CN111317867A

CN111317867A - Nerve conduit and preparation method thereof

Info

Publication number: CN111317867A
Application number: CN202010081724.9A
Authority: CN
Inventors: 王秀梅; 王硕; 段古满
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2020-02-06
Filing date: 2020-02-06
Publication date: 2020-06-23

Abstract

The invention discloses a nerve conduit and a preparation method thereof, wherein the main components of the nerve conduit are collagen which can be desorbed and absorbed in an organism and minerals rich in calcium ions, and the nerve conduit has good biocompatibility and calcium ion release performance. It has a multilayer structure with each layer having a trench-like microstructure. The multilayer catheter is formed by collagen gel film and is curled into a circular tube shape, the manufacturing is simple and convenient, the catheter contains calcium ion mineral materials, inorganic ion elements are released in a micro-scale mode, and the promoting effect is provided for the regeneration of nerves, blood vessels and the like. Clinically, after peripheral nerve injury, the nerve conduit provided by the invention can be used for bypass surgery, and gradually descends and absorbs in organisms while nerve regeneration. The invention has good toughness and strength due to the adoption of a multilayer structure, and has certain porosity by adjusting the moisture content in the forming process, thereby being beneficial to the transportation of nutrient substances and metabolites during nerve repair.

Description

Nerve conduit and preparation method thereof

Technical Field

The invention relates to a biomedical material technology, in particular to a nerve conduit and a preparation method thereof, and especially relates to a collagen nerve conduit with a micro-nano structure and calcium ion slow release and a preparation method thereof.

Background

Peripheral nerve defects are one of the most common clinical wounds, and the number of the incidence cases of the peripheral nerve defects is reported to be 1.5 to 4.0 percent of that of trauma patients every year, and the number of the incidence cases is up to 60 to 90 ten thousand cases per year domestically. Clinically, compared with injuries of skin, soft tissues, joints, bones and the like, the treatment of nerve injuries is more difficult, the recovery period is long, effective measures are lacked, and sometimes the prognosis is not ideal. Nerve injury can cause complete loss of sensory and/or motor functions of the distal limbs innervated by affected nerves, and if the broken ends are injured and are not repaired by effective means, the peripheral connective tissues can block nerve growth and repair to form neuroma-like tissues over time, finally irreversible dysfunction is caused, serious limb disability is caused, and great damage is brought to the work and life of a patient.

The short-distance defect can be directly anastomosed without tension at the nerve broken end. The repair of long-distance defects is clinically difficult. The most common method currently used is to repair nerve endings using autologous nerve graft bridging, which has many drawbacks even though it is considered the "gold standard" for clinical work: such as limited nerve sources, impaired function of donor nerves, scar formation and infection risk in donor area surgery, etc., and studies have shown that only 40-50% of patients can achieve complete recovery of motor function after receiving autologous nerve transplantation, so there is a need to find a method for replacing autologous nerve transplantation.

With the rapid development of biomedical materials and tissue engineering, people turn the research into using nerve conduits to bridge the two broken ends of the defective nerve and promote the repair of peripheral nerves. For example, the chemical materials polyethylene, polyglycolic acid, polylactic acid, etc.; biological materials such as chitosan, gelatin, etc.; the biological tubular stents, arteries, veins and myotubes, have been used in attempts to prepare catheters. However, due to a series of problems of poor biocompatibility of chemical materials, poor mechanical properties of biological materials and biological tubes and the like, the repair effect is poor, and the clinical application is limited.

At present, the focus of research is on a nerve conduit with a relatively simple structure, and the effect of repairing the long nerve defect is definite. And the majority of nerve conduit products approved for clinical use by CFDA are of this type. However, most of these nerve conduits are difficult to combine with good mechanical strength (operation and soft tissue barrier), good biocompatibility (cell and nutrient metabolism transport), controllable in vivo degradation (poor in vivo degradation), or fail to provide biological promotion effect required for repair (single function, only physically bridging nerve broken ends), and cannot better promote nerve regeneration.

The appearance of a peripheral nerve repair material which is simple in preparation process, appropriate in mechanical strength, good in biocompatibility, convenient to degrade in vivo, convenient, safe and effective is expected for a long time in clinic.

Disclosure of Invention

The nerve conduit is formed by collagen gel film and is curled into a round tube shape, the nerve conduit is simple and convenient to manufacture, the conduit contains mineral materials, inorganic ion elements are released in a micro-scale manner, a promoting effect is provided for the regeneration of nerves, blood vessels and the like, the growth of nerve axons is promoted, calcium ions are simultaneously released along with the degradation of the materials, the concentration of local calcium ions is prevented from being increased steeply, the important effect of Schwann cells in nerve regeneration is not influenced, and nerve tissues in the conduit can grow better. Meanwhile, the mineralized collagen catheter is attached to avoid the condition that the pure collagen catheter is degraded too fast, so that the degradation speed of the catheter is easy to regulate and control.

The invention aims to provide a collagen nerve conduit with a micro-nano structure for slowly releasing calcium ions and a preparation method thereof.

The main components of the artificial nerve conduit provided by the invention are collagen which can be desorbed and absorbed in an organism and minerals rich in calcium ions, and the artificial nerve conduit has good biocompatibility and calcium ion release performance. It has a multilayer structure with each layer having a trench-like microstructure.

Recent studies have shown that calcium ions play an important role in nerve repair, and that calcium ions not only accelerate the growth of injured nerve axons, but also participate in other metabolic activities in vivo (Adalbert R, Morleale G, Paizs M, Conforti L, Walker SA, Roderick HL, Bootman MD, Sikl Loideck SiklD, Sikloderick HL, Bootman MD, SiklMD, Sikl Saxotomy in window-type and slow Wallerian degenerationaxons. Neuroscience.2012; 225: 44-54.). However, researchers found that the survival of Schwann cells is affected by the steep increase In Calcium ion concentration In the In Vitro culture medium, and that Schwann cells play an important role In Nerve regeneration and recovery (YangKJ, Yan Y, Zhang LL, Agresti MA, Matlouub HS, LoGiudince JA, Havlik R, YanJG. incorporated Calcium Limits Schwann Cell Numbers In Vitro following sexual desire neural input. journal of biocompatible Micromagery.2017; 33: 435-40.).

The invention provides a nerve conduit, which is a membrane prepared by mixing and solidifying type I collagen hydrogel and a mineral material and then curling the membrane to form the conduit;

optionally, the surface of the prepared membrane is provided with a groove structure with a micrometer scale or nanometer scale, and the surface of the membrane containing the groove structure is used for the inner wall of the nerve conduit;

preferably, the trench structure has a dimension of 25000 μm long, 5-25 μm wide, 5-25 μm deep, and 5-25 μm spacing, or a dimension of 10-20 μm long, 100-200nm wide, 100-200nm deep, and 100-200nm spacing.

In the nerve conduit provided by the invention, the tube wall of the coiled conduit is fixed between different membrane layers in a physical or chemical way;

preferably, the chemical method is cross-linking the collagen using a cross-linking agent.

In the nerve conduit provided by the invention, the inner diameter of the conduit of the nerve conduit is 1-5 mm; the length of the conduit is 10-30 mm; in the curled catheter, the number of layers of the membrane in the catheter wall is 2-6 according to the required strength and degradation efficiency.

In the nerve conduit provided by the invention, the mass ratio of the mineral material to the type I collagen hydrogel is 1 (1-10).

In the nerve conduit provided by the invention, the mineral material comprises one or more of β tricalcium phosphate, nano hydroxyapatite and biomimetic mineralized collagen, and optionally, the mineral material type can also be hydroxyapatite containing other beneficial elements (magnesium, silicon, selenium, zinc and the like).

In the nerve conduit provided by the present invention, preferably, the particle size of the mineral material is 50 to 100 nm.

In the nerve conduit provided by the invention, the type I collagen hydrogel and the mineral material are mixed with water and then are prepared into a film through a mould;

in the nerve conduit provided by the present invention, optionally, the amount of water added is 0% to 50% of the total volume of the collagen type I hydrogel, the mineral material and the water.

In the nerve conduit provided by the invention, the preparation of the film through the mould comprises the following steps,

pouring a solution formed by mixing the type I collagen hydrogel and the mineral material with water into a mold, vacuumizing under negative pressure to remove internal bubbles, and drying to form a film-shaped material;

optionally, the pressure of the negative pressure vacuum pumping is-0 to-0.1 MPa.

In the nerve conduit provided by the invention, the cross-linking agent is selected from one or more of glutaraldehyde, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), epoxy cross-linking agent and genipin;

the epoxy crosslinking agent is selected from one or more of ethylene oxide and epichlorohydrin.

In another aspect, the present invention provides a method for preparing the above nerve conduit, comprising the following steps:

step S1-1: mixing the type I collagen hydrogel with a mineral material to obtain a mineral material/type I collagen hydrogel mixture;

step S1-2: mixing the mineral material/type I collagen hydrogel mixture obtained in the step S1-1 with water to obtain a mineral collagen solution; by adjusting the overall water content, the porosity and degradation rate of the nerve conduit can be adjusted.

Step S2-1: pouring the mineral collagen solution obtained in the step S1-2 into a mould, removing internal bubbles through negative pressure vacuum pumping, and drying to form a film-shaped mineral collagen film;

step S2-2: uniformly coating the mineral collagen film obtained in the step S2-1 with a diluent of the mineral collagen solution obtained in the step S1-2, softening, curling into a tube shape, and drying until the shape is completely fixed; the dried collagen film is rolled into a tube requiring a dilute mineral collagen solution as a binder.

Step S2-3: taking the completely-shaped tubular product obtained in the step S2-2, and crosslinking the tubular product by using a crosslinking agent to obtain the nerve conduit;

optionally, step S2-3 further includes eluting and lyophilizing the product after cross-linking with the cross-linking agent, and then sterilizing the product by irradiation to obtain the collagen multi-layer nerve conduit.

In the preparation method of the nerve conduit provided by the invention, one side of the mould, which is in contact with the mineral collagen solution, is provided with a groove structure with a micron or nanometer scale;

in the preparation method of the nerve conduit provided by the invention, optionally, the material of the mold is monocrystalline silicon, the groove structure is etched on the surface of the monocrystalline silicon by dry etching, or the nano-scale groove structure is added on the mineralized collagen film by adopting a mode of dimethyl siloxane (PDMS) conversion.

In the preparation method of the nerve conduit provided by the invention, in the step S2-1, the thickness of the mineral collagen solution is 3-10mm before the mineral collagen solution is poured into a mould for air drying;

in the preparation method of the nerve conduit provided by the invention, optionally, the content of the mineral collagen solution is 0.5-1g/cm²；

In the preparation method of the nerve conduit provided by the invention, optionally, the drying is air drying, and the air drying temperature is not lower than room temperature and not higher than 30 ℃;

optionally, according to different air drying time, the drying degree can be controlled (the final water content is different), the thickness of the film after air drying is 0.1-1mm, a layered drying process can be adopted, and mineral collagen solutions containing different mineral types (β tricalcium phosphate, nano hydroxyapatite and mineralized collagen) are gradually added to prepare a single film, so that the single film has a higher multi-component structure.

In the preparation method of the nerve conduit provided by the invention, the thickness of the mineral collagen film formed after drying is 0.1-1mm optionally.

In the preparation method of the nerve conduit, in the step S2-2, the mineral collagen solution diluent is diluted by deionized water according to a volume ratio, and the dilution concentration is 1 (1-10).

In the preparation method of the nerve conduit, in the step S2-3, the elution is firstly washed by the ethanol water solution for 3-10 times and then washed by the purified water for 3-10 times, wherein the ethanol concentration of the ethanol water solution is 30-100% by volume.

In the preparation method of the nerve conduit provided by the present invention, the lyophilization process in step S2-3 specifically includes: pre-freezing the product obtained after elution at-30 to-20 ℃, carrying out sublimation pore-forming at-10 to 0 ℃ in vacuum, wherein the pore diameter is 0 to 10 mu m, and finally carrying out vacuum drying at 0 to 50 ℃;

preferably, the pore size ranges from 1 μm to 10 μm.

In the preparation method of the nerve conduit provided by the invention, the crosslinking time of crosslinking in the step S2-3 is 20 min-4 h;

optionally, the reagent used for irradiation is a cobalt-60 sterilizing agent, and the dosage is 15-38 kGy.

The invention has the following beneficial effects:

the multilayer artificial nerve conduit provided by the invention can be implanted into a nerve injury part through a surgery. When the nerve external model suture is used for repairing peripheral nerve injury, a catheter with a proper specification layer thickness can be selected according to the thickness and the defect degree of the injured nerve, the two cut ends of the severed nerve are inserted into the catheter for about 2-5 mm, and the nerve external model suture is fixed by using surgical sutures.

Clinically, the peripheral nerves can be subjected to bridging operation by using the multilayer artificial nerve conduit of the invention, and after nerve regeneration is completed, the peripheral nerves are gradually degraded and absorbed in vivo. Compared with other nerve conduits, the artificial nerve conduit provided by the invention adopts a multilayer structure, has good toughness, can resist the shearing force of suture and can also prevent peripheral soft tissue from growing in to influence the repair effect. The catheter can slowly release calcium ions in a micro-scale manner, the growth of axons is accelerated, and the regeneration speed of nerves after a bridging operation is high and the quality is good. The mineralized collagen catheter is attached to avoid the condition that the pure collagen catheter is degraded too fast, so that the degradation speed of the catheter is easy to regulate and control. In addition, the implant is not required to be taken out after the operation of the invention through a secondary operation, the degradation time is adapted to the growth speed of nerve fibers, and the invention can be applied to the repair of clinical peripheral nerve injury.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Other advantages of the invention may be realized and attained by the instrumentalities and combinations particularly pointed out in the specification.

Drawings

The accompanying drawings are included to provide an understanding of the present invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the examples serve to explain the principles of the invention and not to limit the invention.

FIG. 1 is a schematic diagram of a biomimetic mineralized collagen-collagen multi-layered nerve conduit structure;

FIG. 2 is a schematic representation of a biomimetic mineralized collagen-collagen multilayer nerve conduit prepared in example 1;

FIG. 3 is a scanning electron microscope photograph of the side of the catheter wall prepared in example 1;

FIG. 4 shows an intraoperative catheter implanted in rat sciatic nerve;

FIG. 5 shows postoperative bridging nerve immunofluorescence (neurofilament protein NF200), catheter implantation promoting rat sciatic nerve axonal growth;

FIG. 6 is a transmission electron microscope observation of ultrathin sections of neutral points of rats repaired 12 weeks after operation, wherein the sciatic nerves of the rats are repaired by three groups of different modes. A autologous nerve group; mineralized collagen-collagen multi-layer nerve conduit group; c pure collagen ductal group. (upper: 2550X, lower: 26500X).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention are described in detail below. It should be noted that the embodiments and features of the embodiments of the present invention may be arbitrarily combined with each other without conflict.

The embodiment of the invention provides a nerve conduit, which is a membrane prepared by mixing and solidifying type I collagen hydrogel and a mineral material and a conduit formed by curling;

In the embodiment of the invention, the tube wall of the curled catheter is fixed between different film layers by a physical or chemical method;

In the embodiment of the invention, the inner diameter of the nerve conduit is 1-5 mm; the length of the conduit is 10-30 mm; in the curled catheter, the number of layers of the membrane in the catheter wall is 2-6 according to the required strength and degradation efficiency.

In the embodiment of the invention, the mass ratio of the mineral material to the collagen type I hydrogel is 1 (1-10).

In the embodiment of the invention, the mineral material comprises one or more of β tricalcium phosphate, nano hydroxyapatite and biomimetic mineralized collagen, and optionally, the mineral material category can also be hydroxyapatite containing other beneficial elements (magnesium, silicon, selenium, zinc and the like).

In embodiments of the present invention, preferably, the mineral material has a particle size of 50-100 nm.

In the embodiment of the invention, the type I collagen hydrogel and the mineral material are mixed with water and then are prepared into a film through a mold;

in embodiments of the invention, optionally, the amount of water added is 0% to 50% of the total volume of the collagen I hydrogel, the mineral material and the water.

In the embodiment of the invention, the preparation of the film through the mold comprises the following steps,

In an embodiment of the invention, the cross-linking agent is selected from one or more of glutaraldehyde, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), an epoxy cross-linking agent, and genipin;

In another aspect, an embodiment of the present invention provides a method for preparing the above-mentioned nerve conduit, including the following steps:

In the embodiment of the invention, one side of the mold, which is in contact with the mineral collagen solution, is provided with a groove structure with a micron or nanometer scale;

in the embodiment of the present invention, optionally, the mold is made of monocrystalline silicon, the groove structure is etched on the surface of the monocrystalline silicon by dry etching, or the nano-scale groove structure is attached to the mineralized collagen film by a mode of mold conversion of dimethyl siloxane (PDMS).

In the embodiment of the present invention, the mineral collagen solution is poured into the mold in step S2-1 to be dried to a thickness of 3-10 mm;

in embodiments of the invention, optionally, the mineral collagen solution is present in an amount of 0.5 to 1g/cm²；

In the embodiment of the present invention, optionally, the drying is air drying, and the air drying temperature is not lower than room temperature and not higher than 30 ℃;

In embodiments of the invention, the mineral collagen film formed after drying is optionally 0.1-1mm thick.

In the embodiment of the invention, in the step S2-2, the mineral collagen solution diluent is diluted by deionized water according to a volume ratio, and the dilution concentration is 1 (1-10).

In the embodiment of the invention, in the step S2-3, the elution is firstly washed with an ethanol aqueous solution for 3-10 times and then washed with purified water for 3-10 times, wherein the ethanol volume concentration of the ethanol aqueous solution is 30% -100%.

In the embodiment of the present invention, in step S2-3, the lyophilization process specifically includes: pre-freezing the product obtained after elution at-30 to-20 ℃, carrying out sublimation pore-forming at-10 to 0 ℃ in vacuum, wherein the pore diameter is 0 to 10 mu m, and finally carrying out vacuum drying at 0 to 50 ℃;

preferably, the pore size ranges from 1 μm to 10 μm.

In the embodiment of the invention, the crosslinking time of the crosslinking in the step S2-3 is 20 min-4 h;

In the embodiment of the present invention, the type I collagen is bovine-derived collagen, and is obtained by conventional extraction of bovine achilles tendon, and the specific extraction method can be seen in the following steps: (refer to the preparation and property research of medical composite hydrogel based on Hewei-collagen of bear Yueqin [ J ] modern food technology, 2009,25(12):1454-

1) Cleaning fresh bovine Achilles tendon, scraping off subcutaneous fat, cutting into 1cm × 1cm pieces, soaking in 10% NaCl solution overnight to remove salt soluble protein and other soluble impurities, washing with distilled water for 3 times, and defatting for 1 st time (m bovine Achilles tendon: V defatting solution ═ 1:10, defatting solution is composed of 6% Na₂CO₃Solution and 1% degreasing agent), soaking for 60min at 30 ℃, washing with distilled water for 2-3 times, and then performing the 2 nd degreasing (m bovine achilles tendon: V degreasing solution is 1:10, and the degreasing solution is 3% Na₂CO₃Solution and 1% degreasing agent), stirring at 30 deg.C for 60min, washing with warm water at 30 deg.C for 3 times, and air drying in a fume hood for use.

2) Adding a certain amount of pretreated bovine achilles tendon raw material into an acid solution (pH 2.5-3) containing pepsin for treatment for a period of time, and filtering by using two layers of medical gauze.

3) Adding NaCl solid directly into the filtrate for salting out (NaCl is finally addedThe concentration is less than 3.0mol/L), and the collagen precipitate is obtained by centrifugal separation. The collagen precipitate obtained after salting out was dissolved in the acid solution at 0.02mol/L Na₂HPO₄Dialyzing at pH8.6 for 2d, and dialyzing with distilled water as external dialysate for 1 d. Obtaining the bovine-derived type I collagen hydrogel.

In the present example, the nano-hydroxyapatite was purchased from alatin, cat # H106378;

in the present examples, the biomimetic mineralized collagen (mineralized collagen bone powder) was purchased from Olympic medicine science and technology, Inc.

Example 1 preparation of multilayer Artificial nerve conduit

In this embodiment, the multilayer artificial nerve conduit is prepared by using a mineral collagen solution, and the preparation method of the mineral collagen solution comprises the following steps:

1. mixing the type I collagen hydrogel with a mineral material in a mass ratio of 5: 1; the mineral material is biomimetic mineralized collagen (the granularity is 70 nm);

2. mixing the bionic mineralized collagen and the type I collagen hydrogel in the last step with water, wherein the volume content of the added water is 10% of the total volume of the bionic mineralized collagen, the type I collagen hydrogel and the water, and then obtaining the mineral collagen solution.

The preparation method of the multilayer nerve conduit comprises the following steps:

1. 100g of mineral collagen solution is flatly laid in a mould, the contact surface of the mould and the mineral collagen solution adopts monocrystalline silicon, and parallel grooves with the length equal to that of the mould, the width of 10 microns, the depth of 10 microns and the distance of 5 microns are formed by the monocrystalline silicon through dry etching.

The dimensions of the die were length, width, height, 20, 10, 2cm, the thickness of the solution was 5mm,

the content of the mineral collagen solution is 0.5g/cm²Discharging air bubbles at negative pressure of-0.1 Mp, naturally air drying at room temperature of 25 deg.C until completely dried, and cutting to 20mm x 50 mm; the thickness is 0.5mm, and a film is prepared;

2. uniformly coating a collagen diluent (the collagen diluent is mixed according to the ratio of mineral collagen solution to water (v: v) ═ 1: 5) for softening, curling the softened membrane into a tubular shape (the length is 20mm, the inner diameter is 5mm), wherein in the curled catheter, the number of layers of the membrane in the catheter wall is 4, fixing the tubular shape by a cylindrical mold, and naturally drying at the normal temperature of 25 ℃ until the tubular shape is completely dried and formed to obtain a tubular product;

3. immersing the obtained tubular product into 200mL of EDC aqueous solution, wherein the concentration of the EDC aqueous solution is 10g/mL, crosslinking for 30min, eluting the crosslinked tubular product in 50% (v: v) ethanol aqueous solution, washing with the ethanol aqueous solution for 3 times, pre-freezing at-20 ℃ after washing with purified water for 3 times, carrying out freeze-drying at-5 ℃ under vacuum for sublimation for pore-forming for 48 hours, wherein the pore diameter is 5 mu m, and finally carrying out vacuum drying at 30 ℃. And finally, performing irradiation sterilization, and using a cobalt-60 sterilizing agent with the dose of 15kGy to obtain the multilayer artificial nerve conduit, wherein the inner wall of the nerve conduit is provided with parallel grooves which are as long as the mold, 10 micrometers in width, 10 micrometers in depth and 5 micrometers in distance. The length of the nerve conduit is 20mm, the inner diameter is 5mm, the number of layers of membranes in the conduit wall is 4, and the wall thickness is 2 mm. As shown in fig. 1. The dimensional changes before and after nerve conduit desiccation were negligible.

Example 2

the preparation method of the mineral collagen solution comprises the following steps:

1. mixing the type I collagen hydrogel with a mineral material in a mass ratio of 6: 1; the mineral material is nano hydroxyapatite (the granularity is 85 nm);

2. mixing the nano hydroxyapatite and the type I collagen hydrogel obtained in the last step with water, wherein the volume content of the added water is 20%, and thus obtaining the mineral collagen solution.

1. 100g of mineral collagen solution is flatly laid in a mould, the contact surface of the mould and the mineral collagen solution adopts monocrystalline silicon, and parallel grooves with the length equal to that of the mould, the width of 15 micrometers, the depth of 15 micrometers and the distance of 5 micrometers are formed by the monocrystalline silicon through dry etching.

the content of the mineral collagen solution is 0.5g/cm²Discharging air bubbles at negative pressure of-0.1 Mp, naturally air drying at room temperature of 25 deg.C until completely dried, and cutting to 20mm x 50 mm; the thickness is 0.3mm, and a film is prepared;

2. uniformly coating a collagen diluent (the collagen diluent is mixed according to the ratio of mineral collagen solution to water (v: v) ═ 1: 4) for softening, curling the softened membrane into a tubular shape (the length is 20mm, the inner diameter is 5mm), wherein in the curled catheter, the number of layers of the membrane in the catheter wall is 5, fixing the tubular shape by a cylindrical mold, and naturally drying at the normal temperature of 25 ℃ until the tubular shape is completely dried and formed to obtain a tubular product;

3. immersing the obtained tubular product into 200mL of glutaraldehyde aqueous solution, wherein the volume concentration of the glutaraldehyde aqueous solution is 0.5%, crosslinking for 2h, eluting the crosslinked tubular product in 80% (v: v) ethanol aqueous solution, washing the tubular product for 3 times by using the ethanol aqueous solution, pre-freezing the tubular product at-25 ℃ after washing the tubular product for 3 times by using purified water, carrying out freeze-drying sublimation at-5 ℃ in vacuum for pore-forming for 72 hours, wherein the pore diameter is 5 mu m, and finally carrying out vacuum drying at 30 ℃. And finally, performing irradiation sterilization, and using a cobalt-60 sterilizing agent with the dose of 15kGy to obtain the multilayer artificial nerve conduit, wherein the inner wall of the nerve conduit is provided with parallel grooves which are equal to the mold in length, 15 micrometers in width, 15 micrometers in depth and 5 micrometers in distance. The length of the nerve conduit is 20mm, the inner diameter is 5mm, the number of layers of membranes in the conduit wall is 5, and the wall thickness is 1.5 mm. The dimensional changes before and after nerve conduit desiccation were negligible.

Comparative example 1

The preparation method of the pure collagen multi-layer catheter with the inner diameter of 5mm, the wall thickness of 2mm, the length of 20mm, the number of the membrane layers in the catheter wall of 4 layers and no biomimetic mineralized collagen is as in example 1, except for the difference that no biomimetic mineralized collagen is contained, the other preparation raw materials and the using amount thereof are the same as those in example 1.

Comparative example 2

The procedure of example 2 was followed to prepare a pure collagen multi-layered catheter having an inner diameter of 5mm, a wall thickness of 1.5mm, a length of 20mm, 5 layers of membranes in the wall, and no nano-hydroxyapatite, and the preparation process was the same as that of example 2 except for the difference that no nano-hydroxyapatite was contained in the catheter.

Application example 1

After 5 minutes of saline immersion, the tensile strength of the two samples was measured by a WDW electronic universal tester as shown in table 1:

table 1: mechanical property test statistical table

As can be seen from table 1 and fig. 4, the collagen mineral catheters prepared in examples 1 and 2 can satisfy the requirements of the suture stage on the mechanical properties of the catheters.

Application example 2, multilayer artificial catheter for rat sciatic nerve repair

A multi-layered artificial nerve conduit, in which the length was 20mm and the inner diameter was 5mm, was prepared according to the method of example 1.

In the adult female Spragne-Dawley rat sciatic nerve injury model: the rat is weighed and then is subjected to intraperitoneal injection of pentobarbital sodium for anesthesia, the rat prone position is fixed on an operation table, depilation and skin preparation are carried out in an operation area, alcohol disinfection is carried out, an oblique straight incision with the length of about 2cm is made along the right iliac crest, sciatic nerves are exposed, sharp separation is gently carried out between a nerve coat and peripheral tissues by using fiber scissors so as to fully dissociate the sciatic nerves, the nerves with the length of about 2cm are dissociated from top to bottom, the nerves are cut off along the upper part of the nerve bifurcation, the nerves retract elastically, and the nerve defect of 10mm is excessively caused by short of a fixed. The above catheter was transplanted to the affected part, the nerve endings on both sides were inserted into the above catheter, the part of the catheter overlapping the nerve was sutured with 10-0 nylon thread, and the dynamic observation was carried out for 1 year, and morphological, electrophysiological and behavioral evaluations were carried out.

The results of the study showed that the animals began to recover from behavioral disorders at month 3 after the transplant surgery, had no apparent dyskinesia at month 6, and had returned to normal without weight bearing at month 12. Adopting WGA-HRP to carry out nerve tracing and Neurofilm (NF) immunohistochemical staining to prove that the reconstruction of a morphological structure is realized; gold chloride staining demonstrated recovery of the motor end plates. Restoration of Somatosensory Evoked Potentials (SEPs) and Motor Evoked Potentials (MEPs) has also been demonstrated in electrophysiological studies.

Application example 3

The mineralized collagen-collagen multi-layer nerve conduit prepared in the embodiment 1 of the invention is used for evaluating the effect of repairing 10mm sciatic nerve defects of adult Spragne-Dawley rats, and compared with autologous nerve transplantation repair and collagen catheter repair (comparative example 1), the experimental results are dynamically observed for 12 weeks, and the results of histological, electrophysiological and behavioral evaluation are as follows:

1. histology: the mineralized collagen-collagen multi-layer ductus neurones group had a density of myelinated fibers and a thickness of myelin sheath less than that of autologous nerves, but was significantly better than the pure collagen ductus group (P <0.05), which was not very different in diameter of myelinated fibers (P >0.05) (see table 2, fig. 6). The wet weight recovery rate of gastrocnemius of mineralized collagen-collagen multi-layer nerve conduit group is not statistically different from that of the autologous nerve group (p >0.05), and is significantly better than that of the pure collagen conduit group (p <0.05) (table 3).

2. In electrophysiology, the mineralized collagen-collagen multi-layer nerve conduit group has no statistical difference in nerve conduction rate with the autologous nerve group (p >0.05), and is significantly better than the pure collagen conduit group (p < 0.05); the recovery of the repair nerve amplitude was similar to that of the pure collagen tube group (p >0.05) (Table 4).

4. Behavioral assessment showed that mineralized collagen-collagen multi-layered nerve conduit group had a significantly better sciatic nerve function index (SFI) recovery than the autologous nerve group (P <0.05), but was significantly better than the pure collagen conduit group (P <0.05) (table 5).

Table 2, evaluation of regenerative neurohistology: transmission electron microscope observation of regenerated nerve midpoint cross section

Table 3, evaluation of regenerative muscle histology: wet weight recovery rate of gastrocnemius (%)

	Wet weight recovery rate of gastrocnemius
		Mineralized collagen-collagen multilayer nerve conduit group	39.80
Pure collagen catheter group	36.46
		Autologous nerve group	48.29

Table 4, electrophysiological evaluation: complex Muscle Action Potential (CMAP)

Table 5, assessment of behavioural: sciatic nerve function index (SFI)

	SFI
		Mineralized collagen-collagen multilayer nerve conduit group	-73.75
Pure collagen catheter group	-82.79
		Self-body nerveGroup of	-61.92

Research results show that in 12 weeks after operation, the mineralized collagen-collagen multilayer nerve conduit is superior to a pure collagen conduit group in aspects of histology, electrophysiology, ethology and the like, even is close to an autologous nerve group in individual aspects, and the mineralized collagen-collagen multilayer nerve conduit has an excellent nerve repairing effect compared with the pure collagen conduit.

Although the embodiments of the present invention have been described above, the above description is only for the convenience of understanding the present invention, and is not intended to limit the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A nerve conduit is a conduit formed by mixing and solidifying type I collagen hydrogel and a mineral material to prepare a membrane and then curling the membrane;

2. The nerve conduit of claim 1, wherein the mass ratio of the mineral material to the collagen type I hydrogel is 1 (1-10).

3. A nerve conduit according to claim 1, wherein the mineral material comprises one or more of β tricalcium phosphate, nano-hydroxyapatite and biomimetic mineralized collagen;

preferably, the mineral material has a particle size of 50-100 nm.

4. A nerve conduit according to any one of claims 1 to 3, wherein the wall of the coil-formed conduit is physically or chemically reinforced to enhance fixation between different membrane layers;

5. The nerve conduit of claim 4, wherein the cross-linking agent is selected from a first one or more of glutaraldehyde, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), an epoxy cross-linking agent, and genipin;

6. A nerve conduit according to any one of claims 1 to 3, wherein the inner catheter diameter of the nerve conduit is 1-5 mm; the length of the conduit is 10-30 mm; in the curled catheter, the number of the membrane layers in the catheter wall is 2-6.

7. The nerve conduit of any one of claims 1 to 3, wherein the collagen type I hydrogel and mineral material are mixed with water and then formed into a film by a mold;

optionally, water is added in an amount of 0% to 50% of the total volume of the collagen type I hydrogel, the mineral material and the water.

8. The nerve conduit of claim 7, wherein said preparing a film by a mold comprises the steps of,

9. The method for preparing a nerve conduit according to any one of claims 4 to 8, comprising the following operating steps:

step S1-2: mixing the mineral material/type I collagen hydrogel mixture obtained in the step S1-1 with water to obtain a mineral collagen solution;

step S2-2: uniformly coating the mineral collagen film obtained in the step S2-1 with a diluent of the mineral collagen solution obtained in the step S1-2, softening, curling into a tube shape, and drying until the shape is completely fixed;

10. The method for preparing a nerve conduit according to claim 9, wherein the side of the mold in contact with the mineral collagen solution has a groove structure of a micro or nano scale;

optionally, the mold is made of monocrystalline silicon, the groove structure is etched on the surface of the monocrystalline silicon by dry etching, or the nano-scale groove structure is attached to the mineralized collagen film by a mode of dimethyl siloxane (PDMS) conversion.

11. The method for manufacturing a nerve conduit according to claim 9, wherein the mineral collagen solution has a thickness of 3 to 10mm before being poured into a mold for air-drying in the step S2-1;

optionally, the mineral collagen solution is present in an amount of 0.5-1g/cm²；

Optionally, the drying is air drying, and the air drying temperature is not lower than room temperature and not higher than 30 ℃;

optionally, the mineral collagen film formed after drying has a thickness of 0.1-1 mm.

12. The method for preparing a nerve conduit according to claim 9, wherein in the step S2-2, the mineral collagen solution diluent is diluted by deionized water according to a volume ratio, and the dilution concentration is 1 (1-10).

13. The method for preparing a nerve conduit according to claim 9, wherein in the step S2-3, the elution is washed with an ethanol aqueous solution for 3-10 times and then with purified water for 3-10 times, wherein the ethanol concentration of the ethanol aqueous solution is 30% -100% by volume.

14. The method for manufacturing a nerve conduit according to claim 9, wherein the lyophilization process in step S2-3 is specifically: pre-freezing the product obtained after elution at-30 to-20 ℃, carrying out sublimation pore-forming at-10 to 0 ℃ in vacuum, wherein the pore diameter is 0 to 10 mu m, and finally carrying out vacuum drying at 0 to 50 ℃;

preferably, the pore size ranges from 1 μm to 10 μm.

15. The method for manufacturing a nerve conduit according to claim 9, wherein the crosslinking time of the crosslinking at step S2-3 is 20min to 4 h;