CN110507857A - A kind of engineered nerve graft and preparation method thereof - Google Patents

A kind of engineered nerve graft and preparation method thereof Download PDF

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CN110507857A
CN110507857A CN201910813397.9A CN201910813397A CN110507857A CN 110507857 A CN110507857 A CN 110507857A CN 201910813397 A CN201910813397 A CN 201910813397A CN 110507857 A CN110507857 A CN 110507857A
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nerve
cell
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emscs
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CN110507857B (en
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陆乃彦
张轩
翁雨燕
余雪健
杨国锋
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Jiangnan University
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Abstract

The invention discloses a kind of engineered nerve grafts and preparation method thereof, belong to biomaterial and tissue engineering technique field.The present invention enables striped that EMSCs maximumlly independently to be induced to be divided into myelin and forms cell (schwann cell), obtain EMSCs/ biomaterial scaffolds compound by the specification of optimization striped.The Three-dimensional cell culture model that this EMSCs/ biomaterial scaffolds compound can not only be used for neural stem cell differentiating in vitro study, nerve fibre growth and myelin formation molecular mechanism can be used as tissue-engineering graft constructed for internal skin grafing and mending nervous system injury again.The nerve regeneration conduit that EMSCs/ micro-patterning biological material film is rolled into cylindric Multiple tunnel formula is used for skin grafing and mending injury of sciatic nerve by the present invention, the result shows that the present invention can be transplanted by damage location, promote nerve regneration and lower extremity motor function to restore, there is good potential applicability in clinical practice and research and development value.

Description

A kind of engineered nerve graft and preparation method thereof
Technical field
The present invention relates to a kind of engineered nerve grafts and preparation method thereof, belong to biomaterial and organizational project Technical field.
Background technique
Stem cell/tissue engineering bracket transplanting is to repair the main policies of neural tissue injury.In central nervous system, greatly The nerve cell of brain is interconnected to form network structure by protrusion.After brain tissue impairment, stem cell/structural transplantation is main Purpose is the new neural network formation of promotion to restore the integration of its information and conduction function, therefore to stem cell/bracket of implantation Requirement be that can promote the formation of neural network；Compared with the information integration function of brain tissue, the major function of spinal cord is to pass through Downlink conductive beam if tractus corticospinalis is by the motor neuron of the Information Conduction of brain to spinal cord, and passes through uplink Sensory conduction Beam, as the sensory information that spinal cord receives is uploaded to brain by fasciculus gracilis and cuneate fascicle.Therefore, after spinal cord injury, stem cell/organizational project Structural transplantation is chiefly to facilitate the regeneration of nerve conduction beam and the collimation growth of nerve fibre and is forward determined by damage location To the myeloid tissue for extending to damage distal end (kinesitherapy nerve) or proximal end (sensory nerve), regenerated nerve fibre is along original Channel growth finally re-establishes Synaptic junction with its target cell.At the same time, the stem cell/tissue engineering bracket transplanted is also Should have the function of that newborn nerve fibre is promoted to form myelin.The repair mechanism of peripheral nerve injury and the repair machine of spinal cord injury Similar, stem cell/tissue engineering bracket (conduit) skin grafing and mending peripheral nerve is made, such as injury of sciatic nerve also promotes nerve Fibers parallel effective regeneration and myelin are formed.Therefore, how physics and chemical modification is carried out to bracket and select suitable kind Daughter cell (can be divided into myelin and form cell) plantation in bracket, be formed with the collimation growth of inducing neural fiber and myelin, It is the key factor for improving stem cell/tissue engineering bracket skin grafing and mending spinal cord or peripheral nerve injury therapeutic effect.
At present stem cell/structural transplantation repair spinal cord and peripheral nerve (such as sciatic nerve) damage mainly by stem cell/ Biomaterial is fabricated to hydrogel or nerve trachea is transplanted to damage defect.Especially when wound causes peripheral nerve longer When defect, nerve trachea transplanting will be the maximally efficient treatment method for repairing neurologic defect.The most of nerves applied at present Although conduit can promote nerve regneration, but the striped due to lacking the parallel ordering growth of guidance nerve fibre, lead to nerve fibre The directionality of extension is lost, so that fiber moves ahead, speed is slow, simultaneously because lacking myelin forms cell, so that regenerating nerve is fine The myelinization of dimension is not complete (forming myelin after relying solely on the remaining endogenous Schwann Cell Increase of the neural broken ends of fractured bone).Therefore, it needs One kind is developed in invention can either be obviously promoted regenerated nervous fibers collimation oriented growth again and can provide myelin formation seed cell, The nerve trachea for promoting the regeneration of internal injuring nerve and myelin to be formed, for transplanting, more effectively repairing nerve damage.
Summary of the invention
The object of the present invention is to provide a kind of engineered nerve grafts, and the life of the micro- pattern of striated is introduced with surface Object material is as bracket, using ecto-mesenchymal stem cell EMSCs as seed cell, seed cell is inoculated on bracket and is obtained Engineered nerve graft.
In one embodiment of the invention, the biomaterial surface utilizes micro- patterning technique stamp micro- figure of striated Case；Micro- patterning technique includes but is not limited to photoetching, electron beam exposure or nano impression
In one embodiment of the invention, the width of the micro- pattern of the striated is 1-2 μm, and spacing is 1-2 μm, item Line height is 1-2 μm.
In one embodiment of the invention, with dimethyl silicone polymer (PDMS), polycaprolactone (PCL), chitosan, One of fibrinogen is a variety of as biomaterial.
In one embodiment of the invention, the biomaterial includes chitosan-fibrin.
In one embodiment of the invention, the chitosan-fibrin is to lead to chitosan and fibrinogen The acquisition of Biological cross-linker crosslinking cell growth factor is crossed, the Porcine HGF is one of EGF, FGE, NGF, SHH Or it is a variety of.
In one embodiment of the invention, Biological cross-linker includes Geniposide or/and glutaminase transaminase (TG).
In one embodiment of the invention, the initial cell density of the EMSCs is 104-105A/cm2。
In one embodiment of the invention, the shape of the engineered nerve graft includes membranaceous.
In one embodiment of the invention, it is more to be curled into single-layer or multi-layer for the engineered nerve graft Tunnel nerve trachea.
In one embodiment of the invention, the engineered nerve graft is filled with promotion nerve growth Drug or growth factor slow-release material.
In one embodiment of the invention, the drug or growth factor slow-release material for promoting nerve growth include micro- Ball, nano particle or hydrogel are the drug sustained release system of carrier.
In one embodiment of the invention, the engineered nerve graft is used for repairing nerve damage.
The present invention also provides the methods for preparing above-mentioned engineered nerve graft, and the method includes following steps It is rapid:
(1) biomaterial scaffolds of surface micro-patterning are prepared；The materials culture and amplification of EMSCs:
(2) the EMSCs kind for obtaining step (1) is planted in the biomaterial scaffolds of micro-patterning.
The present invention also provides a kind of nerve tracheas, using the biomaterial of the surface introducing micro- pattern of striated as bracket, Using ecto-mesenchymal stem cell EMSCs as seed cell, seed cell is inoculated on bracket and obtains engineered nerve Graft, the engineered nerve graft curl into single-layer or multi-layer Multiple tunnel nerve trachea.
In one embodiment of the invention, the width of the micro- pattern of the striated is 1-2 μm, and spacing is 1-2 μm, item Line height is 1-2 μm.
In one embodiment of the invention, with dimethyl silicone polymer, polycaprolactone, chitosan, fibrinogen One of or it is a variety of be used as biomaterial.
The present invention also provides above-mentioned engineered nerve grafts or above-mentioned nerve trachea in preparing medical instrument Application.
Beneficial effects of the present invention:
The nerve regeneration conduit that EMSCs/ micro-patterning biological material film is rolled into cylindric Multiple tunnel formula is used for by the present invention Skin grafing and mending injury of sciatic nerve, the results showed that, the mouse injury of sciatic nerve side sciatic nerve function of impassivity conduit processing Index reached -91 ± 25, and use mouse injury of sciatic nerve side sciatic nerve function index after the nerve trachea processing of the application Up to -37 ± 17.Engineered nerve graft provided by the invention can be transplanted by damage location, promote nerve regneration under Limb motor function recovery has good potential applicability in clinical practice and research and development value.
Detailed description of the invention
Fig. 1: the micro- pattern P DMS film of striated surfaceization, striped specification are 0.5 μ m, 0.5 μm of 0.5 μ m (A), 1.0 μ ms 1.0 1.0 μm of μ ms (B), 1.5 μ m, 1.5 μm of 1.5 μ m (C), 2.0 μ m, 2.0 μm of 2.0 μ m (D).
Fig. 2: the fluorescent staining (S100) after the EMSCs of the PDMS film surface of micro-patterning breaks up to schwann cell is planted Scheme (fluorescence microscope shooting).Striped specification be 0.5 μ m, 0.5 μm of 0.5 μ m (A), 1.0 μ m, 1.0 μm of 1.0 μ m (B), 1.5 μ m, 1.5 μm of 1.5 μ m (C), 2.0 μ m, 2.0 μm of 2.0 μ m (D).
Fig. 3: it plants and expresses schwann cell to cell after schwann cell differentiation in the EMSCs of the PDMS film surface of micro-patterning The Western blotting testing result of marker protein level.
Fig. 4: neural stem cell is divided into nerve cell, the radial growth of nerve fibre in no striped PCL film surface (A)；In striped (1.0 μm of 1.0 μ m, 1.0 μ m) PCL film surface nerve fibre Parallel Growth (B)；In order to show item simultaneously Line and nerve fibre plant the neural stem cell of dispersion in striped (1.0 μm of 1.0 μ m, 1.0 μ m) PCL film surface, it is seen that Nerve fibre grows (C) along striped.
Fig. 5: (bottom snow is prosperous in the micro- pattern P CL film surface Parallel Growth of EMSCs (schwann cell)/striped for nerve cell The immunofluorescence dyeing (fluorescein 488, green) of cell marker protein S100；Upper layer nerve fibre marker protein NF-200 Immunofluorescence dyeing (cy3 red).A: 1.0 μm of 1.0 μ m of striped, 1.0 μ m；B: 2.0 μm of 2.0 μ m of striped, 2.0 μ m.
Fig. 6: the EMSCs (schwann cell) of the injury of sciatic nerve rat animal model/micro- pattern film of striped (conduit) moves Plant operation schematic diagram.
Fig. 7: EMSCs (schwann cell)/striped (1.0 μ m, 1.0 μ m of injury of sciatic nerve rat animal model 1.0 μm) micro- pattern P CL composite membrane transfer operation process.A: sciatic nerve transection；B:EMSCs (schwann cell)/striped (1.0 1.0 μm of 1.0 μ m of μ m) micro- pattern P CL composite membrane (since with genipin cross-linked, film is in navy blue) wraps up the neural broken ends of fractured bone (two With suture connection contraposition is absorbed, the gap 5mm is retained in centre at end), and with the Fibrin Glue sealing broken ends of fractured bone；C: film is curled into and is led Pipe, and Fibrin Glue sealing catheter outer surface is used after being sutured with absorbable suture.
Fig. 8: EMSCs (schwann cell)/striped (1.0 μ m, 1.0 μ m of Rats'Sciatic Nerve Injury animal model 1.0 μm) micro- pattern fibrin/chitosan complex film transfer operation process.A: separating and cuts off sciatic nerve；B:EMSCs (snow Prosperous cell)/striped (1.0 μm of 1.0 μ m, 1.0 μ m) the micro- pattern fibrin/neural broken ends of fractured bone of chitosan complex film package, and With the Fibrin Glue sealing broken ends of fractured bone；C: being curled into conduit for film, and is led after being sutured with absorbable suture with Fibrin Glue sealing Tube outer surface.
Fig. 9: fluorogold sciatic nerve injects dorsal root ganglion nerve Cellular tracking as a result, A-F figure is respectively as follows: normal group (A)；Damage non-transplantation group (B)；Damage transplanting EMSCs (schwann cell)/striped (1.0 μm of 1.0 μ m, 1.0 μ m) micro- pattern PCL composite membrane group (C)；Damage transplanting striped (1.0 μm of 1.0 μ m, 1.0 μ m) micro- pattern P CL composite membrane (cell-free) group (D)；Damage transplanting EMSCs (schwann cell)/without striped PCL composite membrane group (E)；Damage transplanting is without striped PCL composite membrane (without thin Born of the same parents) group (F).
Figure 10: normal side lower limb gastrocnemius and other each group injury of sciatic nerve side gastrocnemius general appearances compare.A-F figure It is respectively as follows: normal group (A)；Damage non-transplantation group (B)；Damage transplanting EMSCs (schwann cell)/striped (1.0 1.0 μm of μ ms × 1.0 μm) micro- pattern P CL composite membrane group (C)；Damage transplanting striped (1.0 μm of 1.0 μ m, 1.0 μ m) micro- pattern P CL is compound Film (cell-free) group (D)；Damage transplanting EMSCs (schwann cell)/without striped PCL composite membrane group (E)；Damage transplanting is without striped PCL composite membrane (cell-free) group (F).
Figure 11: normal side lower limb gastrocnemius and other each group injury of sciatic nerve side Gastrocnemius Muscle of Cancer cut HE dyeing observation calf Intestines length of muscle fiber: A-F figure is respectively as follows: normal group (A)；Damage non-transplantation group (B)；Damage transplanting EMSCs (schwann cell)/ Striped (1.0 μm of 1.0 μ m, 1.0 μ m) micro- pattern P CL composite membrane group (C)；Damage transplanting striped (1.0 μ m, 1.0 μ m 1.0 μm) micro- pattern P CL composite membrane (cell-free) group (D)；Damage transplanting EMSCs (schwann cell)/without striped PCL composite membrane group (E)；Damage transplanting is without striped PCL composite membrane (cell-free) group (F).
Figure 12: the micro- pattern P CL composite membrane of EMSCs (schwann cell)/striped of Rats'Sciatic Nerve Injury animal model 16 weeks after transfer operation, sciatic nerve materials are carried out after Animal Anesthesia, the nerve by the nerve trachea of transplanting together with far and near end takes It fixes, is splitted conduit (catheter wall originally is by intracorporal tissue reconstruction) along the longitudinal axis out, observe nerve growth feelings in conduit Condition, A:EMSCs (schwann cell)/striped (1.0 μm of 1.0 μ m, 1.0 μ m) micro- pattern P CL composite membrane transplantation group；B: striped Change (1.0 μm of 1.0 μ m, 1.0 μ m) micro- pattern P CL composite membrane transplantation group (cell-free)；C:EMSCs (schwann cell)/without striped PCL composite membrane transplantation group；D: without striped PCL composite membrane transplantation group (cell-free).
Figure 13: the damage location sciatic nerve tissues slice HE of transplanting and not Nerve Graft conduit dyes observed result, A: The micro- pattern P CL composite membrane transplantation treatment group of EMSCs (schwann cell)/striped, damages the ischium of proximal end after injury of sciatic nerve Nerve, which has been grown, reaches distal side into nerve trachea and by conduit, and absorbable suture has no residual cavity after absorbing；B: ischium The non-treatment group of neurotrosis, it is seen that the serious degeneration of nerve fibre, rarely seen a small amount of regenerated nervous fibers (figure upside), cavity is that can inhale It receives after suture absorbs and is remained.
Figure 14: normal side lower limb sciatic nerve and other each group injury of sciatic nerve positions (conduit including transplanting) are longitudinal Tissue is cut, and nerve fibre marker protein NF-200 immunohistochemical staining observes nerve fiber regeneration situation: A-F figure difference Are as follows: normal group (A)；Damage non-transplantation group (B)；Damage transplanting EMSCs (schwann cell)/striped (1.0 μ m, 1.0 μ m 1.0 μm) micro- pattern P CL composite membrane group (C)；Damage transplanting striped (1.0 μm of 1.0 μ m, 1.0 μ m) micro- pattern P CL composite membrane (nothing Cell) group (D)；Damage transplanting EMSCs (schwann cell)/without striped PCL composite membrane group (E)；Damage transplanting is compound without striped PCL Film (cell-free) group (F).
Figure 15: lower limb sciatic nerve and other each group injury of sciatic nerve position (conduit transplantation site) midpoint cross section groups Slice is knitted, nerve fibre marker protein NF-200 immunohistochemical staining observes the density of regenerated nervous fibers: A-F figure difference Are as follows: normal group (A)；Damage non-transplantation group (B)；Damage transplanting EMSCs (schwann cell)/striped (1.0 μ m, 1.0 μ m 1.0 μm) micro- pattern P CL composite membrane group (C)；Damage transplanting striped (1.0 μm of 1.0 μ m, 1.0 μ m) micro- pattern P CL composite membrane (nothing Cell) group (D)；Damage transplanting EMSCs (schwann cell)/without striped PCL composite membrane group (E)；Damage transplanting is compound without striped PCL Film (cell-free) group (F).
Specific embodiment
The optimum choice of 1 striped of embodiment
1. the micro- pattern production of material surface
Using electron beam exposure and nanometer embossing.First in the silicon chip surface spin coating poly-methyl methacrylate of 3 × 3cm Ester (PMMA) film, applying electronic beam exposure technique are micro- in PMMA film surface etched width, spacing and highly equal stripe Pattern；Using the substrate of above-mentioned micro-patterning as template, by the base-material of dimethyl silicone polymer (PDMS) and curing agent according to 10:1 Ratio mix and be added dropwise in reticle surface (0.5mL/cm2), 60 DEG C of dry 4h in vacuum oven are placed in, PDMS is in template table Face freezing film removes PDMS film from template, and surface is the pattern formed with the micro- pattern complementary of template (see Fig. 1).It is real It tests and uses striped wide, contour, equidistant, specification includes 0.5 μm of 0.5 μ m, 0.5 μ m (0.5 μm), 1.0 μ m, 1.0 μ m 1.0 μm (1.0 μm), 1.5 μm of 1.5 μ m, 1.5 μ m (1.5 μm), 2.0 μm of 2.0 μ m, 2.0 μ m (2.0 μm), 2.5 2.5 μm of μ ms × 2.5 μm (2.5 μm), 3.0 μm of 3.0 μ m, 3.0 μ m (3.0 μm).
Materials, culture, amplification and the identification of 2.EMSCs
SD rat (80-100g) is through 10% chloraldurate (330g/kg) intraperitoneal injection of anesthesia, whole skin disinfection, in nothing Medial canthal is upwardly into along nasal cavity through nostril under the conditions of bacterium and cuts off skin and nasal bone, exposes mucous membrane of nasal septum, 1/3 nasal septum under clip It sets in PBS buffer solution, removes holostrome schneiderian membrance.(contained after taking out SD rat schneiderian membrance with serum-free DMEM/F12 mixed culture medium Penicillin 200U/mL and streptomysin 200U/mL) rinsing remove bloodstain three times, be placed in containing 10% fetal calf serum DMEM/F12 training It supports base (i.e. common complete medium, 100U/mL containing penicillin and streptomysin 100U/mL), is sufficiently shredded with eye scissors, at 37 DEG C 0.25% pancreatin digests 15min, and centrifugation abandons after supernatant and is inoculated in Corning culture bottle with by cell and small tissue blocks, in CO2Training Support in case (37 DEG C, 5%CO2, saturated humidity) and culture.The new DMEM/ containing 10% fetal calf serum is supplemented after cell culture 3 days F12 culture medium.It changes the liquid once in half every three days later, is digested, passed on when cell is paved with bottom of bottle.
By the 5th generation cell inoculation in 24 well culture plates.Respectively with marker protein vimentin, Nestin of EMSCs, CD133, CD44 and antibody carry out immunofluorescence dyeing, identify that the cell that culture obtains is EMSCs.Operating procedure is as follows: cell After 4% paraformaldehyde solution is fixed, 37 DEG C in 0.25%TritonX-100 and 3% bovine serum albumin(BSA) (BSA) mixed liquor 30min is closed, 4 DEG C of incubation 12h of first antibody are incubated for 1h with the corresponding secondary antibody of Cy3 label after PBS buffer solution rinsing at room temperature, PBS is rinsed 3 times, and Hoc hest33342 redyes nucleus, PBS buffer solution rinsing, neutral glycerine mounting, in Leica fluorescence microscopy Under the microscope and piece is taken the photograph, negative control replaces first antibody with PBS buffer solution, remaining step is same as above.Remaining cell is for following Experiment.
3. the optimum choice of film surface striped
Standard of the EMSCs to the effect that the induction of schwann cell like cell is broken up as striped optimum choice is induced using striped. 5th generation EMSCs of above-mentioned culture identification is digested with pancreatin, collecting cell adjustment cell density is about 1 × 105A/mL, with 0.5mL/cm2Plantation amount plantation in the micro-patterning being laid in culture plate PDMS film surface (surrounding have paraffin enclose the ridge limit Culture medium and cell processed are lost).EMSCs/ micro-patterning PDMS film is placed in CO2In incubator (37 DEG C, 5%CO2, it is saturated wet Degree) it is cultivated with the DMEM/F12 (100U/mL containing penicillin and streptomysin 100U/mL) containing 10% fetal calf serum.EMSCs had been both after 2h It is attached at micro-patterning PDMS film surface.Hereafter the new DMEM/F12 culture medium containing 10% fetal calf serum is filled up in culture plate Continue to cultivate, change the liquid once in half every three days.Cell/micro- pattern membrane complex is fixed with 4% paraformaldehyde solution after 14 days, is used The antibody of schwann cell marker protein S100 and MBP carry out immunofluorescence dyeing, and it is prosperous thin to avenging on micro- pattern film to observe EMSCs The case where born of the same parents break up.With Western blotting method detection schwann cell marker protein relative amount, more several Line, to schwann cell differentiation effect, selects pattern of the strongest striped of inducibility as modified biological stock support to EMSCs. During above-mentioned test, only with the DMEM/F12 culture medium culture EMSCs for containing 10% fetal calf serum, any inducer is not added, To obtain the induction differentiation effect of striped single factor test.There is the biological material film plantation of the specification striped with surface or do not plant Timbering material of the EMSCs as production nerve trachea.
4. interpretation of result
The immunofluorescence dyeing of schwann cell marker protein S100 and MBP the result shows that, EMSCs kind is planted in various specifications item Line PDMS film surface, with the DMEM/F12 culture medium culture for containing 10% fetal calf serum, cellular morphology is schwann cell sample, along item Line is arranged in parallel.Different size striped, the form and staining power of the cell of film surface have differences (see Fig. 2)；With Western blotting method detects the relative amount of schwann cell marker protein, and more several stripeds are to EMSCs Xiang Xuewang Cell differentiation effect, it was concluded that the energy that the striped induction EMSCs of 1.0 μm of 1.0 μ m, 1.0 μ m breaks up to schwann cell Power is most strong (being shown in Table 1, Fig. 3).Estriate PDMS film inducibility is most weak.Therefore, 1.0 μ ms are all made of in the test below The striped film of 1.0 1.0 μm of μ ms is as cell growth substrate and nerve trachea material.
Compared with 1 striped of table induces the relative amount (with the ratio of Actin) of EMSCs expression schwann cell marker protein
Illustrate: the relative amount of schwann cell the marker protein MBP and S100 of 1 μm of striped group are obviously higher than other each groups (p < 0.05, n=3)
The practical application of 2 micro-patterning PCL film of embodiment
1. preparing the PCL film of surface micro-patterning
Using electron beam exposure and nanometer embossing.First in the silicon chip surface spin coating poly-methyl methacrylate of 3 × 3cm Ester (PMMA) film, striped of the applying electronic beam exposure technique in 1 μm of PMMA film surface etched width, 1 μm of spacing, 1 μm of height The micro- pattern of type；Using the substrate of above-mentioned micro-patterning as template, the base-material of PDMS and curing agent are mixed simultaneously according to the ratio of 10:1 It is added dropwise in reticle surface, is placed in 60 DEG C of dry 4h in vacuum oven, PDMS is in reticle surface freezing film, by PDMS film from mould It is removed in version, surface is the pattern formed with the micro- pattern complementary of template；It, will using the PDMS film of above-mentioned micro-patterning as template 20% PCL dichloromethane solution mixing is added dropwise in PDMS reticle surface (0.5mL/cm2), it is placed in vacuum oven dry 1h, PCL remove PCL film in PDMS reticle surface freezing film from template, and surface is formed and the micro- pattern complementary of template Pattern.
The materials culture and amplification and identification of 2.EMSCs
SD rat (80-100g) is through 10% chloraldurate (330g/kg) intraperitoneal injection of anesthesia, whole skin disinfection, in nothing Medial canthal is upwardly into along nasal cavity through nostril under the conditions of bacterium and cuts off skin and nasal bone, exposes mucous membrane of nasal septum, 1/3 nasal septum under clip It sets in PBS buffer solution, removes holostrome schneiderian membrance.(contained after taking out SD rat schneiderian membrance with serum-free DMEM/F12 mixed culture medium Penicillin 200U/mL and streptomysin 200U/mL) rinsing remove bloodstain three times, be placed in containing 10% fetal calf serum DMEM/F12 training It supports base (i.e. common complete medium, 100U/mL containing penicillin and streptomysin 100U/mL), is sufficiently shredded with eye scissors, at 37 DEG C 0.25% pancreatin digests 15min, and centrifugation abandons after supernatant and is inoculated in Corning culture bottle with by cell and small tissue blocks, in CO2Training Support in case (37 DEG C, 5%CO2, saturated humidity) and culture.The new DMEM/ containing 10% fetal calf serum is supplemented after cell culture 3 days F12 culture medium.It changes the liquid once in half every three days later, is digested, passed on when cell is paved with bottom of bottle.
By the 5th generation cell inoculation in 24 well culture plates.Marker protein vimentin, Nestin, C of EMSCs are used respectively D133 and CD44 and antibody carry out immunofluorescence dyeing, identify that the cell that culture obtains is EMSCs.Operating procedure is as follows: cell After 4% paraformaldehyde solution is fixed, 37 DEG C in 0.25%TritonX-100 and 3% bovine serum albumin(BSA) (BSA) mixed liquor 30min is closed, 4 DEG C of incubation 12h of first antibody are incubated for 1h with the corresponding secondary antibody of Cy3 label after PBS buffer solution rinsing at room temperature, PBS buffer solution rinses 3 times, and Hoc hest33342 redyes nucleus, and PBS buffer solution rinses, and neutral glycerine mounting is glimmering in Leica Viewed under light microscopy simultaneously takes the photograph piece, and negative control replaces first antibody with PBS buffer solution, remaining step is same as above.Remaining cell is used In following experiment.
3.EMSCs kind is planted in the PCL film surface of micro-patterning
Above-mentioned EMSCs is digested with pancreatin, collecting cell adjustment cell density is about 1 × 105A/mL, with 0.5mL/cm2 Density plantation on the PCL film of above-mentioned micro-patterning (surrounding have paraffin enclose the ridge limitation culture medium and cell be lost), set CO2Training Support in case (37 DEG C, 5%CO2, saturated humidity) and the DMEM/F12 culture medium culture for containing 10% fetal calf serum is used, culture is changed every three days Liquid is primary.The micro- pattern membrane complex of EMSCs/ is fixed with 4% paraformaldehyde solution after culture 14 days, with schwann cell marker protein The antibody of S100 and MBP carries out immunofluorescence dyeing, growing state of the schwann cell that observation EMSCs breaks up in micro- pattern film.
4. the culture of rat embryo neural stem cell
Embryo will be taken out after pregnant 14-16 d SD rat anesthesia, takes two sides brain cortical tissue size about 0.5mm × 1mm Pia mater is rejected completely, is put into serum-free DMEM/F12 mixed culture medium (200U/mL containing penicillin and streptomysin by × 2mm Twice of cleaning in 200U/mL).The group taken is woven in PBS buffer solution wash, shred, pancreatin digestion, the screen to filtrate, be made Single cell suspension.Be seeded in nerve stem cell culture medium (in DMEM/F12 culture medium add 2%B 27mL, 20ng/mL bFGF, 20ng/mL EGF, penicillin and each 100U/mL of streptomysin), inoculum density is 2 × 105A/mL.In order to ensure neural thousand cells The proliferation of ball.Obtained stem cell sphere inoculum density is 2000/mL.Then one was passed on every 1~2 week using mechanical digestion method It is secondary, repeatedly passed on.Nerve ball and noble cells fix 30min with 4% paraformaldehyde solution at room temperature, use neural stem cell Marker protein Nestin antibody carries out immunofluorescence dyeing identification.Remaining neural stem cell is used for following experiment, promotees in analogue body Into nerve regeneration: (1) by nerve ball or the plantation of the neural stem cell of dispersion in striped (1.0 μm of 1.0 μ m, 1.0 μ m) PCL film surface, with being immunized with the antibody of nerve fibre marker protein NF-200 after nerve stem cell culture medium culture 21 days The case where Fluorescent Staining Observation nerve fibre is grown along striped is (see Fig. 4)；(2) neural stem cell is planted micro- in EMSCs/PCL Pattern film observes nerve fibre in cell/striped film surface growing state.
5. neural stem cell is planted in the micro- pattern film of EMSCs/PCL
Plantation sets CO in above-mentioned EMSCs/PCL composite film surface after the neural ball portion of the secondary of above-mentioned culture is broken up2Training Support in case (37 DEG C, 5%CO2, saturated humidity) and with nerve stem cell culture medium culture.It is molten with 4% paraformaldehyde after culture 14 days Liquid fixes the micro- pattern membrane complex of neural stem cell/EMSCs/, with nerve cell/schwann cell marker protein NF-200/MBP Antibody carries out immunofluorescence double staining dyeing, and observation neural stem cell is broken up on the micro- pattern film of EMSCs/ and nerve fibre is flat Row growing state (Fig. 5).
6. transplant experiment in body
(1) experimental animal and transfer operation process
Healthy adult male SD rat 50,250~300g of weight, it is randomly divided into 5 groups, every group 10.1st group is Simple injury of sciatic nerve group；2nd group is injury of sciatic nerve+transplanting merely without striped PCL conduit group；3rd group is ischium mind Through damage+transplanting EMSCs/ without striped PCL conduit/group；4th group has merely striped PCL conduit for injury of sciatic nerve+transplanting Group；5th group has striped PCL conduit group for injury of sciatic nerve+transplanting EMSCs/.
Animal surgery process is as follows: 10% chloraldurate 400mg/kg intraperitoneal anesthesia, stock rear portion median incision, after exposure is right Limb middle section sciatic nerve.1st group, muscle and skin are directly sutured after cutting off sciatic nerve 6mm；2nd group, in Sciatic It transplants merely without striped PCL conduit at position；3rd group, at Sciatic position, transplanting EMSCs/ is without striped PCL conduit；4th Group, at Sciatic position, transplanting has merely striped PCL conduit group；It 5th group, is transplanted at Sciatic position EMSCs/ has striped PCL conduit.Use Fibrin Glue sealing previous anastomotic after conduit transplanting, suture muscle and skin (see Fig. 6,7, 8).Postoperative each group conventinal breeding, periodic measurement sciatic nerve index.
(2) conduit skin grafing and mending neurotrosis Indexes of Evaluation Effect
1. ordinary circumstance observation and sciatic nerve function index (SFI) measurement
Postoperative normal observation rat diet, foot ulcers, limb activity and wound healing situation.Sciatic nerve is measured weekly Function index (SFI): the both ends open trogue of one long 60cm of production, width 10cm, high 20cm, by 70g/m2Blank sheet of paper is cut into and trogue It is isometric it is wide after be laid on slot bottom.After rats with bilateral hind leg is dipped in double ankle-joint colorings with pigment, rat is put in one end of slot, is made It is voluntarily walked to another party of slot, and every side hind leg leaves 5~6 footmarks.Clearly footmark measures just selection trace respectively Normal foot (N) and 3 indexs for hurting parapodum (E): A:PL (footmark length)；B:TS (toes width)；C:IT (intermediate toes width). Above-mentioned index is substituted into Bain formula, calculates 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 be it is normal, -100 for completely damage.Each group ischium mind after animal surgery 16 weeks It is damaged side sciatic nerve function index (SFI) measurement result and is shown in Table 2.
2 each group injury of sciatic nerve side sciatic nerve function index (SFI) of tableCompare
1st group 2nd group 3rd group 4th group 5th group
-91±25 -77±31 -68±19 -57±23 -37±17
Illustrate: the 5th group of damage side sciatic nerve function index is obviously higher than other each groups (p < 0.05, n=9)
2. fluorogold retrograde tracing
3 rats are selected at random for every group carried out fluorogold retrograde tracing (1 week before observation terminal) in postoperative 15 weeks.After anesthesia Sciatic nerve is exposed again, injects 5% fluorogold-phosphate buffer (PBS) with micro syringe at the 5mm of graft distal end 2 μ L of solution.Equally inject the fluorogold of isodose in normal side sciatic nerve corresponding position.Suture operation notch, animal continue to raise It supports.The corresponding L4-L6 of left and right sides is taken out after 1 week, S1-S2 dorsal root ganglion carries out longitudinal section, thickness with freezing microtome It is 10 μm.Under fluorescence microscope respectively at observation 10 open serial section (due to neuromere very little, can be in a low-power field Interior observation overall picture), every is counted with Image-proPlus6.0 and is sliced the positive cell sum of interior fluorogold label, and is calculated flat Mean value.Bilateral label positive cell ratio (positive cell ratio=experimental side label positive cell number/control sides positive cell number × 100%) with positive cell ratio reflection nerve regneration degree, (positive cell sum is positively correlated with conduit skin grafing and mending effect, knot Fruit is shown in Table 3, Fig. 9).
3 each group injury of sciatic nerve side back root neural section fluorogold of table marks positive cell number and normal side cell number ratioCompare
1st group 2nd group 3rd group 4th group 5th group
0.07±0.02 0.13±0.07 0.21±0.15 0.37±0.11 0.57±0.12
Illustrate: the 5th group of damage pleural ganglion positive cell number and normal side ratio obviously higher than other each groups (p < 0.05, n=9)
3. the weight in wet base of gastrocnemius is measured and morphological observation
After animal surgery 16 weeks, anesthetized animal completely cuts bilateral gastrocnemius, and electronic balance weighing (is accurate to 0.001g), calculate groups of animals bilateral wet weight of gastrocnemius muscle ratio (weight in wet base ratio=experimental side muscle weight in wet base/control sides muscle weight in wet base × 100%) 4, be the results are shown in Table.Muscle is fixed with 4% paraformaldehyde solution after weighing, routine paraffin wax embedding, histotomy difference Make H-E dyeing om observation, Leica microimage analysing system measures left and right sides sura length of muscle fiber respectively, and calculates Sectional area ratio (sectional area ratio=experimental side muscle sectional area/control sides muscle sectional area × 100%), the results are shown in Table 5, Figure 10, Figure 11.
4 each group injury of sciatic nerve side wet weight of gastrocnemius muscle of table and normal side wet weight of gastrocnemius muscle ratioCompare
1st group 2nd group 3rd group 4th group 5th group
0.19±0.07 0.31±0.13 0.41±0.13 0.63±0.09 0.77±0.18
Illustrate: the 5th group of wet weight of gastrocnemius muscle ratio is obviously higher than other each groups (p < 0.05, n=9)
5 each group injury of sciatic nerve side sura length of muscle fiber of table and normal side sura length of muscle fiber ratioCompare
1st group 2nd group 3rd group 4th group 5th group
0.21±0.09 0.29±0.13 0.47±0.17 0.75±0.12 0.87±0.23
Illustrate: the 5th group of sura length of muscle fiber ratio is obviously higher than other each groups (p < 0.05, n=9)
4. morphological observation and the metering of sciatic nerve
After animal surgery 16 weeks, sciatic nerve is ibid cut and exposes from former notch after method anesthesia, observation sciatic nerve is again Raw situation (Figure 12).Nerve trachea and non-Nerve Graft conduit are transplanted after choosing Rats'Sciatic Nerve Injury, after injury repair Sciatic nerve, proximal segment (upper section) including damage location, damage section (transfer catheter position) and distal section (lower section), nerve passes through Routine paraffin wax embedding and slice are carried out after 4% paraformaldehyde solution is fixed.Slice direction is longitudinal in parallel with the neural longitudinal axis, is passed through Proximal segment (upper section), damage section (transfer catheter position) and distal section (lower section), to observe feelings of the regenerated nervous fibers by conduit Condition.Histotomy makees H.E dyeing (Figure 13) respectively, carries out immunohistochemistry with the antibody of nerve fibre marker protein NF-200 It dyes (Figure 14).Normal sciatic nerve and other cross-section covering weaves in each group injury of sciatic nerve position (conduit transplantation site) midpoint Slice, nerve fibre marker protein NF-200 immunohistochemical staining observe the density (Figure 15) of regenerated nervous fibers.It is micro- Sem observation and after adopting figure, carries out LI nerve fibers measurement with image analysis system and (chooses groups of animals sciatic nerve longitudinally to cut The most thick section in face is compared), it the results are shown in Table 6.
6 each group injury of sciatic nerve side regenerated nervous fibers cross section quantity of table and normal side cross section quantity ratioCompare
1st group 2nd group 3rd group 4th group 5th group
0.18±0.08 0.21±0.07 0.35±0.17 0.57±0.21 0.69±0.19
Illustrate: the 5th group of damage nervus lateralis fiber number and the ratio of normal side obviously higher than other each groups (p < 0.05, n=9)
Fibrin/chitosan complex film practical application of 3 micro-patterning of embodiment
1. preparing fibrin/chitosan complex film of surface micro-patterning
There is good biocompatibility based on fibrinogen and chitosan, two kinds of material mixing can increase the machinery of composite membrane Intensity, and one or more cells can be crosslinked by Biological cross-linker such as Geniposide or/and glutaminase transaminase (TG) and grown The factor such as EGF, FGE, NGF, SHH etc. put bracket to be built into medicament slow release, further increase its function for promoting nerve regneration Energy.The present invention select first fibrinogen/chitosan complex film as production striped nerve trachea material, plantation or not It is used for internal skin grafing and mending injury of sciatic nerve after planting EMSCs, evaluates application value of the invention.Striped fibrinogen Solution/chitosan complex film manufacturing process is as follows:
The aqueous fibrinogen solution and 2% chitosan-acetic acid solution that compound concentration is 5%, then by fibrinogen solution It is uniformly mixed with chitosan solution 9:1 in mass ratio.Configured solution is added dropwise on the surface being laid in culture plate in advance It is modified with (0.5mL/cm on the PDMS film of 1.0 μm of parallel stripes2, surrounding has paraffin to enclose the ridge to limit fluid loss), liquid flow Fibrin ferment (100U/mL) 50 μ L (5U) is added with micro sprayer after flat, adds 1% Geniposide, 50 μ L after five minutes.It will culture Plate is put into drying box, 37 DEG C of solidifications, and liquid is frozen into gel after 12h.The 50 grams of counterweights that pressurize on gel at this time continue to set 25 Film surface DEG C is dried under vacuum to without working fluid, but keeps film surface wet.Hereafter by 4 DEG C of solidifications in film and template merging refrigerator Stablize for 24 hours.By cured fibrin/chitosan complex film from template slowly carefully removing to guarantee the complete of film and striped Property, fibrin/chitosan complex film surface is the pattern formed with the micro- pattern complementary of template at this time.
2.EMSCs kind is planted in fibrin/chitosan complex film surface of micro-patterning
In order to verify the effect that the equally inducible EMSCs of other materials film surface striped breaks up to schwann cell like cell, The present invention digests above-mentioned EMSCs with pancreatin, and collecting cell adjustment cell density is about 1 × 105A/mL, with 0.5mL/cm2's In fibrin/chitosan complex film surface of above-mentioned micro-patterning, (surrounding, which has, encloses ridge limitation culture medium and cell for density plantation It is lost) set in CO2 incubator (37 DEG C, 5%CO2, saturated humidity) and use culture containing ordinary culture medium.With more than 4% after culture 14 days Polyformaldehyde solution fixes the micro- pattern fibrin/chitosan film compound of EMSCs/, with schwann cell marker protein S100 and MBP Antibody carry out immunofluorescence dyeing, observation EMSCs differentiation schwann cell micro- pattern film growing state.
3. the culture of rat embryo neural stem cell
Embryo will be taken out after pregnant 14-16 d SD rat anesthesia, takes two sides brain cortical tissue size about 0.5mm × 1mm Pia mater is rejected completely, is put into serum-free DMEM/F12 mixed culture medium (200U/mL containing penicillin and streptomysin by × 2mm Twice of cleaning in 200U/mL).The group taken is woven in PBS buffer solution wash, shred, pancreatin digestion, the screen to filtrate, be made Single cell suspension.Be seeded in nerve stem cell culture medium (in DMEM/F12 culture medium add 2%B 27mL, 20ng/mL bFGF, 20ng/mL EGF, penicillin and each 100U/mL of streptomysin), inoculum density is 2 × 105A/mL.In order to ensure neural thousand cells The proliferation of ball.Obtained stem cell sphere inoculum density is 2000/mL.Then one was passed on every 1~2 week using mechanical digestion method It is secondary, repeatedly passed on.Nerve ball and noble cells fix 30min with 4% paraformaldehyde solution at room temperature, use neural stem cell Marker protein Nestin antibody carries out immunofluorescence dyeing identification.Remaining neural stem cell is used for following experiment, promotees in analogue body Into nerve regeneration: (1) by nerve ball or the plantation of the neural stem cell of dispersion in striped (1.0 μm of 1.0 μ m, 1.0 μ m) The micro- pattern film surface of fibrin/chitosan uses nerve fibre marker protein with nerve stem cell culture medium culture after 21 days The case where antibody of NF-200 carries out immunofluorescence dyeing, and observation nerve fibre is grown along striped；(2) neural stem cell is planted In EMSCs/ fibrin/micro- pattern film of chitosan, nerve fibre is observed in cell/striped film surface growing state (following).
4. neural stem cell is planted in EMSCs/ fibrin/micro- pattern film of chitosan
Plantation sets CO in the micro- pattern film surface of above-mentioned EMSCs/PCL after the neural ball portion of the secondary of above-mentioned culture is broken up2 In incubator (37 DEG C, 5%CO2, saturated humidity) and use DMEM/F12 (100U/mL containing penicillin and the chain for containing 10% fetal calf serum Mycin 100U/mL) culture.Neural stem cell/EMSCs/ fibrin/shell is fixed with 4% paraformaldehyde solution after culture 14 days It is double to carry out immunofluorescence with nerve cell/schwann cell marker protein NF-200/MBP antibody for the micro- pattern membrane complex of glycan Label dyeing, observation neural stem cell is broken up on EMSCs/ fibrin/micro- pattern film of chitosan and nerve fibre is parallel Property growth the case where.
5. transplant experiment in body
(1) experimental animal and transfer operation process
Healthy adult male SD rat 50,250~300g of weight, it is randomly divided into 5 groups, every group 10.1st group is Simple injury of sciatic nerve group；2nd group is led without the micro- pattern of striped fibers protein/chitosan merely for injury of sciatic nerve+transplanting Pipe group；3rd group is injury of sciatic nerve+transplanting EMSCs without the micro- pattern conduit/group of striped fibers protein/chitosan；4th group is Injury of sciatic nerve+transplanting has merely the micro- pattern conduit group of striped fibers protein/chitosan；5th group is injury of sciatic nerve+shifting Planting EMSCs/ has the micro- pattern conduit group of striped fibers protein/chitosan.
Animal surgery process is as follows: 10% chloraldurate 400mg/kg intraperitoneal anesthesia, stock rear portion median incision, after exposure is right Limb middle section sciatic nerve.5 experimental groups are set: the 1st group, directly suturing muscle and skin after cutting off sciatic nerve 6mm；2nd group, It transplants at Sciatic position merely without the micro- pattern conduit of striped fibers protein/chitosan；It 3rd group, is lacked in sciatic nerve Position transplanting EMSCs/ fibrin/micro- pattern of chitosan is damaged without streak canal；It 4th group, is transplanted at Sciatic position There is the micro- pattern conduit group of striped fibers protein/chitosan merely；5th group, transplanting EMSCs/ is transplanted at Sciatic position There is the micro- pattern conduit of striped fibers protein/chitosan.Fibrin Glue sealing previous anastomotic is used after conduit transplanting, sutures muscle and skin Skin (surgical procedure is shown in Fig. 8).Postoperative each group conventinal breeding, periodic measurement sciatic nerve index.
(2) conduit skin grafing and mending neurotrosis Indexes of Evaluation Effect
1. ordinary circumstance observation and sciatic nerve function index (SFI) measurement
Postoperative normal observation rat diet, foot ulcers, limb activity and wound healing situation.Sciatic nerve is measured weekly Function index (SFI): the both ends open trogue of one long 60cm of production, width 10cm, high 20cm, by 70g/m2Blank sheet of paper is cut into and trogue It is isometric it is wide after be laid on slot bottom.After rats with bilateral hind leg is dipped in double ankle-joint colorings with pigment, rat is put in one end of slot, is made It is voluntarily walked to another party of slot, and every side hind leg leaves 5~6 footmarks.Clearly footmark measures just selection trace respectively Normal foot (N) and 3 indexs for hurting parapodum (E): A:PL (footmark length)；B:TS (toes width)；C:IT (intermediate toes width). Above-mentioned index is substituted into Bain formula, calculates 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 be it is normal, -100 for completely damage.
2. fluorogold retrograde tracing
3 rats are selected at random for every group carried out fluorogold (Fluorochrome) (1 week before observation terminal) in postoperative 15 weeks Retrograde tracing.Sciatic nerve is exposed after anesthesia again, injects 5% fluorogold-phosphorus with micro syringe at the 5mm of graft distal end 2 μ L of phthalate buffer (PBS) solution.Equally inject the fluorogold of isodose in normal side sciatic nerve corresponding position.Suture operation Notch, animal continue to raise.Take out the corresponding L4-L6 of left and right sides after 1 week, S1-S2 dorsal root ganglion, with freezing microtome into Row longitudinal section, with a thickness of 10 μm.Under fluorescence microscope respectively at observation 10 open serial section (due to neuromere very little, can To observe overall picture in a low-power field), every positive for being sliced interior fluorogold label is counted with Image-proPlus6.0 Total number of cells, and calculate average value.Bilateral marks positive cell ratio (positive cell ratio=experimental side label positive cell number/right According to side positive cell number × 100%) with positive cell ratio reflection nerve regneration degree (positive cell sum and conduit skin grafing and mending Effect is positively correlated.
3. morphological observation and the metering of gastrocnemius
After animal surgery 16 weeks, bilateral gastrocnemius is completely cut, electronic balance is weighed (being accurate to 0.001g), and each group is calculated Animal bilateral wet weight of gastrocnemius muscle ratio (weight in wet base ratio=experimental side muscle weight in wet base/control sides muscle weight in wet base × 100%).By flesh after weighing Meat 4% paraformaldehyde is fixed, routine paraffin wax embedding, and histotomy makees H-E dyeing om observation, Leica micro-image respectively Analysis system measures left and right sides sura length of muscle fiber respectively, and calculates sectional area ratio (sectional area ratio=experimental side muscle section Area/control sides muscle sectional area × 100%).
4. morphological observation and the metering of sciatic nerve
After animal surgery 16 weeks, sciatic nerve is ibid cut and exposes from former notch after method anesthesia, observation sciatic nerve is again Raw situation.Nerve trachea and non-Nerve Graft conduit, the ischium after injury repair are transplanted after choosing Rats'Sciatic Nerve Injury Nerve, proximal segment (upper section), damage section (transfer catheter position) and distal section (lower section) including damage location, nerve is through 4% poly Routine paraffin wax embedding and slice are carried out after formaldehyde is fixed.Slice direction be it is longitudinal in parallel with the neural longitudinal axis, by proximal segment (upper section), Section (transfer catheter position) and distal section (lower section) are damaged, to observe the case where regenerated nervous fibers pass through conduit.Histotomy Make H.E dyeing respectively, carry out immunohistochemical staining with the antibody of nerve fibre marker protein NF-200.Micro- sem observation is simultaneously Figure is adopted, image analysis system carries out LI nerve fibers measurement and (chooses the most thick portion position of groups of animals sciatic nerve longitudinal direction section Cross section is compared).
The result shows that engineered nerve graft provided by the invention can be transplanted by damage location, promote nerve Regeneration and lower extremity motor function restore.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

Claims (10)

1. a kind of engineered nerve graft, which is characterized in that made with the biomaterial that surface introduces the micro- pattern of striated Seed cell is inoculated on bracket and obtains organizational project using ecto-mesenchymal stem cell EMSCs as seed cell for bracket Change nerve graft.
2. engineered nerve graft as described in claim 1, which is characterized in that the surface of the biomaterial utilizes Micro- patterning technique stamp micro- pattern of striated；Micro- patterning technique includes but is not limited to photoetching, electron beam exposure or nanometer pressure Print.
3. engineered nerve graft as claimed in claim 1 or 2, which is characterized in that the micro- pattern of striated Width is 1-2 μm, and spacing is 1-2 μm, and streak height is 1-2 μm.
4. engineered nerve graft as described in claim 1, which is characterized in that with dimethyl silicone polymer, gather oneself One of lactone, chitosan, fibrinogen are a variety of as biomaterial.
5. engineered nerve graft as claimed in claim 4, which is characterized in that the biomaterial includes that shell is poly- Sugar-fibrin, the chitosan-fibrin are that chitosan and fibrinogen is raw by Biological cross-linker crosslinking cell What the long factor obtained, the Porcine HGF is one of EGF, FGE, NGF, SHH or a variety of.
6. engineered nerve graft as claimed in claim 5, which is characterized in that Biological cross-linker includes Geniposide Or/and glutaminase transaminase.
7. engineered nerve graft as described in claim 1, which is characterized in that the initial cell of the EMSCs is close Degree is 104-105A/cm2。
8. engineered nerve graft as described in claim 1, which is characterized in that the engineered nerve-grafting Object is filled with the drug or growth factor slow-release material for promoting nerve growth.
9. engineered nerve graft as claimed in claim 8, which is characterized in that promote drug or the life of nerve growth Long factor slow release material includes the drug sustained release system that microballoon, nano particle or hydrogel are carrier.
10. the engineered nerve graft as described in claim 1-9 is any, which is characterized in that described engineered Nerve graft curls into single-layer or multi-layer Multiple tunnel nerve trachea.
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CN111671975A (en) * 2020-07-01 2020-09-18 江南大学 Composite artificial skin material for repairing skin injury
CN114404653A (en) * 2022-01-18 2022-04-29 中国科学院上海硅酸盐研究所 Multi-cell patterned biological ceramic scaffold and preparation method and application thereof
CN117611584A (en) * 2024-01-22 2024-02-27 广州中大医疗器械有限公司 Tissue engineering peripheral nerve graft culture method and system

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CN105310794A (en) * 2015-02-14 2016-02-10 李贵才 Method for preparing porous artificial nerve conduit with orientation structures on inner walls
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CN109876185A (en) * 2019-03-18 2019-06-14 张海军 A kind of bracket and preparation method thereof that directional induction is neural stem cell differentiating

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CN1580255A (en) * 2004-05-18 2005-02-16 中山大学附属第一医院 Tissue engineered peripheral nerve graft
CN105310794A (en) * 2015-02-14 2016-02-10 李贵才 Method for preparing porous artificial nerve conduit with orientation structures on inner walls
CN106730034A (en) * 2016-11-22 2017-05-31 江苏大学 The artificial nerve graft and preparation method built based on slice type Acellularized valve
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CN111671975A (en) * 2020-07-01 2020-09-18 江南大学 Composite artificial skin material for repairing skin injury
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CN117611584A (en) * 2024-01-22 2024-02-27 广州中大医疗器械有限公司 Tissue engineering peripheral nerve graft culture method and system
CN117611584B (en) * 2024-01-22 2024-04-12 广州中大医疗器械有限公司 Tissue engineering peripheral nerve graft culture method and system

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