KR20150007576A - Multi-channel nerve conduit comprising carbon nanotube coated phosphate glass fiber and preparation method thereof - Google Patents

Abstract

The present invention aims to provide a microstructure by which nerves can be regenerated more efficiently. The present invention relates to: a multi-channel nerve conduit including phosphate glass fiber coated with a carbon nanotube; and a method for manufacturing the same. The method includes the steps of: a) preparing the phosphate glass fiber; b) coating the phosphate glass fiber with the carbon nanotube; c) preparing a porous and biodegradable polymer tube and a porous-biodegradable polymer sheet; and d) inserting the phosphate glass fiber coated with the carbon nanotube in step b) in the porous-biodegradable polymer tube after covering the phosphate glass fiber with the porous-biodegradable polymer sheet.

Description

TECHNICAL FIELD [0001] The present invention relates to a multi-channel nerve conduit including phosphoric acid glass fibers coated with carbon nanotubes, and a method of manufacturing the same. [0002] MULTI- CHANNEL NERVE CONDUIT COMPRISING CARBON NANOTUBE COATED PHOSPHATE GLASS FIBER AND PREPARATION METHOD THEREOF [

The present invention relates to a multi-channel nerve conduit for connecting cut peripheral nerves using a method of filling a carbon nanotube-coated phosphate glass fiber into a nerve conduit, and a method for manufacturing the same.

In the case of motor neurons and sensory nerve damage, autologous nerve grafting has been used as a method to restore continuity of nerve tissue in order to restore its function. However, And it is difficult to apply it to nerve damage with a length that is a real problem, and it is very difficult to match the thickness and shape of the nerve tissue of the injured area with the nerve tissue to be implanted. In addition, there is a disadvantage that the results vary depending on the degree of nerve injury, the duration of injury, or the age of the patient and the degree of recovery is not yet complete.

Although materials with physico - chemical properties of various compositions and substrates have been developed, sufficient material studies have not yet been made for thorough nerve regeneration and appropriate guidelines are needed.

A nerve conduit is a tube that holds both ends of a severed nerve in an artificially created tube and guides the connection of the nerve into the tube. This method has the following advantages. First, it can prevent the infiltration of scar tissue that interferes with nerve regeneration from the periphery. Second, it can induce the growth of axon (component of nerve that plays a pivotal function of information transmission) in the right direction. , The regeneration promoting substances secreted from the nerve itself are maintained in the tube, while the substances interfering with regeneration can be blocked from the outside.

Materials of these nerve conduits are largely composed of human tissues (veins, neurons, cells removed muscle tissue, etc.), natural polymers (collagen, chitosan, etc.) and synthetic polymers (silicone, polylactic acid, PLA, Polyglycolic acid (PGA), polylactic acid-co-glycolic acid (PLGA), polycaprolactone, poly lactic acid-co -caprolactone, polyhydroxybutyric acid-co-hydroxyvaleric acid, and polyphosphoester) have been studied. However, when the human tissue is used, the supply of the tissue is not sufficient, the use of the natural polymer is limited due to the immune response when it is not autograft, and the natural polymer easily absorbs in the body, It is known that there is still a lot of work to be done to meet the functions of In recent years, researches using synthetic polymers that are easy to supply materials and have excellent physical properties have become mainstream.

The conventional nerve conduit used in clinical practice is a tubular tube with no contents in the conduit and an empty hole. In addition, the nonabsorbable silicone conduit is not porous around the conduit, so nutrients can not penetrate and the nerve regeneration is difficult and slow. In order to overcome these disadvantages, in the prior art, the nerve conduit was made of collagen. For example, Japanese Patent Application Laid-Open No. 2009-045221 discloses a neurobiological apparatus comprising a substrate containing an extracellular matrix in which cells that secrete a neural regenerative agent are placed on a surface, a Schwann cell grown on a collagen substrate, Induction ". However, since collagen has a weak strength, it is difficult to maintain its strength for a certain period of time when it comes into contact with water, body fluids, blood or the like, and it has a disadvantage that its decomposition rate is fast.

Currently, the artificial neural conduits used in clinical practice are imported from all over the world, and products from four US companies (NeuraGen®, PGA, and Caprolactone) made from three types of materials approved by the US FDA and European CE (collagen, Neuroflex®, Neurotube®, and Neurolac®).

These products are difficult to apply to nerve damage and have a short recovery time. Therefore, there is a need to develop a nerve conduit that has a quantitative and stable effect by improving the problems of the prior art.

JP 2009-045221

In order to solve the above object,

The present invention aims at solving the problems of conventional methods for treating nerve damage and providing a microstructure in which nerves can be regenerated more efficiently by using materials other than those commercially available.

The present invention also provides a nerve conduit which serves as a pathway for nerve fiber regeneration, which allows the axon to grow in the right direction, prevents infiltration of tissues that interfere with nerve regeneration, and maintains the regenerative factor secreted from the nerve itself The present invention provides a structure for providing a structure.

It is another object of the present invention to provide a nerve conduit having a microstructure produced by filling the inside with a porous and biodegradable material instead of an empty tube shape used in clinical practice.

The present invention provides a multi-channel neural conduit comprising phosphoric acid glass fibers coated with carbon nanotubes.

Preferably, the carbon nanotubes are carboxylated multi-walled carbon nanotubes.

In addition,

In the manufacture of multi-channel neural conduits for connecting cut peripheral nerves,

a) preparing phosphate glass fibers;

b) coating the phosphoric acid glass fibers with carbon nanotubes;

c) preparing a porous-biodegradable polymeric tube and a porous-biodegradable polymeric sheet; And

d) inserting the carbon nanotube-coated phosphate glass fiber into a porous-biodegradable polymer sheet in step b), and then inserting it into the porous-biodegradable polymer tube. .

In the present invention, the nerve conduit is filled with glass fiber to form a microstructure, so that it is possible to expect an improved nerve regeneration effect over the existing neural conduit.

In addition, by coating carbon nanotubes on glass fibers, carbon nanotubes can be stably and stably attached to glass fibers, and carbon nanotubes can be coated to efficiently increase adhesion and growth of nerve cells.

In addition, the sheet or tube in the present invention is a porous, biocompatible and biodegradable polymer having selective permeability. The sheet used for inserting glass fiber into the nerve conduit is utilized as a drug delivery system, It is possible to regulate the secretion time of the drug such as scar formation inhibitor and the effect by addition of various drugs can be expected.

(A) and (B) are photographs of phosphoric acid glass fibers not coated with carbon nanotubes, and (C) and (D) are photographs of carbon nanotubes It is a photograph of coated phosphoric acid glass fiber.
FIG. 2 is a photograph showing in vitro results of observing the influence of axonal growth on nerve fibers using DRG neurons of (A) phosphate glass fibers and (B) carbon nanotube-coated phosphate glass fibers according to the present invention .
FIG. 3 is a SEM photograph of the inside of a nerve conduit according to the present invention, wherein (A) is a cross-sectional view showing the inside of a nerve conduit, (B) shows a microstructure formed by phosphoric acid glass fibers coated with carbon nanotubes (C) is a photograph showing the structure of a PLDLA sheet wrapped with a carbon nanotube-coated phosphoric acid glass fiber.
4A and 4B are photographs (Figs. 4A and 4B) of a nerve conduit made by wrapping a PLLLA sheet with phosphoric acid glass fiber and phosphoric acid glass fiber coated with the carbon nanotube according to the present invention and inserting it into a porous PLDLA tube, (FIG. 4C) implanted with nerve conduits containing uncoated phosphate glass fibers (FIG. 4C) and an experimental group (FIG. 4D) implanted with nerve conduits containing phosphoric acid glass fibers coated with carbon nanotubes.
FIG. 5 shows axon regeneration (FIG. 5A) after 16 weeks of nerve conduit transplantation containing phosphate glass fibers (FIG. 5A) and axon regeneration (FIG. 5B) after 16 weeks of nerve conduit transplantation with carbon nanotube- .

The present invention provides a multi-channel neural conduit comprising phosphoric acid glass fibers coated with carbon nanotubes.

Preferably, the carbon nanotube is a carboxylated multi-walled carbon nanotube.

In addition, preferably, the phosphoric acid glass fiber coated with the carbon nanotube is wrapped in a porous, biodegradable polymer sheet, and is filled in a tube made of a porous and biodegradable polymer.

Preferably, the diameter of the phosphoric acid glass fiber is 28.1 占 6.6 占 퐉.

In addition,

In the manufacture of multi-channel neural conduits for connecting cut peripheral nerves,

a) preparing phosphate glass fibers;

b) coating the phosphoric acid glass fibers with carbon nanotubes;

c) preparing a porous-biodegradable polymeric tube and a porous-biodegradable polymeric sheet; And

d) inserting the carbon nanotube-coated phosphate glass fiber into a porous-biodegradable polymer sheet in step b), and then inserting it into the porous-biodegradable polymer tube. .

Said step a) can be prepared by mixing P ₂ O ₅ -CaO-Na ₂ O-Fe ₂ O ₃ , preferably in a molar ratio of 50: 40: 5: 5.

In the step b), the phosphoric acid glass fiber prepared in the step a) is treated with hydrochloric acid, immersed in 3-aminopropyl-triethoxysilane, dried, preferably heated, and then immersed in a carbon nanotube solution to be.

Preferably, the carbon nanotubes of the present invention are carboxylated multi-wall carbon nanotubes and the phosphate glass fibers of step a) are selected from the group consisting of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride, And is coated by immersing it in a mixed solution of multiple-wall carbon nanotubes.

In addition, the phosphoric acid glass fiber coated with carbon nanotubes in step b) is cross-reacted with ethylenediamine and 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride to amidify the surface.

Porous, biocompatible and biodegradable polymers are all possible, such as collagen, chitosan, silicone, poly (lactic acid), poly (glycolic acid), polylactic acid-glycolic acid copolymer, caprolactone), polylactic acid-caprolactone copolymer, polyhydroxybutyric acid-hydroxyvaleric acid copolymer, polyphosphoester and PLDLA (poly-L / D-lactide) to be.

The step c) is a step of preparing a nerve conduit that serves as a pathway for regenerating nerve fibers with a porous-biodegradable polymer. It prevents axillary tissue from penetrating into the nerve regeneration preventing nerve regeneration, Lt; RTI ID = 0.0 > a < / RTI > regeneration factor. As the porous-biodegradable polymer, the porous-biodegradable polymer sheet used in the present invention refers to a microfine fiber having a diameter of several tens to several hundreds of nanometers, and generally refers to a fiber produced by electrospinning.

In addition, step d) is a step of wrapping the carbon nanotube-coated phosphate glass fiber prepared through steps a) and b) in a PLDLA sheet, and inserting the glass fiber into the nerve conduit.

Preferably, the phosphate glass fibers produced through steps a) and b) above may have 500 to 1500 strands. The greater the number of fibers, the better the direction of regeneration can be induced during nerve regeneration. Therefore, the phosphate glass fiber of the present invention can induce the regeneration direction of nerve more effectively by containing about 1000 fiber strands.

Also preferably, the sheet or tube is a porous, biocompatible and biodegradable polymer with selective permeability. Biodegradable polymeric materials are gradually degraded in shape and weight due to gradual chemical decomposition in vivo, and they can be largely divided into natural polymeric materials and synthetic polymeric materials. Natural biodegradable polymeric materials include collagen, gelatin, chitosan, and hyaluronic acid, and synthetic biodegradable materials include PLA, PGA, and PCL.

Hereinafter, preferred examples, examples and experimental examples are provided to facilitate understanding of the present invention. However, the following Production Examples, Examples and Experimental Examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following Production Examples, Examples and Experimental Examples.

<Production Example> Production of biodegradable glass fiber

In order to produce phosphate glass fibers, P ₂ O ₅ , CaO, Na ₂ O and Fe ₂ O ₃ should be mixed in a molar ratio of 50:40: 5: 5, respectively, and sodium dihydrogen phosphate (NaH ₂ PO ₄ ) , Di-phosphate pentoxide (P ₂ O ₅ ), calcium carbonate (CaCo ₃ ) and iron (iii) oxide (Fe ₂ O ₃ ) were used. The material was melted at 700 ° C. for 30 minutes and at 1100 ° C. for 1 hour. After cooling at room temperature, the spinning method was used to make it into fiber, which was melted again in an oven at 1100 ° C. When the dissolved solution flows downward and comes into contact with the spinning drum, the drum is turned to produce the fiber.

<Examples> Carbon nanotube-coated phosphate glass fiber fabrication and nerve conduit fabrication

Step 1

The phosphate glass fibers were treated with 1N hydrochloric acid for 5 minutes to positively charge the surface, treated with 2.5% of 3-aminopropyltriethoxysilane at pH 5.0 for 10 seconds, To 120 &lt; 0 &gt; C. The above procedure was repeated 10 times.

Step 2

A solution containing 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride and a carboxylated multi-walled carbon nano tube (MWCNT, Iljin Nanotech, purity> 95% And allowed to bind at room temperature for 3 hours.

Step 3

In order to aminize carbon nanotube-coated phosphate glass fibers, 0.1 M ethylenediamine and 0.012 mM 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride were added at pH 5.0 for 2 hours at room temperature Carbodiimide crosslinking reaction. After the reactants were combined, the phosphate glass fibers were washed several times with distilled water and sterilized by treatment with 70% alcohol and UV.

Step 4

Phosphoric acid glass fibers coated with 900 ± 36 strands of phosphoric acid glass fibers and carbon nanotubes were wrapped in a PLDLA sheet and inserted into porous PLDLA tubes with an inner diameter of 0.8 mm, an outer diameter of 1.0 mm and a length of 12 mm.

Experimental Example 1 SEM observation of carbon nanotube-coated phosphoric acid glass fiber

The carbon nanotubes coated on the phosphoric acid glass fibers prepared in Example 1 were confirmed by SEM (scanning electron microscopy; Hitachi 3000, Japan). The results are shown in FIG.

FIGS. 1A and 1B are SEM images and optical images of the phosphoric acid glass fibers of the production example in which the carbon nanotubes are not coated, FIGS. 1C and 1D are SEM images of the carbon nanotube- It is an optical image. The diameters of the phosphoric acid glass fibers in the production example were 28.1 ± 6.6 μm, and it was confirmed that the carbon nanotubes were uniformly coated on the surface of the phosphoric acid glass fiber as shown in FIGS. 1C and 1D.

<Experimental Example 2> In vitro test for measurement of neuronal cell growth

For in vitro experiments, phosphate glass fibers and carbon nanotube-coated phosphate glass fibers were cut to a length of 20 mm and then arranged on the cover slip in the longitudinal direction of the monolayer, and then the both ends were treated with poly-L / D-lactide ). The cover slip with phosphate glass fiber was placed in a 12-well culture dish, coated overnight at 4 ° C with 20 μg / ml poly-D-lysine, and then coated with 10 μg / ml laminin (Sigma) for 2 hours. The dorsal root ganglion (DRG) of thoracic and lumbar spines of 6-week-old Spargue-Dawley rats was removed and membranes surrounding the ganglion ganglion were removed under a dissecting microscope. Then, the cells were treated with collagenase at a concentration of 2.5 mg / ml for 60 minutes in a constant temperature water bath at 37 ° C. Thereafter, centrifugation was performed at low speed to remove the collagenase, and a pre-warmed culture medium was added to wash out the collagenase. The medium was removed and freshly prepared 0.5 to 1.0 ml culture medium was added and DRG neurons were carefully released and then cultured on a cover slip coated with laminin / poly-D-lysine that had been prepared beforehand. The culture medium was changed every 24 hours after the incubation and cultured for 3 days. To observe the effect of phosphoric acid glass fiber and carbon nanotube coated phosphate glass fiber on the growth of neurons, the cells were fixed with 4% paraformaldehyde for 1 hour and then immunostained. The primary antibody was mouse SMI312 monoclonal antibody for axonal staining of neurons. As a result of culturing for 3 days, it was confirmed that the axon length of the DRG neuron cultured in the carbon nanotube-coated phosphate glass fiber grew longer as shown in FIGS. 2A and 2B.

<Experimental Example 3> Confirmation of the structure of the nerve conduit

A cross-sectional photograph of the nerve conduit in which the phosphate glass fiber was inserted was confirmed by SEM (scanning electron microscopy, Hitachi 3000, Japan) (FIG. 3) in order to confirm the nerve conduit inserted with the phosphate glass fiber manufactured in the above example.

&Lt; Experimental Example 4 > In vivo experiment for confirming nerve regeneration effect

To examine the effect of the carbon nanotube-coated phosphate glass fiber according to the present invention on nerve regeneration, an experimental catheter was implanted into an animal body. The nerve conduits were wrapped with PLDLA sheets of phosphate glass fibers coated with 900 ± 36 fibers and carbon nanotubes on average, and inserted into porous PLDLA tubes with an inner diameter of 0.8 mm, an outer diameter of 1.0 mm, and a length of 12 mm, (Figs. 4A and 4B).

First, 12-week-old female Spargue-Dawley rats were randomly selected and sciatic nerves 5 mm below the hips were cut out to a length of 10 mm, and then treated with carbon nanotube-untreated phosphate glass nerve conduits (FIG. 4C) The carbon nanotube-coated phosphate glass fiber nerve conduit of Example 1 according to the present invention was divided into an experimental group (FIG. 4D) implanted into the nerve injury site with a length of 10 mm. At this time, to prevent the neural tube and neuron from separating from each other, the two ends of the neural tube were sutured by using a micro suture (10-0) at intervals of 180 degrees between the nerve and the nerve.

<Experimental Example 5> Confirmation of nerve regeneration effect in an in vivo experiment

Immunostaining was performed to confirm the growth of the sciatic nerve caused by the implant used in the experiment. After 16 weeks of material transplantation, the sciatic nerve including the 12 mm long graft was removed and fixed in 4% paraformaldehyde. This was treated with 30% sucrose for 3 days and then frozen at a thickness of 16 탆. The cut tissues used mouse SMI312 monoclonal antibody for axonal staining of neurons, confirmed the results by confocal microscopy, and confirmed the number of SMI312 positive axons using the imageJ program. The number of distal SMI312-positive axons transplanted in animals implanted with phosphate glass nerve conduit (FIG. 5A) and in the animal transplanted with carbon nanotube-coated phosphate glass nerve conduit (FIG. 5B) More numbers of SMI312 positive axons were observed in the transplanted animals with the tube-coated phosphate glass nerve conduit (Fig. 5B).

As shown in Experimental Example 2, phosphoric acid glass fibers coated with carbon nanotubes grow longer nerve cells than phosphate glass fibers not coated with carbon nanotubes, It can be said that it can contribute to adhesion growth.

Further, as shown in Experimental Example 5, when the phosphoric acid glass fiber coated with the carbon nanotube was implanted, more axons were observed than when the phosphoric acid glass fiber not coated with the carbon nanotube was implanted. By filling the inside of the nerve conduit with carbon fiber coated with phosphoric acid glass fiber, the microstructure was formed and the direction in which the nerve was grown was induced and the efficiency was increased. Further, it has been found that by coating the phosphoric acid glass fiber with carbon nanotubes, the quantitative and stable effect is exhibited and the nerve can be regenerated more efficiently.

Claims (11)

Multichannel neural conduits containing phosphoric acid glass fibers coated with carbon nanotubes. The multi-channel neural conduit of claim 1, wherein the carbon nanotube is a carboxylated multi-walled carbon nanotube. [2] The method of claim 1, wherein the carbon nanotube-coated phosphate glass fibers are wrapped in a porous, biodegradable polymer sheet and filled in a tube made of a porous and biodegradable polymer. . The method according to claim 1, wherein the diameter of the phosphate glass fibers is 28.1 ± 6.6 μm. In the manufacture of multi-channel neural conduits for connecting cut peripheral nerves,
a) preparing phosphate glass fibers;
b) coating the phosphoric acid glass fibers with carbon nanotubes;
c) preparing a porous-biodegradable polymeric tube and a porous-biodegradable polymeric sheet; And
d) inserting the carbon nanotube-coated phosphate glass fiber into a porous-biodegradable polymer sheet in step b), and then inserting it into the porous-biodegradable polymer tube. . 6. The method of claim 5, wherein said step a) comprises producing using P ₂ O ₅ -CaO-Na ₂ O-Fe ₂ O ₃ . [7] The method according to claim 5, wherein the step b) comprises treating the phosphate glass fibers prepared in the step a) with hydrochloric acid, immersing the fiber in 3-aminopropyl-triethoxysilane, drying and then immersing in the carbon nanotube solution Wherein the method comprises the steps of: [6] The method of claim 5, wherein in step d), the inside of the nerve conduit is filled with phosphate glass fiber to form a microstructure, thereby inducing the direction in which the nerve grows. 6. The method of claim 5, wherein the phosphate glass fibers have 500 to 1500 strands. 6. The method of claim 5, wherein the sheet or tube is made of a porous, biocompatible and biodegradable polymer with selective permeability. 10. The method of claim 9, wherein the sheet or tube is comprised of PLDLA.

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