WO2018157847A1 - 一种神经修复材料、制备方法和应用 - Google Patents

一种神经修复材料、制备方法和应用 Download PDF

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WO2018157847A1
WO2018157847A1 PCT/CN2018/077810 CN2018077810W WO2018157847A1 WO 2018157847 A1 WO2018157847 A1 WO 2018157847A1 CN 2018077810 W CN2018077810 W CN 2018077810W WO 2018157847 A1 WO2018157847 A1 WO 2018157847A1
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nerve
solution
growth factor
cleaning
frequency
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PCT/CN2018/077810
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English (en)
French (fr)
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赵博
王洪权
夏磊磊
赵延瑞
李学军
张晋辉
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北京博辉瑞进生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • 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
    • A61L27/20—Polysaccharides
    • 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
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/225—Fibrin; Fibrinogen
    • 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
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3629—Intestinal tissue, e.g. small intestinal submucosa
    • 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
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3675—Nerve tissue, e.g. brain, spinal cord, nerves, dura mater
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    • 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
    • A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification 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
    • 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
    • A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
    • 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
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • 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
    • A61L27/58—Materials at least partially resorbable by the body
    • 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
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    • 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 the technical field of medical biomaterials, in particular to a nerve repairing material, which is used for compounding growth factors into a small intestinal submucosal immunogen to remove matrix, protecting damaged and defective peripheral nerves, preventing tissue adhesion and inducing nerve repair.
  • peripheral nerves spread throughout the skin, mucous membranes, muscles, bones and joints, blood vessels and internal organs. Therefore, human tissues may involve nerve damage caused by peripheral nerves due to traction, cutting, compression, burns, and ischemia.
  • the peripheral nerve is a cell protrusion of neurons, also called nerve fiber, composed of axon, myelin sheath and Schwann sheath.
  • the axonal function is the nerve impulse conduction between the neuron and the neuronal terminal structure, and the myelin sheath prevents the excitatory diffusion.
  • the Schwann sheath is composed of Schwann cells and is a channel for nerve regeneration.
  • the damaged nerve should be protected first, the nerve is isolated from the surrounding tissue, and the surrounding tissues such as muscles, tendons and fascia are prevented from growing into scar tissue, and nutrients are provided for nerve regeneration, and the axons are guided. Regeneration recovery function.
  • the traditional method of repairing nerves is limited to suture stitching. Biomaterials are not used for damage protection. After suture sutures, edema will appear at the anastomosis. Excessive proliferation of fibroblasts may lead to fibrosis at the anastomosis. Or tissue adhesion, which will seriously affect the recovery of nerve function. At the same time, most of the nerve injury is accompanied by muscle, tendon and fascia damage. The repair of these tissues is mainly based on scar repair, which also leads to excessive scar formation around the nerve anastomosis and affects nerve regeneration. And the most important thing is that the repair of Schwann sheath is not considered.
  • the present invention is directed to the above-mentioned deficiencies of the prior art, and provides a nerve repairing material which combines growth factors with a small intestinal mucosa underlying immunogen to remove matrix, protect damaged peripheral nerves, prevent tissue adhesion, and induce nerve repair.
  • the technical solution adopted by the present invention is: a nerve repairing material, characterized in that the nerve repairing material comprises collagen, a polysaccharide substance, an active factor and a nerve regeneration promoting factor.
  • the nerve repairing material of the present invention has a three-dimensional network porous structure, is non-immunogenic, is degradable in vivo, and may be in the form of a sheet or a hollow tube.
  • Nerve damage can be divided into fracture damage and non-fracture damage, in which non-fracture injury such as a part of nerve fiber is damaged or contused, in this case, the nerve is not broken, the hollow tube can be directly used to repair the fracture damage, and the nerve cells are in the middle.
  • the empty tube grows and is isolated from other tissues, thereby controlling local scars and reducing the adhesion of nerves to other tissues; sheet materials are used to treat non-fracture damage, wrapping and isolating nerves.
  • the nerve prosthetic material of the present invention is prepared from a mammalian small intestinal submucosal tissue material, preferably a small intestine submucosal tissue material of pigs or cattle.
  • the collagen of the present invention is a composition comprising collagens of type I, type III, type IV and type VI.
  • the polysaccharide material of the present invention is a composition comprising chondroitin sulfate and hyaluronic acid.
  • the active factor of the present invention is a composition comprising fibronectin, laminin, integrins and growth factors.
  • the promoting nerve regeneration factor of the present invention includes an active substance that induces nerve regeneration, preferably a nerve growth factor.
  • the non-immunogenicity according to the present invention means that the residual amount of cells is 0-10, the residual amount of DNA is less than 10 ng/mg, and the clearance rate of galactosidase ( ⁇ -Gal) is 99% or more.
  • the three-dimensional network porous structure according to the present invention has a porosity of 75 to 90%.
  • the in vivo degradation time of the present invention is 1-3 months.
  • the sheet-like nerve repairing material of the present invention has a length of 1-8 cm, a width of 1-8 cm, and a thickness of 0.1-1 mm.
  • the hollow tubular nerve repairing material of the present invention has a hollow tubular shape of 1-5 cm in length, 0.2-0.9 cm in diameter and 0.1-1 mm in thickness.
  • Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned nerve repairing material, which comprises the use of an animal small intestine submucosal tissue material as a raw material, through tissue pretreatment, virus inactivation, and immunogen elimination. , a composite growth factor and a freeze-drying step; obtaining an animal-derived virus risk, a cellular component, a DNA component, and an ⁇ -Gal antigen, and retaining the extracellular matrix component.
  • the preparation method of the above nerve repair material of the present invention comprises the following steps: (1) tissue pretreatment; (2) virus inactivation: soaking the intestinal submucosal tissue material with peracetic acid-ethanol solution for virus inactivation; (3) (4) Immunogen removal: The immunogen removal solution is a PBS solution containing trypsin and EDTA at pH 6-8, and the immunogen removal process is carried out in a multi-frequency ultrasonic device; (5) cleaning; (6) compounding Growth factor: The small intestinal submucosal matrix material was fixed on a mold, and an aqueous nerve growth factor solution was added; (7) Vacuum freeze-drying: carried out in a vacuum freeze dryer.
  • the volume percentage concentration of peracetic acid in the peracetic acid-ethanol solution is 0.1%-5%, and the volume concentration of ethanol is 5%-40% (dissolved into solution by using water).
  • the volume ratio of the peracetic acid-ethanol solution to the intestinal submucosal tissue material is (3-20)..1, the inactivation time is 2-4 hours, and the temperature range is 10-40 °C.
  • the cleaning solution is a PBS solution having a pH of 6-8, the temperature of the PBS solution is 20 ° C, and the ratio (volume ratio) of the PBS solution to the submucosal tissue material of the small intestine is (20-40).. 1, preferably washed 2-6 times; then washed with purified water, purified water and intestinal submucosal tissue material ratio of (20-40)..1, until the detection conductivity is 10 ⁇ S / cm or less; cleaning process can be ultrasonic cleaning
  • the frequency is 20-80 kHz, preferably 40 kHz, and the power is preferably 3000 W or more.
  • the pH of the immunogen removal solution is 6.0-8.0, preferably 7.2-7.5; including trypsin at a concentration of 0.01-0.2% by mass and a molar concentration of 0.1-1 mmol/L. EDTA; the volume ratio of the immunogen removal solution to the intestinal submucosal tissue material is (20-40)..1, preferably 30:1, and the decellularization process is performed in a multi-frequency ultrasonic device comprising at least two ultrasonic frequencies, wherein the low frequency The frequency range is 20-40KHz, the high frequency frequency is 60-90KHz, wherein the low frequency processing is 5-40min, the high frequency processing is 5-40min, the temperature range is 20-35°C, and the ultrasonic power is 5000W or more.
  • the connection between the cells and the extracellular matrix is disrupted; the cells are disrupted by low-frequency ultrasound, and high-frequency ultrasound is applied to the disrupted cells and extracellular matrix to further separate the cells from the extracellular matrix. Achieve the purpose of decellularization.
  • the various steps in the process of detaching the entire cell from the matrix are enhanced to completely detach the cells from the substrate. Reach the best immunogen removal effect.
  • the cleaning solution is a PBS solution having a pH of 6-8, the temperature of the PBS solution is 20 ° C, and the ratio (volume ratio) of the PBS solution to the submucosal tissue material of the small intestine is (20-40).. 1, preferably washed 2-6 times; then washed with cooling water for injection, the ratio of water for injection to the submucosal tissue of the small intestine is (20-40)..1, until the difference between the conductivity of the water for injection before and after the cleaning is 1 ⁇ S/cm.
  • the cleaning process can be carried out in an ultrasonic cleaner at a frequency of 20-80 kHz, preferably 40 kHz, and a power of preferably 5000 W or more.
  • the concentration percentage of the aqueous solution containing the nerve growth factor, which is an aqueous solution for promoting the nerve regeneration active factor, in the step (6) of the present invention is 0.01% to 0.02%.
  • the solution containing the nerve regeneration promoting factor is added in a ratio of the nerve growth factor to the tissue material mass ratio of 1-2:10000.
  • the animal described above according to the invention is preferably pig or cow.
  • the mold described in the step (6) of the present invention is a stainless steel rod having a diameter of 0.2 to 0.9 cm and a length of 1 to 10 cm, or a stainless steel disc having a length of 1 to 8 cm and a width of 1 to 8 cm.
  • the vacuum freeze-drying in the step (7) of the present invention is specifically: placing the small intestinal submucosal matrix material of the composite growth factor in a vacuum freeze dryer, closing the door of the freeze-drying chamber, opening the circulation pump for about 1 minute, and turning on the compressor to freeze Dry the box, pre-freeze to -45 ° C, keep warm for 2 hours, then turn on the vacuum pump, adjust the temperature to -15 ° C, keep warm for 6 hours, then adjust the temperature to 0 ° C, keep warm for 2 hours, finally adjust the temperature to 25 ° C, keep warm 4h, vacuum freeze-drying is completed, and the lyophilization chamber pressure is 1-50Pa.
  • the method for preparing the above-mentioned nerve repairing material of the present invention further comprises: (8) cutting and packaging: taking the cylindrical nerve repairing material from the stainless steel rod, cutting into a hollow cylindrical sample having a length of 5 cm; or forming a sheet
  • the nerve repair material is cut according to the scope of use; it is packaged in a double-layer special-weiling bag, which requires aseptic transfer and handling; (9) Sterilization analysis: sterilization with ethylene oxide, the sterilization conditions are: The temperature was kept at 40 ° C for 4 hours, the humidity was 70%, and then the concentration was 600 mg / L of ethylene oxide, and sterilized for 6 hours; the analytical process was carried out in a ventilated analysis chamber, and the temperature was controlled at 20 ° C for 14 days.
  • the invention further provides the application of the above nerve repair material, specifically:
  • a neurorestorative material for isolation protection of peripheral nerve injury A neurorestorative material for isolation protection of peripheral nerve injury.
  • a nerve repair material for the bridging of nerve defects is provided.
  • the present invention has the following significant advantages and benefits:
  • the connection between the cells and the extracellular matrix is disrupted; the cells are disrupted by low-frequency ultrasound, and high-frequency ultrasound is applied to the disrupted cells and extracellular matrix to further separate the cells from the extracellular matrix. Achieve the purpose of decellularization.
  • the various steps in the process of detaching the entire cell from the matrix are enhanced to completely detach the cells from the substrate. Reach the best immunogen removal effect;
  • a hollow cylindrical or sheet-like structure is prepared by a mold method and a freeze-drying process to effectively protect the damaged nerve and form a nerve regeneration protection microenvironment;
  • adding a nerve growth factor the freeze-drying process effectively preserves growth factor activity and promotes regeneration of damaged nerves;
  • the degradation process of the product body is adjusted through the sterilization process, and the immunogen removal matrix repair material is gradually degraded, and the regeneration process is basically synchronized with the reconstructed tissue regeneration process, and finally the immunogen removal matrix repair material is completely replaced by the host tissue;
  • peripheral nerve injury and defect repair can not only serve as a barrier, but also effectively isolate the nerve injury site from the surrounding tissue, prevent fibroblasts in the surrounding tissue from invading the nerve injury site, and guide the Schwann sheath.
  • Tissue regeneration repair provides a microenvironment for the recovery, directional growth and regeneration of damaged nerves.
  • FIG. 1 is a schematic view of a cylindrical (hollow tubular) nerve prosthetic material according to an embodiment of the present invention
  • FIG. 2 is a schematic view of a sheet-like nerve repair material according to an embodiment of the present invention.
  • Figure 3 is a SEM photograph of the microstructure of the nerve repair material of the present invention.
  • the submucosal tissue material of the small intestine was divided into the specified size, 10 cm wide and 15 cm long.
  • the lymphoid tissue was removed, washed with tap water for 3 times, rinsed with purified water until the surface was stain-free, and then the cleaned small intestinal submucosal tissue material was placed on the sieve.
  • a water filter such as a net, let stand for more than five minutes to filter the water;
  • the intestine submucosal tissue material is immersed in a peracetic acid-ethanol solution (peracetic acid and ethanol dissolved in water) for virus inactivation, and the process can be carried out in a stainless steel tank.
  • the concentration (volume percentage) of peracetic acid in the peracetic acid-ethanol solution was 1%, the concentration (volume percentage) of ethanol was 24%, and the ratio (volume ratio) of the peracetic acid-ethanol solution to the submucosal tissue material of the small intestine was 9..1, inactivation time 2 hours, temperature range is 20 ° C;
  • the cleaning process is carried out in an ultrasonic cleaner at a frequency of 20-80 kHz, preferably 30. -40 kHz, the ultrasonic power is at least 3000 W or more, and the volume ratio of the PBS solution and the purified water volume to the intestinal submucosal tissue material is 30:1.
  • the immunogen removal solution is a PBS solution containing a mass concentration of 0.02% trypsin and a concentration of 0.5 mmol/L of EDTA at a pH of 7.0, and the ratio of the immunogen removal solution to the intestinal submucosal tissue material (volume ratio) is 30. ..1, the immunogen removal process is carried out in an ultra-dual-frequency sonic cleaning machine, wherein the low-frequency frequency range is 35KHz, the high-frequency frequency is 85KHz, wherein the low-frequency processing is 8min, the high-frequency processing is 10min, and the temperature range of the immunogen removal liquid is 20- At 35 ° C, the ultrasonic power is at least 5000 W or more.
  • the connection between the cells and the extracellular matrix is destroyed; the cells are disrupted by low-frequency ultrasound, and high-frequency ultrasound is applied to the broken cells and extracellular matrix to further separate the cells from the extracellular matrix. Achieve the purpose of decellularization.
  • the various steps in the process of detaching the entire cell from the matrix are enhanced to completely detach the cells from the substrate. Reach the best immunogen removal effect.
  • the immunogen removal is completed, it is washed with a pH 7 PBS solution in an ultrasonic cleaner, and then washed with 24 ° C water for injection until the difference in conductivity of the injection water before and after the cleaning is 1 ⁇ S/cm or less.
  • the cleaning process is in an ultrasonic cleaner.
  • the frequency is 20-80 kHz, preferably 30-40 kHz, more preferably 40 kHz, and the ultrasonic power is at least 3000 W or more.
  • the volume ratio of the PBS solution and the water for injection to the submucosal tissue material is 30:1, and the submucosal matrix material is obtained. ;
  • the small intestinal submucosal matrix material is wrapped on a stainless steel rod having a diameter of 0.1-0.7 cm, a length of 1-10 cm, for example 7 cm, fixed, and then a 8% by weight aqueous solution of nerve growth factor is added.
  • the small intestinal submucosal matrix material is placed in a stainless steel dish and then an aqueous solution of a nerve growth factor at a concentration of 0.02% by mass is added.
  • the aqueous nerve growth factor solution was completely immersed in the intestinal submucosal matrix material, and allowed to stand for 24 hours after mixing; the nerve growth factor was attached to the intestinal submucosal material.
  • the product freeze-drying process needs to be reconfirmed according to different equipment, the mold is laid flat in the vacuum freeze dryer, the door of the freeze-drying chamber is closed, the circulation pump is turned on for about 1 minute, and the compressor is turned on. Dry the box, pre-freeze the material in step (6) to -45 ° C, keep it for 2 hours, then turn on the vacuum pump, adjust the temperature to -15 ° C, keep warm for 6 hours, then adjust the temperature to 0 ° C, keep warm for 2 hours, and finally adjust The temperature was 25 ° C, the temperature was kept for 4 h, and the vacuum freeze-drying was completed.
  • the lyophilization chamber pressure is 1-50 Pa.
  • the preparation method of Embodiment 1 may further include:
  • the cylindrical nerve repair material is taken out from the stainless steel rod and cut into a hollow cylindrical sample having a length of 5 cm; or the sheet-like nerve repair material is cut according to the scope of use; and the double-layer Tyvek packaging bag is used, and the process requires Sterile transport and handling.
  • Epoxidized with ethylene oxide the sterilization conditions are: first temperature 40 ° C for 4 hours, humidity 70%, and then into the concentration of 600mg / L ethylene oxide, sterilized for 6 hours; analytical process in the ventilated analysis room In progress, the temperature is controlled between 20 ° C and 14 days.
  • Fig. 1 and Fig. 2 The structure of the product obtained by the present invention is shown in Fig. 1 and Fig. 2, and the specific microstructure is shown in Fig. 3, which is a three-dimensional network porous structure.
  • I, III, IV and VI collagen monoclonal antibody primary antibody were added dropwise at a concentration of 1:100, incubated at room temperature for 60 min at room temperature overnight, and washed three times with PBS.
  • the Envision reaction solution was added dropwise and incubated at room temperature for 30 min. Wash 3 times with PBS.
  • 0.05% of 3,3 diaminobenzidine + 0.03% H 2 O 2 developed color for 5-10 min.
  • Incremental gradient ethanol dehydration, xylene transparent, conventional resin sealing The results showed that all the four stained specimens under the microscope were stained with brownish yellow, which was positive, indicating that type I, III, IV and VI collagen could be detected in the sample.
  • Determination of polysaccharide content Take 10 samples, sample, extract, test the content of chondroitin sulfate with Biocolor chondroitin sulfate test kit. The average content of chondroitin sulfate in the sample is 4512 ⁇ 524 ⁇ g/g; use hyaluronic acid detection reagent The box was tested for hyaluronic acid (HA) content and the results showed that the average hyaluronic acid (HA) retention of the sample was 187 ⁇ 45 ⁇ g / g.
  • HA hyaluronic acid
  • bFGF alkaline growth factor
  • VEGF vascular endothelial growth factor
  • Virus detection The pseudorabies virus was selected as the indicator virus, and the DNA copy number of the virus was detected by real-time quantitative PCR, and three batches of samples were detected. Result: The viral DNA copy number is zero.
  • DNA residue According to the method for detecting residual DNA of biological agents, the fourth part of the Chinese Pharmacopoeia 2015 edition, the sample DNA residue provided by the examples was detected by fluorescence staining. As a result, the DNA residue of the sample provided in the example was 4.00 on average. ⁇ 0.42 ng/mg.
  • Galactosidase ( ⁇ -Gal) clearance rate Take Gal-positive reference material for animal-derived biological material, 2mg for Gal antigen-negative reference substance, add 1ml of lysate, lyse for 30-90min, and prepare 20, 10, 5, 2.5 , 1.25, 0.625 ⁇ g Gal standard curve sample, test the immunogen before and after the test article taken 50mg, add lysate 2ml, lysis 30-90min; take the lysate and M86 antibody reaction supernatant, add 96-well plate Add the secondary antibody, add the coloring agent, measure the absorbance value by 450nm using ELISA method, calculate the Gal value of the sample according to the standard curve, and calculate the Gal value of the material before the immunogen removal treatment (23.74 ⁇ 2.52) ⁇ 10 14 /mg. In the example, the Gal value of the sample was (0.11 ⁇ 0.01) ⁇ 10 14 /mg, and the clearance rate of galactosidase ( ⁇ -Gal) was 99.52% or more.
  • Bacterial endotoxin According to GB/T 14233.2-2005 "medical infusion, blood transfusion, injector test method part 2: biological test method" for testing, a total of 3 batches of samples, the results: bacterial endotoxin is less than 20EU / packaging.
  • Porosity According to the Archimedes principle, using ethanol as the extraction medium, the porosity of the sample was calculated to be 84.52 ⁇ 8.24%.
  • Stitching tensile strength Prepare the sample according to the example, fix the suture and the other end of the repairing material on the tensioning device at a speed of 20 mm/min with a 3-0 non-absorbable suture at 2 mm of the edge of one end of the repairing material. Stretching until the stitching point is torn, the maximum force value is recorded, and the result shows that the maximum value can reach 10N.
  • Tensile strength Samples were prepared according to the examples, and were tested immediately after being placed at a relative humidity of 40% to 60% and a temperature of 22 ° C ⁇ 2 ° C for 2 hours. The two ends of the sample were fixed on the chuck of the tensile tester, and were sequentially stretched outward at a speed of 100 mm/min until the sample was broken, and the longitudinal sample and the transverse sample were respectively tested. The final measurement showed a longitudinal tensile strength of up to 55 N/cm.
  • Residual ethylene oxide Tested according to the method specified in GB/T14233.1-2008 "Test methods for medical infusion, blood transfusion, and injecting tools - Part 1: Chemical analysis". No more than 10 ⁇ g/package.
  • Heavy metal inspection lead and chromium are tested according to the method specified in 5.9.1 “Test methods for medical infusion, blood transfusion and injecting instruments, Part 1: Chemical analysis method” in GB/T 14233.1-2008. Mercury and arsenic are in accordance with GB/T14233.1- In the method of 5.9.3 “Testing methods for medical infusion, blood transfusion, and injecting tools, Part 1: Chemical analysis method”, the total heavy metal content of lead, chromium, mercury and arsenic in the product test solution was less than 1 ⁇ g/g.
  • the samples in the examples were subjected to biocompatibility experiments including: pyrogen, cytotoxicity, delayed type hypersensitivity reaction, intradermal reaction, acute systemic toxicity, Ames test, mouse lymphoma cell mutation test, chromosome aberration, Implantation, subchronic toxicity.
  • test solution was prepared according to the ratio of the mass ratio of 1:5 leaching medium, 37 ⁇ 1° C., 72 ⁇ 2 hr, and the immersion medium: physiological saline. According to the method specified in GB/T 14233.2-2005, the product has no pyrogen reaction.
  • the test solution was prepared at a mass ratio of 1:5 leaching medium, 37 ⁇ 1° C., 24 ⁇ 2 hr, and the leaching medium: serum-containing MEM medium.
  • the test solution was tested according to the test method specified in GB/T16886.5-2003, and the cytotoxic reaction of the product was not more than grade 1.
  • test solution was prepared according to the ratio of the mass ratio of 1:5 leaching medium, 37 ⁇ 1° C., 72 ⁇ 2 hr, and the immersion medium: physiological saline and cottonseed oil.
  • the test method of GB/T 16886.10-2005 Part 10: Stimulation and delayed type hypersensitivity test method the product has no delayed type hypersensitivity reaction.
  • test solution was prepared according to the ratio of the mass ratio of 1:5 leaching medium, 37 ⁇ 1° C., 72 ⁇ 2 hr, and the immersion medium: physiological saline and cottonseed oil.
  • the test method specified in GB/T 16886.10-2005 Part 10: Stimulation and delayed-type hypersensitivity test the results show that the difference between the average score of the test sample and the solvent control is less than 1.0.
  • the test solution was prepared at a mass ratio of 1:5 leaching medium, 37 ⁇ 1 ° C, 72 ⁇ 2 hr, and the leaching medium: physiological saline and cottonseed oil.
  • the test solution was tested according to the test method specified in GB/T16886.11-2011. The results showed that the product had no acute systemic toxicity.
  • test solution was prepared at a mass ratio of 1:5 leaching medium, 37 ⁇ 1° C., 72 ⁇ 2 hr, and the immersion medium: physiological saline and DMSO. According to the method specified in GB/T16886.3-2008, the result is that the Ames test of the product is negative.
  • the test solution was prepared at a mass ratio of 1:5 leaching medium, 37 ⁇ 1° C., 72 ⁇ 2 hr, and the immersion medium: physiological saline and DMSO. According to the method specified in GB/T16886.3-2008, the results showed that the mouse lymphoma cell mutation test of the product was a negative result.
  • muscle implantation for 1 week neutrophils, lymphocytes and macrophage infiltration around the sample, no cyst formation
  • muscle implantation for 4 weeks A small amount of macrophages and lymphocytes were seen around the sample, collagen fibers and fibroblasts proliferated, and fibrous cysts were formed.
  • the tibialis anterior muscles of the rabbits in each group had different degrees of atrophy, and the elasticity was poor and the joints were stiff.
  • the muscle atrophy of the C group was the most obvious, and the muscle luster and elasticity were not as good as those of the A and B groups.
  • the wet weight of the anterior muscles of each group was 2.56 ⁇ 0.16g, 2.45 ⁇ 0.18g, 1.50 ⁇ 0.11g, and the muscle atrophy rates were 20.83%, 17.45% and 43.40%, respectively.
  • the wet weight recovery rate of group A and group B was significantly different from that of group C. Muscle atrophy was mild in group A and group B, and the degree of muscle atrophy was similar in the two groups. There was no significant difference in the recovery rate of wet weight of tibialis anterior muscle.
  • the sciatic nerve conduction velocity in group A was 33.151 ⁇ 1.434m/s in group A, 34.081 ⁇ 1.116m/s in group B, and 1.0628 ⁇ 1.333m/s in group C.
  • the nerve conduction velocity in group C was smaller than that in group A and B.
  • the nerve repair material of the present invention is a nerve repair material of the present invention.
  • the DNA residue can reach 10 ng/mg or less, 30-50 ng/mg lower than similar products, and the galactosidase removal rate is higher, which can reach more than 99%;
  • NGF nerve growth factor

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Abstract

一种神经修复材料、其制备方法及应用。神经修复材料包含胶原蛋白、多糖物质、活性因子和促进神经再生因子，其具有三维网状多孔结构，无免疫原性、可体内降解，且可以是片状或中空管状。采用小肠粘膜下层组织为原料，经组织前置处理、病毒灭活、免疫原去除、复合生长因子和冷冻干燥等步骤制备而成。用于神经修补医疗器械中，能够有效保护受损神经，形成神经再生保护微环境，促进受损神经再生，还能起到屏障作用，将神经受损部位与周围组织有效隔离，防止周围组织中的成纤维细胞侵入神经损伤部位。

Description

一种神经修复材料、制备方法和应用 技术领域
本发明涉及医用生物材料技术领域，具体为一种神经修复材料，用于将生长因子复合于小肠粘膜下层免疫原去除基质中，保护受损、缺损周围神经，防组织粘连并诱导神经修复。
背景技术
周围神经遍及全身皮肤、粘膜、肌肉、骨关节、血管及内脏等，因此人体组织因牵拉、切割、压迫、烧伤、缺血等原因都会累及周围神经造成神经损伤。周围神经是神经元的细胞突起，又称神经纤维，由轴索、髓鞘和施万鞘组成。轴索功能是神经元和神经终末结构之间神经冲动传导，髓鞘防止兴奋扩散作用，施万鞘由Schwann细胞组成，是神经再生的通道。周围神经鞘受损后即会造成神经难以再生，与周围组织粘连形成瘢痕组织。因此在神经损伤修复过程中，应首先保护受损神经，将神经与周围组织隔离，防止肌肉、肌腱、筋膜等周围组织长入形成瘢痕组织，并为神经再生提供营养物质，引导神经轴索再生恢复功能。
传统修复神经局限于缝线缝合的方法，并未采用生物材料进行损伤保护，缝线缝合后吻合口处会出现水肿，大量成纤维细胞的过度增生会导致吻合口处发生纤维化，若局部瘢痕或组织粘连，则会严重的影响到神经功能的恢复。同时，神经损伤时大多伴有肌肉、肌腱、筋膜损伤，这些组织的修复主要以瘢痕修复为主，这也会导致神经吻合口周围过多的瘢痕形成从而影响神经再生。而且最重要的是未考虑到施万鞘的修复，只有修复施万鞘才能防止周围纤维细胞长入，控制局部瘢痕，减少粘连组织的形成，并为神经再生提供生长微环境与营养成分，有利于神经再生。因此，临床上迫切需要一种可以代替缝合方法的免缝合技术用于修复周围神经的损伤。
发明内容
技术问题
本发明针对现有技术的上述不足，提供一种将生长因子复合于小肠粘 膜下层免疫原去除基质中，保护受损、缺损周围神经，防组织粘连并诱导神经修复的神经修复材料。
解决方案
为了解决上述技术问题，本发明采用的技术方案为：一种神经修复材料，其特征在于：所述神经修复材料包含胶原蛋白、多糖物质、活性因子和促进神经再生因子。
本发明所述的神经修复材料具有三维网状多孔结构，无免疫原性、可体内降解，并且可以是片状或中空管状。神经损伤可以分为断裂损伤和非断裂损伤，其中非断裂伤如神经纤维的一部分受损或挫伤，这种情况下是神经没有断裂，中空管可直接用于修补断裂损伤，神经细胞在中空管中生长并与其它组织相隔离，从而控制局部瘢痕并减少神经与其它组织粘连；片状材料用于处理非断裂损伤，对神经包裹并隔离。
本发明所述的神经修复材料由哺乳动物小肠粘膜下层组织材料制备，优选猪或牛的小肠粘膜下层组织材料。
本发明所述的胶原蛋白为包含I型、III型、IV型和VI型胶原蛋白的组合物。
本发明所述的多糖物质为包含硫酸软骨素和透明质酸的组合物。
本发明所述的活性因子为包含纤维粘连蛋白、层粘连蛋白、整合素及生长因子的组合物。
本发明所述的促进神经再生因子包括诱导神经再生的活性物质，优选神经生长因子。
本发明所述的无免疫原性指细胞残留量为0-10个、DNA残留量小于10ng/mg、半乳糖苷酶(α-Gal)清除率为99％以上。
本发明所述的三维网状多孔结构的孔隙率为75～90％。
本发明所述的体内降解的时间为1-3个月。
本发明所述的片状的神经修复材料长1-8cm，宽1-8cm，厚度0.1-1mm。
本发明所述的中空管状神经修复材料，长1-5cm，直径0.2-0.9cm，厚度0.1-1mm的中空管状。
本发明要解决的另一个技术问题是，提供一种上述神经修复材料的制备方法，其特征在于：包括采用动物小肠粘膜下层组织材料作为原料，通过 组织前置处理，病毒灭活，免疫原消除，复合生长因子和冷冻干燥步骤；获得清除动物源病毒风险、细胞成分、DNA成分和α-Gal抗原，保留细胞外基质成分。
本发明上述神经修复材料的制备方法，具体步骤包括：(1)组织前置处理；(2)病毒灭活：采用过氧乙酸-乙醇溶液浸泡小肠粘膜下层组织材料进行病毒灭活；(3)清洗；(4)免疫原去除：免疫原去除液为含有胰蛋白酶和EDTA的pH值6-8的PBS溶液，免疫原去除过程在多频超声波装置中进行；(5)清洗；(6)复合生长因子：将小肠粘膜下层基质材料固定于模具上，加入神经生长因子水溶液；(7)真空冷冻干燥：在真空冷冻干燥机中进行。
本发明步骤(2)病毒灭活中，过氧乙酸-乙醇溶液中过氧乙酸的体积百分比浓度为0.1％-5％、乙醇的体积百分比浓度为5％-40％(用水配置成溶液)，过氧乙酸-乙醇溶液与小肠粘膜下层组织材料的体积比为(3-20)︰1，灭活时间2-4小时，温度范围为10-40℃。
本发明步骤(3)清洗中，清洗液为pH值为6-8的PBS溶液，PBS溶液温度为20℃，PBS溶液与小肠粘膜下层组织材料的比例(体积比)为(20-40)︰1，优选清洗2-6次；然后用纯化水清洗，纯化水与小肠粘膜下层组织材料比例为(20-40)︰1，至检测电导率为10μS/cm以下终止；清洗过程可在超声波清洗机中进行，频率为20-80kHz，优选40kHz，功率优选3000W以上。
步骤(4)免疫原去除中，免疫原去除液的pH值为6.0-8.0，优选为7.2-7.5；其中包括质量百分比浓度为0.01-0.2％的胰蛋白酶和摩尔浓度为0.1-1mmol/L的EDTA；所述免疫原去除液与小肠粘膜下层组织材料体积比为(20-40)︰1，优选30:1，脱细胞过程在包含至少两个超声频率的多频超声装置中进行，其中低频频率范围为20-40KHz，高频频率为60-90KHz，其中低频处理5-40min，高频处理5-40min，温度范围为20-35℃；超声功率5000W以上。采用胰蛋白酶和EDTA，使细胞与细胞外基质之间的连接被破坏；采用低频超声对细胞进行破碎，同时使用高频超声作用于破碎的细胞及细胞外基质，进一步使细胞脱离细胞外基质，达到脱细胞目的。采用上述方式，对整个细胞脱离基质过程中的各个步骤进行强化，使细胞被从基质上完全脱离。到达最佳的免疫原去除效果。
本发明步骤(5)清洗中，清洗液为pH值为6-8的PBS溶液，PBS溶液温度 为20℃，PBS溶液与小肠粘膜下层组织材料的比例(体积比)为(20-40)︰1，优选清洗2-6次；然后用降温的注射用水清洗，注射用水与小肠粘膜下层组织材料比例为(20-40)︰1，至检测清洗前后注射用水电导率差为1μS/cm以下终止；清洗过程可在超声波清洗机中进行，频率为20-80kHz，优选40kHz，功率优选5000W以上。
本发明步骤(6)所述含促进神经再生活性因子的水溶液即神经生长因子水溶液的质量百分比浓度为0.01％-0.02％。所述含促进神经再生活性因子的溶液按照神经生长因子与组织材料质量比1-2:10000加入。
本发明上述所述的动物优选为猪或牛。
本发明步骤(6)所述的模具为直径0.2-0.9cm、长度1-10cm的不锈钢棒，或者长1-8cm、宽1-8cm的不锈钢盘。
本发明步骤(7)所述真空冷冻干燥具体为：将复合生长因的小肠粘膜下层基质材料放置于真空冷冻干燥机中，关闭冻干室的门，打开循环泵约1min，开启压缩机对冻干箱致冷，预冻至-45℃，保温2小时，然后开启真空泵，调节温度至-15℃，保温6小时，再调节温度至0℃，保温2小时，最后调节温度至25℃，保温4h，真空冷冻干燥完成，冻干室气压为1-50Pa。
本发明上述的神经修复材料的制备方法，具体步骤还包括：(8)切割包装：将圆筒状神经修复材料从不锈钢棒上取出，切割为长度5cm的中空圆筒状样品；或者将片状神经修复材料根据使用范围进行切割；采用双层特卫强包装袋包装，该过程需要无菌转运与操作；(9)灭菌解析：采用环氧乙烷进行灭菌，灭菌条件为：先温度40℃保温4小时，湿度70％，然后通入浓度600mg/L环氧乙烷，灭菌6小时；解析过程在通风的解析室中进行，温度控制在20℃之间，时间14天。
本发明进一步提供上述神经修复材料的应用，具体为：
一种神经修复材料的应用，用于周围神经损伤的隔离保护。
一种神经修复材料的应用，用于神经缺损的桥接。
有益效果
与现有技术相比，本发明具有以下显著优点和有益效果：
采用胰蛋白酶和EDTA，使细胞与细胞外基质之间的连接被破坏；采用低频超声对细胞进行破碎，同时使用高频超声作用于破碎的细胞及细胞外基 质，进一步使细胞脱离细胞外基质，达到脱细胞目的。采用上述方式，对整个细胞脱离基质过程中的各个步骤进行强化，使细胞被从基质上完全脱离。到达最佳的免疫原去除效果；
可选的，对于成型工艺技术：采用模具法、冷冻干燥工艺制备中空圆筒状或片状结构，有效保护受损神经，形成神经再生保护微环境；
可选的，对于添加神经生长因子：添加神经生长因子，冷冻干燥工艺有效保存生长因子活力，促进受损神经再生；
可选的，对于灭菌工艺：通过灭菌过程调整产品体内降解过程，使免疫原去除基质修复材料逐步降解，与重建组织再生过程基本同步，最终免疫原去除基质修复材料完全被宿主组织代替；
可选的，用于周围神经损伤与缺损修复：既能起到屏障作用，可将神经损伤部位与周围组织有效隔离，防止周围组织中的成纤维细胞侵入神经损伤部位，又能引导施万鞘组织再生修复，为受损神经的恢复、定向生长、再生提供微环境。
根据下面参考附图对示例性实施例的详细说明，本发明的其它特征及方面将变得清楚。
附图说明
包含在说明书中并且构成说明书的一部分的附图与说明书一起示出了本发明的示例性实施例、特征和方面，并且用于解释本发明的原理。
图1所示的是根据本发明实施方式的圆筒形(中空管状)神经修复材料的示意图；
图2所示的是根据本发明实施方式的片状神经修复材料的示意图；
图3所示的是本发明神经修复材料的微观结构SEM照片。
具体实施方式
以下将参考附图详细说明本发明的各种示例性实施例、特征和方面。附图中相同的附图标记表示功能相同或相似的元件。尽管在附图中示出了实施例的各种方面，但是除非特别指出，不必按比例绘制附图。
在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为 “示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
另外，为了更好的说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。
实施例1
本实施例神经修复材料的制备方法，包括以下操作步骤：
(1)组织前置处理：
取小肠粘膜下层组织材料分割成规定尺寸，宽10cm，长15cm，剔除淋巴组织，用自来水冲洗3次，再用纯化水冲洗至表面无污渍，然后将清洗后的小肠粘膜下层组织材料置于筛网等滤水装置上，静置五分钟以上，以将水滤干；
(2)病毒灭活：
采用过氧乙酸-乙醇溶液(过氧乙酸和乙醇溶解于水中构成)浸泡小肠粘膜下层组织材料进行病毒灭活，该过程可在不锈钢桶中进行。过氧乙酸-乙醇溶液中过氧乙酸的浓度(体积百分比)为1％、乙醇的浓度(体积百分比)为24％，过氧乙酸-乙醇溶液与小肠粘膜下层组织材料的比例(体积比)为9︰1，灭活时间2小时，温度范围为20℃；
(3)清洗过程：
完成后在超声波清洗机中用pH值为7的PBS溶液清洗，然后用纯化水清洗至检测电导率为10μS/cm以下终止，清洗过程在超声波清洗机中进行，频率为20-80kHz，优选30-40kHz，超声波功率至少3000W以上，所用的PBS溶液和纯化水体积与小肠粘膜下层组织材料体积比为30:1。
(4)免疫原去除：
免疫原去除液为含有质量百分浓度0.02％胰蛋白酶和浓度为0.5mmol/L的EDTA的pH值为7.0的PBS溶液，免疫原去除液与小肠粘膜下层组织材料混合比例(体积比)为30︰1，免疫原去除过程在超双频声波清洗机中进行，其中低频频率范围为35KHz，高频频率为85KHz，其中低频处理8min，高频处理10min，免疫原去除液的温度范围为20-35℃，超声波功率至少在5000W以上。采用胰蛋白酶和EDTA，使细胞与细胞外基质之间的连接被破坏；采用低频超声对细胞进行破碎，同时使用高频超声作用于破碎的细胞及细胞外基质， 进一步使细胞脱离细胞外基质，达到脱细胞目的。采用上述方式，对整个细胞脱离基质过程中的各个步骤进行强化，使细胞被从基质上完全脱离。到达最佳的免疫原去除效果。
(5)清洗过程：
免疫原去除完成后在超声波清洗机中用pH值为7的PBS溶液清洗，然后用24℃注射用水清洗至检测清洗前后注射用水电导率差为1μS/cm以下终止，清洗过程在超声波清洗机中进行，频率为20-80kHz，优选30-40kHz，更优选40kHz，超声波功率至少3000W以上，所用的PBS溶液和注射用水体积与小肠粘膜下层组织材料体积比为30:1，得到小肠粘膜下层基质材料；
(6)复合生长因子：
将小肠粘膜下层基质材料包裹于不锈钢棒上，不锈钢棒的直径可为0.1-0.7cm、长度1-10cm，例如7cm，固定，然后加入质量百分比浓度为0.02％的神经生长因子水溶液。或者，小肠粘膜下层基质材料放置于不锈钢盘中，然后加入质量百分比浓度为0.02％的神经生长因子水溶液。使神经生长因子水溶液完全浸没小肠粘膜下层基质材料，混合后静置24小时；使神经生长因子附着至小肠粘膜下层材料。
(7)真空冷冻干燥：
在真空冷冻干燥机中进行，产品的冷冻干燥工艺需要根据不同的设备重新确认，将模具平铺于真空冷冻干燥机中，关闭冻干室的门，打开循环泵约1min，开启压缩机对冻干箱致冷，将步骤(6)材料预冻至-45℃，保温2小时，然后开启真空泵，调节温度至-15℃，保温6小时，再调节温度至0℃，保温2小时，最后调节温度至25℃，保温4h，真空冷冻干燥完成。冻干室气压为1-50Pa。
实施例1的制备方法中还可以包括：
(8)切割包装：
将圆筒状神经修复材料从不锈钢棒上取出，切割为长度5cm的中空圆筒状样品；或者将片状神经修复材料根据使用范围进行切割；采用双层特卫强包装袋包装，该过程需要无菌转运与操作。
(9)灭菌解析：
采用环氧乙烷进行灭菌，灭菌条件为：先温度40℃保温4小时，湿度70％， 然后通入浓度600mg/L环氧乙烷，灭菌6小时；解析过程在通风的解析室中进行，温度控制在20℃之间，时间14天。
本发明所得产品结构如图1、图2所示，具体微观结构如图3所示，为三维网状多孔结构。
对本发明所得材料的化学成分进行检测，如下表1所示：
表1实施例样品化学成分
蛋白(％) 碳水化合物(％) 脂类(％) 水分 灰分 生长因子
75％-85％ 15％-25％ <1％ <5％ <1％ 0.01-2％
对实施例中样品进行性能检测，检测项目与结果如下：
胶原蛋白亚型鉴别：采用免疫组化染色法检测I、III、IV型和VI型胶原蛋白，3μm厚连续切片，二甲苯脱蜡，梯度乙醇脱水。将切片移入电饭煲水浴中(内含0.01mol/L，pH6.0的枸橼酸三钠缓冲液)，温度保持在95-100℃，煮20min，进行抗原修复，取出后在室温下自然冷却。磷酸盐缓冲液(PBS)洗涤，5min×3次。二步法免疫组化：分别滴加I、III、IV型和VI型胶原蛋白单克隆抗体一抗，浓度1：100，4℃冰箱过夜室温下孵育60min，PBS洗涤3次。滴加Envision反应液，室温下孵育30min。PBS洗涤3次。0.05％的3,3一二氨基联苯胺+0.03％的H 2O 2显色5-10min。流水洗，苏木精衬染。递增梯度乙醇脱水，二甲苯透明，常规树脂封固。结果表明，显微镜下观察四种染色标本皆可见棕黄染色，为阳性，表明样品中可检测到I、III、IV型和VI型胶原蛋白。
多糖物质含量检测：取10个样品，取样，浸提，用Biocolor硫酸软骨素检测试剂盒测试硫酸软骨素含量，样品中硫酸软骨素含量平均值为4512±524μg/g；用透明质酸检测试剂盒测试透明质酸(HA)含量，结果显示，样品的透明质酸(HA)保留量平均值为187±45μg/g。
活性因子种类鉴别：将样品浸泡PBS 24h后，固定于4％多聚甲醛5-10min，用0.1mol/LPBS洗3次，每次5min，然后用玻璃细管转至涂有多聚赖氨酸的玻片上,进行免疫组织化学染色。LN抗体、FN抗体和整合素效价均为1∶100，0.5％胰酶消化3-5min暴露抗原，0.1％Triton X100作用10min增加抗体的穿透性。免疫组织化学染色显阳性，表面样品中包含纤维粘连蛋白、层粘连蛋白、整合素及其配体的等物质。
生长因子含量检测：采用ELISA法检测样品中碱性生长因子(bFGF，见 图1)和血管内皮生长因子(VEGF，见图2)含量，并对免疫原去除前动物组织作为对照。结果发现碱性生长因子(bFGF)免疫原去除前后含量分别为2035±178ng/L、1199±130ng/L，保留生长因子55％以上；血管内皮生长因子(VEGF)含量免疫原去除前后含量分别为731±58ng/L、358±24ng/L，保留生长因子50％以上。
病毒检测：选择伪狂犬病毒为指示病毒，采用实时定量PCR法检测病毒的DNA拷贝数，检测3批样品。结果：病毒DNA拷贝数为0。
DNA残留：依据生物制剂残留DNA检测方法《中国药典》2015年版第四部，采用荧光染色法检测实施例所提供的样品DNA残留量，结果：实施例所提供的样品的DNA残留量平均为4.00±0.42ng/mg。
半乳糖苷酶(α-Gal)清除率：取动物源性生物材料Gal阳性参考品，Gal抗原阴性参考品各2mg，加裂解液1ml，裂解30-90min，配制成20、10、5、2.5、1.25、0.625μg的Gal标准曲线样品，测试免疫原去除前后的测试品各取50mg，加裂解液2ml，裂解30-90min；取裂解液与M86抗体反应后的上清液，加入96孔板，加二抗，加显色剂，采用ELISA方法450nm检测吸光度值，按标准曲线计算出样品的Gal值，免疫原去除处理前材料的Gal值为(23.74±2.52)×10 14/mg，实施例中样品的Gal值为(0.11±0.01)×10 14/mg，半乳糖苷酶(α-Gal)清除率在99.52％以上。
细菌内毒：按照GB/T 14233.2-2005《医用输液、输血、注射器具检验方法第2部分：生物学试验方法》进行检测，共3批样品，结果：细菌内毒小于20EU/包装。
孔隙率：按照阿基米德原理，以乙醇作为浸提介质，计算样品孔隙率为84.52±8.24％。
缝合抗拉强度：按照实施例制备样品，用3-0非吸收缝合线在修修复材料一端边缘2mm处，将缝合线与修修复材料的另一端固定在拉力仪上，以20mm/min的速度进行拉伸，直到缝合点被撕裂，记录最大力值，结果显示，最大值可达10N。
抗张强度：按照实施例制备样品，在相对湿度为40％-60％，温度为22℃±2℃的条件下放置2h后立即进行试验。将试样两端固定在拉伸试验机的夹头上，以100mm/min的速度依次向外拉伸直到试样断裂，纵向试样和横向试 样分别进行试验。最后的测定结果显示纵向抗张强度可达55N/cm。
环氧乙烷残留量：按GB/T14233.1-2008《医用输液、输血、注射器具检验方法第1部分：化学分析法》中9的规定的方法试验，结果：产品环氧乙烷残留量不超过10μg/包装。
重金属检查：铅、铬按GB/T 14233.1-2008中5.9.1《医用输液、输血、注射器具检验方法第1部分：化学分析法》规定的方法试验，汞、砷按GB/T14233.1-2008中5.9.3《医用输液、输血、注射器具检验方法第1部分：化学分析法》规定的方法试验，产品检验液中铅、铬、汞、砷总重金属含量少于1μg/g。
对实施例中样品进行生物相容性实验，检测项目包括：热原、细胞毒性、迟发型超敏反应、皮内反应、急性全身毒性、Ames试验、小鼠淋巴瘤细胞突变试验、染色体畸变、植入、亚慢性毒性。
1)热原
按质量比1：5浸提介质的比例，37±1℃，72±2hr制备试验液，浸提介质：生理盐水。按GB/T 14233.2-2005规定的方法进行，产品无热原反应。
2)细胞毒性
按质量比1：5浸提介质的比例，37±1℃，24±2hr制备试验液，浸提介质：含血清的MEM培养基。取试验液按照GB/T16886.5-2003中规定的试验方法进行试验，结果产品的细胞毒性反应不大于1级。
3)迟发型超敏反应
按质量比1：5浸提介质的比例，37±1℃，72±2hr制备试验液，浸提介质：生理盐水和棉籽油。按照GB/T 16886.10-2005第10部分:刺激与迟发型超敏反应试验方法规定进行试验，结果产品无迟发型超敏反应。
4)皮内反应
按质量比1：5浸提介质的比例，37±1℃，72±2hr制备试验液，浸提介质：生理盐水和棉籽油。按照GB/T 16886.10-2005第10部分:刺激与迟发型超敏反应试验试验方法规定进行试验，结果：试验样品与溶剂对照平均记分之差小于1.0。
5)急性全身毒性
按质量比1：5浸提介质的比例，37±1℃，72±2hr制备试验液，浸提介 质：生理盐水和棉籽油。取试验液按照GB/T16886.11-2011规定的试验方法进行试验，结果：产品无急性全身毒性反应。
6)Ames试验
按质量比1：5浸提介质的比例，37±1℃，72±2hr制备试验液，浸提介质：生理盐水和DMSO。按GB/T16886.3-2008规定的方法进行，结果：产品的Ames试验为阴性。
7)小鼠淋巴瘤细胞突变试验
按质量比1：5浸提介质的比例，37±1℃，72±2hr制备试验液，浸提介质：生理盐水和DMSO。按GB/T16886.3-2008规定的方法进行，结果：产品的小鼠淋巴瘤细胞突变试验为阴性结果。
8)染色体畸变试验
按质量比1：5浸提介质的比例，37±1℃，72±2hr制备试验液，浸提介质：生理盐水和DMSO，按GB/T16886.3-2008规定的方法进行，结果：产品的染色体畸变试验为阴性。
9)植入
按GB/T16886.6-1997规定的方法进行，结果：肌肉植入1周：样品周围可见嗜中性粒细胞、淋巴细胞和巨噬细胞浸润，应无囊腔形成；肌肉植入4周：样品周围可见少量巨噬细胞和淋巴细胞，胶原纤维和纤维母细胞增生，有纤维囊腔形成；肌肉植入12周：样品周围可见少量淋巴细胞、胶原纤维、纤维囊腔较致密规整。
10)亚慢性毒性
按GB/T 16886.11规定的方法进行，结果：无亚慢性毒性反应。
48只同种属新西兰大白兔，雌雄不拘，体重2.5-3KG，随机分成3组。A试验组采用实施例1所制中空管形样品，B对照组自体神经倒置修复，C组为造成神经缺损未修复组。以3％的戊巴比妥钠1ml/kg耳缘静脉缓慢注射麻醉兔，无菌坏境下显露右侧坐骨神经，于梨状肌下缘1cm以远造成坐骨神经1.5cm缺损模型。各组分笼饲养，术后应用抗生素预防感染。48只兔全部进入结果分析，各组兔术后均出现活动困难，精神萎靡，进食、活动少，行走时术肢拖行。数天后兔饮食逐渐恢复正常。A、B组8周后溃疡愈合；针刺各组兔术侧足部时出现挣扎、逃避反应，表明已有痛觉；C组溃 疡愈合时间较迟，约10-12周时愈合(部分未愈合)，且反应较迟钝。术后10周，A、B组步态逐渐恢复正常，肌肉萎缩有所恢复，但C组肌肉恢复不明显，且反应较迟钝。各组兔术侧胫骨前肌均有不同程度的萎缩，且弹性差、关节僵直，C组肌肉萎缩最明显，肌肉光泽与弹性均不及A、B组。各组胚骨前肌湿重分别为2.56±0.16g、2.45±0.18g、1.50±0.11g，肌肉萎缩率分别为20.83％、17.45％、43.40％。A组和B组的湿重恢复率与C组相比，差异有显著性意义。A组和B组肌肉萎缩较轻，两组肌肉萎缩程度相当，其胫骨前肌湿重的恢复率差异无显著性意义。各组兔术侧坐骨神经传导速度A组33.151±1.434m/s、B组34.081±1.116m/s、C组16.028±1.333m/s，C组的神经传导速度要小于A、B组，差异有显著性意义(P<0.05)；A组的神经传导速度与B组相近，差异无显著性意义(P>0.05)。
综上，本发明神经修复材料：
(1)保留细胞外基质中胶原蛋白纤维的三维空间结构；
(2)起到物理隔离作用，为周围神经再生提供微环境；
(3)力学强度高，可控降解，与神经组织再生周期同步；
(4)生物相容性好，促进周围神经缺损的修复，降低引起感染、炎症或纤维包裹的风险；
(5)DNA残留能够达到10ng/mg以下，较同类产品低30-50ng/mg、半乳糖苷酶去除率较高，能够达到99％以上；
(6)保留细胞外基质中活性生长因子；
(7)添加促进神经损伤恢复的活性因子神经生长因子(NGF)。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。

Claims (10)

  1. 一种神经修复材料，其特征在于：所述神经修复材料包含胶原蛋白、多糖物质、活性因子和促进神经再生因子；所述修复材料具有三维网状多孔结构，无免疫原性、能体内降解，为片状或中空管状。
  2. 根据权利要求1所述的神经修复材料，其特征在于：所述的胶原蛋白为包含I型、III型、IV型和VI型胶原蛋白的组合物；所述的多糖物质为包含硫酸软骨素和透明质酸的组合物；所述的活性因子为包含纤维粘连蛋白、层粘连蛋白、整合素及生长因子的组合物；所述的促进神经再生因子包括诱导神经再生的活性物质。
  3. 根据权利要求2所述的神经修复材料，其特征在于：所述的促进神经再生因子为神经生长因子。
  4. 根据权利要求1所述的神经修复材料，其特征在于：所述的无免疫原性指DNA残留量小于10ng/mg、半乳糖苷酶清除率为99％以上；所述的三维网状多孔结构的孔隙率为75～90％。
  5. 根据权利要求1所述的神经修复材料，其特征在于：所述片状为长1-8cm、宽1-8cm，厚度0.1-1mm；所述中空管状为长1-5cm、直径0.2-0.9cm、厚度0.1-1mm的中空管状。
  6. 根据权利要求1-5中任一项权利要求所述的神经修复材料，其特征在于：所述神经修复材料由哺乳动物小肠粘膜下层组织材料制备，优选由猪或牛的小肠粘膜下层组织材料制备。
  7. 一种神经修复材料的制备方法，其特征在于：步骤包括：采用小肠粘膜下层组织材料作为原料，通过组织前置处理、病毒灭活、免疫原去除、复合生长因子和冷冻干燥步骤，清除动物源病毒风险、细胞成分、DNA成分和α-Gal抗原，并且保留细胞外基质成分。
  8. 根据权利要求7所述神经修复材料的制备方法，其特征在于：具体步骤包括：(1)组织前置处理；(2)病毒灭活：采用过氧乙酸-乙醇溶液浸泡小肠粘膜下层组织材料进行病毒灭活；(3)清洗；(4)免疫原去除：免疫原去除液为溶有0.01-0.2％质量百分比浓度的胰蛋白酶和浓度为0.1-1mmol/L的EDTA的pH值6-8的PBS溶液，免疫原去除过程在多频超声波装置中进行；(5)清洗；(6)复合生长因子：将小肠粘膜下层基质材料固定于模具上，加入神经生长因子；(7)真空冷冻干燥：在真空冷冻干燥机中进行。
  9. 根据权利要求7所述神经修复材料的制备方法，其特征在于：步骤(2)病毒灭活中，过氧乙酸-乙醇溶液中过氧乙酸的体积百分比浓度为0.1％-5％、乙醇的体积百分比浓度为5％-40％，过氧乙酸-乙醇溶液与小肠粘膜下层组织材料的体积比为(3-20)︰1，灭活时间2-4小时，温度范围为10-40℃；
    步骤(3)清洗中，清洗液为pH值为6-8的PBS溶液，PBS溶液温度为20℃，PBS溶液与小肠粘膜下层组织材料的体积比为(20-40)︰1，清洗2-6次；然后用纯化水清洗，纯化水与小肠粘膜下层组织材料比例为(20-40)︰1，至检测电导率为10μS/cm以下终止；清洗过程在超声波清洗机中进行，频率为20-80kHz，功率3000W以上；
    步骤(4)免疫原去除中，免疫原去除液的pH值为7.2-7.5；其中包括质量百分比浓度为0.01-0.2％的胰蛋白酶和摩尔浓度为0.1-1mmol/L的EDTA；所述免疫原去除液与小肠粘膜下层组织材料体积比为(20-40)︰1，脱细胞过程在包含至少两个超声频率的多频超声装置中进行，其中低频频率范围为20-40KHz，高频频率为60-90KHz，其中低频处理5-40min，高频处理5-40min，温度范围为20-35℃，超声频率5000W以上；
    步骤(5)清洗中，清洗液为pH值为6-8的PBS溶液，PBS溶液温度为20℃，PBS溶液与小肠粘膜下层组织材料的体积比为(20-40)︰1，清洗2-6次；然后用降温的注射用水清洗，注射用水与小肠粘膜下层组织材料比例为(20-40)︰1，至检测清洗前后注射用水电导率差为1μS/cm以下终止；清洗过程在超声波清洗机中进行，频率为20-80kHz，功率优选3000W以上；
    步骤(6)所述含促进神经再生活性因子的水溶液即神经生长因子水溶液的质量百分比浓度为0.01％-0.02％，所述含促进神经再生活性因子的溶液按照神经生长因子与组织材料质量比1-2:10000加入；步骤(6)所述的模具为直径0.2-0.9cm、长度1-10cm的不锈钢棒，或者长1-8cm、宽1-8cm的不锈钢盘；
    步骤(7)所述真空冷冻干燥具体为：将复合生长因的小肠粘膜下层基质材料放置于真空冷冻干燥机中，关闭冻干室的门，打开循环泵约1min，开启压缩机对冻干箱致冷，预冻至-45℃，保温2小时，然后开启真空泵，调节温度至-15℃，保温6小时，再调节温度至0℃，保温2小时，最后调节温度至25℃，保温4h，真空冷冻干燥完成，冻干室气压为1-50Pa。
  10. 一种神经修复材料的在神经修补医疗器械中的应用，其特征在于：所 述神经补医疗器械用于周围神经损伤的隔离保护，或所述神经修补医疗器械用于神经缺损的桥接。
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