CN110507857B

CN110507857B - Tissue engineering nerve graft and preparation method thereof

Info

Publication number: CN110507857B
Application number: CN201910813397.9A
Authority: CN
Inventors: 陆乃彦; 张轩; 翁雨燕; 余雪健; 杨国锋
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2019-08-30
Filing date: 2019-08-30
Publication date: 2021-01-29
Anticipated expiration: 2039-08-30
Also published as: CN110507857A

Abstract

The invention discloses a tissue engineering nerve graft and a preparation method thereof, belonging to the technical field of biological materials and tissue engineering. The invention optimizes the specification of the stripe to ensure that the stripe can maximally and independently induce the differentiation of the EMSCs into myelinating cells (Schwann cells) to obtain the EMSCs/biomaterial scaffold compound. The EMSCs/biomaterial scaffold compound can be used as a three-dimensional cell culture model for researching neural stem cell differentiation, neural fiber growth and myelination molecular mechanism in vitro and can also be used as a tissue engineering graft for repairing nervous system injury by in vivo transplantation. The EMSCs/micropatterned biological material film is rolled into a cylindrical multi-tunnel nerve regeneration catheter for transplantation and repair of sciatic nerve injury, and the result shows that the nerve regeneration catheter can promote nerve regeneration and recovery of lower limb motor function through transplantation of the injured part, and has good clinical application prospect and research and development values.

Description

Tissue engineering nerve graft and preparation method thereof

Technical Field

The invention relates to a tissue engineering nerve graft and a preparation method thereof, belonging to the technical field of biological materials and tissue engineering.

Background

Stem cell/tissue engineering scaffold transplantation is the main strategy for repairing nerve tissue damage. In the central nervous system, nerve cells of the brain form a network structure by the interconnection of processes. After brain tissue injury, the main purpose of stem cell/scaffold transplantation is to promote the formation of new neural networks to restore their information integration and conduction functions, so the requirement for implanted stem cells/scaffolds is to promote the formation of neural networks; in contrast to the information-integration function of brain tissue, the main functions of the spinal cord are to transmit brain information to motor neurons of the spinal cord through descending sensory conduction tracts such as corticospinal tracts, and to upload sensory information received by the spinal cord to the brain through ascending sensory conduction tracts such as grabs and cuneiform tracts. Therefore, after spinal cord injury, stem cell/tissue engineering scaffold transplantation is mainly to promote regeneration of nerve conduction tracts and parallel growth of nerve fibers and to directionally extend to spinal cord tissues far away from the injury (motor nerves) or near to the injury (sensory nerves) through the injury site, and the regenerated nerve fibers grow along the original channels and finally establish synaptic connection with target cells thereof. Meanwhile, the transplanted stem cell/tissue engineering scaffold also has the function of promoting the myelination of new nerve fibers. The repair mechanism of peripheral nerve injury is similar to that of spinal cord injury, and stem cell/tissue engineering scaffold (catheter) transplantation repairs peripheral nerves such as sciatic nerve injury and promotes effective regeneration of nerve fiber parallelism and myelination. Therefore, how to physically and chemically modify the scaffold and select appropriate seed cells (which can differentiate into myelinating cells) to plant on the scaffold to induce the parallel growth and myelination of nerve fibers is a key factor for improving the treatment effect of stem cell/tissue engineering scaffold transplantation for repairing spinal cord or peripheral nerve injury.

At present, stem cell/scaffold transplantation is mainly used for repairing spinal cord and peripheral nerve (such as sciatic nerve) injuries by preparing stem cells/biological materials into hydrogel or transplanting nerve conduits to injury defect sites. Particularly when trauma causes a long defect in the peripheral nerve, nerve conduit transplantation will be the most effective treatment for repairing the nerve defect. Most nerve conduits used at present can promote nerve regeneration, but lack of stripes for guiding parallel and orderly growth of nerve fibers leads to directional disorientation of nerve fiber extension, so that the fiber advancing speed is slow, and lack of myelinating cells leads to insufficient myelination of regenerated nerve fibers (only depending on the proliferation of endogenous schwann cells at nerve cutting ends for myelination). Therefore, there is a need to develop a nerve conduit capable of obviously promoting the parallel directional growth of regenerated nerve fibers and providing myelinating seed cells to promote the regeneration and myelination of damaged nerves in vivo for transplantation and more effective repair of nerve damage.

Disclosure of Invention

The invention aims to provide a tissue engineering nerve graft, which is obtained by taking a biological material with a striped micro pattern introduced on the surface as a scaffold, taking Ectodermal Mesenchymal Stem Cells (EMSCs) as seed cells and inoculating the seed cells onto the scaffold.

In one embodiment of the present invention, the surface of the biomaterial is imprinted with a striped micropattern using micropatterning techniques; including but not limited to photolithography, electron beam exposure, or nanoimprinting

In one embodiment of the present invention, the width of the striped micro pattern is 1-2 μm, the pitch is 1-2 μm, and the stripe height is 1-2 μm.

In one embodiment of the present invention, one or more of Polydimethylsiloxane (PDMS), Polycaprolactone (PCL), chitosan, fibrinogen are used as the biomaterial.

In one embodiment of the invention, the biomaterial comprises chitosan-fibrin.

In one embodiment of the invention, the chitosan-fibrin is obtained by crosslinking chitosan and fibrinogen with a biological crosslinking agent to obtain a cell growth factor, wherein the cell growth factor is one or more of EGF, FGE, NGF and SHH.

In one embodiment of the invention, the biological cross-linking agent comprises genipin or/and glutamine Transaminase (TG).

In one embodiment of the invention, the EMSCs have an initial cell density of 10⁴-10⁵Per cm²。

In one embodiment of the invention, the shape of the tissue engineered nerve graft comprises a membrane shape.

In one embodiment of the invention, the tissue engineered nerve graft is crimped into a single or multilayer multi-tunnel nerve conduit.

In one embodiment of the invention, the tissue engineered nerve graft is filled with a drug or growth factor slow release material that promotes nerve growth.

In one embodiment of the present invention, the sustained release material of a drug or growth factor for promoting nerve growth comprises a drug sustained release system using microspheres, nanoparticles or hydrogel as a carrier.

In one embodiment of the invention, the tissue engineered nerve graft is used to repair nerve damage.

The invention also provides a method for preparing the tissue engineered nerve graft, which comprises the following steps:

(1) preparing a biomaterial scaffold with a micropatterned surface; and (3) material-taking culture and amplification of EMSCs:

(2) and (2) planting the EMSCs obtained in the step (1) on the micropatterned biomaterial scaffold.

The invention also provides a nerve conduit, which takes the biological material with the surface introduced with the striped micro-pattern as a scaffold, takes the ectodermal mesenchymal stem cells EMSCs as seed cells, and inoculates the seed cells on the scaffold to obtain the tissue engineering nerve graft, wherein the tissue engineering nerve graft is curled to form a single-layer or multi-layer multi-tunnel nerve conduit.

In one embodiment of the invention, one or more of polydimethylsiloxane, polycaprolactone, chitosan, fibrinogen are used as the biomaterial.

The invention also provides application of the tissue engineering nerve graft or the nerve conduit in preparation of medical devices.

The invention has the beneficial effects that:

the invention coils EMSCs/micropatterned biological material film into a cylindrical multi-tunnel nerve regeneration conduit for transplantation and repair of sciatic nerve injury, and the result shows that the sciatic nerve function index of the sciatic nerve injury side of a mouse without nerve conduit treatment reaches-91 +/-25, and the sciatic nerve function index of the sciatic nerve injury side of the mouse after the nerve conduit treatment reaches-37 +/-17. The tissue engineering nerve graft provided by the invention can be transplanted through an injured part, promotes nerve regeneration and lower limb movement function recovery, and has good clinical application prospect and research and development values.

Drawings

FIG. 1: the surface-striped micro-patterned PDMS film has a stripe specification of 0.5 μm × 0.5 μm (A), 1.0 μm × 1.0 μm (B), 1.5 μm × 1.5 μm (C), 2.0 μm × 2.0 μm (D).

FIG. 2: a fluorescent staining (S100) pattern (fluorescence microscopy) of the differentiated emichs planted on the surface of the micropatterned PDMS membrane into schwann cells. The stripe specification is 0.5. mu. m.times.0.5. mu.m (A), 1.0. mu. m.times.1.0. mu.m (B), 1.5. mu. m.times.1.5. mu.m (C), 2.0. mu. m.times.2.0. mu.m (D).

FIG. 3: and (3) Western blotting detection results of the level of the Schwann cell marker protein expressed by the cells after the EMSCs planted on the surface of the micropatterned PDMS membrane differentiate into Schwann cells.

FIG. 4: the neural stem cells are differentiated into nerve cells on the surface of the striation-free PCL membrane, and nerve fibers grow radially (A); parallel nerve fiber growth on the surface of the striped (1.0 μm × 1.0 μm × 1.0 μm) PCL membrane (B); in order to simultaneously display the streaks and the nerve fibers, the scattered neural stem cells were seeded on the surface of the streaks (1.0. mu. m.times.1.0. mu. m) PCL membrane, and the nerve fibers were seen to grow along the streaks (C).

FIG. 5: nerve cells grew in parallel on the surface of the EMSCs (Schwann cells)/striped micropattern PCL membrane (bottom Schwann cells stained with immunofluorescence of marker protein S100 (fluorescein 488, green), top nerve fibers stained with immunofluorescence of marker protein NF-200 (cy3 red). A: striped 1.0. mu. m.times.1.0. mu.m.times.1.0. mu.m, B: striped 2.0. mu. m.times.2.0. mu.m.times.2.0. mu.m.

FIG. 6: schematic diagram of EMSCs (schwann cells)/striped micropatterned membrane (catheter) transplantation surgery in rat animal model of sciatic nerve injury.

FIG. 7: EMSCs (Schwann cells)/striped (1.0 μm × 1.0 μm × 1.0 μm) micropatterned PCL composite membrane transplantation surgical procedure in rat animal model of sciatic nerve injury. A: sciatic nerve cutting; b: EMSCs (Xuewang cells)/striped (1.0 μm × 1.0 μm × 1.0 μm) micro-pattern PCL composite membrane (the membrane is dark blue due to genipin cross-linking) wraps the nerve broken end (two ends are connected and aligned by an absorption suture, a gap of 5mm is reserved in the middle), and the broken end is sealed by fibrin glue; c: the membrane is crimped into a catheter, and the outer surface of the catheter is sealed with fibrin glue after being sutured with absorbable suture.

FIG. 8: EMSCs (Schwann cells)/streaking (1.0 μm × 1.0 μm × 1.0 μm) micropatterned fibrin/chitosan composite membrane transplantation procedure in a rat sciatic nerve injury animal model. A: sciatic nerve was isolated and severed; b: EMSCs (Schwann cells)/striped (1.0 μm multiplied by 1.0 μm) micropatterned fibrin/chitosan composite membrane wraps the nerve broken end and the broken end is sealed by fibrin glue; c: the membrane is crimped into a catheter, and the outer surface of the catheter is sealed with fibrin glue after being sutured with absorbable suture.

FIG. 9: the tracing result of the fluorescence sciatic nerve injection dorsal root ganglion nerve cells is that A-F graphs are respectively as follows: normal group (a); lesion non-transplanted group (B); injury transplant EMSCs (Schwann cells)/striped (1.0 μm × 1.0 μm × 1.0 μm) micro-patterned PCL composite membrane group (C); a lesion graft striped (1.0 μm × 1.0 μm × 1.0 μm) group (D) of micro-patterned PCL composite membranes (cell-free); injury transplanting EMSCs (Schwann cells)/streak-free PCL composite membrane group (E); injury graft striation-free PCL composite membrane (cell-free) group (F).

FIG. 10: the general appearance of the normal lower limb gastrocnemius muscle was compared with that of the other groups of the gastrocnemius muscle on the side with sciatic nerve injury. The A-F diagrams are respectively: normal group (a); lesion non-transplanted group (B); injury transplant EMSCs (Schwann cells)/striped (1.0 μm × 1.0 μm × 1.0 μm) micro-patterned PCL composite membrane group (C); a lesion graft striped (1.0 μm × 1.0 μm × 1.0 μm) group (D) of micro-patterned PCL composite membranes (cell-free); injury transplanting EMSCs (Schwann cells)/streak-free PCL composite membrane group (E); injury graft striation-free PCL composite membrane (cell-free) group (F).

FIG. 11: and (3) observing the fiber sectional area of the gastrocnemius muscle by cutting HE on the gastrocnemius tissue on the side with normal lower limb gastrocnemius and other sciatic nerve injury of each group: the A-F diagrams are respectively: normal group (a); lesion non-transplanted group (B); injury transplant EMSCs (Schwann cells)/striped (1.0 μm × 1.0 μm × 1.0 μm) micro-patterned PCL composite membrane group (C); a lesion graft striped (1.0 μm × 1.0 μm × 1.0 μm) group (D) of micro-patterned PCL composite membranes (cell-free); injury transplanting EMSCs (Schwann cells)/streak-free PCL composite membrane group (E); injury graft striation-free PCL composite membrane (cell-free) group (F).

FIG. 12: rat sciatic nerve injury animal model's EMSCs (schwann cell)/striation micropattern PCL complex film transplantation postoperative 16 weeks, animal anesthesia carries out sciatic nerve and draws materials, takes out the nerve pipe of transplanting together with the nerve of far-near end fixed, cuts open the pipe along the axis of ordinates (original pipe wall has been rebuilt by internal tissue), observes the interior nerve growth condition of pipe, A: EMSCs (Schwann cells)/striped (1.0 μm × 1.0 μm × 1.0 μm) micro-pattern PCL composite membrane transplantation group; b: striped (1.0 μm × 1.0 μm × 1.0 μm) micropatterned PCL composite membrane graft group (cell-free); c: EMSCs (Schwann cells)/streak-free PCL composite membrane transplantation group; d: streak-free PCL composite membrane graft group (cell-free).

FIG. 13: observation results of HE staining of sciatic nerve tissue sections at injured sites with and without nerve conduits transplanted, a: after sciatic nerve injury, the EMSCs (Schwann cells)/striped micro-pattern PCL composite membrane is transplanted to the treatment group, sciatic nerve at the injury proximal end grows into the nerve conduit and reaches the far side through the conduit, and no residual cavity is left after absorbable suture is absorbed; b: in the untreated group of sciatic nerve injury, severe degeneration of nerve fibers was observed, only a few regenerated nerve fibers (upper side of the figure) were observed, and the cavity was left after the absorbable suture was absorbed.

FIG. 14: the normal side lower limb sciatic nerve and other sciatic nerve injury parts (including transplanted catheters) are longitudinally cut, and nerve fiber marker protein NF-200 immunohistochemical staining is used for observing nerve fiber regeneration conditions: the A-F diagrams are respectively: normal group (a); lesion non-transplanted group (B); injury transplant EMSCs (Schwann cells)/striped (1.0 μm × 1.0 μm × 1.0 μm) micro-patterned PCL composite membrane group (C); a lesion graft striped (1.0 μm × 1.0 μm × 1.0 μm) group (D) of micro-patterned PCL composite membranes (cell-free); injury transplanting EMSCs (Schwann cells)/streak-free PCL composite membrane group (E); injury graft striation-free PCL composite membrane (cell-free) group (F).

FIG. 15: the mid-point cross-sectional tissue section of the injury part (catheter transplantation part) of the sciatic nerve of the lower limb and other groups of sciatic nerves, nerve fiber marker protein NF-200 immunohistochemical staining is used for observing the density of regenerated nerve fibers: the A-F diagrams are respectively: normal group (a); lesion non-transplanted group (B); injury transplant EMSCs (Schwann cells)/striped (1.0 μm × 1.0 μm × 1.0 μm) micro-patterned PCL composite membrane group (C); a lesion graft striped (1.0 μm × 1.0 μm × 1.0 μm) group (D) of micro-patterned PCL composite membranes (cell-free); injury transplanting EMSCs (Schwann cells)/streak-free PCL composite membrane group (E); injury graft striation-free PCL composite membrane (cell-free) group (F).

Detailed Description

Example 1 optimal selection of stripes

1. Method for making micro-pattern on material surface

Electron beam exposure and nanoimprint technology are adopted. Firstly, spin-coating a polymethyl methacrylate (PMMA) film on the surface of a silicon wafer of 3 multiplied by 3cm, and etching stripe type micro patterns with equal width, spacing and height on the surface of the PMMA film by using an electron beam exposure technology; taking the micro-patterned substrate as a template, mixing a base material of Polydimethylsiloxane (PDMS) and a curing agent according to a ratio of 10:1, and dripping the mixture on the surface of the template (0.5 mL/cm)²) Drying at 60 deg.c for 4 hr in a vacuum drying oven to solidify PDMS on the template surface and strip the PDMS film from the template to form the pattern complementary to the micro pattern on the template (see FIG. 1). The experimental stripes were of equal width, equal height and equal spacing, and the specifications included 0.5. mu. m.times.0.5. mu.m.times.0.5 μm (0.5 μm), 1.0. mu. m.times. 01.0. mu.m.times.11.0. mu.m (1.0. mu.m), 1.5. mu. m.times.1.5. mu.m.times.1.5 μm (1.5 μm), 2.0. mu. m.times.2.0. mu.m (2.0. mu.m), 2.5. mu. m.times.2.5. mu.m.times.2.5 μm (2.5 μm), 3.0. mu. m.times.3.0. mu.m.times.3.0. mu.m (3.0.

Drawing materials, culturing, amplifying and identifying EMSCs

SD rats (80-100g) are anesthetized by intraperitoneal injection of 10% chloral hydrate (330g/kg), the skin of the whole body is disinfected, the skin and the nasal bone are cut along the nasal cavity upwards to the inner canthus under the aseptic condition, the nasal septum mucosa is exposed, 1/3 nasal septum is cut off and placed in PBS buffer solution, and the whole layer of the nasal mucosa is stripped. After taking out nasal mucosa of SD rat, rinsing with serum-free DMEM/F12 mixed culture medium (containing penicillin 200U/mL and streptomycin 200U/mL) for three times to remove blood stain, placing in DMEM/F12 culture medium (namely common complete culture medium, containing penicillin 100U/mL and streptomycin 100U/mL) containing 10% fetal calf serum,sufficiently cutting with ophthalmic scissors, digesting with 0.25% pancreatin at 37 deg.C for 15min, centrifuging, discarding supernatant, inoculating cells and small tissue blocks into Corning culture flask in CO₂In an incubator (37 ℃, 5% CO)₂Saturated humidity). The cells were supplemented with new DMEM/F12 medium containing 10% fetal bovine serum 3 days after culture. After that, half the amount of the liquid is changed every three days, and when the cells are fully paved on the bottom of the bottle, the cells are digested and passaged.

The 5 th generation cells were seeded in 24-well culture plates. And performing immunofluorescence staining by using marker proteins of the EMSCs, namely vimentin, Nestin, CD133, CD44 and an antibody respectively, and identifying the cultured cells as the EMSCs. The operation steps are as follows: after cells are fixed by 4% paraformaldehyde solution, the cells are blocked for 30min at 37 ℃ in a mixed solution of 0.25% TritonX-100 and 3% Bovine Serum Albumin (BSA), the primary antibody is incubated for 12h at 4 ℃, the cells are incubated for 1h at room temperature by using corresponding secondary antibody marked by Cy3 after being rinsed by PBS buffer solution, the cells are rinsed for 3 times by PBS, the cells are counterstained by Hoc host 33342, the cells are rinsed by PBS buffer solution, neutral glycerol is sealed, the cells are observed and photographed under a Leica fluorescence microscope, the primary antibody is replaced by the PBS buffer solution for negative control, and the other steps are the same as the steps. The remaining cells were used in the following experiments.

3. Optimized selection of film surface stripes

The effect of inducing differentiation from stripe-induced EMSCs to Schwann cell-like cells was used as the standard for optimal selection of stripes. The 5 th-generation EMSCs identified by the above culture were digested with trypsin, and the cells were collected to adjust the cell density to about 1X 10⁵At a concentration of 0.5mL/cm²The amount of the culture medium is planted on the surface of a micro-patterned PDMS membrane (paraffin ridges are arranged around the surface of the PDMS membrane to limit the loss of the culture medium and cells) which is laid in a culture plate. Place the EMSCs/micropatterned PDMS membranes in CO₂In an incubator (37 ℃, 5% CO)₂Saturated humidity) was cultured with DMEM/F12 (containing penicillin 100U/mL and streptomycin 100U/mL) containing 10% fetal bovine serum. After 2h, the EMSCs were attached to the surface of the micro-patterned PDMS film. Thereafter, the culture plate was supplemented with a new DMEM/F12 medium containing 10% fetal bovine serum and the culture was continued by changing the medium half every three days. 14 days later, the cell/micropattern membrane complex was fixed with 4% paraformaldehyde solution, immunofluorescent staining was performed with antibodies against Schwann cell marker protein S100 and MBP, and EMSCs was observedDifferentiation into Schwann cells on the micropattern film. Detecting the relative content of the Schwann cell marker protein by using a Western blotting method, comparing the differentiation effect of the stripes on EMSCs to Schwann cells, and selecting the stripe with the strongest induction capability as a pattern for modifying the biomaterial scaffold. In the above experiment, only DMEM/F12 medium containing 10% fetal calf serum was used to culture EMSCs without any inducer, so as to obtain the effect of inducing differentiation by stripe single factor. The biological material film with the surface having the strip of the specification is planted or not planted with EMSCs as a bracket material for manufacturing the nerve conduit.

4. Analysis of results

Immunofluorescence staining results of the Schwann cell marker protein S100 and MBP show that EMSCs are planted on the surfaces of the striped PDMS membranes of various specifications and cultured by a DMEM/F12 culture medium containing 10% fetal calf serum, and cells are in the form of Schwann cells and are arranged in parallel along the stripes. Different-specification stripes, which have differences in the morphology and staining intensity of cells on the membrane surface (see fig. 2); the relative content of the Schwann cell marker protein is detected by using a Western blotting method, and the differentiation effect of a plurality of stripes on EMSCs to Schwann cells is compared, so that the conclusion is that the stripes with the size of 1.0 mu m multiplied by 1.0 mu m have the strongest capacity of inducing the EMSCs to differentiate to Schwann cells (see table 1 and figure 3). The striation-free PDMS film was the least inducible. Therefore, striped membranes of 1.0 μm × 1.0 μm × 1.0 μm were used as cell growth substrates and nerve conduit materials in the following experiments.

TABLE 1 comparison of relative content (ratio to Actin) of stripe-induced EMSCs expressing Schwann cell marker protein

Description of the drawings: the relative content of the Xuewang cell marker proteins MBP and S100 of the 1 mu m stripe group is obviously higher than that of other groups (p <0.05, n ═ 3)

Example 2 practical application of micro-patterned PCL films

1. Preparation of PCL film with micro-patterned surface

Electron beam exposure and nanoimprint technology are adopted. Firstly, spin-coating a polymethyl methacrylate (PMMA) film on the surface of a silicon wafer of 3 multiplied by 3cm, and etching a stripe type micro pattern with the width of 1 mu m, the interval of 1 mu m and the height of 1 mu m on the surface of the PMMA film by applying an electron beam exposure technology; taking the micro-patterned substrate as a template, mixing a base material of PDMS and a curing agent according to the proportion of 10:1, dripping the mixture on the surface of the template, drying the mixture in a vacuum drying oven at 60 ℃ for 4 hours, solidifying the PDMS on the surface of the template to form a film, stripping the PDMS film from the template, and forming a pattern complementary to the micro-pattern of the template on the surface of the PDMS; taking the micro-patterned PDMS membrane as a template, and mixing and dripping a 20% PCL dichloromethane solution on the surface of the PDMS template (0.5 mL/cm)²) And drying the PCL film in a vacuum drying oven for 1h, solidifying the PCL film on the surface of the PDMS template to form a film, and stripping the PCL film from the template to form a pattern which is complementary with the micro pattern of the template on the surface.

Material-drawing culture, amplification and identification of EMSCs

SD rats (80-100g) are anesthetized by intraperitoneal injection of 10% chloral hydrate (330g/kg), the skin of the whole body is disinfected, the skin and the nasal bone are cut along the nasal cavity upwards to the inner canthus under the aseptic condition, the nasal septum mucosa is exposed, 1/3 nasal septum is cut off and placed in PBS buffer solution, and the whole layer of the nasal mucosa is stripped. Taking out nasal mucosa of SD rat, rinsing with serum-free DMEM/F12 mixed culture medium (containing penicillin 200U/mL and streptomycin 200U/mL) for three times to remove blood stain, placing in DMEM/F12 culture medium (i.e. common complete culture medium containing penicillin 100U/mL and streptomycin 100U/mL) containing 10% fetal calf serum, sufficiently shearing with ophthalmic scissors, digesting with 0.25% pancreatin at 37 deg.C for 15min, centrifuging, discarding supernatant, inoculating cells and small tissue blocks in Corning culture flask, and introducing into CO₂In an incubator (37 ℃, 5% CO)₂Saturated humidity). The cells were supplemented with new DMEM/F12 medium containing 10% fetal bovine serum 3 days after culture. After that, half the amount of the liquid is changed every three days, and when the cells are fully paved on the bottom of the bottle, the cells are digested and passaged.

The 5 th generation cells were seeded in 24-well culture plates. And performing immunofluorescence staining by using marker proteins, namely vimentin, Nestin, C D133 and CD44 of the EMSCs and an antibody respectively, and identifying the cultured cells as the EMSCs. The operation steps are as follows: after cells are fixed by 4% paraformaldehyde solution, the cells are blocked for 30min at 37 ℃ in a mixed solution of 0.25% TritonX-100 and 3% Bovine Serum Albumin (BSA), the primary antibody is incubated for 12h at 4 ℃, the cells are incubated for 1h at room temperature by using corresponding secondary antibody marked by Cy3 after being rinsed by PBS buffer, the cells are rinsed for 3 times by PBS buffer, the cells are counterstained by Hoc host 33342, the cells are rinsed by PBS buffer, neutral glycerol is sealed, the cells are observed and photographed under a Leica fluorescence microscope, the primary antibody is replaced by the PBS buffer for negative control, and the rest steps are the same as the above. The remaining cells were used in the following experiments.

Planting EMSCs on the surface of the micro-patterned PCL film

Digesting the EMSCs with pancreatin, collecting cells, and adjusting cell density to about 1 × 10⁵At a concentration of 0.5mL/cm²The PCL membrane is planted on the micro-patterned PCL membrane (paraffin is arranged around the PCL membrane to limit the loss of culture medium and cells), and CO is placed in the PCL membrane₂In an incubator (37 ℃, 5% CO)₂Saturated humidity) was cultured in DMEM/F12 medium containing 10% fetal bovine serum, and the culture medium was changed every three days. After 14 days of culture, the EMSCs/micropatterned membrane complex was fixed with 4% paraformaldehyde solution, immunofluorescent staining was performed with antibodies against Schwann cell marker protein S100 and MBP, and the growth of the EMSCs-differentiated Schwann cells on the micropatterned membrane was observed.

4. Culture of rat embryonic neural stem cells

Anesthetizing pregnant 14-16 d SD rat, taking out embryo, collecting cerebral cortex tissue of about 0.5mm × 1mm × 2mm, removing pia mater, and washing twice in serum-free DMEM/F12 mixed culture medium (containing penicillin 200U/mL and streptomycin 200U/mL). Washing the tissue in PBS buffer solution, cutting, digesting with pancreatin, and filtering with screen to obtain single cell suspension. The cells were inoculated into neural stem cell culture medium (DMEM/F12 medium supplemented with 2% B27 mL, 20ng/mL bFGF, 20ng/mL EGF, penicillin and streptomycin each 100U/mL) at an inoculation density of 2X 10⁵one/mL. To ensure proliferation of neural cenospheres. The density of the stem cell ball seed obtained is 2000/mL. And then, carrying out passage once every 1-2 weeks by adopting a mechanical digestion method, and carrying out passage for multiple times. Neurospheres and differentiated cells were polymerized at 4%Fixing formaldehyde solution at room temperature for 30min, and performing immunofluorescence staining identification by using neural stem cell marker protein Nestin antibody. The remaining neural stem cells were used in the following experiments, simulating the process of promoting nerve regeneration in vivo: (1) implanting neurospheres or dispersed neural stem cells on the surface of a striped (1.0 μm multiplied by 1.0 μm) PCL membrane, culturing for 21 days by using a neural stem cell culture medium, and performing immunofluorescence staining by using an antibody of a nerve fiber marker protein NF-200 to observe the growth condition of nerve fibers along the stripes (see figure 4); (2) and (3) planting the neural stem cells in an EMSCs/PCL micro-pattern membrane, and observing the growth condition of the neural fibers on the surface of the cell/stripe membrane.

5. Neural stem cells are planted in the EMSCs/PCL micro-pattern membrane

Scattering the cultured secondary neurosphere part, planting on the surface of the EMSCs/PCL composite membrane, and placing CO₂In an incubator (37 ℃, 5% CO)₂Saturated humidity) was cultured with neural stem cell culture medium. After 14 days of culture, fixing the neural stem cell/EMSCs/micropatterned membrane compound by using 4% paraformaldehyde solution, carrying out immunofluorescence double-label staining by using an antibody of the neural cell/Schwann cell marker protein NF-200/MBP, and observing the differentiation of the neural stem cell on the EMSCs/micropatterned membrane and the parallel growth of nerve fibers (figure 5).

6. In vivo transplantation test

(1) Experimental animal and transplantation operation process

Healthy adult male SD rats, weighing 250-300 g, were randomly divided into 5 groups of 10 rats each. Group 1 is the simple sciatic nerve injury group; group 2 is sciatic nerve injury + transplantation simple streak-free PCL catheter group; group 3 is sciatic nerve injury + EMSCs transplantation/striatless PCL catheter/group; group 4 is sciatic nerve injury + PCL catheter with simple striation for transplantation; group 5 is sciatic nerve injury + EMSCs transplanted/striated PCL catheter group.

The animal surgery procedure was as follows: 10% chloral hydrate 400mg/kg abdominal cavity anesthesia, femoral posterior median incision, right hind limb middle sciatic nerve exposure. Group 1, the muscle and skin were sutured directly after sciatic nerve 6mm was excised; group 2, transplanting a pure stripe-free PCL catheter at the sciatic nerve defect part; transplanting EMSCs/streak-free PCL catheter at the sciatic nerve defect part; group 4, transplanting a pure striated PCL catheter group at the sciatic nerve defect part; in group 5, EMSCs/striated PCL catheters were implanted at the site of sciatic nerve defect. After the catheter implantation, the anastomoses were sealed with fibrin glue and the muscles and skin were sutured (see fig. 6, 7, 8). After operation, each group is raised regularly, and sciatic nerve index is measured regularly.

(2) Evaluation index of effect of repairing nerve injury by catheter transplantation

General observation and measurement of sciatic nerve function index (SFI)

The diet, foot ulcer, limb movement and incision healing condition of the rat are observed regularly after the operation. Sciatic nerve function index (SFI) was measured weekly: making a wood groove with 60cm long, 10cm wide and 20cm high and two open ends, and mixing at 70g/m²The white paper is cut into equal length and equal width with the wood groove and then laid at the bottom of the groove. After coloring the bilateral hind limbs of the rat by soaking the bilateral hind limbs in the pigment, placing the rat at one end of the groove, enabling the rat to walk to the other side of the groove by self, and leaving 5-6 footprints on each lateral hind limb. The footprints with clear footprints were selected to measure 3 indices of normal (N) and injured lateral (E) feet, respectively: a: PL (footprint length); b: TS (toe width); c: IT (medial toe width). Substituting the indexes into Bain formula to calculate sciatic nerve function index.

Bain formula: SFI 109.5(ETS-NTS)/NTS-38.3 (EPL-NPL)/NPL +13.3 (EIT-NIT)/NIT-8.8.

Sciatic nerve function index SFI-0 is normal, -100 is complete injury. The results of measuring sciatic nerve function index (SFI) on the sciatic nerve injury side of each group after 16 weeks of animal surgery are shown in table 2.

TABLE 2 sciatic nerve function index (SFI) on sciatic nerve injury side of each group

Comparison

Group 1	Group 2	Group 3	Group 4	Group 5
					-91±25	-77±31	-68±19	-57±23	-37±17

Description of the drawings: the injured sciatic nerve function indexes of the 5 th group were all significantly higher than those of the other groups (p <0.05, n ═ 9)

Fluorescent gold retrograde tracing

3 rats per group were randomly selected for a fluorescent gold retrograde tracing at 15 weeks post-surgery (1 week before the observation endpoint). After anesthesia, the sciatic nerve was again exposed and 2 μ L of 5% fluorescent gold-Phosphate Buffered Saline (PBS) solution was injected 5mm distal to the graft using a microinjector. Equal amounts of fluorogold were also injected at the corresponding sites of the normal lateral sciatic nerve. The surgical incision was sutured and the animals continued to be housed. After 1 week, the dorsal root ganglia corresponding to the left and right sides L4-L6, S1-S2 were removed and sectioned longitudinally with a thickness of 10 μm using a cryomicrotome. 10 serial sections (full view in a low power field due to the very small ganglia) were observed under a fluorescence microscope, and the total number of gold-labeled positive cells in each section was counted using Image-proplus6.0, and the average value was calculated. The bilateral marker-positive cell ratio (positive cell ratio ═ number of test-side marker-positive cells/number of control-side positive cells × 100%) reflected the degree of nerve regeneration as the positive cell ratio (the total number of positive cells positively correlated with the effect of catheter graft repair, and the results are shown in table 3 and fig. 9).

TABLE 3 fluorescence gold labeling positive cell number and normal side cell number ratio of dorsal root ganglion on sciatic nerve injury side of each group

Comparison

Group 1	Group 2	Group 3	Group 4	Group 5
					0.07±0.02	0.13±0.07	0.21±0.15	0.37±0.11	0.57±0.12

Description of the drawings: the ratio of the number of ganglion positive cells on the injured side to the normal side was significantly higher in group 5 than in other groups (p <0.05, n ═ 9)

(iii) Wet weight measurement and morphological observation of gastrocnemius muscle

After 16 weeks of animal surgery, animals were anesthetized, bilateral gastrocnemius muscles were completely excised, weighed on an electronic balance (accurate to 0.001g), and the wet-weight ratio of bilateral gastrocnemius muscles of each group of animals was calculated (wet-weight ratio: wet weight of experimental lateral muscle/wet weight of control lateral muscle × 100%), and the results are shown in table 4. After weighing, the muscle was fixed with 4% paraformaldehyde solution, embedded in normal paraffin, and the tissue sections were observed by H-E staining microscope, and the sectional areas of the fibers of the left and right gastrocnemius muscles were measured by Leica microscopic image analysis system, respectively, and the sectional area ratio (sectional area ratio: experimental side muscle sectional area/control side muscle sectional area × 100%) was calculated, and the results are shown in table 5, fig. 10, and fig. 11.

TABLE 4 Wet-weight ratio of sciatic nerve injury side gastrocnemius muscle to normal side gastrocnemius muscle

Comparison

Group 1	Group 2	Group 3	Group 4	Group 5
					0.19±0.07	0.31±0.13	0.41±0.13	0.63±0.09	0.77±0.18

Description of the drawings: the wet weight ratio of gastrocnemius in group 5 was significantly higher than that in the other groups (p <0.05, n ═ 9)

TABLE 5 ratio of the fiber cross-sectional area of the gastrocnemius muscle on the injured side of the sciatic nerve to the fiber cross-sectional area of the gastrocnemius muscle on the normal side

Comparison

Group 1	Group 2	Group 3	Group 4	Group 5
					0.21±0.09	0.29±0.13	0.47±0.17	0.75±0.12	0.87±0.23

Description of the drawings: the fiber section area ratio of gastrocnemius muscle in group 5 is obviously higher than that in other groups (p <0.05, n ═ 9)

Morphological observation and measurement of sciatic nerve

After 16 weeks of the animal surgery, the sciatic nerve was cut and exposed from the original incision after anesthesia as above, and the sciatic nerve regeneration was observed (fig. 12). Selecting sciatic nerves transplanted with nerve conduits and non-transplanted nerve conduits after sciatic nerve injury of rats, wherein the sciatic nerves after injury repair comprise a near section (upper section), an injury section (transplanted conduit section) and a far section (lower section) of an injury part, and carrying out conventional paraffin embedding and slicing after the nerves are fixed by 4% paraformaldehyde solution. The direction of the section is longitudinally parallel to the longitudinal axis of the nerve and passes through the proximal segment (upper segment), the injury segment (position of the transplanted catheter) and the distal segment (lower segment), so as to observe the condition that the regenerated nerve fiber passes through the catheter. Tissue sections were stained for h.e (fig. 13) and immunohistochemically stained with antibody to the neurofibrillary marker protein NF-200 (fig. 14). Normal sciatic nerve and other groups of sciatic nerve injury sites (catheter transplantation sites) midpoint cross-sectional tissue sections, nerve fiber marker protein NF-200 immunohistochemical staining to observe the density of regenerated nerve fibers (FIG. 15). After microscopic observation and image collection, nerve fiber density measurement was performed using an image analysis system (the thickest section of each group of animals was selected for comparison) and the results are shown in Table 6.

TABLE 6 ratio of the number of regenerated nerve fiber cross sections on the injured side of sciatic nerve to the number of regenerated nerve fiber cross sections on the normal side of sciatic nerve

Comparison

Group 1	Group 2	Group 3	Group 4	Group 5
					0.18±0.08	0.21±0.07	0.35±0.17	0.57±0.21	0.69±0.19

Description of the drawings: the ratio of the number of nerve fibers on the injured side to the normal side was significantly higher in group 5 than in other groups (p <0.05, n ═ 9)

Example 3 practical application of micropatterned fibrin/chitosan composite membrane

1. Preparation of fibrin/chitosan composite membrane with micropatterned surface

Based on good biocompatibility of fibrinogen and chitosan, the mechanical strength of the composite membrane can be increased by mixing the two materials, and one or more cell growth factors such as EGF, FGE, NGF, SHH and the like can be crosslinked by means of a biological crosslinking agent such as genipin or/and glutamine Transaminase (TG) so as to construct a drug slow-release stent and further improve the function of promoting nerve regeneration. The method firstly selects the fibrinogen/chitosan composite membrane as a material for manufacturing the striped nerve conduit, is used for transplanting and repairing sciatic nerve injury in vivo after planting or not planting EMSCs, and evaluates the application value of the method. The preparation process of the striped fibrinogen solution/chitosan composite membrane is as follows:

preparing a 5% fibrinogen raw water solution and a 2% chitosan acetic acid solution, and mixing the fibrinogen solution and the chitosan solution according to a mass ratio of 9: 1, mixing uniformly. The prepared solution is dripped on a PDMS membrane (0.5 mL/cm) which is paved in a culture plate in advance and is decorated with 1.0 mu m parallel stripes on the surface²Around which there is a paraffin ridge to limit the loss of fluid), after leveling the fluid, 50. mu.L (5U) of thrombin (100U/mL) was added using a microsprayer, and after 5 minutes 50. mu.L of 1% genipin was added. Placing the culture plate into a drying oven, curing at 37 deg.C, and solidifying the liquid into gel after 12 hr. At this point, the gel was pressed with a 50 gram weight and dried under vacuum at 25 ℃ until no fluid was on the membrane surface, but the membrane surface was kept wet. After that, the film and the template are placed in a refrigerator for curing and stabilizing at 4 ℃ for 24 hours. And slowly and carefully stripping the cured fibrin/chitosan composite film from the template to ensure the integrity of the film and the stripes, and forming a pattern which is complementary with the micro-pattern of the template on the surface of the fibrin/chitosan composite film.

Planting EMSCs on the surface of the micropatterned fibrin/chitosan composite membrane

In order to verify the effect that the stripes on the surface of other material films can induce the differentiation of the EMSCs into Schwann cell-like cells, the invention uses pancreatin for the EMSCsDigesting, collecting cells, adjusting cell density to about 1 × 10⁵At a concentration of 0.5mL/cm²The cells are planted on the surface of the micropatterned fibrin/chitosan composite membrane (the periphery of the composite membrane is provided with a surrounding ridge for limiting the loss of culture medium and cells) and are placed in a CO2 incubator (37 ℃, 5 percent CO₂Saturated humidity) were cultured in a medium containing a common medium. After 14 days of culture, the EMSCs/micropatterned fibrin/chitosan membrane complex was fixed with 4% paraformaldehyde solution, immunofluorescence staining was performed with antibodies against Schwann cell marker protein S100 and MBP, and the growth of the EMSCs-differentiated Schwann cells on the micropatterned membrane was observed.

3. Culture of rat embryonic neural stem cells

Anesthetizing pregnant 14-16 d SD rat, taking out embryo, collecting cerebral cortex tissue of about 0.5mm × 1mm × 2mm, removing pia mater, and washing twice in serum-free DMEM/F12 mixed culture medium (containing penicillin 200U/mL and streptomycin 200U/mL). Washing the tissue in PBS buffer solution, cutting, digesting with pancreatin, and filtering with screen to obtain single cell suspension. The cells were inoculated into neural stem cell culture medium (DMEM/F12 medium supplemented with 2% B27 mL, 20ng/mL bFGF, 20ng/mL EGF, penicillin and streptomycin each 100U/mL) at an inoculation density of 2X 10⁵one/mL. To ensure proliferation of neural cenospheres. The density of the stem cell ball seed obtained is 2000/mL. And then, carrying out passage once every 1-2 weeks by adopting a mechanical digestion method, and carrying out passage for multiple times. Fixing neurospheres and differentiated cells in 4% paraformaldehyde solution at room temperature for 30min, and performing immunofluorescence staining identification by using a neural stem cell marker protein Nestin antibody. The remaining neural stem cells were used in the following experiments, simulating the process of promoting nerve regeneration in vivo: (1) planting neurospheres or scattered neural stem cells on the surface of a striped (1.0 mu m multiplied by 1.0 mu m) fibrin/chitosan micropattern membrane, culturing by using a neural stem cell culture medium, carrying out immunofluorescence staining by using an antibody of a neural fiber marker protein NF-200 after 21 days, and observing the growth condition of neural fibers along the stripes; (2) neural stem cells were seeded on the EMSCs/fibrin/chitosan micropatterned membrane, and the growth of neural fibers on the cell/streak membrane surface was observed (described below).

4. The neural stem cells are planted in the EMSCs/fibrin/chitosan micro-pattern membrane

Dispersing the cultured secondary neurosphere part, planting on the surface of the EMSCs/PCL micropattern membrane, and placing CO₂In an incubator (37 ℃, 5% CO)₂Saturated humidity) was cultured with DMEM/F12 (containing penicillin 100U/mL and streptomycin 100U/mL) containing 10% fetal bovine serum. After 14 days of culture, fixing the neural stem cell/EMSCs/fibrin/chitosan micropattern membrane compound by using a 4% paraformaldehyde solution, carrying out immunofluorescence double-label staining by using an antibody of the neural cell/Schwann cell marker protein NF-200/MBP, and observing the differentiation of the neural stem cell on the EMSCs/fibrin/chitosan micropattern membrane and the parallel growth of nerve fibers.

5. In vivo transplantation test

(1) Experimental animal and transplantation operation process

Healthy adult male SD rats, weighing 250-300 g, were randomly divided into 5 groups of 10 rats each. Group 1 is the simple sciatic nerve injury group; group 2 is sciatic nerve injury + simple streak-free fibrin/chitosan micropatterned catheter group implanted; group 3 is sciatic nerve injury + transplanted EMSCs striation-free fibrin/chitosan micropatterned catheter/group; group 4 is sciatic nerve injury + implanted simple striated fibrin/chitosan micropatterned catheter group; group 5 is sciatic nerve injury + transplanted EMSCs/striped fibrin/chitosan micropatterned catheter group.

The animal surgery procedure was as follows: 10% chloral hydrate 400mg/kg abdominal cavity anesthesia, femoral posterior median incision, right hind limb middle sciatic nerve exposure. Set up 5 experimental groups: group 1, the muscle and skin were sutured directly after sciatic nerve 6mm was excised; group 2, transplanting pure streak-free fibrin/chitosan micro-pattern catheter at the sciatic nerve defect part; group 3, transplanting EMSCs/fibrin/chitosan micro-pattern non-striated catheter at the sciatic nerve defect part; group 4, transplanting a pure striped fibrin/chitosan micro-pattern catheter group at the sciatic nerve defect part; in group 5, EMSCs/striated fibrin/chitosan micropatterned catheters were implanted at the site of sciatic nerve defect. After the catheter was implanted, the anastomoses were sealed with fibrin glue and the muscles and skin were sutured (see fig. 8 for the procedure). After operation, each group is raised regularly, and sciatic nerve index is measured regularly.

General observation and measurement of sciatic nerve function index (SFI)

Sciatic nerve function index SFI-0 is normal, -100 is complete injury.

Fluorescent gold retrograde tracing

3 rats per group were randomly selected for fluorescent gold (Fluorochrome) retrograde tracing at 15 weeks post-surgery (1 week before the observation endpoint). After anesthesia, the sciatic nerve was again exposed and 2 μ L of 5% fluorescent gold-Phosphate Buffered Saline (PBS) solution was injected 5mm distal to the graft using a microinjector. Equal amounts of fluorogold were also injected at the corresponding sites of the normal lateral sciatic nerve. The surgical incision was sutured and the animals continued to be housed. After 1 week, the dorsal root ganglia corresponding to the left and right sides L4-L6, S1-S2 were removed and sectioned longitudinally with a thickness of 10 μm using a cryomicrotome. 10 serial sections (full view in a low power field due to the very small ganglia) were observed under a fluorescence microscope, and the total number of gold-labeled positive cells in each section was counted using Image-proplus6.0, and the average value was calculated. The bilateral marker positive cell ratio (positive cell ratio ═ number of test-side marker positive cells/number of control-side positive cells × 100%) reflects the degree of nerve regeneration as the positive cell ratio (total number of positive cells positively correlated with the effect of catheter graft repair.

③ morphological Observation and measurement of gastrocnemius muscle

After 16 weeks of animal surgery, bilateral gastrocnemius muscles were completely excised, weighed on an electronic balance (accurate to 0.001g), and the wet-weight ratio of bilateral gastrocnemius muscles of each group was calculated (wet-weight ratio: experimental lateral muscle wet weight/control lateral muscle wet weight × 100%). After weighing, the muscle was fixed with 4% paraformaldehyde, embedded in normal paraffin, and the tissue sections were observed by H-E staining light microscope, and the Leica microscopic image analysis system measured the cross-sectional area of the fibers of the left and right gastrocnemius muscles, respectively, and calculated the cross-sectional area ratio (cross-sectional area ratio of experimental muscle cross-sectional area/control muscle cross-sectional area × 100%).

Morphological observation and measurement of sciatic nerve

After 16 weeks of the animal surgery, the sciatic nerve was cut and exposed from the original incision after anesthesia as above, and the sciatic nerve regeneration was observed. Selecting sciatic nerves transplanted with nerve conduits and non-transplanted nerve conduits after sciatic nerve injury of rats, wherein the sciatic nerves after injury repair comprise a near section (upper section), an injury section (transplanted conduit section) and a far section (lower section) of an injury part, and the nerves are fixed by 4% paraformaldehyde and then are subjected to conventional paraffin embedding and slicing. The direction of the section is longitudinally parallel to the longitudinal axis of the nerve and passes through the proximal segment (upper segment), the injury segment (position of the transplanted catheter) and the distal segment (lower segment), so as to observe the condition that the regenerated nerve fiber passes through the catheter. The tissue sections were stained for h.e, immunohistochemically with antibodies to the neurofibrillary marker protein NF-200, respectively. The nerve fiber density is measured by an image analysis system (the thickest part cross section of the longitudinal section of the sciatic nerve of each group of animals is selected for comparison).

The result shows that the tissue engineering nerve graft provided by the invention can be transplanted through a damaged part, and the nerve regeneration and the recovery of the lower limb motor function are promoted.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A tissue engineering nerve graft is characterized in that a biological material with a striped micropattern introduced on the surface is used as a scaffold, ectodermal mesenchymal stem cells EMSCs are used as seed cells, and the seed cells are inoculated on the scaffold to obtain the tissue engineering nerve graft; the surface of the biological material is imprinted with a stripe-shaped micro pattern by utilizing a micro pattern technology; the micropatterning techniques include, but are not limited to, photolithography, electron beam exposure, or nanoimprinting; the width of the stripe-shaped micro pattern is 1-2 mu m, the interval is 1-2 mu m, and the stripe height is 1-2 mu m; the initial cell density of the EMSCs was 10⁴-10⁵Per cm²。

2. The tissue engineered nerve graft of claim 1, wherein one or more of polydimethylsiloxane, polycaprolactone, chitosan, fibrinogen are used as the biomaterial.

3. The tissue engineered nerve graft of claim 2, wherein the biomaterial comprises chitosan-fibrin, the chitosan-fibrin being obtained by cross-linking chitosan and fibrinogen with a biological cross-linking agent to a cell growth factor, the cell growth factor being one or more of EGF, FGE, NGF, SHH.

4. The tissue engineered nerve graft of claim 3, wherein the biological cross-linking agent comprises genipin or/and glutamine transaminase.

5. The tissue engineered nerve graft of claim 1, wherein the tissue engineered nerve graft is filled with a drug or growth factor slow release material that promotes nerve growth.

6. The tissue engineered nerve graft of claim 5, wherein the nerve growth promoting drug or growth factor sustained release material comprises a drug sustained release system with microspheres, nanoparticles or hydrogel as a carrier.

7. The tissue engineered nerve graft of any one of claims 1-6, wherein the tissue engineered nerve graft is crimped to form a single or multi-layered multi-tunneled nerve conduit.