CN111317867A - Nerve conduit and preparation method thereof - Google Patents

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

A kind of nerve conduit and preparation method thereof

technical field

This article relates to biomedical material technology, in particular to a nerve conduit and a preparation method thereof, in particular to a collagen nerve conduit with a micro-nano structure that slowly releases calcium ions and a preparation method thereof.

Background technique

Peripheral nerve defect is one of the most common clinical traumas. It is reported that the number of cases that occur every year accounts for 1.5% to 4.0% of trauma patients, and the number of cases in China is as high as 600,000 to 900,000 cases per year. Clinically, compared with skin, soft tissue, joint, bone and other injuries, the treatment of nerve injury is more difficult, the recovery period is long, there is a lack of effective measures, and the prognosis is sometimes unsatisfactory. Nerve damage can cause complete loss of sensory and/or motor function in the distal limbs innervated by the affected nerve. If the broken end is damaged and not repaired by effective means, the surrounding connective tissue will hinder nerve growth and repair over time. The formation of neuroma-like tissue will eventually cause irreversible dysfunction, resulting in severe physical disability, and bring huge damage to the patient's work and life.

For short-distance defects, direct tension-free anastomosis of the nerve stump can be performed. The repair of long-distance defects is clinically difficult. At present, the most commonly used method is to use autologous nerve grafts to bridge the repair of nerve stumps. Even though this method is considered to be the "gold standard" for clinical work, it still has many disadvantages: such as limited nerve sources, impaired function of the donor nerve, In addition, studies have shown that only 40% to 50% of patients can achieve complete recovery of motor function after receiving autologous nerve transplantation, so it is necessary to find an alternative to autologous nerve transplantation.

With the rapid development of biomedical materials and tissue engineering, the focus of research has turned to the use of nerve conduits to bridge the two broken ends of defective nerves to promote peripheral nerve repair. For example, chemical materials polyethylene, polyglycolic acid, polylactic acid, etc.; biological materials chitosan, gelatin, etc.; biological tubular stent arteries, veins and sarcolemmal tubes have all been tried to prepare catheters. However, due to a series of problems such as poor biocompatibility of chemical materials, poor mechanical properties of biological materials and biological tubes, etc., the repair effect is not good, and its clinical application is limited.

At present, the research focuses on the relatively simple structure of the nerve conduit, which has a precise effect on repairing long nerve defects. And the vast majority of nerve catheter products approved by CFDA for clinical application are of this type. However, it is difficult for most of these nerve conduits to take into account good mechanical strength (surgical operation and blocking soft tissue ingrowth), good biocompatibility (metabolic transport of cells and nutrients), and controllable in vivo degradation (poor degradation in vivo) Or it cannot provide the biological boosting effect required for repair (single function, only physically bridging nerve stumps), and cannot better promote nerve regeneration.

Clinically, the appearance of a peripheral nerve repair material with simple preparation process, suitable mechanical strength, good biocompatibility, convenient in vivo degradation, and convenient, safe and effective has been long-awaited in clinic.

SUMMARY OF THE INVENTION

The invention provides a nerve conduit and a preparation method thereof. The nerve conduit is formed of collagen gel and rolled into a circular tube, which is simple and convenient to manufacture. The conduit contains mineral materials and releases inorganic ion elements in a small amount, which is used for regeneration of nerves and blood vessels. Provides a stimulatory effect, promotes the growth of nerve axons, and at the same time calcium ions are released with the degradation of the material, avoiding a sudden increase in the local calcium ion concentration, without affecting the important role of Schwann cells in nerve regeneration, so that the nerve tissue in the tube can grow better. At the same time, the mineralized collagen catheter is attached to avoid excessive degradation of the pure collagen catheter, so that the degradation rate of the catheter can be easily regulated.

The purpose of the present invention is to provide a collagen nerve conduit with a micro-nano structure and a preparation method thereof with slow-release calcium ions.

The artificial nerve conduit provided by the present invention is mainly composed of decomposable and absorbable collagen and calcium ion-rich minerals in vivo, and has good biocompatibility and calcium ion release performance. It has a multi-layer structure, each layer has a groove-like microstructure.

The latest research shows that calcium ions play an important role in nerve repair. Calcium ions can not only accelerate the growth of damaged nerve axons, but also participate in other metabolic activities in the body (Adalbert R, Morreale G, Paizs M, Conforti L, Walker SA, Roderick HL, Bootman MD, Siklóikl Sikl D, Sikloderick HL, Bootman MD, Sikl MD, Sikl Saxotomy in wild-type and slow Wallerian degenerationaxons. Neuroscience. 2012;225:44-54.). However, the researchers found that the sharp increase of calcium ion concentration in the in vitro medium will affect the survival rate of Schwann cells, and Schwann cells play an important role in nerve regeneration and recovery (Yang KJ, Yan Y, Zhang LL, Agresti MA, Matloub HS, LoGiudice JA, Havlik R, Yan JG. Increasing Calcium Level Limits Schwann Cell Numbers In Vitro following Peripheral Nerve Injury. Journal of Reconstructive Microsurgery. 2017;33:435-40.).

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

Optionally, the surface of the prepared film has a micro-scale or nano-scale groove structure, and the surface of the film containing the groove structure is used for the inner wall of the nerve conduit;

Preferably, the size of the trench structure is 500-25000 μm in length, 5-25 μm in width, 5-25 μm in depth, and 5-25 μm in pitch, or 10-20 μm in length, 100-200 nm in width, 100-200 nm in depth, and 100- 200nm.

In the nerve conduit provided by the present invention, the wall of the conduit formed by crimping enhances the fixation between different membrane layers through physical or chemical methods;

Preferably, the chemical method is to cross-link the collagen using a cross-linking agent.

In the nerve conduit provided by the present 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 conduit formed by crimping, the number of layers of the membrane in the tube wall depends on the required strength and degradation efficiency For 2-6 layers.

In the nerve conduit provided by the present 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 present invention, the mineral material includes one or more of β-tricalcium phosphate, nano-hydroxyapatite and biomimetic mineralized collagen; optionally, the mineral material may also contain other beneficial Hydroxyapatite of elements (magnesium, silicon, selenium, zinc, etc.).

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

In the nerve conduit provided by the present invention, the type I collagen hydrogel and the mineral material are mixed with water to prepare a film through a mold;

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

In the nerve conduit provided by the present invention, the preparation of the film by the mold comprises the following steps:

The solution formed by mixing type I collagen hydrogel and mineral material with water is poured into the mold, and the internal air bubbles are removed by vacuuming with negative pressure, and then a film-like material is formed after drying;

Optionally, the pressure of the negative pressure vacuuming is -0 to -0.1 MPa.

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

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

On the other hand, the present invention provides the preparation method of the above-mentioned nerve conduit, comprising the following operation steps:

Step S1-1: mixing type I collagen hydrogel and mineral material to obtain a mineral material/type I collagen hydrogel mixture;

Step S1-2: Mix the mineral material/type I collagen hydrogel mixture obtained in 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: take the mineral collagen solution obtained in step S1-2 and pour it into a mold, remove internal air bubbles through negative pressure vacuuming, and then dry to form a film-like mineral collagen film;

Step S2-2: take the mineral collagen film obtained in step S2-1, evenly apply the dilution of the mineral collagen solution obtained in step S1-2 for softening, curl it into a tube shape, and dry it until it is completely shaped; the dried collagen film needs to be diluted to be rolled into a tube of mineral collagen solution as a binder.

Step S2-3: take the fully shaped tubular product obtained in step S2-2, and cross-link with a cross-linking agent to obtain a nerve conduit;

Optionally, step S2-3 further includes eluting and freeze-drying the product cross-linked by the cross-linking agent, and then sterilizing the product by irradiation to prepare a collagen multilayer nerve conduit.

In the preparation method of the nerve conduit provided by the present invention, the side in contact with the mineral collagen solution in the mold has a micro- or nano-scale groove structure;

In the preparation method of the nerve conduit provided by the present invention, optionally, the material of the mold is monocrystalline silicon, and the groove structure is etched on the surface of the monocrystalline silicon by dry etching, or the nanoscale The groove structure is attached to the mineralized collagen film by means of dimethylsiloxane (PDMS) transfer molding.

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

In the preparation method of the nerve conduit provided by the present invention, optionally, the content of the mineral collagen solution is 0.5-1 g/cm ² ;

In the preparation method of the nerve conduit provided by 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°C;

Optionally, depending on the air-drying time, the degree of drying can be controlled (the final water content is different), and the film thickness after air-drying is 0.1-1 mm. The process of layering and drying can be adopted, and the mineral collagen solution containing different mineral species (β-tricalcium phosphate, nano-hydroxyapatite, mineralized collagen) can be gradually added to prepare a single film, so that it has more advanced multi-component properties. structure.

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

In the preparation method of the nerve conduit provided by the present invention, in the step S2-2, the mineral collagen solution diluent is diluted by volume with deionized water, and the dilution concentration is 1:(1-10).

In the preparation method of the nerve conduit provided by the present invention, in the step S2-3, the elution is first 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 range of the ethanol aqueous solution is: 30%-100%.

In the preparation method of the nerve conduit provided by the present invention, the freeze-drying treatment in step S2-3 is specifically: pre-freezing the product obtained after elution at -30 to -20°C, and then Under the conditions of vacuum and -10～0℃, sublimation is carried out to make pores, and the pore size range is 0-10μm, and finally vacuum drying is carried out at 0～50℃;

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

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

Optionally, the reagent used for the irradiation is cobalt-60 sterilant, and the dose is 15-38 kGy.

The beneficial effects of the present invention are as follows:

The multi-layer artificial nerve conduit provided by the present invention can be surgically implanted into the site of nerve injury. When used for the repair of peripheral nerve injury, a catheter with an appropriate thickness can be selected according to the thickness of the injured nerve and the degree of defect, and the two ends of the severed nerve are inserted into the catheter about 2-5 mm, and surgical sutures are used for extra-neural surgery. Die stitched to fix.

Clinically, peripheral nerves can be bridged with the multilayer artificial nerve conduit of the present invention later, and after the nerve regeneration is completed, it is gradually degraded and absorbed in the body. Compared with other nerve conduits, the artificial nerve conduit of the present invention adopts a multi-layer structure, the conduit has good toughness, can withstand the shear force of the suture, and can also hinder the ingrowth of the surrounding soft tissue and affect the repair effect. The catheter can slowly release calcium ions in a small amount, accelerate the growth of axons, and the regeneration of nerves after bridging surgery is fast and of good quality. Attaching the mineralized collagen conduits avoids excessive degradation of pure collagen conduits, making the degradation rate of the conduits easy to control. In addition, the present invention does not require a second operation to take out the implant after operation, the degradation time is adapted to the growth speed of nerve fibers, and can be applied to the repair of clinical peripheral nerve injury.

Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the invention. Other advantages of the present invention may be realized and obtained by means of the arrangements described in the specification.

Description of drawings

The accompanying drawings are used to provide an understanding of the technical solutions of the present invention, and constitute a part of the specification, and together with the embodiments of the present invention, they are used to explain the technical solutions of the present invention, and do not limit the technical solutions of the present invention.

Figure 1 is a schematic diagram of the structure of the biomimetic mineralized collagen-collagen multilayer nerve conduit;

Fig. 2 is the actual picture of the biomimetic mineralized collagen-collagen multilayer nerve conduit prepared in Example 1;

Fig. 3 is the scanning electron microscope picture of the side surface of the catheter wall prepared in Example 1;

Figure 4 shows the intraoperative catheter implantation into the rat sciatic nerve;

Figure 5 shows the post-operative bridging nerve immunofluorescence (neurofilament protein NF200), and catheter implantation promotes the growth of rat sciatic nerve axons;

Fig. 6 The sciatic nerves of rats were repaired in different ways in three groups, and 12 weeks after the operation, the ultrathin sections of the repaired nerves were observed by transmission electron microscopy. A autologous nerve group; B mineralized collagen-collagen multilayer nerve conduit group; C pure collagen conduit group. (Top: 2550x, Bottom: 26500x).

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the embodiments of the present invention are described in detail below. It should be noted that the embodiments of the present invention and the features of the embodiments may be arbitrarily combined with each other unless there is conflict.

In the embodiment of the present invention, a nerve conduit is provided, and the nerve conduit is a membrane prepared by mixing type I collagen hydrogel and a mineral material and then coagulating, and then a conduit formed by crimping;

Optionally, the surface of the prepared film has a micro-scale or nano-scale groove structure, and the surface of the film containing the groove structure is used for the inner wall of the nerve conduit;

Preferably, the size of the trench structure is 500-25000 μm in length, 5-25 μm in width, 5-25 μm in depth, and 5-25 μm in pitch, or 10-20 μm in length, 100-200 nm in width, 100-200 nm in depth, and 100- 200nm.

In the embodiment of the present invention, the wall of the conduit formed by crimping enhances the fixation between different membrane layers through physical or chemical methods;

Preferably, the chemical method is to cross-link the collagen using a cross-linking agent.

In the embodiment of the present 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 conduit formed by crimping, the number of layers of the membrane in the tube wall is 2 according to the required strength and degradation efficiency -layer 6.

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

In the embodiment of the present invention, the mineral material includes one or more of β-tricalcium phosphate, nano-hydroxyapatite and biomimetic mineralized collagen; optionally, the mineral material may also contain other beneficial elements ( hydroxyapatite of magnesium, silicon, selenium, zinc, etc.).

In the embodiment of the present invention, preferably, the particle size of the mineral material is 50-100 nm.

In the embodiment of the present invention, the type I collagen hydrogel and the mineral material are mixed with water to prepare a film through a mold;

In the embodiment of the present invention, optionally, the added amount of water is 0%-50% of the total volume of the type I collagen hydrogel, mineral material and water.

In the embodiment of the present invention, the preparation of the film by the mold includes the following steps:

The solution formed by mixing type I collagen hydrogel and mineral material with water is poured into the mold, and the internal air bubbles are removed by vacuuming with negative pressure, and then a film-like material is formed after drying;

Optionally, the pressure of the negative pressure vacuuming is -0 to -0.1 MPa.

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

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

On the other hand, the embodiment of the present invention provides the preparation method of the above-mentioned nerve conduit, comprising the following operation steps:

Step S1-1: mixing type I collagen hydrogel and mineral material to obtain a mineral material/type I collagen hydrogel mixture;

Step S1-2: Mix the mineral material/type I collagen hydrogel mixture obtained in 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: take the mineral collagen solution obtained in step S1-2 and pour it into a mold, remove internal air bubbles through negative pressure vacuuming, and then dry to form a film-like mineral collagen film;

Step S2-2: take the mineral collagen film obtained in step S2-1, evenly apply the dilution of the mineral collagen solution obtained in step S1-2 for softening, curl it into a tube shape, and dry it until it is completely shaped; the dried collagen film needs to be diluted to be rolled into a tube of mineral collagen solution as a binder.

Step S2-3: take the fully shaped tubular product obtained in step S2-2, and cross-link with a cross-linking agent to obtain a nerve conduit;

Optionally, step S2-3 further includes eluting and freeze-drying the product cross-linked by the cross-linking agent, and then sterilizing the product by irradiation to prepare a collagen multilayer nerve conduit.

In an embodiment of the present invention, the side of the mold in contact with the mineral collagen solution has a micro- or nano-scale groove structure;

In the embodiment of the present invention, optionally, the material of the mold is monocrystalline silicon, and the trench structure is etched on the surface of the monocrystalline silicon by dry etching, or the nanoscale trench structure is made of two The structure was attached to the mineralized collagen film by means of methylsiloxane (PDMS) transfer molding.

In the embodiment of the present invention, in the step S2-1, the mineral collagen solution is poured into the mold and the thickness is 3-10mm before air-drying;

In the embodiment of the present invention, optionally, the content of the mineral collagen solution is 0.5-1 g/cm ² ;

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°C;

Optionally, depending on the air-drying time, the degree of drying can be controlled (the final water content is different), and the film thickness after air-drying is 0.1-1 mm. The process of layering and drying can be adopted, and the mineral collagen solution containing different mineral species (β-tricalcium phosphate, nano-hydroxyapatite, mineralized collagen) can be gradually added to prepare a single film, so that it has more advanced multi-component properties. structure.

In the embodiment of the present invention, optionally, the thickness of the mineral collagen film formed after drying is 0.1-1 mm.

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

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

In the embodiment of the present invention, the freeze-drying treatment in step S2-3 is specifically: pre-freezing the product obtained after elution at -30～-20°C, and then under vacuum, -10～ The sublimation is carried out under the condition of 0 °C to make pores, the pore size is in the range of 0-10 μm, and finally vacuum drying is carried out at 0 to 50 °C;

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

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

Optionally, the reagent used for the irradiation is cobalt-60 sterilant, and the dose is 15-38 kGy.

In the embodiment of the present invention, the type I collagen is bovine-derived collagen, which is obtained by routine extraction from bovine Achilles tendon. The specific extraction method can refer to the following steps: (refer to: Xiong Yueqin, He Xiaowei. Collagen-based medical compound water Preparation and Properties of Gel[J].Modern Food Science and Technology, 2009,25(12):1454-1457.)

1) Wash the fresh bovine Achilles tendon, scrape off the subcutaneous fat, cut into pieces of 1cm×1cm, and soak in 10% NaCl solution overnight to remove salt-soluble protein and other soluble impurities. After washing 3 times with distilled water, carry out the first degreasing (m bovine Achilles tendon: V degreasing solution = 1:10, degreasing solution consists of 6% Na ₂ CO ₃ solution and 1% degreaser), soak at 30 ° C for 60 min and then use Wash with distilled water for 2 to 3 times, and then carry out the second degreasing (m cattle Achilles tendon: V degreasing solution = 1:10, degreasing solution is composed of 3% Na ₂ CO ₃ solution and 1% degreaser), stirring at 30 ° C for 60 min After washing with 30 ℃ warm water 3 times, dry in a fume hood for later use.

2) Add a certain amount of pretreated bovine Achilles tendon raw material to an acid solution (pH 2.5-3) containing pepsin for a period of time, and filter with two layers of medical gauze.

3) NaCl solid is directly added to the filtrate for salting out (the final concentration of NaCl is less than 3.0 mol/L), and centrifugation is performed to obtain collagen precipitation. The collagen precipitate obtained after salting out was dissolved in the acid solution used, dialyzed against 0.02mol/L Na ₂ HPO ₄ (pH 8.6) for 2 d, and then dialyzed with distilled water as the external dialysate for 1 d. A bovine-derived type I collagen hydrogel was obtained.

In the embodiment of the present invention, the nano-hydroxyapatite is purchased from Aladdin Company, item number H106378;

In the examples of the present invention, the biomimetic mineralized collagen (mineralized collagen bone powder) was purchased from Aojing Pharmaceutical Technology Co., Ltd.

Example 1. Preparation of multilayer artificial nerve conduit

In the present embodiment, the multi-layer artificial nerve conduit is prepared by using a mineral collagen solution, and the preparation method of the mineral collagen solution is:

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

2. The biomimetic mineralized collagen and type I collagen hydrogel in the previous step are mixed with water, and the volume content of the added water is 10% of the total volume of the biomimetic mineralized collagen, type I collagen hydrogel and water, That is, the mineral collagen solution is obtained.

The preparation method of the multilayered nerve conduit is:

1. Spread 100 g of the mineral collagen solution in the mold. The contact surface between the mold and the mineral collagen solution is made of single crystal silicon. The single crystal silicon is etched by dry method to have the same length as the mold, with a width of 10 microns and a depth of 10 microns. , parallel trenches with a pitch of 5 microns.

The size of the mold is length*width*height=20*10*2cm, the thickness of the solution is 5mm,

The content of the mineral collagen solution is 0.5g/cm ² , the negative pressure is -0.1Mp to exhaust air bubbles, it is naturally air-dried at room temperature 25°C to completely dry, and cut to 20mm*50mm; its thickness is 0.5mm, and it is prepared into a film ;

2. Evenly apply the collagen dilution solution (the collagen dilution solution is mixed according to the mineral collagen solution: water (v:v)=1:5) for softening, and curl the softened membrane into a tube shape (length 20mm, inner diameter 5mm) , in the tube formed by the curling, the number of layers of the film in the tube wall is 4 layers, and the tubular product is obtained by fixing the cylindrical mold at room temperature and air-drying to complete drying and shaping;

3. Immerse the obtained tubular product in 200 mL of EDC aqueous solution, the concentration of the EDC solution is 10 g/mL, cross-link for 30 min, elute the cross-linked tubular product in 50% (v:v) ethanol aqueous solution, and wash with ethanol aqueous solution for 3 Second, after washing with purified water for 3 times, pre-freezing at -20 °C, freeze-drying and sublimation at -5 °C in vacuum for 48 hours to create pores with a pore size of 5 μm, and finally vacuum drying at 30 °C. Finally, sterilize by irradiation, using cobalt-60 sterilant at a dose of 15kGy to obtain a multilayer artificial nerve conduit. micron parallel grooves. The nerve conduit is 20 mm long and 5 mm in inner diameter. The number of layers of the tunica media is 4, and the wall thickness is 2 mm. As shown in Figure 1. Dimensional changes before and after nerve conduit drying were negligible.

Example 2

In the present embodiment, the multi-layer artificial nerve conduit is prepared by using a mineral collagen solution, and the preparation method of the mineral collagen solution is:

Wherein, the preparation method of mineral collagen solution is:

1. Mix type I collagen hydrogel with a mineral material, and the mass ratio is 6:1; the mineral material is nano-hydroxyapatite (particle size is 85nm);

2. The nano-hydroxyapatite and type I collagen hydrogel obtained in the previous step are mixed with water, and the volume content of the added water is 20% to obtain a mineral collagen solution.

The preparation method of the multilayered nerve conduit is:

1. Spread 100 g of the mineral collagen solution in the mold. The contact surface between the mold and the mineral collagen solution is made of monocrystalline silicon. The monocrystalline silicon is etched by dry method to have the same length as the mold, 15 microns in width and 15 microns in depth. , parallel trenches with a pitch of 5 microns.

The size of the mold is length*width*height=20*10*2cm, the thickness of the solution is 5mm,

The content of the mineral collagen solution is 0.5g/cm ² , the negative pressure is -0.1Mp to exhaust air bubbles, it is naturally air-dried at room temperature 25°C to completely dry, and cut to 20mm*50mm; its thickness is 0.3mm, and it is prepared into a film ;

2. Evenly apply the collagen dilution solution (the collagen dilution solution is mixed according to the mineral collagen solution: water (v:v)=1:4) for softening, and curl the softened membrane into a tube shape (length 20mm, inner diameter 5mm) , in the tube formed by the curling, the number of layers of the film in the tube wall is 5 layers, and the tubular product is obtained by fixing the cylindrical mold at room temperature and air-drying to complete drying and shaping;

3. Immerse the obtained tubular product in 200 mL of glutaraldehyde aqueous solution, the volume concentration of the glutaraldehyde aqueous solution is 0.5%, cross-link for 2 hours, and elute the cross-linked tubular product in 80% (v:v) ethanol aqueous solution, The ethanol solution was washed three times, washed three times with purified water, pre-frozen at -25 °C, and then freeze-dried and sublimated at -5 °C in vacuum for 72 hours to create pores with a pore size of 5 μm, and finally vacuum-dried at 30 °C. Finally, sterilize by irradiation, use cobalt-60 sterilant at a dose of 15kGy, to obtain a multilayer artificial nerve conduit. micron parallel grooves. The nerve conduit is 20 mm long and 5 mm in inner diameter. The number of layers of the tunica media is 5, and the wall thickness is 1.5 mm. Dimensional changes before and after nerve conduit drying were negligible.

Comparative Example 1

The inner diameter of 5mm, the wall thickness of 2mm, and the length of 20mm were prepared according to the method of Example 1. The number of layers in the tube wall was 4 layers. Except for the difference, the rest of the preparation raw materials and their dosages, and the preparation process are the same as those in Example 1.

Comparative Example 2

According to the method of Example 2, the inner diameter of 5mm, the wall thickness of 1.5mm, the length of 20mm, the number of layers of the membrane in the tube wall is 5, and the pure collagen multilayer catheter without nano-hydroxyapatite is prepared, except that it does not contain nano-hydroxyapatite Except this difference of stone, other preparation raw materials and their consumption, preparation process are all identical with embodiment 2.

Application example 1

After being soaked in physiological saline for 5 minutes, the tensile strength of the two samples was tested by WDW electronic universal testing machine, as shown in Table 1:

Table 1: Statistical table of mechanical properties test

It can be seen from Table 1 and Figure 4 that the collagen mineral catheters prepared in Examples 1 and 2 can meet the requirements for the mechanical properties of the catheter in the suturing stage.

Application example 2. Multilayer artificial catheter for the repair of rat sciatic nerve

The multilayer artificial nerve conduit was prepared according to the method of Example 1, wherein the length was 20 mm and the inner diameter was 5 mm.

Taking the adult female Spragne-Dawley rat model of sciatic nerve injury: the rats were weighed and then anesthetized by intraperitoneal injection of sodium pentobarbital, and the rats were fixed on the operating table in a prone position. Make an oblique straight incision about 2 cm long on the spine to expose the sciatic nerve, and use fiber scissors to gently separate the sciatic nerve between the nerve coat and surrounding tissue to fully dissociate the sciatic nerve. , nerve elastic retraction, the proximal end is cut short and a certain length is too long to cause a 10mm nerve defect. The above-mentioned catheter was transplanted to the injured part, and the broken ends of the nerves on both sides were inserted into the above-mentioned catheter, and the overlapping part of the catheter and the nerve was sutured with 10-0 nylon thread, and the dynamic observation was carried out for 1 year. Behavioural evaluation.

The results of the study showed that the behavioral disorders of the animals began to recover in the 3rd month after the transplantation, there was no obvious movement disorder in the 6th month, and the animals returned to normal in the 12th month without weight bearing. WGA-HRP was used for nerve tracing, and Neurfilament (NF) immunohistochemical staining demonstrated the reconstruction of morphological structures; gold chloride staining demonstrated the recovery of motor endplates. Recovery of somatosensory evoked potentials (SEPs) and motor evoked potentials (MEPs) has also been demonstrated in electrophysiological studies.

Application example 3

Using the mineralized collagen-collagen multi-layered nerve conduit prepared in Example 1 of the present invention to evaluate the effect of repairing 10 mm sciatic nerve defect in adult Spragne-Dawley rats, and repairing it with autologous nerve transplantation and collagen conduit repair (Comparative Example 1) , were compared, the experimental results were dynamically observed for 12 weeks, and the evaluation results of histology, electrophysiology and behavior were as follows:

1. Histological aspect: the mineralized collagen-collagen multilayer nerve conduit group was significantly better than the pure collagen conduit group in the density of myelinated fibers and the thickness of the myelin sheath was not as good as that of the autologous nerve (P<0.05). There was little difference between the three groups in terms of fiber diameter (p>0.05) (as shown in Table 2, Figure 6). The recovery rate of gastrocnemius muscle wet weight in the mineralized collagen-collagen multilayer nerve conduit group was not statistically different from that in the autologous nerve group (p>0.05), and was significantly better than that in the pure collagen conduit group (p<0.05) (Table 3).

2. In terms of electrophysiology, the repaired nerve conduction rate of the mineralized collagen-collagen multilayer nerve conduit group was not statistically different from that of the autologous nerve group (p>0.05), and was significantly better than the pure collagen conduit group (p<0.05). The degree of nerve amplitude recovery was similar to that of the pure collagen catheter group (p>0.05) (Table 4).

4. Behavioral evaluation, the recovery degree of sciatic nerve function index (SFI) in the mineralized collagen-collagen multilayer nerve conduit group was not as good as that in the autologous nerve group (P<0.05), but it was significantly better than that in the pure collagen conduit group (P<0.05). )(table 5).

Table 2. Histological evaluation of regenerated nerves: transmission electron microscope observation of the midpoint of regenerated nerves

Table 3. Histological evaluation of regenerated muscle: wet weight recovery rate of gastrocnemius muscle (%)

Gastrocnemius wet weight recovery rate Mineralized Collagen-Collagen Multilayer Nerve Duct Group 39.80 Pure Collagen Catheter Set 36.46 autologous nerve group 48.29

Table 4. Electrophysiological evaluation: compound muscle action potential (CMAP)

Table 5. Behavioral evaluation: sciatic nerve function index (SFI)

SFI Mineralized Collagen-Collagen Multilayer Nerve Duct Group -73.75 Pure Collagen Catheter Set -82.79 autologous nerve group -61.92

The results of the study showed that the mineralized collagen-collagen multilayer nerve conduit was superior to the pure collagen conduit group in histological, electrophysiological and behavioral aspects at 12 weeks after the operation, and even approached the autologous nerve group in some aspects, proving the present invention. Compared with pure collagen catheters, it has excellent nerve repair effect.

Although the embodiments disclosed in the present invention are as above, the described contents are only the embodiments adopted to facilitate the understanding of the present invention, and are not intended to limit the present invention. Any person skilled in the art to which the present invention belongs, without departing from the spirit and scope disclosed by the present invention, can make any modifications and changes in the form and details of the implementation, but the scope of the patent protection of the present invention still needs to be The scope defined by the appended claims shall prevail.

Claims (15)

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;

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.

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;

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

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;

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

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,

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.

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

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;

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.

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;

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

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