A preparation method for composite tissue nerve regeneration and repair catheter

Abstract

The invention discloses a preparation method of a composite tissue nerve regeneration and repair catheter, and the method comprises: preparing solution preparation raw materials and sol preparation raw materials respectively, preparing nerve matrix solution and chitosan sol, preparing spinning nerve fiber membrane and chitosan catheter, compositely preparing nerve repair catheter semi-finished product, and preparing composite tissue nerve regeneration and repair catheter finished product; the invention uses nerve tissue and chitosan powder as main raw materials, polyglycolide crystal, protease solution and anhydrous acetic acid as auxiliary materials, respectively prepares spinning nerve fiber membrane and chitosan catheter, and composites them into composite tissue nerve regeneration and repair catheter, so that the prepared catheter finished product has good formability, certain toughness, good structural stability and biocompatibility, and the use of degradable raw materials does not affect the structure of the nerve itself during the use of the catheter, and there is no potential immune response, which is beneficial to nerve repair and regeneration, and the whole preparation process is also simple and easy to operate.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering materials, and in particular to a method for preparing a composite tissue nerve regeneration and repair conduit. Background Art

[0002] The tissues that make up various organs in the human body are composed of cells. The organizations of countless cells are highly ordered. There are nearly 100 trillion cells in the human body, divided into more than 100 types. They perform their respective missions step by step and combine into four types of tissues: epithelial tissue, connective tissue, muscle tissue and nervous tissue. Complex tissue is a special form of anatomical combination of the above four tissues. It is a tissue that is composed of several simple tissues with different forms and functions to perform a specific function.

[0003] In my country, approximately 2 million cases of peripheral nerve injury occur each year. The repair and reconstruction of peripheral nerve defects is a major problem in the current field of peripheral nerve injury. Long segmental nerve defects cannot be repaired clinically at present. With the development of tissue engineering technology, the use of nerve conduits and seed cells to construct tissue-engineered peripheral nerves in vitro to replace autologous nerve transplantation has become a development direction. The nerve conduit is a carrier for repairing nerve defects. It has the advantages of adjustable tube diameter and length, tension-free suture of the anastomosis, prevention of invasion of surrounding fibrous scar tissue, avoidance of the risks brought by autologous transplantation, and simplified surgical operation.

[0004] Most of the existing nerve repair catheters have single functions, insufficient mechanical properties, lack of good structural stability and biocompatibility, and have a certain impact on the structure of the nerve itself, and there is a potential immune response. In addition, the preparation process is complicated, difficult to operate, and the preparation cost is relatively expensive, which is not convenient for widespread promotion and application. Therefore, the present invention proposes a preparation method of a composite tissue nerve regeneration repair catheter to solve the problems existing in the prior art. Summary of the invention

[0005] In view of the above problems, the purpose of the present invention is to propose a method for preparing a composite tissue nerve regeneration repair catheter to solve the problems that the existing nerve repair catheter does not have good structural stability and biocompatibility, has a certain impact on the structure of the nerve itself, and has potential immune reactions, as well as a complex preparation process, is difficult to operate, and has a relatively high preparation cost.

[0006] In order to achieve the purpose of the present invention, the present invention is implemented by the following technical scheme: a method for preparing a composite tissue nerve regeneration repair conduit, comprising the following steps:

[0007] Step 1: first prepare the solution preparation raw materials according to the following weight parts: 25-35 parts of nerve tissue, 10-15 parts of polyglycolide crystals and 55-60 parts of protease solution, wherein the nerve tissue is collected from the peripheral nerve of an animal, and then prepare the sol preparation raw materials according to the following weight parts: 20-30 parts of chitosan powder, 10-15 parts of anhydrous acetic acid and 60-65 parts of sterile distilled water;

[0008] Step 2: First, the nerve tissue and polyglycolide crystals obtained in step 1 are crushed and mixed, and then the protease solution is added to mix and stir, and the nerve matrix solution is obtained after stirring evenly, and then the chitosan powder obtained in step 1 is added to sterile distilled water and mixed and stirred, and anhydrous acetic acid is evenly added during the mixing and stirring process, and the chitosan sol is obtained after stirring evenly;

[0009] Step 3: firstly, the neural matrix solution prepared in step 2 is placed in an electrospinning instrument for spinning, and the electrospinning parameters are set according to the actual preparation needs to obtain a spun neural fiber membrane, and then the chitosan sol prepared in step 2 is injected into a tube-shaped molding mold for curing and molding to obtain a chitosan catheter;

[0010] Step 4: firstly apply a medical adhesive on the outer wall of the chitosan catheter prepared in step 3, then curl and wrap the spun nerve fiber membrane prepared in step 3 on the outer wall of the chitosan catheter and remove the excess part, and after the medical adhesive is cured, a semi-finished catheter for nerve repair is obtained;

[0011] Step 5: First, fill the interior of the semi-finished nerve repair catheter obtained in step 4 with PGA filaments, and then coat the surface of the semi-finished nerve repair catheter with chitosan polymer. After the chitosan polymer is solidified, a finished composite tissue nerve regeneration repair catheter is obtained.

[0012] A further improvement is that in step one, the solution preparation raw materials are prepared according to the following weight parts: 30 parts of nerve tissue, 13 parts of polyglycolide crystals and 57 parts of protease solution, and the sol preparation raw materials are then prepared according to the following weight parts: 25 parts of chitosan powder, 12 parts of anhydrous acetic acid and 63 parts of sterile distilled water.

[0013] A further improvement is that in the step 2, the nerve tissue is first immersed in a cell membrane lysis solution after being crushed, and then the crushed nerve tissue is rinsed in pure water after the soaking, and then mixed with the crushed polyglycolide crystals after the rinsing.

[0014] A further improvement is that in the step 2, the specific preparation process of the chitosan sol is: first, chitosan powder is added to a stirring container filled with sterile distilled water and mixed and stirred, then sewage acetic acid is added to the stirring container and continued to mix and stir, and then stirring is repeated at a frequency of stirring for 30 minutes and then standing for 30 minutes until the mixture in the stirring container is completely dissolved, and finally the chitosan sol is obtained after being sealed and stored for 12 hours.

[0015] A further improvement is that in step three, the setting values ​​of the electrospinning parameters are: voltage 1-10 kW, collection distance 5-15 cm, propulsion rate 1-2 mL / h, and roller speed 1500-1800 r / min.

[0016] A further improvement is that in step three, after the chitosan sol is injected into the tube-shaped molding mold, it is first placed in a freezing device and frozen at -65°C to -75°C for 12 hours, then freeze-dried by a freeze dryer, and finally demolded.

[0017] A further improvement is that in step 4, the medical adhesive is an α-cyanoacrylate medical adhesive, and the semi-finished nerve repair catheter is sterilized after preparation. The specific steps are: first, the semi-finished nerve repair catheter is placed in a container filled with ethanol and soaked for 20 to 30 minutes, and then the semi-finished nerve repair catheter is washed with PBS buffer after soaking, and finally dried to complete the sterilization.

[0018] A further improvement is that in step five, the finished composite tissue nerve regeneration repair catheter is first inspected for appearance and quality, and then sealed and stored and put into use after passing the inspection.

[0019] The beneficial effects of the present invention are as follows: the present invention uses nerve tissue and chitosan powder as main raw materials, and uses polyglycolide crystals, protease solution and anhydrous acetic acid as auxiliary materials, to prepare spun nerve fiber membrane and chitosan catheter respectively, and composite them into a composite tissue nerve regeneration and repair catheter, so that the prepared catheter product has good formability, certain toughness, and good structural stability and biocompatibility. The use of degradable raw materials does not affect the structure of the nerve itself during the use of the catheter, and there is no potential immune response, which is beneficial to improving the efficiency of nerve repair and regeneration. In addition, the entire preparation process is simple, easy to operate, and has low cost, and is worthy of wide promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0021] Figure 1 It is a schematic flow chart of the preparation method of the present invention. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Embodiment 1

[0024] See also Figure 1 This embodiment provides a method for preparing a composite tissue nerve regeneration repair conduit, comprising the following steps:

[0025] Step 1: first prepare the solution preparation raw materials according to the following weight parts: 25 parts of nerve tissue, 15 parts of polyglycolide crystals and 60 parts of protease solution, wherein the nerve tissue is collected from the peripheral nerve of an animal, and then prepare the sol preparation raw materials according to the following weight parts: 20 parts of chitosan powder, 15 parts of anhydrous acetic acid and 65 parts of sterile distilled water;

[0026] Step 2: First, the nerve tissue and polyglycolide crystals obtained in step 1 are crushed, wherein the nerve tissue is first immersed in a cell membrane lysis solution after being crushed, and then the crushed nerve tissue is rinsed in pure water after soaking, and then mixed with the crushed polyglycolide crystals after rinsing, and then a protease solution is added for mixing and stirring, and a nerve matrix solution is obtained after uniform stirring, and then the chitosan powder obtained in step 1 is added to a stirring container filled with sterile distilled water for mixing and stirring, and then sewage acetic acid is added to the stirring container for continued mixing and stirring, and then the stirring is repeated at a frequency of stirring for 30 minutes and then standing for 30 minutes until the mixture in the stirring container is completely dissolved, and finally the chitosan sol is obtained after being sealed and stored for 12 hours;

[0027] Step 3: First, the neural matrix solution prepared in step 2 is placed in an electrospinning instrument for spinning, and the electrospinning parameters are set according to the actual preparation needs, specifically: voltage 1kW, collection distance 5cm, propulsion rate 1mL / h, roller speed 1500r / min, and the spun neural fiber membrane is obtained after spinning. Then, the chitosan sol prepared in step 2 is injected into the tube-shaped molding mold for curing and molding. After being injected into the tube-shaped molding mold, it is first placed in a freezing device and frozen at -65°C for 12 hours, and then freeze-dried by a freeze dryer, and finally demolded to obtain a chitosan catheter;

[0028] Step 4: firstly, coating the outer wall of the chitosan catheter prepared in step 3 with α-cyanoacrylate medical adhesive, then curling and wrapping the spun nerve fiber membrane prepared in step 3 on the outer wall of the chitosan catheter and removing the excess part, and after the medical adhesive is solidified, a semi-finished product of the nerve repair catheter is obtained, and then sterilization is performed. The specific steps are: firstly, soaking the semi-finished nerve repair catheter in a container containing ethanol for 20 minutes, and then washing the semi-finished nerve repair catheter with PBS buffer after soaking, and finally drying;

[0029] Step 5: First fill the interior of the semi-finished nerve repair catheter obtained in step 4 with PGA filaments, then coat the surface of the semi-finished nerve repair catheter with chitosan polymer, and after the chitosan polymer is solidified, obtain the finished composite tissue nerve regeneration repair catheter. The finished composite tissue nerve regeneration repair catheter is first inspected for appearance and quality, and after passing the inspection, it is sealed and stored and put into use.

[0030] Embodiment 2

[0031] See also Figure 1 This embodiment provides a method for preparing a composite tissue nerve regeneration repair conduit, comprising the following steps:

[0032] Step 1: first prepare the solution preparation raw materials according to the following weight parts: 30 parts of nerve tissue, 13 parts of polyglycolide crystals and 57 parts of protease solution, wherein the nerve tissue is collected from the peripheral nerve of an animal, and then prepare the sol preparation raw materials according to the following weight parts: 25 parts of chitosan powder, 12 parts of anhydrous acetic acid and 63 parts of sterile distilled water;

[0033] Step 2: First, the nerve tissue and polyglycolide crystals obtained in step 1 are crushed, wherein the nerve tissue is first immersed in a cell membrane lysis solution after being crushed, and then the crushed nerve tissue is rinsed in pure water after soaking, and then mixed with the crushed polyglycolide crystals after rinsing, and then a protease solution is added for mixing and stirring, and a nerve matrix solution is obtained after uniform stirring, and then the chitosan powder obtained in step 1 is added to a stirring container filled with sterile distilled water for mixing and stirring, and then sewage acetic acid is added to the stirring container for continued mixing and stirring, and then the stirring is repeated at a frequency of stirring for 30 minutes and then standing for 30 minutes until the mixture in the stirring container is completely dissolved, and finally the chitosan sol is obtained after being sealed and stored for 12 hours;

[0034] Step 3: First, the neural matrix solution prepared in step 2 is placed in an electrospinning instrument for spinning, and the electrospinning parameters are set according to the actual preparation needs, specifically: voltage 5kW, collection distance 10cm, propulsion rate 1.5mL / h, roller speed 1650r / min, and a spun neural fiber membrane is obtained after spinning. Then, the chitosan sol prepared in step 2 is injected into a tube-shaped molding mold for curing and molding. After being injected into the tube-shaped molding mold, it is first placed in a freezing device and frozen at -70°C for 12 hours, and then freeze-dried by a freeze dryer, and finally demolded to obtain a chitosan catheter;

[0035] Step 4: firstly, coating the outer wall of the chitosan catheter prepared in step 3 with α-cyanoacrylate medical adhesive, then curling and wrapping the spun nerve fiber membrane prepared in step 3 on the outer wall of the chitosan catheter and removing the excess part, and after the medical adhesive is solidified, a semi-finished product of the nerve repair catheter is obtained, and then sterilization is performed. The specific steps are: firstly, the semi-finished nerve repair catheter is placed in a container containing ethanol and soaked for 25 minutes, and then the semi-finished nerve repair catheter is washed with PBS buffer after soaking, and finally dried;

[0036] Step 5: First fill the interior of the semi-finished nerve repair catheter obtained in step 4 with PGA filaments, then coat the surface of the semi-finished nerve repair catheter with chitosan polymer, and after the chitosan polymer is solidified, obtain the finished composite tissue nerve regeneration repair catheter. The finished composite tissue nerve regeneration repair catheter is first inspected for appearance and quality, and after passing the inspection, it is sealed and stored and put into use.

[0037] Embodiment 3

[0038] See also Figure 1 This embodiment provides a method for preparing a composite tissue nerve regeneration repair conduit, comprising the following steps:

[0039] Step 1: first prepare the solution preparation raw materials according to the following weight parts: 35 parts of nerve tissue, 10 parts of polyglycolide crystals and 55 parts of protease solution, wherein the nerve tissue is collected from the peripheral nerve of an animal, and then prepare the sol preparation raw materials according to the following weight parts: 30 parts of chitosan powder, 10 parts of anhydrous acetic acid and 60 parts of sterile distilled water;

[0040] Step 2: First, the nerve tissue and polyglycolide crystals obtained in step 1 are crushed, wherein the nerve tissue is first immersed in a cell membrane lysis solution after being crushed, and then the crushed nerve tissue is rinsed in pure water after soaking, and then mixed with the crushed polyglycolide crystals after rinsing, and then a protease solution is added for mixing and stirring, and a nerve matrix solution is obtained after uniform stirring, and then the chitosan powder obtained in step 1 is added to a stirring container filled with sterile distilled water for mixing and stirring, and then sewage acetic acid is added to the stirring container for continued mixing and stirring, and then the stirring is repeated at a frequency of stirring for 30 minutes and then standing for 30 minutes until the mixture in the stirring container is completely dissolved, and finally the chitosan sol is obtained after being sealed and stored for 12 hours;

[0041] Step 3: First, the neural matrix solution prepared in step 2 is placed in an electrospinning instrument for spinning, and the electrospinning parameters are set according to the actual preparation needs, specifically: voltage 10kW, collection distance 15cm, propulsion rate 2mL / h, roller speed 1800r / min, and a spun neural fiber membrane is obtained after spinning. Then, the chitosan sol prepared in step 2 is injected into a tube-shaped molding mold for curing and molding. After being injected into the tube-shaped molding mold, it is first placed in a freezing device and frozen at -75°C for 12 hours, and then freeze-dried by a freeze dryer, and finally demolded to obtain a chitosan catheter;

[0042] Step 4: firstly, coating the outer wall of the chitosan catheter prepared in step 3 with α-cyanoacrylate medical adhesive, then curling and wrapping the spun nerve fiber membrane prepared in step 3 on the outer wall of the chitosan catheter and removing the excess part, and after the medical adhesive is solidified, a semi-finished product of the nerve repair catheter is obtained, and then sterilization is performed. The specific steps are: firstly, the semi-finished nerve repair catheter is placed in a container containing ethanol and soaked for 30 minutes, and then the semi-finished nerve repair catheter is washed with PBS buffer after soaking, and finally dried;

[0043] Step 5: First fill the interior of the semi-finished nerve repair catheter obtained in step 4 with PGA filaments, then coat the surface of the semi-finished nerve repair catheter with chitosan polymer, and after the chitosan polymer is solidified, obtain the finished composite tissue nerve regeneration repair catheter. The finished composite tissue nerve regeneration repair catheter is first inspected for appearance and quality, and after passing the inspection, it is sealed and stored and put into use.

[0044] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a composite tissue nerve regeneration and repair catheter, characterized in that: The following steps are involved: Step 1: first prepare the solution preparation raw materials according to the following weight parts: 25-35 parts of nerve tissue, 10-15 parts of polyglycolide crystals and 55-60 parts of protease solution, wherein the nerve tissue is collected from the peripheral nerve of an animal, and then prepare the sol preparation raw materials according to the following weight parts: 20-30 parts of chitosan powder, 10-15 parts of anhydrous acetic acid and 60-65 parts of sterile distilled water; Step 2: First, the nerve tissue and polyglycolide crystals obtained in step 1 are crushed and mixed, and then the protease solution is added to mix and stir, and the nerve matrix solution is obtained after stirring evenly, and then the chitosan powder obtained in step 1 is added to sterile distilled water and mixed and stirred, and anhydrous acetic acid is evenly added during the mixing and stirring process, and the chitosan sol is obtained after stirring evenly; Step 3: firstly, the neural matrix solution prepared in step 2 is placed in an electrospinning instrument for spinning, and the electrospinning parameters are set according to the actual preparation needs to obtain a spun neural fiber membrane, and then the chitosan sol prepared in step 2 is injected into a tube-shaped molding mold for curing and molding to obtain a chitosan catheter; Step 4: firstly apply a medical adhesive on the outer wall of the chitosan catheter prepared in step 3, then curl and wrap the spun nerve fiber membrane prepared in step 3 on the outer wall of the chitosan catheter and remove the excess part, and after the medical adhesive is cured, a semi-finished catheter for nerve repair is obtained; Step 5: First, fill the interior of the semi-finished nerve repair catheter obtained in step 4 with PGA filaments, and then coat the surface of the semi-finished nerve repair catheter with chitosan sol. After the chitosan sol is solidified, a finished composite tissue nerve regeneration repair catheter is obtained.

2. The method for preparing a composite tissue nerve regeneration and repair catheter according to claim 1, characterized in that: In the step 1, the solution preparation raw materials are prepared according to the following weight parts: 30 parts of nerve tissue, 13 parts of polyglycolide crystals and 57 parts of protease solution, and the sol preparation raw materials are prepared according to the following weight parts: 25 parts of chitosan powder, 12 parts of anhydrous acetic acid and 63 parts of sterile distilled water.

3. The method for preparing a composite tissue nerve regeneration and repair catheter according to claim 1, characterized in that: In the step 2, the nerve tissue is first immersed in a cell membrane lysis solution after being crushed, and then the crushed nerve tissue is rinsed in pure water after the soaking, and then mixed with the crushed polyglycolide crystals after the rinsing.

4. The method for preparing a composite tissue nerve regeneration and repair catheter according to claim 1, characterized in that: In the step 2, the specific preparation process of the chitosan sol is: first, chitosan powder is added to a stirring container filled with sterile distilled water and mixed and stirred, then anhydrous acetic acid is added to the stirring container and continued to mix and stir, and then stirring is repeated at a frequency of stirring for 30 minutes and then standing for 30 minutes until the mixture in the stirring container is completely dissolved, and finally the chitosan sol is obtained after being sealed and stored for 12 hours.

5. The method for preparing a composite tissue nerve regeneration and repair catheter according to claim 1, characterized in that: In the step 3, the setting values ​​of the electrospinning parameters are: voltage 1-10 kW, collection distance 5-15 cm, propulsion rate 1-2 mL / h, and roller speed 1500-1800 r / min.

6. The method for preparing a composite tissue nerve regeneration and repair conduit according to claim 1, characterized in that: In the step three, the chitosan sol is injected into the tube-shaped molding mold and then placed in a freezing device at -65°C to -75°C for 12 hours, then freeze-dried by a freeze dryer, and finally demoulded.

7. The method for preparing a composite tissue nerve regeneration and repair catheter according to claim 1, characterized in that: In the step 4, the medical adhesive is α-cyanoacrylate medical adhesive, and the semi-finished nerve repair catheter is sterilized after preparation. The specific steps are: first, the semi-finished nerve repair catheter is placed in a container filled with ethanol and soaked for 20 to 30 minutes, and then the semi-finished nerve repair catheter is washed with PBS buffer after soaking, and finally dried to complete the sterilization.

8. The method for preparing a composite tissue nerve regeneration and repair catheter according to claim 1, characterized in that: In the step 5, the finished composite tissue nerve regeneration repair catheter is first inspected for appearance and quality, and then sealed and stored and put into use after passing the inspection.

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