Preparation method of nerve scaffold and nerve scaffold

Abstract

The invention relates to the technical field of nerve scaffolds, and particularly provides a preparation method of a nerve scaffold and the nerve scaffold. The preparation method of the nerve scaffold comprises the following steps: transferring a polyurethane solution into a mold, removing an organic solvent, and cleaning to obtain the nerve scaffold, the mold is made of a solid water-soluble polymer material and is insoluble in an organic solvent at the temperature not higher than 50 DEG C. Preferably, the organic solvent is removed by a freeze vacuum drying method. The preparation method disclosed by the invention is simple, the nerve scaffold with a relatively low inner diameter can be obtained, and the nerve scaffold can be provided with a plurality of micropores with open pore structures.

Description

Technical Field

[0001] This application relates to the field of neural scaffold technology, specifically to a method for preparing a neural scaffold and the neural scaffold itself. Background Technology

[0002] Neural scaffolds are a type of tissue engineering scaffold that can promote the proliferation and repair of nerve cells. Polyurethane neural scaffolds are usually formed into tubular materials by extrusion molding. In order to form a porous structure in polyurethane neural scaffolds, sodium chloride is usually added to the polyurethane material. After forming the tubular material, the sodium chloride is removed from the polyurethane tubular material by water extraction. After drying, micropores are formed. However, this method has the following shortcomings: (1) Extrusion molding is not suitable for polyurethane neural scaffolds with a relatively small inner diameter, such as 1-2 mm; (2) The specific gravity of sodium chloride is much higher than that of polyurethane, and it is not easy to disperse evenly in polyurethane, resulting in uneven distribution of micropores; (3) Some sodium chloride is wrapped in closed pores, and water cannot penetrate and extract it, resulting in a reduction of micropores and sodium chloride residue; (4) The water displacement efficiency of sodium chloride is low, which leads to a longer process time.

[0003] Therefore, there is an urgent need to improve the fabrication process of polyurethane neural scaffolds. Summary of the Invention

[0004] To address the aforementioned technical problems in existing polyurethane neural scaffold fabrication methods, the inventors have conducted extensive research and analysis. Therefore, this application is hereby submitted.

[0005] This application proposes a method for preparing a neural scaffold and the neural scaffold itself.

[0006] The technical solution adopted in this application is as follows: A method for preparing a neural scaffold involves transferring a polyurethane solution into a mold, removing the organic solvent, cleaning, and obtaining the neural scaffold. The mold is made of a solid, water-soluble polymer material that is insoluble in the organic solvent at temperatures not exceeding 50°C.

[0007] Preferably, the water-soluble polymer material is selected from one or a combination of several of homopolymer polyacrylic acid and its salts, copolymer polyacrylic acid and its salts, polyvinyl alcohol, polyacrylamide, and sodium chondroitin.

[0008] Preferably, the polyurethane polymer structure used in the polyurethane solution contains at least one first block and at least one second block, the first block and the second block are arranged alternately, and adjacent first blocks and second blocks are connected by urethane bonds; The first block is formed of aliphatic polyester diol and / or alicyclic polyester diol; The second block is formed from an aliphatic polyether diol.

[0009] Preferably, the organic solvent has a melting point of -100 to 20°C.

[0010] Preferably, the concentration of the polyurethane solution is 5-80 wt%.

[0011] Preferably, the cavity of the mold matches the polyurethane neural scaffold.

[0012] Preferably, the removal of organic solvents is selected from one of the following: natural evaporation, heating evaporation, freeze-drying, negative pressure room temperature drying, and negative pressure heating drying.

[0013] More preferably, the temperature of the freeze-drying is lower than the melting point of the organic solvent.

[0014] Preferably, after cleaning, the process further includes: air-drying at room temperature for 1-12 hours.

[0015] A neural scaffold is prepared by the method for preparing a neural scaffold as described in any of the above embodiments.

[0016] The principle of the polyurethane neural scaffold in this application is as follows: a solid, water-soluble polymer material is used directly as the mold material. The mold has a core and a recessed annular cavity, which cannot be dissolved by the organic solvent used in the polyurethane solution. When the polyurethane solution is added to the mold, as the organic solvent evaporates completely, a polyurethane tube is formed, i.e., the polyurethane neural scaffold. The inner diameter of the neural scaffold can be adjusted according to the core size of the mold (i.e., the inner diameter of the annular cavity), and the wall thickness can be adjusted according to the difference between the outer and inner diameters of the annular cavity. Due to the ease of processing the mold, polyurethane neural scaffolds with different inner diameters and wall thicknesses can be obtained, for example, the inner diameter range can be 1-3 mm, and the wall thickness can be 0.2-1 mm, etc.

[0017] When freeze-drying is used, and the temperature is lower than the melting point of the organic solvent in the polyurethane solution, such as 3°C or 5°C lower, the organic solvent occupies a certain volume space of the polyurethane in the frozen state. The frozen organic solvent sublimates, thus forming a large number of open pores inside the polyurethane neural scaffold.

[0018] Because water-soluble polymer materials are used as mold materials, the polyurethane neural scaffold can be easily obtained by washing the mold with water after molding.

[0019] In summary, this application has the following beneficial effects: 1. The preparation method of this application can obtain polyurethane neural scaffolds with different inner diameters, different wall thicknesses and different lengths. The inner diameter can be as low as 1-3 mm or even lower, such as 0.8 mm, and the wall thickness can be as thin as 0.5 mm or even lower, such as 0.2 mm or 0.3 mm.

[0020] 2. By using the freeze-drying method, a large number of open pores can be obtained inside the polyurethane neural scaffold. Attached Figure Description

[0021] Appendix Figure 1 Here is a cross-sectional SEM image of the polyurethane neural scaffold of Example 1; Appendix Figure 2 Photograph of the polyurethane neural scaffold product of Example 1; Appendix Figure 3 A cross-sectional SEM image of the polyurethane neural scaffold in Comparative Example 1. Appendix Figure 4 This is a cross-sectional SEM image of the polyurethane neural scaffold in Comparative Example 3. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below.

[0023] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0024] This application proposes a method for preparing a neural scaffold, which involves transferring a polyurethane solution into a mold, removing the organic solvent, cleaning, and obtaining the neural scaffold. The mold is made of a solid, water-soluble polymer material that is insoluble in organic solvents at temperatures not exceeding 50°C.

[0025] This application uses a solid, water-soluble polymer material as the mold material, which is insoluble in organic solvents at temperatures not exceeding 50°C. This ensures that the mold remains solid at room temperature, and organic solvents will not cause dissolution, corrosion, or other damage to the mold, thus maintaining its function and stability. Furthermore, the water-soluble polymer material is insoluble in organic solvents at temperatures not exceeding 30°C.

[0026] The mold for the water-soluble polymer material of this application can be obtained by direct processing, such as by grinding, etching or carving the block water-soluble polymer material directly, or by preparing an aqueous solution of a certain concentration, transferring it into a molding mold, and removing the water by heating or negative pressure, or by compression molding.

[0027] In a preferred embodiment of this application, the water-soluble polymer material is selected from one or a combination of several of homopolymer polyacrylic acid and its salts, copolymer polyacrylic acid and its salts, polyvinyl alcohol, polyacrylamide, and sodium chondroitin sulfate.

[0028] In a preferred embodiment of this application, the polyurethane polymer structure used in the polyurethane solution contains at least one first block and at least one second block, the first block and the second block are arranged alternately, and adjacent first blocks and second blocks are connected by urethane bonds. The first block is formed from aliphatic polyester diols and / or alicyclic polyester diols; The second block is formed from an aliphatic polyether diol.

[0029] In this application, the average molecular weight range of aliphatic polyester diols, alicyclic polyester diols, and aliphatic polyether diols can be 200-200,000, respectively. To improve the biocompatibility of the neural scaffold, aliphatic polyester diols can be polylactic acid (PLA), polycaprolactone (PCL), polyglycolic acid (PGA), polybutylene succinate (PBS), polybutylene terephthalate (PBAT), etc., and can be one or a combination of two or more of them, such as PLA-b-PCL block copolymers. Alicyclic polyester diols can improve the mechanical strength of polyurethane neural scaffolds; alicyclic polyester diols can be, for example, polycyclohexanediol adipate diol, polycyclohexanediol succinate diol, etc. Polyether diols can be polyethylene glycol, such as PEG400 (400 represents the average molecular weight, and so on), PEG600, PEG800, PEG1000, etc.

[0030] In this application, adjacent first and second blocks are connected by urethane bonds. Therefore, a prepolymer with -NCO end groups can be prepared by reacting an aliphatic polyether diol with a diisocyanate monomer (such as IPDI, HDI, TDI, MDI, HMDI, etc.). The -NCO content in the prepolymer can be 3-10 wt%, or further, the -NCO content in the prepolymer can be 3-8 wt%. The prepolymer is then subjected to an addition reaction with an aliphatic polyester diol and / or an alicyclic polyester diol to obtain a polyurethane polymer material.

[0031] In a preferred embodiment of this application, the organic solvent is a good solvent for polyurethane with a melting point of -100 to 20°C. Examples of organic solvents include tetrahydrofuran, acetone, butyl acetate, ethyl acetate, butanone, 1,1-dichloroethane, 1,2-dichloroethane, chloroform, dichloromethane, 1,1,1-trichloroethane, 1,1,2-trichloroethane, DMSO, DMF, DMAc, etc. More preferably, the organic solvent has a melting point of -60°C to 20°C, or even further, a melting point of -40°C to 20°C, such as one or a combination of 1,2-dichloroethane, nitromethane, N-methylpyrrolidone, 1,4-dioxane, 1,1,1-trichloro-2,2,2-trifluoroethane, 1,1,1-trichloroethane, carbon tetrachloride, 1,1,2-trichloroethane, and DMAc. This application describes various methods for removing organic solvents, such as natural evaporation, heated evaporation, reduced pressure evaporation, or freeze-drying. When using freeze-drying, the temperature is below the melting point of the organic solvent, and the polyurethane solution is in a frozen state. During freeze-drying, the organic solvent sublimates directly. Because the organic solvent occupies a certain volume space of the polyurethane polymer chain in the frozen state, its sublimation forms numerous open-pore micropores within the polyurethane polymer chain, resulting in a polyurethane neural scaffold with many open pores.

[0032] In a preferred embodiment of this application, the concentration of the polyurethane solution is 5-80 wt%. More preferably, from a cost and efficiency perspective, the concentration of the polyurethane solution is 35-70 wt%.

[0033] In a preferred embodiment of this application, the mold cavity is matched with the polyurethane neural scaffold. In this application, the matching of the mold cavity with the polyurethane neural scaffold means that the mold cavity is a concave, hollow circular or annular shape with a circular core in the center. The dimensions of the hollow circular or annular cavity match the inner and outer diameters of the polyurethane neural scaffold, and the diameter of the circular core matches the inner diameter of the polyurethane neural scaffold. The height of the cavity can be greater than or equal to the length of the polyurethane neural scaffold.

[0034] In a preferred embodiment of this application, the organic solvent is removed by one of the following methods: natural evaporation, heated evaporation, freeze-drying, negative pressure at room temperature drying, and negative pressure with heating drying. When natural evaporation, heated evaporation, negative pressure at room temperature drying, or negative pressure with heating drying are used to remove the organic solvent, the polyurethane solution is in a liquid state. As the organic solvent evaporates, a tubular polyurethane neural scaffold gradually forms, and fewer micropores are formed inside the polyurethane neural scaffold. When freeze-drying is used, the temperature is below the melting point of the organic solvent, and the polyurethane solution is in a frozen state. During freeze-drying, the organic solvent sublimates directly. Since the organic solvent occupies a certain volume space of the polyurethane polymer chain in the frozen state, the sublimated organic solvent forms more open micropores inside the polyurethane polymer chain.

[0035] In a more preferred embodiment of this application, the freeze-drying temperature is lower than the melting point of the organic solvent. More preferably, the freeze-drying temperature is 2°C or less lower than the melting point of the organic solvent, such as 3°C or 5°C lower. At this freeze-drying temperature, the polyurethane solution freezes completely, and the organic solvent sublimates during freeze-drying. While forming the polyurethane neural scaffold, a large number of open micropores are formed inside the polyurethane polymer chains.

[0036] In a preferred embodiment of this application, after cleaning, the process further includes: air-drying at room temperature for 1-12 hours. Cleaning can be performed directly with clean water or pure water to remove water-soluble polymer materials from the mold, thus obtaining a polyurethane neural scaffold. Air-drying after cleaning serves two purposes: firstly, it removes moisture from the surface of the neural scaffold, and secondly, it further removes any residual organic solvents that may remain in the neural scaffold.

[0037] In another aspect, this application proposes a polyurethane neural scaffold, prepared by the method for preparing a polyurethane neural scaffold as described in any of the above embodiments. The polyurethane neural scaffold of this invention has an adjustable inner diameter (e.g., within the range of 1-10 mm), an adjustable wall thickness (e.g., within the range of 0.2-1 mm), and an adjustable length (e.g., within the range of 1-10 cm), and can achieve a micropore structure containing a large number of open holes.

[0038] The technical solution of this application will be described in detail below with reference to embodiments and comparative examples.

[0039] The polyurethane materials in the following examples and comparative examples were prepared as follows: PEG1000 and HMDI were added to a dry container at a molar ratio of 0.7:1, nitrogen gas was purged, and 0.18% by weight of PEG1000 and HMDI and dibutyltin dilaurate were added. The mixture was stirred at room temperature for 1 hour, and then heated to 70°C and reacted for another hour to obtain the prepolymer.

[0040] The prepolymer and PCL glycol with an average molecular weight of 1000 were added to a reaction vessel at a -NCO to -OH molar ratio of 0.99:1. The mixture was heated to 80°C and reacted for 5 hours. After cooling, crude polyurethane was obtained. The crude polyurethane was dissolved in butyl acetate to prepare a 25 wt% solution, which was then added to 20 times its volume of methanol for precipitation. The precipitate was collected and dried overnight under vacuum at 50°C to obtain the polyurethane material.

[0041] The above-described methods for preparing polyurethane materials are merely examples and do not represent a limitation of the present invention.

[0042] Example 1 Polyurethane material was dissolved in 1,1,2-trichloroethane to prepare a 45 wt% polyurethane solution. The mold was obtained by compression molding of homopolymer sodium polyacrylate, with a core diameter of 3 mm, an outer diameter of 4 mm for the annular mold cavity, an outer diameter of 2 cm for the mold, and a height of 15 cm.

[0043] The polyurethane solution was transferred into a mold, cooled to -40°C, and dried in a freeze-drying apparatus at -42 to -40°C until it was formed. The mold was then removed, washed with 45°C warm water, and dried overnight at 60°C to obtain a polyurethane neural scaffold with an inner diameter of 3 mm, a wall thickness of 0.5 mm, and a length of 5 cm.

[0044] Example 2 Polyurethane material was dissolved in 1,1,2-trichloroethane to prepare a 40 wt% polyurethane solution. The polyurethane solution was transferred to the mold of Example 1, cooled to -40°C, and dried in a freeze-drying apparatus at -42 to -40°C until it was formed. The mold was then removed, washed with 45°C warm water, and dried overnight at 60°C to obtain a polyurethane neural scaffold with an inner diameter of 3 mm, a wall thickness of 0.5 mm, and a length of 5 cm.

[0045] Example 3 The mold is obtained by compression molding of PVA 17-99, with a core diameter of 2mm, an outer diameter of 4mm for the annular mold cavity, an outer diameter of 2cm for the mold, and a height of 15cm.

[0046] Polyurethane material was dissolved in 1,1,1-trichloroethane to prepare a 55 wt% polyurethane solution. This solution was transferred to the mold described above, cooled to -40°C, and then dried in a freeze-drying apparatus at -42 to -40°C until it was formed. The mold was then removed by washing with 92°C hot water and dried overnight at 60°C to obtain a polyurethane neural scaffold with an inner diameter of 2 mm, a wall thickness of 1 mm, and a length of 8 cm.

[0047] Example 4 Polyurethane material was dissolved in 1,1,1-trichloroethane to prepare a 35 wt% polyurethane solution. This solution was transferred to the mold from Example 3, cooled to -40°C, and dried in a freeze-drying apparatus at -42 to -40°C until it was formed. The mold was then removed, washed with 92°C hot water, and dried overnight at 60°C to obtain a polyurethane neural scaffold with an inner diameter of 2 mm, a wall thickness of 1 mm, and a length of 10 cm.

[0048] Example 5 The mold material is obtained by free radical copolymerization of acrylic acid and butyl methacrylate in a molar ratio of 12:1, followed by neutralization with sodium hydroxide, and then compression molding. The mold core has a diameter of 5mm, the outer diameter of the annular cavity is 7mm, the outer diameter of the mold is 2.5cm, and the height is 15cm.

[0049] Polyurethane material was dissolved in 1,1,2-trichloroethane to prepare a 60wt% polyurethane solution. This solution was transferred to the mold described above, cooled to -40°C, and then dried in a freeze-drying apparatus at -42 to -40°C until it was formed. The mold was then removed by washing with 45°C warm water and dried overnight at 60°C to obtain a polyurethane neural scaffold with an inner diameter of 5 mm, a wall thickness of 1 mm, and a length of 10 cm.

[0050] Example 6 Polyurethane material was dissolved in 1,4-dioxane to prepare a 50 wt% polyurethane solution, which was then transferred to the mold of Example 5. The solution was cooled to -30°C and dried in a freeze-drying apparatus at -35 to -33°C until it was formed. The mold was then removed, washed with 45°C warm water, and dried overnight at 60°C to obtain a polyurethane neural scaffold with an inner diameter of 5 mm, a wall thickness of 1 mm, and a length of 10 cm.

[0051] Comparative Example 1 According to existing technology, 10 wt% sodium chloride particles (average particle size 0.7 μm) were added to polyurethane material, mixed evenly, and melt-extruded into tubular polyurethane tubes with an inner diameter of 6 mm and a wall thickness of 0.5 mm. These tubes were then cut to 5 cm lengths and immersed in clean water for sodium chloride replacement for 72 hours, with the water changed every 12 hours. The tubes were then removed, cleaned, and vacuum-dried overnight at 60°C to obtain the polyurethane neural scaffold.

[0052] Comparative Example 2 The difference between Comparative Example 2 and Example 1: The freeze-drying temperature was adjusted from -42 to -40°C to -22 to -20°C. The remaining steps remained unchanged.

[0053] Comparative Example 3 The difference between Comparative Example 3 and Example 6: The freeze-drying temperature was adjusted from -35 to -33°C to -7 to -5°C. The remaining steps remained unchanged.

[0054] All polyurethane neural scaffolds from Examples 1-6 and Comparative Examples 2 and 3 were cut to a length of 5 cm.

[0055] The density of polyurethane materials and polyurethane neural scaffolds in various embodiments and comparative examples were tested using the water displacement method. The results are shown in Table 1 below.

[0056] Table 1 Density / g / cm³ 3 polyurethane materials Example 1 Example 2 Example 3 Example 4 1.171 0.635 0.646 0.659 0.629 Example 5 Example 6 Comparative Example 1 Comparative Example 2 Comparative Example 3 0.667 0.653 0.804 0.946 0.915 As shown in Table 1, the preparation method of this application can significantly reduce the density of the polyurethane neural scaffold by forming more micropores inside it. For example, compared with Example 6 and Comparative Example 3, the freeze-drying temperature of Example 6 is lower, and the polyurethane polymer chain segments are almost or completely frozen, so they can exist stably after forming micropores. In Comparative Example 3, the freeze-drying temperature is not low enough, and the polyurethane polymer chains still have good mobility. Even if 1,4-dioxane sublimates, the remaining micropores will be gradually filled by the polyurethane polymer chains, resulting in smaller and fewer micropores.

[0057] Appendix Figure 1 Here is a cross-sectional SEM image of the neural scaffold from Example 1, attached. Figure 2 Photographs of the neural scaffold product from Example 1 are attached. Figure 3 Here is a cross-sectional SEM image of the neural scaffold in Comparative Example 1, attached. Figure 4 The cross-sectional SEM image of the neural scaffold in Comparative Example 3 shows that the polyurethane neural scaffold prepared by the method of this application has more and more uniform micropores, and is mainly open-pore, while the micropores prepared by the existing method (Comparative Example 1, sodium chloride as a pore-forming agent) are very uneven and have many closed-pore micropores.

[0058] Comparison Appendix Figure 4 and attached Figure 1 It can also be seen that when the freeze-drying temperature is not low enough, fewer micropores are formed.

[0059] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method of preparing a neural scaffold, characterized by, transferring the polyurethane solution into a mold, removing the organic solvent, and cleaning to obtain the nerve scaffold; the mold is made of a solid water-soluble polymer material which is insoluble in the organic solvent at a temperature not higher than 50°C.

2. The method of claim 1, wherein the neural scaffold is prepared by, the water-soluble polymer material is selected from one or a combination of the following: homopolymer polyacrylic acid and its salt, copolymer polyacrylic acid and its salt, polyvinyl alcohol, polyacrylamide, and sodium chondroitin.

3. The method of claim 1, wherein the neural scaffold is prepared by, the polyurethane polymer structure used in the polyurethane solution comprises at least one first block and at least one second block, the first block and the second block are arranged alternately, and adjacent first block and second block are connected by a urethane bond; the first block is formed by aliphatic polyester diol and / or alicyclic polyester diol; the second block is formed by aliphatic polyether diol.

4. The method of claim 1, wherein the neural scaffold is prepared by, the melting point of the organic solvent is -100-20°C.

5. The method of claim 1, wherein the neural scaffold is prepared by, the concentration of the polyurethane solution is 5-80wt%.

6. The method of claim 1, wherein the neural scaffold is prepared by, the mold cavity and the polyurethane nerve scaffold are matched with each other.

7. The method of claim 1, wherein the neural scaffold is prepared by, the removal of the organic solvent is selected from one of the following: natural evaporation, heating evaporation, freeze vacuum drying, negative pressure normal temperature drying, and negative pressure heating drying.

8. The method of claim 7, wherein the neural scaffold is prepared by, the temperature of the freeze vacuum drying is lower than the melting point of the organic solvent.

9. The method of claim 1, wherein the neural scaffold is prepared by, after the cleaning, the nerve scaffold is left to stand at room temperature for 1-12 hours.

10. A neural scaffold, characterized in that, the nerve scaffold is prepared by the method of any one of claims 1-9.

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