WO2014114043A1 - 用于修复周围神经损伤的细胞基质修饰的组织工程神经移植物及其制备方法 - Google Patents

用于修复周围神经损伤的细胞基质修饰的组织工程神经移植物及其制备方法 Download PDF

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WO2014114043A1
WO2014114043A1 PCT/CN2013/076239 CN2013076239W WO2014114043A1 WO 2014114043 A1 WO2014114043 A1 WO 2014114043A1 CN 2013076239 W CN2013076239 W CN 2013076239W WO 2014114043 A1 WO2014114043 A1 WO 2014114043A1
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nerve
cells
tissue
cell
culture
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PCT/CN2013/076239
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English (en)
French (fr)
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顾晓松
丁斐
顾芸
薛成斌
杨宇民
王勇军
龚蕾蕾
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南通大学
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Priority to EA201591373A priority Critical patent/EA038957B1/ru
Priority to AU2013375655A priority patent/AU2013375655B2/en
Priority to BR112015017174-5A priority patent/BR112015017174B1/pt
Priority to EP13872797.9A priority patent/EP2949349B1/en
Priority to US14/762,243 priority patent/US9492589B2/en
Publication of WO2014114043A1 publication Critical patent/WO2014114043A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—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
    • 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
    • A61L27/14—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
    • A61L27/227—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
    • A61L27/14—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
    • A61L27/36—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3633—Extracellular matrix [ECM]
    • 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
    • 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
    • A61L27/3687—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
    • A61L27/38—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
    • A61L27/36—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
    • A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2527/00—Culture process characterised by the use of mechanical forces, e.g. strain, vibration

Definitions

  • the invention belongs to the technical field of medical biomaterials implantable in human body, and relates to a tissue engineering nerve graft for repairing cell matrix modification of peripheral nerve damage and a preparation method thereof.
  • acellular lularnerve graft which is a decellularized allogeneic nerve.
  • the original nerve cells are removed and the allogeneic cells are removed.
  • the acellular nerve matrix has basic requirements as a scaffold for peripheral nerve tissue engineering and can be used as an alternative to extracellular matrix for tissue engineering research.
  • acellular allogeneic nerve matrix graft on motoneurons of spinal cord anterior horn (Liu Jinchao, Lin Xiaoping, Yan Xiaojie, et al., Advances in Anatomical Sciences, 2005 (3): 206-209) Transplantation protects the motor neurons of the anterior horn of the spinal cord.
  • the other is to use a suitable mold to prepare the nerve conduit, and coat the cell matrix or cells on the inner and outer surfaces of the nerve conduit, such as the literature "Protective effect of tissue engineering artificial nerves on peripheral target organs and spinal cord neurons after rat sciatic nerve defect".
  • OEC olfactory ensheathing cells
  • Schwann cells Rosann Cel l, SC
  • ECM extracelite matrix
  • tissue-engineered peripheral nerves include Schwann cells and various stem cells. Most of these cells are of allogeneic origin and may cause immunogenicity during transplantation which is not conducive to clinical promotion. On the other hand, the specific attribution and biological effects of the cells after transplantation into the body are not completely clear, and may be inactivated due to the environment in the body, failing to achieve the desired biological effects. These have constrained the development of tissue engineering nerve grafts.
  • the present invention provides a cell-matrix-modified tissue engineered nerve graft for repairing peripheral nerve damage and a method for preparing the same, which is directed to the prior art.
  • the invention combines the tissue engineering method, uses the rotating cell culture reactor to culture the supporting cells on the nerve conduit and the scaffold, and then uses the in vitro decellularization technique to remove the supporting cells to form a tissue engineering nerve transplantation containing only the cell matrix secreted by the supporting cells. To repair peripheral nerve defects.
  • the invention can overcome the shortcomings of autologous nerve transplantation, can avoid the immunogenicity of allogeneic cell transplantation, provide an extracellular matrix suitable for nerve cell adhesion, and establish an environment conducive to the regeneration of axons to achieve rapid nerve growth and function. The ideal goal of recovery provides a viable solution for clinical treatment.
  • a tissue engineered nerve graft for repairing a peripheral nerve defect which is constructed by a nerve conduit and a cell matrix obtained by decellularization of an autologous or allogeneic source cell after secretion.
  • the cells are Schwann cells, skin-derived fibroblasts, skin stem cells, bone marrow mesenchymal stem cells or Inducing one or more of pluripotent stem cells.
  • the nerve conduit is composed of a biodegradable material, preferably one or more of silk fibroin, chitosan, collagen, polylactic acid or polyglycolic acid.
  • the above nerve conduit can be a tissue engineered nerve graft commonly used by those skilled in the art, preferably a superficially porous chitosan catheter, such as a 50-90%, 50-300 Mffl porous catheter with a porous structure and high tensile strength, tubular L_3 ⁇
  • the inner diameter of the body is 0. 5-8mm, the wall thickness of 0. l_3mm.
  • a nanofiber silk fibroin catheter which is specifically described in the patent application No. CN200910034583.
  • CN101664346 entitled "Artificial nerve graft prepared by electrospinning, preparation method and special device thereof” Method made.
  • a superficially porous chitosan catheter shell or a nanofiber silk fibroin catheter sheath and the catheter is filled with a composite nerve conduit of silk fibroin fiber.
  • the preparation of the silk fibroin fiber can be referred to the patent application number: CN200910034583.
  • the method disclosed in the publication No. CN101664346 entitled "Artificial nerve graft prepared by electrospinning and its preparation method and special device” preferably comprises 120 silk fibroin monofilaments per catheter.
  • the invention also provides a preparation method of the above-mentioned natural cell matrix modified tissue engineering nerve graft for repairing peripheral nerve defects, which comprises decellularizing treatment to obtain decellularized tissue engineering after preparing tissue-containing nerve graft containing support cells.
  • Nerve graft The above-described support cell-containing tissue engineered nerve graft can be attached to the inner and outer surfaces of the graft by cell culture by means of a nerve graft and a cell well known to those skilled in the art, preferably by three-dimensional microgravity cell culture.
  • the present invention uses a Synthecon microgravity instillation three-dimensional culture system of the type RCMW.
  • tissue engineered nerve grafts containing supporting cells Slowly inject 100 ml of complete medium into the culture vessel, then add 2. 5 X 10 7 cells and aseptically treated nerve conduits, and then slowly inject with a peristaltic pump. After the whole container is filled, the final density of the cells is l X 107ml. After exhausting the air in the system, the microgravity circulation perfusion culture is started. The rotary bioreactor is placed in the C0 2 incubator at 37 ° C, and the rotation speed is 10 rpm in the first 24 hours.
  • the differentiation medium is preferably formulated as H-DMEM + 15% FBS + 50 ng / ml HRG + 2 M forskl in + 50 - g /ml Vc.
  • tissue-engineered nerve graft containing cell matrix secreted by cells Tissue-engineered nerve grafts cultured with cells were decellularized, washed with PBS, deionized sterile water 37 V hypotonic for 10 min; Add the cell extract to lyse the cells at 37 °C for 10-15 min; wash with PBS for 3 times, add Dnase I (4 mg/ml) and digest at 37 °C for 30 min to remove the DNA, store at -80 °C for use, and extract the cells.
  • the liquid formulation is preferably PBS containing 0.5% Triton X-100 and 20 mM ammonia.
  • the cells in the above preparation method are preferably one or more of Schwann cells, skin-derived fibroblasts, skin stem cells, bone marrow mesenchymal stem cells or induced pluripotent stem cells;
  • the nerve conduit is composed of a biodegradable material, preferably One or more of silk fibroin, chitosan, collagen, polylactic acid or polyglycolic acid.
  • the above nerve conduit can be a tissue engineered nerve graft commonly used by those skilled in the art, preferably a superficially porous chitosan catheter, such as a 50-90%, 50-300 Mffl porous catheter with a porous structure and high tensile strength, tubular L_3 ⁇
  • the inner diameter of the body is 0. 5-8mm, the wall thickness of 0.
  • ECM Neuronal extracellular matrix
  • Col collagen
  • LN laminin
  • FN fibronectin
  • hyaluronic acid and proteoglycans eg chondroitin sulfate, heparin sulfate proteoglycan
  • LN, FN and Col provide appropriate "adhesion" for nerve growth, allowing axons to follow the matrix.
  • the bridge grows to guide the directional growth of nerve fibers.
  • the prior art mostly uses one or several components of extracellular matrix, such as laminin (LN), fibronectin (FN), etc., for tissue engineering grafts, and these commercial synthetic ECMs are expensive, and The composition is relatively simple.
  • the natural extracellular matrix obtained by decellularization after culturing cells retains various important components and frameworks of the extracellular matrix, is more conducive to the adhesion of nerve cells, and has a certain guiding effect on the directionality of axon regeneration. Therefore, it is more conducive to adding The process of rapid nerve regeneration.
  • the price is relatively low and is more easily accepted by clinical patients.
  • tissue-engineered peripheral nerves include Schwann cells and various stem cells. Most of these cells are allogeneic sources that may cause immunogenicity during transplantation.
  • the cell matrix in the tissue engineered nerve graft used in the present invention is obtained by decellularization of the cultured cells after formation, and is reduced or non-immunogenic after transplantation, and is suitable for use in a large-scale population.
  • the tissue engineering nerve graft of the present invention adopts a three-dimensional microgravity cell culture method, so that the supporting cells can uniformly adhere to the inner and outer surfaces of the graft, and the extracellular matrix obtained after decellularization is also uniformly distributed.
  • the inner and outer surfaces of the graft facilitate nerve regeneration.
  • the graft used in the present invention does not contain exogenous toxic and side-effect substances brought in by the preparation process, has good biocompatibility and biodegradability, and has good mechanical properties.
  • the used nerve conduit wall has a three-dimensional structure rich in micropores, which provides a necessary route for the transport of nutrients required for nerve cell growth; the silk fibroin filament used is an electrospinning product with a large specific surface area. It provides the necessary induction and necessary growth space for the growth of nerve cells, and the effect is good.
  • FIG. 1 shows Schwann cells (A. Schwann cell light microscopy; B. Schwann cell immunohistochemical identification map, gray long represents S100 immunopositive, gray dot is Ho ec hst33342 labeled nuclei).
  • Figure 2 shows fibroblasts (A. Fibroblasts (100 X); B. Fibroblast immunohistochemistry (100 X), gray for Fibronectin immunopositive, white highlights for Hoechst33342-labeled nuclei).
  • Figure 3 shows skin stem cells (A. skin stem cell spherometry (100 X ); B. skin stem cell sphere immunohistochemical identification map (100 X ), a represents Versican immunopositive, b represents Nestin immunopositive, c is H 0ec Hst33342 labeled nuclei, d is a combination of a, b, c three maps; C. skin stem cell sphere immunohistochemical identification map (100 X ), a represents Vimentin immunopositive, b represents Nestin immunopositive, c is Hoechst33342 labeled nuclei , d is the combination of the three graphs a, b, and c).
  • A skin stem cell spherometry (100 X )
  • B skin stem cell sphere immunohistochemical identification map (100 X )
  • a represents Versican immunopositive
  • b represents Nestin immunopositive
  • c H 0ec Hst33342 labeled nuclei
  • d is
  • Figure 4 shows the directed differentiation of skin stem cells into Schwann cells (A. Schwann cell colony light microscopy (100 X) induced by differentiation of skin stem cells; B. Picking stem cells to induce differentiation into Schwann cells Lack of cell expansion microscopy).
  • Figure 5 shows the immunohistochemical identification of the Schwann cell-derived cells after directed differentiation of skin stem cell spheres (100 X ) (A represents S100 immunopositive; B. is Hoechst33342 labeled nuclei; C. is A, B Merger).
  • Figure 6 shows the identification of bone marrow mesenchymal stem cells and flow cytometry CD molecules.
  • Figure 7 is a cell matrix immunochemical map (A. photomicrograph of cell surface on the surface of cells after decellularization; B. immunoprecipitation of cell matrix FN; C. immunogenicity of cell matrix LN; D. merged map of B and C) .
  • Figure 8 shows the effect of cell matrix on neuronal growth (A. NF immunochemical map of neurons cultured on a cell matrix, where ECM group refers to neurons cultured on purchased ECM; N-ECM group refers to culture in Supports neurons on the cell matrix after cell depletion; B. Detection of neuronal viability; C. Western Blotting detects the expression of neural adhesion molecules (NCAM) and axon growth-related factors (GAP43).
  • NCAM neural adhesion molecules
  • GAP43 axon growth-related factors
  • Figure 9 is a scanning electron micrograph of a tissue engineered nerve graft modified with a natural cell matrix
  • A The inner surface of a nerve tube containing a supporting cell-based tissue engineered nerve graft, showing that the supporting cells uniformly distribute the surface
  • B Decellularized tissue engineering nerve transplantation On the inner surface of the nerve conduit, the extracellular matrix is visible
  • C the silk fibroin scaffold containing the tissue engineered nerve graft of the support cell
  • D the silk fibroin scaffold of the decellularized tissue engineered nerve graft.
  • Figure 10 shows the immunocytochemistry of the cell surface of the silk fibroin scaffold after decellularization (A. indicates that the cell matrix FN immunopositive; B. indicates the cell matrix LN immunopositive; C. is the silk fibroin scaffold; D. is A, B and C Merged diagram).
  • the vertical line in the figure represents the front end of regenerative nerve growth (A is the material group after 1 week, B is the cell matrix group after 1 week, C is 2 After the week, the material group, D is the cell matrix group after 2 weeks).
  • Figure 12 is a graph showing statistical results of regenerated nerve fiber data.
  • the cells were pelleted by trypsinization, resuspended in 1 ml of complete medium containing Thyl. 1 (1:1000), incubated on ice for 2 hours; centrifuged to discard the supernatant, using DMEM and complement.
  • the mixture was resuspended in the mixture (3:1), incubated at 37 °C for 1 hour, centrifuged and completely washed twice in medium, re-inoculated into the culture dish, and changed every other day. After the cells were full, they could be used (cell culture and identification). The results are shown in Figure 1).
  • SD rats were taken for 1 day, and the alcohol was disinfected after sacrifice.
  • the skin on the back was removed and placed in the pre-cooled anatomy to carefully remove the subcutaneous tissue (fat and subcutaneous fascia, blood vessels, etc.); after PBS washing 3 times, use the surgical blade Cut into small pieces ( ⁇ lmmX lmm), completely digested with type I collagenase (1 mg/ml), centrifuge to discard the supernatant, resuspend the cells in complete medium, inoculate the cells in culture, and fuse the cells for 90% and subculture.
  • Fibroblasts are contaminated with epithelial cells during primary culture, and most of the hybrid cells (epithelial and endothelial cells) gradually die after several passages. Therefore, the cells used in the present invention are cells of more than 3 passages (see Figure 2 for cell culture and assay results).
  • Suspension culture Suspension cultured cell spheres can be subcultured to obtain a sufficient number of skin stem cells (see Figure 3 for cell culture and identification results).
  • the obtained skin stem cells are subjected to differentiation culture to obtain Schwann cells, and the specific steps are as follows:
  • the skin stem cells are divided into a differentiation medium I (DMEM/F12 (3:1) containing 0.1% double antibody, 40 ⁇ ⁇ / ⁇ 1 fungizone, 40 ng/ml FGF2, 20 ng/ml EGF, 2% B27 supplement, 10% FBS) cultured for 3 days before differentiation medium (DMEM/F12 (3: 1) contains 0 ⁇ 1% double antibody, 5 Mm forskol In, 50 ng/ml heregul in-1 ⁇ , 50 g/ml, 2% N2 supplement, 1% FBS) Schwann cell colonies can be obtained after 2-3 weeks of culture (see Figure 4. A).
  • Support cells such as Schwann cells
  • Mouse anti laminin ( LN ) rabbit anti fibronectin ( FN ) was incubated overnight at 4 ° C, and washed twice with PBS to add secondary antibody: FITC-conjugated-goat-anti-rabbit-IgG (1: 200) and TRITC-con jugated-goat-anti-mouse-IgG (1:200) was incubated for 2 h at room temperature and examined by fluorescence confocal microscopy (DMR, Leica) (see Figure 7. B, C and D).
  • DMR fluorescence confocal microscopy
  • the dorsal root ganglion neurons were separately cultured into the extracellular matrix containing the decellularized cells of the support cells prepared in Example 5 and coated with the purchased extracellular matrix (Cat. No. E0282; Company: SIGMA), and cultured under the same conditions. After an hour, the activity of neurons was detected by MTT assay, the axon growth of neurons was observed by immunocytochemistry, and the expression of axon growth-related factor GAP43 and NCAM was detected by Western Blotting. As a result, as shown in Fig. 8, there was no statistical difference in the cell viability of neurons on the two extracellular matrices, but immunocytochemical results showed that the neurons were cultured on the extracellular matrix after decellularization of the supporting cells.
  • Example 7 Construction of a tissue engineered nerve graft containing support cells
  • tissue engineered nerve grafts were assembled. After the chitosan nerve conduits and silk fibroin fibers were aseptically treated, 120 silk fibroin monofilaments were inserted into each nerve catheter as a stent to make a nerve conduit. Then, 100 ml of complete medium (DMEM + 10% FBS + 50 ng / ml ⁇ 6 + 2 ⁇ forsklin) was slowly injected into the culture vessel by a peristaltic pump, and then 2. 5 X 10 7 purified Schwann cells and tissue engineered nerve grafts were added. Then, the entire container is slowly filled with a peristaltic pump, and the final density of the cells is l X 10 5 /ml.
  • complete medium DMEM + 10% FBS + 50 ng / ml ⁇ 6 + 2 ⁇ forsklin
  • the microgravity circulation perfusion culture is started.
  • the rotary bioreactor was placed in a 37 ° C (3 ⁇ 4 incubator, rotating at 10 rpm for the first 24 hours to allow the cells to fully contact the graft and attach. Adjust the rotational speed of the rotary bioreactor 24 hours later to
  • the vector can be suspended in the culture medium.
  • the medium is replaced with a differentiation medium (H-DMEM + 15% FBS + 50 ng / ml ⁇ 6 + 2 ⁇ ⁇ forsklin + 5 ( ⁇ g / ml Vc ), and is cultured every three days during the culture. Liquid, replace the 500ml liquid storage bottle.
  • Tissue engineered nerve grafts were fixed with 4% glutaraldehyde at 4 ° C and washed 3 times with PBS; 1% hungry acid at room temperature After 2 hours, it was washed twice with PBS; dehydrated with ethanol gradient (30%, 50%, 70%, 80%, 95%, 100%) after 10 min, replacing lOmin with absolute ethanol: tert-butanol (1:1) ; Then, it was replaced with t-butanol twice for 10 min each; after freeze-drying, platinum was sprayed and observed by scanning electron microscopy (TCS SP2, Leica).
  • Example 8 Construction of tissue engineered nerve graft modified by natural cell matrix
  • the constructed tissue-engineered nerve grafts prepared in Example 7 were subjected to decellularization treatment, washed with PBS and deionized sterile water at 37 ° C for 10 min; and the cell extract was added (including 0. 5% Triton X-100 and 20 mM ammonia in PBS) The cells were lysed at 37 °C for 10-15 min; washed three times with PBS and then added with Dnase I (4 mg/ml). The DNA was removed by digestion at 37 ° C for 30 min. Scanning electron microscopy results It shows that the surface of the nerve conduit and the silk fibroin scaffold uniformly distribute the extracellular matrix components that support cell secretion (see Figure 9. B and D).
  • the prepared natural cell matrix-modified tissue engineered nerve graft can be stored at -80 ° C until use. Scanning electron microscopy and immunochemical methods were used to detect the extracellular matrix components left after decellularization. The scanning electron microscopy method was the same as that of the above Example 7, and the specific method of immunochemistry was the same as in Example 5. The results are shown in Fig. 10.
  • Example 9 Repair of rat sciatic nerve defects with tissue engineered nerve graft modified with natural cell matrix Tissue engineered nerve graft modified with natural cell matrix to repair rat sciatic nerve defects by immunohistochemistry, transmission electron microscopy, electrophysiology, muscle wet weight The rate of nerve regeneration and the recovery of the sciatic nerve were measured by other methods.
  • a 10 mm defect model of rat sciatic nerve was established and randomly divided into 2 groups: a group of tissue engineered nerve grafts modified with natural cell matrix repaired rat sciatic nerve defects, called cell matrix group, and the other group used tissue modified without cell matrix.
  • Engineered nerve grafts repair rat sciatic nerve defects, called the material group.
  • the regenerative nerves were frozen and sectioned 1 week after operation and 2 weeks after operation.
  • the results of NF immunohistochemistry showed that the nerve growth rate of the cell matrix group was faster than that of the material group, and the NF-positive nerve fibers in the cell matrix group were relatively dense and evenly distributed.
  • Figure 11 A is the material group after 1 week, B is the cell matrix group after 1 week, C is the material group after 2 weeks, D is the cell matrix group after 2 weeks); the statistical results after counting the regenerated nerve fibers (Figure 12) ; 3 months after surgery, all animals were exposed to sciatic nerve under moderate anesthesia for neuroelectrophysiological examination.
  • the mean amplitudes of CMAP in the material group and the cell matrix group were 4. 63 ⁇ 0. 13mV, 6. 89 ⁇ 2. 85mV, the difference between the two groups was statistically significant (P ⁇ 0.05); 3 months after surgery, The regenerative nerve interrupted sections were examined by transmission electron microscopy.
  • FIG. 13 A is a material).
  • Group; B is the cell matrix group).
  • the thickness of the neuromyelin sheath of the material group and the cell matrix group were: 0.63 ⁇ 0. 27 ⁇ ⁇ , 0. 82 ⁇ 0. 39 ⁇ ⁇ , and there was a statistical difference between the two groups ( ⁇ ⁇ 0.05).

Abstract

一种用于修复周围神经缺损的组织工程神经移植物，其由神经导管和细胞基质构建而成，所述细胞基质是自体的或同种异体来源细胞分泌形成后脱细胞而获得。一种组织工程神经移植物的制备方法，所述方法包括含支持细胞的组织工程神经移植物的构建及细胞基质修饰的组织工程神经移植物的构建。

Description

用于修复周围神经损伤的细胞基质修饰的组织工程神经移植物及其 制备方法 技术领域
本发明属于可植入人体中的医用生物材料技术领域，涉及一种用于修复周围神 经损伤的细胞基质修饰的组织工程神经移植物及其制备方法。
背景技术
随着社会现代化进程和人们生活节奏的加快，不可避免的会出现更多的交通事 故、 工伤事故、运动意外等事件， 同时频繁的局部战争和暴力事件及地震等自然 灾害， 均会造成周围神经损伤， 临床上当中长距离的神经缺损不能依靠端对端的 缝合来弥补神经缺失时， 就不得不依靠移植物来桥接修复。寻找和研制较为合适 的移植物问题虽然已有一百多年的历史了，但除了自体神经成为首选的神经缺损 桥接移植物外， 至今在人类仍在寻找理想的， 能够在临床上广泛应用的神经移植 替代物。 就自体神经移植中， 因为供移植用的神经来源有限、 组织结构和尺寸难 以匹配、 移植供区长期失神经支配等原因， 未能在临床上广泛使用。
随着组织工程学的出现和发展，为构建自体神经移植替代品提供了一条新的出 路。 目前已有的人工神经移植物主要有两大类， 一类是脱细胞处理的同种异体神 经即脱细胞神经基质（acel lularnerve graft , ANG) , 利用原来神经的管道， 将 异体细胞去掉。脱细胞神经基质具有作为周围神经组织工程支架的基本要求， 可 以成为细胞外基质替代物应用于组织工程研究。如文献《脱细胞神经基质诱导大 鼠骨髓间充质细胞向 Schwann细胞分化的形态学研究》 （何红云， 邓仪昊， 佟晓 杰等，神经解剖学杂志， 2007年第 23卷第 6期）探讨脱细胞神经基质移植物 (ANA) 体外诱导成年大鼠骨髓问充质细胞 （BMSCs ) 向 Schwann细胞分化的可行性， 研 究结果提示 ANA可诱导成年大鼠骨髓问充质细胞分化为 Schwann细胞。如文献《脱 细胞异体神经基质移植物对脊髓前角运动神经元的保护作用》（刘金超，林晓萍， 佟晓杰等， 解剖科学进展， 2005 (3) : 206-209 ) 探讨脱细胞神经基质移植物异体 移植对脊髓前角运动神经元的保护作用。研究结果提示脱细胞异体神经基质移植 物桥接周围神经缺损对运动神经元胞体的存活具有良好的保护作用。虽然目前对 脱细胞神经基质制备技术的研究甚多，进展较快，但是其制备处理方法程序复杂， 而且在处理过程会损伤支架材料的微细结构和受力特性。 另外一类是利用适当的模具制备神经导管，将细胞基质或者细胞包覆在神经导 管的内外表面， 如文献《大鼠坐骨神经缺损后组织工程人工神经对外周靶器官及 脊髓神经元的保护作用》 （游华，矫树生，冯帅南等， 中华创伤杂志， 2010 Vol. 26 No. 3 P. 265-269 ) 公 开 了 一 种 使 用 嗅 球 成 鞘 细 胞 (olfactory ensheathing cell, OEC) -雪旺细胞（Schwann cel l, SC) -细胞外基质 成 分 （extracel lularmatrix，ECM) _ 聚 乳 酸 - 聚 羟 基 乙 酸 共 聚 物
[poly (DLlactide-co-glycol ide acid)， PLGA]桥接体保护大鼠坐骨神经缺损后 外周靶器官及脊髓神经元。 又如申请号为 CN03134541. 7 , 申请公布号为 CN1589913的中国专利 "用于修复周围神经缺损的组织工程化周围神经及制备方 法"公开了一种用于修复周围神经缺损的组织工程化周围神经， 使用神经胶质细 胞或向神经胶质细胞分化的干细胞作为种子细胞，采用生物可降解材料构成的神 经导管， 同时应用复合有神经营养因子的控制释放微球， 并含有细胞外基质。 目前组织工程化周围神经中所用的支持细胞有施万细胞及各种干细胞等。这些 细胞多数是同种异体来源， 移植过程中可能会导致免疫原性不利于临床推广。另 一方面， 细胞移植入体内后具体的归属及生物学效应如何并不完全清楚， 可能会 由于体内的环境而失活， 达不到所期望的生物学效应。这些都制约了组织工程神 经移植物的发展。
发明内容
本发明针对现有技术不足提供了一种利于细胞的黏附，能够促进神经轴突的再 生的用于修复周围神经损伤的细胞基质修饰的组织工程神经移植物及其制备方 法。本发明结合组织工程方法， 利用旋转细胞培养反应器将支持细胞培养于神经 导管及支架上， 然后利用体外脱细胞技术将支持细胞脱去， 形成只含支持细胞分 泌的细胞基质的组织工程神经移植物， 以修复周围神经缺损。本发明可以克服自 体神经移植缺点， 同时可以避免同种异体细胞移植的免疫原性， 提供利于神经细 胞黏附的细胞外基质， 建立一个有利于再生轴突生长的环境， 以达到神经快速生 长、 功能恢复的理想目标， 为临床治疗提供可行方案。
本发明具体技术方案如下：
一种用于修复周围神经缺损的组织工程神经移植物，由神经导管和细胞基质构建 而成， 所述细胞基质是自体的或同种异体来源细胞分泌形成后脱细胞而获得。所 述细胞是施万细胞、 皮肤来源的成纤维细胞、 皮肤干细胞、骨髓间充质干细胞或 诱导多潜能干细胞中的一种或几种。所述神经导管由生物可降解材料构成， 优选 丝素蛋白、 壳聚糖、 胶原、 聚乳酸或聚羟基乙酸中的一种或几种。
上述神经导管可以为本领域技术人员常用的组织工程神经移植物，优选表面多 孔的壳聚糖导管， 如 50-90%、 孔径 50-300Mffl的具有多孔结构、 抗拉强度高的神 经导管， 管状本体内径为 0. 5-8mm， 壁厚 0. l_3mm。 具体可参照专利申请号为 CN201110324474. 8, 名为 "组织工程神经移植物及其用途" 中所述的方法制得。 或者优选纳米纤维蚕丝丝素蛋白导管， 具体可参照专利申请号为 CN200910034583. 9, 公开号为 CN101664346, 名为 "静电纺丝制备的人工神经移 植物及其制备方法和专用装置"中所述的方法制得。或者优选使用表面多孔的壳 聚糖导管外壳或纳米纤维蚕丝丝素蛋白导管外壳，导管内部充填入丝素蛋白纤维 的复合型神经导管，丝素蛋白纤维的制备可参照专利申请号为 CN200910034583. 9， 公开号为 CN101664346, 名为 "静电纺丝制备的人工神经移植物及其制备方法和 专用装置" 中所述的方法制得， 优选每个导管中含有 120根丝素蛋白单丝。
本发明还提供了上述用于修复周围神经缺损的经天然细胞基质修饰的组织工 程神经移植物的制备方法，在于制备含支持细胞的组织工程神经移植物之后进行 脱细胞处理得到去细胞的组织工程神经移植物。上述含支持细胞的组织工程神经 移植物可以采用本领域技术人员熟知的神经移植物和细胞通过细胞培养的方式 使细胞贴附于移植物内外表面生长， 优选采用三维微重力细胞培养的方法。本发 明使用型号为 RCMW的 Synthecon微重力灌入式三维培养系统。
本发明含支持细胞的组织工程神经移植物的制备及去细胞组织工程神经移植 物的构建优选的技术方案如下：
( 1 )含支持细胞的组织工程神经移植物的构建： 将 100ml完全培养基缓慢注入 培养容器，再加入 2. 5 X 107个细胞及无菌处理过的神经导管，再以蠕动泵缓慢注 满整个容器， 细胞最终密度为 l X 107ml， 排尽系统内空气后， 开始旋转微重力 循环灌注培养； 旋转式生物反应器放入 37°C C02培养箱中， 前 24小时转速为 10 转 /分钟， 使细胞与神经导管充分接触， 贴附； 24小时后调整旋转式生物反应器 旋转速度， 使神经导管能悬浮到培养液中； 继续培养 2天后更换为分化培养基培 养， 以促进细胞外基质分泌； 培养 2周后终止培养将表明贴附生长有细胞的神经 导管取出， 其中分化培养基优选配方为 H-DMEM+15%FBS+50ng/ml HRG+2 M forskl in+50 -g/ml Vc。 ( 2) 含细胞自身分泌的细胞基质修饰的组织工程神经移植物的构建： 将培养 有细胞的组织工程神经移植物进行脱细胞处理， PBS清洗后去离子无菌水 37 V 低渗 10 min; 加入细胞抽提液于 37°C裂解细胞 10-15 min; PBS清洗 3次后加入 Dnase I (4 mg/ml) 37 °C消化 30 min去除 DNA， -80 °C保存备用， 其中细胞 抽提液配方优选为含 0. 5% Triton X-100和 20 mM氨水的 PBS。
上述制备方法中所述细胞优选施万细胞、皮肤来源的成纤维细胞、皮肤干细胞、 骨髓间充质干细胞或诱导多潜能干细胞中的一种或几种；神经导管由生物可降解 材料构成，优选丝素蛋白、壳聚糖、胶原、聚乳酸或聚羟基乙酸中的一种或几种。 上述神经导管可以为本领域技术人员常用的组织工程神经移植物，优选表面多 孔的壳聚糖导管， 如 50-90%、 孔径 50-300Mffl的具有多孔结构、 抗拉强度高的神 经导管， 管状本体内径为 0. 5-8mm， 壁厚 0. l_3mm。 具体可参照专利申请号为 CN201110324474. 8, 名为 "组织工程神经移植物及其用途" 中所述的方法制得。 或者优选纳米纤维蚕丝丝素蛋白导管， 具体可参照专利申请号为 CN200910034583. 9, 公开号为 CN101664346, 名为 "静电纺丝制备的人工神经移 植物及其制备方法和专用装置"中所述的方法制得。或者优选使用表面多孔的壳 聚糖导管外壳或纳米纤维蚕丝丝素蛋白导管外壳，导管内部充填入丝素蛋白纤维 的复合型神经导管，丝素蛋白纤维的制备可参照专利申请号为 CN200910034583. 9， 公开号为 CN101664346, 名为 "静电纺丝制备的人工神经移植物及其制备方法和 专用装置" 中所述的方法制得， 优选每个导管中含有 120根丝素蛋白单丝。
本发明技术方案具有如下优点：
( 1 )神经细胞外基质 (ECM)，大体上分化为胶原 (Col)、层粘连蛋白（LN)、 纤维粘 连蛋白（FN)、 透明质酸和蛋白多糖 (如硫酸软骨素、 硫酸肝素蛋白多糖）等。 ECM 是一种为组织器官甚至整个机体的完整性提供力学支持和物理强度的物质， 研 究发现， LN、 FN和 Col等， 能为神经生长提供适当的 "粘着性"， 使轴突沿着 基质桥生长， 引导神经纤维定向生长。现有技术多采用一种或几种细胞外基质的 成分， 如层粘连蛋白（LN)、 纤维粘连蛋白（FN)等用于组织工程移植物， 这些商品 化的人工合成的 ECM价格高昂， 且成分相对单一。本发明通过培养细胞后脱细胞 获得的天然的细胞外基质， 保留了细胞外基质的各种重要的成分以及框架， 更利 于神经细胞的黏附， 并对轴突再生的方向性具有一定的导向作用， 因而更利于加 快神经再生的进程。 且与人工合成的细胞外基质相比， 价格相对低廉， 更容易被 临床病人接受。
( 2 )目前组织工程化周围神经中所用的支持细胞有施万细胞及各种干细胞等。 这些细胞多数是同种异体来源， 移植过程中可能会导致免疫原性。本发明使用的 组织工程神经移植物中的细胞基质是培养的细胞形成后脱细胞获得，移植后降低 或没有免疫原性， 适宜大规模人群的使用。
( 3 ) 本发明所述的组织工程神经移植物采用三维微重力细胞培养的方法， 使 支持细胞能够均匀的粘附于移植物的内外表面，脱细胞后得到的细胞外基质也均 匀的分布于移植物的内外表面， 有利于神经的再生。
( 4) 本发明使用移植物不含有由于制备工艺带入的外源性毒、 副作用物质， 具有良好生物相容性和生物可降解性， 还具有良好的力学性能。使用的神经导管 管壁具有丰富微孔的三维结构，为神经细胞生长过程所需的营养物质的输送提供 了必须的途径； 使用的丝素蛋白丝是静电纺丝产物， 拥有很大的比表面积， 为神 经细胞的生长提供了必要的诱导作用和必须的生长空间， 使用效果好。
下面结合附图和实例对本发明作进一步说明。
附图说明
图 1为施万细胞（A. 施万细胞光镜图； B. 施万细胞免疫组化鉴定图， 灰色长 形代表 S100免疫阳性， 灰色点状为 Hoechst33342标记的细胞核） 。
图 2为成纤维细胞 （A. 成纤维细胞光镜图 （100 X ) ； B. 成纤维细胞免疫组 化鉴定图 ( 100 X ) ， 灰色为 Fibronectin免疫阳性， 白色亮点为 Hoechst33342 标记的细胞核） 。
图 3为皮肤干细胞 （A. 皮肤干细胞球光镜图 （100 X ) ； B. 皮肤干细胞球免 疫组化鉴定图 ( 100 X ) ， a代表 Versican免疫阳性， b代表 Nestin免疫阳性， c为 H0echst33342标记的细胞核， d为 a， b， c三图的合并； C. 皮肤干细胞球 免疫组化鉴定图 ( 100 X )， a代表 Vimentin免疫阳性， b代表 Nestin免疫阳性， c为 Hoechst33342标记的细胞核， d为 a， b， c三图的合并） 。
图 4为皮肤干细胞定向诱导分化为施万细胞 （A. 皮肤干细胞定向诱导分化成 的施万细胞集落光镜图（100 X )； B. 挑取皮肤干细胞球定向诱导分化成的施万 细胞集落后细胞扩增光镜图） 。 图 5 为皮肤干细胞球定向诱导分化成的施万细胞集落后细胞免疫组化鉴定图 ( 100 X ) (A代表 S100免疫阳性.； B.为 Hoechst33342标记的细胞核; C.为 A， B 两图的合并） 。
图 6为骨髓间充质干细胞及流式细胞术 CD分子鉴定图。
图 7为细胞基质免疫化学图 （A. 脱细胞后材料表面细胞基质光镜图； B.表示 细胞基质 FN免疫阳性； C.表示细胞基质 LN免疫阳性； D. 为 B和 C的合并图）。 图 8为细胞基质对神经元生长的影响 （A. 将神经元培养于细胞基质上 NF免 疫化学图， 其中 ECM组是指培养在购买的 ECM上的神经元； N-ECM组是指培养在 支持细胞脱细胞后的细胞基质上的神经元； B. 神经元的活力检测结果； C. Western Blotting检测神经黏附分子 （NCAM) 与轴突生长相关因子 （GAP43 ) 的 表达） 。
图 9为天然细胞基质修饰的组织工程神经移植物扫描电镜图（A.含支持细胞的 组织工程神经移植物神经导管内表面， 可见支持细胞均匀分布其表面； B.脱细胞 的组织工程神经移植物神经导管内表面， 可见细胞外基质； C. 含支持细胞的组 织工程神经移植物的丝素蛋白支架； D. 脱细胞的组织工程神经移植物的丝素蛋 白支架） 。
图 10为脱细胞后丝素支架表面细胞基质免疫化学图 （ A.表示细胞基质 FN免 疫阳性； B.表示细胞基质 LN免疫阳性； C.为丝素蛋白支架； D. 为 A， B和 C的 合并图） 。
图 11为 NF免疫组化显示神经再生速度 （Bar = 200Mm) ， 图中竖划线代表再生 神经生长的最前端 （A为 1周后材料组， B为 1周后细胞基质组， C为 2周后材 料组， D为 2周后细胞基质组） 。
图 12为再生神经纤维数据统计结果图。
图 13为再生神经中段横切面电镜图 （标尺 =5 μ πι) (Α为材料组； B为细胞基质 组） 。
具体实施方式
在本发明中所使用的术语， 除非另有说明， 一般具有本领域普通技术人员通常 理解的含义。
下面结合具体实施例并参照数据进一步详细描述本发明。应理解， 这些实施例 只是为了举例说明本发明， 而非以任何方式限制本发明的范围。 在以下实施例中， 未详细描述的各种过程和方法是本领域中公知的常规方法。 实施例 1 施万细胞的培养及纯化
取新生一天 SD大鼠， 处死后酒精消毒， 取双侧坐骨神经， 置于冰浴操作台去除 神经外膜及黏连组织， 胶原酶 /胰酶混合消化完全后离心弃上清液， 完全培养基 重悬细胞， 接种到 PDL事先包被好的培养皿中培养。 24 小时更换成含有阿糖胞 苷（10 μΜ)的完全培养基培养 48小时，更换为含有 HRG (50 ng/ml)和 Forskl in (2 μΜ)的完全培养基继续培养， 每 3 天换液， 直到细胞融合。 待细胞融合后， 胰酶 消化后离心得到细胞沉淀， 用 1 ml含 Thyl. 1的完全培养基（1： 1000)重悬细胞， 冰上孵育 2小时； 离心弃上清， 用 DMEM和补体的混合物 （3 : 1)重悬细胞， 37°C 孵育 1小时， 离心后完全培养基清洗 2次， 重新接种于培养皿中， 隔天换液， 细 胞长满后即可以使用 （细胞培养及鉴定结果见附图 1 ) 。
实施例 2 皮肤来源的成纤维细胞的培养
取新生 1天的 SD大鼠， 处死后酒精消毒， 取出背部皮肤置于预冷的解剖液中小 心地剔除皮下组织 （脂肪和皮下筋膜层、 血管等） ； PBS清洗 3次后， 用手术刀 片切成小块（〈lmmX lmm)， 以 I 型胶原酶（1 mg/ ml)完全消化后离心弃上清液， 完全培养基重悬细胞， 接种到培养皿培养， 细胞融合 90%后传代培养。 成纤维细 胞在原代培养过程中会有上皮细胞污染， 大多数杂细胞（上皮和内皮细胞）会在 几次传代后逐渐死亡。 因此， 本发明所用细胞为传 3代以上细胞（细胞培养及鉴 定结果见附图 2 ) 。
实施例 3 皮肤干细胞的培养及定向沿施万细胞分化
皮肤干细胞的培养及分化参照文献 《 Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cel l progeny》 ( Jeffrey A Biernaskie, Ian A McKenzie, Jean G Toma & Freda D Mi l ler, NATURE PROTOCOLS, 2006 (1)： 2803-2812 ) ， 简单描述如下： 取新生 1天的 SD 大鼠， 处死后酒精消毒， 取出背部皮肤置于预冷的解剖液中小心地剔除皮下组织 (脂肪和皮下筋膜层、 血管等） ； PBS清洗 3次后， 用手术刀片切成小块 （〈lmm X lmm) ， 以 0· 1%的胰酶或 XI 型胶原酶（1 mg/ ml) 37°C消化 45 - 60 min, 完 培终止消化离心弃上清液， 以增殖培养基 （DMEM/F12 (3 : 1) 包含 0. 1%双抗， 40 g/ml fungizone, 40 ng/ml FGF2, 20 ng/ml EGF, 2% B27 supplement ) 进行 悬浮培养。 悬浮培养的细胞球可以进行传代培养已获得足够数量的皮肤干细胞 (细胞培养及鉴定结果见附图 3 ) 。
将获得的皮肤干细胞进行分化培养以获得施万细胞， 具体步骤如下： 将皮肤干 细胞以分化培养基 I (DMEM/F12 (3 : 1)包含 0. 1%双抗， 40 μδ/πι1 fungizone, 40 ng/ml FGF2, 20 ng/ml EGF, 2% B27 supplement, 10% FBS) 培养 3天后再 于分化培养基 Π (DMEM/F12 (3 : 1) 包含 0· 1%双抗， 5 Mm forskol in ， 50 ng/ml heregul in-1 β， 50 g/ml， 2% N2 supplement, 1% FBS) 中培养 2-3周后即可 获得施万细胞集落 （见附图 4. A) 。 挑取集落后扩增的可得大量皮肤干细胞分 化的施万细胞 （SKP-SC) (见附图 4. B) ， 免疫组化结果显示呈 S100阳性 （见 附图 5 )
实施例 4骨髓间充质干细胞的培养
脱臼处死成年 SD大鼠， 75%酒精浸泡 5 min， 无菌条件下取股骨、 胫骨， 暴露骨 髓腔， IMDM基础培养液冲洗骨髓腔， 收集骨髓。 注射器反复抽吸收获的骨髓， 制成单细胞悬液。 200 目筛网过滤， 置水平离心机中离心， 1000rpmX 5min， 弃 上清。 以 4 X 107 cm2的密度接种于 MDM完全培养液 （含有胎牛血清 10%) ， 置 于 37°C培养。 24 h后全量换液， 去除未贴壁细胞， 以后每 3 d半量换液。 倒置 显微镜下逐日观察细胞形态及生长情况。 当细胞铺满皿底达 90 %融合， 传代培 养 （细胞培养及鉴定结果见附图 6 ) 。
实施例 5 支持细胞的脱细胞方法建立
将支持细胞 （例如施万细胞） 培养于皿中， 当细胞铺满皿底达 90 %融合后以分 化培养基 (H-DMEM+15%FBS+50ng/ml ΗΚ6+2μΜ forskl in+5(^g/ml Vc ) 培养 2周 剌激细胞外基质分泌。 然后进行脱细胞处理， PBS清洗后去离子无菌水 37°C低 渗 10 min; 加入细胞抽提液 （含 0. 5% Triton X-100和 20 mM氨水的 PBS) 于 37°C裂解细胞 10-15 min; PBS清洗 3次后加入 Dnase I (4 mg/ml) 37°C消化 30 111 去除0 。 所得的细胞外基质如 （见附图 7. A) 所示。 通过 LN及 FN免疫 化学鉴定该基质的部分成分： 将所得的细胞外基质以 4 %多聚甲醛室温固定 30 min , 0. 01MPBS 洗涤 3 次后一抗： mouse anti laminin ( LN ) ， rabbit anti fibronectin ( FN ) 4 °C孵育过夜， PBS 洗涤 3 次后加入二抗： FITC-conjugated-goat-anti-rabbit-IgG (1： 200) 及 TRITC-con jugated-goat-anti-mouse-IgG (1： 200)，室温孵育 2h后荧光共聚焦显 微镜（DMR， Leica)检测 （见附图 7. B， C和 D) 。
实施例 6 天然的细胞外基质有利于神经元轴突的生长
将背根节神经元分别种到实施例 5 制备的含有支持细胞脱细胞后的细胞外基质 和包被有购买的细胞外基质 （货号： E0282; 公司： SIGMA) 的皿中， 同样条件培 养 48小时后， MTT法检测神经元的活力， 免疫细胞化学观察神经元轴突生长情 况， Western Blotting检测轴突生长相关因子 GAP43及神经粘附蛋白 NCAM的表 达。结果如附图 8所示， 在两种细胞外基质上神经元的细胞活力在统计学上没有 差异，但是免疫细胞化学结果显示培养于支持细胞脱细胞后的细胞外基质上的神 经元的突起明显比生长于购买的细胞外基质上的神经元的突起要长， TO 结果显 示培养于支持细胞脱细胞后的细胞外基质上的神经元的轴突生长相关因子 GAP43 及神经粘附蛋白 NCAM 的表达高于生长于购买的细胞外基质上的神经元 （附图 8) 。
实施例 7 含支持细胞的组织工程神经移植物的构建
首先进行组织工程神经移植物的组装，即将壳聚糖神经导管与丝素蛋白纤维无菌 处理后， 在每一根神经导管内置入 120根丝素蛋白单丝作为支架制成神经导管。 然后将 100ml完全培养基（DMEM+10%FBS+50ng/ml ΗΚ6+2μΜ forsklin) 通过蠕动 泵缓慢注入培养容器，再加入 2. 5 X 107个已经纯化的施万细胞及组织工程神经移 植物， 再以蠕动泵缓慢注满整个容器， 细胞最终密度为 l X 105/ml， 排尽系统内 空气后， 开始旋转微重力循环灌注培养。 旋转式生物反应器放入 37° C (¾培养 箱中， 前 24小时转速为 10转 /分钟， 使细胞与移植物充分接触， 贴附。 24小时 后调整旋转式生物反应器旋转速度， 使载体能悬浮到培养液中。继续培养 2天后 更换为分化培养基 ( H-DMEM+15%FBS+50ng/ml ΗΚ6+2μΜ forsklin+5(^g/ml Vc ) 培养， 培养期间每三天换液， 更换 500ml储液瓶。 培养 2周后终止培养将移植物 取出， 这样含支持细胞的组织工程神经移植物的构建完成。扫描电镜结果显示其 神经导管的表面及丝素蛋白支架上均匀分布了支持细胞 （见附图 9. A和 C) 。 扫 描电镜实验具体方法如下： 将组织工程神经移植物以 4%戊二醛 4° C固定后以 PBS清洗 3次； 1%饿酸室温固定 2小时后再用 PBS清洗 2次；乙醇梯度脱水（30%， 50%, 70%, 80%, 95%, 100%)各 lOmin后以无水乙醇：叔丁醇 ( 1： 1 )置换 lOmin; 再以叔丁醇置换 2次，每次 lOmin;冷冻干燥后喷铂，扫描电镜 (TCS SP2, Leica) 观察。
实施例 8 经天然细胞基质修饰的组织工程神经移植物的构建
将实施例 7 制得的构建好的培养有支持细胞的组织工程神经移植物进行脱细胞 处理， PBS清洗后去离子无菌水 37°C低渗 10 min; 加入细胞抽提液 （含 0. 5% Triton X-100和 20 mM氨水的 PBS) 于 37°C裂解细胞 10-15 min; PBS清洗 3次 后加入 Dnase I (4 mg/ml) 37°C消化 30 min去除 DNA， 扫描电镜结果显示其神 经导管的表面及丝素蛋白支架上均匀分布了支持细胞分泌的细胞外基质成分（见 附图 9. B和 D)。已制成的经天然细胞基质修饰的组织工程神经移植物可在 -80°C 保存备用。分别用扫描电镜及免疫化学方法检测脱细胞后留下的细胞外基质成分， 其扫描电镜方法同上述实施例 7， 免疫化学的具体方法同实施例 5， 结果见附图 10。
实施例 9 用天然细胞基质修饰的组织工程神经移植物修复大鼠坐骨神经缺损 用天然细胞基质修饰的组织工程神经移植物修复大鼠坐骨神经缺损，通过免疫组 化、透射电镜、 电生理、肌肉湿重比等方法检测神经再生速度及坐骨神经恢复状 况。
建立大鼠坐骨神经 10mm缺损模型并随机分为 2组： 一组用天然细胞基质修饰的 组织工程神经移植物修复大鼠坐骨神经缺损， 称为细胞基质组， 另一组用未经细 胞基质修饰的组织工程神经移植物修复大鼠坐骨神经缺损， 称为材料组。分别于 术后 1周， 2周后灌注取再生神经行冰冻切片， NF免疫组化结果显示细胞基质组 的神经生长速度快于材料组， 并且细胞基质组 NF阳性神经纤维相对密集， 分布 较均匀（图 11， A为 1周后材料组， B为 1周后细胞基质组， C为 2周后材料组， D为 2周后细胞基质组） ； 对再生神经纤维计数后统计结果如 （图 12) ； 术后 3 个月， 所有动物在适度麻醉下暴露术坐骨神经， 行神经电生理学检测。 材料组、 细胞基质组 CMAP平均波幅分别为： 4. 63 ±0. 13mV, 6. 89 ± 2. 85mV, 两组比较有 统计学差异 （P〈0. 05) ； 术后 3个月， 取再生神经中断切片行透射电镜检测， 电 镜下，细胞基质组和材料组的再生神经横切面上均可见散在分布的再生的有髓神 经纤维， 还可见少量无髓神经纤维 （图 13， A为材料组； B为细胞基质组） 。 材 料组、 细胞基质组神经髓鞘厚度分别为： 0. 63 ±0. 27 μ πι、 0. 82 ±0. 39 μ πι， 两组 之间有统计学差异（Ρ〈0. 05)。

Claims

利 要 求 书
1. 一种用于修复周围神经缺损的组织工程神经移植物， 由神经导管和细胞基质 构建而成，其特征在于所述细胞基质是自体的或同种异体来源细胞分泌形成后脱 细胞而获得。
2. 如权利要求 1所述的组织工程神经移植物，其特征在于所述细胞是施万细胞、 皮肤来源的成纤维细胞、皮肤干细胞、骨髓间充质干细胞或诱导多潜能干细胞中 的一种或几种。
3. 如权利要求 1所述的组织工程神经移植物， 其特征在于所述神经导管由生物 可降解材料构成， 所述的生物可降解材料为丝素蛋白、 壳聚糖、 胶原、 聚乳酸或 聚羟基乙酸中的一种或几种。
4. 如权利要求 1-3之一所述的组织工程神经移植物的制备方法， 其特征在于制 备含支持细胞的组织工程神经移植物之后进行脱细胞处理得到含细胞自身分泌 的细胞基质修饰的组织工程神经移植物。
5. 如权利要求 4所述的组织工程神经移植物的制备方法， 其特征在与于所述含 支持细胞的组织工程神经移植物采用三维微重力旋转培养得到。
6. 如权利要求 5所述的组织工程神经移植物的制备方法， 其特征在于含支持细 胞的组织工程神经移植物的制备及去细胞组织工程神经移植物的构建具体为：
( 1 ) 含支持细胞的组织工程神经移植物的构建： 将完全培养基缓慢注入培养容 器， 再加入 2. 5 X 107个细胞及无菌处理过的神经导管， 再将完全培养基注满整 个容器， 控制细胞最终密度为 l X 105/ml， 排尽系统内空气后， 开始旋转微重力 循环灌注培养； 旋转式生物反应器放入 37°C C02培养箱中， 前 24小时转速为 10 转 /分钟， 使细胞与神经导管充分接触， 贴附； 24小时后调整旋转式生物反应器 旋转速度， 使神经导管能悬浮到培养液中； 继续培养 2天后更换为分化培养基培 养， 以促进细胞外基质分泌； 培养 2周后终止培养将表明贴附生长有细胞的神经 导管取出；
( 2) 含细胞自身分泌的细胞基质修饰的组织工程神经移植物的构建： 将培养有 细胞的组织工程神经移植物进行脱细胞处理， PBS清洗后去离子无菌水 37 °。低 渗 10 min; 加入细胞抽提液于 37°C裂解细胞 10-15 min; PBS清洗 3次后加入 Dnase I (4 mg/ml) 37 °C消化 30 min去除 DNA， -80 °C保存备用。
7. 如权利要求 4-6之一所述的组织工程神经移植物的制备方法， 其特征在于所 述细胞是施万细胞、皮肤来源的成纤维细胞、 皮肤干细胞、骨髓间充质干细胞或 诱导多潜能干细胞中的一种或几种。
8. 如权利要求 4-6之一所述的组织工程神经移植物的制备方法， 其特征在于所 述神经导管由生物可降解材料构成，所述的生物可降解材料为丝素蛋白、壳聚糖、 胶原、 聚乳酸或聚羟基乙酸中的一种或几种。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016191073A1 (en) * 2015-05-26 2016-12-01 Mayo Foundation For Medical Education And Research Decellularized nerve allografts
CN115337458A (zh) * 2022-08-16 2022-11-15 尧舜泽生物医药(南京)有限公司 一种修复周围神经损伤的细胞基质神经移植物及其制备方法

Families Citing this family (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103041450B (zh) 2013-01-25 2015-06-10 南通大学 用于修复周围神经损伤的细胞基质修饰的组织工程神经移植物及其制备方法
CN103230623A (zh) * 2013-05-10 2013-08-07 南通大学 一种体外构建组织工程化神经的方法
CN103877623B (zh) * 2014-02-19 2016-03-02 西南大学 一种组织工程化的人工神经及其制备方法
CN103877622B (zh) * 2014-03-26 2016-04-20 中山大学 一种静电纺丝纳米纤维-细胞外基质复合材料及其制备方法和应用
CN104161771A (zh) * 2014-04-09 2014-11-26 大连医科大学 骨髓间充质干细胞来源的神经细胞作为治疗正己烷致周围性神经病的药物的应用
CN103919806A (zh) * 2014-04-09 2014-07-16 大连医科大学 骨髓间充质干细胞作为治疗正己烷致周围性神经病的药物的应用
CN106913908B (zh) * 2015-12-25 2020-05-26 北京瑞健高科生物科技有限公司 一种具有结构记忆特性的细胞生长支架
WO2018013612A1 (en) * 2016-07-11 2018-01-18 Trustees Of Tufts College Artificial brain tissue
CN106421912A (zh) * 2016-10-13 2017-02-22 中山大学 一种基质化去细胞神经支架的制备和应用
CN106474549B (zh) * 2016-11-21 2019-05-03 南通大学 MicroRNA基因介导的新型组织工程化神经的构建及其在修复神经缺损的应用
CN106421899B (zh) * 2016-12-02 2019-07-05 上海其胜生物制剂有限公司 一种盐致相变制备外周神经导管的方法
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CN107384862B (zh) * 2017-08-23 2020-08-04 北京再生生物科技研究院有限公司 MSCs来源的雪旺细胞的制备方法及试剂盒
CN110624133B (zh) * 2019-09-25 2021-08-24 重庆理工大学 一种用于神经修复的神经基质导管及其制备方法
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CN114958745A (zh) * 2022-04-28 2022-08-30 复旦大学附属儿科医院 一种适宜儿童使用的组织工程神经及其制备方法
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1589913A (zh) 2003-09-02 2005-03-09 中国人民解放军第四军医大学口腔医学院 用于修复周围神经缺损的组织工程化周围神经及制备方法
WO2005046457A2 (en) * 2003-11-05 2005-05-26 Texas Scottish Rite Hospital For Children A biomimetic biosynthetic nerve implant
CN101434934A (zh) * 2008-12-26 2009-05-20 哈尔滨工业大学 聚羟基乙酸-猪胰岛复合物的制备方法
CN101616698A (zh) * 2006-10-23 2009-12-30 库克生物科技公司 组分特性增强的处理的ecm材料
CN101664346A (zh) 2009-09-02 2010-03-10 南通大学 静电纺丝制备的人工神经移植物及其制备方法和专用装置
WO2011119804A2 (en) * 2010-03-25 2011-09-29 Lifecell Corporation Preparation of regenerative tissue scaffolds
CN102343114A (zh) * 2011-10-24 2012-02-08 南通大学 组织工程神经移植物及其用途
WO2012094611A1 (en) * 2011-01-06 2012-07-12 Humacyte Tissue-engineered constructs
CN103041450A (zh) * 2013-01-25 2013-04-17 南通大学 用于修复周围神经损伤的细胞基质修饰的组织工程神经移植物及其制备方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002014480A2 (en) * 2000-08-16 2002-02-21 Duke University Decellularized tissue engineered constructs and tissues
US7402319B2 (en) * 2002-09-27 2008-07-22 Board Of Regents, The University Of Texas System Cell-free tissue replacement for tissue engineering
US20110143429A1 (en) * 2008-04-30 2011-06-16 Iksoo Chun Tissue engineered blood vessels

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1589913A (zh) 2003-09-02 2005-03-09 中国人民解放军第四军医大学口腔医学院 用于修复周围神经缺损的组织工程化周围神经及制备方法
WO2005046457A2 (en) * 2003-11-05 2005-05-26 Texas Scottish Rite Hospital For Children A biomimetic biosynthetic nerve implant
CN101616698A (zh) * 2006-10-23 2009-12-30 库克生物科技公司 组分特性增强的处理的ecm材料
CN101434934A (zh) * 2008-12-26 2009-05-20 哈尔滨工业大学 聚羟基乙酸-猪胰岛复合物的制备方法
CN101664346A (zh) 2009-09-02 2010-03-10 南通大学 静电纺丝制备的人工神经移植物及其制备方法和专用装置
WO2011119804A2 (en) * 2010-03-25 2011-09-29 Lifecell Corporation Preparation of regenerative tissue scaffolds
WO2012094611A1 (en) * 2011-01-06 2012-07-12 Humacyte Tissue-engineered constructs
CN102343114A (zh) * 2011-10-24 2012-02-08 南通大学 组织工程神经移植物及其用途
CN103041450A (zh) * 2013-01-25 2013-04-17 南通大学 用于修复周围神经损伤的细胞基质修饰的组织工程神经移植物及其制备方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HE HONGYUN; DENG YIHAO; KE XIAOJIE ET AL.: "Morphologic Study of Bone Marrow Stroma Cells of Rat Differentiating into Schwann Cells By Acellularnerve Grafts", CHINESE JOURNAL OF NEUROANATOMY, vol. 23, no. 6, 2007
JEFFREY A BIERNASKIE; LAH A MCKENZIE; JEAN G TOMA; FREDA D MILLER: "Skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny", NATURE PROTOCOLS, 2006, pages 2803 - 2812, XP000152647
LIU JINCHAO; LIN XIAOPING; KE XIAOJIE ET AL., THE PROTECTIVE EFFECTS OF ACELLULARNERVE MATRIX ALLOGRAFTS ON THE MOTOR NEURONS OF THE ANTERIOR HORN OF SPINAL CORD PROGRESS OF ANATOMICAL SCIENCES, 2005, pages 206 - 209
YOU HUA; JIAO SHUSHENG; FENG SHUAINAN ET AL.: "The Protective Effects of Tissue Engineering Artificial Nerves on Peripheral Target Organs and Spinal Cord Neurons after Sciatic Nerve Defect", CHINESE JOURNAL OF TRAUMA, vol. 26, no. 3, 2010, pages 265 - 269

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016191073A1 (en) * 2015-05-26 2016-12-01 Mayo Foundation For Medical Education And Research Decellularized nerve allografts
CN115337458A (zh) * 2022-08-16 2022-11-15 尧舜泽生物医药(南京)有限公司 一种修复周围神经损伤的细胞基质神经移植物及其制备方法
CN115337458B (zh) * 2022-08-16 2023-08-22 尧舜泽生物医药(南京)有限公司 一种修复周围神经损伤的细胞基质神经移植物及其制备方法

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