CN104623738B

CN104623738B - Tissue engineering nerve graft with suspension fibrous framework and preparation method thereof

Info

Publication number: CN104623738B
Application number: CN201510015281.2A
Authority: CN
Inventors: 杨宇民; 顾晓松; 丁斐; 刘炎; 刘梅; 王勇军
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2015-01-12
Filing date: 2015-01-12
Publication date: 2016-08-24
Anticipated expiration: 2035-01-12
Also published as: CN104623738A

Abstract

The invention discloses a kind of tissue engineering nerve graft with suspension fibrous framework, including fibrous framework in conduit and pipe, it is characterized in that the some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, fibrous framework is hung in conduit by suspension distribution.The present invention utilizes biometric print technology, the built-in microfilament stable and uniform of tissue engineering nerve graft is distributed in the catheter by the support force utilizing suspension, every fibrous framework has independent space, ensure that nerve cell can greatly improve the success rate of operation along fibrous framework oriented growth without the bridge joint made a mistake.

Description

Tissue engineering nerve graft with suspension fibrous framework and preparation method thereof

Technical field

The present invention relates to medical science and biomedical engineering field, be specifically related to a kind of organizational project with suspension fibrous framework Nerve graft and preparation method thereof.

Background technology

The events such as traffic accident in society, industrial accident, motion accident, earthquake, war and clinical operation all can be made Become peripheral nerve injury, clinically but when the neurologic defect of middle and long distance cannot rely on end-to-end stitching to make up neurological deficits Time, would have to rely on graft to bridge reparation.It is best that current nerve autograft the most clinically remains this kind of disease for the treatment of Method.Because for the autologous nerve limited source transplanted, producing new wound, institutional framework and size and be difficult to match and move Plant for reasons such as head of district's phase denervations, cause the biggest limitation that nerve autograft is applied.In recent years, based on life section Learning the organizational engineering grown up with the principle of engineering science and technology is to build nerve-grafting substitute to provide new going out Road.The research using tissue engineering technique repairing nerve damage is concentrated mainly on design and builds new tissue engineering nerve bridge joint dress Putting, current this kind of bridge material is typically individually to use biomaterial, by being processed into the nerve guidance channels of tubular structure, Suitable space and guiding function is provided for neural regeneration.Prepared by Traditional Man nerve graft many employings injection molding method, or adopt The method combined with biometric print technology by Static Spinning technology produces artificial graft's material of nanofiber.China Patent Publication No. CN101829361A discloses a kind of nano-bionic material for tissue repair and preparation method thereof, described nano-bionic material Including nano bionic support and the hydrosol attached to it, in the hydrosol, it is coated with one or more trophic factors and/or thin Born of the same parents.Its described preparation method includes preparing electrospun solution and containing trophic factors and/or the hydrosol solution of cell；Use Static Spinning The prepared nano bionic support of silk；With ink-jet printer, the hydrosol solution containing trophic factors and/or cell is printed to nanometer to imitate The raw first-class step of support.It is engineered around that the Chinese patent literature of Application No. CN200410027221.4 discloses one Nerve graft, uses the pencil Subfilament Structure being made up of microfilament prepared by biodegradation material as support, and its outer surface is coated with One tunic, forms tubular bracket.It is implanted into fibrous framework at conduit, it is therefore intended that guide Scs to migrate under prior art, Be conducive to nerve-selective growth, reduce motion and sensory nerve misorientation grows (as shown in Figure 1).But the transplanting of nerve is again Life is not simple " repairing ", but needs to guide Proximal fiber alignment length to target organ by " bridge ".Due to god Many through fibre number, bridge joint difficulty is big (Fig. 2 is nerve anatomies schematic diagram), and neural transplanting regeneration remains numerous section Institute needs the difficult problem captured.Nerve graft under prior art, in conduit, simple built-in microfilament is difficult to be distributed in uniformly lead Inside pipe, the microfilament as " bridge " is easy to mutually flock together, the most in actual applications, and the effect that orientation guides The most limited, affect result for the treatment of.

Inkjet technology is ink droplet to be ejected into acceptor form image or the untouchable printing technique of word, and it is not only It is applied to ink-jet printer of handling official business, and is used successfully in medical science and biomedical engineering, define biometric print skill Art.Biometric print can be accurately positioned according to schedule, and the scraps of paper of biometric print are designed as the most degradable life of one in theory The thing scraps of paper；The ink of biometric print is designed as the cell solution of speciality in theory or has bioactive trophic factors solution.By this Plant special solution to be ejected on the biodegradable biological scraps of paper.Again the scraps of paper are stacked in certain sequence after printing.Use biological beating Print technology, can be by cell or/and trophic factors be attached to predetermined position accurately.

Along with the research of this technology deepens continuously, research shows that inkjet technology is applicable to print cell, biological support material Material and cell active factor, its application in Organ printing is the most of increasing concern, and this is that organizational project is building fibr tissue New hope is brought with the Successful utilization in organ.

Summary of the invention

It is an object of the invention to provide a kind of tissue engineering nerve with suspension fibrous framework based on biometric print technology Graft and preparation method thereof, described tissue engineering nerve graft is to be prepared by biometric print by degradable polymeric material, Including fibrous framework and suspension in conduit, pipe.Described preparation method is: the accurately different neural three-D space structure of simulation, profit Use biometric print technology, by adjusting the size of nozzle of ink-jet printer, quantity, the distance of nozzle to bottom, booster arteries and veins Rush frequency and work out the control program of specific printing, the nerve-grafting become by polymer material solution 3 D-printing with ink-jet printer Thing.

Another object of the present invention is to provide a kind of fibrous framework surface and/or conduit surfaces externally and internally to be also wrapped on trophic factors And/or the tissue engineering nerve graft with suspension fibrous framework of cell and preparation method thereof, will be above by biometric print The nerve graft that technology prepares is immersed in the nutrient solution containing cell and/or trophic factors, makes nerve graft support Inside or surface and conduit surfaces externally and internally are coated with one or more cells and/or trophic factors, or, utilize biometric print Technology, by design corresponding control software and adjust the size of nozzle of ink-jet printer, quantity, nozzle to powder bed away from From, shower nozzle translational speed and layer interlude, with ink-jet printer by polymer material solution and cell and/or trophic factors Hydrosol solution 3 D-printing shaping.

The concrete technical scheme of the present invention is as follows:

A kind of tissue engineering nerve graft with suspension fibrous framework, including fibrous framework in conduit and pipe, it is characterised in that every The some suspensions that are provided with along fibre length direction of fibrous framework are connected with pipe inner wall, and fibrous framework is hung by suspension distribution In conduit, perfectly even distribution hangs in conduit.

One preferred version of above-mentioned tissue engineering nerve graft, the suspension of every fibrous framework is circumferentially distributed, and not Suspension position and/or the circumference angle of same fibrous framework are different, it is ensured that between suspension and other fibrous framework and other suspension It is not in contact with, and is evenly distributed in conduit, thus fully guide peripheral nerve regeneration.Further, above-mentioned organizational project Multiple micropore being also distributed on the catheter wall of nerve graft, provides space for nerve regneration, tube wall is conducive to mass exchange and blood Pipe regenerates.Preferred microporous aperture is 10-100 μm, porosity 50%-90%.

One preferred version of above-mentioned tissue engineering nerve graft, described conduit diameter 1-9mm, conduit wall thickness is 0.5- 2mm, a diameter of 0.05-0.3mm of fibrous framework in described pipe, the radical of support is 3-30 root, and described suspension is a diameter of 0.05-0.3mm。

Preferably, the quantity of suspension is 2-5 root.

Above-mentioned nerve graft rack surface and/or conduit surfaces externally and internally can also be coated with trophic factors and/or cell, institute State cell to include deriving from autologous or allosome, do thin for schwann cell, fibroblast, mesenchymal stem cells MSCs, Cord blood One or more combination in born of the same parents, skin progenitor cell, the cell density being coated on described conduit and the interior fibrous framework surface of pipe is 1 ×10⁶-1×10⁸/ml；Described trophic factors is Basic Fibroblast Growth Factor (bFGF), brain derived neurotrophic factor (BDNF), the one in NT3 (NT-3), glial cell line-derived neurotrophic (GDNF) or Several, trophic factors amount on nerve graft timbering material is 1 μ g/g-10mg/g.

In above-mentioned nerve graft conduit and pipe, fibrous framework is degradable polymeric material, preferably PLA, PVOH A kind of or the most several mixture in acid, collagen, gelatin, shitosan, sodium alginate, fibroin albumen, keratin.

Present invention also offers the preparation method of a kind of above-mentioned tissue engineering nerve graft with suspension fibrous framework, bag Include following steps:

(1) preparation solution containing nerve graft material is placed in ink-cases of printers；

(2) adjusting printer nozzle needle diameter is 2-200 μm, and syringe needle quantity is 4-16, and the distance of nozzle to bottom is 10- 50mm, booster pulse frequency 1-50v；

(3) by the different neural shape of accurately simulation and three-D space structure, the shape of design organization engineering nerve graft, profit Use biometric print technology, carry out high-true emulation according to neural actual form demand and print, use step (2) parameter to carry out Print, in forming conduit, suspension and pipe, be uniformly distributed the tissue engineering nerve graft of fibrous framework.

(1) preparation is containing the solution of nerve graft material, cell and/or trophic factors solution, is respectively placed in independent print cartridge；

(2) adjusting with the printer nozzle needle diameter being connected equipped with nerve graft material print cartridge is 2-200 μm, and syringe needle quantity is 2-10, the distance of nozzle to bottom is 10-50mm, booster pulse frequency 1-50v； Adjusting the printer nozzle needle diameter being connected with the print cartridge equipped with cell and/or trophic factors is 50-200 μm, and syringe needle quantity is 2-6, the distance of nozzle to bottom is 10-50mm, booster pulse frequency 1-50v；

(3) different neural shape and three-D space structure, the shape of design organization engineering nerve graft, Yi Jishe are accurately simulated Meter cell and/or trophic factors cladding form, work out certain print routine, and threedimensional model preset by printer, uses step (2) parameter prints, and forms equally distributed fibrous framework in conduit, suspension and pipe, internal stent or surface and leads Pipe surfaces externally and internally is coated with one or more cells and/or trophic factors.

(1) preparation solution containing nerve graft material is placed in print cartridge；

(3) different neural shape and three-D space structure, the shape of design organization engineering nerve graft, Yi Jixi are accurately simulated Born of the same parents and/or trophic factors cladding form, work out certain print routine, and threedimensional model preset by printer, uses step (2) ginseng Number prints, and forms equally distributed fibrous framework in conduit, suspension and pipe.

(4) nerve graft that step (3) prepares is immersed in the nutrient solution containing cell and/or trophic factors In, make nerve graft internal stent or surface and conduit surfaces externally and internally be coated with one or more cells and/or nutrition because of Son.

In above-mentioned preparation method, nerve graft material is degradable polymeric material, preferably PLA, polyglycolic acid, A kind of or the most several mixture in collagen, gelatin, shitosan, sodium alginate, fibroin albumen, keratin；Degradable Polymer material solution solvent is selected from acetic acid, oxalic acid, benzoic acid, the aqueous solution of one or more arbitrary proportions in citric acid.

The above-mentioned cell in preparation method includes deriving from autologous or allosome, for schwann cell, fibroblast, marrow One or more combination in mescenchymal stem cell, cord blood stem cell, skin progenitor cell.

The above-mentioned preparation method Middle nutrition factor is Basic Fibroblast Growth Factor (bFGF), brain derived neurotrophic factor (BDNF), the one in NT3 (NT-3), glial cell line-derived neurotrophic (GDNF) or Several.

Above-mentioned tissue engineering nerve graft is used for peripheral nerve defection and spinal cord injury.

Hewlett-Packard's printer 55C ink-jet printer that described ink-jet printer is preferably reequiped, method of modifying is with reference to United States Patent (USP) US7051654, employing cartridge model is HP51626A

Compared with prior art, the method have the advantages that

(1) present invention utilizes biometric print technology, and the support force utilizing suspension is stable by the built-in microfilament of tissue engineering nerve graft It is uniformly distributed in the catheter, has broken the technical bottleneck that conventional process technology such as mould is processed, electrostatic spinning is difficult to.This Inventing described tissue engineering nerve graft, the fibrous framework as nerve growth " bridge " is suspended from conduit by suspension, Every fibrous framework has independent space, it is ensured that nerve cell can be along fibrous framework oriented growth without making a mistake Bridge joint, greatly improves the success rate of operation；

(2) present invention utilizes biometric print technology, can be according to the structure of patient's defect nerve and size, the size that designs a model and knot Structure, precise Printing obtains tissue engineering nerve graft for peripheral nerve defection and the treatment of spinal cord injury；

(3) degradable polymer material used by the nerve graft of the present invention is harmless to human non-toxic, will not bring immunity row Scold, all multi-risk Systems of viral transmission, disease propagation；

(4) nerve graft of the present invention is degraded according to CO2 laser weld process automatic safe, and catabolite is fully absorbed by human body, keeps away Mian Liao immuning tissue reacts；

(5) the biometric print technology that the present invention uses is simple, and cost is relatively low, and production process is safely controllable；

(6) present invention use seed cell relative in prior art use NSC and Deiter's cells for, marrow Mescenchymal stem cell has the stem cell feature such as self, hyperproliferation, and has the potential being divided into Schwann cell, bone Bone marrow-drived mesenchymal stem and Schwann cell abundance, draw materials conveniently, can be easily separated purifying, and is that same body cell of the same race comes Source, without immunological rejection, is more suitable as good seed cell, is used for building engineered nerve, especially for Defect and the reparation of old peripheral nerve defection to thick nerve cord distance.Additionally, mesenchymal stem cells MSCs and Xue Wang Schwann Cells has the function of secreting, expressing neurotrophic factor, compared with prior art, uses organizational project of the present invention god Just can improve, without the neurotrophic factor of the most additional external source, the repairing effect that defect is neural through graft, its preparation technology is with existing Technology is had to compare the easiest；

(7) it is activated protein or peptides due to trophic factors, it is easy to being lost activity by ambient influnence, the present invention utilizes biology Trophic factors is coated on nerve graft conduit and inner tube fibrous framework surface by printing technique immediately, or is printing nerve graft After be soaked in trophic factors solution, be directly used in operation, this can preferably ensure the activity of trophic factors.

Accompanying drawing explanation

Fig. 1 is nerve cell oriented growth schematic diagram.

Fig. 2 is nerve anatomies schematic diagram.

Fig. 3 is the cross sectional representation of tissue-engineering graft constructed of the present invention.

Fig. 4 is the position view of two different fibrous framework suspensions of tissue-engineering graft constructed of the present invention.

Fig. 5 is postoperative 24 weeks compound muscle action potential measurement results of peripheral nerve defect in rats model.(A is answering of normal side Closing MAP measurement result, B is autologous group of compound muscle action potential measurement result, and C is that organizational project of the present invention is moved Plant group compound muscle action potential measurement result, (SF-graft group is this to scheme the comparison that D is compound muscle action potential amplitude Invent described tissue-engineering graft constructed group, autograft group be autologous group and contralateral unoperated side be normal Side)).

Fig. 6 is the spinal motor god of peripheral nerve defect in rats model confocal laser scanning microscope fluorogold retrogradation marker Through unit and DRGs sensory neuron.(a is tissue-engineering graft constructed group of the present invention, and b is autologous group；A is Dorsal root Neuromere sensory neuron (Hoechst lining dye nucleus)；B is that the DiD in the A visual field redyes photo；C is gray nucleus anterior angle Motor neuron；D is the partial enlargement of boxed area in C.Bar=40 μm (A, B, D)；Bar=300 μm (C)).

Fig. 7 is the optical, electrical mirror structure observation of postoperative 24 weeks rat regenerating nerves of peripheral nerve defect in rats model.(A is just Often side sciatic nerve, B is autologous nerve group, and C is tissue-engineering graft constructed group of the present invention；A is Meyer ' s tri-look dye Look, Bar=10 μm；B is anti-NF-200 immunocytochemical stain, Bar=20 μm；C is transmission electron microscope photo, Bar=5 μm).

Detailed description of the invention

Technical scheme is further illustrated below by way of specific embodiment.

The term used in the present invention, except as otherwise noted, typically has those of ordinary skill in the art and is generally understood that Implication.

Below in conjunction with specific embodiment and with reference to data, the present invention is described in further detail.Should be understood that these embodiments are simply In order to demonstrate the invention, rather than limit the scope of the present invention by any way.

In the examples below, the various processes not described in detail and method are conventional methods as known in the art.

Embodiment 1

(1) prepare the benzoic acid solution 50ml (benzoic acid solution concentration is 0.1mol/L) of 3% PLA and be loaded on model and be In the print cartridge of HP51626A；

(2) adjusting printer nozzle needle diameter is 2 μm, and syringe needle quantity is 4, and the distance of nozzle to bottom is 10mm, Booster pulse frequency 1v；

(3) three-D space structure that accurately simulation difference is neural, establishment controls program accordingly, and threedimensional model preset by printer, The shape of design organization engineering nerve graft: tissue engineering nerve graft include conduit and pipe in fibrous framework, as Fig. 3, Shown in Fig. 4, the some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, and fibrous framework leads to Crossing suspension distribution and hang in conduit, the suspension of every fibrous framework is circumferentially distributed, and the company of suspension on different fibrous frameworks Connecing position different with circumference angle, wherein conduit diameter 1mm, wall thickness is 0.5mm, and in pipe, fibrous framework is a diameter of 0.05mm, the radical of fibrous framework is 3, a diameter of 0.05-0.3mm of suspension, and suspension quantity is 2, and fibrous framework is at pipe Inside it is uniformly distributed.Use step (2) parameter to print, form equally distributed fibrous framework in conduit, suspension and pipe.

Or, the accurately different neural three-D space structure of simulation, establishment controls program accordingly, and three-dimensional preset by printer Model, the shape of design organization engineering nerve graft: tissue engineering nerve graft includes fibrous framework in conduit and pipe, often The some suspensions that are provided with along fibre length direction of root fibrous framework are connected with pipe inner wall, and fibrous framework is outstanding by suspension distribution Being hung in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and circumference on different fibrous frameworks Angle is different, wherein conduit diameter 5mm, and wall thickness is 1.2mm, a diameter of 0.15mm of fibrous framework, fibrous framework in pipe Radical be 15, a diameter of 0.15mm of suspension, suspension quantity is 3.Fibrous framework is uniformly distributed in pipe.Use step (2) parameter prints, and forms fibrous framework in conduit, suspension and pipe.

Or, the accurately different neural three-D space structure of simulation, establishment controls program accordingly, and three-dimensional preset by printer Model, the shape of design organization engineering nerve graft: tissue engineering nerve graft includes fibrous framework in conduit and pipe, often The some suspensions that are provided with along fibre length direction of root fibrous framework are connected with pipe inner wall, and fibrous framework is outstanding by suspension distribution Being hung in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and circumference on different fibrous frameworks Angle is different, wherein conduit diameter 9mm, and wall thickness is 2mm, a diameter of 0.3mm of fibrous framework in pipe.Fibrous framework Radical is 30, a diameter of 0.3mm of suspension, and suspension quantity is 5.Fibrous framework is uniformly distributed in pipe.Use step (2) parameter prints, and forms fibrous framework in conduit, suspension and pipe.

According to above-mentioned preparation method, can respectively obtain:

A kind of tissue engineering nerve graft with suspension fibrous framework includes fibrous framework in conduit and pipe, the edge of every fibrous framework Fibre length direction is provided with some suspensions and is connected with pipe inner wall, and fibrous framework hangs in conduit, often by suspension distribution The suspension of root fibrous framework is circumferentially distributed, and on different fibrous frameworks, the link position of suspension is different, wherein with circumference angle Conduit diameter 1mm, wall thickness is 0.5mm, a diameter of 0.05mm of fibrous framework in pipe, and the radical of fibrous framework is 3, The a diameter of 0.05-0.3mm of suspension, suspension quantity is 2, and fibrous framework equally distributed PLA tissue engineering nerve in pipe moves Plant.

Or, a kind of tissue engineering nerve graft with suspension fibrous framework includes fibrous framework in conduit and pipe, often The some suspensions that are provided with along fibre length direction of root fibrous framework are connected with pipe inner wall, and fibrous framework is outstanding by suspension distribution Being hung in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and circumference on different fibrous frameworks Angle is different, wherein conduit diameter 5mm, and wall thickness is 1.2mm, a diameter of 0.15mm of fibrous framework, fibrous framework in pipe Radical be 15, a diameter of 0.15mm of suspension, suspension quantity is 3, fibrous framework pipe in equally distributed PLA group Weaver's journey nerve graft.

Or, a kind of tissue engineering nerve graft with suspension fibrous framework includes fibrous framework in conduit and pipe, often The some suspensions that are provided with along fibre length direction of root fibrous framework are connected with pipe inner wall, and fibrous framework is outstanding by suspension distribution Being hung in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and circumference on different fibrous frameworks Angle is different, wherein conduit diameter 9mm, and wall thickness is 2mm, a diameter of 0.3mm of fibrous framework in pipe.Fibrous framework Radical is 30, a diameter of 0.3mm of suspension, and suspension quantity is 5, and fibrous framework is equally distributed PLA tissue in pipe Engineering nerve graft.

Embodiment 2

(1) prepare the citric acid solution 50ml (citric acid solution concentration is 0.05mol/L) of 5% sodium alginate and be loaded on model and be In the print cartridge of HP51626A；

(2) adjusting printer nozzle needle diameter is 100 μm, and syringe needle quantity is 16, and the distance of nozzle to bottom is 25mm, booster pulse frequency 15v；

(3) three-D space structure that accurately simulation difference is neural, establishment controls program accordingly, and threedimensional model preset by printer, The shape of design organization engineering nerve graft: tissue engineering nerve graft include conduit and pipe in fibrous framework, as Fig. 3, Shown in Fig. 4, the some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, and fibrous framework leads to Crossing suspension distribution and hang in conduit, the suspension of every fibrous framework is circumferentially distributed, and the company of suspension on different fibrous frameworks Connecing position different with circumference angle, wherein conduit diameter 1mm, wall thickness is 0.5mm, and in pipe, fibrous framework is a diameter of 0.05mm, the radical of fibrous framework is 3, a diameter of 0.05-0.3mm of suspension, and suspension quantity is 2, and fibrous framework is at pipe Inside it is uniformly distributed.Use step (2) parameter to print, form fibrous framework in conduit, suspension and pipe.

Or, the accurately different neural three-D space structure of simulation, establishment controls program accordingly, and three-dimensional preset by printer Model, being shaped as of design organization engineering nerve graft: tissue engineering nerve graft includes fibrous framework in conduit and pipe, The some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, and fibrous framework is distributed by suspension Hanging in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and week on different fibrous frameworks Different to angle, wherein conduit diameter 5mm, wall thickness is 1.2mm, and a diameter of 0.15mm of fibrous framework in pipe, fiber props up The radical of frame is 15, a diameter of 0.15mm of suspension, and suspension quantity is 3, and fibrous framework is uniformly distributed in pipe.Use step Suddenly (2) parameter prints, and forms fibrous framework in conduit, suspension and pipe.

Or, the accurately different neural three-D space structure of simulation, establishment controls program accordingly, and three-dimensional preset by printer Model, being shaped as of design organization engineering nerve graft: tissue engineering nerve graft includes fibrous framework in conduit and pipe, The some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, and fibrous framework is distributed by suspension Hanging in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and week on different fibrous frameworks Different to angle, wherein conduit diameter 9mm, wall thickness is 2mm, a diameter of 0.3mm of fibrous framework in pipe.Fibrous framework Radical be 30, a diameter of 0.3mm of suspension, suspension quantity is 5, and fibrous framework is uniformly distributed in pipe.Use step (2) parameter prints, and forms fibrous framework in conduit, suspension and pipe.

According to above-mentioned preparation method, can respectively obtain:

A kind of tissue engineering nerve graft with suspension fibrous framework, including fibrous framework in conduit and pipe, every fibrous framework Being provided with some suspensions to be connected along fibre length direction with pipe inner wall, fibrous framework hangs in conduit by suspension distribution, The suspension of every fibrous framework is circumferentially distributed, and on different fibrous frameworks, the link position of suspension is different with circumference angle, its Middle conduit diameter 1mm, wall thickness is 0.5mm, and a diameter of 0.05mm of fibrous framework in pipe, the radical of fibrous framework is 3 Root, a diameter of 0.05-0.3mm of suspension, suspension quantity is 2, and fibrous framework is equally distributed sodium alginate organizational project in pipe Nerve graft.

Or, a kind of tissue engineering nerve graft with suspension fibrous framework, including fibrous framework in conduit and pipe, The some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, and fibrous framework is distributed by suspension Hanging in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and week on different fibrous frameworks Different to angle, wherein conduit diameter 5mm, wall thickness is 1.2mm, and a diameter of 0.15mm of fibrous framework in pipe, fiber props up The radical of frame is 15, a diameter of 0.15mm of suspension, and suspension quantity is 3, and fibrous framework is equally distributed alginic acid in pipe Sodium tissue engineering nerve graft.

Or, a kind of tissue engineering nerve graft with suspension fibrous framework, including fibrous framework in conduit and pipe, The some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, and fibrous framework is distributed by suspension Hanging in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and week on different fibrous frameworks Different to angle, wherein conduit diameter 9mm, wall thickness is 2mm, a diameter of 0.3mm of fibrous framework in pipe.Fibrous framework Radical be 30, a diameter of 0.3mm of suspension, suspension quantity is 5, fibrous framework pipe in equally distributed sodium alginate Tissue engineering nerve graft.

Embodiment 3

(1) prepare the acetum 50ml (acetum concentration is 0.1mol/L) of 5% shitosan and be loaded on model and be In the print cartridge of HP51626A；

(2) adjusting printer nozzle needle diameter is 50 μm, and syringe needle quantity is 9, and the distance of nozzle to bottom is 40mm, booster pulse frequency 10v；

Or, the accurately different neural three-D space structure of simulation, establishment controls program accordingly, and three-dimensional preset by printer Model, being shaped as of design organization engineering nerve graft: tissue engineering nerve graft includes fibrous framework in conduit and pipe, The some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, and fibrous framework is distributed by suspension Hanging in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and week on different fibrous frameworks Different to angle, wherein conduit diameter 9mm, wall thickness is 2mm, a diameter of 0.3mm of fibrous framework in pipe.Fibrous framework Radical be 30, a diameter of 0.3mm of suspension, suspension quantity is 5.Fibrous framework is uniformly distributed in pipe.Use step (2) parameter prints, and forms fibrous framework in conduit, suspension and pipe.

According to above-mentioned preparation method, can respectively obtain:

Tissue engineering nerve graft include conduit and pipe in fibrous framework, every fibrous framework be provided with some along fibre length direction Root suspension is connected with pipe inner wall, and fibrous framework hangs in conduit by suspension distribution, and the suspension of every fibrous framework is circumference Distribution, and on different fibrous frameworks, the link position of suspension is different with circumference angle, wherein conduit diameter 1mm, wall thickness is 0.5mm, a diameter of 0.05mm of fibrous framework in pipe, the radical of fibrous framework is 3, a diameter of 0.05-of suspension 0.3mm, suspension quantity is 2, and fibrous framework is equally distributed shitosan tissue engineering nerve graft in pipe.

Or, tissue engineering nerve graft include conduit and pipe in fibrous framework, every fibrous framework along fibre length Direction is provided with some suspensions and is connected with pipe inner wall, and fibrous framework hangs in conduit by suspension distribution, and every fiber props up The suspension of frame is circumferentially distributed, and on different fibrous frameworks, the link position of suspension is different with circumference angle, wherein conduit diameter 5mm, wall thickness is 1.2mm, and a diameter of 0.15mm of fibrous framework in pipe, the radical of fibrous framework is 15, and suspension is straight Footpath is 0.15mm, and suspension quantity is 3.Fibrous framework is equally distributed shitosan tissue engineering nerve graft in pipe.

Or, tissue engineering nerve graft include conduit and pipe in fibrous framework, every fibrous framework along fibre length Direction is provided with some suspensions and is connected with pipe inner wall, and fibrous framework hangs in conduit by suspension distribution, and every fiber props up The suspension of frame is circumferentially distributed, and on different fibrous frameworks, the link position of suspension is different with circumference angle, wherein conduit diameter 9mm, wall thickness is 2mm, a diameter of 0.3mm of fibrous framework in pipe.The radical of fibrous framework is 30, and suspension is a diameter of 0.3mm, suspension quantity is 5.Fibrous framework is equally distributed shitosan tissue engineering nerve graft in pipe.

Embodiment 4

(1) prepare the acetum 50ml (acetum concentration is 0.1mol/L) of 5% fibroin albumen and be loaded on model and be In the print cartridge of HP51626A；

(2) adjusting printer nozzle needle diameter is 200 μm, and syringe needle quantity is 16, and the distance of nozzle to bottom is 50mm, booster pulse frequency 50v；

Or, the accurately different neural three-D space structure of simulation, establishment controls program accordingly, and three-dimensional preset by printer Model, being shaped as of design organization engineering nerve graft: tissue engineering nerve graft includes fibrous framework in conduit and pipe, The some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, and fibrous framework is distributed by suspension Hanging in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and week on different fibrous frameworks Different to angle, wherein conduit diameter 9mm, wall thickness is 2mm, a diameter of 0.3mm of fibrous framework in pipe.Fibrous framework Radical be 20, a diameter of 0.3mm of suspension, suspension quantity is 5.Fibrous framework is uniformly distributed in pipe.Use step (2) parameter prints, and forms fibrous framework in conduit, suspension and pipe.

According to above-mentioned preparation method, can respectively obtain:

A kind of tissue engineering nerve graft with suspension fibrous framework, including fibrous framework in conduit and pipe, every fibrous framework Being provided with some suspensions to be connected along fibre length direction with pipe inner wall, fibrous framework hangs in conduit by suspension distribution, The suspension of every fibrous framework is circumferentially distributed, and on different fibrous frameworks, the link position of suspension is different with circumference angle, its Middle conduit diameter 1mm, wall thickness is 0.5mm, and a diameter of 0.05mm of fibrous framework in pipe, the radical of fibrous framework is 3 Root, a diameter of 0.05-0.3mm of suspension, suspension quantity is 2, and fibrous framework is equally distributed fibroin albumen organizational project in pipe Nerve graft.

Or, a kind of tissue engineering nerve graft with suspension fibrous framework, including fibrous framework in conduit and pipe, The some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, and fibrous framework is distributed by suspension Hanging in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and week on different fibrous frameworks Different to angle, wherein conduit diameter 5mm, wall thickness is 1.2mm, and a diameter of 0.15mm of fibrous framework in pipe, fiber props up The radical of frame is 15, a diameter of 0.15mm of suspension, and suspension quantity is 3.Fibrous framework is equally distributed fibroin egg in pipe White tissue engineering nerve graft.

Or, a kind of tissue engineering nerve graft with suspension fibrous framework, including fibrous framework in conduit and pipe, The some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, and fibrous framework is distributed by suspension Hanging in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and week on different fibrous frameworks Different to angle, wherein conduit diameter 9mm, wall thickness is 2mm, a diameter of 0.3mm of fibrous framework in pipe.Fibrous framework Radical be 20, a diameter of 0.3mm of suspension, suspension quantity is 5.Fibrous framework is equally distributed fibroin albumen in pipe Tissue engineering nerve graft.

The fibroblastic preparation of embodiment 5

Take conceived 12.5-14.5d dams, take out tire mouse, with Sterile ophthalmic scissors or blade, mouse embryo trunk is cut into 1mm³Following is broken Block, adds trypsinization, centrifugal, abandons supernatant, adds nutrient solution piping and druming, is dispensed in T75, puts 37 DEG C, 5%CO₂, Saturated humidity incubator is cultivated.Pass on 1:3-1:6 after 3-7 days.Typically pass to heteroproteose cell during the third generation reduce.Development of Mouse Embryos Tire fibroblast is shuttle shape.Using nutrient solution suspension cell, making cell density is 1 × 10⁶-1×10⁸/ml。

The preparation of embodiment 6 autologous bone marrow mesenchymal stem cells (MSCs) solution

(1) cell separation

Extraction experimenter's marrow about 40ml, anticoagulant heparin, note avoiding grumeleuse to be formed as far as possible, marrow is transferred in super-clean bench 2 In 50ml sterile centrifugation tube, respectively by PBS proportional diluted and mix, draw FICOLL10-20ml/ pipe × 4 pipe, tilt Centrifuge tube, is added slowly to FICOLL surface along wall respectively by bone marrow suspension, is allowed to be formed interface clearly, 22 DEG C, and 1350 Rev/min, centrifugal 30 minutes, steadily take out centrifuge tube, by centre tunica albuginea layer sucking-off in super-clean bench, as sterile centrifugation tube In, add PBS to 40ml mixing, 1500 revs/min, centrifugal 5 minutes, abandon supernatant, add MESENCULT nutrient solution 10ml, uniformly, obtained cell suspension 20 μ l, in EP pipe, adds leucocyte dilution 180ul dilution, in vibration in piping and druming Device mixes, and takes 20 μ l and is added on cell counting count board, counting: the mean × 10-E of four-quadrant TCS⁶For total cell number. By 2-5 × 10E⁵/ ware seeds cells in culture dish, and adds fresh medium, makes final volume reach 7-8ml, puts into and incubate Case.Within the most every 3 days, changing liquid once, half amount changes liquid every time, until the cell growth 70-80% reached bottom culture dish passes on. 3-4 days half amounts of cell after passing on change liquid 1 time, and cell growth area reaches 80-90% bottom culture dish and need to pass on again Through the MSCs of washing after directly collecting MSCs during passage or collecting defrosting, add containing 20% albuminous life It is standby that cell suspension made by reason salt solution.Making cell density is 1 × 10⁶-1×10⁸/ml。

MSCs needs to carry out flow cytometry cell type before being used for assisting treatment；Chromosome detection is got rid of dliploid and is become Different；Infusion moves ahead pathogeny detection；Cell suspension skin test.

The preparation of embodiment 7 Schwann cell solution

Take the SD rats with bilateral sciatic nerve of 10 new lives 2～3d, divest perilemma epineurium, be cut into about 1cm³The fragment of size, uses 0.03% clostridiopetidase A and 0.25% trypsase are pressed 1:2 and neural fragment are digested 12min, add on a small quantity containing 10% hyclone DMED culture medium carefully blows and beats cell and tissue block, replants and cultivates in culture dish.Snow is identified with S-100 immunohistochemical staining Prosperous cell, calculates its purity in conjunction with Hoechst3342 dyeing, determines cell quantity under the microscope.Suspend with nutrient solution thin Born of the same parents, making cell density is 1 × 10⁶-1×10⁸/ml。

Embodiment 8

Embodiment 3 is printed the nerve graft obtained be immersed in the nutrient solution containing Schwann cell so that it is fiber in conduit and pipe Support surfaces externally and internally is adsorbed with cell, and cell density is 1 × 10⁸/ml。

According to above-mentioned preparation method, can respectively obtain:

A kind of tissue engineering nerve graft with suspension fibrous framework, including fibrous framework in conduit and pipe, every fibrous framework Being provided with some suspensions to be connected along fibre length direction with pipe inner wall, fibrous framework hangs in conduit by suspension distribution, The suspension of every fibrous framework is circumferentially distributed, and on different fibrous frameworks, the link position of suspension is different with circumference angle, its Middle conduit diameter 1mm, wall thickness is 0.5mm, and a diameter of 0.05mm of fibrous framework in pipe, the radical of fibrous framework is 3 Root, a diameter of 0.05-0.3mm of suspension, suspension quantity is 2, and fibrous framework is uniformly distributed in pipe, and rack surface cladding is snowy The shitosan tissue engineering nerve graft of prosperous Schwann Cells.

Or, a kind of tissue engineering nerve graft with suspension fibrous framework, including fibrous framework in conduit and pipe, The some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, and fibrous framework is distributed by suspension Hanging in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and week on different fibrous frameworks Different to angle, wherein conduit diameter 5mm, wall thickness is 1.2mm, and a diameter of 0.15mm of fibrous framework in pipe, fiber props up The radical of frame is 15, a diameter of 0.15mm of suspension, and suspension quantity is 3.Fibrous framework is uniformly distributed in pipe, support table Bread is covered with the shitosan tissue engineering nerve graft of Schwann cell.

Or, a kind of tissue engineering nerve graft with suspension fibrous framework, including fibrous framework in conduit and pipe, The some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, and fibrous framework is distributed by suspension Hanging in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and week on different fibrous frameworks Different to angle, wherein conduit diameter 9mm, wall thickness is 2mm, a diameter of 0.3mm of fibrous framework in pipe.Fibrous framework Radical be 30, a diameter of 0.3mm of suspension, suspension quantity is 5.Fibrous framework is uniformly distributed in pipe, rack surface It is coated with the shitosan tissue engineering nerve graft of Schwann cell.

Embodiment 9

Embodiment 4 is printed the nerve graft obtained and is immersed in the nutrient solution containing autologous bone marrow mesenchymal stem cells (MSCs) In so that it is in conduit and pipe, fibrous framework surfaces externally and internally is adsorbed with cell, and cell density is 1 × 10⁶/ml。

According to above-mentioned preparation method, can respectively obtain:

A kind of tissue engineering nerve graft with suspension fibrous framework, including fibrous framework in conduit and pipe, every fibrous framework Being provided with some suspensions to be connected along fibre length direction with pipe inner wall, fibrous framework hangs in conduit by suspension distribution, The suspension of every fibrous framework is circumferentially distributed, and on different fibrous frameworks, the link position of suspension is different with circumference angle, its Middle conduit diameter 1mm, wall thickness is 0.5mm, and a diameter of 0.05mm of fibrous framework in pipe, the radical of fibrous framework is 3 Root, a diameter of 0.05-0.3mm of suspension, suspension quantity is 2, and fibrous framework is uniformly distributed in pipe, and rack surface is coated with certainly The fibroin albumen tissue engineering nerve graft of body mesenchymal stem cells MSCs.

Or, a kind of tissue engineering nerve graft with suspension fibrous framework, including fibrous framework in conduit and pipe, The some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, and fibrous framework is distributed by suspension Hanging in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and week on different fibrous frameworks Different to angle, wherein conduit diameter 5mm, wall thickness is 1.2mm, and a diameter of 0.15mm of fibrous framework in pipe, fiber props up The radical of frame is 15, a diameter of 0.15mm of suspension, and suspension quantity is 3.Fibrous framework is uniformly distributed in pipe, support table Bread is covered with the fibroin albumen tissue engineering nerve graft of autologous bone marrow mesenchymal stem cells.

Or, a kind of tissue engineering nerve graft with suspension fibrous framework, including fibrous framework in conduit and pipe, The some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, and fibrous framework is distributed by suspension Hanging in conduit, the suspension of every fibrous framework is circumferentially distributed, and the link position of suspension and week on different fibrous frameworks Different to angle, wherein conduit diameter 9mm, wall thickness is 2mm, a diameter of 0.3mm of fibrous framework in pipe.Fibrous framework Radical be 20, a diameter of 0.3mm of suspension, suspension quantity is 5.Fibrous framework is uniformly distributed rack surface bag in pipe It is covered with the fibroin albumen tissue engineering nerve graft of autologous bone marrow mesenchymal stem cells.

Embodiment 10

(2) adjusting the printer nozzle needle diameter being connected with equipped with shitosan print cartridge is 50 μm, and syringe needle quantity is 6, spray Mouth is 40mm to the distance of bottom, booster pulse frequency 10v；Adjust with equipped with schwann cell (cell density is 1 × 10⁸/ ml) print cartridge connect printer nozzle needle diameter be 150 μm, syringe needle quantity is 3, nozzle to bottom away from From for 40mm, booster pulse frequency 10v；

(3) three-D space structure that accurately simulation difference is neural, the shape of design organization engineering nerve graft, and cell bag Covering form, establishment controls program accordingly, and printer presets threedimensional model, the shape of design organization engineering nerve graft: group Weaver's journey nerve graft include conduit and pipe in fibrous framework, every fibrous framework be provided with some along fibre length direction Suspension is connected with pipe inner wall, and fibrous framework hangs in conduit by suspension distribution, and the suspension of every fibrous framework is that circumference is divided On cloth, and different fibrous frameworks, the link position of suspension is different with circumference angle, and wherein conduit diameter 1mm, wall thickness is 0.5mm, a diameter of 0.05mm of fibrous framework in pipe, the radical of fibrous framework is 3, a diameter of 0.05-of suspension 0.3mm, suspension quantity is 2, and fibrous framework is uniformly distributed in pipe.Using step (2) parameter to print, formation is led Fibrous framework in pipe, suspension and pipe, rack surface is coated with schwann cell.

Or, the accurately different neural three-D space structure of simulation, the shape of design organization engineering nerve graft, and Cell cladding form, establishment controls program accordingly, and printer presets threedimensional model, the shape of design organization engineering nerve graft Shape: tissue engineering nerve graft includes fibrous framework in conduit and pipe, being provided with along fibre length direction of every fibrous framework Some suspensions are connected with pipe inner wall, fibrous framework by suspension distribution hang in conduit, the suspension of every fibrous framework in On circumferentially distributed and different fibrous framework, the link position of suspension is different with circumference angle, wherein conduit diameter 5mm, wall Thickness is 1.2mm, and a diameter of 0.15mm of fibrous framework in pipe, the radical of fibrous framework is 15, and suspension is a diameter of 0.15mm, suspension quantity is 3.Fibrous framework is uniformly distributed in pipe.Using step (2) parameter to print, formation is led Fibrous framework in pipe, suspension and pipe, rack surface is coated with schwann cell.

Or, the accurately different neural three-D space structure of simulation, the shape of design organization engineering nerve graft, and Cell cladding form, establishment controls program accordingly, and printer presets threedimensional model, the shape of design organization engineering nerve graft Shape: tissue engineering nerve graft includes fibrous framework in conduit and pipe, being provided with along fibre length direction of every fibrous framework Some suspensions are connected with pipe inner wall, fibrous framework by suspension distribution hang in conduit, the suspension of every fibrous framework in On circumferentially distributed and different fibrous framework, the link position of suspension is different with circumference angle, wherein conduit diameter 9mm, wall Thickness is 2mm, a diameter of 0.3mm of fibrous framework in pipe.The radical of fibrous framework is 30, a diameter of 0.3mm of suspension, Suspension quantity is 5.Fibrous framework is uniformly distributed in pipe.Use step (2) parameter to print, form conduit, suspension With fibrous framework in pipe, rack surface is coated with schwann cell.

According to above-mentioned preparation method, can respectively obtain:

Tissue engineering nerve graft include conduit and pipe in fibrous framework, every fibrous framework be provided with some along fibre length direction Root suspension is connected with pipe inner wall, and fibrous framework hangs in conduit by suspension distribution, and the suspension of every fibrous framework is circumference Distribution, and on different fibrous frameworks, the link position of suspension is different with circumference angle, wherein conduit diameter 1mm, wall thickness is 0.5mm, a diameter of 0.05mm of fibrous framework in pipe, the radical of fibrous framework is 3, a diameter of 0.05-of suspension 0.3mm, suspension quantity is 2, and fibrous framework is uniformly distributed in pipe, and rack surface is coated with the shitosan group of Schwann cell Weaver's journey nerve graft.

Or, tissue engineering nerve graft include conduit and pipe in fibrous framework, every fibrous framework along fibre length Direction is provided with some suspensions and is connected with pipe inner wall, and fibrous framework hangs in conduit by suspension distribution, and every fiber props up The suspension of frame is circumferentially distributed, and on different fibrous frameworks, the link position of suspension is different with circumference angle, wherein conduit diameter 5mm, wall thickness is 1.2mm, and a diameter of 0.15mm of fibrous framework in pipe, the radical of fibrous framework is 15, and suspension is straight Footpath is 0.15mm, and suspension quantity is 3.Fibrous framework is uniformly distributed in pipe, and rack surface is coated with the shell of Schwann cell Glycan tissue engineering nerve graft.

Or tissue engineering nerve graft include conduit and pipe in fibrous framework, every fibrous framework along fibre length side Upwards being provided with some suspensions to be connected with pipe inner wall, fibrous framework hangs in conduit by suspension distribution, every fibrous framework Suspension be circumferentially distributed, and on different fibrous frameworks the link position of suspension with circumference angle different, wherein conduit diameter 9mm, wall thickness is 2mm, a diameter of 0.3mm of fibrous framework in pipe.The radical of fibrous framework is 30, and suspension is a diameter of 0.3mm, suspension quantity is 5.Fibrous framework is uniformly distributed in pipe, and rack surface is coated with the shitosan group of Schwann cell Weaver's journey nerve graft.

Embodiment 11

(2) adjusting the printer nozzle needle diameter being connected with equipped with sodium alginate print cartridge is 100 μm, and syringe needle quantity is 10 Individual, the distance of nozzle to bottom is 25mm, booster pulse frequency 15v；

Adjusting the printer nozzle needle diameter being connected with the print cartridge equipped with NT3 (NT-3) is 100 μm, syringe needle Quantity is 6, and the distance of nozzle to bottom is 25mm, booster pulse frequency 15v；

(3) three-D space structure that accurately simulation difference is neural, the shape of design organization engineering nerve graft, and trophic factors Cladding form, establishment controls program accordingly, and threedimensional model preset by printer, the shape of design organization engineering nerve graft: Tissue engineering nerve graft include conduit and pipe in fibrous framework, every fibrous framework be provided with some along fibre length direction Root suspension is connected with pipe inner wall, and fibrous framework hangs in conduit by suspension distribution, and the suspension of every fibrous framework is circumference Distribution, and on different fibrous frameworks, the link position of suspension is different with circumference angle, wherein conduit diameter 1mm, wall thickness is 0.5mm, a diameter of 0.05mm of fibrous framework in pipe, the radical of fibrous framework is 3, a diameter of 0.05-of suspension 0.3mm, suspension quantity is 2, and fibrous framework is uniformly distributed in pipe.Using step (2) parameter to print, formation is led Fibrous framework in pipe, suspension and pipe, rack surface is coated with NT3 (NT-3), and NT3 exists Amount on nerve graft timbering material is 500 μ g/g.

Or, the accurately different neural three-D space structure of simulation, the shape of design organization engineering nerve graft, and Trophic factors cladding form, establishment controls program accordingly, and printer presets threedimensional model, design organization engineering nerve graft Shape: tissue engineering nerve graft include conduit and pipe in fibrous framework, every fibrous framework along fibre length direction Being provided with some suspensions to be connected with pipe inner wall, fibrous framework hangs in conduit by suspension distribution, hanging of every fibrous framework On the fibrous framework that silk is in circumferentially distributed and different, the link position of suspension is different with circumference angle, wherein conduit diameter 5mm, wall thickness is 1.2mm, and a diameter of 0.15mm of fibrous framework in pipe, the radical of fibrous framework is 15, and suspension is straight Footpath is 0.15mm, and suspension quantity is 3.Fibrous framework is uniformly distributed in pipe.Step (2) parameter is used to print, shape Becoming fibrous framework in conduit, suspension and pipe, rack surface is coated with NT3 (NT-3), NT3 Amount on nerve graft timbering material is 500 μ g/g.

Or, the accurately different neural three-D space structure of simulation, the shape of design organization engineering nerve graft, and Trophic factors cladding form, establishment controls program accordingly, and printer presets threedimensional model, design organization engineering nerve graft Shape: tissue engineering nerve graft include conduit and pipe in fibrous framework, every fibrous framework along fibre length direction Being provided with some suspensions to be connected with pipe inner wall, fibrous framework hangs in conduit by suspension distribution, hanging of every fibrous framework On the fibrous framework that silk is in circumferentially distributed and different, the link position of suspension is different with circumference angle, wherein conduit diameter 9mm, wall thickness is 2mm, a diameter of 0.3mm of fibrous framework in pipe.The radical of fibrous framework is 30, and suspension is a diameter of 0.3mm, suspension quantity is 5.Fibrous framework is uniformly distributed in pipe.Using step (2) parameter to print, formation is led Fibrous framework in pipe, suspension and pipe, rack surface is coated with NT3 (NT-3), and NT3 exists Amount on nerve graft timbering material is 500 μ g/g.

According to above-mentioned preparation method, can respectively obtain:

Tissue engineering nerve graft include conduit and pipe in fibrous framework, every fibrous framework be provided with some along fibre length direction Root suspension is connected with pipe inner wall, and fibrous framework hangs in conduit by suspension distribution, and the suspension of every fibrous framework is circumference Distribution, and on different fibrous frameworks, the link position of suspension is different with circumference angle, wherein conduit diameter 1mm, wall thickness is 0.5mm, a diameter of 0.05mm of fibrous framework in pipe, the radical of fibrous framework is 3, a diameter of 0.05-of suspension 0.3mm, suspension quantity is 2, and fibrous framework is uniformly distributed in pipe, and rack surface is coated with NT3 (NT- 3) sodium alginate tissue engineering nerve graft, NT3 amount on nerve graft timbering material is 500 μg/g。

Or, tissue engineering nerve graft include conduit and pipe in fibrous framework, every fibrous framework along fibre length Direction is provided with some suspensions and is connected with pipe inner wall, and fibrous framework hangs in conduit by suspension distribution, and every fiber props up The suspension of frame is circumferentially distributed, and on different fibrous frameworks, the link position of suspension is different with circumference angle, wherein conduit diameter 5mm, wall thickness is 1.2mm, and a diameter of 0.15mm of fibrous framework in pipe, the radical of fibrous framework is 15, and suspension is straight Footpath is 0.15mm, and suspension quantity is 3.Fibrous framework is uniformly distributed in pipe, and rack surface is coated with NT3 (NT-3) sodium alginate tissue engineering nerve graft, NT3 amount on nerve graft timbering material It is 500 μ g/g.

Or tissue engineering nerve graft include conduit and pipe in fibrous framework, every fibrous framework along fibre length side Upwards being provided with some suspensions to be connected with pipe inner wall, fibrous framework hangs in conduit by suspension distribution, every fibrous framework Suspension be circumferentially distributed, and on different fibrous frameworks the link position of suspension with circumference angle different, wherein conduit diameter 9mm, wall thickness is 2mm, a diameter of 0.3mm of fibrous framework in pipe.The radical of fibrous framework is 30, and suspension is a diameter of 0.3mm, suspension quantity is 5.Fibrous framework is uniformly distributed in pipe, and rack surface is coated with NT3 (NT- 3) sodium alginate tissue engineering nerve graft, NT3 amount on nerve graft timbering material is 500 μg/g。

Embodiment 12

The shitosan tissue engineering nerve graft selecting method described in embodiment 3 to prepare carries out repairing peripheral nerve defect in rats Research, described conduit diameter 5mm, wall thickness is 1.2mm, a diameter of 0.15mm of fibrous framework, the root of fibrous framework in pipe Number is 15, a diameter of 0.15mm of suspension, and suspension quantity is 3, and fibrous framework is uniformly distributed in pipe.

SD rat anesthesia state descending Sciatic art, sets up rat sciatic nerve 10mm defect model and divides at random It is 3 groups, tissue engineering nerve graft group, autologous nerve bridge joint group (the sciatic nerve suture in situ of cut-out) and with silicone tube Carry out the control group bridged.

All there is not obvious immunological rejection or inflammatory reaction in postoperative three groups of rat groups.Postoperative 1～June, respectively organize rat Carry out footprint respectively, observe sciatic nerve functional rehabilitation situation.Result show nerve graft bridge joint group of the present invention with Autologous group of effect is suitable.

Postoperative 6 months, fluorogold retrograde tracing result of the test showed, nerve graft bridge joint group of the present invention and autologous The most visible neuron marked by HRP in neural group rat spinal cord anterior horn or DRGs respective slice.

Postoperative 6 months, exposed Rats sciatic nerve, nerve graft bridge joint group of the present invention had more rich blood vessel raw Long.

Regenerating nerve om observation and count results show, nerve graft bridge joint group of the present invention and autologous nerve group Regenerating nerve is dry complete membrane bound, and quantity and the distribution of nerve fibre bundle are similar to Proximal, and the connective tissue of bundle film is relatively Thin；Regeneration axon through bridge material total length in bundle, arranges more neat, intensive.After special trichrome stain, regenerating nerve Proximal segment, has the axon periphery of more than nearly half red myelin occur；The distal section of regenerating nerve, it is seen that be wrapped thin layer myelin on a small quantity Axon and more " regeneration unit ".Electronic Speculum result shows, nerve graft bridge joint group of the present invention is compared and silicone tube The regenerating nerve axon of group is relatively thick, has outward relatively thin myelin to be formed, and myelin queueing discipline and electron density are higher.

Embodiment 12

The fibroin albumen tissue engineering nerve graft selecting method described in embodiment 4 to prepare carries out repairing peripheral nerve defect in rats Research, described graft tubing diameter 9mm, wall thickness is 2mm, a diameter of 0.3mm of fibrous framework in pipe.Fiber props up The radical of frame is 20, a diameter of 0.3mm of suspension, and suspension quantity is 5, and fibrous framework is uniformly distributed in pipe.

SD rat anesthesia state descending Sciatic art, sets up rat sciatic nerve 10mm defect model and divides at random It is 3 groups, tissue engineering nerve graft group, autologous nerve bridge joint group (the sciatic nerve suture in situ of cut-out) and defect group, Using autologous nerve bridge joint group and defect group as comparison, within postoperative 24 weeks, carry out a series of evaluation analysis.

(1) electrophysiology detection

Postoperative 24 weeks, again expose field of operation sciatic nerve, at bridge joint proximal stimulus, in gastrocnemius record compound muscle action potential (compound muscle action potentials,CMAPs).Tissue engineering nerve graft group recorded CMAPs, and it shakes There was no significant difference compared with autologous nerve bridge joint group for width, as it is shown in figure 5, A is normal side compound muscle action potential, B is certainly Somatic nerves bridge joint group compound muscle action potential, C is tissue engineering nerve graft group compound muscle action potential, D be normal side, Comparison (the SF-graft group of the compound muscle action potential amplitude of autologous group and tissue-engineering graft constructed group three of the present invention For tissue-engineering graft constructed group of the present invention, autograft group is that autologous group and contralateral unoperated side is Normal side).Fig. 5 shows that the art side of normal side (A) and autologous group (B) and tissue engineering nerve graft group (C) all recorded CMAPs, defect group art side all not recorded CMAPs.D figure is the comparison (n=8) of compound muscle action potential amplitude.* show with P < 0.05 is compared in No operation side (normal side).Autologous group and fibroin graft group do not have marked difference (p > 0.05).Result shows silk Rat 10mm Sciatic repaired by element conduit bridge joint, and sciatic target flesh has regained the domination of nerve, and And regenerating nerve is provided with basic electrophysiological function, there is no significant difference compared with nerve autograft group.

(2) fluorogold retrograde tracing test

After electro physiology, at the joint portion 5mm of bridge joint sections distally, in nerve tract, inject 5% fluorogold solution 5 μ l.Animal survival 5 After it, conventional perfusion, take spinal cord L4～6 sections and the DRGs (DRGs) of corresponding sections art side.Tissue is at routine Frozen section after reason, spinal cord slice dyes with Hoechst 33342, and DRG section is redyed with DiD with Hoechst 33342, Leica DMR basis of microscopic observation counts, the FG mark rate of DRG sensory neuron and anterior horn motor neurons two groups Relatively p > 0.05, no difference of science of statistics, as shown in Figure 6, (a) is tissue engineering nerve graft group to result；B () is autologous Group.(A) DRGs sensory neuron (Hoechst lining dye nucleus)；(B) DiD for (A) visual field redyes photo；(C) Gray nucleus Motoneurons；(D) it is the partial enlargement of boxed area in (C).Bar=40 μm (A, B, D)； Bar=300 μm (C).Result shows that tissue engineering nerve graft group regenerating nerve has the nervous physiology of basic axonal transport Function, also further illustrates neurologic defect and is repaired.

(3) histological observation

After animal perfusion, after taking the section of graft stage casing, carry out Mayer ' s nerve trichrome stain and NF immunohistochemical staining, Om observation, another label taking originally carries out electron microscopic observation.Take gastrocnemius belly of muscle tissue and carry out Masson trichrome stain, and press Karnovsky-Roots method carries out acetylcholine ester enzyme staining, om observation.Result is as it is shown in fig. 7, (A) is normal Side sciatic nerve；(B) autologous nerve group；(C) tissue engineering nerve graft group.(a) Meyer ' s trichrome stain, Bar=10 μm；(b) anti-NF-200 immunocytochemical stain, Bar=20 μm；(c) transmission electron microscope photo, Bar=5 μm.Result Showing, tissue engineering nerve graft group regeneration medullated fibers is dense, and cluster is distributed, with a large amount of Remak's nerve fibers, Although its myelin is relatively thin compared with normal neuronal, but medullated fibers compact structure is homogeneous, and myelin is high electron density, structure Clearly, Scs basilar memebrane is complete.The optical, electrical mirror histological findings of regenerating nerve further illustrates graft bridge joint sections By the nerve fiber reparation regenerated, there is complete neuromechanism.

Comprehensive above electrophysiology, neurotracer and Histological results show, tissue engineering nerve of the present invention moves Plant can preferably bridge reparation rat 10mm peripheral nerve defection.

Claims

1. the tissue engineering nerve graft with suspension fibrous framework, including fibrous framework in conduit and pipe, it is characterized in that the some suspensions that are provided with along fibre length direction of every fibrous framework are connected with pipe inner wall, fibrous framework is hung in conduit by suspension.

2. nerve graft as claimed in claim 1, it is characterised in that the suspension of described every fibrous framework is circumferentially distributed, and on different fibrous frameworks, link position and/or the circumference angle of suspension are different.

3. nerve graft as claimed in claim 2, it is characterised in that multiple micropore is also distributed on described catheter wall.

4. nerve graft as claimed in claim 2, it is characterised in that described conduit diameter is 1-9mm, and conduit wall thickness is 0.5-2mm, a diameter of 0.05-0.3mm of fibrous framework in described pipe, and the radical of support is 3-30 root, a diameter of 0.05-0.3mm of described suspension.

5. nerve graft as claimed in claim 1, it is characterised in that in described conduit and pipe, nano fiber scaffold is degradable polymeric material.

6. the nerve graft as described in any one of claim 1-5, it is characterised in that described fibrous framework surface and/or conduit surfaces externally and internally are also wrapped on trophic factors and/or cell.

7. nerve graft as claimed in claim 6, it is characterized in that described cell includes deriving from autologous or allosome, for the one or more combination in schwann cell, fibroblast, mesenchymal stem cells MSCs, cord blood stem cell, skin progenitor cell, described trophic factors is one or more in nerve growth factor, Basic Fibroblast Growth Factor, brain derived neurotrophic factor, NT3, glial cell line-derived neurotrophic.

8. the preparation method with the tissue engineering nerve graft of suspension fibrous framework, it is characterised in that comprise the following steps:

(2) adjusting printer nozzle needle diameter is 2-200 μm, and syringe needle quantity is 4-16, and the distance of nozzle to bottom is 10-50mm, and booster pulse frequency is 1-50v；

(3) by the different neural shape of accurately simulation and three-D space structure, the shape of design tissue engineering nerve graft as described in any one of claim 1-5, utilize biometric print technology, carry out high-true emulation according to neural actual form demand to print, use step (2) parameter to print, in forming conduit, suspension and pipe, be uniformly distributed the tissue engineering nerve graft of fibrous framework.

9. the preparation method with the tissue engineering nerve graft of suspension fibrous framework, it is characterised in that comprise the following steps:

(2) adjusting with the printer nozzle needle diameter being connected equipped with nerve graft material print cartridge is 2-200 μm, and syringe needle quantity is 2-10, and the distance of nozzle to bottom is 10-50mm, and booster pulse frequency is 1-50v；

Adjusting the printer nozzle needle diameter being connected with the print cartridge equipped with cell and/or trophic factors is 50-200 μm, and syringe needle quantity is 2-6, and the distance of nozzle to bottom is 10-50mm, and booster pulse frequency is 1-50v；

(3) different neural shape and three-D space structure are accurately simulated, design the shape of tissue engineering nerve graft as claimed in claim 6, and design cell and/or trophic factors are coated with form, work out corresponding print routine, threedimensional model preset by printer, using step (2) parameter to print, form equally distributed fibrous framework in conduit, suspension and pipe, internal stent or surface and conduit surfaces externally and internally are coated with one or more cells and/or trophic factors.

10. the preparation method of a tissue engineering nerve graft, it is characterised in that comprise the following steps:

(3) different neural shape and three-D space structure are accurately simulated, design the shape of tissue engineering nerve graft as claimed in claim 6, work out corresponding print routine, threedimensional model preset by printer, use step (2) parameter to print, form equally distributed fibrous framework in conduit, suspension and pipe；

(4) nerve graft that step (3) prepares is immersed in the nutrient solution containing cell and/or trophic factors, makes nerve graft fibrous framework surface and/or conduit surfaces externally and internally be coated with one or more cells and/or trophic factors.