CN108452377A - Growth factor composite collagen timbering material and function collagen as tissue engineering scaffold for repairing nerve damage - Google Patents
Growth factor composite collagen timbering material and function collagen as tissue engineering scaffold for repairing nerve damage Download PDFInfo
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- CN108452377A CN108452377A CN201710092354.7A CN201710092354A CN108452377A CN 108452377 A CN108452377 A CN 108452377A CN 201710092354 A CN201710092354 A CN 201710092354A CN 108452377 A CN108452377 A CN 108452377A
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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
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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
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
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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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
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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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
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Abstract
The invention discloses the growth factor composite collagen timbering materials and function collagen as tissue engineering scaffold for repairing nerve damage.Growth factor composite collagen timbering material disclosed by the invention is made of function collagen as tissue engineering scaffold with growth factor；Function collagen as tissue engineering scaffold is made of the collagen of neurofilament and coating neurofilament；Growth factor is nerve growth factor or basic fibroblast growth factor；Collagen is collagen tube.Experiments have shown that, the function collagen as tissue engineering scaffold (LOCC) of the present invention can be used for repairing nerve damage, growth factor composite collagen timbering material can also be used for repairing animal nerve damage, show that the function collagen as tissue engineering scaffold of the present invention can be applied to animal nerve injury repair with growth factor composite collagen timbering material.
Description
Technical field
The present invention relates in biotechnology, for repairing nerve damage growth factor composite collagen timbering material and
Function collagen as tissue engineering scaffold.
Background technology
Neurotrosis is clinical common one of disease, the direct broken ends of fractured bone suturing skill clinically applied at present or self god
Clinical dilemma cannot fully be solved due to the limitation with its application through transplantation method.With the development of organizational project, adopt
Nerve regneration is promoted to become research hotspot with repairing nerve damage with Method of Tissue Engineering.Suitable injury repair material can not only
The holder of regenerating tissues growth is provided, and can be to providing guiding function with the special tissue with directional growth.
But lacks suitable repairing of neural injury material at present, be badly in need of a kind of suitable repairing of neural injury material at present.
Invention content
The technical problem to be solved by the present invention is to how repair animal nerve damage.
In order to solve the above technical problems, present invention firstly provides the growth factor compound adhesives that can be used for repairing nerve damage
Former timbering material.
Growth factor composite collagen timbering material provided by the present invention, by function collagen as tissue engineering scaffold and growth factor group
At；
The function collagen as tissue engineering scaffold, entitled LOCC are made of neurofilament with the collagen for being coated with the neurofilament.
The neurofilament can be Filamentous collagen-based materials.The neurofilament can be prepared by vitro fascia.The fascia
It can be bovine fascia.The preparation method of the neurofilament may include：By the fascia successively through acetone, NaTDC,
TritonX-100 and/or Papain enzymatic treatment obtain the neurofilament.
In above-mentioned growth factor composite collagen timbering material, the growth factor can be nerve growth factor (Nerve
Growth factor, NGF) or basic fibroblast growth factor (basic fibroblast growth factor,
bFGF)。
In above-mentioned growth factor composite collagen timbering material, the collagen can be collagen tube (i.e. tubulose collagen-based materials).
The preparation method of the function collagen as tissue engineering scaffold may include：The neurofilament is filled in the collagen tube and is obtained
To the function collagen as tissue engineering scaffold.The diameter of the collagen tube can determine according to specific needs, such as according to damage to be repaired god
The diameter of warp determines.
The proportioning of the neurofilament and the collagen tube can be determined according to specific needs.The amount of the neurofilament meets
The collagen tube is filled up radially.
In one embodiment of the invention, the proportioning of the collagen tube and the neurofilament is a collagen tube：
30 neurofilaments.
The collagen tube can be identical with the length of the neurofilament, and the both ends of the collagen tube also can be slightly longer than the god
Organizine.The length of the collagen tube can be determined according to the breaking length of neurotrosis.
In one embodiment of the invention, the breaking length of the neurotrosis is 3.5cm, the collagen tube used
Length is 4cm.
In above-mentioned growth factor composite collagen timbering material, the collagen tube is according to the method system that may include following steps
It is standby：The mold of winding collagem membrane is obtained with collagem membrane Wound Dies, with containing collagen on the mold of the winding collagem membrane
More than the wrapped circle of liquid or a circle, the mold is taken out after dry, obtains the collagen tube.
The material of the mold can be glass or plastics.The mold can be cylindric.The diameter of the mold can basis
Specifically it needs to be determined that, such as determined according to the determination of the diameter of injured nerve to be repaired or according to the diameter of required collagen tube.
The collagem membrane can be membranaceous collagen-based materials.The collagem membrane can be prepared by vitro skin histology.It is described
Skin histology can be ox-hide skin tissue.The preparation method of the collagem membrane may include：By the skin histology successively through SDS, spit
Temperature 80 and/or the wooden acetone treatment obtain the collagem membrane.The thickness of the collagem membrane can be 0.2-0.4mm, such as 0.3mm.
The collagem membrane Wound Dies may also include the softening collagem membrane.The softening collagem membrane can specifically wrap
It includes and impregnates the collagem membrane with water (such as tri-distilled water).
It is more than a circle that the use collagem membrane Wound Dies concretely wind the mold with the collagem membrane.It is described to use glue
Former film Wound Dies concretely wind the mold 5/4 with the collagem membrane and enclose.
Collagen in the liquid containing collagen it is different from the collagen in the collagen of the coating neurofilament it
It place can be only morphologically different.The liquid containing collagen can be prepared by the collagem membrane.The liquid containing collagen
The collagem membrane is concretely dissolved in the liquid obtained in 1M acetic acid aqueous solutions, the collagem membrane and 1M acetic acid aqueous solutions by body
Proportioning can be 1 gram：500 milliliters.The preparation method of the liquid containing collagen includes：The collagem membrane is dissolved in 1M acetic acid water
In solution, in 4 degree it is lower impregnate 24 hours, 10000g centrifuges 20 minutes removal precipitations, obtains the liquid containing collagen.
The drying can specifically air-dry at room temperature.
In above-mentioned growth factor composite collagen timbering material, the function collagen as tissue engineering scaffold can be handled with growth factor and obtained
To the growth factor composite collagen timbering material.
It is described to handle the function collagen as tissue engineering scaffold with growth factor in above-mentioned growth factor composite collagen timbering material
It may include：Liquid containing the growth factor is added on the function collagen as tissue engineering scaffold, it is multiple to obtain the growth factor
Close collagen as tissue engineering scaffold.
It is described to add to the liquid containing the growth factor on the function collagen as tissue engineering scaffold, it can make described containing
The liquid for stating growth factor is full of the internal clearance of the function collagen as tissue engineering scaffold and/or is covered with the function collagen scaffold material
The outer surface of material.
The liquid containing the growth factor is made of the growth factor as solute with the water as solvent.
Collagen tube described in the growth factor composite collagen timbering material, the growth factor are matched with the neurofilament
Than that can be 1 collagen tube：Growth factor described in 20-40 μ g：30 neurofilaments.The growth factor composite collagen branch
Collagen tube described in frame material, the growth factor and the proportioning of the neurofilament can be (20-100) mg：(20-40)μg:
(100-500)mg.Collagen tube described in the growth factor composite collagen timbering material, the growth factor and the neurofilament
Proportioning concretely (20-100) mg：40μg:(100-500) mg, such as 60mg：40μg:300mg.The growth factor is compound
Collagen tube described in collagen as tissue engineering scaffold, the growth factor and the proportioning of the neurofilament are specifically alternatively (20-100) mg：
20μg:(100-500) mg, such as 60mg：20μg:300mg.
In order to solve the above technical problems, the present invention also provides the function collagen as tissue engineering scaffold.
In order to solve the above technical problems, the present invention also provides the substances for repairing animal nerve damage.
Substance provided by the present invention for repairing animal nerve damage, by collagen-based materials and the growth factor group
At.
In above-mentioned substance, the collagen-based materials and the proportioning of the growth factor can be (100-500) mg：(20-40) μ g,
Such as 360mg：(20-40)μg.
In above-mentioned substance, the collagen-based materials can be Filamentous, tubulose and/or membranaceous collagen-based materials.The collagen-based materials tool
Body can be prepared by the Filamentous, tubulose and/or membranaceous collagen-based materials.
In above-mentioned substance, the collagen-based materials are specifically alternatively function as described above collagen as tissue engineering scaffold.
In order to solve the above technical problems, the present invention also provides the function collagen as tissue engineering scaffold, the growth factor are multiple
Close collagen as tissue engineering scaffold or following any applications for repairing the substance of animal nerve damage：
X1, the application in repairing animal nerve damage product is being prepared；
X2, the application in repairing animal nerve damage；
X3, the application in promoting animal nerve reconstituted product is being prepared；
X4, the application in promoting animal nerve regeneration；
X5, the application in promoting animal Remyelination product is being prepared；
X6, the application in promoting animal Remyelination.
In the present invention, the animal can be mammal.The mammal can be beasle dog.
In the present invention, the neural concretely sciatic nerve.The myelin concretely sciatic nerve myelin.
In the present invention, the neurotrosis can be long cross-section neurotrosis.The length of the cross-section neurotrosis of length can be
3-3.8cm such as 3.5cm.
It is demonstrated experimentally that the present invention is successfully prepared the function collagen as tissue engineering scaffold (LOCC) that can be used for repairing of neural injury,
Pass through the neurotrosis for repairing animal nerve damage model, it was demonstrated that the material can be used for repairing nerve damage, utilize growth
The effect that the factor repairs animal nerve damage after compound LOCC respectively shows that the LOCC after composite growth factor can also be used for repairing
Animal nerve damages, and repairing effect is substantially better than LOCC.Show the function collagen as tissue engineering scaffold and growth factor of the present invention
Composite collagen timbering material can be applied to animal nerve injury repair.
Description of the drawings
Fig. 1 is that CO2 laser weld is performed the operation schematic diagram and the material used.(A) it is CO2 laser weld operation schematic diagram；(B) it is
LOCs；(C) it is collagen tube.
Fig. 2 is compound muscle action potential peak value ratio of the Ipsilateral than strong side.* it indicates compared with blank control group, difference
The level of signifiance (p is reached<0.05), * * indicate that compared with blank control group, difference has reached the level of signifiance (p<0.01).
Fig. 3 is expression of the NF albumen in each group.* indicates that compared with blank control group, difference has reached the level of signifiance
(p<0.01), * * * indicate that compared with blank control group, difference has reached the level of signifiance (p<0.001).
Fig. 4 is expression of the S100 albumen in each group.* indicates that compared with blank control group, difference has reached notable water
Flat (p<0.01), * * * indicate that compared with blank control group, difference has reached the level of signifiance (p<0.001).
Fig. 5 is regeneration situation of the newborn myelin of fast blue dyeing detection in each group.* is indicated compared with blank control group, poor
It is different to have reached the level of signifiance (p<0.01), * * * indicate that compared with blank control group, difference has reached the level of signifiance (p<0.001).
Fig. 6 is regeneration situation of the newborn myelin of transmission electron microscope detection in each group.* expressions are compared with blank control group, difference
The level of signifiance (p is reached<0.01), * * * indicate that compared with blank control group, difference has reached the level of signifiance (p<0.001).
Specific implementation mode
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Beasle dog in following embodiments is Nanjing An Limo Science and Technology Ltd.s product.
Embodiment 1, function collagen as tissue engineering scaffold and growth factor composite collagen timbering material can treat the length of beasle dog
Apart from cross-section injury of sciatic nerve
Function collagen as tissue engineering scaffold (LOCC) provided by the present invention is in a branch of directive neurofilament (LOCs) of tool
The material that one collagen of outer wrapping is in control, growth factor composite collagen timbering material are the LOCs handled by a branch of growth factor
The material that one collagen of outer wrapping is in control.
1, the preparation of function collagen as tissue engineering scaffold and growth factor composite collagen timbering material
The preparation of LOCs：
In vitro Cowhells membrane tissue is removed into muscle and fat, bovine fascia after being handled, is soaked in acetone 24 hours, is used
Deionized water is cleaned 3 times, every time 2 hours；Bovine fascia after acetone treatment is soaked in the deoxycholic acid of mass percentage 3% again
24 hours in sodium water solution, deionized water is cleaned 3 times, every time 2 hours；Bovine fascia is soaked in matter after NaTDC is handled again
It measures in the TritonX-100 aqueous solutions of percentage composition 1% 24 hours, is cleaned 3 times, every time 2 hours with deionized water；Again will
Bovine fascia is soaked in 1% Papain enzyme aqueous solution of mass percentage 12 hours after TritonX-100 processing, uses deionization
Water cleans 3 times, every time 2 hours；It is again that bovine fascia after Papain enzymatic treatment is water-soluble in the pepsin of mass percentage 1%
It is impregnated 20 minutes in liquid, deionized water is cleaned 3 times, every time 2 hours；Then it is lyophilized 48 hours, obtains at vacuum degree 0.1Mpa
Neurofilament (LOCs), LOCs are shown in B in Fig. 1.
The preparation of collagem membrane：
In vitro ox-hide skin tissue is taken, is lost hair or feathers, cuticula and epidermis is removed, obtains dermal tissue, dermal tissue is soaked in
In the SDS aqueous solutions of mass percentage 5%, after impregnating 48 hours, deionized water is cleaned 3 times, every time 2 hours；It takes out at SDS
Dermal tissue is soaked in 1% Tween 80 aqueous solution of mass percentage 24 hours again after reason, then deionized water cleaning 5 times,
2 hours every time；Dermal tissue is soaked in acetone 5 hours again after taking out Tween 80 processing, and deionized water is cleaned 3 times, and 2 is small every time
When；Dermal tissue freezes 24 hours in -80 degree refrigerators after then taking out acetone treatment, is put in 16 degree of water and melts, multigelation
Three times, it is finally lyophilized 48 hours under 0.1Mpa again, obtains collagem membrane.
The preparation of collagen tube：
It selects radius for the glass mold of 2.5mm, collagem membrane (thickness 0.3mm) is cut into suitable size, steamed with three
Water logging maceration is saturating, and by collagem membrane around 5/4 circle of mold winding, collagen solution (collagen solution preparation method is used after winding：By 1 gram of glue
Former film is dissolved in 500 milliliters of 1M acetic acid aqueous solutions, is impregnated 24 hours under 4 degree, and 10000g centrifuges 20 minutes removal precipitations, takes
It is collagen solution clearly) a wrapped circle, ambient temperature overnight air-dries, mold taken out to get to collagen tube (C in Fig. 1), collagen tube freezes
It is spare after dry, irradiation.
The preparation of LOCC：
LOCs is filled in collagen tube, the proportioning of the both ends slightly longer than LOCs of collagen tube, LOCs and collagen tube is 30
Root collagen silk：1 collagen tube is to get to LOCC.
To contain 40 μ g nerve growth factors (Nerve growth factor, NGF) (Life Technologies companies,
Article No. 13257019) NGF solution (NGF solution is that the obtained liquid of NGF is added into deionized water) drop in a branch of LOCC
(4cm long) (proportioning of NGF, LOCs and collagen tube is 40 μ gNGF：30 collagen silks (300mg/30 roots)：1 collagen tube
(60mg/)) on, so that NGF solution is full of the internal clearance of entire LOCC, be incubated after twenty minutes to get to the compound LOCC of NGF,
It is named as NGF-LOCC.
20 μ g basic fibroblast growth factors (basic fibroblast growth factor, bFGF) will be contained
(bFGF suspensions are that bFGF is added into deionized water to the bFGF solution of (Life Technologies companies, article No. 13256029)
Obtained liquid) dropping in a branch of LOCC (4cm long), (proportioning of bFGF, LOCs and collagen tube is 20 μ g bFGF：30 collagen silks
(300mg/30 roots)：1 collagen tube (60mg/)) on, so that NGF solution is full of the internal clearance of entire LOCC, is incubated 20 minutes
Afterwards to get to the compound LOCC of bFGF, it is named as bFGF-LOCC.
2, function collagen as tissue engineering scaffold and growth factor composite collagen timbering material can be used for treating the length of beasle dog away from
From cross-section injury of sciatic nerve
Animal packet：15 adult healthy beasle dogs are randomly divided into five groups, i.e., blank control group, LOCC groups, NGF groups,
BFGF groups and autotransplantation group (as positive control), every group 3.
2.1 prepare the sciatic nerve transection lesion model of beasle dog (3.5cm) over long distances, and neurotrosis function is repaired glue
Former timbering material bridge joint is at neurotrosis both ends.
Beasle dog sciatic nerve transection lesion model is prepared first：After beasle dog neuromuscular block, by the sciatic nerve of dog
(radius 2.5mm) free cut-out 3.5cm, makes the long range injury of sciatic nerve model of beasle dog.Then pass through suture
Mode carry out CO2 laser weld operation (A in Fig. 1), by LOCC (collagen length of tube be 4cm, neurofilament length be 4cm) bridge joint exist
The proximal end (DNS) and distal end (PNS) of LOCC group beasle dog neurotrosises, make two at beasle dog neurotrosis broken ends cover respectively
LOCs is longer than in the part of collagen tube in LOCC；By NGF-LOCC bridge joints in the proximal end of NGF group beasle dog neurotrosises and far
End；By bFGF-LOCC bridge joint the neurotrosis of bFGF group beasle dogs proximally and distally；Compare lattice for every of autotransplantation group
Dog, the sciatic nerve scaled off is bridged again respectively beasle dog neurotrosis proximally and distally, every beasle dog bridge joint
The sciatic nerve that sciatic nerve scales off in being prepared both from itself sciatic nerve transection lesion model, the bridge joint of sciatic nerve
Direction is consistent with the direction of growth of itself script；Blank control group does not do any CO2 laser weld operation.
2.2 repair collagen material after postoperative nine months through the multinomial detection such as electro physiology, immunohistochemistry, evaluation neurotrosis function
Expect that holder promotes beasle dog injury of sciatic nerve recovery situation
2.2.1. electrophysiologic study：
Compound muscle action potential (compound muscle action were carried out to beasle dog in postoperative nine months
Potential, cMAP) test.Used instrument is the RM6240USB2.0Z type Muscle Electricity Level Inducing Apparatus of Chengdu Instruement Factory
Retouch instrument.Operating method is as follows：Method anesthesia before beasle dog is pressed, cuts skin and exposes sciatic nerve again, use plastics
Gloves pad makes nerve and the tissue of surrounding separate to reduce interference under sciatic nerve.The various parameters of instrument are adjusted, it will be double
Stimulating electrode one end is placed on sciatic nerve, and the other end is placed on gastrocnemius.It is stimulated in sciatic nerve side, records answering on muscle
Close the compound muscle action potential peak value ratio of muscle action potential value and Ipsilateral than strong side.As a result (Fig. 2) is prompted, LOCC
The compound muscle action potential peak value ratio of group, NGF groups and bFGF groups is obviously higher than blank control group, NGF groups and bFGF groups
Middle-end and distal end compound muscle action potential peak value ratio also obviously higher than LOCC groups.
2.2.2 immunohistochemistry detection damage schwann cell Specific marker S100 and neurofilament protein NF expression.
After putting to death beasle dog, nerve is taken out rapidly, 48 hours are fixed in 10% formalin fixer.Paraffin embedding
After be sliced, carry out neurofilament protein NF and S100 albumen immunohistochemical staining.It is processed using poly-D-lysine
Slide bonding die toasts slice 3 hours on roasting piece machine with 56 DEG C.It carries out needing to undergo following steps when immunohistochemical staining
Suddenly：It is followed successively by dimethylbenzene I, 15 minutes；Dimethylbenzene II, 15 minutes；Dimethylbenzene/ethyl alcohol (1:1), 5 minutes；100% ethyl alcohol, 5 points
Clock；95% ethyl alcohol, 5 minutes；80% ethyl alcohol, 5 minutes；70% ethyl alcohol, 5 minutes；50% ethyl alcohol, 5 minutes；Tap water, 5 minutes；
Distilled water, 5 minutes.PBS impregnates 3 times, every time 5 minutes.Antigen retrieval buffers are boiled, tissue is put into the antigen retrieval buffers boiled
In and maintain 10 minutes, allow tissue to be slowly cooled to room temperature.It is washed 3 times, every time 3 minutes with PBS.With immunohistochemistry pen in group
It draws a circle around knitting, is blotted with blotting paper around sample.3%H is added dropwise2O2Deionized water is put into wet box at 37 DEG C and is incubated 10 points
Clock, to block Endogenous peroxidase.PBS is rinsed 3 times, every time 5 minutes.Every tissue is added dropwise Normal Goat Serum and closes fluid-tight
It closes, is put into wet box at 37 DEG C and is incubated 15 minutes.Closing serum is sucked, not washed.Add the primary antibody anti-in mouse source
Neurofilement(1:Or anti-S100 (1 100):100), after 4 DEG C of overnight incubations, PBS is rinsed 3 times, every time 5 minutes.Add
Biotinylation secondary antibody, be put into wet box 37 DEG C be incubated 15 minutes after, PBS is rinsed 3 times, every time 5 minutes.Add three to resist, is put into wet box
After interior 37 DEG C are incubated 15 minutes, each sample adds the DAB solution that proper amount of fresh configures, and reacts at room temperature 10 minutes, discards reaction solution,
Tap water terminates.It is placed 5 minutes in 50% ethyl alcohol；It is placed 5 minutes in 70% ethyl alcohol；It is placed 5 minutes in 80% ethyl alcohol；95% second
It is placed 5 minutes in alcohol；It is placed 5 minutes in 95% ethyl alcohol；It is placed 5 minutes in 100% ethyl alcohol；It is placed 5 minutes in 100% ethyl alcohol；
Ethyl alcohol/dimethylbenzene 1:It is placed 5 minutes in 1；It is placed 10 minutes in dimethylbenzene I；It is placed 5 minutes in dimethylbenzene II；Neutral gum seals
Piece, micro- sem observation, photograph.ImmunohistochemistryResults Results (Fig. 3 and Fig. 4) prompt, LOCC groups, NGF groups and bFGF groups S100 and NF
Expression be all remarkably higher than blank control group, the expression of the S100 and NF of NGF groups and bFGF groups are also all remarkably higher than LOCC groups.
2.2.3 fast blue dyeing and transmission electron microscope observing myelin diameter and myelin wall thickness
After beasle dog is put to death, newborn sciatic nerve is removed for doing histological stain and transmission electron microscope observing.Fast blue
Staining procedure is as follows：Slice is impregnated into 3min in 95% ethanol solution；It is placed in fast blue dye liquor, at a temperature of 57 DEG C, incubates
It educates 4 hours.Slice is taken out, embathes 3min in 95% ethanol solution.Slice is taken out, 3min is embathed in deionized water.Slice
15s is quickly embathed in 0.05% Lithium carbonate solution, is taken out slice immediately, is placed in 70% ethanol solution and breaks up, until grey matter
Taking-up slice can be clearly distinguished with white matter, 3min is rinsed in deionized water, and slice is placed in the solid blue solution of Luxol,
At a temperature of 57 DEG C, it is incubated 10s, slice, which is placed in 95% ethyl alcohol, impregnates 3min, is placed in 100% ethyl alcohol and impregnates 3min, dimethylbenzene
Mounting (Fig. 5) after transparent.Transmission electron microscope detecting step is as follows：(1) nerve is placed in 2.5% glutaraldehyde solution and fixes 2 hours；
(2) rinsing of 0.1M phosphoric acid rinsing liquids is used three times, 15 minutes every time；(3) 2 hours are fixed in 1% osmic acid fixer；(4) it uses
0.1M phosphoric acid rinsing liquids rinse three times, 15 minutes every time；(5) group is woven in 4 DEG C of refrigerators in the alcohol of various concentration and is carried out
Serial dehydration：It places in 50% alcohol 15-20 minutes；It is placed 15-20 minutes in 70% alcohol；15- is placed in 90% alcohol
20 minutes；+ 90% acetone (1 of (6) 90% alcohol：1) it is placed 15-20 minutes in alcohol；15- is placed in (7) 90% acetone alcohol
20 minutes；(8) it is placed altogether three times in 100% acetone in being placed at room temperature for 15-20 minutes；(9) organization embedding：Pure acetone+packet
Bury liquid (2：1) room temperature 4 hours；(10) pure acetone+embedding liquid (1：2) ambient temperature overnight；(11) pure embedding liquid, 37 DEG C 3 hours；(12)
Solidification：In 37 DEG C of baking ovens overnight；(13) 45 DEG C of baking ovens 12 hours, 24 hours in 60 DEG C of baking ovens；(14) with ultramicrotome by group
Knit the slice for being cut into 50-60nm.Use the double dyeing of 3% acetic acid uranium-lead citrate.It completes to use transmission electron microscope after above-mentioned steps
It observes and takes pictures (Fig. 6).
As a result (Fig. 6) is shown, the new myelinogenetic diameter of LOCC groups, NGF groups and bFGF groups is all remarkably higher than blank control
The thickness of the newborn myelin wall of group, NGF groups and bFGF groups is also all remarkably higher than LOCC groups.It is big by comparing the myelin diameter of each group
Therapeutic effect (Fig. 6) discovery of small and myelin wall thickness evaluation each group, the myelin diameter of LOCC groups, NGF groups and bFGF groups
It is all remarkably higher than blank control group, the myelin diameter and myelin wall thickness of NGF groups and bFGF groups with myelin wall thickness
It is all remarkably higher than LOCC groups.
The present invention is successfully prepared the function collagen as tissue engineering scaffold (LOCC) that can be used for repairing of neural injury, by repairing ratio
Lattice dog grows the neurotrosis of cross-section injury of sciatic nerve model, it was demonstrated that the material can be used for repairing nerve damage, utilize life
Reparation beasle dog grows the with obvious effects better than LOCC of cross-section injury of sciatic nerve after long factor NGF and bFGF distinguishes compound LOCC.
Claims (10)
1. growth factor composite collagen timbering material is made of function collagen as tissue engineering scaffold with growth factor；
The function collagen as tissue engineering scaffold is made of neurofilament with the collagen for being coated with the neurofilament.
2. growth factor composite collagen timbering material according to claim 1, it is characterised in that：The growth factor is god
Through growth factor or basic fibroblast growth factor.
3. growth factor composite collagen timbering material according to claim 1 or 2, it is characterised in that：The collagen is glue
Original pipe.
4. growth factor composite collagen timbering material according to claim 3, it is characterised in that：The collagen tube is according to packet
It is prepared by the method for including following steps：The mold of winding collagem membrane is obtained with collagem membrane Wound Dies, in the winding collagem membrane
It is wrapped with the liquid containing collagen on mold, the mold is taken out, the collagen tube is obtained.
5. growth factor composite collagen timbering material according to any one of claims 1-4, it is characterised in that：With growth because
Function collagen as tissue engineering scaffold described in subprocessing obtains the growth factor composite collagen timbering material.
6. growth factor composite collagen timbering material according to claim 5, it is characterised in that：It is described at growth factor
Managing the function collagen as tissue engineering scaffold includes：Liquid containing the growth factor is added into the function collagen as tissue engineering scaffold
On, obtain the growth factor composite collagen timbering material.
7. any function collagen as tissue engineering scaffold in claim 1-3.
8. the substance for repairing animal nerve damage, is made of collagen-based materials and growth factor described in claims 1 or 2.
9. any described in any the growth factor composite collagen timbering material or claim 1-3 in claim 1-6
Function collagen as tissue engineering scaffold or substance according to any one of claims 8 following any applications：
X1, the application in repairing animal nerve damage product is being prepared；
X2, the application in repairing animal nerve damage；
X3, the application in promoting animal nerve reconstituted product is being prepared；
X4, the application in promoting animal nerve regeneration；
X5, the application in promoting animal Remyelination product is being prepared；
X6, the application in promoting animal Remyelination.
10. the application described in substance according to claim 8 or claim 9, it is characterised in that：The animal is lactation
Animal.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710092354.7A CN108452377A (en) | 2017-02-21 | 2017-02-21 | Growth factor composite collagen timbering material and function collagen as tissue engineering scaffold for repairing nerve damage |
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| CN201710092354.7A CN108452377A (en) | 2017-02-21 | 2017-02-21 | Growth factor composite collagen timbering material and function collagen as tissue engineering scaffold for repairing nerve damage |
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Application publication date: 20180828 |