CN110624133B

CN110624133B - Nerve matrix catheter for nerve repair and preparation method thereof

Info

Publication number: CN110624133B
Application number: CN201910913789.2A
Authority: CN
Inventors: 陈国宝; 黄湘; 付强; 熊建; 夏斌; 王富平; 陈忠敏
Original assignee: Chongqing University of Technology
Current assignee: Chongqing University of Technology
Priority date: 2019-09-25
Filing date: 2019-09-25
Publication date: 2021-08-24
Anticipated expiration: 2039-09-25
Also published as: CN110624133A

Abstract

The invention discloses a nerve matrix catheter for nerve repair and a preparation method thereof. The natural gel matrix catheter prepared by the invention has a stable structure, is not easy to deform, has gel resilience, has smooth and through inner diameter holes, has good temperature sensitivity and plasticity on the basis of meeting good biocompatibility, is immediately gelated when the temperature is raised to the physiological body temperature of 37 ℃, can be stored at room temperature for a long time, and can meet different tissue requirements of a human body; has degradation controllability and is suitable for repairing different types of nerve defects; has low immunity, can induce nerve regeneration, and is beneficial to cell adhesion and accelerate nerve regeneration. The preparation method is simple in preparation process, easy to operate and control and wide in application prospect.

Description

Nerve matrix catheter for nerve repair and preparation method thereof

Technical Field

The invention relates to the technical field of tissue engineering materials, in particular to a nerve matrix catheter for nerve repair and a preparation method thereof.

Background

Repair and reconstruction of peripheral nerve defects is a major problem in the current field of peripheral nerve injury. For short segment nerve lesions, the severed nerves can be anastomosed directly to one another, allowing the regenerated nerve fibers at the proximal end to grow into the distal end. But for long-segment neurological defects, repair is not clinically available at present. With the development of tissue engineering technology, the construction of tissue engineered peripheral nerves in vitro using nerve conduits and seed cells to replace autologous nerve transplantation has become the development direction.

The nerve conduit is a carrier for repairing nerve defects, and has the advantages of adjustable pipe diameter and length, tension-free suture of an anastomotic stoma, prevention of invasion of surrounding fibrous scar tissues, avoidance of risks caused by autografting, simplification of operation and the like. However, since some catheters are made of non-degradable materials, toxicity and foreign body reactions may occur after implantation, and a secondary operation is required to remove them, which increases the cost and risk of the operation for the patient. Other degradable catheter materials such as Polyglycolic acid (PGA) are only suitable for short and thin nerve defect repair due to their too fast degradation rate (Dienstknecht T, Klein S, Vykoukal J, et al. Type I collagen nerve derivatives for medium nerve repairs in the for about. J Hand Surg Am. 2013;38(6): 1119. sub. 1124.). Therefore, the research on the degradation material adaptive to the growth rate of the nerve is the key point of nerve repair. The Extracellular matrix (ECM) of nerve tissue is a microenvironment for the growth of Schwann cells serving as seed cells, contains biological macromolecules such as fibrin, collagen, endostatin and the like and growth factors, not only provides a support structure and an attachment site for the cells, but also contains a plurality of biological information, can provide signals required by the cells, has important and significant effects on the adhesion, migration, proliferation, differentiation and gene expression regulation of the cells, and has obvious guiding and promoting effects on nerve regeneration. However, the acellular nerve matrix alone has high degradation speed and is difficult to form, so that the acellular nerve matrix is not beneficial to long-segment nerve repair. Acellular neural matrix gels, which are well biocompatible, adjustable and contain tissue-specific ECM components, also promote migration of human mesenchymal stem cells and differentiation of neural stem cells, as well as axonal growth in vitro, have been widely used for short-segment nerve repair (Lin T, Liu S, Chen S, et al. Hydrogel derived from porous neural tissue as a formulating biological material for repaying neural nerve repair, Acta biometer 2018;73: 326-. However, DNM, like ECM, still suffers from too fast a degradation rate.

In order to solve the above problems, a large amount of research has been conducted by domestic and foreign scholars. "protective action of tissue engineered artificial nerves on peripheral target organs and spinal cord neurons after rat sciatic nerve defect" (2010, vol.26 No.3 p.265-269) protection of peripheral target organs and spinal cord neurons after rat sciatic nerve defect using olfactory ensheathing cells-schwann cells-extracellular matrix components (ECM) -polylactic acid-polyglycolic acid copolymer bridge; the invention patent CN03134541.7 discloses a tissue engineering peripheral nerve product and a preparation method thereof, the tissue engineering peripheral nerve is constructed by nerve conduits and glial cells or stem cells; the nerve conduit is made of biodegradable materials; the glial cells or stem cells are autologous or allogeneic glial cells or stem cells differentiated towards nervous system cells, and the inside of the glial cells or stem cells contains a cell or neurotrophic factor controlled release system and extracellular matrix; the invention patent CN201010147529.8 discloses a preparation method and application of a neural tissue matrix-derived tissue engineering scaffold material, wherein the neural tissue matrix-derived material can be further prepared into a three-dimensional porous oriented scaffold through directional crystallization, freeze drying and crosslinking by alone or by being matched with other high polymer materials, or a nano-level film is prepared by an electrospinning technology and then wound to form a neural regeneration catheter, but the preparation process of the method is complex. The electrospun layer of the outer layer of the catheter is easy to break, has poor tensile property and has higher difficulty in operation. The degradation rate is fast under the action of biological enzymes in vivo, and the biological enzymes are difficult to match with the growth rate of own nerves.

Chitosan is a good repairing material, and the degradation speed can be adjusted by changing the deacetylation degree, the molecular weight and the crosslinking method of raw materials. The chitosan has good biocompatibility, can promote the growth of endothelial cells, can inhibit the growth of fibroblasts, and can reduce the formation of scars, but the chitosan has poor formability. At present, the temperature-sensitive gel catheter with controllable degradation rate is not reported to be used for nerve repair.

Disclosure of Invention

In view of the above defects in the prior art, the present invention aims to provide a neural matrix catheter for long-distance nerve repair, which solves the problems of the lack of source, the mismatch of the sizes of the donor and the damaged nerve, the requirement of secondary operation for the non-degradable neural repair catheter material, etc. in autologous nerve transplantation. The long-distance nerve repair catheter prepared by the invention has good biocompatibility, low immunity, temperature sensitivity, degradability and plasticity.

In order to solve the technical problems, the invention adopts the following technical scheme: a nerve matrix catheter for nerve repair is prepared from acellular nerve matrix, chitosan and sodium glycerophosphate through compounding and solidifying.

Further, the mass ratio of the acellular nerve matrix to the chitosan to the sodium glycerophosphate is 0.1-0.5: 3-9: 47-53.

The invention also provides a preparation method of the nerve matrix catheter for nerve repair, which comprises the following steps:

1) crushing the acellular nerve tissues, adding a pepsin solution, and continuously stirring until the pepsin solution is dissolved to obtain an acellular nerve matrix solution;

2) mixing a chitosan solution and a sodium glycerophosphate solution to obtain a mixed solution, adding the acellular nerve matrix solution prepared in the step 1) into the mixed solution, and uniformly mixing to obtain a composite nerve hydrogel;

3) and (3) injecting the composite nerve gel obtained in the step 2) into a self-made mould, and heating in a water bath to solidify the composite nerve gel to obtain the nerve matrix catheter for nerve repair.

Further, the decellularization of the neural tissue specifically comprises the following steps: taking nerve tissues of healthy animals, bathing in Triton X-100 solution, shaking, degreasing with methanol, incubating with DNA enzyme diluent, washing with absolute ethanol and water in sequence, and drying to obtain the acellular nerve tissues.

Further, the mass concentration of the Triton X-100 solution is 0.5-5%.

Further, the oscillation speed is 20 r/min to 80 r/min.

Further, the concentration of the pepsin solution is 0.1-5 mg/mL, and the stirring speed is 100-400 r/min.

Further, the pH value of the acellular nerve matrix solution is 5-9, and the concentration of the acellular nerve matrix solution is 1 mg/mL-20 mg/mL.

Further, the concentration of the chitosan solution is 1% -7% (m/v); the concentration of the sodium glycerophosphate solution is 20-80% (m/v).

Further, the volume ratio of the chitosan solution to the sodium glycerophosphate solution in the mixed solution is 1-4: 0.1-3.

Further, the volume ratio of the acellular nerve matrix solution to the mixed solution is 0.1-2: 20-60.

Further, the water bath heating temperature is 30-45 ℃.

Compared with the prior art, the invention has the following beneficial effects:

1. the nerve matrix catheter is prepared by taking the acellular nerve matrix, the chitosan and the sodium glycerophosphate as raw materials, and the hydrogel system of the chitosan and the sodium glycerophosphate carries the acellular nerve matrix to carry out gelation in situ to form a porous structure on the surface, so that the fixation force of the acellular nerve matrix on the hydrogel is increased, and the porous structure is more favorable for the adhesion of cells on the bracket and the growth of the cells in the depth direction. The acellular nerve matrix is a completely natural cell matrix, is more beneficial to cell adhesion and has obvious guiding and promoting effects on nerve regeneration, so that the process of nerve regeneration is more beneficial to acceleration, and meanwhile, the immunogenicity of allogeneic cell transplantation can be reduced or avoided after the cells are removed, so that the functions of rapid nerve growth and function recovery are achieved; the chitosan is compounded, and the degradation speed is controllable by changing the deacetylation degree, the molecular weight and the crosslinking method of the raw materials; and the plasticity and the temperature sensitivity of the composite material are improved by compounding sodium glycerophosphate, so that the three components have synergistic interaction and have synergistic effect, and the nerve conduit has the advantages of promotion and induction of nerve regeneration, good biocompatibility, controllable degradation speed, no immunogenicity and excellent mechanical property.

2. The nerve matrix catheter is prepared by taking acellular nerve matrix, chitosan and sodium glycerophosphate as raw materials and compounding and curing the raw materials, and mutual reaction and breakage recombination of chemical bonds exist among the materials in the compounding process instead of simple physical mixing. The catheter has good temperature sensitivity and plasticity on the basis of meeting good biocompatibility, gelation can occur immediately when the temperature is raised to the physiological body temperature range, and the catheter after gelation can be stored at room temperature for a long time, so that different tissue requirements of a human body can be met; has degradation controllability and is suitable for repairing different types of nerve defects; has low immunity, can induce nerve regeneration, and is beneficial to cell adhesion and accelerate nerve regeneration. The nerve conduit is an ideal novel nerve repair material and has good application prospect.

3. The natural gel matrix catheter prepared by the invention has good biocompatibility, mechanical property matched with implanted tissues, stable structure, difficult deformation, gel resilience, smooth and through inner diameter holes of the catheter, favorable guidance and promotion of nerve regeneration, and good temperature sensitivity and degradation controllability. Compared with other similar nerve matrix catheters, the nerve matrix catheter has the degradation rate matched with the nerve growth rate, and can be used for long-distance nerve repair. The preparation method is simple in preparation process, easy to operate and control and wide in application prospect.

Drawings

FIG. 1 is a diagram of the ischial nerve of the rat after decellularization prepared in example 1;

FIG. 2 is a diagram of the acellular neural matrix prepared in example 1;

FIG. 3 is a graph comparing H-E staining before and after neural decellularization prepared in example 2; panel a is the pre-decellularized neural tissue and panel B is the post-decellularized neural tissue;

FIG. 4 is a graph of the effect of acellular neural matrix on temperature-sensitive curing of composite neural hydrogel;

FIG. 5 is a photograph of the neural substrate catheter for nerve repair prepared in example 2;

fig. 6 is an infrared spectrum of the complex nerve gel matrix prepared in example 2.

Detailed Description

The present invention will be described in further detail with reference to examples. The reagents used in the examples are not specifically described and are commercially available.

Example 1

A natural nerve matrix catheter is prepared by the following method:

1) taking sciatic nerve from healthy rat, bathing in Triton X-100 solution (requiring 0.22 μm filter membrane for sterilization) with concentration of 3% (m/v), shaking at 50 r/min for 12 hr, and changing water every 4 hr; after 12h, washing the nerves with distilled water for 2-3 times, and then placing the nerves in a Triton X-100 solution of 3% (m/v) to shake for 12 h; the neural tissue was further subjected to degreasing treatment with methanol for 24 hours (during which the methanol solution was changed 2 times), and then treated with a DNase dilution at a concentration of 2. mu.L/mL (v/v) at 37 ℃ for 2 hours. And finally, sequentially washing and drying with absolute ethyl alcohol and water to obtain the acellular nerve tissue, wherein after the acellular treatment, the nerve tissue becomes loose and is in a white strip shape, and the volume is slightly reduced (figure 1).

2) Crushing the decellularized nerve tissue, adding a pepsin solution with the concentration of 10 mg/mL, continuously stirring at the speed of 200 r/min until the solution is dissolved, and adjusting the pH of the digested solution to 7.4 by using NaOH to obtain a decellularized nerve matrix solution with the concentration of 10 mg/mL, wherein the decellularized nerve matrix solution is milky white transparent gel (figure 2).

3) Preparing a chitosan solution with the mass concentration of 3%, adding 0.1 mol/L dilute hydrochloric acid in the chitosan dissolving process to completely dissolve the chitosan, then placing at 0 ℃, continuously stirring by using a magnetic stirrer, and slowly dropwise adding a sodium glycerophosphate solution with the mass concentration of 50% to ensure that the volume ratio of the chitosan solution to the sodium glycerophosphate solution is 2.3: 1 until the solution is completely mixed and the pH is adjusted to 7.2 using 0.1 mol/L NaOH solution to give a hydrogel matrix.

4) And (3) adding 0 mu L of the acellular nerve matrix solution prepared in the step 2) into 4.5 mL of the hydrogel matrix obtained in the step 3), and uniformly mixing to obtain the composite nerve hydrogel.

5) And (3) injecting the composite nerve hydrogel obtained in the step 4) into a self-made mould, and heating in a water bath at 37 ℃ to solidify the composite nerve hydrogel to obtain the gel catheter for nerve repair.

Examples 2 to 4 were the same as in example 1, except that the amount of the acellular nerve matrix solution added was different, as shown in table 1.

TABLE 1

Examples	Chitosan solution (mL)	Sodium glycerophosphate solution (mL)	DNM（μL）
				Example 1	10.35	4.5	0
Example 2	10.35	4.5	100
				Example 3	10.35	4.5	200
Example 4	10.35	4.5	300

1. The rat sciatic nerve from example 2 was analyzed for H-E staining before and after decellularization. The result of the hematoxylin staining solution staining the cell nucleus blue and the eosin staining solution staining the cytoplasm pink is shown in FIG. 3.

As can be seen from the figure, after fresh rat sciatic nerve tissues are stained by hematoxylin eosin staining solution, cell nuclei and cytoplasm can be observed under a microscope, and after the nerve tissue samples are subjected to decellularization, almost no blue cell nuclei and only pink cytoplasm exist after the nerve tissue samples are stained by the staining solution, which indicates that the invention can effectively remove cells and completely reserve extracellular matrix.

2. The composite nerve hydrogel prepared in examples 1 to 4 was placed in a test tube, cured by heating in a water bath at 37 ℃, and inverted, and the results are shown in fig. 4.

As can be seen from the figure, the composite nerve hydrogel prepared by the invention can be solidified, and does not fall off when being inverted, which shows that the composite nerve hydrogel has good temperature sensitivity near the body temperature (37 ℃), and the temperature sensitivity is not changed when 0-400 mu L of acellular nerve matrix solution is added.

3. The gel tube prepared in example 2 was observed, and the results are shown in fig. 5.

As can be seen from the figure, the natural nerve stroma catheter prepared by the invention has stable structure, is not easy to deform, has the resilience of gel, and has clear and through inner diameter holes, the inner diameter of the catheter is 1mm, and the outer diameter of the catheter is about 4 mm.

4. The results of infrared spectroscopic analysis of Chitosan (CS), sodium Glycerophosphate (GP), acellular nerve matrix (DNM), and the complex nerve gel matrix of the present invention are shown in fig. 6.

As can be seen from the figure, the composite nerve hydrogel matrix prepared by the invention contains characteristic absorption peaks of CS, GP and DNM, and also has a new characteristic absorption peak, which indicates that mutual reaction and breakage and recombination of chemical bonds exist among materials in the composite process, but not simple physical mixing.

The above description is only exemplary of the present invention and should not be taken as limiting, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A preparation method of a nerve matrix catheter for nerve repair is characterized in that the catheter is formed by compounding and solidifying acellular nerve matrix, chitosan and sodium glycerophosphate, and is prepared by the following steps:

2. The method for preparing a nerve stroma catheter for nerve repair according to claim 1, wherein the mass ratio of the acellular nerve stroma, the chitosan and the sodium glycerophosphate is 0.1-0.5: 3-9: 47-53.

3. The method for preparing a neural substrate conduit for nerve repair as claimed in claim 1, wherein the decellularization of the neural tissue specifically comprises the steps of: taking nerve tissues of healthy animals, bathing in Triton X-100 solution, shaking, degreasing with methanol, incubating with DNA enzyme diluent, washing with absolute ethanol and water in sequence, and drying to obtain the acellular nerve tissues.

4. The method for preparing a neural substrate conduit for nerve repair according to claim 3, wherein the mass concentration of the Triton X-100 solution is 0.5% to 5%.

5. The method for preparing a nerve matrix conduit for nerve repair according to claim 3, wherein the oscillation speed is 20 r/min to 80 r/min.

6. The method for preparing a nerve matrix catheter for nerve repair according to claim 1, wherein the pepsin solution has a concentration of 0.1 to 5 mg/mL and a stirring speed of 100 to 400 r/min.

7. The method for preparing a nerve matrix catheter for nerve repair according to claim 1, wherein the acellular nerve matrix solution has a pH of 5 to 9 and a concentration of 1 to 20 mg/mL.

8. The method of preparing a neural substrate catheter for nerve repair of claim 1, wherein the concentration of the chitosan solution is 1% to 7% (m/v); the concentration of the sodium glycerophosphate solution is 20-80% (m/v).

9. The method for preparing a neural substrate catheter for nerve repair of claim 1, wherein the water bath heating temperature is 30 ℃ to 45 ℃.