CN114796628B - Melatonin nerve conduit and preparation method thereof - Google Patents
Melatonin nerve conduit and preparation method thereof Download PDFInfo
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- CN114796628B CN114796628B CN202210207175.4A CN202210207175A CN114796628B CN 114796628 B CN114796628 B CN 114796628B CN 202210207175 A CN202210207175 A CN 202210207175A CN 114796628 B CN114796628 B CN 114796628B
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/148—Materials at least partially resorbable by the body
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/12—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
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- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/146—Porous materials, e.g. foams or sponges
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
- A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L31/16—Biologically active materials, e.g. therapeutic substances
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- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y10/00—Processes of additive manufacturing
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y70/00—Materials specially adapted for additive manufacturing
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
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- B33Y80/00—Products made by additive manufacturing
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- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0015—Electro-spinning characterised by the initial state of the material
- D01D5/003—Electro-spinning characterised by the initial state of the material the material being a polymer solution or dispersion
- D01D5/0038—Electro-spinning characterised by the initial state of the material the material being a polymer solution or dispersion the fibre formed by solvent evaporation, i.e. dry electro-spinning
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0061—Electro-spinning characterised by the electro-spinning apparatus
- D01D5/0076—Electro-spinning characterised by the electro-spinning apparatus characterised by the collecting device, e.g. drum, wheel, endless belt, plate or grid
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- D—TEXTILES; PAPER
- D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
- D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
- D01D5/00—Formation of filaments, threads, or the like
- D01D5/0007—Electro-spinning
- D01D5/0061—Electro-spinning characterised by the electro-spinning apparatus
- D01D5/0092—Electro-spinning characterised by the electro-spinning apparatus characterised by the electrical field, e.g. combined with a magnetic fields, using biased or alternating fields
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/204—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with nitrogen-containing functional groups, e.g. aminoxides, nitriles, guanidines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/602—Type of release, e.g. controlled, sustained, slow
- A61L2300/604—Biodegradation
Abstract
The invention relates to the field of biological materials, in particular to a melatonin nerve conduit and a preparation method thereof. The invention provides a preparation method of a melatonin nerve conduit, which comprises the following steps: preparing biodegradable material and melatonin suspension or adding melatonin into the melted biodegradable material and mixing uniformly to obtain a mixture; and adding the suspension or the mixture into 3D printing or electrostatic spinning equipment, and printing or electrostatic spinning to obtain the melatonin nerve conduit. The melatonin nerve conduit prepared by the preparation method provided by the invention has ideal biomedical functional material. The rat sciatic nerve defect repair experiment shows that the melatonin nerve conduit provided by the invention promotes nerve regeneration, improves the maturity of regenerated nerves and the number of nerve fibers playing a role, promotes the sciatic nerve function to recover to be normal, has better effect than autologous nerve transplantation repair, and has good application prospect.
Description
The application is a divisional application of an invention patent with the application date of 2018, the application number of 201810921130.7 and the invention name of melatonin nerve conduit composition, nerve conduit and preparation method and application thereof.
Technical Field
The invention relates to the field of biological materials, in particular to a melatonin nerve conduit and a preparation method thereof.
Background
Various biomedical catheters, including degradable and non-degradable catheters, such as polyethylene tubes, are used as nerve catheters for researching nerve regeneration, and the clinical application effect is not ideal because the catheters are not degraded, and if the catheters are not treated after nerve regeneration is completed, the catheters can cause tissue fibrosis and cause toxic and side effects such as inflammation. Various biodegradable materials are currently developed to prepare various biomedical catheters, such as collagen, polylactic acid and the like, but the materials have the defects of strength, nerve regeneration speed, toxic and side effects and the like due to the independent use of the materials. Further studies on these catheters have been recently conducted, such as performing various regeneration on the surface or inside of the catheter, which is advantageous for various lumen injuries in the body, such as performing cell modification on the surface of the catheter, carrying various active substances for promoting lumen growth, such as nerve growth factors, blood-profound cells, etc. of nerve catheters.
The electrostatic spinning is to apply high-voltage direct current power to a capillary containing polymer solution, under the action of an electric field, the liquid drop hung at the tail end of the capillary deforms to form a Taylor cone, when the applied electric field exceeds a certain critical value, the electric field force overcomes the surface tension of the solution to form thinner jet flow with charged surface, the jet flow moves to a grounded collector under the action of the electric field, in the moving process, the jet flow is continuously stretched, and in some cases, the jet flow is split into thinner jet flow, and meanwhile, the solvent is continuously volatilized, and finally, the fiber with the diameter of several micrometers to tens of nanometers or even several nanometers is obtained on the collector. The nerve conduit stent material prepared by electrostatic spinning not only can simulate the collagen fiber structure in extracellular matrix, but also has high porosity and specific surface area, and is beneficial to cell adhesion, growth and proliferation.
Although a nerve conduit has been studied to some extent, a method for preparing the nerve conduit is disclosed in chinese patent No. CN100479785C, but there is a certain distance from autologous nerve repair. In addition, several patents disclose different preparation methods (such as chinese patent No. CN101439205a, publication No. 2009.5.27; CN101507842a, publication No. 2009.8.19, CN106668938A, publication No. 2017.05.17; CN106924820a, publication No. 2017.07.07), but these catheters have problems of strength, toxic side effects, quality control or cost. Chinese patent 2010800025691 discloses a method and apparatus for preparing a three-dimensional porous tubular scaffold, and in particular discloses that a 3D tubular scaffold may be manufactured from non-biodegradable polymers including, for example, polylactic acid (PLA), polyglycolic acid (PGA), etc., or biodegradable polymers including, for example, polylactic acid (PLA), polyglycolic acid (PGA), etc., 3D tubular scaffolds may incorporate hormones, examples of which are melatonin, etc., and that 3D tubular scaffolds may also be used as peripheral nerve conduits to guide damaged nerve regeneration; although the patent discloses the preparation of nerve conduits using melatonin and polylactic acid, the effect of melatonin in promoting nerve repair is not disclosed. None of the nerve catheters disclosed in the prior art have ideal biomedical catheters. An ideal catheter should have: has enough strength, elasticity, hardness and the like; degradable and in vivo until the damaged tissue is completely regenerated, and completely degraded, and the operation is not needed to be taken out again; the material has no toxic or side effect as much as possible; a suitable degradation period; can guide the tissue to grow towards a proper direction; there are prevention of unwanted tissue regeneration, etc.
Disclosure of Invention
The invention aims to provide a melatonin nerve conduit and a preparation method thereof, and the melatonin nerve conduit obtained by the invention has enough strength, elasticity, hardness and degradability, can promote nerve regeneration, and improves the maturity of regenerated nerves and the number of nerve fibers playing a role.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of a melatonin nerve conduit, which comprises the following steps: preparing biodegradable material and melatonin suspension or adding melatonin into the melted biodegradable material and mixing uniformly to obtain a mixture; and adding the suspension or the mixture into 3D printing or electrostatic spinning equipment, and printing or electrostatic spinning to obtain the melatonin nerve conduit.
Preferably, the weight percentage of the biodegradable material is 90-99.5%, and the weight percentage of the melatonin is 0.5-10%.
Preferably, the weight percentage of the biodegradable material is 95-99%, and the weight percentage of the melatonin is 1-5%.
Preferably, the biodegradable material is one or more of polylactic acid, polylactic acid-polyglycolic acid, polycaprolactone, silk protein, collagen, gelatin, hyaluronic acid and chitosan;
the melatonin is a single-layer melatonin and/or a multi-layer melatonin.
Preferably, the electrospinning comprises the steps of: dissolving the biodegradable material with 3-6 times of organic solvent, adding melatonin, mixing, injecting into an injector, forming a tube by using an electrostatic spinning process, spraying the tube onto a continuously rotating rod-shaped die by using direct current voltage, removing the nerve conduit from the die after forming the tube, drying the volatilized solvent, and cutting into melatonin nerve conduits with different length specifications;
the organic solvent is dichloromethane or ethyl acetate.
Preferably, when the diameter of the bar-shaped mold is 3mm, the rotational speed of the bar-shaped mold is 800rpm;
when the diameter of the rod-shaped mold is 5mm, the rotating speed of the rod-shaped mold is 100rpm;
the voltage in the electrostatic spinning process is 20kV, and the receiving distance is 20cm; the moving speed of the spray head in the horizontal direction is 10cm/min or 15cm/min.
Preferably, the obtaining the melatonin nerve conduit further comprises: printing or electrostatic spinning a layer of bioadhesive substance in the melanin nerve conduit, and performing crosslinking and curing; the bioadhesive substance is one or more of dopamine, bioadhesive peptide and extracellular matrix.
The invention also provides the melatonin nerve conduit prepared by the preparation method.
Preferably, the melatonin nerve conduit further comprises a bioadhesive layer inside.
Preferably, the average pore diameter of the outer surface of the melatonin nerve conduit is 0.01-10 μm; the bioadhesive layer has an average pore size of 10-1000 μm.
The invention has the advantages that:
1. a catheter stent having a biologically desirable shape and having an ideal biomedical function is provided.
2. The nerve conduit has enough strength, elasticity, hardness and degradability, the damaged tissues in the body and the like are completely regenerated after the implantation, the nerve conduit is completely degraded, and the nerve conduit does not need to be taken out by operation again;
3. the rat sciatic nerve defect repair experiment shows that the melatonin nerve conduit can promote nerve regeneration, improve the maturity of regenerated nerves and the number of nerve fibers playing a role, improve the sciatic nerve function index of the rat, promote the sciatic nerve function to recover to be normal, has better effect than autologous nerve transplantation repair, and has good application prospect.
Drawings
FIG. 1 is a topography of a nerve conduit prepared in accordance with an embodiment of the present invention;
FIG. 2 is a graph of the results of a pure PLA catheter regenerated nerve transmission electron microscopy;
FIG. 3 is a graph showing the result of the regenerative nerve transmission electron microscopy of autologous nerve grafting;
FIG. 4 is a graph showing the results of a regenerative nerve transmission electron microscopy of a nerve conduit group prepared from a combination of melatonin and PCL in an amount of 1%.
Detailed Description
The invention is further described below in conjunction with the detailed description. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications of the present invention may be made by those skilled in the art after reading the description of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
EXAMPLE 1 nerve conduit biodegradable Material composition
Taking polylactic acid with the weight ratio of 99% and melatonin with the weight ratio of 1%, dissolving the polylactic acid with 3-6 times of dichloromethane, and then adding the melatonin for fully and uniformly mixing to prepare the nerve conduit biodegradable material composition.
Example 2 nerve conduit biodegradable Material composition
Taking polylactic acid-polyglycolic acid with the weight ratio of 99.5% and melatonin with the weight ratio of 0.5%, dissolving the polylactic acid-polyglycolic acid with ethyl acetate with the weight of 3-6 times, and then adding melatonin for fully and uniformly mixing to prepare the nerve conduit biodegradable material composition.
EXAMPLE 3 nerve conduit biodegradable Material composition
Taking 98% of polycaprolactone by weight and 2% of melatonin by weight, dissolving the polycaprolactone by 3-6 times of dichloromethane by weight, and then adding the melatonin and fully mixing uniformly to prepare the nerve conduit biodegradable material composition.
Example 4 nerve conduit biodegradable Material composition
Taking 95% of polylactic acid and 5% of melatonin by weight, adding melatonin into the melted polylactic acid, and fully and uniformly mixing to prepare the nerve conduit biodegradable material composition.
Example 5 nerve conduit biodegradable Material composition
Taking polylactic acid-polyglycolic acid with the weight ratio of 90 percent and melatonin with the weight ratio of 10 percent, and adding the melatonin into the melted polylactic acid-polyglycolic acid for fully and uniformly mixing to prepare the nerve conduit biodegradable material composition.
EXAMPLE 6 preparation of nerve conduit
Preparing a catheter from the biodegradable material composition according to any one of examples 1 to 5, and preparing various needed catheters by adopting a conventional electrostatic spinning technology; or adopting 3D printing technology to print various catheters; or a catheter mold is used to prepare various desired catheters. The melatonin PCL nerve conduit as 3D printed and 1% had good nerve regeneration properties such as better elastic modulus (48.32 and 31.77MPa respectively) than pure PCL printed, etc.
Example 7 preparation of nerve conduit
On the basis of the nerve conduit of example 6, an internal printing or electrospinning of a layer of bioadhesive substance was performed and crosslinked and cured. The bioadhesive substance may be any of dopamine, bioadhesive peptide or extracellular matrix, or a mixture thereof in any ratio.
Example 8 preparation of nerve conduit
Catheters were prepared using the biodegradable material composition of example 1, the specific steps were as follows: taking a nerve conduit biodegradable material composition, removing bubbles, taking 100ml of the composition, injecting the composition into an injector, outputting the composition at the speed of 10ml/h, forming a tube by using an electrostatic spinning process, spraying the composition onto a rod-shaped die with the rotating speed of 800rpm and the diameter of 3mm by using direct current voltage, and receiving the voltage of 20KV and the distance of 20cm. The electrostatic spinning nozzle moves back and forth at a speed of 15cm/min in the horizontal direction, and the length of the tube is 15-20cm. And after the solution is sprayed, the rod-shaped mold continues to rotate for 5-10 hours, so that the solvent volatilizes. And removing the nerve conduit from the mold, drying to completely volatilize the solvent, and cutting the nerve conduit into different length specifications.
Example 9 preparation of nerve conduit
Catheters were prepared using the biodegradable material composition of example 1, the specific steps were as follows: taking polylactic acid with the weight ratio of 99% and dopamine with the weight ratio of 1%, dissolving the polylactic acid with 3-6 times of dichloromethane, and then adding dopamine for fully and uniformly mixing to obtain a mixture. Taking 10ml of the mixture after bubble removal, injecting the mixture into an injector, outputting the mixture at the speed of 10ml/h, forming a tube by using an electrostatic spinning process, spraying the tube onto a rod-shaped die with the rotating speed of 800rpm and the diameter of 3mm by using direct current voltage, and receiving the voltage of 20KV for 20cm. The electrostatic spinning nozzle moves back and forth at a speed of 15cm/min in the horizontal direction, and the length of the tube is 15-20cm. And after the solution is sprayed, the rod-shaped mold continues to rotate for 5-10 hours, so that the solvent volatilizes. Taking the nerve conduit biodegradable material composition, removing bubbles, taking 100ml of the composition, injecting the composition into an injector, outputting the composition at the speed of 10ml/h, forming a tube by using an electrostatic spinning process, spraying the composition onto the surface of a rod-shaped die tube body with the rotating speed of 800rpm through direct current voltage, and receiving the voltage of 20KV and the distance of 20cm. The electrostatic spinning nozzle moves back and forth at a speed of 15cm/min in the horizontal direction, and the length of the tube is 15-20cm. And removing the nerve conduit from the mold, drying to completely volatilize the solvent, and cutting the nerve conduit into different length specifications.
Example 10 preparation of nerve conduit
Catheters were prepared using the biodegradable material composition of example 2, the specific steps were as follows: taking 100ml of nerve conduit biodegradable material composition after removing bubbles, injecting the composition into an injector, outputting the composition at the speed of 5ml/h, forming a tube by using an electrostatic spinning process, spraying the composition onto a rod-shaped die with the rotating speed of 1000rpm and the diameter of 5mm by using direct current voltage, and receiving the voltage of 20KV for 20cm. The electrostatic spinning nozzle moves back and forth at a speed of 10cm/min in the horizontal direction, and the length of the tube is 15-20cm. And after the solution is sprayed, the rod-shaped mold continues to rotate, so that the solvent volatilizes. And removing the nerve conduit from the mold, drying to completely volatilize the solvent, and cutting the nerve conduit into different length specifications.
EXAMPLE 11 preparation of nerve conduit
Catheters were prepared using the biodegradable material composition of example 2, the specific steps were as follows: taking polylactic acid-polyglycolic acid with the weight ratio of 99.5% and bioadhesive peptide with the weight ratio of 0.5%, dissolving the polylactic acid-polyglycolic acid with 3-6 times of ethyl acetate, and then adding bioadhesive peptide for fully mixing to obtain a mixture. Taking 10ml of the mixture after bubble removal, injecting the mixture into an injector, outputting the mixture at a flow rate of 5ml/h, forming a tube by using an electrostatic spinning process, spraying the tube onto a rod-shaped die with a rotating speed of 1000rpm and a diameter of 5mm by using direct current voltage, and receiving the voltage of 20KV for 20cm. The electrostatic spinning nozzle moves back and forth at a speed of 10cm/min in the horizontal direction, and the length of the tube is 15-20cm. And after the solution is sprayed, the rod-shaped mold continues to rotate, so that the solvent volatilizes. Taking the nerve conduit biodegradable material composition, removing bubbles, taking 100ml of the composition, injecting the composition into an injector, outputting the composition at a flow rate of 5ml/h, forming a tube by using an electrostatic spinning process, spraying the composition onto the surface of a rod-shaped die tube body with a rotating speed of 1000rpm through direct current voltage, and receiving the voltage of 20KV and the distance of 20cm. The electrostatic spinning nozzle moves back and forth at a speed of 10cm/min in the horizontal direction, and the length of the tube is 15-20cm. And after the solution is sprayed, the rod-shaped mold continues to rotate, so that the solvent volatilizes. And removing the nerve conduit from the mold, drying to completely volatilize the solvent, and cutting the nerve conduit into different length specifications.
Example 12 preparation of nerve conduit
Catheters were prepared using the biodegradable material composition of example 3, the specific steps were as follows: taking a nerve conduit biodegradable material composition, removing bubbles, taking 100ml of the composition, injecting the composition into an injector, outputting the composition at the speed of 15ml/h, forming a tube by using an electrostatic spinning process, spraying the composition onto a rod-shaped die with the rotating speed of 1000rpm and the diameter of 5mm by using direct current voltage, and receiving the voltage of 20KV and the distance of 20cm. The electrostatic spinning nozzle moves back and forth at a speed of 15cm/min in the horizontal direction, and the length of the tube is 15-20cm. And after the solution is sprayed, the rod-shaped mold continues to rotate, so that the solvent volatilizes. And removing the nerve conduit from the mold, drying to completely volatilize the solvent, and cutting the nerve conduit into different length specifications.
Example 13 preparation of nerve conduit
Catheters were prepared using the biodegradable material composition of example 3, the specific steps were as follows: taking 98% of polycaprolactone by weight and 2% of dopamine by weight, dissolving the polycaprolactone by 3-6 times of dichloromethane by weight, and then adding the dopamine and fully mixing to obtain a mixture. Taking 10ml of the mixture after bubble removal, injecting the mixture into an injector, outputting the mixture at the speed of 15ml/h, forming a tube by using an electrostatic spinning process, spraying the tube onto a rod-shaped die with the rotating speed of 1000rpm and the diameter of 5mm by using direct current voltage, and receiving the voltage of 20KV and the distance of 20cm. The electrostatic spinning nozzle moves back and forth at a speed of 15cm/min in the horizontal direction, and the length of the tube is 15-20cm. And after the solution is sprayed, the rod-shaped mold continues to rotate, so that the solvent volatilizes. Taking the nerve conduit biodegradable material composition, removing bubbles, taking 100ml of the composition, injecting the composition into an injector, outputting the composition at the speed of 15ml/h, forming a tube by using an electrostatic spinning process, spraying the composition onto the surface of a rod-shaped die tube body with the rotating speed of 1000rpm through direct current voltage, and receiving the voltage of 20KV and the distance of 20cm. The electrostatic spinning nozzle moves back and forth at a speed of 15cm/min in the horizontal direction, and the length of the tube is 15-20cm. And after the solution is sprayed, the rod-shaped mold continues to rotate, so that the solvent volatilizes. And removing the nerve conduit from the mold, drying to completely volatilize the solvent, and cutting the nerve conduit into different length specifications.
Example 14 animal experiments
The nerve conduit prepared in example 8 was implanted into the sciatic nerve (repair of 15MM injury) of a rat, and a conduit of PCL material of 1% melatonin was selected as shown in fig. 1. As shown in fig. 2-4, the myelin sheath thickness is an important index reflecting the maturation of regenerated nerves, and the results in the figure show that 1% of melatonin and PCL compositions prepared by the nerve conduit have greater myelin sheath thickness than pure PLA conduit groups and autologous nerve graft groups at the 12 th week after operation, which indicates that the nerve conduit of the present invention has a significant effect of promoting nerve regeneration, and the effect is superior to that of autologous nerves and pure PCL conduits.
Example 15 animal experiment
Healthy adult male SD rats were divided into 30 groups of A, B, 15 per group, according to the random number table method. All rats were right-hand side of the experiment and left-hand side of the normal control. Under the anesthesia of ketamine intraperitoneal injection, the rat right thigh is longitudinally notched on the outer side, and enters the exposed sciatic nerve from the muscle gap. Cutting a sciatic nerve trunk 10mm in the middle thigh, and performing in-situ nerve transplantation on the group A by using an autologous nerve line; group B was repaired by bridging the nerve conduit prepared in example 9, two nerve endings were each inserted 1mm into the conduit, leaving 10mm gaps between the nerve defects, suturing the wall of the conduit and the adventitia with 9-0 silk threads, and 3-4 needles at each end. And (5) cage-separated feeding after operation. The sciatic nerve function index (SFI) values of each group of rats were examined at weeks 4, 6, 8, and 10, with SFI values near 0 indicating normal sciatic nerve function and SFI values at-100 indicating complete loss of sciatic nerve function. The calculation is performed according to the SFI formula: SFI= -38.3 (EPL-NPL)/NPL+109.5 (ETS-NTS)/NTS+13.3 (EIT-NIT)/NIT-8.8 (E: experimental trial lateral foot; N: normal lateral foot). The results are shown in the following table. The second week after operation, the two SFI values have no significant difference (P > 0.05), and from week 8, the two SFI values show significant difference, and the SFI value of group B is significantly improved compared with that of group A, which indicates that the nerve conduit can significantly promote the recovery of sciatic nerve function.
Table 1 comparison of two sets of sciatic nerve function index SFI after surgery
| Postoperative time | Group A | Group B |
| For 2 weeks | -87±2.90 | -85±2.90 |
| 4 weeks of | -74±2.65 | -70±2.45 |
| For 6 weeks | -65±2.40 | -59±2.30 |
| 8 weeks of | -51±2.35 | -43±2.25 |
| For 10 weeks | -48±2.25 | -35±2.10 |
The regenerated nerve was cut at the position of 2mm of the sciatic nerve distal end anastomotic orifice at week 12, fixed with 4% glutaraldehyde, fixed with 1% osmium acid, embedded, sectioned, stained, observed under a transmission electron microscope for five unit fields of left upper, left lower, center, right upper and right lower of the cross section, counted for the number of medullary nerve fibers, and measured for axon diameter and myelin thickness. The results are shown in the following table. Axon diameter and myelin thickness are the primary morphological markers of nerve maturation, with more mature medullary nerve fibers in the regenerated nerve being effectively regenerated. From the results in the table, group B is significantly higher than group a in terms of regenerated nerve fiber number, axon diameter and myelin thickness, indicating that the nerve conduit of the present invention can promote nerve regeneration, increase regenerated nerve maturity and the number of functional nerve fibers.
Table 2 comparison of the number of regenerated nerve fibers, axon diameter and myelin thickness in two groups 12 weeks after surgery
| Number of nerve fibers | Axon diameter (μm) | Myelin thickness (mum) | |
| Group A | 225.1±13.5 | 3.61±0.64 | 1.21±0.08 |
| Group B | 242.7±15.2* | 4.28±0.76* | 1.36±0.06* |
Note that: * Represents P < 0.05 compared to group A.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and additions may be made to those skilled in the art without departing from the method of the present invention, which modifications and additions are also to be considered as within the scope of the present invention.
Claims (1)
1. A preparation method of a melatonin nerve conduit, which is characterized by comprising the following steps: taking polylactic acid with the weight ratio of 99% and dopamine with the weight ratio of 1%, dissolving the polylactic acid with 3-6 times of dichloromethane, and then adding dopamine for fully and uniformly mixing to obtain a mixture;
taking 10mL of the mixture after bubble removal, injecting the mixture into an injector, outputting the mixture at the speed of 10mL/h, forming a tube by using an electrostatic spinning process, spraying the tube onto a rod-shaped die with the rotating speed of 800rpm and the diameter of 3mm by using direct current voltage, and receiving the voltage of 20KV and the distance of 20cm; the electrostatic spinning nozzle moves back and forth at a speed of 15cm/min in the horizontal direction, and the length of the tube is 15-20cm;
continuously rotating the rod-shaped mold for 5-10h after the solution is sprayed, volatilizing dichloromethane to obtain the polylactic acid tube containing dopamine;
taking a nerve conduit biodegradable material composition, removing bubbles, taking 100mL of the composition, injecting the composition into an injector, outputting the composition at the speed of 10mL/h, forming a tube by using an electrostatic spinning process, and spraying the composition onto the surface of a rod-shaped die tube body with the rotating speed of 800rpm and the surface coated with the dopamine-containing polylactic acid tube by using direct-current voltage, wherein the voltage is 20KV, and the receiving distance is 20cm; the electrostatic spinning nozzle moves back and forth at a speed of 15cm/min in the horizontal direction, and the length of the tube is 15-20cm;
removing the nerve conduit from the mold, drying to completely volatilize dichloromethane, and cutting the nerve conduit into different length specifications;
the preparation method of the nerve conduit biodegradable material composition comprises the following steps: taking polylactic acid with the weight ratio of 99% and melatonin with the weight ratio of 1%, dissolving the polylactic acid with 3-6 times of dichloromethane, and then adding the melatonin for fully and uniformly mixing to prepare the nerve conduit biodegradable material composition.
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