CN105597148B - A kind of Nerve Scaffold, preparation method and application for repairing of neural injury - Google Patents
A kind of Nerve Scaffold, preparation method and application for repairing of neural injury Download PDFInfo
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Abstract
A kind of Nerve Scaffold, preparation method and application for repairing of neural injury, the Nerve Scaffold includes collagen scaffold, neural stem cell of the culture on collagen scaffold and the growth factor being fixed on collagen scaffold by heparin, the growth factor has heparin binding domain, first collagen solution is lyophilized before crosslinking when preparation, the collagen sponge obtained after freeze-drying is immersed in crosslinker solution again and is crosslinked, growth factor is fixed on collagen scaffold using heparin, maintain neural stem cell activity or promotes nerve stem cell proliferation.Preparation method of the invention is drawn materials convenient, step is simple, avoid the complex steps such as acid-base neutralization, dialysis, reduce the chance of collagen-based materials contact harmful reagent, the safety is improved for Nerve Scaffold obtained, can according to need and fashions into different shape, for filling up nerve fiber defect and the Bridging nerve broken ends of fractured bone, nerve growth is guided, to promote repairing of neural injury and functional rehabilitation, and there is good stability.
Description
Technical field
The invention belongs to nerve regneration field of biology, and in particular to a kind of Nerve Scaffold for repairing of neural injury,
Preparation method and application.
Background technique
Nervous system injury mainly includes spinal cord injury, cerebral injury, peripheral nerve injury, and consequence is serious, and treatment is always
It is research hotspot.Nervous system injury disease incidence increases year by year with economic and traffic development, increasingly causes clinic
The attention of doctor and scientific research scholar.
Lesion once occurs for nervous system, and recovery extent is limited, and therapeutic effect is not good enough, for a long time, the nervous system disease
After traditional treatment, still some still there are serious sequelae, therefore, select a kind of exogenous substance substitution
The tissue thoroughly damaged promotes the nerve fiber regeneration of remaining, becomes the main thought for capturing the nervous system disease.
Along with the development of bioengineered tissue, organizational project has consequence in neurotrosis treatment.Collagen
Material source is abundant and has many advantages, such as, for example, degradability, biocompatibility, its hypoimmunity and can preferably repair
Decorations property, and it is widely used in organizational project.Studies have shown that collagen can promote the differentiation of neuron；Experiment in vitro is ground
Study carefully display, collagen can promote the axon regeneration of dorsal root ganglion, and therefore, collagen can be used as trophic factors or drug
Carrier is widely used in nerve regneration engineering to play the application effect of collaboration in organizational project.
When producing collagen-based materials in the prior art, usual collagen tissue needs to be dissolved in acetic acid, if desired obtains solid-state
Collagen-based materials, it usually needs alkaline solution neutralizes acetic acid, and the salinity that generates mixes in the solution after neutralizing, thus need into
One step removes wherein salinity by dialysis, and therefore, collagen-based materials production method used at present is complicated, higher cost.
However, merely cannot effective repairing nerve damage using carrier material.In recent years, research neural stem cell gradually at
It when repairing the tissue of damage, needs to be made in collagen-based materials in nerve regneration engineering to treat the hot spot of the nervous system disease
Supported on carriers play the neural stem cell of repair, neural stem cell is latent with self-renewal capacity and Multidirectional Differentiation
A kind of cell of energy, has plasticity, its achievable specific nervous function is divided into the thin of internal specific nervous function
Born of the same parents have directed differentiation potentiality.Neural stem cells transplantation art is that the neural stem cell of in vitro culture and induction differentiation is passed through hand
Art mode is transplanted to the process that encephalic a part reaches reparation, rebuilds nervous function.The neural stem cell cultivated in vitro enters
Differentiation, proliferation and directional migration after in vivo, and integrated with the nerve cell of human body itself, it is finally reached and repairs impaired nerve,
Rebuild partly or completely nervous function.
In nerve regneration engineering, being implanted into the intracorporal neural stem cell of host can become to diseased region where nervous system
Change, migration and assemble, can survive, Proliferation, Differentiation is neuron and Deiter's cells, to promote patient's missing function
Recovery, however, the studies have shown that neural stem cell of early period after heteroplastic transplantation due to immune response etc., survival rate is very low, because
This, the utilization rate of neural stem cell is lower.
Summary of the invention
The purpose of the present invention is to provide a kind of Nerve Scaffold, preparation method and application for repairing of neural injury,
Materials are convenient, and preparation method is simple, can obtain and need to fashion into Nerve Scaffold of different shapes according to different, improve nerve cord
Cell enhances repair of the cell to neurotrosis, is used to fill up neural group for the Nerve Scaffold in the quantity of damage part
Defect and the Bridging nerve broken ends of fractured bone are knitted, nerve growth is guided, to promote repairing of neural injury and functional rehabilitation, and is had good
Stability.
In order to achieve the above object, technical solution provided by the invention is as follows:
A kind of Nerve Scaffold for repairing of neural injury, the nerve cord including collagen scaffold, culture on collagen scaffold
Cell and the growth factor on collagen scaffold is fixed on by heparin, the growth factor has heparin binding domain.
The preparation method of Nerve Scaffold of the present invention for repairing of neural injury, includes the following steps:
1) collagen solution is prepared
Animal collagen tissue is obtained, is dissolved in 0.05~0.1% glacial acetic acid-PBS (v/v), it is molten to be configured to collagen
Liquid；
2) it is lyophilized, sterilizes
Collagen solution is poured into mold, then mold is put into liquid nitrogen frozen, is then lyophilized with freeze dryer, freeze-drying time is
8~12 hours, collagen sponge is obtained, then carry out sterilization treatment；The collagen sponge is three-dimensional collagen-based materials, aperture is 30~
200μm；
3) it is crosslinked, washs
Heparin is dissolved in crosslinker solution, the collagen sponge of sterilizing is immersed in the crosslinker solution containing heparin and is handed over
Connection, crosslinking time 3~4 hours, is washed 4~8 hours after crosslinking with phosphate buffered saline solution by 30~40 DEG C of crosslinking temperature,
It is washed with deionized again 4~8 hours；
Wherein, the crosslinker solution is 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide that concentration is 1~25mM
The mixed solution of the N- hydroxysuccinimide of hydrochloride and 1~12.5mM；
4) it forms
Collagen sponge after crosslinking is sucked into moisture, is immersed in growth factor solution and forms, 30~40 DEG C of forming temperature,
Molding time 0.5~2 hour, obtain collagen scaffold；Wherein, the growth factor has heparin binding domain；
5) culture of neural stem cells neural
By Primary culture of neural stem cells on the collagen scaffold that step 4) obtains, the Nerve Scaffold is obtained.
Further, the concentration of collagen is 5~20mg/ml in the collagen solution obtained in step 1).
Preferably, sterilization treatment carried out to collagen sponge using electron beam irradiation in step 2), treatment conditions for 6~
12kGy 60Co。
Further, in step 3) in the crosslinker solution containing heparin, the concentration of heparin is 2~4mg/mL.
Further, the concentration of growth factor is 50~500 μ g/mL in growth factor solution described in step 4).
Preferably, growth factor described in step 4) is basic fibroblast growth factor or vascular endothelial growth factor
Son.
Further, in step 5) by neural stem cell after Hoechst is marked, be further cultured on collagen scaffold, nerve cord
The number of cell is 5~10 × 105A, Nerve Scaffold obtained is nerve trachea or circle, ellipse, rectangular or irregular
Cavernous nerve bracket.
0.05~0.1% glacial acetic acid-PBS (v/v) of the invention, which refers to, contains volume in PBS (phosphate buffered saline solution)
The glacial acetic acid that score is 0.05~0.1%.
Nerve Scaffold of the invention is applied to repairing of neural injury, especially for repairing spinal cord injury or repairing facial nerve
Damage, in addition to being suitable for repairing of neural injury, it can also be used to repair peripheral nerve injury and its hetero-organization damage.
Collagen sponge prepared by the present invention is three-dimensional collagen-based materials, its aperture is adjusted at 30~200 μm, to be used for cell
Growth and tissue repair, and different shapes can be modelled as by mold and be suitable for different repairing of neural injury.
The present invention collectively promotes nerve regneration using growth factor and neural stem cell, improves neural stem cell in damage office
The quantity in portion enhances repair of the neural stem cell to neurotrosis, also, growth factor itself can promote neurotrosis
It repairs.
In Nerve Scaffold of the invention, by heparin crosslinking on collagen sponge, and by heparin to heparin binding domain
Growth factor high-affinity, growth factor is fixed on collagen sponge, to control the rate of release of growth factor, is prevented
Growth factor diffusion, avoid growth factor after body fluid washes away a large amount of diffusions due to caused by loss, meanwhile, be fixed on collagen sponge
On growth factor on the one hand itself can more effectively promote repairing of neural injury, on the other hand can give neural stem cell provide battalion
Nerve stem cell proliferation is supported or promoted, neural stem cell is improved in the quantity of damage part, enhances neural stem cell to nerve
The repair of damage collectively promotes repairing of neural injury with neural stem cell.
In Nerve Scaffold of the invention, growth factor is not easy to spread after fixing by heparin, therefore can be reduced because spreading band
The side effect come, fixed growth factor itself can be partially formed effective concentration in treatment, can promote repairing of neural injury.
When growth factor be basic fibroblast growth factor (basic fibroblast growth factor,
When bFGF), bFGF can promote a large amount of proliferation of mesoderm and neural endoderm cell in embryo development procedure, and in difference
Proliferation and atomization in play adjustment effect.Basic fibroblast growth factor is added in the proximal end energy of nerves transected wound
Enough prevent the death of sensory neuron in dorsal root ganglion after damaging.In addition to this, in the position of damage, basic fibroblast
Growth factor can promote axon regeneration and revascularization, and regenerated nerve fibre is promoted to pass through the nearly distal end of neurologic defect.Alkali
Property fibroblast growth factor is widely used in neural restoration, and neural stem cell, which is directly used at peripheral nerve injury, to divide
Turn to the cell of schwann cells shape, can secretory nerve trophic factors, promote nerve regneration, the present invention consolidates bFGF by heparin
It is scheduled in Nerve Scaffold, controls its rate of release.
In addition to this, fixed bFGF can promote nerve stem cell proliferation and maintain neural stem cell activity, and nerve cord
Cell can also be effectively facilitated repairing of neural injury, therefore, fixed bFGF and neural stem cell in Nerve Scaffold of the present invention
Synergistic effect can be played, nerve stem cell proliferation is promoted, improves neural stem cell in the quantity of damage part, enhancing nerve cord is thin
Repair of the born of the same parents to neurotrosis, the reparation after collectively promoting neurotrosis, therefore, the present invention contain bFGF and neural stem cell
Nerve Scaffold can effectively facilitate nerve regneration and functional rehabilitation.
By the present invention in that the collagen solution for being dissolved in acetic acid is directly lyophilized with freeze dryer, collagen sponge is obtained, is made a living
It is multiple to avoid acid-base neutralization, dialysis etc. using the method being crosslinked after freeze-drying to it for the carrier of the long factor and neural stem cell
Miscellaneous process, and the aperture of collagen sponge can be adjusted by different collagen concentrations, when crosslinking, crosslinking agent passes through in catalytic molecular
Carboxyl and amino reaction are crosslinked, and also known as " zero-length cross-linkers ", the tension and stabilization of collagenous fibres are improved after crosslinking
Property, crosslinking agent is 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and N- hydroxysuccinimide, EDC/NHS.
Compared with prior art, beneficial effects of the present invention:
1) Nerve Scaffold of the invention, raw material materials are convenient, and preparation process is simple, by using heparin by growth factor into
Row is fixed, and is controlled the rate of release of growth factor, is prevented growth factor from spreading under the impact of body fluid, so that growth factor be consolidated
It is scheduled on damage part, reaches treatment concentration in damage part, meanwhile, fixed growth factor can effectively facilitate refreshing in Nerve Scaffold
Survival through stem cell, or promote nerve stem cell proliferation, so that damage local nerve stem cell is reached more effectively treatment number
Amount, enhances repair of the neural stem cell to neurotrosis, the neural stem cell and growth factor in bracket collectively promote
Nerve regneration, so that repairing of neural injury effect is more significant compared with conventional method.
2) in preparation method of the invention, collagen solution is prepared using rat-tail and is drawn materials conveniently, the collagen solution of acquisition is used
The step of freeze dryer is directly lyophilized, and manufacturing process is simple, eliminates collagen through acid-base neutralization, the complex process such as dialysis, is reduced
The chance of collagen-based materials contact harmful reagents, therefore its safety in utilization greatly improves.
3) neural stem cell is marked using Hoechst by the present invention, can track the change of neural stem cell amount in vivo
Change situation.
Detailed description of the invention
Fig. 1 is the collagen sponge aspect graph of the embodiment of the present invention 1.
Fig. 2 is the nerve trachea of the embodiment of the present invention 2.
Fig. 3 is to detect cell Proliferation and expression activitiy figure by CCK-8 in the embodiment of the present invention 2.
Fig. 4 is to detect cell Proliferation and expression activitiy figure by CCK-8 in the embodiment of the present invention 3.
Fig. 5 is to detect cell Proliferation and expression activitiy figure by CCK-8 in the embodiment of the present invention 4.
Fig. 6 is that functional rehabilitation compares figure after facial nerve injury in the embodiment of the present invention 5.
Specific embodiment
Below in conjunction with specific embodiments and the drawings, the invention will be further described, and the content of present invention is not limited to listed reality
Example is applied, the non-intrinsically safe modifications and adaptations that researcher carries out the Nerve Scaffold according to above content still fall within guarantor of the invention
Protect range.
SD rat is purchased from Fudan University experimental animal portion, and animal hearts perfusion is purchased from boster engineering finite with paraformaldehyde
Company, deionized water are purchased from invitrogen, and glacial acetic acid is purchased from Sinopharm Chemical Reagent Co., Ltd., and PBS is purchased from
Hyclone, EDC and NHS are purchased from sigma；BFGF and VEGF is purchased from peprotech；Hoechst is purchased from sigma.
Embodiment 1
1) rat is put to death by heart perfusion, cuts rat-tail, extract the silver in rat-tail by the rat for buying 180~220g
Color tail tendon is put into deionized water, is put on shaking table, 4 DEG C of temperature, 50~200rpm of revolving speed, washs 5 times repeatedly, the tail tendon after washing
It is dissolved in 0.05%~0.1% glacial acetic acid-PBS, 4 DEG C of temperature, stands 2~5 days, adjustment collagen concentration to 10mg/ml.
2) take upper layer collagen solution, be put into mold, mold is put into liquid nitrogen frozen, then be put in 2.5L freeze dryer through 8~
It is lyophilized within 12 hours, obtains collagen sponge, for three-dimensional collagen-based materials, aperture is 30~200 μm, and the collagen sponge form is referring to Fig. 1.
3) sterilization treatment, treatment conditions are as follows: 12kGy are carried out to collagen sponge using electron beam irradiation60Co。
4) heparin being dissolved in the crosslinker solution containing 4mM EDC and 2.5mM NHS, the concentration of heparin is 4mg/mL,
Collagen sponge is immersed in crosslinker solution, is placed 3~4 hours at 37 DEG C, finally washs 4~8 hours with PBS, deionization
Water washing 4~8 hours.
5) collagen scaffold, is immersed in the blood vessel endothelium of 450 μ g/mL by the moisture after sucking crosslinking with filter paper in collagen sponge
Collagen scaffold is constructed in growth factor (VEGF) solution, 37 DEG C, 1 hour, obtains collagen scaffold.
6) originally culture neural stem cell
Pregnant 19 days or so pregnant mouse are taken, is put to death in super-clean bench inner termination, takes out akrencephalon, shredded akrencephalon using curved scissors, be added
0.05% pancreatin digests 30 minutes, is terminated and is digested using pancreatin inhibitor, and postdigestive mixed liquor is passed through 200 mesh filter screens,
After collecting filtered solution, revolving speed 1800rpm is centrifuged 5 minutes, removes supernatant, and culture solution culture is added, uses after obtaining nerve ball
Nerve ball digestion is opened the neural stem cell dispersed by 0.05% pancreatin, and neural stem cell and Hoechst are cultivated at 37 DEG C
Case is incubated for 10 minutes, neural stem cell is marked, by 5~10 × 105A Culture of neural stem cells on collagen scaffold,
Obtain the Nerve Scaffold.
Embodiment 2
1. including the following steps: for the preparation method for the nerve trachea that repairing of neural injury is put into
1) rat is put to death by heart perfusion, cuts rat-tail, extract the silver in rat-tail by the rat for buying 180~220g
Color tail tendon is put into deionized water, is put on shaking table, 4 DEG C of temperature, 50~200rpm of revolving speed, washs 5 times repeatedly, the tail tendon after washing
It is dissolved in 0.05%~0.1% glacial acetic acid-PBS, 4 DEG C of temperature, stands 2~5 days, adjustment collagen concentration to 10mg/ml.
2) upper layer collagen solution is taken, is put into tubular die, mold is put into liquid nitrogen frozen, then is put in 2.5L freeze dryer warp
It is lyophilized within 8~12 hours, obtains the collagen sponge of hollow tubular, i.e. collagen tube.
Collagen sponge is that the carrier of growth factor and neural stem cell therefore can be in the hollow portion of collagen tube obtained
Divide filling some collagen sponges, to adhere to more neural stem cell.
3) sterilization treatment, treatment conditions are as follows: 6kGy are carried out to collagen tube using electron beam irradiation60Co。
4) heparin being dissolved in the crosslinker solution containing 4mM EDC and 2.5mM NHS, the concentration of heparin is 2mg/mL,
Collagen tube is immersed in the crosslinker solution, is placed 3~4 hours at 37 DEG C, finally washs 4~8 hours with PBS, deionization
Water washing 4~8 hours.
5) collagen tube is immersed in the bFGF of 50~500 μ g/mL after the moisture after sucking crosslinking with filter paper in collagen tube
Molding, temperature are 37 DEG C, and the time is 1 hour, obtain collagen scaffold, i.e. collagen catheter.
6) originally culture neural stem cell
Pregnant 19 days or so pregnant mouse are taken, is put to death in super-clean bench inner termination, takes out akrencephalon, shredded akrencephalon using curved scissors, be added
0.05% pancreatin digests 30 minutes, is terminated and is digested using pancreatin inhibitor, and postdigestive mixed liquor is passed through 200 mesh filter screens,
After collecting filtered solution, revolving speed 1800rpm is centrifuged 5 minutes, removes supernatant, and culture solution culture is added, uses after obtaining nerve ball
Nerve ball digestion is opened the neural stem cell dispersed by 0.05% pancreatin, and neural stem cell and Hoechst are cultivated at 37 DEG C
Case is incubated for 10 minutes, neural stem cell is marked, by 5~10 × 105A Culture of neural stem cells in collagen catheter,
Nerve trachea is obtained, sees Fig. 2.
2. detecting cell Proliferation and activity by CCK-8.
If 3 groups of Nerve Scaffold parallel laboratory tests, first group is the present embodiment group, and in the Nerve Scaffold of the group, passing through heparin will
BFGF is fixed on collagen scaffold, and culture has neural stem cell (abbreviation HS-bFGF group)；Second group is comparative example group, the group
Nerve Scaffold in, the fixed bFGF of heparin is not used, but bFGF is simply incubated on collagen scaffold, culture has neural stem cell
(abbreviation bFGF group)；Third group is control group, in the Nerve Scaffold of the group, does not contain heparin and bFGF, only right as feminine gender using PBS
According to incubating on collagen scaffold, and cultivates and have neural stem cell (abbreviation PBS group).
Neural stem cell is cultivated 7 days on above-mentioned three groups of Nerve Scaffold, liquid is changed daily, passed through CCK-8 at the 7th day
Detect the proliferation and activity of neural stem cell.
Detecting step is as follows:
1) above-mentioned three groups of samples are respectively put into porous plate, 10%CCK8 mixed liquor is added.
2) 37 DEG C of incubator cultures 2 hours are put into.
3) mixed liquor is sucked out, measures 450nm absorbance.
As a result referring to Fig. 3, * * indicates P < 0.01 in figure, and as seen from Figure 3, the proliferation and activity of neural stem cell exist
HS-bFGF group will be apparently higher than bFGF group and PBS group, and due to changing liquid daily, loose bFGF is continuous in bFGF group and PBS group
Spread, lost, cannot be effectively facilitated nerve stem cell proliferation, bFGF group and PBS group nerve stem cell proliferation and activity compared with
Difference.
Embodiment 3
1. the preparation method for the nerve trachea being put into the present embodiment for repairing of neural injury is led with nerve in embodiment 2
The difference of the preparation method of pipe is that collagen concentration is 20mg/ml, the concentration of crosslinking agent in crosslinker solution are as follows: 10mM EDC and
5mM NHS, the concentration of heparin are 4mg/mL, and the concentration of bFGF is 500 μ g/mL, remaining operating procedure is the same as embodiment 2.
2. detecting cell Proliferation and activity by CCK-8.
If 3 groups of Nerve Scaffold parallel laboratory tests, first group is the present embodiment group, and in the Nerve Scaffold of the group, passing through heparin will
BFGF is fixed on collagen scaffold, and culture has neural stem cell (abbreviation HS-bFGF group)；Second group is comparative example group, the group
Nerve Scaffold in, the fixed bFGF of heparin is not used, but bFGF is simply incubated on collagen scaffold, culture has neural stem cell
(abbreviation bFGF group)；Third group is control group, in the Nerve Scaffold of the group, does not contain heparin and bFGF, only right as feminine gender using PBS
According to incubating on collagen scaffold, and cultivates and have neural stem cell (abbreviation PBS group).
Neural stem cell is cultivated 7 days on above-mentioned three groups of Nerve Scaffold, liquid is changed daily, passed through CCK-8 at the 7th day
Detect the proliferation and activity of neural stem cell.
Detecting step is as follows:
1) above-mentioned three groups of samples are respectively put into porous plate, 10%CCK8 mixed liquor is added.
2) 37 DEG C of incubator cultures 2 hours are put into.
3) mixed liquor is sucked out, measures 450nm absorbance.
As a result referring to fig. 4, * * indicates P < 0.01 in figure, and as seen from Figure 4, the proliferation and activity of neural stem cell exist
HS-bFGF group will be apparently higher than bFGF group and PBS group, and due to changing liquid daily, loose bFGF is continuous in bFGF group and PBS group
Spread, lost, cannot be effectively facilitated nerve stem cell proliferation, bFGF group and PBS group nerve stem cell proliferation and activity compared with
Difference.
Embodiment 4
1. nerve trachea in the preparation method and embodiment 2 of the nerve trachea that the present embodiment is put into for repairing of neural injury
The difference of preparation method be that collagen concentration is 7mg/ml, the concentration of crosslinking agent in crosslinker solution are as follows: 1mM EDC and 10mM
The concentration of NHS, heparin are 3mg/mL, and the 100 μ g/mL of concentration of bFGF, remaining operating procedure is the same as embodiment 2.
2. detecting cell Proliferation and activity by CCK-8.
If 3 groups of Nerve Scaffold parallel laboratory tests, first group is the present embodiment group, and in the Nerve Scaffold of the group, passing through heparin will
BFGF is fixed on collagen scaffold, and culture has neural stem cell (abbreviation HS-bFGF group)；Second group is comparative example group, the group
Nerve Scaffold in, the fixed bFGF of heparin is not used, but bFGF is simply incubated on collagen scaffold, culture has neural stem cell
(abbreviation bFGF group)；Third group is control group, in the Nerve Scaffold of the group, does not contain heparin and bFGF, only right as feminine gender using PBS
According to incubating on collagen scaffold, and cultivates and have neural stem cell (abbreviation PBS group).
Neural stem cell is cultivated 7 days on above-mentioned three groups of Nerve Scaffold, liquid is changed daily, passed through CCK-8 at the 7th day
Detect the proliferation and activity of neural stem cell.
Detecting step is as follows:
1) above-mentioned three groups of samples are respectively put into porous plate, 10%CCK8 mixed liquor is added.
2) 37 DEG C of incubator cultures 2 hours are put into.
3) mixed liquor is sucked out, measures 450nm absorbance.
As a result referring to Fig. 5, * * indicates P < 0.01 in figure, and as seen from Figure 5, the proliferation and activity of neural stem cell exist
HS-bFGF group will be apparently higher than bFGF group and PBS group, and due to changing liquid daily, loose bFGF is continuous in bFGF group and PBS group
Spread, lost, cannot be effectively facilitated nerve stem cell proliferation, bFGF group and PBS group nerve stem cell proliferation and activity compared with
Difference.
As it can be seen that changing collagen solution concentration, heparin concentration and the obtained Nerve Scaffold of growth factor concentration in the present invention
In neural stem cell proliferation and activity be superior to simply use growth factor and negative control group.
5. experiment in vivo of embodiment verifies the repairing of neural injury effect of Nerve Scaffold of the invention
Adult female SD rat is selected, routine disinfection after rat anesthesia is spread list by 200~220g of weight, takes left side face
Row notch cuts skin, separates muscle, and connective tissue exposes the nervus buccinatorius branch of facial nerve, cuts off 8mm long nerve,
Then the nerve trachea of the embodiment of the present invention 2 is sewn to the neural broken ends of fractured bone with needle suture with 9-0, neurologic defect is connected
Come.
8 weeks after surgery observation rat two sides whisker movement opposite neurological functional recoveries are evaluated through row.Ipsilateral beard without motion
It is chosen as 1 point；Ipsilateral beard has slight movement to be chosen as 2 points；Ipsilateral whisker movement is comparatively close to strong side whisker movement amplitude and frequency
Rate is chosen as 3 points；Ipsilateral whisker movement and strong side whisker movement are completely the same, and being chosen as 4 points, (i.e. facial nerve function has normally restored afterwards
Entirely).Statistical analysis is done into Ipsilateral scoring and ratio (vibrissae movement ratio) i.e. recovery rate of strong side scoring,
Scoring is double blind experiment, as a result referring to Fig. 6.
From fig. 6, it can be seen that HS-bFGF group postoperative function recovery rate is apparently higher than other two groups, the result with pass through
CCK-8 detection cell Proliferation is identical as active experimental result trend, and in HS-bFGF group, on the one hand fixed bFGF can promote
Nerve stem cell proliferation maintains neural stem cell activity, enhances repair of the neural stem cell to neurotrosis, on the other hand
Fixed bFGF can effectively facilitate nerve regneration from the effective concentration being partially formed in damage, and bFGF and neural stem cell are mutual
Collaboration collectively promotes neural restoration after rat facial nerve damage, and in bFGF group, since growth factor is without fixation, in vivo
A large amount of diffusions, thus the concentration in damage part is lower, cannot effectively facilitate promotion nerve stem cell proliferation, damages to nerve
The repair of wound is limited.
Claims (9)
1. a kind of Nerve Scaffold for repairing of neural injury, which is characterized in that including collagen scaffold, culture on collagen scaffold
Neural stem cell and the growth factor on collagen scaffold is fixed on by heparin, the growth factor has Heparin-binding
Domain, the heparin are crosslinked on collagen scaffold by crosslinker solution；Wherein, the crosslinker solution be concentration be 1~
The N- hydroxysuccinimide of 1- (3- the dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride and 1~12.5mM of 25mM
Mixed solution.
2. a kind of preparation method of the Nerve Scaffold for repairing of neural injury, includes the following steps:
1) collagen solution is prepared
Animal collagen tissue is obtained, is dissolved in 0.05~0.1% glacial acetic acid-PBS (v/v), is configured to collagen solution；
2) it is lyophilized, sterilizes
Collagen solution is poured into mold, then mold is put into liquid nitrogen frozen, is then lyophilized with freeze dryer, freeze-drying time be 8~
12 hours, collagen sponge is obtained, then carry out sterilization treatment；The collagen sponge is three-dimensional collagen-based materials, and aperture is 30~200
μm；
3) it is crosslinked, washs
Heparin is dissolved in crosslinker solution, the collagen sponge of sterilizing is immersed in the crosslinker solution containing heparin and is crosslinked,
It 30~40 DEG C of crosslinking temperature, crosslinking time 3~4 hours, is washed 4~8 hours after crosslinking with phosphate buffered saline solution, then use
Deionized water is washed 4~8 hours；
Wherein, the crosslinker solution is 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride that concentration is 1~25mM
The mixed solution of the N- hydroxysuccinimide of salt and 1~12.5mM；
4) it forms
Collagen sponge after crosslinking is sucked into moisture, is immersed in growth factor solution and forms, 30~40 DEG C of forming temperature, molding
Time 0.5~2 hour, obtain collagen scaffold；Wherein, the growth factor has heparin binding domain；
5) culture of neural stem cells neural
By Primary culture of neural stem cells on the collagen scaffold that step 4) obtains, the Nerve Scaffold is obtained.
3. the preparation method for the Nerve Scaffold of repairing of neural injury according to claim 2, which is characterized in that step 1)
The concentration of collagen is 5~20mg/mL in the collagen solution of middle acquisition.
4. the preparation method for the Nerve Scaffold of repairing of neural injury according to claim 2, which is characterized in that step 2)
Middle to carry out sterilization treatment to collagen sponge using electron beam irradiation, treatment conditions are 6~12kGy60Co。
5. the preparation method for the Nerve Scaffold of repairing of neural injury according to claim 2, which is characterized in that step 3)
In in the crosslinker solution containing heparin, the concentration of heparin is 2~4mg/mL.
6. the preparation method for the Nerve Scaffold of repairing of neural injury according to claim 2, which is characterized in that step 4)
The concentration of growth factor is 50~500 μ g/mL in the growth factor solution.
7. the preparation method according to claim any one of 2-6 for the Nerve Scaffold of repairing of neural injury, feature exist
In growth factor described in step 4) is basic fibroblast growth factor or vascular endothelial growth factor.
8. the preparation method according to claim any one of 2-6 for the Nerve Scaffold of repairing of neural injury, feature exist
In in step 5) by neural stem cell after Hoechst is marked, being further cultured on collagen scaffold, the number of neural stem cell is 5
~10 × 105It is a.
9. the preparation method according to claim any one of 2-6 for the Nerve Scaffold of repairing of neural injury, feature exist
In, Nerve Scaffold obtained be nerve trachea or circle, ellipse, rectangular or irregular cavernous nerve bracket.
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| CN108324993B (en) * | 2018-01-15 | 2020-11-03 | 朱剑虹 | Stem cell complex for inducing hair regeneration, preparation method and application thereof |
| CN108653193A (en) * | 2018-06-15 | 2018-10-16 | 大连医科大学附属第医院 | A kind of stem cell medicine sponge patch complex, preparation method and application for treating brain diseases |
| CN109464700B (en) * | 2018-11-22 | 2021-09-21 | 深圳先进技术研究院 | Paste for 3D printing, 3D structure and preparation method and application thereof |
| CN110917386B (en) * | 2019-12-10 | 2021-12-07 | 康妍葆(北京)干细胞科技有限公司 | Stem cell sponge scaffold, preparation method and application |
| CN111500522A (en) * | 2020-04-12 | 2020-08-07 | 江苏安泰康健康科技有限公司 | Culture system based on pigskin collagen and three-dimensional tissue culture method |
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