CN112546303A

CN112546303A - Nerve repair material and preparation method thereof

Info

Publication number: CN112546303A
Application number: CN202011496454.4A
Authority: CN
Inventors: 梁嘉美; 王丹妍; 毛诗哲; 尹家瑞; 谭荣伟; 佘振定
Original assignee: Shenzhen Lando Biomaterials Co ltd
Current assignee: Shenzhen Lando Biomaterials Co ltd
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2021-03-26
Anticipated expiration: 2040-12-17
Also published as: CN112546303B

Abstract

The invention relates to a nerve repair material and a preparation method thereof, wherein the nerve repair material comprises an outer tube and an inner core matrix; the outer tube comprises a multi-layer round tube which is formed by coiling an animal-derived acellular matrix material and performing vacuum pressing; the inner core matrix comprises a natural polymer material and axially oriented microchannels distributed in the natural polymer material. The nerve repair material has good biocompatibility, mechanical property and appropriate degradation rate, and the outer tube with a compact structure formed by vacuum pressing has good barrier function on peripheral fibrous tissues, can prevent the peripheral fibrous tissues from growing in and prevent nerve fibers from escaping; the inner core matrix distributed with the axial orientation micro-channel provides a template for migration and proliferation of nerve cells, and can guide and promote directional regeneration of nerve fibers. The invention reduces the formation of the repair sectional neuroma and promotes the recovery of the nerve function through the synergistic effect of the outer tube and the inner core matrix.

Description

Nerve repair material and preparation method thereof

Technical Field

The invention belongs to the technical field of medical biomaterials, particularly relates to a nerve repair material and a preparation method thereof, and particularly relates to a nerve repair material capable of reducing the formation of repair sectional neuroma, guiding and promoting directional regeneration of nerve fibers and a preparation method thereof.

Background

Repair and reconstruction of peripheral nerve defects is a difficult problem in the field of peripheral nerve injury. When peripheral nerves are separated, the two broken ends are directly anastomosed and repaired, so that a satisfactory function recovery effect cannot be achieved, nerve fibers (sensory and motor nerve fibers) with different properties are easily mismatched, and the anastomotic stoma is easily subjected to problems of escape of nerve fibers, proliferation of connective tissues and the like. Autonerve graft vegetation is considered the gold standard for nerve defect repair, but requires donor nerve sacrifice, has limited sources, and causes donor area damage and loss of function. Therefore, the nerve conduit repair is a promising repair means for nerve defects. The nerve conduit establishes a nerve fiber regeneration bridge between two nerve broken ends, allows selective regeneration and butt joint of nerve fibers, reduces escape of regenerated nerve fibers, prevents growth of peripheral connective tissues, reduces loss of nutrient substances, and provides a relatively closed regeneration microenvironment for the nerve fibers.

The animal derived acellular matrix is a material which retains the natural scaffold structure of the extracellular matrix after acellular treatment. The animal derived acellular matrix has good biocompatibility, degradability and induced tissue regeneration capability, and is widely applied to tissue defect repair. At present, a plurality of animal derived acellular products are approved to enter clinical application and are mainly used as wound repair materials, hernia repair patches, dural patches and the like. In the field of repair of peripheral nerve defects, animal-derived acellular matrices have also begun to attract much attention as nerve conduit constituting materials, but only Cook Biotech company has currently attracted attention

Entering a clinical application stage of nerve repair.

The nerve catheters that are available on the market are generally used for repairing nerve defects with defect lengths of less than 3cm, but have poor repairing effect on longer nerve defects. The reason for this is that the existing nerve conduit has a larger difference from the natural nerve structure, and is mostly a hollow tubular structure, which is not favorable for the adhesion and growth of nerve fibers. In addition, the proximal axon of the severed nerve grows to the distal end at a speed of 1-3mm every day, and partial nerve conduits have the problems of loose tube wall, too fast degradation and the like in clinical application, so that the barrier effect is weakened, and neuroma is easily formed.

Patent 201710124327.3 discloses a nerve repair material composed of decellularized small intestine submucosa, the nerve repair material comprises collagen, polysaccharide substance, active factor and nerve regeneration promoting factor, the repair material has three-dimensional net porous structure, is non-immunogenic, is degradable in vivo, and has a sheet-like or hollow tubular structure; patent 201611209243.1 discloses a biofilm made of a stack of layers of decellularized material. When the material disclosed in the patent is applied to nerve repair, a hollow tubular structure is formed, and the effects of attaching, directionally guiding and promoting growth of nerve fibers are limited.

Patent 200410009259.9 discloses a neural tissue engineering tubular scaffold, which is composed of an outer tube wall of chitosan and a filling matrix with axial multiple channels; patent 201810148281.3 discloses a nerve conduit that enhances the order of axonal regeneration. The outer tube wall of the stent prepared by the methods has weak effect on shielding the ingrowth of fibrous tissues around the nerve endings.

Therefore, the key to determine the clinical application of the nerve repair material is to improve the barrier function of the nerve repair material on peripheral fibrous tissues and improve the promotion effect on the directional regeneration of nerve fibers.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a nerve repair material and a preparation method thereof, in particular to a nerve repair material which can reduce the formation of repair sectional neuroma, guide and promote the directional regeneration of nerve fibers and a preparation method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a nerve repair material comprising an outer tube and an inner core matrix; the outer tube comprises a multi-layer round tube which is formed by coiling an animal-derived acellular matrix material and performing vacuum pressing; the inner core matrix comprises a natural polymer material and axially oriented microchannels distributed in the natural polymer material.

The nerve repair material has good biocompatibility, mechanical property and appropriate degradation rate, and the outer tube with a compact structure formed by vacuum pressing has good barrier function on peripheral fibrous tissues, can prevent the peripheral fibrous tissues from growing in and prevent nerve fibers from escaping; the inner core matrix distributed with the axial orientation micro-channel makes up the application limitation of the hollow nerve conduit, provides a template for the migration and proliferation of nerve cells, and can guide and promote the directional regeneration of nerve fibers. The invention reduces the formation of the repair sectional neuroma and promotes the recovery of the nerve function through the synergistic effect of the outer tube and the inner core matrix.

Preferably, the number of layers of the multilayer round tube is 2 to 12, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 layers.

Preferably, the animal derived acellular matrix material comprises small intestine submucosa, peritoneum, pericardium or dermis derived from animals.

Preferably, the thickness of the outer tube is 250-800 μm, such as 250 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, or 800 μm, and other specific values within the value range can be selected, which is not described herein again, and is preferably 400-800 μm.

The specific selection of the thickness of the tube body of the outer tube is 250-800 μm because the semi-permeability of the outer tube is reduced if the thickness is further increased, and the exchange of substances inside and outside the tube is influenced; further reduction reduces the barrier function of the outer tube to the surrounding fibrous tissue, which is susceptible to invasion by inflammatory cells or surrounding fibrous tissue. Wherein 400-600 μm is the range where the effect is optimal.

Preferably, the inner diameter of the outer tube is 2-7mm, such as 2mm, 3mm, 4mm, 5mm, 6mm or 7mm, and other specific values within the numerical range can be selected, and are not described in detail herein.

Preferably, the length of the outer tube is 10-60mm, for example 10mm, 20mm, 30mm, 40mm, 50mm or 60mm, and other specific points in the numerical range can be selected, and are not described in detail herein.

The length of the outer tube is specially selected to be 10-60mm because the existing hollow nerve conduit can be generally used for repairing the nerve defect smaller than 30mm, and the repairing effect of the long-section nerve defect can be improved after the hollow nerve conduit is compounded with the inner core matrix; when the length of the nerve defect is too large, the nerve defect needs to be repaired by means of autologous nerve transplantation and the like. The length of the outer tube according to the invention is therefore selected in particular to be 10-60 mm.

In the present invention, the outer tube includes a tube body, an inner surface and an outer surface, and the tube body and the outer surface are independently distributed with active factors for inhibiting escape of nerve fibers.

In a more preferred embodiment of the present invention, the tube body and the outer surface of the outer tube are further independently distributed with active factors for inhibiting escape of nerve fibers, and further perform a repulsive barrier function, and the migration direction of axons is adjusted to guide the nerve fibers to accurately reach the target organ.

Preferably, the active factor inhibiting escape of nerve fibers comprises chondroitin sulfate proteoglycan and/or myelin associated glycoprotein. The chondroitin sulfate proteoglycan CSPGs are a group of proteoglycan formed by covalently combining core protein and chondroitin sulfate, can play a repulsive barrier function on a key node of axon extension, and adjust the migration direction of axon to guide nerve fibers to accurately reach target organs; myelin-associated glycoprotein MAG is a major component of myelin-derived nerve growth inhibitor, which inhibits axon growth.

In the present invention, both ends of the inner core matrix are independently retracted into the outer tube by 2-4mm, such as 2mm, 2.5mm, 3mm, 3.5mm or 4mm, and other specific values within the value range can be selected, which is not described herein again.

The two ends of the inner core matrix are independently retracted into the outer tube by 2-4mm because the nerve broken end can be conveniently placed in the nerve repairing material during nerve repairing, the nerve broken end is effectively protected, and the growth of peripheral fibrous tissues into the nerve broken end is reduced.

Preferably, the natural polymer material comprises any one or a combination of at least two of collagen, gelatin, chitosan, silk fibroin or hyaluronic acid; the combination of at least two of the above-mentioned compounds, such as the combination of collagen and gelatin, the combination of chitosan and silk fibroin, the combination of silk fibroin and hyaluronic acid, etc., can be selected in any combination manner, and will not be described in detail herein.

Preferably, the inner core matrix further comprises an active factor for promoting the growth of nerve fibers distributed in the natural polymer material.

In a more preferred embodiment of the present invention, the inner core matrix further comprises an active factor distributed in the natural polymer material for promoting the growth of nerve fibers, so as to further promote tissue regeneration.

Preferably, the mass ratio of the natural polymer to the active factor for promoting the growth of nerve fibers is (50-100):1, for example, 50:1, 60:1, 70:1, 80:1, 90:1 or 100:1, and other specific values in the numerical range can be selected, and are not repeated herein.

Preferably, the nerve fiber growth promoting factor includes amorphous polyphosphate, the number of repeating units of the amorphous polyphosphate is 10-100, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100, and other specific values within the range of the values can be selected, and are not repeated herein.

The amorphous polyphosphate is preferred as the active factor for promoting the growth of the nerve fibers because the amorphous polyphosphate is inorganic salt and has relatively stable property, and the amorphous polyphosphate is not easy to lose activity in the process of preparing the nerve repair material; and exists in cells and outside cells in a large amount, and can release energy after being decomposed by human alkaline phosphatase ALP to promote tissue regeneration.

Preferably, the amorphous polyphosphate comprises any one of amorphous magnesium polyphosphate, amorphous zinc polyphosphate or amorphous calcium polyphosphate, or a combination of at least two of them; the combination of at least two of the above-mentioned compounds, such as the combination of amorphous magnesium polyphosphate and amorphous zinc polyphosphate, the combination of amorphous zinc polyphosphate and amorphous calcium polyphosphate, the combination of amorphous magnesium polyphosphate and amorphous calcium polyphosphate, and the like, can be selected in any combination manner, and will not be described in detail herein.

In summary, the nerve repair material according to the present invention utilizes the animal-derived acellular matrix material with good biocompatibility to construct the outer tube of the nerve repair material, wherein the outer tube is a multi-layer circular tube formed by coiling the animal-derived acellular matrix material, and the outer tube has a compact structure through vacuum pressing, and carries active factors for inhibiting escape of nerve fibers, so as to synergistically reduce neuroma formation. Introducing an inner core matrix into the hollow tube, wherein the inner core matrix comprises natural polymers and active factors for promoting the growth of nerve fibers, and synergistically promotes the growth of the nerve fibers; the inner core matrix is provided with a plurality of micro-channels with axial orientation, which can guide the nerve fiber to grow directionally.

Preferably, the number of the microchannels is 5-50, for example, 5, 10, 20, 30, 40 or 50, and other specific point values within the range of values can be selected, and are not described herein again.

Preferably, the pore diameter of the micro channel is 100-1000 μm, such as 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm, etc., and other specific values within the numerical range can be selected, which is not described herein again.

The specific selection of the pore diameter of the microchannel is 100-. .

Preferably, the inner core matrix further comprises radially oriented microchannels distributed in the natural polymeric material.

Preferably, the porosity of the inner core matrix is not lower than 95%, such as 95%, 96%, 97%, 98%, or 99%, and other specific values within the numerical range can be selected, which is not described in detail herein.

In a second aspect, the present invention provides a method for preparing the nerve repair material according to the second aspect, the method comprising the steps of:

(1) laying and placing the animal-derived acellular matrix material, winding the animal-derived acellular matrix material around a rod core material to form a multi-layer circular tube, and then performing vacuum pressing, vacuum freeze drying and demoulding to obtain a hollow outer tube;

(2) mixing and reacting a natural polymer material and a cross-linking agent in a solvent to obtain an inner core matrix solution; then carrying out deaeration treatment on the inner core matrix liquid, injecting the inner core matrix liquid into a cylindrical mold, sealing and freezing, carrying out vacuum freeze drying, and demoulding; finally, perforating the inner core matrix by using ultraviolet laser to form an inner core matrix containing the micro-channel;

(3) respectively cutting the outer tube and the inner core matrix, and pushing the inner core matrix into the outer tube;

(4) carrying out vacuum high-temperature crosslinking treatment on the material obtained in the step (3);

(5) and (5) sterilizing the material obtained in the step (4) to obtain the nerve repairing material.

In the invention, after the animal-derived acellular matrix material is curled around the rod core material by one layer in the step (1), the inner side surface of the residual animal-derived acellular matrix material is coated with an active factor solution for inhibiting escape of nerve fibers, and then the animal-derived acellular matrix material is continuously curled into a multilayer round tube.

Preferably, the concentration of the solution of the active factor for inhibiting escape of nerve fibers is 10-30 μ g/mL, such as 10 μ g/mL, 15 μ g/mL, 20 μ g/mL, 25 μ g/mL or 30 μ g/mL, and other specific points in the numerical range can be selected, and are not repeated herein.

Preferably, the solution of the active factor for inhibiting escape of nerve fibers is coated on the inner side surface in an amount of 60-200 μ L/mm²E.g. 60 μ L/mm²、80μL/mm²、100μL/mm²、120μL/mm²、150μL/mm²、200μL/mm²And other specific point values in the numerical range can be selected, and are not described in detail herein.

Preferably, step (1) is to coat the outer surface of the round tube with a solution of an active factor for inhibiting escape of nerve fibers after rolling the round tube into a multi-layer round tube.

Preferably, the coating amount of the solution of the active factor for inhibiting escape of nerve fibers on the outer surface of the round tube is 60-200 mu L/mm²E.g. 60 μ L/mm²、80μL/mm²、100μL/mm²、120μL/mm²、150μL/mm²、200μL/mm²And other specific point values in the numerical range can be selected, and are not described in detail herein.

Preferably, the rod core material in the step (1) is a stainless steel rod core coated with polytetrafluoroethylene.

Preferably, the vacuum drying time in step (1) is 16-24h, such as 16h, 18h, 20h, 22h or 24h, and other specific values in the numerical range can be selected, and are not repeated herein.

In the invention, the preparation method of the animal derived acellular matrix material in the step (1) comprises the following steps:

and (3) cleaning the target tissue material, removing redundant tissues, performing virus inactivation treatment, and then performing cell removal treatment to obtain the tissue material.

Preferably, the virus inactivation treatment comprises: soaking the pretreated target tissue material in peroxyacetic acid-ethanol solution for 1-3h (e.g. 1h, 2h, 3h, etc.), and washing in phosphate buffer solution for 3-5 times (e.g. 3 times, 4 times, 5 times, etc.) with shaking for 15-30min (e.g. 15min, 20min, 25min, 30min, etc.) each time.

Preferably, the concentration of the peracetic acid in the peracetic acid-ethanol solution is 0.1-1%, for example, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, or 1%, etc., and the concentration of the ethanol is 5-25%, for example, 5%, 10%, 15%, 20%, or 25%, etc., and other specific values within the above numerical range can be selected, and are not repeated herein.

Preferably, the weight ratio of the peracetic acid-ethanol solution to the target tissue material is (8-12):1, for example, 8:1, 9:1, 10:1, 11:1 or 12:1, and other specific points within the numerical range can be selected, and are not described in detail herein.

Preferably, the weight ratio of the phosphate buffer solution to the target tissue material is (20-30):1, for example, 20:1, 22:1, 24:1, 26:1, 28:1 or 30:1, and other specific points in the numerical range can be selected, and are not described in detail herein.

Preferably, the decellularization treatment comprises: soaking the target tissue material subjected to virus inactivation treatment in strong alkali for 1-24h (e.g. 1h, 5h, 10h, 15h, 18h, 20h, 24h, etc.), and then washing in phosphate buffer solution with shaking for 3-5 times (e.g. 3 times, 4 times, 5 times), each time for 15-30min (e.g. 15min, 20min, 25min, 30min, etc.). Other specific point values within the above numerical range can be selected, and are not described in detail herein.

Preferably, the strong alkaline solution is a sodium hydroxide-EDTA solution, wherein the sodium hydroxide concentration is 0.01-0.2M (e.g., 0.01M, 0.02M, 0.05M, 0.1M, 0.15M, 0.2M, etc.) and the EDTA concentration is 0.08-0.15M (e.g., 0.08M, 0.10M, 0.12M, 0.14M, 0.15M, etc.).

In the invention, the mixed reaction system in the step (2) further comprises an active factor for promoting the growth of nerve fibers.

Preferably, the mixed reaction system in the step (2) further comprises fibrin glue as a binder.

Preferably, the cross-linking agent in step (2) comprises any one of glutaraldehyde, formaldehyde, genipin or 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide or a combination of at least two of them.

Preferably, the solvent in step (2) is an acetic acid solution of 0.03-0.05M, and the concentration of the acetic acid solution may be 0.03M, 0.04M, or 0.05M, and other specific values within the range may be selected, which is not described herein again.

Preferably, the reaction temperature in step (2) is 4-10 ℃, for example, 4 ℃, 5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃ or 10 ℃, and the like, and the time is 2-5h, for example, 2h, 3h, 4h or 5h, and other specific values in the above numerical range can be selected, and are not repeated herein.

Preferably, after the reaction in step (2) is finished, the mixture is allowed to stand for 12 to 24 hours, for example, 12 hours, 15 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours, and other specific values in the numerical range can be selected, and thus, the details are not repeated.

Preferably, the concentration of the natural polymer material in the inner core matrix solution in step (2) is 3-12mg/mL, such as 3mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 10mg/mL or 12mg/mL, etc., and the concentration of the cross-linking agent is 0.01-5mg/mL, such as 0.01mg/mL, 0.1mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL or 5mg/mL, etc., and other specific values in the above numerical range can be selected, which is not repeated herein.

Preferably, the temperature of the seal freezing in the step (2) is-80 to-60 ℃, such as-80 ℃, 75 ℃, 70 ℃, 65 ℃ or-60 ℃, and the like, and the time is 4 to 8 hours, such as 4 hours, 5 hours, 6 hours, 7 hours or 8 hours, and other specific values in the above numerical value range can be selected, and are not repeated herein.

Preferably, the vacuum freeze-drying time in step (2) is 16-24h, such as 16h, 18h, 20h, 22h or 24h, and other specific values in the value range can be selected, and are not described in detail herein.

Preferably, the parameters of the ultraviolet laser drilling in the step (2) are as follows: a speed of 200-; current 37A; the frequency is 20-30kHz, such as 20kHz, 22kHz, 25kHz, 28kHz, 30kHz, and the like.

In the present invention, the temperature of the vacuum high-temperature crosslinking treatment in step (4) is 100-.

The outer tube in the nerve repairing material provided by the invention is subjected to vacuum pressing and physical crosslinking treatment, so that the mechanical property is enhanced, and the nerve repairing material is more favorable for surgical suture.

The outer tube in the nerve repair material is subjected to specific vacuum pressing and physical crosslinking treatment, and the inner core matrix is subjected to specific chemical crosslinking treatment and physical crosslinking treatment, so that the degradation time of the nerve repair material can be comprehensively adjusted, the degradation rate of the final nerve repair material is matched with the nerve repair regeneration rate, and the template effect of guiding nerve fiber regeneration is played.

Preferably, the sterilization treatment in step (5) comprises ethylene oxide sterilization, high-energy electron beam irradiation sterilization or gamma ray sterilization.

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

(1) the nerve repair material provided by the invention comprises an outer tube and an inner core matrix, wherein the outer tube can prevent peripheral fibrous tissues from growing in and prevent nerve fibers from escaping, the inner core matrix provides a template for migration and proliferation of nerve cells, the regeneration of the nerve fibers is facilitated, and the repair of the sectional neuroma is prevented and the recovery of the nerve function is promoted through the synergistic effect of the outer tube and the inner core matrix.

(2) The outer tube body is formed into a compact structure by vacuum pressing 2-12 layers of animal-derived acellular matrix materials, so that the barrier function can be enhanced, and the growth of surrounding fibrous tissues can be effectively prevented. Active factors for inhibiting escape of nerve fibers can be further distributed on the outer surface of the outer tube and in the tube body, and the new axons can be prevented from escaping outwards of the tube.

(3) The inner core matrix makes up the application limitation of the hollow nerve conduit and is beneficial to the migration and proliferation of nerve cells. The inner core matrix comprises natural macromolecules or further contains active factors for promoting the growth of nerve fibers, and is provided with a plurality of axially oriented microchannels, so that the nerve fibers can be guided and promoted to directionally regenerate, and the innervation to target organs is restored.

(4) The active factors for promoting the growth of nerve fibers contained in the inner core matrix of the nerve repair material are amorphous polyphosphate which is inorganic salt, so that the property is relatively stable, and the activity is not easy to lose in the process of preparing the nerve repair material.

(5) The outer tube of the nerve repair material is subjected to vacuum pressing and physical crosslinking treatment, and the inner core matrix is subjected to physical crosslinking and chemical crosslinking treatment, so that the degradation time of the nerve repair material can be adjusted, the degradation rate of the nerve repair material is matched with the nerve repair regeneration rate, and the template function of guiding nerve fiber regeneration is played.

(6) After the outer tube is subjected to vacuum pressing and physical crosslinking treatment, the mechanical property is enhanced, and the surgical suture is facilitated.

(7) The invention adopts the laser drilling process to form a plurality of axially oriented microchannels, which can guide the directional regeneration of nerve fibers and recover the domination of target organs. Compared with some methods for manufacturing the axial orientation micro-channel by mold forming (the micro-channel needs to be demoulded after forming), the laser punching method has small damage to the inner core matrix structure and higher yield.

(8) The nerve repair material does not contain chemically synthesized macromolecules and has better biocompatibility.

Drawings

FIG. 1 is a schematic view showing the preparation of a neural restoration material according to the present invention in which an outer tube is rolled into a tube; wherein, 10-acellular matrix material; 11-a rod core; 12-a solution of an active factor that inhibits escape of nerve fibers; 13-outer surface of outer tube.

Fig. 2 is a schematic view of a 3-layer structure of a radial section of an outer tube of a nerve repair material according to the present invention, which is not vacuum-compressed.

FIG. 3 is a schematic structural view of an outer tube of the nerve repair material according to the present invention; wherein, 101-a pipe body; 102-an outer surface; 103-inner surface.

FIG. 4 is a schematic structural view of an inner core matrix in the nerve repair material according to the present invention; wherein, 201-natural polymer material; 202-axially oriented microchannels.

FIG. 5 is a schematic radial cross-sectional view of an arrangement of three axially oriented microchannels in an inner core matrix of a nerve repair material according to the present invention, wherein 201 is a natural polymer material; 202-axially oriented microchannels.

Fig. 6 is a schematic structural view of the nerve repair material after the outer tube and the inner core matrix of the nerve repair material according to the present invention are combined.

FIG. 7 is a schematic cross-sectional view of a nerve repair material according to the present invention in an axial direction; wherein, 101-a pipe body; 201-natural polymer material; 202-axially oriented microchannels.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

FIG. 1 is a schematic view showing the preparation of a neural restoration material according to the present invention in which an outer tube is rolled into a tube; wherein, 10-acellular matrix material; 11-a rod core; 12-a solution of an active factor that inhibits escape of nerve fibers; 13-outer surface of outer tube. The outer tube of the nerve repairing material is formed by coiling acellular matrix material 10 around a rod core 11 into a tube, after the acellular matrix material 10 is coated with 1 layer of the rod core 11, the inner side surface of the acellular matrix material 10 is coated with active factors 12 for inhibiting escape of nerve fibers. After the curling is continued to form a 2-12-layer structure, the outer surface 13 of the material is coated with active factors 12 for inhibiting escape of nerve fibers.

FIG. 3 is a schematic structural view of an outer tube of the nerve repair material according to the present invention; wherein, 101-a pipe body; 102-an outer surface; 103-inner surface. The tube 101, the outer surface 102 contains an active factor that inhibits escape of nerve fibers, while the inner surface 103 does not contain an active factor that inhibits escape of nerve fibers.

FIG. 4 is a schematic structural view of an inner core matrix in the nerve repair material according to the present invention; wherein, 201-natural polymer material; 202-axially oriented microchannels. The core matrix comprises a natural polymer material 201, a plurality of axially oriented microchannels 202. The natural polymer material 201 also contains an active factor for promoting the growth of nerve fibers.

FIG. 7 is a schematic cross-sectional view of a nerve repair material according to the present invention in an axial direction; wherein, 101-a pipe body; 201-natural polymer material; 202-axially oriented microchannels. The two ends of the inner core matrix are respectively retracted into the outer tube by 2-4 mm.

The collagen involved in the following examples is provided by Shenzhen orchid degree biomaterial GmbH, and the involved amorphous polyphosphate is derived from Germany ocean nanotechnology GmbH. Other starting materials and reagents required for the assay are also commercially available.

Example 1

The embodiment provides a nerve repair material, which is prepared by the following steps:

(1) washing fresh small intestines of the pigs with clear water, mechanically removing a serosal layer, a muscular layer and a mucous layer to obtain a submucosal tissue of the small intestines of the pigs, and weighing the submucosal tissue of the small intestines of the pigs by using a balance. Cutting porcine small intestinal submucosa tissue into small sections of about 10cm, cutting the small intestinal submucosa tissue into sheet-shaped materials along an axis, soaking the sheet-shaped materials in 0.1% peroxyacetic acid-10% ethanol solution for 3h, carrying out virus inactivation treatment, wherein the weight of the soaking solution is 10 times of the weight of the tissue, and then shaking and cleaning the tissue materials by using phosphate buffer solution with the pH value of 7 for 5 times, 20min each time, and the weight of the cleaning solution is 20 times of the weight of the tissue. After the tissue material was slightly drained, the tissue material was soaked in 0.03M sodium hydroxide-0.1M EDTA solution for 12 hours for decellularization, and the tissue material was washed with phosphate buffer at pH 7 by shaking for 20min each time for 5 times.

(2) Chondroitin sulfate proteoglycan is weighed and dissolved in phosphate buffer solution, and the concentration is 10 mug/mL. Laying down the acellular small intestine submucosa tissue, curling around a stainless steel rod core (the inner diameter is 3mm) coated with polytetrafluoroethylene, coating the material on the rod core by one layer, and coating chondroitin sulfate proteoglycan solution (the coating amount is 200 mu L/mm) on the inner side surface of the rest laid material²). Continuously curling into a tube to form 4 layers of round tubes, coating chondroitin sulfate proteoglycan solution (coating amount is 200 mu L/mm) on the outer surface of the round tube²) Pressing with cylindrical stainless steel fixture with holes on the wall, drying in vacuum freeze drier for 24 hr, and demolding to obtain hollow cylindrical outer tube with wall thickness of 300 μm.

(3) Weighing collagen, adding 60mL of 0.05M acetic acid solution, and stirring uniformly to make the concentration of the collagen be 10 mg/mL. 12mg of amorphous magnesium polyphosphate (degree of polymerization 10-100) powder was weighed and dispersed in 30mL of 0.05M acetic acid solution. The amorphous magnesium polyphosphate suspension was added to the collagen solution at a rate of 0.5mL/min and the system was mixed well at 4 ℃.5mg of glutaraldehyde crosslinking agent was weighed, dissolved in 10mL of 0.05M acetic acid solution, and stirred well. Adding the glutaraldehyde solution into the collagen solution, continuously stirring the system for 4h at 4 ℃, placing the system in a refrigerator at 4 ℃ for 12h, taking out the system, continuously stirring for 1h, and carrying out vacuum defoaming treatment to obtain the inner core matrix solution.

(4) Extracting the inner core matrix solution with a syringe, injecting into a cylindrical mold with an inner diameter of 3mm, freezing in a refrigerator at-80 deg.C for 4h, and lyophilizing in a vacuum freeze-drying machine for 24 h. After demolding, the molded inner core matrix is placed in an ultraviolet laser marking machine, the equipment parameters are set to be 300mm/s of speed, 37A of current and 20kHz of frequency, and laser punching is carried out along the axial direction of the inner core matrix to form 9 through micro channels (the aperture is 300 mu m).

(5) The outer tube is cut to 30mm, the inner core matrix is cut to 24mm, and the inner core matrix is slowly pushed into the outer tube by using forceps, so that two ends of the inner core matrix are retracted into the outer tube by 3mm respectively. And (3) placing the combined nerve repairing material in a vacuum drying box, and performing vacuum high-temperature crosslinking for 24 hours at 120 ℃ with the vacuum degree of 100 Pa. And finally, sterilizing by using ethylene oxide to obtain the nerve repairing material.

The nerve repair material prepared by the embodiment is provided with an outer tube and an inner core matrix, wherein the tube body of the outer tube is formed into a compact structure by vacuum pressing of 4 layers of animal-derived acellular matrix materials, so that the barrier function can be enhanced, peripheral fibrous tissues are effectively prevented from growing into the outer tube, and escape of nerve fibers is prevented; the outer surface of the outer tube and the inside of the tube body are also distributed with chondroitin sulfate proteoglycan which is an active factor for inhibiting escape of nerve fibers, and can prevent the new axons from escaping outwards. The inner core matrix consists of natural high molecular collagen and amorphous magnesium polyphosphate as an active factor for promoting the growth of nerve fibers, and is provided with 9 axially oriented microchannels, which can guide and promote the directional regeneration of the nerve fibers, recover the domination of target organs, provide a template for the migration and proliferation of nerve cells and facilitate the regeneration of the nerve fibers. In conclusion, the synergistic effect of the outer tube and the inner core matrix prevents the repair of the sectional neuroma and promotes the recovery of the nerve function.

The outer tube of the nerve repair material is subjected to vacuum pressing and physical crosslinking treatment under specific conditions, and the inner core matrix is subjected to physical crosslinking and chemical crosslinking treatment under specific conditions, so that the degradation time of the nerve repair material can be comprehensively adjusted, the degradation rate of the nerve repair material is matched with the nerve repair regeneration rate, and the template effect of guiding nerve fiber regeneration is played. And after the outer tube is subjected to vacuum pressing and physical crosslinking treatment, the mechanical property is enhanced, and the surgical suture is facilitated. The nerve repair material does not contain chemically synthesized macromolecules and has better biocompatibility.

Example 2

(1) washing fresh small intestines of the pigs with clear water, mechanically removing a serosal layer, a muscular layer and a mucous layer to obtain a submucosal tissue of the small intestines of the pigs, and weighing the submucosal tissue of the small intestines of the pigs by using a balance. Cutting porcine small intestine submucosa tissue into small sections of about 10cm, cutting the small intestine submucosa tissue into sheet-shaped materials along an axis, soaking the sheet-shaped materials in 0.5 percent peroxyacetic acid-15 percent ethanol solution for 2h for virus inactivation treatment, wherein the weight of the soaking solution is 8 times of the weight of the tissue, and then shaking and cleaning the tissue materials by phosphate buffer solution with the pH value of 7 for 3 times, 30min each time, and the weight of the cleaning solution is 30 times of the weight of the tissue. After the tissue material was slightly drained, the tissue material was soaked in 0.08M sodium hydroxide-0.08M EDTA solution for 18h for decellularization, and the tissue material was washed with phosphate buffer at pH 7 by shaking for 30min 3 times.

(2) Myelin associated glycoprotein was weighed and dissolved in phosphate buffer at a concentration of 20. mu.g/mL. Laying the acellular small intestine submucosa tissue, coiling around a stainless steel rod core (with the inner diameter of 5mm) coated with polytetrafluoroethylene, coating a layer of the material on the rod core, and coating myelin sheath related glycoprotein solution (the coating amount is 150 mu L/mm) on the inner side surface of the rest of the laid material²). Continuously curling into tubes to form 8 layers of round tubes, and coating myelin associated glycoprotein solution (coating amount is 150 mu L/mm) on the outer surfaces of the round tubes²) Pressing with cylindrical stainless steel fixture (with hole on wall), drying in vacuum freeze drier for 24 hr, and demolding to obtain hollow cylindrical outer tube with wall thickness of 500 μm.

(3) Gelatin is weighed and added with 60mL of 0.05M acetic acid solution to be uniformly stirred, so that the concentration of the gelatin is 10 mg/mL. 12mg of amorphous zinc polyphosphate (degree of polymerization: 10-100) powder was weighed out and dispersed in 30mL of a 0.05M acetic acid solution. The amorphous zinc polyphosphate suspension is added into the gelatin solution at the rate of 0.5mL/min, and the system is mixed uniformly at 4 ℃.5mg of formaldehyde crosslinking agent was weighed, dissolved in 10mL of 0.05M acetic acid solution, and stirred well. Adding the formaldehyde solution into the gelatin solution, continuously stirring the system for 4h at 4 ℃, placing the system in a refrigerator at 4 ℃ for 12h, taking out the system, continuously stirring for 1h, and carrying out vacuum defoaming treatment to obtain the inner core matrix solution.

(4) Extracting the inner core matrix solution with a syringe, injecting into a cylindrical mold with an inner diameter of 5mm, freezing in a refrigerator at-70 deg.C for 4h, and lyophilizing in a vacuum freeze-drying machine for 24 h. After demolding, the molded inner core matrix is placed in an ultraviolet laser marking machine, the equipment parameters are set to be 300mm/s of speed, 37A of current and 20kHz of frequency, and laser punching is carried out along the axial direction of the inner core matrix to form 15 through micro channels (the aperture is 300 mu m).

(5) The outer tube is cut to 50mm, the inner core matrix is cut to 44mm, and the inner core matrix is slowly pushed into the outer tube by using forceps, so that two ends of the inner core matrix are retracted into the outer tube by 3mm respectively. And (3) placing the combined nerve repairing material in a vacuum drying box, and performing vacuum high-temperature crosslinking for 24 hours at 120 ℃ with the vacuum degree of 100 Pa. And finally, sterilizing by high-energy electron beam irradiation to obtain the nerve repair material.

The nerve repair material prepared by the embodiment is provided with an outer tube and an inner core matrix, wherein the tube body of the outer tube is formed into a compact structure by 8 layers of animal-derived acellular matrix materials through vacuum pressing, so that the barrier function can be enhanced, peripheral fibrous tissues are effectively prevented from growing into the outer tube, and escape of nerve fibers is prevented; myelin sheath related glycoprotein which is an active factor for inhibiting escape of nerve fibers is also distributed on the outer surface of the outer tube and in the tube body, and can prevent the new axons from escaping outwards. The inner core matrix consists of natural polymer gelatin and active factor amorphous zinc polyphosphate for promoting the growth of nerve fibers, is provided with 15 axially oriented microchannels, can guide and promote the directional regeneration of the nerve fibers, recovers the innervation of target organs, provides a template for the migration and proliferation of nerve cells and is favorable for the regeneration of the nerve fibers. In conclusion, the synergistic effect of the outer tube and the inner core matrix prevents the repair of the sectional neuroma and promotes the recovery of the nerve function.

Example 3

(1) washing fresh small intestines of the pigs with clear water, mechanically removing a serosal layer, a muscular layer and a mucous layer to obtain a submucosal tissue of the small intestines of the pigs, and weighing the submucosal tissue of the small intestines of the pigs by using a balance. Cutting porcine small intestinal submucosa tissue into small sections of about 10cm, cutting the small intestinal submucosa tissue into sheet-shaped materials along an axis, soaking the sheet-shaped materials in 0.8% peroxyacetic acid-20% ethanol solution for 1h, performing virus inactivation treatment, wherein the weight of the soaking solution is 10 times of the weight of the tissue, and then shaking and cleaning the tissue materials by using phosphate buffer solution with the pH value of 7 for 4 times, 25min each time, and the weight of the cleaning solution is 25 times of the weight of the tissue. After the tissue material was slightly drained, the tissue material was soaked in 0.15M sodium hydroxide-0.12M EDTA solution for 12 hours for decellularization, and the tissue material was washed with phosphate buffer at pH 7 by shaking 4 times for 25min each time.

(2) Myelin associated glycoprotein was weighed and dissolved in phosphate buffer at a concentration of 20. mu.g/mL. Laying and placing the acellular small intestine submucosa tissue, coiling the acellular small intestine submucosa tissue around a stainless steel rod core (the inner diameter is 7mm) coated with polytetrafluoroethylene, coating a layer of material on the rod core, and coating myelin sheath related glycoprotein solution (the coating amount is 200 mu L/mm)²). Continuously curling into tube to form 10 layers of round tubes, and coating myelin associated glycoprotein solution (coating amount is 200 mu L/mm) on the outer surface of the round tubes²) Pressing with cylindrical stainless steel fixture (with hole on wall), drying in vacuum freeze drier for 24 hr, and demolding to obtain hollow cylindrical outer tube with wall thickness of 700 μm.

(3) Weighing chitosan, adding 60mL of 0.05M acetic acid solution, and uniformly stirring to ensure that the concentration of the chitosan is 10 mg/mL. 12mg of amorphous calcium polyphosphate (degree of polymerization 10-100) powder was weighed out and dispersed in 30mL of 0.05M acetic acid solution. Adding the amorphous calcium polyphosphate suspension into the chitosan solution at the speed of 0.5mL/min, and uniformly mixing the system at 4 ℃.5mg of genipin cross-linking agent is weighed and dissolved in 10mL of 0.05M acetic acid solution, and the mixture is stirred uniformly. Adding the genipin solution into the chitosan solution, continuously stirring the system at 4 ℃ for 4h, placing the system in a refrigerator at 4 ℃ for 12h, taking out the system, continuously stirring for 1h, and carrying out vacuum defoaming treatment to obtain the inner core matrix solution.

(4) Extracting the inner core matrix solution with a syringe, injecting into a cylindrical mold with an inner diameter of 7mm, freezing in a refrigerator at-60 deg.C for 4h, and lyophilizing in a vacuum freeze-drying machine for 24 h. After demolding, the molded inner core matrix is placed in an ultraviolet laser marking machine, the equipment parameters are set to be 300mm/s of speed, 37A of current and 20kHz of frequency, and laser punching is carried out along the axial direction of the inner core matrix to form 20 through micro channels (the aperture is 300 mu m).

(5) The outer tube is cut to 40mm, the inner core matrix is cut to 36mm, and the inner core matrix is slowly pushed into the outer tube by using forceps, so that two ends of the inner core matrix are respectively retracted into the outer tube by 2 mm. And (3) placing the combined nerve repairing material in a vacuum drying box, and performing vacuum high-temperature crosslinking for 24 hours at 120 ℃ with the vacuum degree of 100 Pa. And finally, sterilizing by gamma rays to obtain the nerve repairing material.

The nerve repair material prepared by the embodiment is provided with an outer tube and an inner core matrix, wherein the tube body of the outer tube is formed into a compact structure by vacuum pressing of 10 layers of animal-derived acellular matrix materials, so that the barrier function can be enhanced, peripheral fibrous tissues are effectively prevented from growing into the outer tube, and escape of nerve fibers is prevented; myelin sheath related glycoprotein which is an active factor for inhibiting escape of nerve fibers is also distributed on the outer surface of the outer tube and in the tube body, and can prevent the new axons from escaping outwards. The inner core matrix consists of natural high molecular chitosan and amorphous calcium polyphosphate as an active factor for promoting the growth of nerve fibers, and is provided with 20 axially oriented microchannels, so that the directional regeneration of the nerve fibers can be guided and promoted, the innervation to target organs is recovered, a template is provided for the migration and proliferation of nerve cells, and the regeneration of the nerve fibers is facilitated. In conclusion, the synergistic effect of the outer tube and the inner core matrix prevents the repair of the sectional neuroma and promotes the recovery of the nerve function.

The applicant states that the present invention is illustrated by the above examples to a nerve repair material and a method for preparing the same, but the present invention is not limited to the above examples, that is, it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims

1. A nerve repair material, comprising an outer tube and an inner core matrix; the outer tube comprises a multi-layer round tube which is formed by coiling an animal-derived acellular matrix material and performing vacuum pressing; the inner core matrix comprises a natural polymer material and axially oriented microchannels distributed in the natural polymer material.

2. The nerve repair material of claim 1, wherein the number of layers of the multilayer circular tube is 2-12;

preferably, the animal derived acellular matrix material comprises small intestine submucosa, peritoneum, pericardium or dermis which is derived from animals;

preferably, the thickness of the outer tube is 250-;

preferably, the inner diameter of the outer tube is 2-7 mm;

preferably, the length of the outer tube is 10-60 mm.

3. The nerve repair material of claim 1 or 2, wherein the outer tube comprises a tube body, an inner surface and an outer surface, the tube body and the outer surface being independently distributed with active factors that inhibit escape of nerve fibers;

preferably, the active factor inhibiting escape of nerve fibers comprises chondroitin sulfate proteoglycan and/or myelin associated glycoprotein.

4. The nerve repair material of any one of claims 1 to 3, wherein both ends of the inner core matrix are independently retracted into the outer tube by 2-4 mm;

preferably, the natural polymer material comprises any one or a combination of at least two of collagen, gelatin, chitosan, silk fibroin or hyaluronic acid;

preferably, the inner core matrix further comprises active factors distributed in the natural polymer material for promoting the growth of nerve fibers;

preferably, the mass ratio of the natural polymer to the active factor for promoting the growth of the nerve fibers is (50-100): 1;

preferably, the nerve fiber growth promoting factor comprises amorphous polyphosphate, the number of repeating units of the amorphous polyphosphate is 10 to 100;

preferably, the amorphous polyphosphate salt comprises any one of amorphous magnesium polyphosphate, amorphous zinc polyphosphate or amorphous calcium polyphosphate, or a combination of at least two thereof.

5. The nerve repair material of any one of claims 1 to 4, wherein the number of the microchannels is 5 to 50;

preferably, the pore diameter of the micro-channel is 100-1000 μm;

preferably, the inner core matrix further comprises radially oriented microchannels distributed in the natural polymeric material;

preferably, the porosity of the inner core matrix is not less than 95%.

6. The method for producing a nerve repair material according to any one of claims 1 to 5, comprising the steps of:

7. The method for preparing a nerve repair material according to claim 6, wherein the animal-derived acellular matrix material in the step (1) is coiled by a layer around the rod core material, and then a solution of an active factor for inhibiting escape of nerve fibers is coated on the inner side surface of the remaining animal-derived acellular matrix material, and then the coiling is continued to form a multi-layer round tube;

preferably, the concentration of the active factor solution for inhibiting escape of nerve fibers is 10-30 mug/mL;

preferably, the solution of the active factor for inhibiting escape of nerve fibers is coated on the inner side surface in an amount of 60-200 μ L/mm²；

Preferably, step (1) is to coat the external surface of the round tube with an active factor solution for inhibiting escape of nerve fibers after the round tube is curled into a multi-layer round tube;

preferably, the coating amount of the solution of the active factor for inhibiting escape of nerve fibers on the outer surface of the round tube is 60-200 mu L/mm²；

Preferably, the rod core material in the step (1) is a stainless steel rod core coated with polytetrafluoroethylene;

preferably, the vacuum drying time in the step (1) is 16-24 h.

8. The method for preparing a nerve repair material according to claim 6 or 7, wherein the method for preparing the animal-derived acellular matrix material according to the step (1) comprises the following steps:

cleaning a target tissue material, removing redundant tissues, performing virus inactivation treatment, and then performing cell removal treatment to obtain the tissue material;

preferably, the virus inactivation treatment comprises: placing the pretreated target tissue material in a peroxyacetic acid-ethanol solution for soaking for 1-3h, and then shaking and cleaning for 3-5 times in a phosphate buffer solution, wherein each time lasts for 15-30 min;

preferably, the concentration of the peroxyacetic acid in the peroxyacetic acid-ethanol solution is 0.1-1%, and the concentration of the ethanol is 5-25%;

preferably, the weight ratio of the peroxyacetic acid-ethanol solution to the target tissue material is (8-12) to 1;

preferably, the weight ratio of the phosphate buffer to the target tissue material is (20-30): 1;

preferably, the decellularization treatment comprises: soaking the target tissue material subjected to virus inactivation treatment in strong alkali for 1-24h, and then washing in phosphate buffer solution by shaking for 3-5 times, each time for 15-30 min;

preferably, the strong alkaline solution is a sodium hydroxide-EDTA solution, wherein the concentration of sodium hydroxide is 0.01-0.2M, and the concentration of EDTA is 0.08-0.15M.

9. The method for preparing a nerve repair material according to any one of claims 6 to 8, wherein the mixed reaction system of the step (2) further comprises an active factor for promoting the growth of nerve fibers;

preferably, the solvent in the step (2) is 0.03-0.05M acetic acid solution;

preferably, the reaction temperature of the step (2) is 4-10 ℃, and the reaction time is 2-5 h;

preferably, standing for 12-24h after the reaction in the step (2) is finished;

preferably, the concentration of the natural polymer material in the inner core matrix solution in the step (2) is 3-12mg/mL, and the concentration of the cross-linking agent is 0.01-5 mg/mL;

preferably, the temperature of the sealed refrigeration in the step (2) is-80 to-60 ℃, and the time is 4 to 8 hours;

preferably, the vacuum freeze-drying time of the step (2) is 16-24 h.

10. The method for preparing the nerve repair material according to any one of claims 6 to 9, wherein the temperature of the vacuum high-temperature crosslinking treatment in the step (4) is 100-120 ℃, the time is 12-24h, and the vacuum degree is less than 150 Pa;