CN110038166B

CN110038166B - Self-curling film agent applied to nerve repair and preparation method thereof

Info

Publication number: CN110038166B
Application number: CN201910375017.8A
Authority: CN
Inventors: 赵应征; 鲁翠涛; 虞希冲; 金冰慧; 林蒙婷; 王丽芬; 黄志伟
Original assignee: Wenzhou Medical University
Current assignee: Wenzhou Medical University
Priority date: 2019-04-24
Filing date: 2019-04-24
Publication date: 2021-06-22
Anticipated expiration: 2039-04-24
Also published as: CN110038166A

Abstract

The invention provides a self-curling film agent applied to nerve repair and a preparation method thereof, the self-curling film agent is a film agent which is composed of A, B layers and C layers and has the thickness of 60-120 mu m, the main components of the A layer are indocyanine green and silk fibroin, the main components of the B layer comprise cyclic dipeptide and calcium alginate, the main components of the C layer comprise cyclic dipeptide and sodium alginate, the film agent is directionally curled in water to form a continuous hollow tube, the A layer is arranged inside, the C layer is arranged outside, and the film agent rapidly becomes a closed tube cavity structure under the irradiation of near-red light. The invention provides a new method for nerve butt joint and sleeve repair in nerve defects, meets the requirements of simple and convenient manufacture, convenient tube formation, controllable size and convenient application, and has wide application prospect.

Description

Self-curling film agent applied to nerve repair and preparation method thereof

Technical Field

The invention relates to a pellicle for nerve repair treatment, in particular to a composition, a preparation and an application of a self-curling pellicle capable of maintaining a favorable microenvironment of a nerve injury part for a long time and ensuring a nerve repair process.

Background

At present, global terrorist attacks and natural disasters are increasingly frequent, and according to incomplete statistics, about 100 million cases of peripheral nerve injury are newly added in China every year, wherein about 30 ten thousand cases of nerve injury are caused, and in clinical work, the prognosis of nerve injury caused by long-section nerve injury is poor, the function recovery of postoperative patients is poor, and a heavy burden is brought to the society, families and patients. In the existing research, a nerve conduit is often used as a scaffold material to combine with cells or growth factors to repair nerve injury, but the wide application of the nerve conduit is limited due to the defects of poor tube formation, fixed tube formation size, high operation requirement of practical application and the like. Therefore, it is urgently needed to prepare a nerve conduit which is simple and convenient to manufacture, convenient to form a tube, controllable in size and convenient to apply.

Sodium alginate is a natural polysaccharide compound extracted from kelp or seaweed, and shows special physicochemical properties due to a unique three-dimensional structure: biocompatibility, degradability, and adherability. Sodium alginate was first studied in 1881 and the british chemist Stanford found that brown alginate thickens solutions, forms gels, is biodegradable and easily forms films. The research on sodium alginate membranes was started in 1990, and after Koen Dewettinck et al found the foaming and film forming ability of sodium alginate, researchers at home and abroad began to extensively research on sodium alginate membranes. Sodium alginate has good film forming property, is a commonly used good film forming material at present, but is not further applied in the field of biomedical materials due to the limitation of mechanical property. At present, no sodium alginate membrane agent capable of being applied to nerve repair is developed.

From the perspective of clinical treatment, due to the obvious difference between the nerve injury part and the injury degree, a nerve conduit meeting the requirement of pipe diameter cannot be prepared in advance, in addition, the existing nerve conduit cannot form a closed pipe cavity aiming at the nerve which is not separated, and a film agent which can be quickly self-curled to form a pipe cavity structure meeting the requirement of repairing the defective nerve is clinically needed, so that the quick nerve protection and treatment are realized.

Ideally such films should simultaneously satisfy the following conditions: good biocompatibility, in-situ rapid directional molding, good durability, appropriate physical and chemical parameters such as mechanical strength and elasticity, and easy and rapid formation of a closed tube cavity with appropriate tube diameter.

At present, a preparation for intrauterine administration which can simultaneously meet the requirements is not available.

Disclosure of Invention

The invention aims to overcome the bottleneck that the existing preparation can not meet all requirements of rapid repair and treatment of nerve injury at the same time, provides a membrane with a lumen structure which is rapidly self-curled to meet the requirements of defective nerve repair, and provides an optimal condition for realizing rapid repair of nerve injury.

Therefore, what this application protected is applied to neural restoration from curling film agent, the film agent that the thickness is 60 ~ 120 μm that comprises A, B and C three-layer, and A layer principal ingredients are green and the silk fibroin of indocyanine, and B layer principal ingredients include cyclic dipeptide and calcium alginate, and C layer principal ingredients include cyclic dipeptide and sodium alginate, the film agent meet water and orient to curl and form continuous hollow tube, A layer is inside, and C layer is outside, under near red light irradiation, becomes confined lumen structure rapidly.

The cyclic dipeptide is preferably a cyclic (aspartic acid-proline) dipeptide.

The layer A is adhered to the surface of the layer B in a parallel arrangement of silk columns.

The layer A is adhered to the surface of the layer B in a single-layer parallel-arranged silk column mode, and the single-layer parallel-arranged silk column mode is preferred.

The thickness of the film agent is preferably 80 to 90 μm.

And glycerin and disodium ethylene diamine tetraacetate are further added into the two-layer components B and C.

One or more of electrolyte, antioxidant, adhesive, pH regulator, stem cell, therapeutic factor and therapeutic medicine are further added into the two-layer components B and C.

The film agent of the self-curling film agent is prepared according to the following scheme:

(1) mixing 0.5g of sodium alginate, 0.01g of cyclic dipeptide, 0.2ml of glycerol and 0.072g of ethylene diamine tetraacetic acid in a beaker, adding 24ml of distilled water, sealing the beaker by using a sealing film, standing for fully swelling, stirring for 2 hours for uniformly dissolving, standing for 24 hours at 4 ℃, and removing bubbles in the solution.

(2) And (3) extracting 10mL of sodium alginate solution by using an injector, casting the solution on a die with a surrounding edge to form a film, drying the film in a constant-temperature oven at 60 ℃, and drying the film to obtain a smooth and flat film agent.

(3) Preparing 2% calcium chloride solution, pouring the calcium chloride solution onto the film agent in the step (2), completely immersing the film agent, performing crosslinking treatment for 30 seconds, immediately pouring out the calcium chloride solution after the crosslinking treatment is finished, sucking the solution on the surface of the film by using filter paper, and placing the film agent in a fume hood for air drying to obtain the film agent with the B layer and the C layer.

(4) A 4% silk fibroin solution was prepared, with an open cell as per green indole: adding green indole phthalocyanine in a mass ratio of 1: 400 of silk fibroin, uniformly mixing to obtain a solution, carrying out parallel spinning on the surface of the film agent B layer in the step (3) by using an electrostatic spinning process, and fully drying to obtain a film agent which is self-curled when meeting water to form a tube cavity structure, wherein the thickness of the film agent is 60-100 micrometers.

In the preparation process of the film agent, one or more of electrolyte, antioxidant, adhesive, pH regulator, stem cells, therapeutic factors and therapeutic drugs are further added.

The self-curling film agent is used as a nerve sleeve material to be applied to repair of defective nerves.

In the technical scheme protected by the application, all components are in a synergistic complementary and necessary relationship and play a role together.

Drawings

FIG. 1 is a plan view of a self-curling pellicle of the invention for use in nerve repair

FIG. 2 is a lumen structure formed by the curled self-curling membrane applied to nerve repair

FIG. 3 is a schematic diagram of a three-layer structure of a self-curling membrane applied to nerve repair according to the present invention

Detailed Description

Hereinafter, specific embodiments of the present invention will be described in detail. It should be noted that technical features or combinations of technical features described in the following embodiments should not be considered as being isolated, and they may be combined with each other to achieve better technical effects.

Example 1 preparation of self-curling film of silk fibroin

The preparation method comprises the following steps: the experimental group of self-curling film was formulated according to the following protocol, designed according to table 1.

(3) Preparing 2% calcium chloride solution, pouring the calcium chloride solution onto the film agent in the step (2), completely immersing the film agent, performing crosslinking treatment for 30 seconds, immediately pouring out the calcium chloride solution after crosslinking is completed, sucking the solution on the surface of the film by using filter paper, and placing the film agent in a fume hood for air drying to obtain the film agent with the A layer and the B layer.

A control of self-curling film was prepared according to the design of table 1, in a similar manner as described above.

TABLE 1 compositions of the test and control groups of self-curling film

Note: "/": represents the vacancy of the component; "()": representing that the item of content is replaced by parenthesized content; the method comprises the following steps: cyclo (aspartic acid-proline) dipeptide; secondly, the step of: cyclo (proline-leucine) dipeptide; ③: cyclo (aspartate-glycine) dipeptide; fourthly, the method comprises the following steps: cyclo (tryptophan-tryptophan) dipeptide; fifthly: cyclo (phenylalanine-tryptophan) dipeptide; sixthly, the method comprises the following steps: glycine-proline dipeptide; seventh, the method comprises the following steps: arginine-glycine-aspartic acid tripeptide.

Example 2 physical characteristics of self-curling film

The test group and the control group of the self-curling film agent of example 1 were sprayed with physiological saline under 785nm infrared light irradiation, the tube forming ability of the self-curling film agent to form a closed tube cavity was observed, the tube forming time was recorded, and the elastic modulus of the closed tube was measured, respectively, and the results are shown in table 2.

TABLE 2 physical characteristics of the test and control groups of self-curling film agents

Example 3 Effect of self-curling film Agents in vivo application to nerve repair

(1) Rat sciatic nerve defect model

Animal preparation: healthy adult male SD rats weighing 220-250 g are fed freely for water intake and are bred adaptively for 1 week.

Modeling operation: the animals are fasted for 12 hours before operation and are not forbidden to drink water. After 10% chloral hydrate (0.3mL/100g) was anesthetized in the abdominal cavity, the hair on the right leg was shaved off with a small animal shaver, and the animals were fixed on the operating table of the experimental animals in the prone position. After the right leg operation area was disinfected with iodophor, the patient was wiped with alcohol and the skin was cut parallel to the femur in the right hip area of the rat with surgical scissors. After the skin is cut open, a firm and movable sheath of subcutaneous connective tissue is seen, which covers the sarcolemma, which is opened with blunt scissors to separate the biceps femoris muscle covered by the sarcolemma in the direction of blunt longitudinal dissection, and the sciatic nerve of the right leg is exposed. After the muscles on both sides were retracted with hemostats to expose a larger visual field, the nerves were carefully dissected with a glass needle, and sciatic nerve defects of 1cm were cut off with the starting end at a distance of approximately 0.5cm below the greater trochanter of the femur from the anterior border of the piriformis. The aseptic operation principle is strictly followed in the whole operation process.

(2) Nerve repair effect of self-curling film

The established sciatic nerve defect model is applied, each group of self-curling film agents are cut into rectangles with the size of 1.0cm x 0.5cm before use, the severed nerve at two sides is placed in a film after sciatic nerve defects of rats, preheated physiological saline at 37 ℃ is extracted by an injector to infiltrate the film agents to enable the film agents to wrap the severed nerve at two sides in a self-winding mode, 785nm near infrared light is used for irradiating for 20s, and the two ends of a tube are sutured and reinforced through operation sutures. After 8 weeks, the repair effect of the self-curling membrane agent on the defective nerve of each group is comparatively analyzed.

The foot print test was performed on the molded rats at week 8 after the operation. Before the test was started, both soles of the hind limbs of the rats were stained with red ink and then placed on a 1m long and 1.5cm wide runway to be walked forward. Each pair of footsteps will be measured and the sciatic nerve functional index (SFI) calculated according to the following formula. PL is the total sole length, TS is the length between the first toe and the fifth toe, IT is the length between the second toe and the fourth toe, E represents the experimental side, and N represents the normal side. The closer to-100% the SFI represents the total loss of function, whereas the closer to 0% the better.

After the behavioral test, the rats were subjected to abdominal anesthesia with 10% chloral hydrate (0.3mL/100g) and then to cardiac perfusion and sampling. The sciatic nerve on the right side of the experiment was removed, fixed with 4% paraformaldehyde for 4h, subjected to gradient dehydration with 20% and 30% sucrose solutions for 12h each, then subjected to OCT embedding and liquid nitrogen quick freezing, and then stored overnight in a refrigerator at-20 ℃ and then sliced with a freezing microtome, wherein the thickness of the slices was 5 μm. Bilateral gastrocnemius muscles of each group of rats were taken and measured for weight, and the gastrocnemius index (GMI), i.e., the ratio of the weight of the injured gastrocnemius muscle to the weight of the uninjured gastrocnemius muscle, was calculated to evaluate muscular atrophy caused by injury of the sciatic nerve.

Determining the repairing effect of the damaged nerve by HE staining and immunofluorescence method on the pathological section of the sciatic nerve of the rat; transmission Electron Microscopy (TEM) is used for observing and cutting sciatic nerve electron microscopy specimens of each group, and the tissue morphological changes of the nerve myelin sheaths after different groups of self-curling film agents are applied are compared.

Based on the results, the application effect of each group of self-curling film agents is evaluated by a double-blind method, and the evaluation is respectively carried out on the aspects of sciatic nerve function index, gastrocnemius atrophy degree (negative evaluation index), histology evaluation (whether nerve axon growth and blood vessel regeneration are promoted), nerve myelin sheath recovery degree and the like, so that a comprehensive score (10 points full) is given, and the higher the score is, the better the effect is. The results of the experiment are shown in table 3.

TABLE 3 application Effect of self-curling film agent test group and control group

Experimental groups: the behavioral foot prints and the calculated sciatic nerve function index results show that the injured side of the rats in each experimental group, namely the right sole, is healthy in skin, the toes are differentiated, and the SFI is about-50%. Evaluation of the degree of the gastrocnemius atrophy shows that the wet weight ratio of the damaged gastrocnemius muscle to the undamaged gastrocnemius muscle is about 70% more, and no obvious atrophy of the damaged gastrocnemius muscle is observed. Histological observation shows that the injured sciatic nerves are connected together, and the growth of nerve axons and the regeneration of blood vessels are obviously promoted. It was found from the degree of recovery of the nerve myelin that the myelin morphology was intact with some reduction in thickness. In each experimental group, the composite score was best for experimental group 1, followed by experimental group 6, followed by experimental groups 2, 3, 7, 8.

The application effect of each control group is obviously lower than that of the experimental group. According to the behavioral foot print and the calculated sciatic nerve function index, the injured side of the control group of rats, namely the right sole, is ulcerated to different degrees, toes of the rats are all in a close and curly state, and SFI is about-10%. Evaluation of the degree of the gastrocnemius atrophy found that the wet weight ratio of the damaged gastrocnemius muscle to the undamaged gastrocnemius muscle was about 20% more, and significant atrophy of the damaged gastrocnemius muscle was observed. Histological observation shows that the injured sciatic nerve is still in a defect state without connection, the axon growth is disordered, and the nerve vessel regeneration rarely exists. It was found from the degree of recovery of the nerve myelin that myelin morphology was incomplete and both the number and thickness were greatly reduced. Of the controls, control 14 scored the highest, followed by controls 2, 3, 9, 10, while controls 4-6, 11-13 had no effect.

The results show that the self-curling film agent has good repairing effect on the defective nerve, the structures and the components of the self-curling film agent are complementary with each other, and the self-curling film agent is absent and can not be used, so that the synergistic effect is exerted.

The above detailed description is specific to possible embodiments of the invention, and the embodiments are not intended to limit the scope of the invention, and all equivalent implementations or modifications that do not depart from the scope of the invention should be construed as being included within the scope of the invention.

In addition, various modifications, additions and substitutions in other forms and details may occur to those skilled in the art within the scope and spirit of the invention as disclosed in the claims. It is understood that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention as disclosed in the accompanying claims.

Claims

1. A self-curling film agent for nerve repair, comprising: the self-curling film agent is a film agent which is composed of A, B layers and C layers and has the thickness of 60-120 mu m, the main components of the layer A comprise indocyanine green and silk fibroin, the main components of the layer B comprise cyclic dipeptide and calcium alginate, the main components of the layer C comprise cyclic dipeptide and sodium alginate, the layer A is adhered to the surface of the layer B in a parallel wire column mode, the film agent is directionally curled in water to form a continuous hollow tube, the layer A is arranged inside, the layer C is arranged outside, and the film agent rapidly becomes a closed tube cavity structure under the irradiation of near red light.

2. A self-curling film for use in nerve repair according to claim 1, wherein: the cyclic dipeptide is cyclo (aspartic acid-proline) dipeptide.

3. A self-curling film for use in nerve repair according to claim 1, wherein: the layer A is adhered to the surface of the layer B in a single-layer parallel arrangement silk column mode.

4. A self-curling film for use in nerve repair according to claim 1, wherein: the thickness of the film agent is preferably 80-90 μm.

5. A self-curling film for use in nerve repair according to claim 1, wherein: and glycerin and disodium ethylene diamine tetraacetate are further added into the two-layer component B and the component C.

6. A self-curling film for use in nerve repair according to claim 1, wherein: one or a plurality of combinations of electrolytes, antioxidants, adhesives, pH regulators, stem cells, therapeutic factors and therapeutic drugs are further added into the B and C two-layer components.

7. A method for preparing a self-curling film agent for nerve repair according to claim 1, which comprises the following steps: the film agent of the self-curling film agent is prepared according to the following scheme:

(1) mixing 0.5g of sodium alginate, 0.01g of cyclic dipeptide, 0.2mL of glycerol and 0.072g of ethylene diamine tetraacetic acid in a beaker, adding 24mL of distilled water, sealing the beaker by using a sealing film, standing for fully swelling, stirring for 2 hours for uniformly dissolving, standing for 24 hours at 4 ℃, and removing bubbles in the solution;

(2) extracting 10mL of sodium alginate solution by using an injector, casting the solution on a mould with a surrounding edge to form a film, drying the film in a constant-temperature oven at 60 ℃, and drying the film to form a smooth and flat film agent;

(3) preparing 2% calcium chloride solution, pouring the calcium chloride solution onto the film agent obtained in the step (2), completely immersing the film agent, performing crosslinking treatment for 30 seconds, immediately pouring out the calcium chloride solution after the crosslinking treatment is finished, sucking the solution on the surface of the film by using filter paper, and placing the film agent in a fume hood for air drying to obtain the film agent with the B layer and the C layer;

8. A process for preparing a self-curling film according to claim 7, wherein: during the preparation process of the film agent, one or a combination of more of electrolyte, antioxidant, adhesive, pH regulator, stem cells, therapeutic factors and therapeutic drugs is further added.

9. A self-curling film for use in nerve repair according to claim 1, wherein: the self-curling film agent is used as a nerve sleeve material to be applied to repair of defective nerves.