Cylindrical composite nerve scaffold, and preparation method therefor and use thereof

Abstract

The present invention belongs to the technical field of medical biological materials. Disclosed in the present invention are a cylindrical composite nerve scaffold, and a preparation method therefor and the use thereof. The method for preparing the cylindrical composite nerve scaffold comprises: preparing a degradable bio-responsive material film and an extracellular matrix-like composite hydrogel; and covering the outer surface of the extracellular matrix-like hydrogel with the degradable bio-responsive material film to obtain the cylindrical composite nerve support. Further disclosed in the present invention is the use of the cylindrical composite nerve scaffold in a medical instrument for guiding nerve regeneration. The present invention has excellent safety and biocompatibility, adopts a degradable bio-responsive material certified by the American FDA, and can be used clinically, and extracellular matrix-like substances are natural substances, thus greatly reducing the toxicity and side effects.

Description

A cylindrical composite nerve stent and its preparation method and application Technical Field

[0001] The present invention relates to the technical field of medical biomaterials, and in particular to a cylindrical composite nerve stent and a preparation method and application thereof. Background Art

[0002] The regeneration and repair of tissue defects is a widely discussed issue. Autologous tissue transplantation has limitations due to limited donor site selection and dysfunction. Allogeneic tissue transplantation also faces numerous challenges due to insufficient sources and immune rejection. One of the key elements of tissue engineering research is the development of scaffolds, materials that support cellular processes. In tissue engineering research, scaffolds provide an artificial extracellular matrix, building a support system that promotes cell adhesion, proliferation, metabolism, and new tissue formation. Neural scaffolds can be hollow, multi-channel, or filled with biodegradable materials. The walls can be single-layer or multi-layered. Conduit materials primarily consist of natural materials such as proteins (e.g., silk fibroin, collagen) and polysaccharides (e.g., gelatin, chitosan, cellulose), as well as artificial materials such as polylipids, conductive and non-conductive polymers, and hydrogels. Currently, research using neural scaffolds or conduits to repair nerve defects and promote regeneration has made some progress. However, inefficient nerve growth and high levels of scar infiltration remain challenges that need to be addressed in the repair of peripheral nerve injuries. The repair strategies in the prior art are all focused on providing a biocompatible catheter or stent surface, but still do not have good results.

[0003] Therefore, how to establish a microenvironment similar to the extracellular matrix and reduce scar infiltration during the regeneration process to achieve better nerve regeneration is a technical problem that technicians in this field urgently need to solve.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to provide a cylindrical composite nerve stent and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing a cylindrical composite nerve scaffold, comprising the following steps:

[0008] (1) dissolving the bioresponsive degradable material in a mixed solvent of dichloromethane and N,N-dimethylformamide, and then electrospinning the bioresponsive degradable material film under positive and negative high voltage conditions;

[0009] (2) Carboxymethyl chitosan, sodium alginate and N-acetyl-5-methoxytryptamine were added to water and dissolved, and stirred for 6-8 hours until the mixture was uniformly mixed. The solution was then transferred to a substrate and evenly spread. Calcium chloride solution was then added thereto to cover the surface of the hydrogel to obtain an extracellular matrix-like composite hydrogel.

[0010] (3) The extracellular matrix-like composite hydrogel is rolled into a tube shape, and the bioresponsive degradable material film is covered on the outer surface of the extracellular matrix-like hydrogel to obtain the cylindrical composite neural scaffold.

[0011] Furthermore, in step (1), the ratio of the added amounts of the bioresponsive degradable material, dichloromethane and N,N-dimethylformamide is 1.5 g:7 ml:3 ml.

[0012] Furthermore, the bioresponsive degradable material includes one or more of polylactic acid, polycaprolactone, polytrimethylene carbonate, polyethylene glycol, and polylactic acid-glycolic acid copolymer.

[0013] Furthermore, the electrospinning conditions in step (2) are: positive high voltage 12kV and negative high voltage -4kV.

[0014] Furthermore, the ratio of the added amounts of carboxymethyl chitosan, sodium alginate, N-acetyl-5-methoxytryptamine and water in step (2) is 0.224 g: 0.224 g: 0.056 g: 5.6 ml;

[0015] The concentration of the calcium chloride solution is 0.1 mol / L.

[0016] The present invention also provides a cylindrical composite nerve stent obtained by a preparation method of the cylindrical composite nerve stent.

[0017] The present invention also provides the use of the cylindrical composite nerve scaffold in a medical device for guiding the regeneration of nerves and nerve-related muscle and tendon tissues.

[0018] Beneficial Effects: The present invention establishes an inner layer that resembles an extracellular matrix microenvironment, promoting efficient nerve growth, while also creating a relatively rigid, biocompatible, hydrophobic interface to prevent high levels of scar infiltration. The inner and outer layers combine to form a cylindrical composite neural scaffold. The cylindrical composite neural scaffold in the present invention is layered, with a hard exterior and a soft interior. A soft, hydrophilic interface is created within the scaffold to simulate the extracellular matrix microenvironment, facilitating cell adhesion and migration. Simultaneously, a relatively rigid, hydrophobic interface is created on the outer layer of the scaffold to meet the mechanical requirements for structural integrity during long-term nerve regeneration, preventing fibroblast attachment or infiltration and thus avoiding scar formation.

[0019] The bioresponsive biodegradable materials used in this solution have excellent safety and biocompatibility. These bioresponsive biodegradable materials are US FDA-approved and can be used clinically. Among them, a polycaprolactone-related product, the "Polycaprolactone Microsphere Facial Filler for Injection," has been approved by my country's National Medical Products Administration.

[0020] The above-mentioned electrospinning conditions can provide electric field supplementation, improve spinning efficiency, enhance fiber pulling orientation, and facilitate more concentrated deposition of fibers in the receiving area.

[0021] The extracellular matrix-like substances in the present invention are all natural substances, have good biocompatibility, biodegradability and remodeling ability, can better support cell migration, proliferation, differentiation and adhesion, and greatly reduce toxic side effects.

[0022] The present invention discloses a cylindrical composite nerve scaffold and its preparation method and application. The present invention constructs a nerve regeneration channel to guide nerve regeneration according to the requirements of the nerve regeneration microenvironment and the adjacent structure design, and simultaneously performs interface differentiation design to promote nerve growth and reduce scar infiltration. In addition, the present invention uses bioresponsive degradable materials to prepare a relatively hard hydrophobic interface outer layer, constructs a regeneration space to meet the mechanical requirements for structural integrity in long-term nerve regeneration, prevents fibroblast attachment or infiltration, thereby avoiding scar formation and further resisting tissue adhesion. In addition, the present invention uses extracellular matrix microenvironment materials to prepare a soft inner layer, simulates the extracellular matrix microenvironment, and is composed of a bioactive substance N-acetyl-5-methoxytryptamine and an extracellular matrix-like substance. It has the function of promoting cell proliferation adhesion at the regeneration interface, anti-inflammatory, and angiogenesis. The present invention has excellent safety and biocompatibility. It uses bioresponsive degradable materials certified by the US FDA and can be used in clinical practice. Moreover, the extracellular matrix-like substances are all natural substances, which greatly reduces toxic and side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] FIG1 is a flow chart of electrospinning preparation in Example 1;

[0025] FIG2 is a schematic diagram of the equipment principle for preparing the cylindrical composite nerve scaffold in Example 1;

[0026] FIG3 is a scanning electron micrograph of a cross section of the cylindrical composite nerve scaffold of Example 1;

[0027] FIG4 is a scanning electron micrograph of the differentiated interface of the cylindrical composite nerve scaffold in Example 1;

[0028] FIG5 is a stress-strain curve of the tensile modulus of the outer layer of the cylindrical composite nerve stent in Example 1;

[0029] Figure 6 shows in vivo validation in an animal model;

[0030] Among them, a is the composite nerve scaffold intervention group provided in Example 1 of the present invention, b is the polycaprolactone nerve scaffold group, and c is the autologous nerve transplantation group. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] Example 1

[0034] A method for preparing a cylindrical composite neural scaffold by electrospinning, comprising the following steps:

[0035] step:

[0036] 1) At room temperature, 1500 mg of polycaprolactone was dissolved in a mixed solvent of 7 mL of dichloromethane and 3 mL of N,N-dimethylformamide and stirred until completely dissolved. The mixed solution was then electrospun at a positive high voltage of 12 kV and a negative high voltage of -4 kV. After spinning, a 15 mm x 15 cm rectangular spinning membrane was cut to obtain a polycaprolactone electrospun membrane.

[0037] 2) Weigh 0.224 g of carboxymethyl chitosan, 0.224 g of sodium alginate, and 0.056 g of N-acetyl-5-methoxytryptamine separately, place them in a container, add 5.6 mL of water to dissolve, and stir overnight with a magnetic stirrer until uniform. Then, use a 1 mL syringe to draw 0.2 mL of the solution into a six-well plate and spread it evenly. Then, add about 3 mL of a 0.1 M calcium chloride aqueous solution as a crosslinker until the entire hydrogel is completely covered, thereby obtaining an extracellular matrix-like composite hydrogel;

[0038] 3) The extracellular matrix-like composite hydrogel and the polycaprolactone electrospun membrane were rolled up separately using a round rod with a diameter of 2.6 mm to form a cylindrical biomimetic interface cylindrical composite neural scaffold with the hydrogel inside and the electrospun membrane outside.

[0039] Comparative Example 1

[0040] A method for preparing a polycaprolactone neural scaffold comprises the following steps:

[0041] The polycaprolactone electrospun membrane obtained in step (1) of Example 1 was rolled into a round rod with a diameter of 2.6 mm to form a cylindrical nerve scaffold.

[0042] Technical Effects

[0043] 1. As can be seen from the scanning electron microscopy images in Figures 3 and 4, the nanofibers are relatively straight and stacked in multiple layers, providing a good mechanical performance foundation for the outer layer of the catheter, which can prevent the catheter from collapsing due to structural performance problems when the catheter is connected to the nerve, thereby affecting nerve repair.

[0044] 2. Stress-strain curve of tensile modulus

[0045] The polycaprolactone electrospun membrane obtained in step (1) of Example 1 was cut into rectangular shapes (35 mm × 12.5 mm, L × W) and subjected to tensile modulus measurement using a dynamic mechanical analyzer (DMAQ850 / TA / China). The DMA machine was set to 3 mm·min - 1 The tensile modulus is determined at room temperature using a tensile speed of 100 nm. The stress-strain curve is plotted in Origin, and the slope within the elastic range is the tensile modulus.

[0046] The results are shown in Figure 5, which shows the stress-strain curves during DMA stretching. Slope analysis revealed a tensile modulus of 3.12 ± 0.06 MPa (n = 3) for the polycaprolactone electrospun membrane, indicating good tensile strength. Furthermore, the polycaprolactone electrospun membrane exhibited Hookean elastic behavior throughout the stretching phase, breaking directly after passing the yield point. This suggests that the fibers of the nerve conduit outer membrane are well aligned locally and can rupture suddenly along the alignment direction.

[0047] 3. Animal Experimentation

[0048] (1) Validation test design:

[0049] SPF male Sprague-Dawly (SD) rats (weighing 200-250 g) were randomly divided into three groups, with 5 rats in each group:

[0050] Group A: Cylindrical composite nerve scaffold group (using the product obtained in Example 1)

[0051] Group B: polycaprolactone nerve scaffold group (product obtained in Comparative Example 1);

[0052] Group C: autologous nerve transplantation group.

[0053] (2) Methods and procedures:

[0054] Housing conditions: temperature 21°C-25°C, humidity 50%-60%. After anesthesia, the three groups of rats under sterile conditions underwent midline incisions on the right thigh and buttocks to free and expose the sciatic nerve. A 10 mm segment of the midsegment of the sciatic nerve was resected to create sciatic nerve defect models. Groups A, B, and C each received their own nerve stent for bridge repair, with the epineurium and stent sutured to each end using microsutures.

[0055] (3) Technical information:

[0056] A single polycaprolactone nerve scaffold lacks a differentiated interface design and an extracellular matrix-like hydrogel inner layer (i.e., no extracellular matrix-like material and N,N-dimethylformamide), resulting in poor nerve growth promotion. Figure 6 shows HE staining images of the three animal models A, B, and C for in vivo validation, analyzing the differences in regenerated nerve fibers between the two groups from a histological perspective. It can be seen that the cylindrical composite nerve scaffold group and the autologous nerve transplant group regenerated nerves better than the polycaprolactone nerve scaffold group (experimental animal sample size (SD rats) n = 5).

[0057] The present invention established a 10 mm sciatic nerve defect model in SD rats. After intervention with the cylindrical composite nerve scaffold obtained in Example 1, both sciatic nerve function index and neuroelectrophysiological parameters were improved, demonstrating that the cylindrical composite nerve scaffold provided by the present invention can improve neurological function. Furthermore, the interaction between N-acetyl-5-methoxytryptamine and the extracellular matrix-like material also reduced inflammation and oxidative stress levels.

[0058] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

[0059] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a cylindrical composite nerve scaffold, characterized in that: The following steps are involved: (1) dissolving the bioresponsive degradable material in a mixed solvent of dichloromethane and N,N-dimethylformamide, and then electrospinning the bioresponsive degradable material film under positive and negative high voltage conditions; (2) Carboxymethyl chitosan, sodium alginate and N-acetyl-5-methoxytryptamine were added to water and dissolved, and stirred for 6-8 hours until the mixture was uniformly mixed. The solution was then transferred to a substrate and evenly spread. Calcium chloride solution was then added thereto to cover the surface of the hydrogel to obtain an extracellular matrix-like composite hydrogel. (3) The extracellular matrix-like composite hydrogel is rolled into a tube shape, and the bioresponsive degradable material film is covered on the outer surface of the extracellular matrix-like hydrogel to obtain the cylindrical composite neural scaffold.

2. The method for preparing a cylindrical composite nerve scaffold according to claim 1, characterized in that: The ratio of the added amounts of the bioresponsive degradable material, dichloromethane and N,N-dimethylformamide in step (1) is 1.5 g:7 ml:3 ml.

3. The method for preparing a cylindrical composite nerve scaffold according to claim 2, characterized in that: The bioresponsive degradable material includes one or more of polylactic acid, polycaprolactone, polytrimethylene carbonate, polyethylene glycol, and polylactic acid-glycolic acid copolymer.

4. The method for preparing a cylindrical composite nerve scaffold according to claim 1, characterized in that: The electrospinning conditions in step (2) are: positive high voltage 12kV and negative high voltage -4kV.

5. The method for preparing a cylindrical composite nerve scaffold according to claim 1, characterized in that: The ratio of the added amounts of carboxymethyl chitosan, sodium alginate, N-acetyl-5-methoxytryptamine and water in step (2) is 0.224 g: 0.224 g: 0.056 g: 5.6 ml; The concentration of the calcium chloride solution is 0.1 mol / L. 6 . The cylindrical composite nerve scaffold obtained by the preparation method of the cylindrical composite nerve scaffold according to any one of claims 1 to 5 .

7. Use of the cylindrical composite nerve scaffold as claimed in claim 6 in a medical device for guiding the regeneration of nerves and nerve-related muscle and tendon tissues.

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