WO2021035679A1 - 一种组织工程化神经移植物及其制备方法 - Google Patents

一种组织工程化神经移植物及其制备方法 Download PDF

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WO2021035679A1
WO2021035679A1 PCT/CN2019/103649 CN2019103649W WO2021035679A1 WO 2021035679 A1 WO2021035679 A1 WO 2021035679A1 CN 2019103649 W CN2019103649 W CN 2019103649W WO 2021035679 A1 WO2021035679 A1 WO 2021035679A1
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nerve
emscs
tissue
cells
nerve graft
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PCT/CN2019/103649
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English (en)
French (fr)
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陆乃彦
张轩
翁雨燕
余雪健
杨国锋
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江南大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • 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
    • 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

Definitions

  • the invention relates to a tissue engineered nerve graft and a preparation method thereof, and belongs to the technical field of biological materials and tissue engineering.
  • Stem cell/tissue engineering scaffold transplantation is the main strategy to repair nerve tissue damage.
  • the nerve cells of the brain are connected to each other to form a network structure.
  • the main purpose of stem cell/stent transplantation is to promote the formation of new neural networks to restore its information integration and conduction functions. Therefore, the requirements for implanted stem cells/scaffolds can promote the formation of neural networks;
  • the main function of the spinal cord is to transmit information from the brain to the motor neurons of the spinal cord through the downward conduction bundles, such as the corticospinal tract, and to receive the sensation of the spinal cord through the upward sensory conduction bundles, such as the thin bundle and the wedge bundle The information is uploaded to the brain.
  • stem cell/tissue engineering scaffold transplantation mainly promotes the regeneration of nerve conduction bundles and the parallel growth of nerve fibers and extends through the injury site to the distal end (motor nerve) or proximal end (sensory nerve) of the injury.
  • the regenerated nerve fibers grow along the original channels and eventually re-establish synaptic connections with their target cells.
  • the transplanted stem cell/tissue engineering scaffold should also have the effect of promoting the formation of myelin sheath of new nerve fibers.
  • the repair mechanism of peripheral nerve injury is similar to that of spinal cord injury.
  • Stem cell/tissue engineering scaffold (catheter) transplantation repairs peripheral nerves, such as sciatic nerve injury, and also promotes parallel and effective regeneration of nerve fibers and myelination. Therefore, how to physically and chemically modify the scaffold and choose suitable seed cells (differentiated into myelin forming cells) to plant on the scaffold to induce parallel growth and myelination of nerve fibers is to improve stem cell/tissue engineering scaffold transplantation The key factor in repairing spinal cord or peripheral nerve injury treatment effect.
  • stem cell/scaffold transplantation repairs spinal cord and peripheral nerve (such as sciatic nerve) injury mainly by making stem cells/biological materials into hydrogels or transplanting nerve conduits to the injured defect site.
  • spinal cord and peripheral nerve such as sciatic nerve
  • nerve catheter transplantation will be the most effective treatment method to repair nerve defects.
  • most of the nerve conduits currently in use can promote nerve regeneration, the lack of stripes that guide the parallel and orderly growth of nerve fibers leads to the loss of the directionality of nerve fiber extension, which makes the forward speed of fibers slow.
  • the purpose of the present invention is to provide a tissue-engineered nerve graft, which uses biomaterials with striped micro-patterns introduced on the surface as a scaffold, ectoembryonic mesenchymal stem cells EMSCs as seed cells, and seed cells are inoculated on the scaffold to obtain tissue engineering. Nerve graft.
  • the surface of the biological material is imprinted with striped micropatterns using micropatterning technology;
  • the micropatterning technology includes, but is not limited to, photolithography, electron beam exposure, or nanoimprinting
  • the stripe-shaped micropattern has a width of 1-2 ⁇ m, an interval of 1-2 ⁇ m, and a stripe height of 1-2 ⁇ m.
  • one or more of polydimethylsiloxane (PDMS), polycaprolactone (PCL), chitosan, and fibrinogen are used as the biological material.
  • the biological material includes chitosan-fibrin.
  • the chitosan-fibrin is obtained by cross-linking cell growth factors between chitosan and fibrinogen through a biological cross-linking agent, and the cell growth factors are EGF, FGE, One or more of NGF and SHH.
  • the biological crosslinking agent includes genipin or/and glutamylaminotransferase (TG).
  • the initial cell density of the EMSCs is 10 4 -10 5 cells/cm 2 .
  • the shape of the tissue engineered nerve graft includes a membranous shape.
  • the tissue-engineered nerve graft is crimped to form a single-layer or multi-layer multi-tunnel nerve conduit.
  • the tissue-engineered nerve graft is filled with drugs or growth factor slow-release materials that promote nerve growth.
  • the drug or growth factor sustained-release material that promotes nerve growth includes a drug sustained-release system with microspheres, nanoparticles, or hydrogels as carriers.
  • the tissue engineered nerve graft is used to repair nerve damage.
  • the present invention also provides a method for preparing the above-mentioned tissue engineered nerve graft, the method comprising the following steps:
  • the present invention also provides a nerve conduit, using biomaterials with striped micro-patterns introduced on the surface as a scaffold, and ectoembryonic mesenchymal stem cell EMSCs as seed cells.
  • the seed cells are seeded on the scaffold to obtain a tissue engineered nerve graft.
  • the tissue engineered nerve graft is crimped to form a single-layer or multilayer multi-tunnel nerve conduit.
  • the stripe-shaped micropattern has a width of 1-2 ⁇ m, an interval of 1-2 ⁇ m, and a stripe height of 1-2 ⁇ m.
  • one or more of polydimethylsiloxane, polycaprolactone, chitosan, and fibrinogen are used as the biological material.
  • the present invention also provides the application of the above-mentioned tissue engineered nerve graft or the above-mentioned nerve catheter in the preparation of medical devices.
  • the EMSCs/micro-patterned biomaterial film is rolled into a cylindrical multi-tunnel nerve regeneration catheter for transplantation and repair of sciatic nerve injury.
  • the results show that the sciatic nerve function index of the mouse sciatic nerve injury side without nerve catheter treatment reaches -91 ⁇ 25, and the functional index of the sciatic nerve on the injured side of the sciatic nerve of the mouse after treatment with the nerve catheter of the present application reached -37 ⁇ 17.
  • the tissue engineered nerve graft provided by the present invention can be transplanted through the injured part to promote nerve regeneration and lower limb motor function recovery, and has good clinical application prospects and research and development value.
  • Figure 1 Surface striped micro-patterned PDMS film, stripe specification is 0.5 ⁇ m ⁇ 0.5 ⁇ m ⁇ 0.5 ⁇ m (A), 1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m (B), 1.5 ⁇ m ⁇ 1.5 ⁇ m ⁇ 1.5 ⁇ m (C), 2.0 ⁇ m ⁇ 2.0 ⁇ m ⁇ 2.0 ⁇ m (D).
  • Figure 2 Fluorescence staining (S100) image of EMSCs planted on the surface of micropatterned PDMS membrane after differentiation into Schwann cells (photographed by fluorescence microscope). Stripe specification is 0.5 ⁇ m ⁇ 0.5 ⁇ m ⁇ 0.5 ⁇ m (A), 1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m (B), 1.5 ⁇ m ⁇ 1.5 ⁇ m ⁇ 1.5 ⁇ m (C), 2.0 ⁇ m ⁇ 2.0 ⁇ m ⁇ 2.0 ⁇ m (D).
  • S100 Fluorescence staining
  • Figure 3 Western blotting detection results of Schwann cell marker protein levels of EMSCs planted on the surface of the micropatterned PDMS membrane after differentiation into Schwann cells.
  • FIG 4 Neural stem cells differentiate into nerve cells on the surface of the non-striated PCL membrane, and the nerve fibers grow radially (A); ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) PCL membrane surface nerve fibers grow parallel (B); in order to display the stripes and nerve fibers at the same time, the scattered neural stem cells are planted on the stripes (1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) on the surface of the PCL membrane, nerve fibers can be seen growing along the stripes (C).
  • FIG. 5 Nerve cells grow parallel on the surface of EMSCs (Schwann cells)/striped micro-patterned PCL membrane (the bottom layer of Schwann cells is stained with the marker protein S100 immunofluorescence (fluorescein 488, green); the upper layer of nerve fibers uses the marker protein NF -200 immunofluorescence staining (cy3 red).
  • FIG. 6 Schematic diagram of the transplantation of EMSCs (Schwann cells)/striped micro-patterned membrane (catheter) in a rat animal model of sciatic nerve injury.
  • FIG. 7 EMSCs (Schwann cells)/striated (1.0) in a rat model of sciatic nerve injury ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) micro-patterned PCL composite membrane transplantation surgery.
  • A sciatic nerve cut
  • B EMSCs (Schwann cells)/striated (1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) micro-patterned PCL composite film (due to cross-linking with genipin, the film is dark blue) wraps the nerve stump (both ends are connected by absorption sutures, leaving a 5mm gap in the middle), and uses fibers Seal the broken ends with protein glue
  • C Curl the membrane into a catheter, suture it with absorbable sutures, and seal the outer surface of the catheter with fibrin glue.
  • FIG. 8 EMSCs (Schwann cells)/striated (1.0) in an animal model of rat sciatic nerve injury ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) micro-patterned fibrin/chitosan composite membrane transplantation operation process.
  • A Separate and cut off the sciatic nerve;
  • B EMSCs (Schwann cells)/striated (1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m)
  • the micro-patterned fibrin/chitosan composite film wraps the nerve stump and seals the stump with fibrin glue;
  • C The film is crimped into a catheter and sutured with absorbable sutures, then fibrin is used Glue seals the outer surface of the catheter.
  • FIG. 9 Fluorescent gold sciatic nerve injection dorsal root ganglion nerve cell tracing results
  • A-F diagrams are: normal group (A); injury untransplanted group (B); injury transplanted EMSCs (Schwann cells)/striated (1.0) ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) micro-patterned PCL composite film group (C); damage transplantation striped (1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) micro-patterned PCL composite film (cell-free) group (D); damage transplantation EMSCs ( Schwann cell)/non-stripe PCL composite membrane group (E); injury transplantation non-stripe PCL composite membrane (cell-free) group (F).
  • Figure 10 Comparison of the general appearance of the gastrocnemius muscle on the normal side of the lower limbs and the other groups on the sciatic nerve injury side.
  • the pictures A-F are: normal group (A); injury untransplanted group (B); injury transplanted EMSCs (Schwann cells)/striated (1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) micro-patterned PCL composite film group (C); damage transplantation striped (1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) micro-patterned PCL composite film (cell-free) group (D); damage transplantation EMSCs ( Schwann cell)/non-stripe PCL composite membrane group (E); injury transplantation non-stripe PCL composite membrane (cell-free) group (F).
  • Figure 11 HE staining of the gastrocnemius muscle tissue of the normal lower limb gastrocnemius muscle and other groups of the sciatic nerve injury side gastrocnemius muscle tissues to observe the cross-sectional area of gastrocnemius muscle fibers:
  • AF diagrams are: normal group (A); injury without transplantation group (B); injury transplanted EMSCs (Schwann) Cell)/Stripe (1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) micro-patterned PCL composite film group (C); damage transplantation striped (1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) micro-patterned PCL composite film (cell-free) group (D); damage transplantation EMSCs ( Schwann cell)/non-stripe PCL composite membrane group (E); injury transplantation non-stripe PCL composite membrane (cell-free) group (F).
  • Figure 12 16 weeks after the transplantation of EMSCs (Schwann cells)/stripe micro-patterned PCL composite membrane in the rat model of sciatic nerve injury, the animal was anesthetized and the sciatic nerve was taken out. The transplanted nerve conduit together with the distal and proximal nerves were taken out and fixed.
  • EMSCs Stemcell cells
  • Figure 13 HE staining observation results of sciatic nerve tissue slices at the injured site with and without nerve duct transplantation.
  • A EMSCs (Schwann cells)/stripe micro-patterned PCL composite membrane transplantation treatment group after sciatic nerve injury.
  • the sciatic nerve at the proximal end of the injury has grown Entered into the nerve catheter and passed through the catheter to the distal side, and there was no residual cavity after absorption of the absorbable suture;
  • B In the untreated group of sciatic nerve injury, the nerve fiber was severely ulcerated, and only a small amount of regenerated nerve fiber was seen (upper side of the figure). The cavity was Can absorb the residue left after the suture is absorbed.
  • Figure 14 The normal lower limb sciatic nerve and other groups of sciatic nerve injury sites (including transplanted catheters) longitudinal tissue section, nerve fiber marker protein NF-200 immunohistochemical staining to observe the state of nerve fiber regeneration: AF respectively: normal group (A ); Damaged non-transplanted group (B); Damaged transplanted EMSCs (Schwann cells)/striated (1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) micro-patterned PCL composite film group (C); damage transplantation striped (1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) micro-patterned PCL composite film (cell-free) group (D); damage transplantation EMSCs ( Schwann cell)/non-stripe PCL composite membrane group (E); injury transplantation non-stripe PCL composite membrane (cell-free) group (F).
  • A normal group
  • B Damaged non-transplanted group
  • EMSCs Damaged transplanted EMSCs (Schwann cells)/striated (1.0 ⁇ m ⁇ 1.0
  • Figure 15 The sciatic nerve of the lower limbs and other groups of the sciatic nerve injury site (catheter transplantation site) midpoint cross-sectional tissue section, nerve fiber marker protein NF-200 immunohistochemical staining to observe the density of regenerated nerve fibers: AF respectively: normal group ( A); Damaged non-transplanted group (B); Damaged transplanted EMSCs (Schwann cells)/striated (1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) micro-patterned PCL composite film group (C); damage transplantation striped (1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) micro-patterned PCL composite film (cell-free) group (D); damage transplantation EMSCs ( Schwann cell)/non-stripe PCL composite membrane group (E); injury transplantation non-stripe PCL composite membrane (cell-free) group (F).
  • A normal group
  • B Damaged non-transplanted group
  • EMSCs Damaged transplanted EMSCs (Schwann cells)
  • a polymethyl methacrylate (PMMA) film is spin-coated on the surface of a 3 ⁇ 3 cm silicon wafer, and the electron beam exposure technology is used to etch striped micro patterns with equal width, spacing and height on the surface of the PMMA film; use the above-mentioned micro-patterning
  • the base plate is a template, the base material of polydimethylsiloxane (PDMS) and the curing agent are mixed in a ratio of 10:1 and dropped on the surface of the template (0.5 mL/cm 2 ), and placed in a vacuum drying oven After drying at 60°C for 4 hours, the PDMS solidified into a film on the surface of the stencil.
  • PDMS polydimethylsiloxane
  • the PDMS film was peeled from the stencil, and the surface formed a pattern complementary to the micropattern of the stencil (see Figure 1).
  • the strips used in the experiment are of equal width, equal height and equal spacing.
  • the specifications include 0.5 ⁇ m ⁇ 0.5 ⁇ m ⁇ 0.5 ⁇ m (0.5 ⁇ m), 1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m (1.0 ⁇ m), 1.5 ⁇ m ⁇ 1.5 ⁇ m ⁇ 1.5 ⁇ m (1.5 ⁇ m), 2.0 ⁇ m ⁇ 2.0 ⁇ m ⁇ 2.0 ⁇ m (2.0 ⁇ m), 2.5 ⁇ m ⁇ 2.5 ⁇ m ⁇ 2.5 ⁇ m (2.5 ⁇ m), 3.0 ⁇ m ⁇ 3.0 ⁇ m ⁇ 3.0 ⁇ m (3.0 ⁇ m).
  • SD rats (80-100 g) were anesthetized by intraperitoneal injection of 10% chloral hydrate (330 g/kg), and their skin was disinfected. Under aseptic conditions, cut the skin and nasal bones through the nostrils and up the nasal cavity to the inner canthus. For the nasal septum mucosa, cut off 1/3 of the nasal septum and place it in PBS buffer to peel off the full-thickness nasal mucosa. After removing the nasal mucosa of SD rats, rinse with serum-free DMEM/F12 mixed medium (containing 200 U/mL penicillin and 200 U/mL streptomycin) three times to remove blood stains, and culture in DMEM/F12 containing 10% fetal bovine serum Base (i.e.
  • the 5th passage cells were seeded on a 24-well culture plate. Immunofluorescence staining was performed with the marker proteins vimentin, Nestin, CD133, CD44 and antibodies of EMSCs, respectively, and the cultured cells were identified as EMSCs.
  • the operation steps are as follows: After the cells are fixed in 4% paraformaldehyde solution, they are blocked in a mixture of 0.25% TritonX-100 and 3% bovine serum albumin (BSA) at 37°C for 30 min, incubate the primary antibody for 12 h at 4°C, rinse with PBS buffer and incubate with Cy3 labeled corresponding secondary antibody for 1 h at room temperature, rinse with PBS 3 times, Hoc Hest33342 counter-stained the nucleus, rinsed with PBS buffer, mounted with neutral glycerol, observed and photographed under a Leica fluorescence microscope, the negative control replaced the primary antibody with PBS buffer, and the rest of the steps were the same as above. The remaining cells were used in the following experiments.
  • BSA bovine serum albumin
  • the effect of stripes inducing EMSCs to differentiate into Schwann cell-like cells was used as the criterion for optimal selection of stripes.
  • the fifth-generation EMSCs identified in the above-mentioned culture were trypsinized, and the cells were collected to adjust the cell density to about 1 ⁇ 10 5 cells/mL, and planted in the micro-patterning flat in the culture plate at a planting amount of 0.5 mL/cm 2
  • the surface of the PDMS membrane (there are paraffin ridges around to limit culture medium and cell loss).
  • EMSCs/micro-patterned PDMS membrane Place the EMSCs/micro-patterned PDMS membrane in a CO 2 incubator (37°C, 5% CO 2 , saturated humidity) with DMEM/F12 containing 10% fetal bovine serum (containing 100 U/mL penicillin and 100 U/mL streptomycin) U/mL) culture. After 2h, the EMSCs were attached to the surface of the micro-patterned PDMS film. After that, add enough new DMEM/F12 medium containing 10% fetal bovine serum to the culture plate to continue the culture, and change the medium in half every three days.
  • the cell/micropatterned membrane complex was fixed with 4% paraformaldehyde solution, and immunofluorescence staining was performed with antibodies to Schwann cell marker protein S100 and MBP to observe the differentiation of EMSCs into Schwann cells on the micropatterned membrane.
  • Western blotting was used to detect the relative content of Schwann cell marker protein, compare the effects of several stripes on the differentiation of EMSCs into Schwann cells, and select the stripes with the strongest inducibility as the pattern for modifying the biomaterial scaffold.
  • DMEM/F12 medium containing 10% fetal bovine serum was used to culture EMSCs without any inducer, in order to obtain the effect of single factor inducing differentiation of stripes.
  • the biomaterial membrane with this specification stripe on the surface is planted or not planted with EMSCs as the scaffold material for making nerve catheters.
  • the immunofluorescence staining results of Schwann cell marker protein S100 and MBP showed that EMSCs were planted on the surface of striped PDMS membranes of various specifications and cultured in DMEM/F12 medium containing 10% fetal bovine serum.
  • the cell morphology was Schwann cell-like. Arrange in parallel along the stripes.
  • the morphology and staining intensity of the cells on the membrane surface are different (see Figure 2); use Western The relative content of Schwann cell marker protein was detected by blotting method, and the effect of several stripes on the differentiation of EMSCs into Schwann cells was compared, and the conclusion was 1.0 ⁇ m ⁇ 1.0
  • the stripes of ⁇ m ⁇ 1.0 ⁇ m have the strongest ability to induce EMSCs to differentiate into Schwann cells (see Table 1, Figure 3).
  • the PDMS membrane without streaks has the weakest inducing ability. Therefore, 1.0 ⁇ m ⁇ 1.0 is used in the subsequent experiments
  • the striped membrane of ⁇ m ⁇ 1.0 ⁇ m is used as the cell growth substrate and nerve conduit material.
  • a polymethyl methacrylate (PMMA) film was spin-coated on the surface of a 3 ⁇ 3 cm silicon wafer, and electron beam exposure technology was used to etch a striped micro pattern with a width of 1 ⁇ m, a spacing of 1 ⁇ m, and a height of 1 ⁇ m on the surface of the PMMA film;
  • the PDMS base material and curing agent were mixed in a ratio of 10:1 and dropped on the surface of the template, placed in a vacuum drying oven at 60°C for 4 hours, and the PDMS solidified on the surface of the template
  • peel off the PDMS film from the stencil, and its surface will form a pattern complementary to the micro-pattern of the stencil; take the micro-patterned PDMS film as the stencil, mix and drop 20% PCL dichloromethane solution on the surface of the PDMS stencil (0.5 mL/cm 2 ),
  • SD rats (80-100 g) were anesthetized by intraperitoneal injection of 10% chloral hydrate (330 g/kg), and their skin was disinfected. Under aseptic conditions, cut the skin and nasal bones through the nostrils and up the nasal cavity to the inner canthus. For the nasal septum mucosa, cut off 1/3 of the nasal septum and place it in PBS buffer to peel off the full-thickness nasal mucosa. After removing the nasal mucosa of SD rats, rinse with serum-free DMEM/F12 mixed medium (containing 200 U/mL penicillin and 200 U/mL streptomycin) three times to remove blood stains, and culture in DMEM/F12 containing 10% fetal bovine serum Base (i.e.
  • the 5th passage cells were seeded on a 24-well culture plate. Use the marker proteins vimentin, Nestin, and C of EMSCs D133, CD44 and antibody were used for immunofluorescence staining, and the cultured cells were identified as EMSCs.
  • the operation steps are as follows: After the cells are fixed in 4% paraformaldehyde solution, they are blocked in a mixture of 0.25% TritonX-100 and 3% bovine serum albumin (BSA) at 37°C for 30 minutes, and the primary antibody is incubated at 4°C for 12 h.
  • BSA bovine serum albumin
  • EMSCs are planted on the surface of the micro-patterned PCL membrane
  • the EMSCs/micropatterned membrane complex was fixed with 4% paraformaldehyde solution, and immunofluorescence staining was performed with antibodies against Schwann cell marker protein S100 and MBP to observe the growth of Schwann cells differentiated by EMSCs on the micropatterned membrane.
  • the embryos were taken out, and the size of the cerebral cortex on both sides was about 0.5 mm ⁇ 1 mm ⁇ 2 mm.
  • the pia mater was removed and put into serum-free DMEM/F12 mixed medium (containing penicillin). Wash twice in 200 U/mL and 200 U/mL streptomycin). The taken tissue is washed in PBS buffer, cut into pieces, trypsinized, and filtered through a mesh to prepare a single cell suspension.
  • the inoculation density is 2 ⁇ 10 5 pcs/mL.
  • the seeding density of the obtained stem cell pellets was 2000 cells/mL.
  • Subsequent passages were carried out by mechanical digestion every 1 to 2 weeks for multiple passages.
  • Neurospheres and differentiated cells were fixed with 4% paraformaldehyde solution for 30 min at room temperature, and then identified by immunofluorescence staining with Nestin antibody, a marker protein of neural stem cells.
  • the remaining neural stem cells were used in the following experiments to simulate the process of promoting nerve regeneration in vivo: (1) The neurospheres or scattered neural stem cells were planted on the surface of the striped (1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) PCL membrane and cultured with neural stem cell culture medium. After 21 days, immunofluorescence staining was performed with the antibody of the nerve fiber marker protein NF-200 to observe the growth of nerve fibers along the stripes (see Figure 4); (2) The neural stem cells were planted on the EMSCs/PCL micro-patterned membrane, and the nerve fibers were observed in the cell /Growth of stripe film surface.
  • Neural stem cells are planted on EMSCs/PCL micro-patterned membrane
  • the above-mentioned cultured secondary neurospheres were partially broken up and planted on the surface of the above-mentioned EMSCs/PCL composite membrane, and placed in a CO 2 incubator (37° C., 5% CO 2 , saturated humidity) and cultured with neural stem cell culture medium. After 14 days of culture, the neural stem cell/EMSCs/micropatterned membrane complex was fixed with 4% paraformaldehyde solution, and immunofluorescence double-labeled staining was performed with the antibody of neural cell/Schwann cell marker protein NF-200/MBP to observe the neural stem cells in EMSCs. /Differentiation on the micropatterned membrane and parallel growth of nerve fibers (Figure 5).
  • the first group is a simple sciatic nerve injury group; the second group is a sciatic nerve injury + transplantation of simple non-stripe PCL catheter group; the third group is a sciatic nerve injury + transplantation of EMSCs/non-stripe PCL catheter/group; the fourth group is a sciatic nerve injury + transplantation simple Striped PCL catheter group; Group 5 is the sciatic nerve injury + transplanted EMSCs/stripe PCL catheter group.
  • the animal surgery procedure is as follows: 10% chloral hydrate 400 mg/kg intraperitoneal anesthesia, a median incision on the posterior thigh, exposing the middle sciatic nerve of the right hind limb. Group 1, removal of the sciatic nerve 6
  • the muscle and skin were sutured directly after mm; the second group, the sciatic nerve defect site was transplanted with simple non-stripe PCL catheter; the third group, the sciatic nerve defect site was transplanted with EMSCs/non-stripe PCL catheter; the fourth group, the sciatic nerve defect site was transplanted with simple Striped PCL catheter group; Group 5, EMSCs/stripe PCL catheters were transplanted to the sciatic nerve defect. After the catheter is transplanted, the anastomosis is sealed with fibrin glue, and the muscle and skin are sutured (see Figure 6, 7, 8). Each group was reared routinely after operation, and the sciatic nerve index was measured regularly.
  • Sciatic nerve function index SFI 0 means normal, -100 means complete injury.
  • Table 2 shows the measurement results of the sciatic nerve function index (SFI) on the injured side of the sciatic nerve in each group 16 weeks after the animal operation.
  • Table 3 The ratio of the number of fluorescent gold-labeled positive cells in the dorsal root ganglion of each group of sciatic nerve injury to the number of cells on the normal side ( ⁇ X ⁇ SD ) Compare
  • Table 5 The ratio of the cross-sectional area of gastrocnemius muscle fiber on the side of sciatic nerve injury to that of normal side gastrocnemius muscle fiber in each group ( ⁇ X ⁇ SD ) Compare
  • the sciatic nerve was incised and exposed from the original incision after anesthesia with the same method to observe the regeneration of the sciatic nerve (Figure 12).
  • the sciatic nerve after the injury of the rat sciatic nerve is selected and the nerve catheter is transplanted and the nerve catheter is not transplanted.
  • the injured sciatic nerve includes the proximal (upper), injured (transplanted catheter site) and distal (lower) sections of the injury site.
  • the nerve channel is 4% After fixation in paraformaldehyde solution, routine paraffin embedding and sectioning were performed.
  • the slice direction is longitudinally parallel to the longitudinal axis of the nerve, passing through the proximal section (upper section), the injured section (transplant catheter site), and the distal section (lower section) in order to observe the regenerated nerve fibers passing through the catheter.
  • Tissue sections were stained with H.E ( Figure 13) and immunohistochemical stained with antibodies to the neurofiber marker protein NF-200 ( Figure 14).
  • H.E Figure 13
  • Figure 14 immunohistochemical stained with antibodies to the neurofiber marker protein NF-200
  • the image analysis system was used to measure the density of nerve fibers (the thickest section of the longitudinal section of the sciatic nerve of each group of animals was selected for comparison). The results are shown in Table 6.
  • the mixing of the two materials can increase the mechanical strength of the composite membrane, and can be cross-linked with bio-crosslinking agents such as genipin or/and glutamylaminotransferase (TG) Combined with one or more cell growth factors such as EGF, FGE, NGF, SHH, etc., to construct a slow-release drug scaffold to further improve its function of promoting nerve regeneration.
  • bio-crosslinking agents such as genipin or/and glutamylaminotransferase (TG) Combined with one or more cell growth factors such as EGF, FGE, NGF, SHH, etc.
  • TG glutamylaminotransferase
  • the present invention first selects the fibrinogen/chitosan composite film as the material for making the striped nerve conduit, and after planting or not planting EMSCs, it is used in vivo transplantation to repair the sciatic nerve injury, and the application value of the present invention is evaluated.
  • a 5% fibrinogen aqueous solution and a 2% chitosan acetic acid solution were prepared, and then the fibrinogen solution and the chitosan solution were mixed uniformly in a mass ratio of 9:1.
  • Add 50 ⁇ L (5 U) of thrombin (100 U/mL) to the micro-sprayer, and then add 50 ⁇ L of 1% genipin after 5 minutes. Put the culture plate into the drying box and solidify at 37°C. After 12 hours, the liquid solidified into a gel.
  • EMSCs are planted on the surface of micro-patterned fibrin/chitosan composite membrane
  • the present invention digests the above-mentioned EMSCs with trypsin, collects cells and adjusts the cell density to about 1 ⁇ 10 5 cells/mL, and 0.5 mL/mL.
  • the density of cm 2 is planted on the surface of the above-mentioned micro-patterned fibrin/chitosan composite membrane (there are surrounding ridges to limit culture medium and cell loss) and placed in a CO2 incubator (37°C, 5% CO 2 , saturated humidity). Contain ordinary culture medium.
  • the EMSCs/micropatterned fibrin/chitosan membrane complex was fixed with 4% paraformaldehyde solution, and immunofluorescence staining was performed with antibodies against Schwann cell marker protein S100 and MBP to observe whether the Schwann cells differentiated by EMSCs were in the microstructure.
  • the growth of patterned film was performed with antibodies against Schwann cell marker protein S100 and MBP to observe whether the Schwann cells differentiated by EMSCs were in the microstructure. The growth of patterned film.
  • the embryos were taken out, and the size of the cerebral cortex on both sides was about 0.5 mm ⁇ 1 mm ⁇ 2 mm.
  • the pia mater was removed and put into serum-free DMEM/F12 mixed medium (containing penicillin). Wash twice in 200 U/mL and 200 U/mL streptomycin). The taken tissue is washed in PBS buffer, cut into pieces, trypsinized, and filtered through a mesh to prepare a single cell suspension.
  • the inoculation density is 2 ⁇ 10 5 pcs/mL.
  • the seeding density of the obtained stem cell pellets was 2000 cells/mL.
  • Subsequent passages were carried out by mechanical digestion every 1 to 2 weeks for multiple passages.
  • Neurospheres and differentiated cells were fixed with 4% paraformaldehyde solution for 30 min at room temperature, and then identified by immunofluorescence staining with Nestin antibody, a marker protein of neural stem cells.
  • the remaining neural stem cells were used in the following experiments to simulate the process of promoting nerve regeneration in vivo: (1) Planting neurospheres or scattered neural stem cells on the surface of striped (1.0 ⁇ m ⁇ 1.0 ⁇ m ⁇ 1.0 ⁇ m) fibrin/chitosan micro-patterned membrane , Cultured with neural stem cell culture medium, 21 days later, immunofluorescence staining was carried out with an antibody against the nerve fiber marker protein NF-200 to observe the growth of nerve fibers along the stripes; (2) Neural stem cells were planted in EMSCs/fibrin/chitosan Micro-pattern membrane to observe the growth of nerve fibers on the surface of the cell/striated membrane (below).
  • Neural stem cells are planted on EMSCs/fibrin/chitosan micro-patterned membrane
  • the above-mentioned cultured secondary neurospheres were partially broken up and planted on the surface of the above-mentioned EMSCs/PCL micro-patterned membrane, and placed in a CO 2 incubator (37°C, 5% CO 2 , saturated humidity) with DMEM containing 10% fetal bovine serum /F12 (containing 100 U/mL penicillin and 100 U/mL streptomycin) culture.
  • the neural stem cell/EMSCs/fibrin/chitosan micro-patterned membrane complex was fixed with 4% paraformaldehyde solution, and immunofluorescence double-labeled staining was performed with an antibody against neural cell/Schwann cell marker protein NF-200/MBP , To observe the differentiation of neural stem cells on the EMSCs/fibrin/chitosan micropatterned membrane and the parallel growth of nerve fibers.
  • the first group is the simple sciatic nerve injury group; the second group is the sciatic nerve injury + transplantation of simple non-striated fibrin/chitosan micropattern catheter group; the third group is the sciatic nerve injury + the transplantation of EMSCs without striated fibrin/chitosan micropattern Catheter/group; the fourth group is the sciatic nerve injury + transplantation of simple striped fibrin/chitosan micro-patterned catheter group; the fifth group is the sciatic nerve injury + transplantation of EMSCs/stripe fibrin/chitosan micro-patterned catheter group.
  • the animal surgery procedure is as follows: 10% chloral hydrate 400 mg/kg intraperitoneal anesthesia, a median incision on the posterior thigh, exposing the middle sciatic nerve of the right hind limb.
  • Sciatic nerve function index SFI 0 means normal, -100 means complete injury.
  • wet weight ratio wet weight of the experimental side muscle/wet weight of the control side muscle ⁇ 100%.
  • the muscle was fixed with 4% paraformaldehyde, embedded in conventional paraffin, and the tissue sections were observed with HE staining light microscope.
  • the Leica microscopic image analysis system measured the cross-sectional area of the left and right gastrocnemius muscle fibers, and calculated the cross-sectional area ratio (cross-sectional area).
  • Area ratio cross-sectional area of experimental side muscle / cross-sectional area of control side muscle ⁇ 100%).
  • the sciatic nerve after the injury of the rat sciatic nerve is selected and the nerve catheter is transplanted and the nerve catheter is not transplanted.
  • the injured sciatic nerve includes the proximal (upper), injured (transplanted catheter site) and distal (lower) sections of the injury site.
  • the nerve channel is 4% After paraformaldehyde fixation, routine paraffin embedding and sectioning were performed.
  • the slice direction is longitudinally parallel to the longitudinal axis of the nerve, passing through the proximal section (upper section), the injured section (transplant catheter site), and the distal section (lower section) in order to observe the regenerated nerve fibers passing through the catheter.
  • the tissue sections were stained with H.E and immunohistochemical staining with antibodies to the neurofiber marker protein NF-200. Observe and take pictures under a microscope, and measure the density of nerve fibers with an image analysis system (select the thickest part of the longitudinal section of the sciatic nerve in each group for comparison).
  • tissue-engineered nerve graft provided by the present invention can be transplanted through the injured site to promote nerve regeneration and lower limb motor function recovery.

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Abstract

本发明公开了一种组织工程化神经移植物及其制备方法，属于生物材料和组织工程技术领域。本发明通过优化条纹的规格，使条纹能够最大化地独立诱导EMSCs分化为髓鞘形成细胞（雪旺细胞），得到EMSCs/生物材料支架复合物。这种EMSCs/生物材料支架复合物既可作为体外研究神经干细胞分化、神经纤维生长和髓鞘形成分子机制的三维细胞培养模型又可用作为组织工程移植物用于体内移植修复神经系统损伤。本发明将EMSCs/微图案化生物材料膜卷成圆筒状多隧道式的神经再生导管用于移植修复坐骨神经损伤，结果表明本发明能通过损伤部位移植，促进神经再生和下肢运动功能恢复，具有良好的临床应用前景和研究开发价值。

Description

一种组织工程化神经移植物及其制备方法 技术领域
本发明涉及一种组织工程化神经移植物及其制备方法，属于生物材料和组织工程技术领域。
 背景技术
干细胞/组织工程支架移植是修复神经组织损伤的主要策略。在中枢神经系统，大脑的神经细胞通过突起的相互连接形成网络结构。脑组织损伤后，干细胞/支架移植的主要目的是促进新的神经网络形成以恢复其信息整合和传导功能，因此对植入的干细胞/支架的要求是可促进神经网络的形成；与脑组织的信息整合功能相比，脊髓的主要功能是通过下行传导束，如皮质脊髓束将大脑的信息传导到脊髓的运动神经元，并通过上行感觉传导束，如薄束和楔束将脊髓接受的感觉信息上传到大脑。因此，脊髓损伤后，干细胞/组织工程支架移植主要是促进神经传导束的再生和神经纤维的平行性生长并顺向通过损伤部位定向延伸至损伤远端（运动神经）或近端（感觉神经）的脊髓组织，再生的神经纤维沿着原有的通道生长最终与其靶细胞重新建立突触连接。与此同时，所移植的干细胞/组织工程支架还应具有促进新生神经纤维形成髓鞘的作用。周围神经损伤的修复机制与脊髓损伤的修复机制相类似，干细胞/组织工程支架（导管）移植修复外周神经，如坐骨神经损伤，亦促进神经纤维平行性有效再生和髓鞘形成。因此，如何对支架进行物理和化学修饰并选择合适的种子细胞（可分化为髓鞘形成细胞）种植于支架，以诱导神经纤维的平行性生长和髓鞘形成，是提高干细胞/组织工程支架移植修复脊髓或外周神经损伤治疗效果的关键因素。
目前干细胞/支架移植修复脊髓和周围神经（如坐骨神经）损伤主要是将干细胞/生物材料制作成水凝胶或神经导管移植到损伤缺损部位。尤其是当创伤造成外周神经较长缺损时，神经导管移植将是修复神经缺损的最为有效的治疗方法。目前应用的大多数神经导管虽然可促进神经再生，但由于缺少引导神经纤维平行有序生长的条纹，导致神经纤维延伸的方向性迷失，使得纤维前行速度缓慢，同时由于缺少髓鞘形成细胞，使得再生神经纤维的髓鞘化不全（仅仅依靠神经断端残留的内源性雪旺细胞增殖后形成髓鞘）。因此，亟需发明研制一种既能够明显促进再生神经纤维平行性定向生长又能提供髓鞘形成种子细胞，促进体内损伤神经再生和髓鞘形成的神经导管，以供移植，更有效地修复神经损伤。
 技术解决方案
本发明的目的是提供一种组织工程化神经移植物，以表面引入条纹状微图案的生物材料作为支架，以外胚间充质干细胞EMSCs作为种子细胞，将种子细胞接种到支架上得到组织工程化神经移植物。
在本发明的一种实施方式中，所述生物材料表面利用微图案技术印刻条纹状微图案；所述微图案技术包括但不限于光刻、电子束曝光或纳米压印
在本发明的一种实施方式中，所述条纹状微图案的宽度为1-2 μm，间距为1-2 μm，条纹高度为1-2 μm。
在本发明的一种实施方式中，以聚二甲基硅氧烷（PDMS）、聚己内酯（PCL）、壳聚糖、纤维蛋白原中的一种或多种作为生物材料。
在本发明的一种实施方式中，所述生物材料包括壳聚糖-纤维蛋白。
在本发明的一种实施方式中，所述壳聚糖-纤维蛋白是将壳聚糖和纤维蛋白原通过生物交联剂交联细胞生长因子获得的，所述细胞生长因子为EGF、FGE、NGF、SHH中的一种或多种。
在本发明的一种实施方式中，生物交联剂包括京尼平或/和谷氨酰氨转氨酶（TG）。
在本发明的一种实施方式中，所述EMSCs的初始细胞密度为10 4-10 5个/cm 2。
在本发明的一种实施方式中，所述组织工程化神经移植物的形状包括膜状。
在本发明的一种实施方式中，所述组织工程化神经移植物卷曲形成单层或多层多隧道神经导管。
在本发明的一种实施方式中，所述组织工程化神经移植物填充有促进神经生长的药物或生长因子缓释材料。
在本发明的一种实施方式中，促进神经生长的药物或生长因子缓释材料包括微球、纳米颗粒或水凝胶为载体的药物缓释系统。
在本发明的一种实施方式中，所述组织工程化神经移植物用于修复神经损伤。
本发明还提供了制备上述组织工程化神经移植物的方法，所述方法包括以下步骤：
（1）制备表面微图案化的生物材料支架；EMSCs的取材培养和扩增：
（2）将步骤（1）得到的EMSCs种植于微图案化的生物材料支架。
本发明还提供了一种神经导管，以表面引入条纹状微图案的生物材料作为支架，以外胚间充质干细胞EMSCs作为种子细胞，将种子细胞接种到支架上得到组织工程化神经移植物，所述组织工程化神经移植物卷曲形成单层或多层多隧道神经导管。
在本发明的一种实施方式中，所述条纹状微图案的宽度为1-2 μm，间距为1-2 μm，条纹高度为1-2 μm。
在本发明的一种实施方式中，以聚二甲基硅氧烷、聚己内酯、壳聚糖、纤维蛋白原中的一种或多种作为生物材料。
本发明还提供了上述组织工程化神经移植物或上述神经导管在制备医疗器械中的应用。
 有益效果
本发明将EMSCs/微图案化生物材料膜卷成圆筒状多隧道式的神经再生导管用于移植修复坐骨神经损伤，结果表明，无神经导管处理的小鼠坐骨神经损伤侧坐骨神经功能指数达-91±25，而使用本申请的神经导管处理后小鼠坐骨神经损伤侧坐骨神经功能指数达-37±17。本发明提供的组织工程化神经移植物能通过损伤部位移植，促进神经再生和下肢运动功能恢复，具有良好的临床应用前景和研究开发价值。
 附图说明
图1：表面条纹化微图案PDMS膜，条纹规格为0.5 μm×0.5 μm×0.5 μm（A），1.0 μm×1.0 μm×1.0 μm（B），1.5 μm×1.5 μm×1.5 μm（C），2.0 μm×2.0 μm×2.0 μm（D）。
图2：种植于微图案化的PDMS膜表面的EMSCs向雪旺细胞分化后的荧光染色（S100）图（荧光显微镜拍摄）。条纹规格为0.5 μm×0.5 μm×0.5 μm（A），1.0 μm×1.0 μm×1.0 μm（B），1.5 μm×1.5 μm×1.5 μm（C），2.0 μm×2.0 μm×2.0 μm（D）。
图3：种植于微图案化的PDMS膜表面的EMSCs向雪旺细胞分化后细胞表达雪旺细胞标志蛋白水平的Western blotting检测结果。
图4：神经干细胞在无条纹PCL膜表面分化为神经细胞，神经纤维呈放射状生长（A）；在条纹化（1.0 μm×1.0 μm×1.0 μm）PCL膜表面神经纤维平行生长（B）；为了同时显示条纹和神经纤维，将分散的神经干细胞种植于条纹（1.0 μm×1.0 μm×1.0 μm）PCL膜表面，可见神经纤维沿条纹生长（C）。
图5：神经细胞在EMSCs（雪旺细胞）/条纹化微图案PCL膜表面平行生长（底层雪旺细胞用标志蛋白S100的免疫荧光染色（荧光素488，绿色）；上层神经纤维用标志蛋白NF-200的免疫荧光染色（cy3红色）。A：条纹1.0 μm×1.0 μm×1.0 μm；B：条纹2.0 μm×2.0 μm×2.0 μm。
图6：坐骨神经损伤大鼠动物模型的EMSCs（雪旺细胞）/条纹化微图案膜（导管）移植手术示意图。
图7：坐骨神经损伤大鼠动物模型的EMSCs（雪旺细胞）/条纹化（1.0 μm×1.0 μm×1.0 μm）微图案PCL复合膜移植手术过程。A：坐骨神经切断；B：EMSCs（雪旺细胞）/条纹化（1.0 μm×1.0 μm×1.0 μm）微图案PCL复合膜（由于用京尼平交联，膜呈深蓝色）包裹神经断端（两端用吸收缝线连接对位，中间留存5mm间隙），并用纤维蛋白胶封固断端；C：将膜卷曲成导管，并用可吸收缝线缝合后用纤维蛋白胶封固导管外表面。
图8：大鼠坐骨神经损伤动物模型的EMSCs（雪旺细胞）/条纹化（1.0 μm×1.0 μm×1.0 μm）微图案纤维蛋白/壳聚糖复合膜移植手术过程。A：分离并切断坐骨神经；B：EMSCs（雪旺细胞）/条纹化（1.0 μm×1.0 μm×1.0 μm）微图案纤维蛋白/壳聚糖复合膜包裹神经断端，并用纤维蛋白胶封固断端；C：将膜卷曲成导管，并用可吸收缝线缝合后用纤维蛋白胶封固导管外表面。
图9：荧光金坐骨神经注射背根神经节神经细胞示踪结果，A-F图分别为：正常组（A）；损伤未移植组（B）；损伤移植EMSCs（雪旺细胞）/条纹化（1.0 μm×1.0 μm×1.0 μm）微图案PCL复合膜组（C）；损伤移植条纹化（1.0 μm×1.0 μm×1.0 μm）微图案PCL复合膜（无细胞）组（D）；损伤移植EMSCs（雪旺细胞）/无条纹PCL复合膜组（E）；损伤移植无条纹PCL复合膜（无细胞）组（F）。
图10：正常侧下肢腓肠肌和其他各组坐骨神经损伤侧腓肠肌大体外观比较。A-F图分别为：正常组（A）；损伤未移植组（B）；损伤移植EMSCs（雪旺细胞）/条纹化（1.0 μm×1.0 μm×1.0 μm）微图案PCL复合膜组（C）；损伤移植条纹化（1.0 μm×1.0 μm×1.0 μm）微图案PCL复合膜（无细胞）组（D）；损伤移植EMSCs（雪旺细胞）/无条纹PCL复合膜组（E）；损伤移植无条纹PCL复合膜（无细胞）组（F）。
图11：正常侧下肢腓肠肌和其他各组坐骨神经损伤侧腓肠肌组织切HE染色观察腓肠肌纤维截面积：A-F图分别为：正常组（A）；损伤未移植组（B）；损伤移植EMSCs（雪旺细胞）/条纹化（1.0 μm×1.0 μm×1.0 μm）微图案PCL复合膜组（C）；损伤移植条纹化（1.0 μm×1.0 μm×1.0 μm）微图案PCL复合膜（无细胞）组（D）；损伤移植EMSCs（雪旺细胞）/无条纹PCL复合膜组（E）；损伤移植无条纹PCL复合膜（无细胞）组（F）。
图12：大鼠坐骨神经损伤动物模型的EMSCs（雪旺细胞）/条纹化微图案PCL复合膜移植手术后16周，动物麻醉后进行坐骨神经取材，将移植的神经导管连同远近端的神经取出固定，沿纵轴剖开导管（原来的导管壁已经被体内的组织改建），观察导管内神经生长情况，A：EMSCs（雪旺细胞）/条纹化（1.0 μm×1.0 μm×1.0 μm）微图案PCL复合膜移植组；B：条纹化（1.0 μm×1.0 μm×1.0 μm）微图案PCL复合膜移植组（无细胞）；C：EMSCs（雪旺细胞）/无条纹PCL复合膜移植组；D：无条纹PCL复合膜移植组（无细胞）。
图13：移植和不移植神经导管的损伤部位坐骨神经组织切片HE染色观测结果，A：坐骨神经损伤后EMSCs（雪旺细胞）/条纹化微图案PCL复合膜移植治疗组，损伤近端的坐骨神经已生长入神经导管内并通过导管达到远侧，可吸收缝线吸收后未见残留空洞；B：坐骨神经损伤未治疗组，可见神经纤维严重溃变，仅见少量再生神经纤维（图上侧），空洞为可吸收缝线吸收后所残留。
图14：正常侧下肢坐骨神经和其他各组坐骨神经损伤部位（包括移植的导管）纵向组织切，神经纤维标志蛋白NF-200免疫组织化学染色观察神经纤维再生情况：A-F图分别为：正常组（A）；损伤未移植组（B）；损伤移植EMSCs（雪旺细胞）/条纹化（1.0 μm×1.0 μm×1.0 μm）微图案PCL复合膜组（C）；损伤移植条纹化（1.0 μm×1.0 μm×1.0 μm）微图案PCL复合膜（无细胞）组（D）；损伤移植EMSCs（雪旺细胞）/无条纹PCL复合膜组（E）；损伤移植无条纹PCL复合膜（无细胞）组（F）。
图15：下肢坐骨神经和其他各组坐骨神经损伤部位（导管移植部位）中点横断面组织切片，神经纤维标志蛋白NF-200免疫组织化学染色观察再生神经纤维的密度：A-F图分别为：正常组（A）；损伤未移植组（B）；损伤移植EMSCs（雪旺细胞）/条纹化（1.0 μm×1.0 μm×1.0 μm）微图案PCL复合膜组（C）；损伤移植条纹化（1.0 μm×1.0 μm×1.0 μm）微图案PCL复合膜（无细胞）组（D）；损伤移植EMSCs（雪旺细胞）/无条纹PCL复合膜组（E）；损伤移植无条纹PCL复合膜（无细胞）组（F）。
 本发明的实施方式
实施例1 条纹的优化选择
1. 材料表面微图案制作
采用电子束曝光及纳米压印技术。首先在3×3 cm的硅片表面旋涂聚甲基丙烯酸甲酯（PMMA）薄膜，应用电子束曝光技术在PMMA膜表面蚀刻宽度、间距和高度相等的条纹型微图案；以上述微图案化的基片为模版，将聚二甲基硅氧烷（PDMS）的基料与固化剂按照10:1的比例混合并滴加在模版表面（0.5 mL/cm 2），置于真空干燥箱内60℃干燥4 h，PDMS在模版表面凝固成膜，将PDMS膜从模版上剥离，其表面即形成与模版微图案互补的图案（见图1）。实验用条纹等宽、等高、等间距，规格包括0.5 μm×0.5 μm×0.5 μm（0.5 μm），1.0 μm×1.0 μm×1.0 μm（1.0 μm），1.5 μm×1.5 μm×1.5 μm（1.5 μm），2.0 μm×2.0 μm×2.0 μm（2.0 μm），2.5 μm×2.5 μm×2.5 μm（2.5 μm），3.0 μm×3.0 μm×3.0 μm（3.0 μm）。
2. EMSCs的取材、培养、扩增和鉴定
SD大鼠（80-100 g）经10%水合氯醛（330 g/kg）腹腔注射麻醉，全身皮肤消毒，在无菌条件下经鼻孔沿鼻腔向上至内眦部剪开皮肤及鼻骨，暴露鼻中隔黏膜，剪取下1/3鼻中隔置PBS缓冲液中，剥离全层鼻黏膜。取出SD大鼠鼻黏膜后用无血清DMEM/F12混合培养基（含青霉素200 U/mL和链霉素200 U/mL）漂洗三次去除血迹，置于含10%胎牛血清的DMEM/F12培养基（即普通完全培养基，含青霉素100 U/mL和链霉素100 U/mL），用眼科剪充分剪碎，37℃下0.25%胰酶消化15min，离心弃上清后用将细胞和小组织块接种于Corning培养瓶，于CO 2培养箱内（37℃，5%CO 2，饱和湿度）培养。在细胞培养3天后补充新的含10%胎牛血清的DMEM/F12培养基。以后每三天半量换液一次，当细胞铺满瓶底时进行消化、传代。
将第5代细胞接种于24孔培养板。分别用EMSCs的标志蛋白vimentin、Nestin、CD133、CD44和抗体进行免疫荧光染色，鉴定培养得到的细胞为EMSCs。操作步骤如下：细胞经4%多聚甲醛溶液固定后，37℃于0.25%TritonX-100和3%牛血清白蛋白（BSA）混合液中封闭30 min，第一抗体4℃孵育12 h，PBS缓冲液漂洗后用Cy3标记的相应二抗室温下孵育1 h，PBS漂洗3遍，Hoc hest33342复染细胞核，PBS缓冲液漂洗，中性甘油封片，于Leica荧光显微镜下观察并摄片，阴性对照用PBS缓冲液代替第一抗体，其余步骤同上。其余细胞用于以下实验。
3. 膜表面条纹的优化选择
以条纹诱导EMSCs向雪旺细胞样细胞诱导分化的效果作为条纹优化选择的标准。将上述培养鉴定的第5代EMSCs用胰酶消化，收集细胞调整细胞密度约为1×10 5个/mL，以0.5 mL/cm 2的种植量种植于平铺在培养板内的微图案化的PDMS膜表面（四周有石蜡围埂限制培养基和细胞流失）。将EMSCs/微图案化PDMS膜置于CO 2培养箱内（37℃，5%CO 2，饱和湿度）用含10%胎牛血清的DMEM/F12（含青霉素100 U/mL和链霉素100 U/mL）培养。2h 后EMSCs既贴附于微图案化PDMS膜表面。此后在培养板内加足新的含10%胎牛血清的DMEM/F12培养基继续培养，每三天半量换液一次。14天后用4%多聚甲醛溶液固定细胞/微图案膜复合物，用雪旺细胞标志蛋白S100和MBP的抗体进行免疫荧光染色，观察EMSCs在微图案膜上向雪旺细胞分化的情况。用Western blotting 方法检测雪旺细胞标志蛋白的相对含量，比较几种条纹对EMSCs向雪旺细胞分化效果，选择诱导能力最强的条纹作为修饰生物材料支架的图案。在上述试验过程中，只用含10%胎牛血清的DMEM/F12培养基培养EMSCs，不加任何诱导剂，以获得条纹单因素的诱导分化效果。以表面具有该规格条纹的生物材料膜种植或不种植EMSCs作为制作神经导管的支架材料。
4.结果分析
雪旺细胞标志蛋白S100和MBP的免疫荧光染色结果表明，EMSCs种植于各种规格条纹化PDMS膜表面，用含10%胎牛血清的DMEM/F12培养基培养，细胞形态为雪旺细胞样，沿条纹平行排列。不同规格条纹，其膜表面的细胞的形态和染色强度存在差异（见图2）；用Western blotting方法检测雪旺细胞标志蛋白的相对含量，比较几种条纹对EMSCs向雪旺细胞分化效果，得出的结论是1.0 μm×1.0 μm×1.0 μm的条纹诱导EMSCs向雪旺细胞分化的能力最强（见表1，图3）。无条纹的PDMS膜诱导能力最弱。因此，在后面的试验中均采用1.0 μm×1.0 μm×1.0 μm的条纹膜作为细胞生长基底和神经导管材料。
表1 条纹诱导EMSCs表达雪旺细胞标志蛋白的相对含量（与Actin的比值）比较（`X±SD）
Figure dest_path_image001
说明：1 μm条纹组的雪旺细胞标志蛋白MBP和S100的相对含量均明显高于其他各组（ p<0.05，n=3）
实施例2微图案化PCL膜的实际应用
1. 制备表面微图案化的PCL膜
采用电子束曝光及纳米压印技术。首先在3×3 cm的硅片表面旋涂聚甲基丙烯酸甲酯（PMMA）薄膜，应用电子束曝光技术在PMMA膜表面蚀刻宽度1 μm、间距1 μm、高度1 μm的条纹型微图案；以上述微图案化的基片为模版，将PDMS的基料与固化剂按照10:1的比例混合并滴加在模版表面，置于真空干燥箱内60℃干燥4 h，PDMS在模版表面凝固成膜，将PDMS膜从模版上剥离，其表面即形成与模版微图案互补的图案；以上述微图案化的PDMS膜为模版，将20%的PCL二氯甲烷溶液混合滴加在PDMS模版表面（0.5 mL/cm 2），置于真空干燥箱内干燥1 h，PCL在PDMS模版表面凝固成膜，将PCL膜从模版上剥离，其表面即形成与模版微图案互补的图案。
2. EMSCs的取材培养和扩增和鉴定
SD大鼠（80-100 g）经10%水合氯醛（330 g/kg）腹腔注射麻醉，全身皮肤消毒，在无菌条件下经鼻孔沿鼻腔向上至内眦部剪开皮肤及鼻骨，暴露鼻中隔黏膜，剪取下1/3鼻中隔置PBS缓冲液中，剥离全层鼻黏膜。取出SD大鼠鼻黏膜后用无血清DMEM/F12混合培养基（含青霉素200 U/mL和链霉素200 U/mL）漂洗三次去除血迹，置于含10%胎牛血清的DMEM/F12培养基（即普通完全培养基，含青霉素100 U/mL和链霉素100 U/mL），用眼科剪充分剪碎，37℃下0.25%胰酶消化15min，离心弃上清后用将细胞和小组织块接种于Corning培养瓶，于CO 2培养箱内（37℃，5%CO 2，饱和湿度）培养。在细胞培养3天后补充新的含10%胎牛血清的DMEM/F12培养基。以后每三天半量换液一次，当细胞铺满瓶底时进行消化、传代。
将第5代细胞接种于24孔培养板。分别用EMSCs的标志蛋白vimentin、Nestin、C D133和CD44和抗体进行免疫荧光染色，鉴定培养得到的细胞为EMSCs。操作步骤如下：细胞经4%多聚甲醛溶液固定后，37℃于0.25%TritonX-100和3%牛血清白蛋白（BSA）混合液中封闭30min，第一抗体4℃孵育12 h，PBS缓冲液漂洗后用Cy3标记的相应二抗室温下孵育1 h，PBS缓冲液漂洗3遍，Hoc hest33342复染细胞核，PBS缓冲液漂洗，中性甘油封片，于Leica荧光显微镜下观察并摄片，阴性对照用PBS缓冲液代替第一抗体，其余步骤同上。其余细胞用于以下实验。
3. EMSCs种植于微图案化的PCL膜表面
将上述EMSCs用胰酶消化，收集细胞调整细胞密度约为1×10 5个/mL，以0.5 mL/cm 2的密度种植于上述微图案化的PCL膜上（四周有石蜡围埂限制培养基和细胞流失），置CO 2培养箱内（37℃，5%CO 2，饱和湿度）用含10%胎牛血清的DMEM/F12培养基培养，每三天换培养液一次。培养14天后用4%多聚甲醛溶液固定EMSCs/微图案膜复合物，用雪旺细胞标志蛋白S100和MBP的抗体进行免疫荧光染色，观察EMSCs分化的雪旺细胞在微图案膜的生长情况。
4. 大鼠胚胎神经干细胞的培养
将孕14-16 d SD大鼠麻醉以后取出胚胎，取两侧大脑皮层组织大小约0.5 mm×1 mm×2 mm，将软脑膜剔除干净，放入无血清DMEM/F12混合培养基（含青霉素200 U/mL和链霉素200 U/mL）中清洗两遍。将所取的组织在PBS缓冲液中洗涤、剪碎、胰酶消化、筛网过滤，制得单细胞悬液。接种在神经干细胞培养基（DMEM／F12培养基中添加2% B 27 mL、20 ng/mL bFGF、20 ng/mL EGF、青霉素和链霉素各100 U/mL），接种密度是2×10 5个/mL。为了确保神经千细胞球的增殖。得到的干细胞球接种密度为2000个/mL。随后采用机械消化法每隔1~2周传代一次，进行多次传代。神经球及分化细胞以4％多聚甲醛溶液室温下固定30 min，用神经干细胞标志蛋白Nestin抗体进行免疫荧光染色鉴定。其余神经干细胞用于以下实验，模拟体内促进神经再生过程：（1）将神经球或分散的神经干细胞种植于条纹化（1.0 μm×1.0 μm×1.0 μm）PCL膜表面，用神经干细胞培养基培养21天后用神经纤维标志蛋白NF-200的抗体进行免疫荧光染色观察神经纤维沿条纹生长的情况（见图4）；（2）将神经干细胞种植于EMSCs/PCL微图案膜，观察神经纤维在细胞/条纹膜表面生长情况。
5.神经干细胞种植于EMSCs/PCL微图案膜
将上述培养的次级神经球部分打散后种植于上述EMSCs/PCL复合膜表面，置CO 2培养箱内（37℃，5%CO 2，饱和湿度）用神经干细胞培养基培养。培养14天后用4%多聚甲醛溶液固定神经干细胞/EMSCs/微图案膜复合物，用神经细胞/雪旺细胞标志蛋白NF-200/MBP的抗体进行免疫荧光双标记染色，观察神经干细胞在EMSCs/微图案膜上分化以及神经纤维平行生长情况（图5）。
6.体内移植试验
（1）实验动物及移植手术过程
健康成年雄性SD大鼠50只，体重250~300 g，将其随机分为5组，每组10只。第1组为单纯坐骨神经损伤组；第2组为坐骨神经损伤+移植单纯无条纹PCL导管组；第3组为坐骨神经损伤+移植EMSCs/无条纹PCL导管/组；第4组为坐骨神经损伤+移植单纯有条纹PCL导管组；第5组为坐骨神经损伤+移植EMSCs/有条纹PCL导管组。
动物手术过程如下：10%水合氯醛400 mg/kg腹腔麻醉，股后部正中切口，暴露右后肢中段坐骨神经。第1组，切除坐骨神经6 mm后直接缝合肌肉和皮肤；第2组，在坐骨神经缺损部位移植单纯无条纹PCL导管；第3组，在坐骨神经缺损部位移植EMSCs/无条纹PCL导管；第4组，在坐骨神经缺损部位移植单纯有条纹PCL导管组；第5组，在坐骨神经缺损部位移植EMSCs/有条纹PCL导管。导管移植后用纤维蛋白胶封固吻合口，缝合肌肉和皮肤（见图6，7，8）。术后各组常规饲养，定期测量坐骨神经指数。
（2）导管移植修复神经损伤效果评价指标
①一般情况观察及坐骨神经功能指数（SFI）测定
术后常规观察大鼠饮食、足部溃疡、肢体活动及切口愈合情况。每周测定坐骨神经功能指数（SFI）：制作一长60 cm、宽10 cm、高20 cm的两端开口木槽，将70 g/m 2白纸裁成与木槽等长等宽后铺于槽底。大鼠双侧后肢用颜料浸于双踝关节着色后，将大鼠放于槽的一端，使其自行向槽的另一方行走，每侧后肢各留下5～6个足印。选择印迹清晰的足印分别测量正常足（N）和伤侧足（E）的3个指标：A：PL（足印长度）；B：TS（足趾宽度）；C：IT（中间足趾宽度）。将上述指数代入Bain公式，计算出坐骨神经功能指数。
Bain公式：SFI=109.5（ETS-NTS）/NTS-38.3（EPL—NPL）/NPL+13.3（EIT—NIT）/NIT-8.8。
坐骨神经功能指数SFI=0为正常，-100为完全损伤。动物手术16周后各组坐骨神经损伤侧坐骨神经功能指数（SFI）测量结果见表2。
表2各组坐骨神经损伤侧坐骨神经功能指数（SFI）（`X±SD ）比较
第1组 第2组 第3组 第4组 第5组
-91±25 -77±31 -68±19 -57±23 -37±17
说明：第5组的损伤侧坐骨神经功能指数均明显高于其他各组（ p<0.05，n=9）
②荧光金逆行示踪
每组随机挑选3只大鼠于术后15周（观察终点前1周）进行荧光金逆行示踪。麻醉后再次暴露坐骨神经，于移植物远端5 mm处用微量注射器注射5%荧光金-磷酸盐缓冲液（PBS）溶液2 μL。正常侧坐骨神经相应位置同样注射同等量的荧光金。缝合手术切口，动物继续饲养。1周后取出左右侧相对应的L4-L6，S1-S2背根神经节，用冰冻切片机进行纵向切片，厚度为10 μm。在荧光显微镜下分别于观察10张连续切片（由于神经节很小，可以在一个低倍视野内观察全貌），用Image-proPlus6.0计数每张切片内荧光金标记的阳性细胞总数，并计算平均值。双侧标记阳性细胞比（阳性细胞比=实验侧标记阳性细胞数/对照侧阳性细胞数×100%）以阳性细胞比反映神经再生程度（阳性细胞总数与导管移植修复效果成正相关，结果见表3，图9）。
表3各组坐骨神经损伤侧背根神经节荧光金标记阳性细胞数与正常侧细胞数比值（`X±SD ）比较
Figure dest_path_image002
说明：第5组的损伤侧神经节阳性细胞数与正常侧比值均明显高于其他各组（ p<0.05，n=9）
③腓肠肌的湿重计量和形态学观察
动物手术16周后，麻醉动物，完整切取双侧腓肠肌，电子天平称重（精确至0.001g），计算各组动物双侧腓肠肌湿重比（湿重比=实验侧肌肉湿重/对照侧肌肉湿重×100%），结果见表4。称重后将肌肉用4%多聚甲醛溶液固定，常规石蜡包埋，组织切片分别作 H-E染色光镜观察，Leica显微图像分析系统分别测量左右侧腓肠肌纤维截面积，并计算截面积比（截面积比=实验侧肌肉截面积/对照侧肌肉截面积×100%），结果见表5，图10，图11。
表4各组坐骨神经损伤侧腓肠肌湿重与正常侧腓肠肌湿重比值（`X±SD ）比较
Figure dest_path_image003
说明：第5组的腓肠肌湿重比值均明显高于其他各组（ p<0.05，n=9）
表5各组坐骨神经损伤侧腓肠肌纤维截面积与正常侧腓肠肌纤维截面积比值（`X±SD ）比较
Figure dest_path_image004
说明：第5组的腓肠肌纤维截面积比值均明显高于其他各组（ p<0.05，n=9）
④坐骨神经的形态学观察和计量
动物手术16周后，同上法麻醉后自原切口切开并暴露坐骨神经，观察坐骨神经再生情况（图12）。选取大鼠坐骨神经损伤后移植神经导管和未移植神经导管的，损伤修复后的坐骨神经，包括损伤部位的近段（上段）、损伤段（移植导管部位）和远段（下段），神经经4%多聚甲醛溶液固定后进行常规石蜡包埋和切片。切片方向为纵向平行与神经纵轴，通过近段（上段）、损伤段（移植导管部位）和远段（下段），以便观察再生神经纤维通过导管的情况。组织切片分别作 H.E染色（图13）、用神经纤维标志蛋白NF-200的抗体进行免疫组织化学染色（图14）。正常坐骨神经和其他各组坐骨神经损伤部位（导管移植部位）中点横断面组织切片，神经纤维标志蛋白NF-200免疫组织化学染色观察再生神经纤维的密度（图15）。显微镜观察并采图后，用图像分析系统进行神经纤维密度测定（选取各组动物坐骨神经纵向切面的最粗切面进行比较），结果见表6。
表6各组坐骨神经损伤侧再生神经纤维横断面数量与正常侧横断面数量比值（`X±SD）比较
Figure dest_path_image005
说明：第5组的损伤侧神经纤维数量与正常侧的比值均明显高于其他各组（ p<0.05，n=9）
实施例3微图案化的纤维蛋白/壳聚糖复合膜的实际应用
1. 制备表面微图案化的纤维蛋白/壳聚糖复合膜
基于纤维蛋白原和壳聚糖具有生物相容性好，两种材料混合可增加复合膜的机械强度，并可借助生物交联剂如京尼平或/和谷氨酰氨转氨酶（TG）交联一种或多种细胞生长因子如EGF、FGE、NGF、SHH等，以构建成药物缓释放支架，进一步提高其促进神经再生的功能。本发明首先选择纤维蛋白原/壳聚糖复合膜作为制作条纹化神经导管的材料，种植或不种植EMSCs后用于体内移植修复坐骨神经损伤，评价本发明的应用价值。条纹化纤维蛋白原溶液/壳聚糖复合膜的制作过程如下：
配制浓度为5％的纤维蛋白原水溶液和2％壳聚糖醋酸溶液，再将纤维蛋白原溶液和壳聚糖溶液按质量比9：1混合均匀。将配置好的溶液滴加在预先平铺在培养板内的表面修饰有1.0μm平行条纹的PDMS膜上（0.5 mL/cm 2，四周有石蜡围埂以限制液体流失），液体流平后用微量喷雾器加入凝血酶（100 U/mL）50 µL（5 U），5分钟后再加入1%京尼平50 µL。将培养板放入干燥箱内，37℃固化，12h后液体凝固成凝胶。此时在凝胶上加压50克砝码继续置25℃真空干燥至膜表面无流动液体，但保持膜表面湿润。此后将膜和模版置入冰箱内4℃固化稳定24 h。将固化的纤维蛋白/壳聚糖复合膜从模版上缓慢仔细剥离以保证膜和条纹的完整性，此时纤维蛋白/壳聚糖复合膜表面即形成与模版微图案互补的图案。
2. EMSCs种植于微图案化的纤维蛋白/壳聚糖复合膜表面
为了验证其他材料膜表面条纹同样可诱导EMSCs向雪旺细胞样细胞分化的效果，本发明将上述EMSCs用胰酶消化，收集细胞调整细胞密度约为1×10 5个/mL，以0.5 mL/cm 2的密度种植于上述微图案化的纤维蛋白/壳聚糖复合膜表面（四周有围埂限制培养基和细胞流失）置CO2培养箱内（37℃，5%CO 2，饱和湿度）用含普通培养基培养。培养14天后用4%多聚甲醛溶液固定EMSCs/微图案纤维蛋白/壳聚糖膜复合物，用雪旺细胞标志蛋白S100和MBP的抗体进行免疫荧光染色，观察EMSCs分化的雪旺细胞在微图案膜的生长情况。
3. 大鼠胚胎神经干细胞的培养
将孕14-16 d SD大鼠麻醉以后取出胚胎，取两侧大脑皮层组织大小约0.5 mm×1 mm×2 mm，将软脑膜剔除干净，放入无血清DMEM/F12混合培养基（含青霉素200 U/mL和链霉素200 U/mL）中清洗两遍。将所取的组织在PBS缓冲液中洗涤、剪碎、胰酶消化、筛网过滤，制得单细胞悬液。接种在神经干细胞培养基（DMEM／F12培养基中添加2% B 27 mL、20 ng/mL bFGF、20 ng/mL EGF、青霉素和链霉素各100 U/mL），接种密度是2×10 5个/mL。为了确保神经千细胞球的增殖。得到的干细胞球接种密度为2000个/mL。随后采用机械消化法每隔1~2周传代一次，进行多次传代。神经球及分化细胞以4％多聚甲醛溶液室温下固定30 min，用神经干细胞标志蛋白Nestin抗体进行免疫荧光染色鉴定。其余神经干细胞用于以下实验，模拟体内促进神经再生过程：（1）将神经球或分散的神经干细胞种植于条纹化（1.0 μm×1.0 μm×1.0 μm）纤维蛋白/壳聚糖微图案膜表面，用神经干细胞培养基培养，21天后用神经纤维标志蛋白NF-200的抗体进行免疫荧光染色，观察神经纤维沿条纹生长的情况；（2）将神经干细胞种植于EMSCs/纤维蛋白/壳聚糖微图案膜，观察神经纤维在细胞/条纹膜表面生长情况（下述）。
4.神经干细胞种植于EMSCs/纤维蛋白/壳聚糖微图案膜
将上述培养的次级神经球部分打散后种植于上述EMSCs/PCL微图案膜表面，置CO 2培养箱内（37℃，5%CO 2，饱和湿度）用含10%胎牛血清的DMEM/F12（含青霉素100 U/mL和链霉素100 U/mL）培养。培养14天后用4%多聚甲醛溶液固定神经干细胞/EMSCs/纤维蛋白/壳聚糖微图案膜复合物，用神经细胞/雪旺细胞标志蛋白NF-200/MBP的抗体进行免疫荧光双标记染色，观察神经干细胞在EMSCs/纤维蛋白/壳聚糖微图案膜上分化以及神经纤维平行性生长的情况。
5.体内移植试验
（1）实验动物及移植手术过程
健康成年雄性SD大鼠50只，体重250~300 g，将其随机分为5组，每组10只。第1组为单纯坐骨神经损伤组；第2组为坐骨神经损伤+移植单纯无条纹纤维蛋白/壳聚糖微图案导管组；第3组为坐骨神经损伤+移植EMSCs无条纹纤维蛋白/壳聚糖微图案导管/组；第4组为坐骨神经损伤+移植单纯有条纹纤维蛋白/壳聚糖微图案导管组；第5组为坐骨神经损伤+移植EMSCs/有条纹纤维蛋白/壳聚糖微图案导管组。
动物手术过程如下：10%水合氯醛400 mg/kg腹腔麻醉，股后部正中切口，暴露右后肢中段坐骨神经。设置5个实验组：第1组，切除坐骨神经6 mm后直接缝合肌肉和皮肤；第2组，在坐骨神经缺损部位移植单纯无条纹纤维蛋白/壳聚糖微图案导管；第3组，在坐骨神经缺损部位移植EMSCs/纤维蛋白/壳聚糖微图案无条纹导管；第4组，在坐骨神经缺损部位移植单纯有条纹纤维蛋白/壳聚糖微图案导管组；第5组，在坐骨神经缺损部位移植移植EMSCs/有条纹纤维蛋白/壳聚糖微图案导管。导管移植后用纤维蛋白胶封固吻合口，缝合肌肉和皮肤（手术过程见图8）。术后各组常规饲养，定期测量坐骨神经指数。
（2）导管移植修复神经损伤效果评价指标
①一般情况观察及坐骨神经功能指数（SFI）测定
术后常规观察大鼠饮食、足部溃疡、肢体活动及切口愈合情况。每周测定坐骨神经功能指数（SFI）：制作一长60 cm、宽10 cm、高20 cm的两端开口木槽，将70 g/m 2白纸裁成与木槽等长等宽后铺于槽底。大鼠双侧后肢用颜料浸于双踝关节着色后，将大鼠放于槽的一端，使其自行向槽的另一方行走，每侧后肢各留下5～6个足印。选择印迹清晰的足印分别测量正常足（N）和伤侧足（E）的3个指标：A：PL（足印长度）；B：TS（足趾宽度）；C：IT（中间足趾宽度）。将上述指数代入Bain公式，计算出坐骨神经功能指数。
Bain公式：SFI=109.5（ETS-NTS）/NTS-38.3（EPL—NPL）/NPL+13.3（EIT—NIT）/NIT-8.8。
坐骨神经功能指数SFI=0为正常，-100为完全损伤。
②荧光金逆行示踪
每组随机挑选3只大鼠于术后15 周（观察终点前1周）进行荧光金（Fluorochrome）逆行示踪。麻醉后再次暴露坐骨神经，于移植物远端5 mm处用微量注射器注射5%荧光金-磷酸盐缓冲液（PBS）溶液2 μL。正常侧坐骨神经相应位置同样注射同等量的荧光金。缝合手术切口，动物继续饲养。1周后取出左右侧相对应的L4-L6，S1-S2背根神经节，用冰冻切片机进行纵向切片，厚度为10 μm。在荧光显微镜下分别于观察10张连续切片（由于神经节很小，可以在一个低倍视野内观察全貌），用Image-proPlus6.0计数每张切片内荧光金标记的阳性细胞总数，并计算平均值。双侧标记阳性细胞比（阳性细胞比=实验侧标记阳性细胞数/对照侧阳性细胞数×100%）以阳性细胞比反映神经再生程度（阳性细胞总数与导管移植修复效果成正相关。
③腓肠肌的形态学观察和计量
动物手术16周后，完整切取双侧腓肠肌，电子天平称重（精确至0.001g），计算各组动物双侧腓肠肌湿重比（湿重比=实验侧肌肉湿重/对照侧肌肉湿重×100%）。称重后将肌肉用4%多聚甲醛固定，常规石蜡包埋，组织切片分别作H-E染色光镜观察，Leica显微图像分析系统分别测量左右侧腓肠肌纤维截面积，并计算截面积比（截面积比=实验侧肌肉截面积/对照侧肌肉截面积×100%）。
④坐骨神经的形态学观察和计量
动物手术16周后，同上法麻醉后自原切口切开并暴露坐骨神经，观察坐骨神经再生情况。选取大鼠坐骨神经损伤后移植神经导管和未移植神经导管的，损伤修复后的坐骨神经，包括损伤部位的近段（上段）、损伤段（移植导管部位）和远段（下段），神经经4%多聚甲醛固定后进行常规石蜡包埋和切片。切片方向为纵向平行与神经纵轴，通过近段（上段）、损伤段（移植导管部位）和远段（下段），以便观察再生神经纤维通过导管的情况。组织切片分别作 H.E染色、用神经纤维标志蛋白NF-200的抗体进行免疫组织化学染色。显微镜观察并采图，图像分析系统进行神经纤维密度测定（选取各组动物坐骨神经纵向切面的最粗部位横切面进行比较）。
结果表明，本发明提供的组织工程化神经移植物能通过损伤部位移植，促进神经再生和下肢运动功能恢复。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (16)

  1. 一种组织工程化神经移植物，其特征在于，以表面引入条纹状微图案的生物材料作为支架，以外胚间充质干细胞EMSCs作为种子细胞，将种子细胞接种到支架上得到组织工程化神经移植物。
  2. 如权利要求1所述的组织工程化神经移植物，其特征在于，所述生物材料表面利用微图案技术印刻条纹状微图案。
  3. 如权利要求2所述的组织工程化神经移植物，其特征在于，所述微图案技术包括但不限于光刻、电子束曝光或纳米压印。
  4. 如权利要求1-3任一所述的组织工程化神经移植物，其特征在于，所述条纹状微图案的宽度为1-2 μm，间距为1-2 μm，条纹高度为1-2 μm。
  5. 如权利要求1所述的组织工程化神经移植物，其特征在于，以聚二甲基硅氧烷、聚己内酯、壳聚糖、纤维蛋白原中的一种或多种作为生物材料。
  6. 如权利要求1所述的组织工程化神经移植物，其特征在于，所述生物材料包括壳聚糖-纤维蛋白。
  7. 如权利要求6所述的组织工程化神经移植物，其特征在于，所述壳聚糖-纤维蛋白是将壳聚糖和纤维蛋白原通过生物交联剂交联细胞生长因子获得的，所述细胞生长因子为EGF、FGE、NGF、SHH中的一种或多种。
  8. 如权利要求7所述的组织工程化神经移植物，其特征在于，生物交联剂包括京尼平或/和谷氨酰氨转氨酶。
  9. 如权利要求1所述的组织工程化神经移植物，其特征在于，所述EMSCs的初始细胞密度为10 4-10 5个/cm 2。
  10. 如权利要求1所述的组织工程化神经移植物，其特征在于，所述组织工程化神经移植物填充有促进神经生长的药物或生长因子缓释材料。
  11. 如权利要求10所述的组织工程化神经移植物，其特征在于，促进神经生长的药物或生长因子缓释材料包括微球、纳米颗粒或水凝胶为载体的药物缓释系统。
  12. 如权利要求1-11任一所述的组织工程化神经移植物，其特征在于，所述组织工程化神经移植物的制备方法包括以下步骤：
    （1）制备表面微图案化的生物材料支架；EMSCs的取材培养和扩增：
    （2）将步骤（1）得到的EMSCs种植于微图案化的生物材料支架。
  13. 一种神经导管，其特征在于，以表面引入条纹状微图案的生物材料作为支架，以外胚间充质干细胞EMSCs作为种子细胞，将种子细胞接种到支架上得到组织工程化神经移植物，所述组织工程化神经移植物卷曲形成单层或多层多隧道神经导管。
  14. 如权利要求13所述的神经导管，其特征在于，所述条纹状微图案的宽度为1-2 μm，间距为1-2 μm，条纹高度为1-2 μm。
  15. 如权利要求13所述的神经导管，其特征在于，以聚二甲基硅氧烷、聚己内酯、壳聚糖、纤维蛋白原中的一种或多种作为生物材料。
  16. 权利要求1-12任一所述的组织工程化神经移植物或权利要求13-15任一所述的神经导管在制备医疗器械中的应用。
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