CN110935068A - Double-layer nerve conduit and preparation method thereof - Google Patents
Double-layer nerve conduit and preparation method thereof Download PDFInfo
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- CN110935068A CN110935068A CN201911322232.8A CN201911322232A CN110935068A CN 110935068 A CN110935068 A CN 110935068A CN 201911322232 A CN201911322232 A CN 201911322232A CN 110935068 A CN110935068 A CN 110935068A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3633—Extracellular matrix [ECM]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
Abstract
The invention provides a preparation method of a double-layer nerve conduit, wherein the design of a double-layer structure achieves the unification of the mechanical property requirement and the biological function requirement of the nerve conduit, a acellular matrix of an inner layer can endow the nerve conduit with high biological functionality, an oriented fiber structure can guide the directional rapid growth of nerve axons and the directional rapid migration of Schwann cells, so that the rapid and accurate repair of nerves is promoted, and a compact porous structure of the outer layer not only allows the sufficient exchange of substances, but also can play a role of a barrier and prevent the formation of scars. The strong mechanical property of the implant can meet the suturability of the implant in vivo transplantation and provide necessary support for the regeneration space of nerves.
Description
Technical Field
The invention relates to the technical field of nerve conduits, in particular to a double-layer nerve conduit and a preparation method thereof.
Background
Nerve conduits are commonly used in the repair of peripheral nerve defects and are generally hollow tubular structures that bridge the proximal and distal ends of the damaged nerve to provide a suitable microenvironment for nerve regeneration. An excellent nerve conduit needs: 1. promoting the rapid regeneration of nerves; 2. effectively block the invasion of external fibroblasts, inflammatory cells and the like and maintain the space for nerve growth; 3. the exchange of internal and external substances is fully realized. The nerve conduit on the market at present is generally a conduit with a single structure made of a single material (such as polyglycolic acid, collagen and the like), and although the nerve conduit has good effects in clinical application, the nerve conduit has fatal defects, such as too fast degradation, too hard material, more complications, fragile material, difficult suture and the like, and is difficult to meet various requirements.
Electrospinning is a technique that can process polymers into micro/nano fiber structures, which can process materials close to the natural neural extracellular matrix structure, and have porosity and large specific surface area, allowing sufficient exchange of substances. In addition, the electrostatic spinning can also construct directionally arranged nano fibers, can guide the directional rapid growth of nerve axons and the directional rapid migration of Schwann cells, thereby realizing the accurate and rapid repair of nerves. However, a single oriented fiber film (tube) has weak mechanical properties in the transverse direction, and it is difficult to maintain its spatial structure. Therefore, constructing a multi-layered nerve conduit, the inner layer of which guides nerve growth and the outer layer of which provides support protection, is an important design.
In addition, the introduction of bioactive components is important for promoting nerve regeneration, and natural macromolecules such as collagen or conductive substances are often introduced in the prior art, but the functions of the substances are single. The acellular matrix is derived from animal natural tissues, is a complex substance consisting of a plurality of polysaccharides, proteins and growth factors, is close to natural human tissue components, and has important significance for nerve regeneration when being applied to the construction of nerve conduits.
Disclosure of Invention
The invention provides a preparation method of a double-layer nerve conduit, wherein the design of a double-layer structure achieves the unification of the mechanical property requirement and the biological function requirement of the nerve conduit, the inner layer is endowed with high biological functionality by a acellular matrix, an oriented fiber structure can guide the directional rapid growth of nerve axons and the directional rapid migration of Schwann cells, so that the rapid and accurate repair of nerves is promoted, and the compact porous structure of the outer layer not only allows the sufficient exchange of substances, but also can play a role of a barrier and prevent the formation of scars. The strong mechanical property of the implant can meet the suturability of the implant in vivo transplantation and provide necessary support for the regeneration space of nerves.
The invention relates to a preparation method of a double-layer nerve conduit, which comprises the following steps:
(1) preparing a cell matrix removing solution;
taking a certain mass of acellular matrix powder, and adding the acellular matrix powder into an organic solvent to prepare an acellular matrix solution;
(2) preparing a polyester solution;
dissolving a certain mass of degradable polyester in an organic solvent, and stirring for 1-2 days to fully dissolve the degradable polyester to prepare a polyester solution;
(3) mixing the cell matrix removing solution and the polyester solution to obtain a mixed solution;
(4) processing an oriented fiber membrane by electrostatic spinning;
carrying out electrostatic spinning processing on the mixed solution to prepare an oriented fiber membrane;
(5) separating and cutting the oriented fiber film;
placing the oriented fiber film in a vacuum drying box, separating the oriented fiber film after vacuum drying for 24 hours, and cutting the oriented fiber film into a rectangle with the length of 5-15 cm and the width of 1-5 cm;
(6) preparing an inner layer of the nerve conduit;
covering the rectangular oriented fiber film on a metal rod with the diameter of 0.5-60 mm, and coiling the metal rod into a tube to obtain the inner layer of the nerve conduit;
(7) preparing a solution of the outer layer material of the nerve conduit;
dissolving degradable polyester with certain mass in an organic solvent to prepare a nerve conduit outer layer material solution;
(8) preparing a double-layer nerve conduit;
wrapping the inner layer of the nerve conduit on a receiver of an electrostatic spinning processing device, and simultaneously carrying out electrostatic spinning processing on the material solution of the outer layer of the nerve conduit to obtain a double-layer nerve conduit;
(9) post-processing of the double-layer nerve conduit;
and (3) placing the double-layer nerve conduit in a vacuum drying box, performing vacuum drying for 24 hours, then demolding, and cutting into the double-layer nerve conduit with the length of 5-60 mm.
Preferably, in the step (1), a certain mass of cell matrix-removed powder is added into an organic solvent, the mass concentration of the organic solvent is 1-15% w/v, the organic solvent is stirred for 4-6 days, and then the organic solvent is placed into a ball mill at the temperature of-10 ℃ to be ball-milled for 5-10 minutes at the power of 25-75 Hz for 2 times; transferring the solution after ball milling into a centrifuge tube, putting the centrifuge tube into an ultra-high speed centrifuge, placing the centrifuge tube into a centrifuge with the experimental condition of 5000-10000 rpm for ultracentrifugation for 3-10 minutes, and absorbing the upper layer solution to prepare the cell matrix removing solution.
Preferably, sources of the acellular matrix powder include brain, spinal cord, nerves, skin, small intestinal mucosa, adipose tissue of human, pig and cow mammals; the organic solvent comprises 2,2, 2-trifluoroethanol and hexafluoroisopropanol.
Preferably, in the step (2), a certain mass of degradable polyester is dissolved in an organic solvent, and the mixture is stirred for 1 to 2 days to be fully dissolved, so that a polyester solution is prepared.
Preferably, the degradable polyester comprises polylactic acid, polyglycolic acid, poly (D, L-lactic acid-co-glycolic acid), poly (D, L-lactic acid-co-caprolactone), poly (D, L-lactic acid-co-trimethylene carbonate); the organic solvent comprises 2,2, 2-trifluoroethanol, hexafluoroisopropanol, chloroform and dichloromethane.
Preferably, in the step (4), the mixed solution is transferred to a syringe and placed on a syringe pump for electrostatic spinning, wherein the injection rate of the solution is 0.5-4 ml/h; the inner diameter of the injection needle is 0.20-1.60 mm, the voltage connected with the needle is 5-20 kv, and the injection needle performs left-right reciprocating scanning at the speed of 2-5 cm/s; the receiver is a metal roller rotating at a high speed, the diameter of the metal roller is 5-15 cm, the rotating speed is 1500-3000 rpm, the metal roller is connected with a high-voltage power supply, the voltage is 0-minus 3kv, the material fiber is deposited on the roller, and the fiber direction is arranged along the rotating direction.
Preferably, in the step (8), the outer layer material solution is transferred into an injector, is arranged on an injection pump, and is subjected to electrostatic spinning processing, wherein the injection rate of the solution is 0.5-4 ml/h; the inner diameter of the injection needle is 0.20-1.60 mm, the voltage connected with the needle is 5-20 kv, and the injection needle performs left-right reciprocating scanning at the speed of 2-5 cm/s; the receiver is the metal rod wrapped by the inner layer of the nerve conduit, rotates at the speed of 60-500 rpm, and is connected with a high-voltage power supply, and the voltage is 0-minus 3 kv.
The invention also protects the double-layer nerve conduit prepared by the method.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts
FIG. 1 is a schematic illustration of the preparation of an oriented fiber film;
FIG. 2 is a schematic diagram of the preparation of a double-layer nerve conduit;
FIG. 3 is a scanning electron microscope image of the double-layer nerve conduit (B is a partial enlarged view of A);
FIG. 4 is the outer surface of the double-layer nerve conduit
Figure 5 is a double layer nerve conduit inner surface.
Detailed Description
The preparation method of the double-layer nerve conduit comprises the following steps:
(1) preparation of acellular matrix solution
Adding a certain mass of cell matrix-removed powder into an organic solvent, stirring for 4-6 days at a mass concentration of 1-15% w/v, and then placing the mixture into a ball mill at the temperature of-10 ℃ to perform ball milling for 5-10 minutes at the power of 25-75 Hz for 2 times. Transferring the ball-milled solution into a centrifugal tube, putting the centrifugal tube into an ultra-high speed centrifuge, placing the centrifugal tube into a centrifugal machine with the rotating speed of 5000-10000 rpm for ultracentrifugation for 3-10 minutes, and absorbing the upper layer solution for later use.
Sources of the acellular matrix include different tissues of brain, spinal cord, nerve, skin, small intestinal mucosa, fat and the like of mammals such as human, pig, cattle and the like.
The organic solvent includes 2,2, 2-trifluoroethanol, hexafluoroisopropanol, etc.
(2) Preparation of polyester solution
Dissolving a certain mass of degradable polyester in an organic solvent, and stirring for 1-2 days to fully dissolve the degradable polyester.
The degradable polyester comprises polylactic acid, polyglycolic acid, poly (D, L-lactic acid-co-glycolic acid), poly (D, L-lactic acid-co-caprolactone), poly (D, L-lactic acid-co-trimethylene carbonate) and the like.
The organic solvent includes 2,2, 2-trifluoroethanol, hexafluoroisopropanol, chloroform, dichloromethane, etc.
(3) Preparation of nerve conduit inner layer material solution
Mixing the cell matrix removing solution and the polyester solution according to a certain ratio (ratio is 1: 1-1: 5), fully stirring for 1-2 days to uniformly mix to obtain a mixed solution, and using the mixed solution to prepare the inner layer material of the nerve conduit.
(4) Electrostatic spinning processing oriented fiber membrane
The mixed solution was transferred to a syringe and mounted on a syringe pump as shown in FIG. 1, and subjected to an electrospinning process. Wherein the injection rate of the solution is 0.5-4 ml/h; the inner diameter of an injection needle connected with the injector is 0.20-1.60 mm, the voltage connected with the needle is 5-20 kv, and the needle performs left-right reciprocating scanning at the speed of 2-5 cm/s; the receiver is a metal roller rotating at a high speed, the diameter of the metal roller is 5-15 cm, the rotating speed is 1500-3000 rpm, and the metal roller is connected with a high-voltage power supply, and the voltage is 0-3 kv. The material fibers are deposited on a metal drum with the fiber direction aligned along the direction of rotation.
(5) Separation and cutting of oriented fibrous membranes
And after the electrostatic spinning processing is finished, placing the metal roller and the received oriented fiber film in a vacuum drying box, separating the oriented fiber film after vacuum drying for 24 hours, and cutting the oriented fiber film into a rectangle with the length of 5-15 cm and the width of 1-5 cm.
(5) Preparation of inner layer of nerve conduit
And covering the rectangular oriented fiber film on a metal rod with the diameter of 0.5-60 mm, and coiling the rectangular oriented fiber film into a tube, wherein the fiber orientation is consistent with the axial direction of the metal rod.
(6) Preparation of solution of nerve conduit outer layer material
Dissolving a certain mass of degradable polyester in an organic solvent, and stirring for 1-2 days to fully dissolve the degradable polyester.
The degradable polyester comprises polylactic acid, polyglycolic acid, poly (D, L-lactic acid-co-glycolic acid), poly (D, L-lactic acid-co-caprolactone), poly (D, L-lactic acid-co-trimethylene carbonate), polylactic acid-polytrimethylene carbonate and the like.
The organic solvent includes 2,2, 2-trifluoroethanol, hexafluoroisopropanol, chloroform, dichloromethane, etc.
(7) Preparation of double-layer nerve conduit
As shown in fig. 2, the outer layer material solution was transferred to a syringe and mounted on a syringe pump to perform an electrospinning process. Wherein the injection rate of the solution is 0.5-4 ml/h; the inner diameter of the injection needle is 0.20-1.60 mm, the voltage connected with the needle is 5-20 kv, and the injection needle performs left-right reciprocating scanning at the speed of 2-5 cm/s; the receiver is the metal rod wrapped by the inner layer of the nerve conduit, rotates at the speed of 60-500 rpm, and is connected with a high-voltage power supply, and the voltage is 0-minus 3 kv.
(8) Post-treatment of double-layer nerve conduits
And after the electrostatic spinning processing is finished, placing the metal round rod and the received material in a vacuum drying box, carrying out vacuum drying for 24 hours, then demoulding the sleeve, and cutting the sleeve into a double-layer nerve conduit with the length of 5-60 mm.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example preparation of double-layer nerve conduit containing pig-derived acellular nerve matrix (pDMM)
pDMM was dissolved in hexafluoroisopropanol, PLLA in 2,2, 2-trifluoroethanol and mixed well to give a final PLLA concentration of 7.5% w/v and pDMM concentration of 2.5% w/v. And (4) carrying out electrostatic spinning processing on the fiber by utilizing the steps 4-6 to obtain the inner layer of the nerve conduit consisting of the oriented fibers. Then poly (lactic acid-co-trimethylene carbonate) (P (LA-TMC)) is dissolved in 2,2, 2-trifluoroethanol to prepare a solution of the outer layer material of the nerve conduit with the concentration of 15% w/v, and the double-layer nerve conduit is prepared according to the steps 7 to 9.
The scanning electron microscope picture is shown in fig. 3, and the fiber has a double-layer structure, wherein the outer layer shown in fig. 4 is a structure formed by stacking randomly arranged fibers, and the inner layer shown in fig. 5 is PLLA/pDMN oriented fibers.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (8)
1. A method for preparing a double-layer nerve conduit, which is characterized by comprising the following steps:
(1) preparing a cell matrix removing solution;
taking a certain mass of acellular matrix powder, and adding the acellular matrix powder into an organic solvent to prepare an acellular matrix solution;
(2) preparing a polyester solution;
dissolving a certain mass of degradable polyester in an organic solvent, and stirring for 1-2 days to fully dissolve the degradable polyester to prepare a polyester solution;
(3) mixing the cell matrix removing solution and the polyester solution to obtain a mixed solution;
(4) processing an oriented fiber membrane by electrostatic spinning;
carrying out electrostatic spinning processing on the mixed solution to prepare an oriented fiber membrane;
(5) separating and cutting the oriented fiber film;
placing the oriented fiber film in a vacuum drying box, separating the oriented fiber film after vacuum drying for 24 hours, and cutting the oriented fiber film into a rectangular oriented fiber film with the length of 5-15 cm and the width of 1-5 cm;
(6) preparing an inner layer of the nerve conduit;
covering a metal rod with the diameter of 0.5-60 mm with a rectangular oriented fiber membrane, and coiling the rectangular oriented fiber membrane into a tube to obtain an inner layer of the nerve conduit;
(7) preparing a solution of the outer layer material of the nerve conduit;
dissolving degradable polyester with certain mass in an organic solvent to prepare a nerve conduit outer layer material solution;
(8) preparing a double-layer nerve conduit;
wrapping the inner layer of the nerve conduit on a receiver of an electrostatic spinning processing device, and simultaneously carrying out electrostatic spinning processing on the material solution of the outer layer of the nerve conduit to obtain a double-layer nerve conduit;
(9) post-processing of the double-layer nerve conduit;
and (3) placing the double-layer nerve conduit in a vacuum drying box, performing vacuum drying for 24 hours, then demolding, and cutting into the double-layer nerve conduit with the length of 5-60 mm.
2. The preparation method of the double-layer nerve conduit according to claim 1, wherein in the step (1), a certain mass of cell matrix-removed powder is taken, added into an organic solvent with the mass concentration of 1-15% w/v, stirred for 4-6 days, and then placed into a ball mill with the temperature of-10 ℃ for ball milling for 5-10 minutes at the power of 25-75 Hz for 2 times; transferring the solution after ball milling into a centrifuge tube, putting the centrifuge tube into an ultra-high speed centrifuge, placing the centrifuge tube into a centrifuge with the experimental condition of 5000-10000 rpm for ultracentrifugation for 3-10 minutes, and absorbing the upper layer solution to prepare the cell matrix removing solution.
3. The method for preparing a double-layered nerve conduit according to claim 2, wherein the sources of the acellular matrix powder include brain, spinal cord, nerves, skin, small intestinal mucosa, adipose tissue of human, pig, and bovine mammals; the organic solvent comprises 2,2, 2-trifluoroethanol and hexafluoroisopropanol.
4. The method for preparing the double-layer nerve conduit according to claim 1, wherein in the step (2), a certain mass of degradable polyester is dissolved in an organic solvent, and the mixture is stirred for 1 to 2 days to be fully dissolved to prepare a polyester solution.
5. The method for preparing a double-layered nerve conduit according to claim 4, wherein the degradable polyester comprises polylactic acid, polyglycolic acid, poly (D, L-lactic acid-co-glycolic acid), poly (D, L-lactic acid-co-caprolactone), poly (D, L-lactic acid-co-trimethylene carbonate); the organic solvent comprises 2,2, 2-trifluoroethanol, hexafluoroisopropanol, chloroform and dichloromethane.
6. The method for preparing the double-layered nerve conduit according to claim 1, wherein in the step (4), the mixed solution is transferred to a syringe and placed on a syringe pump to be processed by electrospinning, wherein the injection rate of the solution is 0.5 to 4 ml/h; the inner diameter of the injection needle is 0.20-1.60 mm, the voltage connected with the needle is 5-20 kv, and the injection needle performs left-right reciprocating scanning at the speed of 2-5 cm/s; the receiver is a metal roller rotating at a high speed, the diameter of the metal roller is 5-15 cm, the rotating speed is 1500-3000 rpm, the metal roller is connected with a high-voltage power supply, the voltage is 0-minus 3kv, the material fiber is deposited on the roller, and the fiber direction is arranged along the rotating direction.
7. The method for preparing the double-layer nerve conduit according to claim 1, wherein in the step (8), the outer layer material solution is transferred into a syringe, is arranged on a syringe pump, and is subjected to an electrospinning process, wherein the injection rate of the solution is 0.5-4 ml/h; the inner diameter of the injection needle is 0.20-1.60 mm, the voltage connected with the needle is 5-20 kv, and the injection needle performs left-right reciprocating scanning at the speed of 2-5 cm/s; the receiver is the metal rod wrapped by the inner layer of the nerve conduit, rotates at the speed of 60-500 rpm, and is connected with a high-voltage power supply, and the voltage is 0-minus 3 kv.
8. A double-layered nerve conduit produced by the method of any one of claims 1 to 7.
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| CN201911322232.8A CN110935068A (en) | 2019-12-20 | 2019-12-20 | Double-layer nerve conduit and preparation method thereof |
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