin and a lower layer of KGM, indicating phase separation and the inability to form a composite gel.

[0065] Comparative Example 4

[0066] Collagen was dispersed in distilled water and heated at 60°C for 1 hour to prepare a 20% (W / V) stock solution. A 0.6% (W / V) KGM stock solution was further heated at 60°C for 8 hours. Equal amounts of collagen and KGM aqueous solution were then mixed and heated at 40°C with magnetic stirring for 30 minutes, resulting in a final concentration of 5% collagen and 0.3% KGM. The system was observed to have a transparent upper layer of collagen and a lower layer of KGM, indicating phase separation and failure to form a composite gel.

[0067] In Examples 1-9, after equal masses of type B gelatin or collagen were mixed with the KGM aqueous solution, the mixture was allowed to stand above the gelation temperature of type B gelatin or collagen, and the state of the mixed system was observed to see whether phase separation occurred. Gel scaffolds were further prepared. The types and pore size ranges of the gel scaffolds prepared in Examples 1-9 and the gels obtained in Comparative Examples 1-4 are shown in Table 1.

[0068] Table 1 Parameters and pore size range of composite gel scaffolds formed by directional freezing

[0069]

[0070]

[0071] As shown in Table 1, when the gelatin concentration (0.5%-3.5%) and collagen concentration (2.5%-3.5%) are low, the type B gelatin or collagen and KGM systems are uniform and no phase separation occurs. At this time, KGM and type B gelatin or collagen are homogeneously distributed, forming a uniform composite gel scaffold. As the concentration of type B gelatin or collagen increases, phase separation gradually occurs, with the lower layer being a KGM-rich phase and the upper layer being a type B gelatin or collagen-rich phase. Phase separation becomes more likely to occur as the gelatin concentration increases, making it even more difficult to form a composite gel, and therefore, a uniform and stable composite gel scaffold cannot be formed. At the same time, when the PCNT concentration is 0.01-0.1%, a uniform and stable conductive gel scaffold can be formed in the above systems, and the pore size distribution of the scaffold can be controlled by the freezing temperature and the concentration of the system components.

[0072] Figure 1 Macroscopic images of a mixture of type B gelatin (2.5-10% w / w) and KGM (0.3 wt%) before and after standing at 40°C for 24 hours are shown. In samples with 5-10% gelatin and 0.3% KGM, a clear upper gelatin phase and a turbid lower KGM phase are clearly visible. No phase separation was observed in samples with 2.5-3.5% and 0.3% KGM concentrations.

[0073] Figure 2 The following microrheological plots show the dynamic changes in gel formation at different gelatin concentrations. In the initial stage, the tracer particles move freely, and the MSD curves for both concentrations increase linearly, indicating that viscosity dominates the KGM-B gelatin system. In the second stage, the slope of the MSD curve gradually decreases, eventually reaching a plateau, indicating that the Brownian motion of the particles in the system is restricted, trapped in a three-dimensional network structure, and their movement slows. The plateau corresponds to the elastic properties of the sample. The lower the gel plateau formed at a 3.5% concentration, the stronger the elasticity. The EI value corresponds to the inverse of the plateau height in the MSD curve. As the gelatin concentration increases, hydrogen bonds and amide bonds between KGM and gelatin form a denser gel network, and the EI value of the system increases. At the beginning, the SLB was 1.2, and the gel system exhibited fluid behavior at this stage. In the next 1.5 hours, the SLB dropped to 0.5, indicating that the viscous solution was transformed into an elastic gel at this stage. Finally, after 19 hours, the SLB value was balanced, and the gel had a stronger solid-like behavior, indicating a stable bonding effect between type B gelatin and KGM.

[0074] Figure 3The swelling ratios of the composite gel scaffolds were shown. Overall, scaffolds formed with different gelatin concentrations (2.5% and 3.5%) exhibited relatively high swelling ratios. Gelatin concentration influenced the scaffold swelling ratio. At a gelatin concentration of 2.5%, multidirectional freezing (R) swelling was higher, while at a gelatin concentration of 3.5%, bidirectional freezing (BI) swelling was higher, facilitating water and nutrient transport.

[0075] Figure 4 All scaffolds exhibited high porosity (>91%) (Figure B), and the porosity of scaffolds formed with different gelatin concentrations did not change significantly. As for the density of the scaffolds, the density increased with increasing gelatin concentration (Figure A).

[0076] comprehensive Figures 1 to 4 It can be seen that a stable three-dimensional gel structure can be formed between the type B gelatin and KGM molecules. The scaffold has good swelling water absorption and high porosity, showing good potential in cell culture.

[0077] Figure 5 The absorbance increased significantly over time, indicating that the scaffold has good biocompatibility and great potential as a scaffold for culturing cells in tissue engineering. After 5 days of growth, gel scaffolds formed by different freezing directions showed significant differences, with the scaffolds formed by unidirectional freezing having the best cell proliferation ability, indicating that unidirectional freezing scaffolds have a more suitable microenvironment for cell growth and are better suited for culturing neural cells.

[0078] The detection method adopted by the present invention is:

[0079] (1) Preparation of type B gelatin or collagen-KGM mixed system

[0080] KGM was dispersed in distilled water and heated at 80°C for 4 hours to prepare a 0.3% (W / V) stock solution. A series of type B gelatin or collagen stock solutions with concentrations ranging from 1 to 20% (W / V) were prepared by further heating at 60°C for 8 hours.

[0081] Then, equal amounts of type B gelatin or collagen and KGM aqueous solution are mixed and heated at 40-50°C with magnetic stirring for 30 minutes. The final concentration of type B gelatin or collagen is controlled to be 0.5-10%, and the final concentration of KGM is controlled to be 0.3%. The mixture (above the gelation temperature of type B gelatin or collagen) is allowed to stand at 40-50°C for 24-36 hours. A gel scaffold is prepared by selecting a system that does not undergo phase separation.

[0082] (2) Preparation of type B gelatin or collagen-KGM-based gel scaffolds

[0083] The mixed system was prepared according to the method (1), and different concentrations (0.01-0.1%) of PCNT and genipin biocrosslinker were added to a final concentration of 0.03-0.05%. After heating at 40-50°C for 30 minutes, the mixed system was transferred into a food-grade polytetrafluoroethylene mold with an inner diameter of 40 mm and gelled at room temperature for 24 hours. The gel was then placed in a directional freezing device for freezing (-20°C, -40°C, -60°C, and -80°C) and freeze-dried in a freeze dryer.

[0084] (3) Water absorption rate

[0085] A certain mass of dried gel scaffold was immersed in simulated biological fluid (pH 7.4, 37°C). At regular intervals, the scaffold was removed, the surface moisture was wiped with filter paper, and then the scaffold was weighed. The water absorption rate of the gel scaffold was calculated according to the following formula:

[0086] Water absorption rate (%) = (W S -W d ) / W d ×100

[0087] Among them, W S and W d are the swelling mass and dry weight of the gel scaffold after a certain period of time, respectively.

[0088] (4) Apparent density and porosity

[0089] The apparent density and porosity of the gel scaffolds were determined according to standard methods for cellular materials. Density is the ratio of the mass of the gel scaffold to its volume.

[0090] ρ s =m / (Π×D / 2) 2 ×H)

[0091] P(%)=V P / V×100=((Vm / ρ skeletal ) / V)×100

[0092] Where m is the mass, V is the total volume of the stent, D is the diameter, and H is the thickness of the stent. s is the density of the scaffold, the pore volume (V p ) and the density of the skeleton material (ρskeleton) was calculated as the mass-weighted arithmetic mean of the materials, ρ 明胶 =1.35g / cm 3 ,ρ KGM =1.19g / cm 3 ,ρ genipin =1.4g / cm 3 . Calculate the mean and standard deviation (SD).

[0093] (5) Cell proliferation ability on gel scaffolds

[0094] After the gel scaffold was sterilized under UV irradiation for 4 h, the gel scaffold was balanced in PBS for 12 h and then balanced in complete culture medium for 12 h. Cells were plated at a density of 5.0 × 10 5 Cells were plated onto 12-well plates at 100 μg / mL. After culturing on the gel scaffolds for 1 day, the scaffolds were transferred to new 12-well plates to remove cells not adhering to the scaffolds. Cell proliferation activity was determined by MTT assay at specified time intervals.

Claims

1. A method for preparing a conductive three-dimensional biomimetic oriented gel scaffold, characterized in that: The steps include: S1: adding equal mass of type B gelatin aqueous solution or collagen aqueous solution dropwise to konjac glucomannan (KGM) aqueous solution, heating to above the gelling temperature of type B gelatin or collagen, and mixing to obtain a first mixed solution, wherein the final concentration of type B gelatin or collagen is 0.5%-3.5%, and the final concentration of konjac glucomannan (KGM) is 0.3%; S2 is maintained above the gelation temperature of type B gelatin or collagen, and polydopamine-coated carbon nanotubes (PCNT) and a cross-linking agent are added to the first mixed solution to obtain a third mixed solution; In S3, the third mixed solution is gelled at room temperature, and then unidirectionally frozen and freeze-dried to obtain a conductive three-dimensional biomimetic oriented gel scaffold. In S2, the concentration of the polydopamine-coated carbon nanotubes (PCNT) is 0.01-0.1%.

2. The method for preparing a conductive three-dimensional biomimetic oriented gel scaffold according to claim 1, characterized in that: In S2, the concentration of the cross-linking agent added is 0.05%~0.1%.

3. The method for preparing a conductive three-dimensional biomimetic oriented gel scaffold according to claim 1, characterized in that: In S2, the cross-linking agent is genipin cross-linking agent.

4. The method for preparing a conductive three-dimensional biomimetic oriented gel scaffold according to claim 1, characterized in that: In S2, the third mixed liquid is placed in a mold for gelation, and the mold is a cylindrical food-grade polytetrafluoroethylene mold.

5. The method for preparing a conductive three-dimensional biomimetic oriented gel scaffold according to claim 1, characterized in that: In S3, the temperature of one-way freezing is -20~-80℃.

6. A conductive three-dimensional biomimetic oriented gel scaffold, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 5, and has a directionally arranged microporous structure inside.

7. An application of a conductive three-dimensional biomimetic oriented gel scaffold, characterized in that: The nerve cell suspension is spread on a conductive three-dimensional biomimetic oriented gel scaffold to culture the nerve cells. The conductive three-dimensional biomimetic oriented gel scaffold is the conductive three-dimensional biomimetic oriented gel scaffold according to claim 6.

8. The use according to claim 7, characterized in that The proliferation state of nerve cells is regulated by adjusting the pore size and PCNT concentration of the conductive three-dimensional biomimetic oriented gel scaffold.

9. A stent for treating peripheral nerve damage, characterized in that: The invention comprises the scaffold according to claim 6 or the scaffold prepared by the method according to any one of claims 1 to 5, and neural cells cultured on the scaffold.

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