CN106913393B - Artificial nerve scaffold and preparation method and application thereof - Google Patents
Artificial nerve scaffold and preparation method and application thereof Download PDFInfo
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- CN106913393B CN106913393B CN201511003594.2A CN201511003594A CN106913393B CN 106913393 B CN106913393 B CN 106913393B CN 201511003594 A CN201511003594 A CN 201511003594A CN 106913393 B CN106913393 B CN 106913393B
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Abstract
The invention discloses an artificial nerve scaffold with nerve regeneration inducing activity, which is characterized by comprising three components of a double-layer nerve conduit, an oriented nanofiber bundle and a natural polymer adhesive, wherein the double-layer nerve conduit is directly prepared outside the oriented nanofiber bundle, and then the natural polymer adhesive is poured into gaps of the oriented nanofiber bundle to prepare the artificial nerve scaffold. The oriented nanofiber bundle is formed by rolling a double-component nanofiber membrane, and the sparsely arranged nanofiber bundle is beneficial to the growth of nerve cells; the 'outer layer' of the nerve conduit is composed of a compact nanofiber membrane tube with strong hydrophobicity, and can prevent connective tissues from growing in, and the 'inner layer' is composed of a double-component nanofiber membrane tube, so that the reconstruction of capillary vessels is facilitated; the natural polymer adhesive is water solution prepared from natural polymer material, and stem cells can be added. The invention also discloses a preparation method of the artificial nerve scaffold and application of the artificial nerve scaffold in repairing peripheral nerve injury.
Description
Technical Field
The invention belongs to the field of tissue engineering, and relates to an artificial nerve repair material filled with active oriented nanofibers, a preparation method thereof, and application of the artificial nerve repair material in peripheral nerve injury repair.
Background
Peripheral nerve injury is a common clinical disease, is poor after healing, has high disability rate, and brings great economic loss and mental burden to patients, families and society. Therefore, how to promote nerve regeneration and restore the function of target organs has been a focus of social attention. For peripheral nerve defects, autologous nerve transplantation remains the current "gold standard" for repair. The main disadvantages are: the autologous nerve has limited material sources, the diameters of the donor nerve and the receptor nerve are not matched, and the injury of the donor area and certain dysfunction are caused when the autologous nerve is cut. Finding alternatives to autologous nerves and promoting nerve regeneration is therefore a hot spot of research. The nerve regeneration catheter constructed by the biological material not only can provide a favorable space for obtaining nutrition, growth and metabolism of nerve cells, but also has the advantages of reducing the tension of a suture opening, guiding the growth of nerve fibers, improving the accuracy of nerve bundle involution, preventing scar tissues from invading regenerated nerve fibers and the like.
At present, four kinds of materials of regenerative nerve conduits are mainly found internationally, including polyglycolic acid nerve conduits, collagen nerve conduits, small intestine mucosa nerve conduits, and P (LA-CL) nerve conduits. However, the collagen nerve conduit has poor mechanical properties, is degraded too fast, has poor biocompatibility and guided regeneration activity of the artificially synthesized material nerve conduit, has limited raw material sources of the acellular matrix nerve conduit, and has risks of rejection, virus transmission and the like. An ideal nerve conduit must satisfy a number of conditions: 1. with a suitable degradation rate, the degradation of the nerve conduit is synchronized with the regeneration of the nerve. 2. The material has good biocompatibility, nerve cells can grow along the tube wall, and nerves can be regenerated quickly. 3. The structure which is arranged in parallel along the axial direction is beneficial to guiding the nerve to grow directionally. 4. Can slowly release nerve nourishing medicines such as nerve growth factor, etc., and promote nerve regeneration speed. Therefore, the development of the regeneration nerve conduit which has excellent mechanical property and biocompatibility and can quickly repair long-distance nerve defects has important practical application value and social benefit.
The prior report of developing nerve conduits by adopting an electrostatic spinning technology in China, and patent 201410279780.8 reports a preparation method of an axially oriented nanofiber nerve conduit, wherein the degradable axially oriented nanofiber nerve conduit is obtained by utilizing electrostatic spinning controlled by a magnetic field, has good biocompatibility, can guide nerve cells to grow along the orientation, and promotes the regeneration and repair of damaged nerves. Patent 201010598436.7 reports a degradable nerve conduit with a highly oriented tube-in-tube structure and a preparation method thereof, comprising an outer layer tubular substrate and an inner small tube, wherein the inner small tube is formed by coiling an electrostatic spinning nanofiber felt on a tubular mold, and is beneficial to guiding nerve cell regeneration. Patent 201410561695.0 reports a multichannel nerve repair conduit with tissue inducing function and a mold, wherein a multi-aperture columnar nerve conduit core layer is prepared by the mold, a nanofiber nerve conduit shell layer is prepared by an electrostatic spinning technology, and the core layer and the shell layer are nested, so that the multichannel nerve repair conduit with the tissue inducing function is obtained.
However, the currently marketed nerve conduit products have a number of drawbacks, such as: firstly, a refined mould is used, and large-scale production is difficult; the form structure and the material formula of the product need to be optimized to ensure that the nerve conduit has enough mechanical strength and does not swell or collapse; and the biocompatibility and the bioactivity of the product need to be improved so as to meet the clinical requirement of repairing long-distance peripheral nerve defects.
Disclosure of Invention
Aiming at various problems of the existing nerve conduit, the invention prepares the artificial nerve repair material composition with nerve regeneration induction activity, which comprises three components of a double-layer nerve conduit, an oriented nanofiber bundle and a natural polymer binder (figure 2), wherein the double-layer nerve conduit is directly prepared outside the oriented nanofiber bundle, the natural polymer binder can be directly poured into the oriented nanofiber bundle during clinical use, or stem cells and the natural polymer binder are mixed and then poured into the oriented nanofiber bundle, and the prepared composition can be used for repairing long-distance peripheral nerve defects.
The invention provides an artificial nerve scaffold with nerve regeneration inducing activity, which is characterized in that: the nerve conduit is of a double-layer membrane structure, the outer layer is composed of a compact nanofiber membrane tube with strong hydrophobicity (attached figure 5), connective tissues can be prevented from growing in, and the inner layer is composed of a double-component nanofiber membrane tube, wherein the rapidly degraded biological fibers are rapidly degraded after being implanted into a body, so that the aperture of the membrane is increased (attached figure 4), and the nerve conduit is beneficial to the growth and reconstruction of cells and capillaries, and further promotes nerve regeneration. The oriented nanofiber bundle is formed by rolling a bicomponent nanofiber membrane (figure 1), the nanofibers are arranged in an axial orientation manner (figure 3), and the rapidly degraded biological fibers are rapidly degraded after being implanted into a body to form the sparsely arranged nanofiber bundle, so that the large gaps are beneficial to the growth of nerve cells, and the nerve regeneration is guided. Particularly, the oriented nanofiber bundle can be compounded with a drug through an electrostatic spinning technology, so that the slow-release active drug is endowed with biological activity such as promotion of nerve repair and the like.
The natural polymer adhesive is an aqueous solution prepared from a natural polymer material, and comprises one or any combination of gelatin, collagen, silk fibroin, chitosan, modified chitosan, hyaluronic acid and alginate, wherein the mass concentration of the solution is 1-10 mg/ml. Preferably, the natural polymer material is type I collagen.
One or any combination of embryonic stem cells, induced pluripotent stem cells, bone marrow mesenchymal stem cells, neural stem cells and Schwann cells is added into the natural polymer adhesive. Preferably, the patient autologous nerve fragments or stem cells can be uniformly mixed with a 'natural polymer binder', poured into an 'oriented nanofiber bundle', and then filled in a 'double-layer nerve conduit' to prepare the tissue-engineered nerve scaffold.
The medicine is selected from one or any combination of the following medicines, including nerve growth factor, fibroblast growth factor, transforming growth factor family, vascular endothelial growth factor, platelet derived growth factor, neuropeptide, ganglioside and sex hormone.
The raw material of the nano-fiber is selected from one or more synthetic absorbable polymer materials, one or more natural polymer materials, or the combination thereof. The synthetic absorbable polymer material comprises polylactic acid, polyglycolic acid, polycaprolactone, polylactic-glycolic acid copolymer, polylactic-caprolactone copolymer and polyethylene glycol copolymer. The natural polymer material comprises gelatin, collagen, silk fibroin, chitosan, modified chitosan, hyaluronic acid, alginate, fibrin and cellulose. Preferably, the synthetic absorbable polymer material is polylactic acid or polylactic-co-glycolic acid, the natural polymer material is collagen or silk fibroin, and further the natural polymer material is type I collagen derived from fish skin, so that the risk of infecting the zoonosis of people and livestock is reduced.
The invention also discloses a preparation method of the artificial nerve scaffold, which is characterized by comprising the following steps: comprises the following steps:
(1) preparing liquid: preparing spinning solution I, spinning solution O, spinning solution Y and a natural high-molecular binder which are required by electrostatic spinning;
(2) preparing a two-component nanofiber membrane: and (3) applying positive voltage and negative voltage to the spinning solution I and the spinning solution O respectively by adopting a conjugate electrostatic spinning device, and carrying out electrostatic spinning to obtain the nanofiber membrane with certain orientation.
(3) Preparing an axially oriented nanofiber bundle: and (3) flatly paving the bicomponent nanofiber membrane obtained in the step (2) on a working table, then taking a filament to be placed in parallel with the oriented fiber of the fiber membrane, sticking the filament to one end of the fiber membrane by using a natural polymer adhesive, and rolling the bicomponent nanofiber membrane into a nanofiber bundle by taking the filament as a reel.
(4) Preparing a double-layer nerve conduit: performing electrostatic spinning on the axially oriented nanofiber bundle in the step 3 by using the spinning solution I and the spinning solution O by adopting a bidirectional electrostatic spinning device to prepare an inner layer of the nerve conduit; then, the spinning solution O is adopted to carry out electrostatic spinning on the inner layer of the catheter, and the outer layer of the nerve catheter is prepared.
(5) And (3) post-treatment: demoulding, cutting, subpackaging and sterilizing the double-layer nerve conduit containing the nanofiber bundle; then the natural polymer binder is poured into the oriented nano fiber bundle to obtain the nano fiber.
Preferably, stem cells or autologous nerve fragments of a patient are uniformly mixed with a 'natural polymer binder' and then are poured into an 'oriented nanofiber bundle', so that the tissue-engineered artificial nerve scaffold is prepared.
According to the method for preparing the artificial nerve scaffold, the spinning solution O mainly comprises a synthetic absorbable polymer material, the auxiliary component comprises a natural polymer material, and the mass ratio of the main component to the auxiliary component is 10: 0-5: 5; the spinning solution I comprises a main component and an auxiliary component, wherein the main component is a natural polymer material, the auxiliary component is a synthetic absorbable polymer material, and the mass ratio of the main component to the auxiliary component is 10: 0-5: 5; the solvent of the spinning solution is selected from hexafluoroisopropanol, trifluoroethanol, dichloromethane, chloroform, tetrahydrofuran, formic acid, water, or any combination thereof.
In the method, the ratio of the spinning speed of the spinning solution I to the spinning speed of the spinning solution O in the step (2) is 50: 1-1: 1; and (4) when the inner layer of the nerve conduit is spun, the ratio of the spinning speed of the spinning solution I to the spinning speed of the spinning solution O is 50: 1-1: 1.
The spinning solution Y mainly comprises the components of medicines, and the mass concentration of the medicines is 10-50 mu g/ml; and (2) preparing the drug-included nanofiber by adopting a coaxial electrostatic spinning technology and taking the spinning solution Y as a core layer solution and the spinning solution O as a shell layer solution.
The post-treatment step comprises drying, packaging and sterilization, and the sterilization step can adopt irradiation sterilization or ethylene oxide sterilization, and preferably adopts gamma irradiation sterilization.
The application of the artificial nerve scaffold developed by the invention in peripheral nerve defects also belongs to the protection scope of the invention.
Compared with the common artificial nerve conduit, the artificial nerve scaffold provided by the invention has the following innovation points and advantages:
(1) the design concept of three-in-one effect of double-layer nerve conduits, oriented nanofiber bundles and natural polymer binders is innovatively provided, and the product is highly bionic of the nerve extracellular matrix: the oriented nanofiber yarn bundle simulates nerve fibers, and the large-gap oriented fibers can provide a scaffold for nerve regeneration; the natural polymer adhesive is in a hydrogel shape and provides nutrition for regeneration of a nerve intima and a nerve bundle membrane; the double-layer nerve conduit simulates a nerve sheath. The product of the patent overcomes the defects of easy collapse, no induced nerve regeneration activity and the like of the traditional nerve conduit, can promote the nerve regeneration speed, and makes substantial progress compared with the existing product.
(2) The invention designs the nerve conduit into a double-layer membrane tube structure: the inner layer is composed of a bicomponent nanofiber membrane tube, wherein the rapidly degraded biological fiber is rapidly degraded after being implanted into a body, so that the aperture of the membrane is increased, the growth and reconstruction of capillary vessels are facilitated, and the nerve regeneration is further promoted; the 'outer layer' is formed by a compact nanofiber membrane tube with strong hydrophobicity, and can prevent connective scar tissue from growing in.
(3) The oriented nanofiber bundle is formed by rolling a bicomponent nanofiber membrane, the nanofibers are arranged in an axial orientation manner, and the rapidly degraded biological fibers are rapidly degraded after being implanted into a body to form the sparsely arranged nanofiber bundle, so that the large gaps are favorable for the growth of nerve cells, and the nerve regeneration is guided; the oriented nanofiber bundle can be compounded with a drug by an electrostatic spinning technology, and the slow-release drug is endowed with the biological activity of inducing nerve regeneration; in addition, the oriented nanofiber bundle is formed by rolling fiber membranes, large gaps are formed among layers, stem cells or patient autologous nerve fragments can be poured into the oriented nanofiber bundle, and the tissue-engineered nerve scaffold is prepared and used for repairing long-distance peripheral nerve defects.
(4) The natural polymer adhesive is hydrogel prepared from natural polymer materials such as collagen, hyaluronic acid and the like, on one hand, the three-dimensional network structure and the neurotrophic effect of the natural polymer adhesive can promote the regeneration of nerves, and on the other hand, the natural polymer adhesive is used as a cell culture solution to uniformly fill bioactive substances such as stem cells and the like into oriented nanofiber bundles to promote the homing, proliferation and differentiation of the stem cells.
(5) The electrostatic spinning technology adopted by the invention is an advanced processing technology for internationally preparing tissue engineering scaffold materials, avoids the complexity of the traditional die manufacturing process, has the advantages of high production efficiency, easiness in large-scale production and the like, and represents an advanced productivity.
Drawings
Fig. 1 is a schematic view of a preparation process of an "oriented nanofiber bundle", in which a is a filament, B is a natural polymer binder, and C is a bicomponent nanofiber membrane, and fibers of the bicomponent nanofiber membrane have certain directionality. Placing A at one end of C, wherein the direction of A is consistent with the fiber direction of C, bonding one side of C with A by using B, taking A as a scroll, rolling C into a bundle, and obtaining D in the figure, namely the oriented nanofiber bundle. The figure is a schematic diagram and only represents the manufacturing process of a product, and the size of the figure has no actual reference value.
Fig. 2 is a schematic cross-sectional view of an artificial nerve scaffold, in which a component 1 is an "oriented nanofiber bundle", a component 2 is an "inner nerve conduit layer" wrapping the fiber bundle, and an outermost component 3 is an "outer nerve conduit layer". In clinical use, the natural polymer binder (component 4) is poured into the oriented nanofiber bundle (component 1) to obtain the composite artificial nerve scaffold. After implantation in vivo, some of the fibers in the oriented nanofiber bundle (component 1) are rapidly degraded, resulting in the formation of sparsely arranged nanofiber bundles (component 5). The figure is a schematic diagram and only represents a product combination form, and the size of the figure has no actual reference value.
FIG. 3 is a scanning electron micrograph of mouse fibroblasts L929 cultured on "oriented nanofiber membrane" for four days
FIG. 4 is the scanning electron micrograph of the inner layer of the double-layer nerve conduit after being soaked in normal saline for 2 weeks
FIG. 5 is a scanning electron micrograph of the outer layer of the double-layer nerve conduit
Detailed Description
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified. Unless otherwise specified, the concentration of the solution prepared with a liquid is expressed in terms of volume ratio (v/v), and the concentration of the solution prepared with a solid is expressed in terms of weight to volume ratio (w/v).
Example 1
1. Liquid preparation
Dissolving collagen and polylactic acid (PLA) in Hexafluoroisopropanol (HFIP) according to the mass ratio of 1:9, wherein the material-liquid ratio is 1:10, and stirring until the collagen and the PLA are completely dissolved to obtain uniform nerve conduit outer layer spinning solution (O);
dissolving collagen in Hexafluoroisopropanol (HFIP) at a ratio of 1:10, and stirring to dissolve completely to obtain uniform spinning solution (I) for inner layer of nerve conduit.
Dissolving collagen in pure water to reach final concentration of 1mg/ml to obtain natural polymer binder, filtering to remove bacteria, and storing at 4 deg.C.
2. Preparation of two-component nanofiber membranes
The nanofiber membrane with certain orientation is prepared by adopting a conjugate electrostatic spinning device to carry out electrostatic spinning, namely, nozzles of two spinning solutions are respectively arranged at two sides above a rotating roller receiving device, and the two nozzles jet oppositely on the same horizontal line and respectively apply positive voltage and negative voltage. The specific method comprises the following steps: and (2) respectively filling the spinning solution I and the spinning solution O into electrostatic spinning injectors I and O, adjusting the jet flow speed of the injector O to be 0.2 ml/h, and adjusting the ratio of the jet flow speed of the injector I to the jet flow speed of the injector O to be 19:1, namely, 5% of a fiber membrane is the fiber which is spun by the spinning solution O and has longer degradation time, and 95% of the fiber membrane is the fiber which is spun by the spinning solution I and has shorter degradation time. The distance between the two spray heads is 20cm, voltage of 5KV and-5 KV is applied respectively, the distance between the receiving device and the plane where the two spray heads are located is 10cm, two bicomponent fiber membranes with different degradation properties are prepared by spinning the two materials simultaneously, and spinning is stopped after the fiber membranes reach the thickness of 20 microns.
3. Preparation of "oriented nanofiber Beam"
And (3) flatly paving the two-component nanofiber membrane obtained in the step (2) on a working table, placing a stainless steel filament coated with a natural polymer binder at one end of the fiber membrane to enable the direction of the filament to be consistent with the fiber direction of the fiber membrane, then rolling up one side of the fiber membrane to wrap the filament therein (shown in figure 1), carrying out vacuum drying for 24 hours, then taking the filament as a reel, and rolling the two-component nanofiber membrane into a bundle until the diameter of the fiber bundle reaches 2 mm.
4. Preparation of double-layer nerve conduit
Firstly, a bidirectional electrostatic spinning device is adopted to prepare an inner layer of a nerve conduit, namely nozzles of two spinning solutions are respectively arranged at two sides above a rotating roller receiving device, the two nozzles simultaneously spray to the receiving device and apply positive voltage, and the specific method comprises the following steps: firstly, respectively filling the spinning solution I and the spinning solution O into electrostatic spinning injectors I and O, adjusting the jet flow speed of the injector O to be 0.2 ml/h, and adjusting the ratio of the jet flow speed of the injector I to the jet flow speed of the injector O to be 19: 1. The distance between the receiving device and the plane where the two nozzles are located is 10cm, voltage is applied for 10KV, electrostatic spinning is carried out on the nanofiber bundle in the step 3, and the spinning is stopped after the thickness of the nanofiber bundle reaches 0.1mm, so that the inner layer of the nerve conduit is obtained;
then, the outer layer of the nerve conduit is prepared by adopting a traditional electrostatic spinning device: and (3) filling the spinning solution O into an injector, adjusting the speed of a micro injection pump to be 5 ml/h, adjusting the voltage of a high-voltage electrostatic generator to be 20KV, adjusting the receiving distance of a receiving device to be 20cm, performing electrostatic spinning on the inner layer of the guide pipe, stopping spinning when the total thickness of the guide pipe reaches 0.2mm to obtain the outer layer of the nerve guide pipe, and then performing vacuum drying for 48 hours to obtain the double-layer nerve guide pipe.
5. Post-treatment
Rinsing the double-layer nerve conduit containing the nanofiber bundle in a 20% ethanol water solution, removing residual solvent, dissolving natural high molecular binder on the filaments, drawing out the filaments to complete demolding, cutting into 5cm length and performing irradiation sterilization; and (3) pouring the natural polymer binder into the oriented nanofiber bundle to obtain the artificial nerve scaffold.
Example 2
1. Liquid preparation
Dissolving gelatin and polylactic acid caprolactone copolymer in trifluoroethanol according to the mass ratio of 2:8, wherein the material-liquid ratio is 1:20, and stirring until the gelatin and the polylactic acid caprolactone copolymer are completely dissolved to obtain uniform nerve conduit outer layer spinning solution (O);
dissolving gelatin and polylactic acid caprolactone in the trifluoroethanol according to the mass ratio of 50:1, wherein the material-liquid ratio is 1:20, and stirring until the gelatin and the polylactic acid caprolactone are completely dissolved to obtain the uniform nerve conduit inner layer spinning solution (I).
Dissolving gelatin in pure water to make final concentration reach 3mg/ml to obtain "natural polymer binder", filtering, sterilizing, and storing at 4 deg.C.
2. Preparation of two-component nanofiber membranes
The nanofiber membrane with certain orientation is prepared by adopting a conjugate electrostatic spinning device to carry out electrostatic spinning, namely, nozzles of two spinning solutions are respectively arranged at two sides above a rotating roller receiving device, and the two nozzles jet oppositely on the same horizontal line and respectively apply positive voltage and negative voltage. The specific method comprises the following steps: and (2) respectively filling the spinning solution I and the spinning solution O into electrostatic spinning injectors I and O, adjusting the jet flow speed of the injector O to be 0.1 ml/h, and adjusting the ratio of the jet flow speed of the injector I to the jet flow speed of the injector O to be 49:1, namely, 2% of a fiber membrane is the fiber which is spun by the spinning solution O and has longer degradation time, and 98% of the fiber membrane is the fiber which is spun by the spinning solution I and has shorter degradation time. Respectively applying voltage of 10KV and-10 KV, wherein the distance between the two nozzles is 30cm, the distance between the receiving device and the plane where the two nozzles are located is 20cm, preparing two bicomponent fiber membranes with different degradation properties by simultaneously spinning the two materials, and stopping spinning after the thickness of the bicomponent fiber membranes reaches 50 micrometers.
3. Preparation of "oriented nanofiber Beam"
And (3) flatly paving the two-component nanofiber membrane obtained in the step (2) on a working table, placing a polytetrafluoroethylene filament coated with a natural polymer binder at one end of the fiber membrane to enable the direction of the filament to be consistent with the fiber direction of the fiber membrane, then rolling up one side of the fiber membrane to wrap the filament therein (shown in figure 1), carrying out vacuum drying for 36 hours, then taking the filament as a reel, and rolling the two-component nanofiber membrane into a bundle until the diameter of the fiber bundle reaches 3 mm.
4. Preparation of double-layer nerve conduit
Firstly, a bidirectional electrostatic spinning device is adopted to prepare an inner layer of a nerve conduit, namely nozzles of two spinning solutions are respectively arranged at two sides above a rotating roller receiving device, the two nozzles simultaneously spray to the receiving device and apply positive voltage, and the specific method comprises the following steps: and (3) respectively filling the spinning solution I and the spinning solution O into electrostatic spinning injectors I and O, adjusting the jet flow rate of the injector O to be 2 ml/h, and adjusting the ratio of the jet flow rate of the injector I to the jet flow rate of the injector O to be 1: 1. The distance between the receiving device and the plane where the two nozzles are located is 20cm, voltage is applied for 20KV, electrostatic spinning is carried out on the nanofiber bundle in the step 3, and the spinning is stopped after the thickness of the nanofiber bundle reaches 0.05mm, so that the inner layer of the nerve conduit is obtained;
then, the outer layer of the nerve conduit is prepared by adopting a traditional electrostatic spinning device: and (3) filling the spinning solution O into an injector, adjusting the speed of a micro injection pump to be 2 ml/h, adjusting the voltage of a high-voltage electrostatic generator to be 15KV, adjusting the receiving distance of a receiving device to be 15m, performing electrostatic spinning on the inner layer of the guide pipe, stopping spinning when the total thickness of the guide pipe reaches 0.1mm to obtain the outer layer of the nerve guide pipe, and then performing vacuum drying for 36 hours to obtain the double-layer nerve guide pipe.
5. Post-treatment
Rinsing the double-layer nerve conduit containing the nanofiber bundle in a 10% ethanol water solution, wherein the step is to remove residual solvent, dissolve natural polymer binder, and complete demoulding after filaments are slowly drawn out; then cutting the mixture into 3m length specification for ethylene oxide sterilization; mixing the above natural polymer binder with Schwann cells to obtain a mixture with a cell concentration of 2 × 105Then pouring the mixture into the oriented nanofiber bundle to obtain the artificial nerve scaffold.
Example 3
1. Liquid preparation
Dissolving chitosan and polylactic acid caprolactone copolymer in an acetone/acetic acid mixed solvent according to a mass ratio of 4:6 (volume ratio of 20:1), wherein the material-liquid ratio is 1:8, and stirring until the chitosan and polylactic acid caprolactone copolymer are completely dissolved to obtain a uniform nerve conduit outer layer spinning solution (O);
dissolving collagen and polyglycolic acid (PGA) in Hexafluoroisopropanol (HFIP) according to a mass ratio of 10:1, wherein the material-liquid ratio is 1:8, and stirring until complete dissolution to obtain uniform nerve conduit inner layer spinning solution (I).
The nerve growth factor was dissolved in pure water to a final concentration of 50. mu.g/ml, and stirred to be completely dissolved to obtain a uniform core layer solution (Y).
Dissolving sodium hyaluronate in pure water to make the final concentration reach 10mg/ml, and filtering to remove bacteria, and storing at 4 deg.C.
2. Double-component nano-fiber membrane for preparing sustained-release medicine
The method adopts a conjugate electrostatic spinning device and a coaxial spinning nozzle to carry out electrostatic spinning to prepare the oriented nanofiber membrane with the drug sustained-release activity, and comprises the following specific steps: the spinning solution Y is used as a core layer solution for coaxial electrospinning, the spinning solution O is used as a shell layer solution for coaxial electrospinning, and the spinning solution Y and the spinning solution O are respectively filled into electrostatic spinning injectors Y and O, and corresponding spray heads of the two injectors are coaxial spinning spray heads; and the spinning solution I is loaded into a spinning injector I, corresponding spray heads and coaxial spinning spray heads are respectively arranged at two sides above the rotating roller receiving device, and the two spray heads oppositely spray on the same horizontal line and respectively apply positive voltage and negative voltage. The jet flow rates of the injector O and the injector I are both adjusted to be 2 ml/h, and the jet flow rate of the injector Y is 0.2 ml/h. Respectively applying voltage of 15KV and 15KV, wherein the distance between the two nozzles is 15cm, the distance between the receiving device and the plane where the two nozzles are located is 30cm, preparing two bicomponent fiber membranes with different degradation properties by spinning the two materials simultaneously, and stopping spinning after the thickness of the bicomponent fiber membranes reaches 50 microns.
3. Preparation of "oriented nanofiber Beam"
And (3) flatly paving the two-component nanofiber membrane obtained in the step (2) on a working table, placing a stainless steel filament coated with a natural polymer binder at one end of the fiber membrane to enable the direction of the filament to be consistent with the fiber direction of the fiber membrane, then rolling up one side of the fiber membrane to wrap the filament therein (shown in figure 1), carrying out vacuum drying for 48 hours, then taking the filament as a reel, and rolling the two-component nanofiber membrane into a bundle until the diameter of the fiber bundle reaches 5 mm.
4. Preparation of double-layer nerve conduit
Firstly, a bidirectional electrostatic spinning device is adopted to prepare an inner layer of a nerve conduit, namely nozzles of two spinning solutions are respectively arranged at two sides above a rotating roller receiving device, the two nozzles simultaneously spray to the receiving device and apply positive voltage, and the specific method comprises the following steps: and (2) respectively filling the spinning solution I and the spinning solution O into electrostatic spinning injectors I and O, adjusting the jet flow speed of the injector O to be 2 ml/h, and adjusting the ratio of the jet flow speed of the injector I to the jet flow speed of the injector O to be 1:1, namely, 50% of a fiber membrane is the fiber which is spun by the spinning solution O and has longer degradation time, and 50% of the fiber membrane is the fiber which is spun by the spinning solution I and has shorter degradation time. The distance between the receiving device and the plane where the two nozzles are located is 30cm, voltage is applied for 30KV, electrostatic spinning is carried out on the nanofiber bundle in the step 3, spinning is stopped after the thickness of the nanofiber bundle reaches 0.2mm, and the inner layer of the nerve conduit is obtained;
then, the outer layer of the nerve conduit is prepared by adopting a traditional electrostatic spinning device: and (3) filling the spinning solution O into an injector, adjusting the speed of a micro injection pump to be 0.5 ml/h, adjusting the voltage of a high-voltage electrostatic generator to be 10KV, adjusting the receiving distance of a receiving device to be 10m, performing electrostatic spinning on the inner layer of the guide pipe, stopping spinning when the total thickness of the guide pipe reaches 0.4mm to obtain the outer layer of the nerve guide pipe, and performing vacuum drying for 60 hours to obtain the double-layer nerve guide pipe.
5. Post-treatment
Rinsing the double-layer nerve conduit containing the nanofiber bundle in pure water, wherein the step is used for removing residual solvent, dissolving natural polymer binder, and completing demoulding after filaments are slowly drawn out; cutting into 1.5m length for radiation sterilization; mixing the natural polymer binder with bone marrow mesenchymal stem cells to obtain a mixture with a cell concentration of 1 × 105Then pouring the mixture into the oriented nanofiber bundle to obtain the artificial nerve scaffold.
The inventor of the present invention performs scanning electron microscope observation on the artificial nerve scaffold material prepared in example 1, and details are shown in figures 3-5. In addition, in order to verify the practical effect, the following animal experiment was performed on the artificial nerve scaffold prepared in the above example 1, and the specific experimental scheme was:
10 healthy adult beagle dogs are taken as research objects, a bilateral sciatic nerve injury model of a dog with the length of 40mm is constructed after 3% of sodium pentobarbital is injected into an abdominal cavity for general anesthesia, the right side is an experimental side, the artificial nerve scaffold described in example 1 is implanted to bridge a nerve amputation, and the left side is an autologous nerve transplantation bridge nerve amputation. The regeneration condition of the bridged nerve is measured at 3 months after the operation, the number, the diameter, the myelin sheath thickness and the axon diameter of myelinated nerve fibers of the regenerated nerve are mainly considered, and the morphology analysis is carried out on the regenerated nerve.
The test results show that the experimental group and the control group realize the regeneration of the defective nerve, and the difference between the two groups has no statistical significance. The artificial nerve scaffold developed by the invention can replace autologous nerve transplantation to repair peripheral nerve defects within 40mm, and has great clinical development value.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of this invention, and is not intended to limit the invention, so that any modification, equivalent replacement, or improvement made within the spirit and principle of the invention will fall within the protection scope of the claims.
Claims (8)
1. An artificial neural scaffold having an activity of inducing nerve regeneration, characterized in that: the artificial nerve scaffold comprises three components of a double-layer nerve conduit, an oriented nanofiber bundle and a natural polymer adhesive, wherein the double-layer nerve conduit is directly prepared outside the oriented nanofiber bundle, then the natural polymer adhesive is poured into gaps in the oriented nanofiber bundle to prepare the oriented nanofiber bundle, a double-component nanofiber membrane is continuously wound into a bundle to prepare the oriented nanofiber bundle, the double-layer nerve conduit is prepared by an electrostatic spinning technology, the outer layer of the double-layer nerve conduit is composed of compact nanofiber membrane tubes with strong hydrophobicity, the inner layer of the double-component nanofiber membrane is composed of double-component nanofiber membrane tubes, the double-component nanofiber membrane is prepared by the electrostatic spinning technology, nanofibers of the double-component nanofiber membrane are arranged in an axial orientation mode, the double-component nanofiber membrane can be used for compounding medicines by the electrostatic spinning technology, and the medicines are selected from one or any combination of the following medicines, the double-component nanofiber membrane tube and the double-component nanofiber membrane both comprise two fibers with different degradation times, one of the fibers can be degraded quickly, so that the aperture of the fiber membrane is increased, the natural polymer adhesive is an aqueous solution prepared from natural polymer materials, the components of the natural polymer adhesive comprise one or any combination of gelatin, collagen, silk fibroin, chitosan, modified chitosan, hyaluronic acid and alginate, and the mass concentration of all the components of the natural polymer materials in the solution is 1-10 mg/ml.
2. The artificial neural scaffold of claim 1, wherein: one or any combination of embryonic stem cells, induced pluripotent stem cells, bone marrow mesenchymal stem cells, neural stem cells and Schwann cells is added into the natural polymer adhesive.
3. A method of preparing the artificial neural scaffold of any one of claims 1 to 2, comprising: comprises the following steps:
(1) preparing liquid: preparing spinning solution I, spinning solution O, spinning solution Y and a natural high-molecular adhesive which are required by electrostatic spinning;
(2) preparing a two-component nanofiber membrane: respectively applying positive voltage and negative voltage to the spinning solution I and the spinning solution O by adopting a conjugate electrostatic spinning device, and carrying out electrostatic spinning to prepare a nanofiber membrane with certain orientation;
(3) preparing oriented nanofiber bundles: flatly paving the two-component nanofiber membrane in the step (2) on a working table, then taking a filament to be placed in parallel to the oriented fiber of the fiber membrane, sticking the filament to one end of the fiber membrane by using a natural polymer adhesive, taking the filament as a reel, and rolling the two-component nanofiber membrane into a nanofiber bundle;
(4) preparing a double-layer nerve conduit: performing electrostatic spinning on the axially oriented nanofiber bundle in the step (3) by using a spinning solution I and a spinning solution O by using a bidirectional electrostatic spinning device to prepare an inner layer of the nerve conduit; then, carrying out electrostatic spinning on the inner layer of the catheter by adopting a spinning solution O to prepare the outer layer of the nerve catheter;
(5) and (3) post-treatment: demoulding, cutting, subpackaging and sterilizing the double-layer nerve conduit containing the nanofiber bundle; then the natural polymer adhesive is poured into the oriented nano fiber bundle to obtain the fiber.
4. The method of claim 3, wherein: the spinning solution O in the step (1) is mainly made of synthetic absorbable polymer materials, the auxiliary components are natural polymer materials, and the mass ratio of the main components to the auxiliary components is 10: 0-5: 5; the spinning solution I comprises a main component and an auxiliary component, wherein the main component is a natural polymer material, the auxiliary component is a synthetic absorbable polymer material, and the mass ratio of the main component to the auxiliary component is 10: 0-5: 5; the solvent of the spinning solution is selected from hexafluoroisopropanol, trifluoroethanol, dichloromethane, chloroform, tetrahydrofuran, formic acid, water, or any combination thereof.
5. The method of claim 4, wherein: the main component of the spinning solution Y in the step (1) is a medicament; and (2) preparing the drug-included nanofiber by adopting a coaxial electrostatic spinning technology and using the spinning solution Y as a core layer solution and the spinning solution O as a shell layer solution.
6. The method of claim 4, wherein: the ratio of the spinning speed of the spinning solution I to the spinning speed of the spinning solution O in the step (2) is 50: 1-1: 1; and (4) when the inner layer of the nerve conduit is spun, the ratio of the spinning speed of the spinning solution I to the spinning speed of the spinning solution O is 50: 1-1: 1.
7. The method of claim 4, wherein: the natural polymer adhesive in the steps (3) and (5) is an aqueous solution prepared from a natural polymer material, the components of the aqueous solution comprise one or any combination of gelatin, collagen, silk fibroin, chitosan, modified chitosan, hyaluronic acid and alginate, and the mass concentration of all the components of the natural polymer material in the solution is 1-10 mg/ml.
8. The method of claim 4, wherein: the synthetic absorbable polymer material is selected from one or any combination of the following compounds, including polylactic acid, polyglycolic acid, polycaprolactone, polylactic-glycolic acid copolymer, polylactic-caprolactone copolymer and polyethylene glycol copolymer, and the natural polymer material is selected from one or any combination of the following compounds, including gelatin, collagen, silk fibroin, chitosan, modified chitosan, hyaluronic acid and alginate.
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| CN109847104B (en) * | 2019-03-07 | 2022-05-03 | 宁波光远致信生物科技有限公司 | Nerve repair membrane and preparation method and application thereof |
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| CN115444614A (en) * | 2022-09-15 | 2022-12-09 | 青岛大学 | Nanofiber yarn filling type nerve conduit and preparation method and application thereof |
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