CN105497979B - Controllable functional material of guiding nerve regneration of degradation rate and preparation method and application - Google Patents
Controllable functional material of guiding nerve regneration of degradation rate and preparation method and application Download PDFInfo
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- CN105497979B CN105497979B CN201510984759.2A CN201510984759A CN105497979B CN 105497979 B CN105497979 B CN 105497979B CN 201510984759 A CN201510984759 A CN 201510984759A CN 105497979 B CN105497979 B CN 105497979B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/24—Collagen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L89/00—Compositions of proteins; Compositions of derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
Abstract
The invention discloses controllable functional material of guiding nerve regneration of degradation rate and preparation method and application.Functional material provided by the present invention, to be crosslinked the material that N HOSu NHSs and 1 (3 dimethylamino-propyl) 3 ethyl-carbodiimide hydrochlorides are obtained with collagen as tissue engineering scaffold, obtained using crosslinked fluid immersion collagen as tissue engineering scaffold, crosslinked fluid is made up of solvent and solute, solvent is water, solute and its concentration are respectively the 1mg/ml of N HOSu NHSs 0.025, the 4mg/ml of 1 (3 dimethylamino-propyl) 3 ethyl-carbodiimide hydrochloride 0.1.The functional material of the present invention has reached the requirement of preferable nerve regneration biomaterial, can be for reparation injured nerve, it is also possible to prepare the product for repairing injured nerve.
Description
Technical field
The present invention relates to the functional material of the controllable guiding nerve regneration of degradation rate in biological technical field and its preparation
Method and application.
Background technology
Collagen is the important feature albumen in mammal body, not only rich content, and with good biocompatibility,
Low immunogenicity and biological degradability, are a kind of preferable biomaterial for medical purpose sources.The timbering material prepared using collagen
Certain filling and supporting role can be provided for injury tissue, adhesion, propagation and the migration of cell is effectively facilitated.At present, glue
Former class biomaterial is had been widely used in Various Tissues injury repair research, including skin, bone, cartilage, tendon etc..But,
For neurotrosis, particularly central nervous system injury (such as spinal cord injury), the collagen-based materials of nerve regneration can be guided by still lacking.
Preferable nerve regneration biomaterial needs have guide effect to nerve growth, can be implanted into nerve damage portion
The pathological death of neuron cell body is reduced afterwards, and being grown to distal end for guiding fracture aixs cylinder promotes synapse correctly to connect
Connect.Need simultaneously with the degradation characteristic being adapted with nerve regneration speed, could so reduce damage location cicatrization, and
And can progressively degrade, space is provided for regeneration, while can be carried for the sustained release of foreign cell grappling and growth factor and medicine
It is final to provide a kind of suitable reparation microenvironment for neurotrosis for carrier, promote repairing of neural injury.
Glial scar is spongiocyte malignant proliferation generation after spinal cord injury, is to suppress neural regeneration after spinal cord injury
Major obstacle.Nestin belongs to Intermediate filament (nestin), is expressed in undifferentiated neural multipotential stem cell, it is considered to be
The label of brain Tumor Stem Cells.Spongiocyte GFAP (glial fibrillary acidic
Protein, GFAP) be nervous system Activation of Astrocytes mark.Chondroitin sulfate proteoglycan
(chondroitin sulphate peoteoglycan, CSPG), early detection spinal cord of adult rats and brain a group into
Expressed in ripe spongiocyte.
The content of the invention
The technical problems to be solved by the invention are the function materials for how preparing the controllable guiding nerve regneration of degradation rate
Material, and how to repair spinal cord injury.
In order to solve the above technical problems, present invention firstly provides the functional material available for guiding nerve regneration.
Functional material provided by the present invention, to be crosslinked n-hydroxysuccinimide (NHS) and 1- with collagen as tissue engineering scaffold
The material that (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) are obtained.
In above-mentioned material, the functional material can be prepared according to the preparation method of following functional materials.
In order to solve the above technical problems, present invention also offers the preparation method of functional material.
The preparation method of functional material provided by the present invention, including obtain described with crosslinked fluid immersion collagen as tissue engineering scaffold
Functional material；The crosslinked fluid is made up of solvent and solute, and the solvent is water, and the solute and its concentration are respectively EDC
0.1-4mg/ml, NHS 0.025-1mg/ml.
In the above method, EDC and NHS mass ratio can be 4 in the crosslinked fluid:1.EDC and NHS in the crosslinked fluid
Concentration can be respectively 1-4mg/ml (such as 2mg/ml) and 0.25-1mg/ml (such as 0.5mg/ml).
In the above method, the collagen as tissue engineering scaffold can be 20000 with the EDC and NHS mass ratio in the crosslinked fluid：
(1-200):(0.25-50), such as 20000:100:25.
In the above method, the diameter of the nerve regneration collagen scaffold can be 2-4mm, and length can be 10cm.
In the above method, the temperature of the immersion can be 37 DEG C, and the time of the immersion can be 8-24 hours, and such as 16 is small
When.
In the above method, methods described, which may also include, cleans the functional material with deionized water.
In the above method, methods described, which may also include, freezes the functional material.It is described to freeze under 0.1Pa to enter
OK.Freeze-drying time can be 24 hours.
In the above method, the collagen as tissue engineering scaffold can be prepared as follows：
1) with tributyl phosphate (TnBP) processing manadesma, the material of TnBP processing is obtained；
2) material of the TnBP processing is handled with NaCl, the material of NaCl processing is obtained；
3) with the material of NaCl processing described in trypsin treatment, collagen as tissue engineering scaffold is obtained.
In the above method, the manadesma can be Cowhells film.
In the above method, the time that manadesma is handled with tributyl phosphate can be 36-72 hours, such as 48 hours.It is described
The time that the material of the TnBP processing is handled with NaCl can be 12-48 hours, such as 36 hours.It is described to use trypsin treatment institute
The time for stating the material of NaCl processing can be 1-4 hours, such as 2.5 hours.
In the above method, it is described can be with tributyl phosphate processing manadesma with concentration expressed in percentage by volume be 1-1.5% (quality
Percent concentration) tributyl phosphate solution processing manadesma；The solvent of the tributyl phosphate solution can be deionized water.
The material that the TnBP processing is handled with NaCl can be the material that the TnBP processing is handled with NaCl solution；
NaCl is added in the Tris-HCl buffer solutions that it is 7.6-8.5 to 25-100mmol/L, pH that the NaCl solution, which can be, to obtain
NaCl concentration is 0.5-1.5mol/L in solution, NaCl solution.
The material that NaCl described in the use trypsin treatment is handled can be to be handled with trypsin solution at the NaCl
The material of reason；The trypsin solution can be to be added into 25-100mmol/L Tris-HCl buffer solution (pH is 7-8)
The concentration of trypsase is 0.5-1.5g/100ml in the solution that trypsase is obtained, trypsin solution.
In the above method, step 1) and step 2) can be carried out under 4-20 degree；Step 3) it can be carried out under 37 degree.
In the above method, methods described may also include step 2) the material deionized water of obtained NaCl processing and/
Or 3% (concentration of volume percent) Tween20 aqueous cleanings the step of.The cleaning can be carried out under 4-20 degree.
In the above method, methods described may also include step 3) obtained collagen as tissue engineering scaffold deionized water and/or
The step of water for injection is cleaned.The cleaning can be carried out under 4-20 degree.
In the above method, the manadesma will also remove the musculature of internal layer adhesion and the fat of outer layer adhesion before treatment
Fat.
In order to solve the above technical problems, present invention also offers the composition for preparing the functional material.
It is provided by the present invention to be used to prepare the composition of the functional material, by the collagen as tissue engineering scaffold, NHS and
EDC is constituted；The mass ratio of the collagen as tissue engineering scaffold, EDC and NHS can be 20000：(1-200):(0.25-50), such as 20000:
100:25。
In order to solve the above technical problems, present invention also offers the functional material, methods described or the composition
Following any applications：
X1, the application in spinal cord injury is repaired；
X2, the application in reparation spinal cord injury product is prepared；
X3, the application in spinal cord injury is repaired；
X4, the application in reparation spinal cord injury product is prepared；
X5, the application in repairing nerve damage；
X6, the application in repairing nerve damage product is prepared；
X7, the effect in site spinal cord injury cicatrization is suppressed；
X8, the effect in suppression site spinal cord injury cicatrization product is prepared；
X9, the effect in site spinal cord injury cicatrization is suppressed；
X10, the effect in suppression site spinal cord injury cicatrization product is prepared；
X11, the effect in nerve damage portion cicatrization is suppressed；
X12, the effect in suppression nerve damage portion cicatrization product is prepared；
X13, the application in nerve growth is oriented to；
X14, the application in guiding nerve growth product is prepared；
X15, the effect in promoting and/or guiding axon growth；
X16, the effect in promotion and/or the exsule long products of leading axle are prepared；
X17, the effect in synapse connection is promoted；
X18, the effect in promotion synapse connection product is prepared；
X19, the effect in site spinal cord injury two ends cell migration is promoted；
X20, the effect in promotion site spinal cord injury two ends cell migration product is prepared；
X21, the effect in site spinal cord injury two ends cell migration is promoted；
X22, the effect in promotion site spinal cord injury two ends cell migration product is prepared；
X23, the effect in nerve damage portion two ends cell migration is promoted；
X24, the effect in promotion nerve damage portion two ends cell migration product is prepared；
X25, the application in spinal cord injury animal movement functional rehabilitation is promoted；
X26, the application in promotion spinal cord injury animal movement functional rehabilitation product is prepared；
X27, the application in nerve injury animals motor function recovery is promoted；
X28, the application in promotion nerve injury animals motor function recovery product is prepared.
In above-mentioned application, the nerve can be nervous centralis, such as spinal cord.The aixs cylinder can be fracture aixs cylinder.The animal
Can be mammal, such as people or rat.
In the present invention, NHS can be sigma-aldrich Products, and article No. is 130672.EDC can be the production of sigma companies
Product, article No. is E6383.
Applicant is groped by many experiments, by the optimization of neural guidance quality and the regulation and control of degradability to collagen, is prepared
A kind of functional material of the controllable bootable nerve regneration of degradation rate, the material has certain mechanics elasticity, can be preferable
Stretch, can be grown, can be progressively degraded in nerve damage portion along material bearing of trend with Induction of neuronal aixs cylinder, suppress pars affecta
Position glial scar is formed, and can be promoted the recovery of animal sports ability after spinal cord injury after transplanting, be provided newly for repairing of neural injury
Method.
It is demonstrated experimentally that the preparation method of the functional material of the present invention can prepare the functional material of different degradation rates：
In collagenase solution, uncrosslinked NHS and EDC 20mg collagen as tissue engineering scaffold are degradable within 4 hours, and be crosslinked NHS and
The functional material degradation rate that EDC is obtained is substantially slack-off, and the concentration of degradation rate and NHS and EDC becomes in dose dependent
Change, the degradation rate of the functional material obtained in the crosslinked fluid that EDC and NHS concentration is respectively 1mg/ml and 0.25mg/ml is
0.78 ± 0.12mg/h, the functional material obtained in the crosslinked fluid that EDC and NHS concentration is respectively 2mg/ml and 0.5mg/ml
Degradation rate is 0.16 ± 0.02mg/h, the work(obtained in the crosslinked fluid that EDC and NHS concentration is respectively 4mg/ml and 1mg/ml
The degradation rate of energy material is 0.078 ± 0.0013mg/h.During damage spinal cord in repairing double action object, with collagen as tissue engineering scaffold
Compare, functional material degraded is slack-off, it is possible to is preferably merged with in-vivo tissue, plays filling guiding function.In practical application
In, the degradation rate of functional material can be further adjusted by adjusting the concentration of NHS and EDC in crosslinked fluid.
The functional material of the present invention has reached the requirement of preferable nerve regneration biomaterial, can be refreshing for repairing damage
Through, it is also possible to prepare the product for repairing injured nerve.
Brief description of the drawings
Fig. 1 is the feature of functional material.Wherein, A is the outward appearance photo of functional material；B is the ESEM of functional material
Photo；C is the transmission electron microscope photo of functional material.
Fig. 2 is degraded situation analysis of the material of different crosslinking agents processing in clostridiopetidase A.
Fig. 3 is the mechanical strength of collagen as tissue engineering scaffold and functional material.Wherein, A is the mechanical strength of collagen as tissue engineering scaffold；
B is the mechanical strength of functional material.
Fig. 4 is degraded situation of the functional material in Spinal Cord Injury in Rats is repaired.Wherein, A damages for control rats spinal cord
The HE dyeing coloration results of collagen as tissue engineering scaffold during wound is repaired, B is the HE of functional material during experimental group Spinal Cord Injury in Rats is repaired
Dye coloration result；Scale is 1mm.
Fig. 5 is part myeloid tissue of the functional material in experimental group rat body together with the functional material two ends being attached thereto
Nestin immunostaining and the DAPI coloration results of nucleus.Wherein, the red immunostaining for Nestin, blueness is thin
Karyon DAPI is dyed, and scale is 1mm.
Fig. 6 is the glial scar formational situation at Spinal Cord Injury in Rats.Wherein, A is does not repair control group, and B is experimental group.
Red is glial scar CSPG positive stainings, and green is spongiocyte GFAP dyeing, and scale is 500 μm.
Fig. 7 is experimental group rat and the BBB neurological deficit scores for not repairing control rats.Wherein, control represents not repair
Control group, material transplantation group represents experimental group.
Embodiment
The present invention is further described in detail with reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.
Experimental method in following embodiments, is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
N-hydroxysuccinimide (NHS) in following embodiments is sigma Products, and article No. is 130672；1-(3-
Dimethylamino-propyl) -3- ethyl-carbodiimide hydrochlorides (EDC) be simga Products, article No. is E6383.
Rat in following embodiments is Beijing Vital River Experimental Animals Technology Co., Ltd.'s Products, catalogue
Number be 101.
The preparation of embodiment 1, functional material
1st, manadesma is pre-processed
The fresh Adult Bovine muscle with white manadesma is taken, with cold deionized water rinsing 3 times；With tweezers and scalpel point
Manadesma is separated out, the tissue such as the musculature of internal layer adhesion and the fat of outer layer is removed as far as possible, obtains pre-processing manadesma.
2nd, prepared by functional material
Pretreatment manadesma is handled as follows, wherein step 1) and 2) and step 4) and the 6) progress at 4 DEG C,
Step 3) and the 5) progress at 37 DEG C：
1) in TnBP (tributyl phosphate) solution, (TnBP solution is to 50mMTris- to the pretreatment manadesma for obtaining step 1
During 1%) TnBP concentration of volume percent is in the solution that addition TnBP is obtained in HCl buffer solutions (pH=7.6), TnBP solution
It is incubated 48 hours, changes liquid once within every 12 hours, obtains the material of TnBP processing；
2) by step 1) (NaCl solution is slow to 25mM Tris-HCl in NaCl solution for the material of obtained TnBP processing
The concentration that NaCl in the solution that NaCl is obtained, NaCl solution is added in fliud flushing (pH=7.6) is 1.5M) in be incubated 36 hours, often
Liquid was changed every 3 hours once, obtained the material of NaCl processing；The material that NaCl is handled first cleans 20 times with deionized water, every time
20 minutes, then handled 35 minutes with the Tween20 aqueous solution of 3% (concentration of volume percent), finally clean 10 with deionized water
It is secondary, 20 minutes every time；
3) by step 2) cleaning after NaCl processing material trypsin solution (trypsin solution be to
Trypsase in the solution that trypsase is obtained, trypsin solution is added in 25mMTris-HCl buffer solutions (pH=7.6)
Concentration is 1.5g/100ml) in be incubated 2.5 hours, obtain the material of trypsin treatment；
4) by step 3) the obtained material of trypsin treatment cleans 10 times with deionized water, and 20 minutes every time, then
Cleaned and stayed overnight with deionized water, cleaned with water for injection within second day 5 times, 30 minutes every time, obtain collagen as tissue engineering scaffold；
5) by step 4) cleaning after 20g collagen as tissue engineering scaffold in crosslinked fluid 1, (crosslinked fluid 1 is made up of solvent and solute, molten
Agent is water, and solute and its concentration are respectively EDC 1mg/ml, NHS 0.25mg/ml) in 37 degree of crosslinking Treatments 16 hours, obtain
Cross-linking products 1；
By step 4) cleaning after 20g collagen as tissue engineering scaffold in crosslinked fluid 2, (crosslinked fluid 2 is made up of solvent and solute, solvent
For water, solute and its concentration are respectively EDC 2mg/ml, NHS 0.5mg/ml) in 37 degree of crosslinking Treatments 16 hours, handed over
Co-product 2；
By step 4) cleaning after 20g collagen as tissue engineering scaffold in crosslinked fluid 3, (crosslinked fluid 3 is made up of solvent and solute, solvent
For water, solute and its concentration are respectively EDC 4mg/ml, NHS 1mg/ml) in 37 degree of crosslinking Treatments 16 hours, be crosslinked
Product 3；
6) respectively by step 5) in cross-linking products 1, cross-linking products 2 and cross-linking products 3 clean 20 with deionized water respectively
It is secondary, 20 minutes every time, respectively obtain pre- functional material 1, pre- functional material 2 and pre- functional material 3；
7) it is respectively that pre- functional material 1, pre- functional material 2 and pre- functional material 3 is lyophilized 24 hours in 0.1Pa, respectively
To functional material 1, functional material 2 and functional material 3, bunchy is tied up.
Step 7) obtained functional material 1-3 outward appearances be white pencil arrangement filament, be 2-4mm per beam diameter,
0.1-0.2g, length is 10cm (A in Fig. 1).Using scanning electron microscopic observation it can be seen that functional material internal fiber longitudinal direction is arranged in order
Arrange (B in Fig. 1).Utilize transmission electron microscope observing, it can be seen that material internal collagenous fibres are in typical three light and dark spiral knots
Structure (C in Fig. 1).
The characteristic of embodiment 2, the functional material of embodiment 1
1st, degradation characteristic
Respectively according to following operating procedure detect embodiment 1 collagen as tissue engineering scaffold and with functional material 1, functional material
2 and the degradation characteristic of functional material 3, experiment is in triplicate：
0.02g above-mentioned materials are soaked in into 2ml collagens enzyme aqueous solution, and (collagen enzyme aqueous solution is that glue is added into deionized water
The mass percent concentration for the clostridiopetidase A that protoenzyme is obtained is 0.1% liquid) in, soaking 1,2,4,8,16,32,64 and respectively
Shown in the residual mass (table 1) for weighing material in 128 hours, residual mass such as Fig. 2 and table 2.
The residual mass (g) of table 1, functional material
| Soak time (hour) | Collagen as tissue engineering scaffold | Functional material 1 | Functional material 2 | Functional material 3 |
| 0 | 0.02±0.0001 | 0.0188±0.0007 | 0.02±0.0001 | 0.02±0.0001 |
| 1 | 0.01±0.0015 | 0.0159±0.0016 | 0.0199±0.0001 | 0.0199±0.0001 |
| 2 | 0.0043±0.0014 | 0.014±0.002 | 0.0195±0.00076 | 0.0199±0.0001 |
| 4 | 0 | 0.011±0.0014 | 0.0187±0.00076 | 0.0199±0.0001 |
| 8 | 0 | 0.0049±0.0014 | 0.0175±0.0009 | 0.0198±0.0002 |
| 16 | 0 | 0 | 0.0171±0.001 | 0.0193±0.0004 |
| 32 | 0 | 0 | 0.0134±0.0012 | 0.0182±0.0003 |
| 64 | 0 | 0 | 0.0045±0.0012 | 0.0141±0.0016 |
| 128 | 0 | 0 | 0 | 0.012±0.0016 |
The residual mass ratio (%) of table 2, functional material
| Soak time (hour) | Collagen as tissue engineering scaffold | Functional material 1 | Functional material 2 | Functional material 3 |
| 0 | 1 | 0.94 | 1 | 1 |
| 1 | 0.5 | 0.795 | 0.995 | 0.995 |
| 2 | 0.215 | 0.7 | 0.975 | 0.995 |
| 4 | 0 | 0.55 | 0.935 | 0.995 |
| 8 | 0 | 0.245 | 0.875 | 0.99 |
| 16 | 0 | 0 | 0.855 | 0.965 |
| 32 | 0 | 0 | 0.67 | 0.91 |
| 64 | 0 | 0 | 0.225 | 0.705 |
| 128 | 0 | 0 | 0 | 0.6 |
As a result show, uncrosslinked NHS and EDC collagen as tissue engineering scaffold are degradable within 4 hours, and be crosslinked NHS and
The functional material degradation rate that EDC is obtained is substantially slack-off, and the concentration of degradation rate and NHS and EDC becomes in dose dependent
Change (Fig. 2), in 0-16 hours, the degradation rate of functional material 1 is 0.78 ± 0.12mg/h, in 0-128 hours, function material
The degradation rate of material 2 is 0.16 ± 0.02mg/h, in 0-128 hour, the degradation rate of functional material 3 is 0.078 ±
0.0013mg/h。
2nd, mechanical strength
The functional material that crosslinking NHS and EDC in uncrosslinked NHS and EDC collagen as tissue engineering scaffold and embodiment 1 is obtained
2 are soaked in physiological saline after 10 minutes respectively, are pressed from both sides with tweezers and lift material in the middle part of material, collagen as tissue engineering scaffold is more soft,
It is affected by gravity two ends substantially sagging (A in Fig. 3), and is crosslinked functional material that NHS and EDC obtain then after physiological saline immersion
Still there is certain mechanics elasticity, can preferably stretch (B in Fig. 3).Show that crosslinking agent processing can improve the mechanical strength of material, this
It is beneficial to after transplanting preferably keep the original form of material, promotes the migration of the teloblast of material two and the growth of aixs cylinder.
Embodiment 3, embodiment 1 functional material repair Spinal Cord Injury in Rats in application
Mouse spinal cord injury model is prepared from 24 200g or so rat, preparation method is as follows：After rat anesthesia, beat
Open T11-T13 vertebral plate exposing spinal cord tissues, by T12 sections of spinal cord cut off 2-3mm (because spinal cord bounces back, the length between two broken ends of fractured bone
Degree is about 5mm), mouse spinal cord injury model is obtained, 24 mouse spinal cord injury models are obtained.By this 24 mouse spinal cord injury models
Three groups are randomly divided into, every group 8, one group is control group, and another group is experimental group, and the 3rd group is not repair control group.It will prepare
Mouse spinal cord injury model as Zhou Jiwei the 1st week.
At the 1st week, the functional material Bridge 2 of embodiment 1 is connected on to every rat spinal cord of experimental group by way of filling
Functional material proximally and distally, is then tightly sewn on endorchis by damage, by this group of rat in nursing after prosthesis
Under the conditions of feed 3 months, obtain postoperative experimental group rat.
At the 1st week, the collagen as tissue engineering scaffold of embodiment 1 is bridged into every rat ridge in control group by way of filling
Marrow is damaged proximally and distally, is then tightly sewn to functional material on endorchis, by this group of rat in shield after prosthesis
Fed 3 months under the conditions of reason, obtain postoperative control rats.
Reparation of the control group without any spinal cord injury is not repaired, and this group of rat is only fed 3 under nursing condition
Month, obtain postoperative not repairing control rats.
1st, degraded situation of the functional material in Spinal Cord Injury in Rats is repaired
Respectively by partial ridge of the functional material in postoperative experimental group rat body together with the functional material two ends being attached thereto
The portion of collagen as tissue engineering scaffold in myeloid tissue and postoperative control rats body together with the collagen as tissue engineering scaffold two ends being attached thereto
Costula myeloid tissue is taken out, and carries out HE staining analysis respectively.
As a result show, the collagen as tissue engineering scaffold degraded in postoperative control rats body is obvious (A in Fig. 4), postoperative experimental group
Functional material degraded in rat body is slack-off, is preferably merged with in-vivo tissue, plays filling guiding function (B in Fig. 4).
2nd, functional material can guide the growth of nerve cell
Respectively by partial ridge of the functional material in postoperative experimental group rat body together with the functional material two ends being attached thereto
The portion of collagen as tissue engineering scaffold in myeloid tissue and postoperative control rats body together with the collagen as tissue engineering scaffold two ends being attached thereto
Costula myeloid tissue is taken out, and carries out the DAPI dyeing of Nestin immunostaining and nucleus.
As a result show, the nerve cell at spinal cord injury two ends can prolong prolonging for functional material in postoperative experimental group rats body
Stretch direction growth (Fig. 5), and the nerve cell at spinal cord injury the two ends not growth characteristics, explanation in postoperative control rats body
Functional material has the characteristic of guiding nerve growth.
3rd, functional material can suppress cicatrization
Respectively by partial ridge of the functional material in postoperative experimental group rat body together with the functional material two ends being attached thereto
Myeloid tissue and the postoperative part myeloid tissue for not repairing two ends at spinal cord injury in control rats body take out, and carry out colloid
The dyeing of scar CSPG positive stainings and spongiocyte GFAP.
As a result show, spinal cord injury two ends are almost without glial scar (B in Fig. 6), art in postoperative experimental group rats body
Do not repair in control rats body that there is substantial amounts of scar (A in Fig. 6) at two ends at spinal cord injury afterwards, show, functional material can press down
The formation of scar processed.
4th, functional material can promote the recovery of rats with spinal cord injury motor function
From the 1st week to the 12nd week weekly respectively to experimental group rat and do not repair control rats carry out BBB behaviouristics comment
Point, as a result as shown in Table 3 and Figure 7.
Table 3, the BBB neurological deficit score results of rat
| Time | Experimental group | Control group is not repaired |
| 1st week | 0.33±0.58 | 0.33±0.58 |
| 2nd week | 1.33±0.58 | 0.33±0.58 |
| 3rd week | 2.33±1.53 | 1.33±0.58 |
| 4th week | 4.33±0.58 | 3±1 |
| 5th week | 4.67±0.58 | 3.33±0.58 |
| 6th week | 4.67±0.58 | 3.67±1.53 |
| 7th week | 4.67±0.58 | 4±1 |
| 8th week | 5±1 | 4±1 |
| 9th week | 5±1 | 4±1 |
| 10th week | 5.33±1.53 | 4.33±0.58 |
| 11st week | 5.33±0.58 | 4.33±0.58 |
| 12nd week | 5.33±0.58 | 4.33±1.53 |
As a result show, with non-portability function material do not repair group rat compared with, the control rats of portability function material
Motor behavior recovers preferably (Fig. 7).
Claims (10)
1. a kind of functional material for guiding nerve regneration, for n-hydroxysuccinimide and 1- (3- dimethylamino-propyls) -3-
The material that ethyl-carbodiimide hydrochloride crosslinked with collagen timbering material is obtained；
The preparation method of the functional material, including obtain the functional material with crosslinked fluid immersion collagen as tissue engineering scaffold；It is described
Crosslinked fluid is made up of solvent and solute, and the solvent is water, and the solute and its concentration are respectively EDC 1-4mg/ml, NHS
0.25-1mg/ml；
In the above method, EDC and NHS mass ratio is 4 in the crosslinked fluid:1；
In the above method, the collagen as tissue engineering scaffold is 20000 with the EDC and NHS mass ratio in the crosslinked fluid：（1-
200）:（0.25-50）；
The collagen as tissue engineering scaffold is prepared as follows：
1）Manadesma is handled with tributyl phosphate, the material of TnBP processing is obtained；
2）The material of the TnBP processing is handled with NaCl, the material of NaCl processing is obtained；
3）The material handled with NaCl described in trypsin treatment, obtains collagen as tissue engineering scaffold.
2. the functional material according to claim 1, it is characterised in that：The temperature of the immersion is 37 DEG C；The leaching
The time of bubble is 8-24 hours.
3. the functional material according to claim 2, it is characterised in that：The preparation method of the functional material also includes
The functional material is freezed.
4. the functional material according to claim 1, it is characterised in that：The manadesma is Cowhells film.
5. functional material described in claim 1 is preparing the application in repairing spinal cord injury product.
6. functional material described in claim 1 is preparing the effect in suppressing site spinal cord injury cicatrization product.
7. effect of the functional material described in claim 1 in promotion and/or the exsule long products of leading axle are prepared.
8. functional material described in claim 1 is preparing the effect in promoting synapse connection product.
9. functional material described in claim 1 is preparing the effect in promoting site spinal cord injury two ends cell migration product.
10. functional material described in claim 1 is preparing the application in promoting spinal cord injury animal movement functional rehabilitation product.
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