WO2023001261A1 - 外周神经复合导管及其制备方法与应用 - Google Patents

外周神经复合导管及其制备方法与应用 Download PDF

Info

Publication number
WO2023001261A1
WO2023001261A1 PCT/CN2022/107235 CN2022107235W WO2023001261A1 WO 2023001261 A1 WO2023001261 A1 WO 2023001261A1 CN 2022107235 W CN2022107235 W CN 2022107235W WO 2023001261 A1 WO2023001261 A1 WO 2023001261A1
Authority
WO
WIPO (PCT)
Prior art keywords
oriented
nanofiber
outer tube
nerve
peripheral nerve
Prior art date
Application number
PCT/CN2022/107235
Other languages
English (en)
French (fr)
Inventor
郭杰
冷鸿飞
徐小雨
陶秀梅
Original Assignee
北京诺康达医药科技股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京诺康达医药科技股份有限公司 filed Critical 北京诺康达医药科技股份有限公司
Publication of WO2023001261A1 publication Critical patent/WO2023001261A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/0059—Cosmetic or alloplastic implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/0077—Special surfaces of prostheses, e.g. for improving ingrowth
    • 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
    • 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
    • A61L27/38—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 containing added animal cells
    • A61L27/3804—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 containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/383—Nerve cells, e.g. dendritic cells, Schwann cells
    • 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
    • A61L27/38—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 containing added animal cells
    • A61L27/3804—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 containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • 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
    • A61L27/38—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 containing added animal cells
    • A61L27/3839—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 containing added animal cells characterised by the site of application in the body
    • A61L27/3878—Nerve tissue, brain, spinal cord, nerves, dura mater
    • 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
    • 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
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A61L2300/414—Growth factors
    • 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
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/602—Type of release, e.g. controlled, sustained, slow
    • 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
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
    • 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

Definitions

  • the invention relates to a peripheral nerve composite catheter and a preparation method and application thereof.
  • Peripheral nerve injury is a common type of injury with a high incidence and disability rate, among which defect injury is the most common type of injury. For a small number of defects, tension-free suturing at the broken ends is possible. For larger defects, autologous nerve transplantation is still the first choice for clinical treatment, but the results after repair are not ideal, especially for long-term defects (>6mm).
  • Nerve bridging catheters can be used to repair long-distance peripheral nerve defects, which is a hot research direction at present, and various nerve catheters and optimization design ideas are emerging.
  • the recovery effect of the existing nerve conduits on regenerative nerve function is not ideal.
  • patent CN 202010415408.0 adopts electrospinning and coaxial co-spinning to prepare oriented nanomembranes with a shell-core structure, and then winds them into bionic nerve bridges, which cannot load drugs, Cell and growth factor components.
  • Patent CN 201510822838.3 Disperses the nerve growth factor in the polymer to prepare an inner and outer cannula nerve bridge tube.
  • the outer layer has no orientation to play a supporting role, and the inner layer is oriented to guide cell growth.
  • nerve growth The slow release of factors affects the effect of nerve repair.
  • the embodiment of the present invention provides a peripheral nerve composite catheter and its preparation method and application.
  • the peripheral nerve composite catheter provided by the embodiment of the present invention can self-regulate and release blood-prosperous cells or neurotrophic factors according to the characteristics of nerve growth, and can solve the current problem of nerve fiber regeneration and repair after nerve defect.
  • a composite catheter for peripheral nerves comprising:
  • Nanofiber porous hollow outer tube as a support cavity
  • An oriented nanofiber membrane attached to the inner wall of the porous hollow outer tube of the nanofiber to form a cavity, is used to promote directional docking and regeneration of nerves;
  • Hollow oriented tubules built in the cavity formed by the oriented nanofiber membrane, are used to strengthen the pressure resistance and orientation of the peripheral nerve composite catheter, and guide the orderly growth of nerves;
  • the nanofiber porous hollow outer tube, the oriented nanofiber membrane and the hollow oriented small tube are all prepared by an electrospinning process.
  • the fiber filaments in the nanofiber porous hollow outer tube are in a non-oriented state.
  • the fiber filaments in the oriented nanofibrous membrane are in an oriented state.
  • the fiber filaments in the hollow oriented tubules are in an oriented state.
  • the hydrogel column is made by reacting multi-arm polyethylene glycol succinimide glutarate and trilysine salt.
  • the multi-arm polyethylene glycol succinimide glutarate preferably includes two-arm, four-arm and eight-arm polyethylene glycol succinimide glutarate.
  • the trilysine salt is preferably trilysine acetate.
  • the hydrogel column is loaded with nerve growth factor, plasmin-sensitive microsphere components of neuron cells, brain-derived neurotrophic factor, neuron cells, and stem cells.
  • nerve growth factor nerve growth factor
  • plasmin-sensitive microsphere components of neuron cells neuron cells
  • brain-derived neurotrophic factor neuron cells
  • stem cells stem cells.
  • the raw materials used for electrospinning include: one of silk fibroin, chitosan, gelatin, polylactic acid, polycaprolactone, and lactide-glycolide copolymer Or a blend of several materials; solvents used include: dichloromethane, chloroform, N,N-dimethylformamide, hexafluoroisopropanol, dimethyl sulfoxide, ethyl acetate.
  • the raw material for preparing the nanofiber porous hollow outer tube is poly(lactic acid polycaprolactone) copolymer, wherein the molar content of polycaprolactone is 10-25%; more preferably tetrahydrofuran and N,N-dimethyl A mixed solution of methyl formamide with a volume ratio of 5:5-8:2 is used as a solvent, more preferably a volume ratio of 6:4.
  • the raw material for preparing the oriented nanofiber membrane and the hollow oriented tubules is poly(lactic acid polycaprolactone) copolymer, wherein the molar content of polycaprolactone is 10-25%; more preferably tetrahydrofuran and N,N -Dimethylformamide is used as a solvent in a mixed solution with a volume ratio of 6:4-7:3.
  • the present invention also provides a preparation method of a peripheral nerve composite catheter, comprising:
  • a hydrogel column is provided, embedded in the cavity of the hollow alignment tubule.
  • the electrospinning parameters for preparing the nanofiber porous hollow outer tube are as follows: voltage 10-30kv, pushing speed 0.5-2ml/h, receiving distance 8-15cm, rotating receiver diameter 2.1-11mm, rotating speed 50-200rpm.
  • the electrospinning parameters for preparing the oriented nanofiber membrane and the hollow oriented tubule are as follows: voltage 10-30kv, pushing speed 0.5-3ml/h, receiving distance 8-15cm, rotating receiver diameter 10- 20cm, speed 1200-3000rpm.
  • the raw material viscosity for preparing the nanofiber porous hollow outer tube, the oriented nanofiber membrane and the hollow oriented small tube is 0.5-4.0dl/g; the electrospinning solution with a concentration of 8-15% is prepared with a solvent, and the Electrospinning.
  • the present invention also includes the peripheral nerve composite catheter prepared by the above method.
  • the peripheral nerve composite catheter of the embodiment of the present invention from the outside to the inside, the first layer is a nanofiber porous hollow tube prepared by electrospinning as a supporting cavity; the second layer is an oriented nanofiber film prepared by electrospinning, which is wound into an orientation tube Promote the directional regeneration of nerves; the third part is the built-in hollow oriented small tube, which strengthens the pressure resistance and orientation of the catheter; the fourth part is the plasmin-sensitive gel loaded with nerve growth factor or neuron cells in the cavity of the built-in fiber catheter The column, which plays the role of controlled release and orientation at the same time.
  • the four parts improve the microenvironment of nerve regeneration in many ways, and accelerate nerve regeneration and repair.
  • the invention also provides the application of the peripheral nerve composite conduit in preparing materials for promoting nerve repair.
  • the present invention has the following beneficial effects:
  • the first layer of nanofiber porous hollow outer tube prepared by the electrospinning technology of the peripheral nerve composite catheter of the embodiment of the present invention can provide the catheter with space for growth and material exchange, and at the same time provide high mechanical compressive strength.
  • the second layer of the composite catheter for peripheral nerves in the embodiment of the present invention is an oriented nanofiber membrane.
  • the plasminogen activator secreted by the growth cone head of nerve regeneration can promote the transformation of plasminogen into plasmin.
  • the embodiment of the present invention prepares a plasmin-sensitive drug delivery system.
  • the hydrogel column is compounded with plasmin-sensitive microspheres loaded with Schwann cells or neurotrophic factors, which can control the growth of trophic factors or neuron cells along with nerve regeneration. It is continuously released locally according to the physiological function to achieve the effect of controlled release.
  • the hydrophilic gel column can enhance the adhesion and migration of neuron cells, and can also intelligently release Schwann cells and neurotrophic factors according to the increase in the expression of plasmin after nerve damage, so as to speed up the process of nerve repair.
  • the orientation of the gel column and the spinning alignment of the hollow oriented tubules all increase the orientation of the composite catheter.
  • Fig. 1 is a schematic cross-sectional view of a peripheral nerve compound catheter according to an embodiment of the present invention.
  • FIG. 2 is a photomicrograph of the nanofiber porous hollow outer tube prepared in Example 1.
  • FIG. 3 is a photomicrograph of the oriented nanofiber membrane prepared in Example 1.
  • Figure 6 and Figure 7 are micrographs of two kinds of oriented nanofiber membranes prepared in Example 2, respectively.
  • Figure 8 and Figure 9 are the HE staining diagrams of the regenerated nerve cross-section of the peripheral nerve composite catheter in the first group and the second group in Experimental Example 1, respectively.
  • Fig. 10, Fig. 11 and Fig. 12 are the HE staining images of the peripheral nerve composite catheters in the first group, the second group and the third group in the experimental example 2 respectively.
  • Fig. 13 is the completion diagram of the first group of peripheral nerve composite catheter bridging.
  • a peripheral nerve composite catheter includes a nanofiber porous hollow outer tube 1 as a supporting cavity; an oriented nanofiber membrane 2 is attached to the inner wall of the nanofiber porous hollow outer tube 1 to form The cavity is used to promote the directional docking and regeneration of nerves; the hollow oriented small tube 3 is built (or set) in the cavity formed by the oriented nanofiber membrane 2, and is used to strengthen the pressure resistance and orientation of the peripheral nerve composite catheter The property guides the orderly growth of nerves; the hydrogel column 4 is built (or arranged) in the cavity of the hollow oriented tubule 3 .
  • the nanofiber porous hollow outer tube 1 , the oriented nanofiber membrane 2 and the hollow oriented small tube 3 can all be prepared by an electrospinning process.
  • the peripheral nerve compound catheter can improve the microenvironment of nerve regeneration in many aspects, and accelerate nerve regeneration and repair.
  • no hydrogel column 4 is disposed in the cavity formed by the oriented nanofiber membrane 2 .
  • the hydrogel column 4 can be loaded with one of the gel columns of nerve growth factor, plasmin-sensitive microsphere components of neuron cells, brain-derived neurotrophic factor, neuron cells, and stem cells or several.
  • the hydrogel column 4 can be loaded with nerve growth factor and/or plasmin-sensitive microsphere components of neuron cells, and at the same time play the roles of controlled drug release and orderly growth of nerves.
  • the hydrogel column 4 can be loaded with gel columns of neurotrophic factors (NGF, brain-derived neurotrophic factor BDNF, etc.), neuron cells, and stem cells.
  • NGF neurotrophic factors
  • BDNF brain-derived neurotrophic factor
  • the hydrogel column 4 has a length of 1-5 cm and a diameter of 0.2-0.8 mm.
  • the number of hydrogel columns in each peripheral nerve composite catheter is 3-500 pieces, or the number of hydrogel columns can be adjusted according to the drug concentration requirements for clinical repair of nerve defects. This will not only play a certain role in tube support, but also reserve enough space for nerve regeneration.
  • the peripheral nerve composite catheter can be prepared by electrospinning technology and in-situ hydrogel carrier technology.
  • the nanofiber porous hollow outer tube 1 , the oriented nanofiber membrane 2 and the hollow oriented small tube 3 are prepared by electrospinning technology.
  • the raw materials used in electrospinning include: silk fibroin, chitosan, gelatin, polylactic acid, polycaprolactone, lactide-glycolide copolymer or a blend of several materials;
  • the solvent includes: at least one of dichloromethane, chloroform, N,N-dimethylformamide (DMF), hexafluoroisopropanol, dimethyl sulfoxide (DMSO), ethyl acetate and the like.
  • the nanofiber porous hollow outer tube 1 is a nanofiber porous hollow tube prepared by electrospinning technology, with an inner diameter of 2.1-11 mm, a length of 2-6 cm, and a thickness (referring to wall thickness, the same below) of 0.25-1.0 mm.
  • Peripheral nerves vary in thickness at different locations, the thickest can reach 10mm, and the thinnest is less than 2mm.
  • the nanofiber porous hollow outer tube 1 with an inner diameter of 2.1-11 mm can satisfy nerve repair in various parts.
  • the nanofiber porous hollow outer tube 1 with a length of 2-6cm can be adapted to repair most peripheral nerves.
  • the defect is too long, and the result of nerve regeneration and repair is not ideal, and the nerve autograft is usually used to replace it.
  • the thickness of the nanofiber porous hollow outer tube 1 reaches 0.25-1.0 mm, so that even if the inner diameter is 10 mm, it can also achieve a certain compressive performance.
  • the fiber filaments in the nanofiber porous hollow outer tube 1 are in a non-oriented state.
  • the non-oriented state of the fiber filaments in the nanofiber porous hollow outer tube 1 can make the prepared peripheral nerve composite catheter have higher The mechanical compression resistance allows the catheter to maintain a certain support space in the muscle gap after transplantation and avoid squeezing the regeneration channel.
  • the preparation method of the nanofiber porous hollow outer tube 1 includes using the above-mentioned poly(lactic acid polycaprolactone) copolymer (polycaprolactone (PCL) molar content is 10-25%) as the electrospinning raw material,
  • the viscosity is preferably 0.5-4.0dl/g
  • the above-mentioned one solvent or a mixture of multiple solvents is preferably prepared into an electrospinning solution with a concentration of 8-15% (mass volume fraction, W/V, g/ml) for electrospinning .
  • Electrospinning parameters are as follows: voltage 10-30kv, pushing speed 0.5-2ml/h, receiving distance 8-15cm, rotating receiver diameter 2.1-11mm, rotating speed 50-200rpm, and the thickness of the spun hollow tube is 0.25-1.0mm.
  • the raw material for preparing the nanofiber porous hollow outer tube 1 such as the above-mentioned poly(lactic acid polycaprolactone copolymer), can ensure that the nanofiber porous hollow outer tube 1 has certain mechanical properties and degradation when the viscosity is 0.5-4.0dl/g Cycle, but also conducive to spinning.
  • the raw material for preparing the porous hollow outer tube 1 of nanofibers such as the above-mentioned poly(lactic acid polycaprolactone) copolymer
  • a concentration of 8-15% mass volume fraction, W/V, g/ml
  • the concentration of the solution is related to the viscosity of the solution. The higher the concentration, the greater the viscosity, and it is difficult for the electric field force to overcome the viscous spinning inside the solution; the smaller the solution concentration, the smaller the viscosity, and it is difficult to spin or the spinning is too short to cause broken filaments.
  • a mixture of tetrahydrofuran and N,N-dimethylformamide with a volume ratio of 5:5-8:2 is used as a solvent, preferably with a volume ratio of 6 :4.
  • the filaments in the oriented nanofiber film 2 are in an oriented state.
  • the oriented nanofiber membrane 2 is an oriented nanofiber membrane made of filaments and wound.
  • the length of the oriented nanofiber membrane 2 is 2-6 cm.
  • the thickness of the oriented nanofiber membrane 2 is 0.05-0.5mm, and the inner diameter of the cavity formed by it is 2-10mm. Different parts of the nerve thickness is different. The inner diameter of the cavity formed by the oriented nanofiber membrane 2 is within 2-10 mm, which can meet the socket of most peripheral nerves. If the thickness of the oriented nanofiber membrane is too thick, it is not easy to adhere to itself; if the thickness is too thin, it is easy to wrinkle during the process of inlaying or attaching the inner wall of the nanofiber porous hollow outer tube 1; a large number of experiments have found that the orientation of the nanofiber membrane 2 A thickness of 0.05-0.5mm is more appropriate.
  • the filaments in the oriented nanofibrous membrane 2 are in an orderly orientation state, which can promote the rapid migration of cells from the proximal end to the distal end during the nerve cell migration process in the early stage of nerve repair, thus speeding up the process of nerve cell migration.
  • the preparation method of the oriented nanofiber film 2 includes, using the above-mentioned one or more polymers as the electrospinning raw material, such as poly(lactic acid polycaprolactone copolymer) (PCL molar content is 10- 25%), the viscosity is preferably 0.5-4.0dl/g, and the electrospinning solution is preferably prepared with a concentration of 8-15% (mass volume fraction, W/V, g/ml) with the above-mentioned solvent or a mixture of multiple solvents, Perform electrospinning.
  • the electrospinning raw material such as poly(lactic acid polycaprolactone copolymer) (PCL molar content is 10- 25%)
  • the viscosity is preferably 0.5-4.0dl/g
  • the electrospinning solution is preferably prepared with a concentration of 8-15% (mass volume fraction, W/V, g/ml) with the above-mentioned solvent or a mixture of multiple solvents, Perform electrospinning.
  • Electrospinning parameters are as follows: voltage 10-30kv, pushing speed 0.5-3ml/h, receiving distance 8-15cm, rotating receiver diameter 10-20cm, rotating speed 1200-3000rpm, and the thickness of the oriented nanofiber film spun is 0.05-0.5 mm, followed by winding and forming along the vertical direction of the rotating receiver.
  • the orientation nanofiber membrane can be obtained by controlling the rotation speed of electrospinning and the shape of the rotating receiver to make the fiber filaments in an ordered orientation state.
  • the rotational speed of electrospinning should be controlled at 1200-3000 rpm, and the shape of the rotating receiver is preferably cylindrical.
  • a mixture of tetrahydrofuran and N,N-dimethylformamide in a volume ratio of 6:4-7:3 is used as a solvent, and the volume of the two can be 6 :4 or 7:3.
  • the filaments in the hollow oriented tubule 3 are in an oriented state.
  • the hollow oriented tubule 3 is wound from an oriented nanofiber film made of fiber filaments.
  • the length of the hollow alignment tubule 3 is 1-5 cm.
  • the inner diameter of the hollow alignment tubule 3 is 0.5-2.0 mm, and the thickness is 0.05-0.5 mm.
  • the thickness of the oriented nanofiber film is too thick, it is not easy to adhere to itself; if the thickness is too thin, the compression resistance after the film is rolled into a tube is too poor to meet the needs of use.
  • the inner diameter of the hollow oriented small tube 3 is too small, it is easy to cause the inner space of the catheter to be overfilled when more hydrogel columns 4 are placed inside; if the inner diameter is too large, the pressure resistance of the fiber membrane will be very small after the tube is formed, so the inner diameter is 0.5 -2.0mm is suitable.
  • the outer diameter of the hollow oriented tubule 3 is less than or equal to the inner diameter of the cavity formed by the oriented nanofiber membrane 2, so that the hollow oriented tubule 3 can be built into (or built into) the cavity formed by the oriented nanofiber membrane 2. inside the cavity formed.
  • the number of hollow oriented tubules 3 in each peripheral nerve composite conduit is 3-60, for example 3, 6, 10, 12, 20, 30, 40, 50 a, 60. If the number of hollow oriented tubules 3 is too small, the number of channels decreases, which affects the oriented repair and growth of nerves; in addition, if the hollow oriented tubules 3 are too many, the inside of the catheter will be overfilled, leaving no room for growth. Since the peripheral nerve composite catheter is sleeved on the nerve stump, there will be a suture site of about 5 mm, and the two ends are about 1 cm shorter, so the length of the hollow oriented small tube 3 is about 1 cm shorter than that of the nanofiber porous hollow outer tube 1 (see Figure 13 ).
  • the preparation method of the hollow oriented tubule 3 can refer to the preparation method of the oriented nanofiber membrane 2 above.
  • the hollow alignment tubule 3 also has thermal memory. After being squeezed, the hollow oriented small tube 3 is not easy to rebound under low temperature conditions, but can quickly rebound to the original tubular shape at about 37°C.
  • the hydrogel column 4 is made by reacting multi-arm polyethylene glycol succinimide glutarate with trilysine salt.
  • the molar ratio of N-hydroxysuccinimide group (-NHS) in the multi-arm polyethylene glycol succinimide glutarate to the amino group (-NH 2 ) in the trilysine salt is preferably 1: 1 ⁇ 1:4.
  • the multi-arm polyethylene glycol succinimide glutarate includes two-arm, four-arm and eight-arm polyethylene glycol succinimide glutarate.
  • the prepared gel column is elongated to 2-4 times and dried as an oriented gel column, which is built into the cavity of the hollow oriented tubule 3 .
  • the trilysine salt is trilysine acetate.
  • the peripheral nerve composite catheter can be made by assembling the nanofiber porous hollow outer tube 1 , the oriented nanofiber membrane 2 , the hollow oriented small tube 3 and the hydrogel column 4 .
  • the viscosity refers to the viscosity of the raw material mass volume concentration of 0.5% (trichloromethane is used as the solvent), which can be detected by the second method of the Pharmacopoeia 0633 viscosity measurement method (Ubbelohde capillary viscometer method).
  • the peripheral nerve composite catheter provided by the embodiment of the present invention is a high-sensitivity self-regulating slow-release directional composite bionic catheter, which can self-regulate the release of blood-prosperous cells or neurotrophic factors according to the characteristics of nerve growth, and can solve the current problem of nerve fiber regeneration after nerve defects
  • the problem of disordered growth and slow regeneration speed after providing the physical scaffold and space for nerve regeneration, give cells and growth factor components that promote nerve repair, so that the body can self-regulate the release of neurotrophic factors after peripheral nerve damage Or neuron cells, guide the orderly and precise docking and regeneration of nerves.
  • the NGF (nerve growth factor) used below is Enjingfu (mouse nerve growth factor for injection), from Weiming Biomedical Co., Ltd., 18ug/bottle.
  • the present embodiment provides a composite catheter for peripheral nerves, including a nanofiber porous hollow outer tube as a supporting cavity; an oriented nanofiber membrane, attached to the inner wall of the nanofiber porous hollow outer tube to form a cavity, It is used to promote the directional docking and regeneration of nerves; the hollow oriented small tube is built into the cavity formed by the oriented nanofiber membrane, which is used to strengthen the pressure resistance and orientation of the peripheral nerve composite conduit, and guide the orderly growth of nerves; Hydrogel columns embedded in hollow oriented tubules.
  • Adopt polylactic acid-polycaprolactone copolymer (PCL molar content 10%) as starting raw material, viscosity 1.0dl/g, THF and N,N-dimethylformamide as solvent (volume ratio of two kinds of solvents 6: 4), the concentration of the prepared electrospinning solution is 15% (mass volume fraction, W/V, g/ml).
  • Electrospinning parameters are as follows: voltage 15kv, pushing speed 1.5ml/h, receiving distance 10cm, rotating receiver diameter 2mm, rotating speed 50rpm.
  • the spun nanofiber porous hollow outer tube has an inner diameter of 2.1 mm, a length of 2 cm, and a thickness of 0.45 mm.
  • the photomicrograph of the nanofiber porous hollow outer tube prepared in Example 1 is shown in FIG. 2 .
  • Adopt polylactic acid-polycaprolactone copolymer (PCL molar content 10%) as starting raw material, viscosity 1.0dl/g, THF and N,N-dimethylformamide as solvent (volume ratio of two kinds of solvents 6: 4), the concentration of the prepared electrospinning solution is 10% (mass volume fraction, W/V, g/ml).
  • Electrospinning parameters are as follows: voltage 12kv, pushing speed 2ml/h, receiving distance 10cm, rotating receiver diameter 10cm, rotating speed 1500rpm, spun oriented fiber film with a thickness of 0.05mm, and then wound along the vertical direction of the cylindrical rotating receiver On the mold, it is wound into an oriented nanofiber film with a thickness of 0.05 mm and a length of 2 cm, and the inner diameter of the cavity formed by the winding is 2 mm.
  • the photomicrograph of the oriented nanofiber membrane prepared in this example is shown in FIG. 3 .
  • step 2 The same method as step 2) was used to prepare the oriented nanofiber film, which was wound on the mold (0.9 mm in diameter) along the vertical direction of the rotating receiver to prepare a hollow oriented tube with an inner diameter of 0.9 mm, a length of 1 cm, and a thickness of 0.05 mm.
  • the present embodiment provides a composite catheter for peripheral nerves, including a nanofiber porous hollow outer tube as a supporting cavity; an oriented nanofiber membrane, attached to the inner wall of the nanofiber porous hollow outer tube to form a cavity, It is used to promote the directional docking and regeneration of nerves; the hollow oriented small tube is built into the cavity formed by the oriented nanofiber membrane, which is used to strengthen the pressure resistance and orientation of the peripheral nerve composite conduit, and guide the orderly growth of nerves; Hydrogel columns embedded in hollow oriented tubules.
  • the viscosity is 1.0dl/g; using tetrahydrofuran and N,N-dimethylformamide as solvents, the prepared electrospinning solution has a concentration of 12 % (mass volume fraction, W/V, g/ml).
  • Sample S21 The volume ratio of tetrahydrofuran and N,N-dimethylformamide in the solvent is 8.5:1.5;
  • Sample S22 The volume ratio of tetrahydrofuran and N,N-dimethylformamide in the solvent is 6:4.
  • Electrospinning parameters are as follows: voltage 23kv, pushing speed 1ml/h, receiving distance 10cm, rotating receiver diameter 6mm, rotational speed 50rpm.
  • the spun nanofiber porous hollow outer tube has an inner diameter of 6.2 mm, a length of 3 cm, and a thickness of 0.6 mm.
  • Adopt polylactic acid-polycaprolactone copolymer (PCL molar content 10%) as starting material, viscosity 1.0dl/g, tetrahydrofuran and N,N-dimethylformamide as solvent (two kinds of solvent volume ratio 7:3 ), the concentration of the prepared electrospinning solution is 10-15% (mass volume fraction, W/V, g/ml).
  • the electrospinning parameters are as follows: voltage 12kv, push speed 2ml/h, receiving distance 12cm, rotating receiver diameter 10cm, set speed, spin an oriented fiber film with a thickness of 0.1mm, and then wind it in the vertical direction along the rotating receiver. On the mold, it is wound into an oriented nanofiber film with a thickness of 0.10 mm and a length of 3 cm, and the inner diameter of the cavity formed by the winding is 6 mm.
  • Sample S24 The rotational speed is 3100 rpm.
  • Adopt polylactic acid-polycaprolactone copolymer (PCL molar content 10%) as starting material, viscosity 1.0dl/g, tetrahydrofuran and N,N-dimethylformamide as solvent (two kinds of solvent volume ratio 7:3 ), the concentration of the prepared electrospinning solution was 10% (mass volume fraction, W/V, g/m).
  • the electrospinning parameters are as follows: voltage 15kv, push speed 2ml/h, receiving distance 10cm, rotating receiver diameter 10cm, rotating speed 1500rpm, spun oriented fiber film with a thickness of 0.1mm, and then wound it on the mold along the vertical direction of the rotating receiver ( 0.9 mm in diameter), to prepare hollow oriented tubules with an inner diameter of 0.9 mm, a length of 2 cm, and a thickness of 0.10 mm.
  • NGF 500ng/ml
  • four-armed polyethylene glycol succinimide glutarate 4-armed polyethylene glycol succinimide glutarate
  • NGF was loaded on the hydrogel .
  • Hydrogel column diameter 0.5mm, length 3cm.
  • This embodiment provides a peripheral nerve composite catheter, which differs from Embodiment 1 only in that the preparation method of the hydrogel column is different, as follows: four-arm polyethylene glycol succinimide glutarate and trilysine Acid acetate was dissolved separately in buffered saline solution, the two components were mixed, vortexed for 20s, injected into a silica gel tube, and cross-linked and solidified to prepare a hydrogel column.
  • the molar ratio of N-hydroxysuccinimide group (-NHS) in four-arm polyethylene glycol succinimide glutarate to amino group (-NH 2 ) in trilysine acetate is 1:1 .
  • NGF 500ng/ml
  • was added was added, and then cross-linked with four-armed polyethylene glycol succinimide glutarate, and NGF was loaded on the hydrogel .
  • a composite catheter for peripheral nerves the difference from Example 3 is that the fiber filaments of the hollow oriented tubules are in a non-oriented state, and the specific preparation method is as follows:
  • Adopt polylactic acid-polycaprolactone copolymer (PCL molar content 10%) as starting raw material, viscosity 1.0dl/g, THF and N,N-dimethylformamide as solvent (volume ratio of two kinds of solvents 6: 4), the concentration of the prepared electrospinning solution is 10% (mass volume fraction, W/V, g/ml).
  • Electrospinning parameters are as follows: voltage 12kv, push speed 2ml/h, receiving distance 10cm, rotating receiver diameter 10cm, rotating speed 50rpm, spun oriented fiber film with a thickness of 0.05mm, and then wound along the vertical direction of the cylindrical rotating receiver On the mold, a non-oriented nanofiber film is wound, with an inner diameter of 0.9 mm, a length of 1 cm, and a thickness of 0.05 mm.
  • a composite catheter for peripheral nerves the only difference from Embodiment 2 is: there is no hollow oriented small tube; the hydrogel column is directly placed in the cavity formed by the oriented nanofiber membrane.
  • a composite catheter for peripheral nerves the only difference from Embodiment 2 is that it does not contain a hydrogel column.
  • a composite catheter the only difference from Example 1 is: the molar content of PCL in the polylactic acid-polycaprolactone copolymer, the starting material for preparing the nanofiber porous hollow outer tube, is 8%.
  • a composite catheter the only difference from Example 1 is: in the polylactic acid-polycaprolactone copolymer, the starting material for preparing the nanofiber porous hollow outer tube, the molar content of PCL is 30%.
  • Group 1 is the peripheral nerve composite catheter prepared in Example 3.
  • Group 2 is the peripheral nerve composite catheter prepared in Comparative Example 1.
  • the animal operation process is as follows: 10% chloral hydrate 400mg/kg intraperitoneal anesthesia, femoral rear midline incision, exposes the sciatic nerve in the middle section of right hind limb, excises sciatic nerve 1cm, transplants embodiment 3 (the first group) and sciatic nerve defect position respectively Comparative example 1 (group 2) peripheral nerve composite catheter. After the catheter is transplanted, the anastomosis is sutured with surgical thread, and the muscle and skin are sutured. After the operation, the rats in each group were reared routinely. Three months later, the animals were collected, cut longitudinally, and immunofluorescence staining.
  • Group 1 is the peripheral nerve composite catheter prepared in Example 2.
  • Group 2 is the peripheral nerve composite catheter prepared in Comparative Example 2.
  • Group 3 is the peripheral nerve composite catheter prepared in Comparative Example 3.
  • the animal operation process is as follows: 10% chloral hydrate 400mg/kg intraperitoneal anesthesia, femoral rear portion median incision, exposes right hindlimb midsection sciatic nerve, excises sciatic nerve 2cm, transplants embodiment 2 (the 1st group) respectively in sciatic nerve defect site Comparative example 2 (group 2) and comparative example 3 (group 3) peripheral nerve composite catheter. After the catheter is transplanted, the anastomosis is sutured with surgical thread, and the muscle and skin are sutured. After the operation, each group was reared routinely, and the nerve tissue was taken out 90 days after the operation, about 3 cm in length, fixed, dehydrated, transparent, dipped in wax, embedded, and cut transversely. HE staining.
  • Figure 10, Figure 11 and Figure 12 show the HE staining images of the composite catheters of peripheral nerves in Group 1, Group 2 and Group 3, respectively.
  • the axons of the first group of cross-sectional facial nerves are located in the center of each nerve and are in the shape of red dots.
  • the composite catheter socketed with an oriented hollow inner tube has a better repair effect.
  • the nanofiber porous hollow outer tube (inner diameter 2.1 mm, thickness 0.45 mm, cut length 1 cm) was prepared by the method of Example 1, and the number is C tube.
  • the nanofiber porous hollow outer tube (inner diameter 2.1 mm, thickness 0.45 mm, cut length 1 cm) was prepared by the method of Comparative Example 4, and the number is D tube.
  • the nanofiber porous hollow outer tube (inner diameter 2.1 mm, thickness 0.45 mm, cut length 1 cm) was prepared by the method of Comparative Example 5, and the number was E tube.
  • the three kinds of tubes C, D, and E were measured by a universal tensile testing machine (compressed to 60% of the outer diameter of the catheter) and the elastic recovery rate of the catheter (the degree of recovery of the outer diameter of the catheter after 5 minutes after being compressed to 60%) .
  • the results are shown in the table below.
  • the 60% compression force (N) can characterize the anti-compression of the catheter in the muscle space, and the 5min elastic recovery rate can characterize the self-recovery ability of the catheter after compression.
  • N 60% compression force
  • D tube has less elastic recovery rate.
  • the anti-pressure of E tube is 0.166N, which is less than 0.25N (as mentioned in Reaxon Plus 510(k) Summary of similar listed nerve guides), so polylactic acid polycaprolactone copolymer (PCL molar content 10%-25%) is used. It is appropriate.
  • the invention provides a peripheral nerve composite catheter, a preparation method and application thereof.
  • a composite catheter for peripheral nerves comprising: a nanofiber porous hollow outer tube as a supporting cavity; an oriented nanofiber membrane attached to the inner wall of the nanofiber porous hollow outer tube to form a cavity for promoting directional docking and regeneration of nerves;
  • the hollow oriented tubule is built into the cavity formed by the oriented nanofiber membrane, which is used to strengthen the pressure resistance and orientation of the peripheral nerve composite catheter and guide the orderly growth of nerves;
  • the hydrogel column is built into the said In the cavity of the hollow oriented small tube; wherein, the nanofiber porous hollow outer tube, the oriented nanofiber membrane and the hollow oriented small tube are all prepared by an electrospinning process.
  • the peripheral nerve composite catheter provided by the present invention can self-regulate and release blood-prosperous cells or neurotrophic factors according to the characteristics of nerve growth, can solve the current problem of nerve fiber regeneration and repair after nerve defect, and has good economic value and application prospect.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Medicinal Chemistry (AREA)
  • Dermatology (AREA)
  • Epidemiology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Vascular Medicine (AREA)
  • Botany (AREA)
  • Urology & Nephrology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Neurology (AREA)
  • Immunology (AREA)
  • Neurosurgery (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Developmental Biology & Embryology (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Materials For Medical Uses (AREA)

Abstract

本发明涉及外周神经复合导管及其制备方法与应用。外周神经复合导管，包括：纳米纤维多孔中空外管，作为支撑腔体；取向纳米纤维膜，贴附于所述纳米纤维多孔中空外管的内壁形成空腔，用于促进神经定向对接和再生；中空取向性小管，内置于由所述取向纳米纤维膜所形成的空腔中，用于加强外周神经复合导管抗压能力和取向性，引导神经有序生长；水凝胶柱，内置于所述中空取向性小管的空腔中；其中，所述纳米纤维多孔中空外管、取向纳米纤维膜和中空取向性小管均通过静电纺丝工艺制备而成。本发明提供的外周神经复合导管可根据神经生长特性自调释放血旺细胞或者神经营养因子，能够解决目前神经缺损后神经纤维再生修复问题。

Description

外周神经复合导管及其制备方法与应用
交叉引用
本申请要求2021年7月23日提交的专利名称为“外周神经复合导管及其制备方法与应用”的第202110837273.1号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及外周神经复合导管及其制备方法与应用。
背景技术
周围神经损伤是一类发生率和致残率较高的常见损伤，其中，缺损性损伤是最常见的损伤类型。对少量缺损可行断端无张力的缝合，对于较大范围的缺损，自体神经移植仍是目前临床治疗的首选方法，但修复后结果不甚理想，尤其是较长段的缺损(>6mm)。
神经桥接导管可以用于长距离的周围神经缺损修复，是目前研究的热点方向，各种神经导管和优化设计思路不断出现。但现有神经导管的再生神经功能恢复效果还不理想，如专利CN 202010415408.0采用静电纺丝同轴共纺制备具有壳芯结构的取向纳米膜，再卷绕成仿生神经桥接管，无法负载药物、细胞及生长因子类成分。专利CN 201510822838.3将神经生长因子分散在聚合物中制备一种内外套管式神经桥接管，外层无取向起支撑作用，内层取向引导细胞生长，但由于聚合物的亲水性限制，神经生长因子释放缓慢影响神经修复效果。
综上，急需开发一种性能更优异的神经导管，解决目前神经缺损后神经再生修复问题。
发明内容
本发明实施例提供外周神经复合导管及其制备方法与应用。本发明实施例提供的外周神经复合导管可根据神经生长特性自调释放血旺细胞或者神经营养因子，能够解决目前神经缺损后神经纤维再生修复问题。
一种外周神经复合导管，包括：
纳米纤维多孔中空外管，作为支撑腔体；
取向纳米纤维膜，贴附于所述纳米纤维多孔中空外管的内壁形成空腔，用于促进神经定向对接和再生；
中空取向性小管，内置于由所述取向纳米纤维膜所形成的空腔中，用于加强外周神经复合导管抗压能力和取向性，引导神经有序生长；
水凝胶柱，内置于所述中空取向性小管的空腔中；
其中，所述纳米纤维多孔中空外管、取向纳米纤维膜和中空取向性小管均通过静电纺丝工艺制备而成。
根据本发明实施例的所述外周神经复合导管，纳米纤维多孔中空外管中的纤维丝呈无取向性状态。
根据本发明实施例的所述外周神经复合导管，取向纳米纤维膜中的纤维丝呈取向性状态。
根据本发明实施例的所述外周神经复合导管，中空取向性小管中的纤维丝呈取向性状态。
根据本发明实施例的所述外周神经复合导管，水凝胶柱由多臂聚乙二醇琥珀酰亚胺戊二酸酯与三赖氨酸盐反应而制成。优选地，所述多臂聚乙二醇琥珀酰亚胺戊二酸酯中的N-羟基琥珀酰亚胺基(-NHS)与三赖氨酸盐中的氨基(-NH 2)的摩尔比为1:1～1:4；所述多臂聚乙二醇琥珀酰亚胺戊二酸酯优选包括二臂、四臂、八臂聚乙二醇琥珀酰亚胺戊二酸酯。所述三赖氨酸盐优选为三赖氨酸醋酸盐。
根据本发明实施例的所述外周神经复合导管，所述水凝胶柱负载神经生长因子、神经元细胞的纤溶酶敏感微球组分、脑源性神经营养因子、神经元细胞、干细胞的凝胶柱中的一种或几种。
根据本发明实施例的所述外周神经复合导管，静电纺丝所用原料包括：丝素蛋白、壳聚糖、明胶、聚乳酸、聚己内酯、丙交酯乙交酯共聚物中的一种或几种材料的共混物；所用溶剂包括：二氯甲烷、三氯甲烷、 N,N-二甲基甲酰胺、六氟异丙醇、二甲基亚砜、乙酸乙酯。
优选地，制备所述纳米纤维多孔中空外管的原料为聚乳酸聚己内酯共聚物，其中，聚己内酯的摩尔含量为10-25％；更优选以四氢呋喃和N,N-二甲基甲酰胺按体积比为5:5-8:2的混合液作为溶剂，更优选体积比为6:4。
优选地，制备所述取向纳米纤维膜和中空取向性小管的原料为聚乳酸聚己内酯共聚物，其中，聚己内酯的摩尔含量为10-25％；更优选以四氢呋喃和N,N-二甲基甲酰胺按体积比为6:4-7:3的混合液作为溶剂。
本发明还提供一种外周神经复合导管的制备方法，包括：
提供纳米纤维多孔中空外管；
提供取向纳米纤维膜，贴附于所述纳米纤维多孔中空外管的内壁形成空腔；
提供中空取向性小管，内置于由所述取向纳米纤维膜所形成的空腔中；
提供水凝胶柱，内置于所述中空取向性小管的空腔中。
根据本发明实施例，制备所述纳米纤维多孔中空外管的静电纺丝参数如下：电压10-30kv，推速0.5-2ml/h，接收距离8-15cm，旋转接收器直径2.1-11mm，转速50-200rpm。
根据本发明实施例，制备所述取向纳米纤维膜和中空取向性小管的静电纺丝参数如下：电压10-30kv，推速0.5-3ml/h，接收距离8-15cm，旋转接收器直径10-20cm，转速1200-3000rpm。
根据本发明实施例，制备所述纳米纤维多孔中空外管、取向纳米纤维膜和中空取向性小管的原料粘度为0.5-4.0dl/g；以溶剂制成浓度8-15％电纺液，进行静电纺丝。
本发明还包括上述方法制备的外周神经复合导管。
本发明实施例外周神经复合导管，由外向内第一层为静电纺丝制备的纳米纤维多孔中空管作为支撑腔体；第二层为静电纺丝制备的取向纳米纤 维膜卷绕成取向管促进神经定向再生；第三部分为内置中空取向小管，加强导管抗压能力和取向性；第四部分为在内置纤维导管腔体内载有神经生长因子或神经元细胞的纤溶酶敏感凝胶柱，同时起到控释释药和取向的作用。四部分从多方面改善神经再生的微环境，加速神经再生修复。
本发明还提供所述外周神经复合导管在制备用于促进神经修复的材料中的应用。
与现有技术相比，本发明具有如下有益效果：
1)本发明实施例外周神经复合导管采用静电纺丝技术制备的第一层纳米纤维多孔中空外管可以给导管生长空间的和物质交换的同时，又提供较高的机械抗压强度。
2)本发明实施例外周神经复合导管第二层为取向纳米纤维膜，通过对纤维丝的排列方式进行改善，进一步巩固导管强度并引导神经定向再生。
3)神经再生生长锥头部分泌纤溶酶原激活剂能够促使纤溶酶原转化成为纤溶酶。本发明实施例制备纤溶酶敏感的给药系统，水凝胶柱复合有载有雪旺细胞或者神经营养因子的纤溶酶敏感微球，可以控制营养因子或者神经元细胞随着神经再生而不断地根据生理机能局部释放，达到控释效果。同时，亲水性的凝胶柱可以增强神经元细胞的粘附和迁移，还可在神经受损后根据体内纤溶酶表达的增高，智能释放雪旺细胞和神经营养因子，加快神经修复的效果，另外凝胶柱的方向和中空取向性小管的纺丝排列方向，都增加了复合导管的取向性。
附图说明
图1为本发明实施例外周神经复合导管的横截面示意图。
图2为实施例1制备的纳米纤维多孔中空外管的显微照片。
图3为实施例1制备的取向纳米纤维膜的显微照片。
图4和图5分别为实施例2制备的纳米纤维多孔中空外管的显微照片。
图6和图7分别实施例2制备的两种取向纳米纤维膜的显微照片。
图8和图9分别为实验例1中第1组、第2组外周神经复合导管的再生神经横切面HE染色图。
图10、图11和图12分别为实验例2中第1组、第2组及第3组外周神经复合导管的HE染色图。
图13为第1组外周神经复合导管桥接完成图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购买得到的常规产品。
如图1所示，本发明实施例一种外周神经复合导管，包括纳米纤维多孔中空外管1，作为支撑腔体；取向纳米纤维膜2，贴附于纳米纤维多孔中空外管1的内壁形成空腔，用于促进神经定向对接和再生；中空取向性小管3，内置于(或设置于)由取向纳米纤维膜2所形成的空腔中，用于加强外周神经复合导管抗压能力和取向性，引导神经有序生长；水凝胶柱4，内置于(或设置于)中空取向性小管3的空腔中。纳米纤维多孔中空外管1、取向纳米纤维膜2和中空取向性小管3均可通过静电纺丝工艺制备而成。
所述外周神经复合导管可从多方面改善神经再生的微环境，加速神经再生修复。
在一些实施例中，由所述取向纳米纤维膜2所形成的空腔中不设置有水凝胶柱4。
在一些实施例中，中空取向性小管3的内部留有空隙。研究发现，通过在由所述取向纳米纤维膜2所形成的空腔中及中空取向性小管3的空腔留有空隙，有利于促进细胞迁移和神经生长。
在一些实施例中，水凝胶柱4可负载神经生长因子、神经元细胞的纤 溶酶敏感微球组分、脑源性神经营养因子、神经元细胞、干细胞的凝胶柱中的一种或几种。
在一些实施例中，水凝胶柱4可负载神经生长因子和/或神经元细胞的纤溶酶敏感微球组分，同时起到控释释药和引导神经有序生长的作用。
在一些实施例中，水凝胶柱4可负载神经营养因子(NGF、脑源性神经营养因子BDNF等)、神经元细胞、干细胞的凝胶柱。
在一些实施例中，水凝胶柱4的长度为1-5cm，直径0.2-0.8mm。每个外周神经复合导管中水凝胶柱数量为3-500根，或可根据临床修复神经缺损的药物浓度需求而调整水凝胶柱数量。这样既起到一定的管支撑作用，还预留足够的空间给与神经再生。
所述外周神经复合导管可通过静电纺丝技术和原位水凝胶载体技术制备。
在一些实施例中，纳米纤维多孔中空外管1、取向纳米纤维膜2和中空取向性小管3，通过静电纺丝技术制备而成。其中，静电纺丝所用原料包括：丝素蛋白、壳聚糖、明胶、聚乳酸、聚己内酯、丙交酯乙交酯共聚物中的一种或者以上几种材料的共混物；所用溶剂包括：二氯甲烷、三氯甲烷、N,N-二甲基甲酰胺(DMF)、六氟异丙醇、二甲基亚砜(DMSO)、乙酸乙酯等中的至少一种。
在一些实施例中，纳米纤维多孔中空外管1为静电纺丝技术制备的纳米纤维多孔中空管，内径为2.1-11mm，长度2-6cm，厚度(指壁厚，下同)0.25-1.0mm。外周神经各位置粗细不同，最粗可到10mm，最细的小于2mm。内径2.1-11mm的纳米纤维多孔中空外管1可以满足各部位神经修复。长度2-6cm的纳米纤维多孔中空外管1可以适应大多外周神经的修复。大于6cm的神经，缺损过长，神经再生修复结果不理想，通常由神经自体移植替代。另外，纳米纤维多孔中空外管1的厚度达到0.25-1.0mm，这样即使其为内径10mm时也可达到一定抗压性能。
如图2所示，纳米纤维多孔中空外管1中的纤维丝呈无取向性状态。 实验证明，与由呈取向性状态的纤维丝所制成的中空外管相比，纳米纤维多孔中空外管1中的纤维丝呈无取向性状态可以使制备的外周神经复合导管具有更高的机械抗压性能，让导管移植后在肌肉间隙中保持一定的支持空间，避免挤压再生通道。
在一些实施例中，纳米纤维多孔中空外管1的制备方法包括，采用上述聚乳酸聚己内酯共聚物(聚己内酯(PCL)摩尔含量为10-25％)作为静电纺丝原料，粘度优选0.5-4.0dl/g，用上述一种溶剂或者多种溶剂混合液，优选制备成浓度8-15％(质量体积分数，W/V，g/ml)电纺液，进行静电纺丝。静电纺丝参数如下：电压10-30kv，推速0.5-2ml/h，接收距离8-15cm，旋转接收器直径2.1-11mm，转速50-200rpm，纺出的中空管厚度0.25-1.0mm。
实验发现，制备纳米纤维多孔中空外管1的原料例如上述聚乳酸聚己内酯共聚物，粘度为0.5-4.0dl/g时既可以保证纳米纤维多孔中空外管1有一定的力学性能和降解周期，又利于纺丝。
实验发现，制备纳米纤维多孔中空外管1的原料例如上述聚乳酸聚己内酯共聚物，以溶剂制备成浓度8-15％(质量体积分数，W/V，g/ml)电纺液。溶液的浓度对溶液粘度有关，浓度越高，粘度越大，电场力难以克服溶液内部黏力纺丝；溶液浓度越小，粘度越小，不易纺丝或纺丝太短为断丝。
在一些实施例纳米纤维多孔中空外管1的制备方法中，以四氢呋喃和N,N-二甲基甲酰胺按体积比为5:5-8:2的混合液作为溶剂，优选体积比为6:4。
在一些实施例中，取向纳米纤维膜2中的纤维丝呈取向性状态。
在一些实施例中，取向纳米纤维膜2为纤维丝制备的取向纳米纤维膜卷绕成。
在一些实施例中，取向纳米纤维膜2的长度为2-6cm。
在一些实施例中，取向纳米纤维膜2的厚度是0.05-0.5mm，其形成 的空腔的内径为2-10mm。不同部位的神经粗细不同。取向纳米纤维膜2形成的空腔的内径在2-10mm内可以满足多数外周神经的套接。若取向纳米纤维膜厚度太厚，不易于自身粘合；厚度太薄，则在内接或贴附纳米纤维多孔中空外管1内壁的过程中容易褶皱；大量实验发现，取向纳米纤维膜2的厚度在0.05-0.5mm是较为合适的。
如图3所示，取向纳米纤维膜2中的纤维丝呈有序的取向性状态，这样可以使神经在修复早期的神经细胞迁移过程中，促进细胞快速从近端向远端迁移，从而加快神经再生修复的效果。
在一些实施例中，取向纳米纤维膜2的制备方法包括，采用上述一种聚合物或多种聚合物作为静电纺丝原料，具体例如聚乳酸聚己内酯共聚物(PCL摩尔含量为10-25％)，粘度优选0.5-4.0dl/g，用上述一种溶剂或者多种溶剂混合液，优选制备成浓度8-15％(质量体积分数，W/V，g/ml)电纺液，进行静电纺丝。静电纺丝参数如下：电压10-30kv，推速0.5-3ml/h，接收距离8-15cm，旋转接收器直径10-20cm，转速1200-3000rpm，纺出的取向纳米纤维膜厚度为0.05-0.5mm，随后沿着旋转接收器垂直方向卷绕成型。
研究发现，可以通过控制静电纺丝的转速及旋转接收器的形状使纤维丝呈有序的取向性状态，从而获得取向纳米纤维膜。具体来说，静电纺丝的转速应控制在1200-3000rpm，优选旋转接收器的形状为圆柱形。
在一些实施例取向纳米纤维膜2的制备方法中，以四氢呋喃和N,N-二甲基甲酰胺按体积比6:4-7:3的混合液作为溶剂，二者的体积可选为6:4或7:3。
在一些实施例中，中空取向性小管3中的纤维丝呈取向性状态。
在一些实施例中，中空取向性小管3为纤维丝制备的取向纳米纤维膜卷绕成。
在一些实施例中，中空取向性小管3的长度为1-5cm。
在一些实施例中，中空取向性小管3的内径为0.5-2.0mm，厚度是 0.05-0.5mm。研究发现，对于中空取向性小管3，若取向纳米纤维膜厚度太厚，不易于自身粘合；若厚度太薄，则卷膜成管后抗压性能太差，不能满足使用需要。若中空取向性小管3的内径太小，内部放较多的水凝胶柱4时容易造成导管内部空间填充过满；若内径太大，纤维膜成管后抗压力会很小，因此内径0.5-2.0mm为宜。
可以理解，中空取向性小管3的外径小于或等于由取向纳米纤维膜2所形成的空腔的内径，从而可以使得中空取向性小管3内置于(或内置于)由取向纳米纤维膜2所形成的空腔内部。
在一些实施例中，每个外周神经复合导管中的中空取向性小管3的数量为3-60个，例如3个、6个、10个、12个、20个、30个、40个、50个、60个。中空取向性小管3数目过少导致通道数减少，影响神经的取向性修复生长；另外，若中空取向性小管3太多，会使导管内部填充过满，而无生长的空间。由于外周神经复合导管套接在神经断端会有约5mm的缝合部位，两端少1cm左右，故中空取向性小管3的长度要比纳米纤维多孔中空外管1短1cm左右(可参见图13)。
中空取向性小管3的制备方法可参照上文取向纳米纤维膜2的制备方法。
在一些实施例中，中空取向性小管3还具有热记性。中空取向性小管3在受到挤压后，在低温条件下，不易回弹，而在37℃左右时，能够较快的回弹至原始管状形态。
在一些实施例中，水凝胶柱4由多臂聚乙二醇琥珀酰亚胺戊二酸酯与三赖氨酸盐反应而制成。其中，多臂聚乙二醇琥珀酰亚胺戊二酸酯中的N-羟基琥珀酰亚胺基(-NHS)与三赖氨酸盐中的氨基(-NH 2)摩尔比优选为1:1～1:4。所述多臂聚乙二醇琥珀酰亚胺戊二酸酯包括二臂、四臂、八臂聚乙二醇琥珀酰亚胺戊二酸酯。
在一些实施例中，将制备的凝胶柱拉长至2-4倍干燥作为取向凝胶柱，内置于中空取向性小管3的空腔中。
在一些实施例中，所述三赖氨酸盐为三赖氨酸醋酸盐。
在一些实施例中，将纳米纤维多孔中空外管1、取向纳米纤维膜2、中空取向性小管3和水凝胶柱4组装，即可制成外周神经复合导管。
本文中，所述粘度是指原料质量体积浓度0.5％的粘度(溶剂采用三氯甲烷)可采用药典0633粘度测定法第二法(乌式毛细管黏度计测定法)检测。
本发明实施例提供的外周神经复合导管是一种高灵敏度自调控缓释取向性复合仿生导管，可根据神经生长特性自调释放血旺细胞或者神经营养因子，能够解决目前神经缺损后神经纤维再生的无序生长和再生速度较慢的问题，在提供神经再生的物理支架和空间后，给予促进神经修复的细胞和生长因子组分，使机体在外周神经受损后，自调控释放神经营养因子或者神经元细胞，引导神经定向有序和精准对接再生。
以下所用NGF(神经生长因子)为恩经复(注射用鼠神经生长因子)，未名生物医药有限公司，18ug/瓶。
实施例1
如图1所示，本实施例提供一种外周神经复合导管，包括纳米纤维多孔中空外管，作为支撑腔体；取向纳米纤维膜，贴附于纳米纤维多孔中空外管的内壁形成空腔，用于促进神经定向对接和再生；中空取向性小管，内置于由所述取向纳米纤维膜所形成的空腔中，用于加强外周神经复合导管抗压能力和取向性，引导神经有序生长；水凝胶柱，内置于中空取向性小管中。
本实施例外周神经复合导管具体制备方法如下：
1)纳米纤维多孔中空外管的制备
采用聚乳酸-聚己内酯共聚物(PCL摩尔含量10％)作为起始原料，粘度1.0dl/g，四氢呋喃和N,N-二甲基甲酰胺作为溶剂(两种溶剂的体积比6:4)，制备的电纺液浓度15％(质量体积分数，W/V，g/ml)。
静电纺丝参数如下：电压15kv，推速1.5ml/h，接收距离10cm，旋 转接收器直径2mm，转速50rpm。纺出的纳米纤维多孔中空外管，内径2.1mm，长度2cm，厚度0.45mm。
本实施例1制备的纳米纤维多孔中空外管的显微照片见图2。
2)取向纳米纤维膜的制备
采用聚乳酸-聚己内酯共聚物(PCL摩尔含量10％)作为起始原料，粘度1.0dl/g，四氢呋喃和N,N-二甲基甲酰胺作为溶剂(两种溶剂的体积比6:4)，制备的电纺液浓度10％(质量体积分数，W/V，g/ml)。
静电纺丝参数如下：电压12kv，推速2ml/h，接收距离10cm，旋转接收器直径10cm，转速1500rpm，纺出厚度0.05mm的取向纤维膜，随后沿着圆柱形旋转接收器垂直方向卷绕在模具上，卷绕成取向纳米纤维膜，厚度0.05mm，长度2cm，其卷绕形成的空腔的内径为2mm。
本实施例制备的取向纳米纤维膜的的显微照片见图3。
3)中空取向性小管的制备
采用与步骤2)相同的方法制备取向纳米纤维膜，沿着旋转接收器垂直方向卷绕在模具(直径0.9mm)制备成中空取向性小管，内径0.9mm、长度1cm、厚度0.05mm。
4)水凝胶柱的制备
四臂聚乙二醇琥珀酰亚胺戊二酸酯与三赖氨酸醋酸盐采用缓冲盐溶液分别溶解，两组分混合，涡旋20s，注入硅胶管中，交联固化制备水凝胶柱。其中，四臂聚乙二醇琥珀酰亚胺戊二酸酯中的N-羟基琥珀酰亚胺基(-NHS)与三赖氨酸醋酸盐中的氨基(-NH 2)摩尔比为1:1。水凝胶柱：直径0.5mm、长度2cm。
组装：将纳米纤维多孔中空外管、取向纳米纤维膜、中空取向性小管和水凝胶柱组装，每个外周神经复合导管中的中空取向性小管的数量为3个，每个中空取向性小管中水凝胶柱的数量为3根，即可制成外周神经复合导管，其横截面示意图参见图1。
实施例2
如图1所示，本实施例提供一种外周神经复合导管，包括纳米纤维多孔中空外管，作为支撑腔体；取向纳米纤维膜，贴附于纳米纤维多孔中空外管的内壁形成空腔，用于促进神经定向对接和再生；中空取向性小管，内置于由所述取向纳米纤维膜所形成的空腔中，用于加强外周神经复合导管抗压能力和取向性，引导神经有序生长；水凝胶柱，内置于中空取向性小管中。
本实施例外周神经复合导管具体制备方法如下：
1)纳米纤维多孔中空外管的制备
采用聚乳酸-聚己内酯共聚物(PCL摩尔含量10％)作为起始原料，粘度1.0dl/g；以四氢呋喃和N,N-二甲基甲酰胺作为溶剂，制备的电纺液浓度12％(质量体积分数，W/V，g/ml)。
样品S21：溶剂中四氢呋喃和N,N-二甲基甲酰胺的体积比为8.5:1.5；
样品S22：溶剂中四氢呋喃和N,N-二甲基甲酰胺的体积比为6:4。
静电纺丝参数如下：电压23kv，推速1ml/h，接收距离10cm，旋转接收器直径6mm，转速50rpm。纺出的纳米纤维多孔中空外管，内径6.2mm、长度3cm、厚度0.6mm。
样品S21、样品S22的显微照片分别见图4和图5。
结果可见，与样品S22相比，样品S21的显微镜下微结构纤维丝不够光滑，孔结构较小，中间掺有碎珠。
进一步实验结果表明，溶剂中四氢呋喃和N,N-二甲基甲酰胺的体积比为5:5-8:2是较为合适的，所制备的导管显微结构纤维丝光滑，孔结构适中。
2)取向纳米纤维膜的制备
采用聚乳酸-聚己内酯共聚物(PCL摩尔含量10％)作为起始原料，粘度1.0dl/g，四氢呋喃和N,N-二甲基甲酰胺作为溶剂(两种溶剂体积比7:3)，制备的电纺液浓度10-15％(质量体积分数，W/V，g/ml)。
静电纺丝参数如下：电压12kv，推速2ml/h，接收距离12cm，旋转 接收器直径10cm，设定转速，纺出厚度0.1mm的取向纤维膜，随后沿着旋转接收器垂直方向卷绕在模具上，卷绕成取向纳米纤维膜，厚度0.10mm，长度3cm，其卷绕形成的空腔的内径为6mm。
样品S23：转速为1500rpm；
样品S24：转速为3100rpm。
样品S23、样品S24的显微照片分别见图6和图7。
结果可见，与样品S24相比，取向纤维膜样品S23的电镜显微结构的取向性更高。进一步实验结果表明，为保证纤维膜的取向性更高，应使转速控制在1200-3000rpm。
3)中空取向性小管的制备
采用聚乳酸-聚己内酯共聚物(PCL摩尔含量10％)作为起始原料，粘度1.0dl/g，四氢呋喃和N,N-二甲基甲酰胺作为溶剂(两种溶剂体积比7:3)，制备的电纺液浓度10％(质量体积分数，W/V，g/m)。
静电纺丝参数如下：电压15kv，推速2ml/h，接收距离10cm，旋转接收器直径10cm，转速1500rpm，纺出厚度0.1mm取向纤维膜，随后沿着旋转接收器垂直方向卷绕在模具(直径0.9mm)，制备中空取向性小管，内径0.9mm、长度2cm、厚度0.10mm。
4)水凝胶柱的制备
四臂聚乙二醇琥珀酰亚胺戊二酸酯与三赖氨酸醋酸盐采用缓冲盐溶液(磷酸盐缓冲液，pH＝7.3)分别溶解，两组分混合，涡旋20s，注入硅胶管中，交联固化制备水凝胶柱。其中，四臂聚乙二醇琥珀酰亚胺戊二酸酯中的N-羟基琥珀酰亚胺基(-NHS)与三赖氨酸醋酸盐中的氨基(-NH 2)摩尔比为1:1。其中，在溶解三赖氨酸醋酸盐过程中，加入NGF(500ng/ml)，随后与四臂聚乙二醇琥珀酰亚胺戊二酸酯反应交联，NGF便负载在水凝胶上。水凝胶柱，直径0.5mm、长度3cm。
组装：将纳米纤维多孔中空外管、取向纳米纤维膜(样品S22)、中空取向性小管(样品S23)和水凝胶柱组装，每个外周神经复合导管中的 中空取向性小管的数量为12个，每个中空取向性小管中水凝胶柱的数量为3根，即可制成外周神经复合导管，横截面示意图参见图1。
实施例3
本实施例提供一种外周神经复合导管，其与实施例1的区别仅在于水凝胶柱的制备方法不同，具体如下：四臂聚乙二醇琥珀酰亚胺戊二酸酯与三赖氨酸醋酸盐采用缓冲盐溶液分别溶解，两组分混合，涡旋20s，注入硅胶管中，交联固化制备水凝胶柱。四臂聚乙二醇琥珀酰亚胺戊二酸酯中的N-羟基琥珀酰亚胺基(-NHS)与三赖氨酸醋酸盐中的氨基(-NH 2)摩尔比为1:1。其中，在溶解三赖氨酸醋酸盐过程中，加入NGF(500ng/ml)，随后与四臂聚乙二醇琥珀酰亚胺戊二酸酯反应交联，NGF便负载在水凝胶上。
对比例1
一种外周神经复合导管，与实施例3的区别仅在于中空取向性小管的纤维丝呈无取向性状态，具体制备方法如下：
采用聚乳酸-聚己内酯共聚物(PCL摩尔含量10％)作为起始原料，粘度1.0dl/g，四氢呋喃和N,N-二甲基甲酰胺作为溶剂(两种溶剂的体积比6:4)，制备的电纺液浓度10％(质量体积分数，W/V，g/ml)。
静电纺丝参数如下：电压12kv，推速2ml/h，接收距离10cm，旋转接收器直径10cm，转速50rpm，纺出厚度0.05mm的取向纤维膜，随后沿着圆柱形旋转接收器垂直方向卷绕在模具上，卷绕成无取向纳米纤维膜，内径0.9mm，长度1cm，厚度0.05mm。
对比例2
一种外周神经复合导管，与实施例2的区别仅在于：没有中空取向性小管；水凝胶柱直接放置于由取向纳米纤维膜所形成的空腔中。
对比例3
一种外周神经复合导管，与实施例2的区别仅在于：不含有水凝胶柱。
对比例4
一种复合导管，与实施例1的区别仅在于：制备纳米纤维多孔中空外管的起始原料聚乳酸-聚己内酯共聚物中，PCL摩尔含量为8％。
对比例5
一种复合导管，与实施例1的区别仅在于：制备纳米纤维多孔中空外管的起始原料聚乳酸-聚己内酯共聚物中，PCL摩尔含量为30％。
实验例1体内动物实验
(1)实验动物及移植手术过程
健康成年雄性SD大鼠12只，体重250-300g，将其随机分为2组，每组6只。
第1组为实施例3制备的外周神经复合导管。
第2组为对比例1制备的外周神经复合导管。
(2)动物手术过程如下：10％水合氯醛400mg/kg腹腔麻醉，股后部正中切口，暴露右后肢中段坐骨神经，切除坐骨神经1cm，分别在坐骨神经缺损部位移植实施例3(第1组)和对比例1(第2组)外周神经复合导管。导管移植后手术线缝合吻合口，缝合肌肉和皮肤。术后各组常规饲养，三个月后取材，行纵切，免疫荧光染色。
第1组及第2组中典型的神经再生横切面HE染色图分别见图8和图9。
由图8可见，横切面神经轴突位于每条神经中央，呈深红色圆点状，其外层为粉红色髓鞘结构，髓鞘外层为神经膜。由图9可以看出，部分神经纤维轴突消失，髓鞘结构不清晰，神经膜结构不完整，神经束内可见较多新生毛细血管。
实验例2体内动物实验
(1)实验动物及移植手术过程
比格犬，9只，体重7-8kg，将其随机分为3组，每组3只。
第1组为实施例2制备的外周神经复合导管。
第2组为对比例2制备的外周神经复合导管。
第3组为对比例3制备的外周神经复合导管。
(2)动物手术过程如下：10％水合氯醛400mg/kg腹腔麻醉，股后部正中切口，暴露右后肢中段坐骨神经，切除坐骨神经2cm，分别在坐骨神经缺损部位移植实施例2(第1组)、对比例2(第2组)和对比例3(第3组)外周神经复合导管。导管移植后手术线缝合吻合口，缝合肌肉和皮肤。术后各组常规饲养，于术后90d将神经组织取出，长约3cm，固定、脱水、透明、浸蜡、包埋，横切。HE染色。
第1组、第2组及第3组外周神经复合导管的HE染色图分别见图10、图11和图12。
由图10可见，第1组横切面神经轴突位于每条神经中央，呈红色圆点状，其外层为粉红色髓鞘结构，髓鞘外层为神经膜，病理结果显示，神经组织走行正常，排列整齐，在神经束膜包绕下，形成圆形或椭圆形神经束，套接取向性中空内管的复合导管其修复效果更佳。
由图11可见，第2组神经束内神经纤维排列紊乱，神经轴突消失或偏离中央位置，髓鞘结构不清晰，神经膜结构不完整。
由图12可见，第3组神经组织走行正常，排列略显紊乱，神经束膜明显增厚，排列形状不规则，疏松弯曲。
HE染色显示，第1组比第2组和第3组神经再生更好。
第1组外周神经复合导管桥接完成(两端预留缝线距离)可参见图13。
实验例3机械性能实验
以实施例1的方法制备纳米纤维多孔中空外管(内径2.1mm，厚度0.45mm，裁剪成长度1cm)，编号为C管。
以对比例4的方法制备纳米纤维多孔中空外管(内径2.1mm，厚度0.45mm，裁剪成长度1cm)，编号为D管。
以对比例5的方法制备纳米纤维多孔中空外管(内径2.1mm，厚度0.45mm，裁剪成长度1cm)，编号为E管。
将C、D、E三种管采用万能拉力试验机测量抗压性能(压缩至导管外径的60％力的大小)和导管弹性回复率(导管压缩至60％后5min后外径恢复程度)。结果见下表。
导管编号 60％压缩力(N) 5min弹性回复率
C 0.332 92％
D 1.264 75％
E 0.166 99％
结果：60％压缩力(N)可以表征导管在肌肉间隙中的抗压力，5min弹性回复率可以表征导管受压后自身恢复能力。避免神经再生空间减少，理想条件应该抗压力和弹性回复率尽可能高为好。D管弹性恢复率较少。E管抗压力为0.166N，小于0.25N(同类已上市神经导管Reaxon Plus 510(k)Summary中所提)，因此以选用聚乳酸聚己内酯共聚物(PCL摩尔含量10％-25％)为宜。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供一种外周神经复合导管及其制备方法与应用。外周神经复合导管，包括：纳米纤维多孔中空外管，作为支撑腔体；取向纳米纤维膜，贴附于所述纳米纤维多孔中空外管的内壁形成空腔，用于促进神经定向对接和再生；中空取向性小管，内置于由所述取向纳米纤维膜所形成的空腔中，用于加强外周神经复合导管抗压能力和取向性，引导神经有序生长；水凝胶柱，内置于所述中空取向性小管的空腔中；其中，所述纳米纤维多孔中空外管、取向纳米纤维膜和中空取向性小管均通过静电纺丝工艺制备而成。本发明提供的外周神经复合导管可根据神经生长特性自调释放血旺细胞或者神经营养因子，能够解决目前神经缺损后神经纤维再生修复问题，具有较好的经济价值和应用前景。

Claims (18)

  1. 一种外周神经复合导管，其包括：
    纳米纤维多孔中空外管，作为支撑腔体；
    取向纳米纤维膜，贴附于所述纳米纤维多孔中空外管的内壁形成空腔，用于促进神经定向对接和再生；
    中空取向性小管，内置于由所述取向纳米纤维膜所形成的空腔中，用于加强外周神经复合导管抗压能力和取向性，引导神经有序生长；
    水凝胶柱，内置于所述中空取向性小管的空腔中；
    其中，所述纳米纤维多孔中空外管、取向纳米纤维膜和中空取向性小管均通过静电纺丝工艺制备而成。
  2. 根据权利要求1所述的外周神经复合导管，其中，所述纳米纤维多孔中空外管中的纤维丝呈无取向性状态；和/或，
    所述取向纳米纤维膜中的纤维丝呈取向性状态；和/或，
    所述中空取向性小管中的纤维丝呈取向性状态。
  3. 根据权利要求1或2所述的外周神经复合导管，其中，所述水凝胶柱由多臂聚乙二醇琥珀酰亚胺戊二酸酯与三赖氨酸盐反应而制成。
  4. 根据权利要求3所述的外周神经复合导管，其中，所述多臂聚乙二醇琥珀酰亚胺戊二酸酯中的N-羟基琥珀酰亚胺基(-NHS)与三赖氨酸盐中的氨基(-NH2)的摩尔比为1:1～1:4。
  5. 根据权利要求3所述的外周神经复合导管，其中，所述多臂聚乙二醇琥珀酰亚胺戊二酸酯包括二臂、四臂、八臂聚乙二醇琥珀酰亚胺戊二酸酯。
  6. 根据权利要求3所述的外周神经复合导管，其中，所述三赖氨酸盐为三赖氨酸醋酸盐。
  7. 根据权利要求1-6任一项所述的外周神经复合导管，其中，所述水凝胶柱负载神经生长因子、神经元细胞的纤溶酶敏感微球组分、脑源性神经营养因子、神经元细胞、干细胞的凝胶柱中的一种或几种。
  8. 根据权利要求1-7任一项所述的外周神经复合导管，其中，静电 纺丝所用原料包括：丝素蛋白、壳聚糖、明胶、聚乳酸、聚己内酯、丙交酯乙交酯共聚物中的一种或几种材料的共混物；所用溶剂包括：二氯甲烷、三氯甲烷、N,N-二甲基甲酰胺、六氟异丙醇、二甲基亚砜、乙酸乙酯。
  9. 根据权利要求8所述的外周神经复合导管，其中，制备所述纳米纤维多孔中空外管的原料为聚乳酸聚己内酯共聚物，其中，聚己内酯的摩尔含量为10-25％。
  10. 根据权利要求9所述的外周神经复合导管，其中，在制备所述纳米纤维多孔中空外管中，以四氢呋喃和N,N-二甲基甲酰胺按体积比为5:5-8:2的混合液作为溶剂。
  11. 根据权利要求10所述的外周神经复合导管，其中，在制备所述纳米纤维多孔中空外管中，以四氢呋喃和N,N-二甲基甲酰胺按体积比为6:4的混合液作为溶剂。
  12. 根据权利要求8所述的外周神经复合导管，其中，制备所述取向纳米纤维膜和中空取向性小管的原料为聚乳酸聚己内酯共聚物，其中，聚己内酯的摩尔含量为10-25％。
  13. 根据权利要求12所述的外周神经复合导管，其中，在制备所述取向纳米纤维膜和中空取向性小管中，以四氢呋喃和N,N-二甲基甲酰胺按体积比为6:4-7:3的混合液作为溶剂。
  14. 权利要求1-13任一项所述的外周神经复合导管的制备方法，包括：
    提供纳米纤维多孔中空外管；
    提供取向纳米纤维膜，贴附于所述纳米纤维多孔中空外管的内壁形成空腔；
    提供中空取向性小管，内置于由所述取向纳米纤维膜所形成的空腔中；
    提供水凝胶柱，内置于所述中空取向性小管的空腔中。
  15. 根据权利要求14所述的外周神经复合导管的制备方法，其中，制备所述纳米纤维多孔中空外管的静电纺丝参数如下：电压10-30kv，推速0.5-2ml/h，接收距离8-15cm，旋转接收器直径2.1-11mm，转速 50-200rpm；和/或，
    制备所述取向纳米纤维膜和中空取向性小管的静电纺丝参数如下：电压10-30kv，推速0.5-3ml/h，接收距离8-15cm，旋转接收器直径10-20cm，转速1200-3000rpm。
  16. 根据权利要求14或15所述的外周神经复合导管的制备方法，其中，制备所述纳米纤维多孔中空外管、取向纳米纤维膜和中空取向性小管的原料粘度为0.5-4.0dl/g；以溶剂制成浓度8-15％电纺液，进行静电纺丝。
  17. 权利要求14-16任一项所述方法制备的外周神经复合导管。
  18. 权利要求1-13、17任一项所述外周神经复合导管在制备用于促进神经修复的材料中的应用。
PCT/CN2022/107235 2021-07-23 2022-07-22 外周神经复合导管及其制备方法与应用 WO2023001261A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110837273.1 2021-07-23
CN202110837273.1A CN113633430B (zh) 2021-07-23 2021-07-23 外周神经复合导管及其制备方法与应用

Publications (1)

Publication Number Publication Date
WO2023001261A1 true WO2023001261A1 (zh) 2023-01-26

Family

ID=78418270

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/107235 WO2023001261A1 (zh) 2021-07-23 2022-07-22 外周神经复合导管及其制备方法与应用

Country Status (2)

Country Link
CN (1) CN113633430B (zh)
WO (1) WO2023001261A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116139335A (zh) * 2023-03-19 2023-05-23 西安交通大学 具有定向输送能力的多通道孔径渐变水凝胶支架及其制备方法
CN116196472A (zh) * 2023-02-23 2023-06-02 武汉理工大学 一种双导电载药复合神经导管及其制备方法
CN116899014A (zh) * 2023-07-24 2023-10-20 上海工程技术大学 一种三维拓扑结构多通道神经导管及其制备方法
CN117018290A (zh) * 2023-10-10 2023-11-10 常州丝波敦生物科技有限公司 一种具有仿生取向结构的丝蛋白纳米纤维神经导管及其制备方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113633430B (zh) * 2021-07-23 2023-09-19 北京诺康达医药科技股份有限公司 外周神经复合导管及其制备方法与应用
CN117618657A (zh) * 2022-08-25 2024-03-01 北京化工大学 一种超多孔水凝胶填充神经导管及其制备方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120221025A1 (en) * 2009-09-14 2012-08-30 David Simpson Electrospun nerve guides for nerve regeneration designed to modulate nerve architecture
CN108187148A (zh) * 2018-02-13 2018-06-22 中山大学附属第医院 一种能提高轴突再生有序性的神经导管及其制备方法
CN110694104A (zh) * 2019-10-21 2020-01-17 深圳市立心科学有限公司 复合人工纤维元及人工韧带
CN110869063A (zh) * 2017-06-12 2020-03-06 博洛尼亚大学 用于肌腱/韧带组织的再生和/或置换的分层多尺度电纺支架及其生产方法
CN110975016A (zh) * 2019-12-25 2020-04-10 武汉理工大学 一种神经修复导管及其制备方法
KR20200119919A (ko) * 2019-03-19 2020-10-21 고려대학교 산학협력단 바이오 프린팅 기술을 이용한 신경도관의 제조방법 및 이에 따라 제조된 신경도관
CN112915255A (zh) * 2021-01-20 2021-06-08 浙江大学 一种多尺度生物支架及其制造方法与应用
CN113633430A (zh) * 2021-07-23 2021-11-12 北京诺康达医药科技股份有限公司 外周神经复合导管及其制备方法与应用

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120221025A1 (en) * 2009-09-14 2012-08-30 David Simpson Electrospun nerve guides for nerve regeneration designed to modulate nerve architecture
CN110869063A (zh) * 2017-06-12 2020-03-06 博洛尼亚大学 用于肌腱/韧带组织的再生和/或置换的分层多尺度电纺支架及其生产方法
CN108187148A (zh) * 2018-02-13 2018-06-22 中山大学附属第医院 一种能提高轴突再生有序性的神经导管及其制备方法
KR20200119919A (ko) * 2019-03-19 2020-10-21 고려대학교 산학협력단 바이오 프린팅 기술을 이용한 신경도관의 제조방법 및 이에 따라 제조된 신경도관
CN110694104A (zh) * 2019-10-21 2020-01-17 深圳市立心科学有限公司 复合人工纤维元及人工韧带
CN110975016A (zh) * 2019-12-25 2020-04-10 武汉理工大学 一种神经修复导管及其制备方法
CN112915255A (zh) * 2021-01-20 2021-06-08 浙江大学 一种多尺度生物支架及其制造方法与应用
CN113633430A (zh) * 2021-07-23 2021-11-12 北京诺康达医药科技股份有限公司 外周神经复合导管及其制备方法与应用

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116196472A (zh) * 2023-02-23 2023-06-02 武汉理工大学 一种双导电载药复合神经导管及其制备方法
CN116196472B (zh) * 2023-02-23 2024-03-26 武汉理工大学 一种双导电载药复合神经导管及其制备方法
CN116139335A (zh) * 2023-03-19 2023-05-23 西安交通大学 具有定向输送能力的多通道孔径渐变水凝胶支架及其制备方法
CN116139335B (zh) * 2023-03-19 2023-10-31 西安交通大学 具有定向输送能力的多通道孔径渐变水凝胶支架及其制备方法
CN116899014A (zh) * 2023-07-24 2023-10-20 上海工程技术大学 一种三维拓扑结构多通道神经导管及其制备方法
CN116899014B (zh) * 2023-07-24 2024-05-07 上海工程技术大学 一种三维拓扑结构多通道神经导管及其制备方法
CN117018290A (zh) * 2023-10-10 2023-11-10 常州丝波敦生物科技有限公司 一种具有仿生取向结构的丝蛋白纳米纤维神经导管及其制备方法
CN117018290B (zh) * 2023-10-10 2024-01-05 常州丝波敦生物科技有限公司 一种具有仿生取向结构的丝蛋白纳米纤维神经导管及其制备方法

Also Published As

Publication number Publication date
CN113633430A (zh) 2021-11-12
CN113633430B (zh) 2023-09-19

Similar Documents

Publication Publication Date Title
WO2023001261A1 (zh) 外周神经复合导管及其制备方法与应用
US5925053A (en) Multi-lumen polymeric guidance channel, method for promoting nerve regeneration, and method of manufacturing a multi-lumen nerve guidance channel
JP5638554B2 (ja) 治療薬デリバリー用ドラッグ放出生分解性繊維
US8652215B2 (en) Nanofilament scaffold for tissue regeneration
AU2007211018B2 (en) Biomimetic scaffolds
CN102525689B (zh) 取向纳米纤维仿生神经导管及其制作方法
CN106913393B (zh) 一种人工神经支架及其制备方法与应用
AU772047B2 (en) Multi-channel bioresorbable nerve regeneration conduit and process for preparing the same
CN106729980B (zh) 一种用于外周神经修复的仿生神经移植物及其制备方法
US20050161857A1 (en) Polymeric fibre and method for making same
KR102265640B1 (ko) 바이오 프린팅 기술을 이용한 신경도관의 제조방법 및 이에 따라 제조된 신경도관
EP1454641A2 (en) Porous implantable medical device seeded with mammalian cells
US20030203003A1 (en) Drug releasing biodegradable fiber implant
CN103920194B (zh) 一种多层神经导管及其制备方法
MX2011009282A (es) Duramadre artificial y su metodo de fabricacion.
JP2008513051A5 (zh)
CN101543645A (zh) 聚己内酯pcl静电纺丝神经导管及其制备和应用
WO2016192733A1 (en) Conduit for regeneration of biological material
CN110755684A (zh) 负载外泌体和生长因子的微球/纳米纱复合支架及其制备方法
WO2020062976A1 (zh) 一种具有生物活性的可降解手术缝线及其制备方法
ES2430353T3 (es) Banda de nanofibras biomiméticas y método y dispositivo para fabricar la misma
CN105828847B (zh) 由纱线制备可吸收的聚合物管
US11369717B2 (en) Fibrous nerve conduit for promoting nerve regeneration
Biazar et al. Nanotechnology for peripheral nerve regeneration
Cirillo et al. 16.1 Pathophysiology of nerve degeneration and regeneration

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22845424

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE