CN106730034A - The artificial nerve graft and preparation method built based on slice type Acellularized valve - Google Patents
The artificial nerve graft and preparation method built based on slice type Acellularized valve Download PDFInfo
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- A—HUMAN NECESSITIES
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- 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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- 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
- A61L27/225—Fibrin; Fibrinogen
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
- A61L27/3675—Nerve tissue, e.g. brain, spinal cord, nerves, dura mater
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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Abstract
Artificial nerve graft and preparation method the present invention relates to be based on slice type Acellularized valve structure, belong to field of tissue engineering technology；The present invention is as support with the section of heterogenous animal spinal cord, skeletal muscle or tendon de-cellular system, with the Geniposide crosslinking cell factor, autologous ecto-mesenchymal stem cell is planted on support, and artificial nerve graft is built as adhesive using fibrin, promote nerve fiber reparation；The present invention has that preparation cost is cheap, cycle is short, preparing raw material wide material sources, it is simple and easy to operate many advantages, such as；Constructed artificial graft's thing has low immunogenicity, containing a large amount of autologous stem cells and cell factor, help to adjust neurotrosis microenvironment, promote nerve regneration, myelin is formed；The present invention can be used to repair peripheral nerve and Spinal Cord Defect, have broad application prospects.
Description
Technical field
The invention belongs to field of tissue engineering technology, and in particular to based on slice type Acellularized valve build artificial nerve graft and
Preparation method.
Background technology
Can cause empty generation after defect, particularly spinal cord injury are often resulted in after neurotrosis, how fill defect, reduce
Cavity formation is the popular problem in repairing of neural injury field.Although nerve autograft can treat neurologic defect, significantly
Side effect and limited source seriously limit its application.It is studying at present to be moved it is important that building nerve using the idea of organizational project
Plant fills up defect, promotes repairing of neural injury.
Support, cell factor and seed cell are three key elements of organizational project.Artificial macromolecular material such as poly- breast
Acid etc., although wide material sources, but exist biocompatibility it is undesirable the shortcomings of.From the group of mammal such as rat and pig
Convenient material drawing is knitted, it is cheap, there is good biocompatibility after Cell extraction.But prepared by current Acellularized valve
Technique is overall materials, and cell is removed after being processed through multiple compounds such as detergents.Technology present in current technology lacks
Falling into has：（1）Global tissue goes cell to exist and goes the too small easy residual of cell intensity, and intensity is excessive, crushes；（2）On global tissue
Plantation seed cell, must rely on seed cell and attack migration to organization internal, and otherwise only rack surface has the load of cytoskeleton
Cell ability is relatively low；（3）Similarly, the global tissue crosslinking cell factor also must dependent factor infiltrate through organization internal, its influence because
Element is more.
Numerous studies show, except the chemical signal of cytokine class has a significant impact to the Growth and Differentiation of cell, machinery
The topological structure of sexual stimulus and material surface is also the key factor for influenceing cell fate, and tissue can be exposed after tissue is sliced
Interior fine structure, contributes to cell attachment, migration, or even control cell growth direction, cellular morphology, or even influence cell point
Change direction.As the stringer fibre bundle in substantia alba medullae spinalis can induce stem cell along its side with the stringer sarcostyle in skeletal muscle
It is raised to extending, to reach the connection upper and lower fiber of fracture location.In addition, fixer there is stronger crosslinking to make protein molecular
With the protein molecular major part in donor tissue is cross-linked into interconnection architecture, can play the effect of blocking antigen, so as to reduce shifting
The immunogenicity of plant.
Cell factor is cross-linked to the crosslinking agent commonly used on support at present includes glutaraldehyde, peroxidase, polyphenol oxidase
And carbodiimide etc.；Wherein there is cytotoxicity in glutaraldehyde, and peroxidase and polyphenol oxidase can significantly affect cell factor
Function, carbodiimide is not only expensive but also can equally influence cytokine activity.And Geniposide is Gardenoside through β-grape
Product after glucosides enzyme hydrolysis, with extensive bioactivity including antitumor, anti-oxidant etc..Numerous studies show Geniposide
Cell factor can be crosslinking in collagen, gelatin and shitosan surface as protein-crosslinking agent.Geniposide crosslinking protein
It is related to there are two aldehyde structures in mechanism to it.
Fibrin is clinical conventional surgical adhesive, also has commercialized product for repairing skin wounds.Fiber
Albumen can be formed by catalyzed by thrombin fibrinogen, be a kind of excellent hydrogel, can bond slices holder, be rebuild
Artificial nerve graft.
The content of the invention
The present invention is intended to provide a kind of tissue engineering nerve graft rebuild based on slice type Acellularized valve and its system
Preparation Method, the method for the present invention has the advantages that low cost, cycle is short, method are easy, easily operated；Prepared nerve-grafting
Thing loads high concentration cell factor and autologous stem cells, and cell arrangement direction disclosure satisfy that connection damages the requirement of the broken ends of fractured bone.
Present invention firstly provides a kind of tissue engineering nerve graft, the nerve graft goes cell branch based on slice type
Frame is built-up；
Further, the nerve graft is loaded from soma by slice type Acellularized valve and the Geniposide crosslinking cell factor
Cell construction is formed；
Further, ecto-mesenchymal stem cell of the autologous stem cells from nose breathing portion mucomembranous cell.
The present invention also provides a kind of preparation method of tissue engineering nerve graft, and methods described is：Fixed with fixer
Tissue, carries out Cell extraction after frozen section, with the Geniposide crosslinking cell factor, prepare on Geniposide crosslinking support；Culture
Autologous patient ecto-mesenchymal stem cell, is planted on the support of Geniposide crosslinking, and cell section branch is carried with fibrin bonding
Framework builds nerve graft.Concrete operation step is as follows：
S1. the preparation of slice type Acellularized valve：
Processed 24 hours with fixer after the spinal cord of mammal, skeletal muscle or tendon are removed into surface connective tissue, by defect
Demand prunes tissue, carries out frozen section, longitudinal section, and slice thickness is 60 microns.With deionization ultra-pure water and phosphoric acid buffer
Liquid embathes standby repeatedly successively；Then with 3%（wt.%）Triton X-100,1M NaOH, 10%（wt.%）Dodecyl sulphur
Sour sodium and 4%（wt.%）NaTDC soaks removal section cellular constituent successively, then with 4%（wt.%）Paraformaldehyde soaked
At night, aseptically cleaned with the phosphate buffer that largely sterilizes and obtain aseptic Acellularized valve, refrigerated standby.
S2. the Geniposide crosslinking cell factor：
Cell factor is cross-linked on Acellularized valve using the protein-crosslinking function of Geniposide, by step（1）Going for obtaining is thin
Born of the same parents' support by volume 1：20 are soaked in DMEM/F-12 nutrient solutions, and EGF is premixed in nutrient solution（EGF, 50-
100ng/mL）, basic fibroblast growth factor（BFGF, 50-100ng/mL）, neurotrophic factor（NGF, 50-100ng/
mL）And Neurotrophin3（NT-3,50-100ng/mL）, it is subsequently added pre-sterilized 1%（w/v）The capital that phosphate buffer is prepared
The flat solution of Buddhist nun（Add about 1mL ethanol dissolutions）, in 37 DEG C of culture 12h in cell culture incubator, there is color reaction, solution is in dark blue
Color, section is changed into blue from white.
S3. factor-containing slices holder loads autologous stem cells：
Take patient's nose breathing portion mucomembranous cell and ecto-mesenchymal stem cell is purified with limited medium culture in vitro, go forward side by side
Row amplification identification, then plants on Geniposide crosslinking support the ecto-mesenchymal stem cell after identification, just can enter within 1-2 days
Row next step is operated.
S4. body ecto-mesenchymal stem cell support is downloaded from fibrin bonding and builds artificial nerve graft, by as follows
Step is carried out：
（1）Simple die is prepared, the similar PVC moulds of synthesis can be also designed, in order to which it is arc to prepare containing cross section
The carrier of groove.
（2）Cell section support will be carried to be layered in semi-circular recesses, or be placed in groove after curling, add 8-10mg/
ML fibrinogens and 50-100 IU/mL fibrin ferments, build artificial nerve graft, and this is final finished.
Compared with prior art, advantage of the invention is that：
（1）Slice type support prepared by the present invention goes the cell more thorough, it is to avoid global tissue goes cell to exist and removes cell intensity
Too small easy residual, intensity is excessive, the shortcoming crushed.
（2）Present invention can assure that the intra-graft rebuild has a large amount of seed cells, the load cell ability of support shows
Write and improve.（3）Present invention can assure that the intra-graft rebuild contains the cell factor of higher concentration.
（4）The fine structure in tissue can be exposed after tissue is sliced, contributes to cell attachment, migration, or even control
Cell
The direction of growth, cellular morphology, or even influence cell differentiation direction.In the stringer fibre bundle and skeletal muscle in substantia alba medullae spinalis
Stringer sarcostyle can induce stem cell to extend projection along its direction, with reach connection the upper and lower fiber of fracture location.
In addition, fixer has stronger crosslinked action to protein molecular, the protein molecular major part in donor tissue is cross-linked into interconnection
Structure, can play the effect of blocking antigen, so as to reduce the immunogenicity of graft.
（5）EGF, basic fibroblast growth factor, neurotrophic factor and nerve are used in the present invention
Nutrient 3, it is induced to Neural Differentiation while stem cells hyperplasia is promoted.Geniposide is Gardenoside through beta-glucosidase water
Product after solution, with extensive bioactivity including antitumor, anti-oxidant etc..Numerous studies show that Geniposide can be as
Protein-crosslinking agent, can be crosslinking in collagen, gelatin and shitosan surface by cell factor.
（6）Fibrin can be formed by catalyzed by thrombin fibrinogen, be a kind of excellent hydrogel, can be glued
Slices holder is closed, artificial nerve graft is rebuild.
Brief description of the drawings
Fig. 1 is slice type Acellularized valve photo prepared by the present invention；Before wherein A is the Geniposide crosslinking cell factor, B
After the Geniposide crosslinking cell factor, C is the cylinder shape groove prepared using Ago-Gel, and D is to be handed over using fibrin
Nerve graft finished product obtained by connection slice type support.
Fig. 2 is the microphoto of slice type support；Wherein A and B is fluorescence photo, and C and D is stereoscan photograph；A shows
Support after Geniposide treatment has a red fluorescence, B display GFP transgenosis ecto-mesenchymal stem cells be grown on slices holder it
On, C is empty support, and D is load ecto-mesenchymal stem cell support.
Specific embodiment
The present invention is further illustrated below in conjunction with specific embodiment：
With chloraldurate deep anaesthesia rat（400-500 g Sprague Dawley rats；From Jiangsu University, experiment is dynamic
Thing center）, after the mL of heart perfusion phosphate buffer about 500, perfusion 4%（wt.%）The mL of paraformaldehyde 500 is fully fixed big
Rat tissue.Rat canalis spinalis is dissected, carefulness separates spinal cord, after the spinal meninges on careful removal surface, after being carried out in 4% paraformaldehyde of input
Fixed about 2h.It is embedded in OCT embedding mediums and rip cutting is carried out at -15 DEG C using coming card freezing microtome, slice thickness elects 60 μ as
m.It is placed in phosphate buffer and tentatively cleans after fishing for section, into removes programmed cell.
With 3%（wt.%）After Triton X-100 immersion section 2-3 h, a large amount of phosphate buffer cleanings；1M hydroxides
Cleaned after sodium immersion 1-2 h； 10% （wt.%）Cleaned after dodecyl sodium sulfate immersion 2-3 h； 4% （wt.%）Deoxidation courage
Sour sodium soaks 1h；With 4% after a large amount of phosphate buffer cleanings（wt.%）Paraformaldehyde immersion 2h sterilizings and blocking antigen material.
All of above immersion is shaken in 37 DEG C of baking ovens and is completed.Now section can be stored in 4% for a long time under the conditions of 4 DEG C（wt.%）
In paraformaldehyde solution.Used time to be needed, fixed section is transferred in a large amount of phosphate buffers that sterilized, by multiple tracks cleaning with
Remove cytotoxic paraformaldehyde.
1% is prepared with phosphate buffer（w/v）Genipin solution（Add about 1mL ethanol dissolutions）, 0.1 μm of filter filtering
It is degerming stand-by.By Acellularized valve by volume 1：20 are soaked in DMEM/F-12 nutrient solutions, be previously added in nutrient solution EGF,
BFGF, NGF and NT-3（Concentration is 50-100ng/mL）, above-mentioned genipin solution is subsequently added, in effect in cell culture incubator
12-24h, whole solution can switch to navy blue from water white transparency, and section switchs to navy blue by milky white.So far, factor-containing is cut
Plate rack is prepared and finished.
Take patient（Spinal cord injury model rat）Nose breathing portion mucous membrane, with uterus tissue pieces after aseptically shredding
Method culture purified ecto-mesenchymal stem cell, carries out streaming to it or exempts from using nestin, HNK-1, vimentin, CD133
Epidemic disease Fluorescence Identification.With pancreatin EDTA solution digestion ecto-mesenchymal stem cells, single cell suspension is formed, cell is counted,
By 1 × 106The ratio of/support is planted in factor-containing slices holder.After 1 day, cell just can securely be attached to section branch
On frame, next step operation just can be carried out.
Simple die is prepared, such as a cylindrical object is bonded in culture dish interior surface, the shaft of cylindrical object is horizontal
Covered in culture dish, it just constitutes a simple die with culture dish body, can also design the similar PVC moulds of synthesis.High temperature is disappeared
After poison 3%（W/V）Agarose phosphate buffered solutions, down to sterile petri dish in from bottleneck about 5mm, cover and be stained with cylinder
The culture dish lid of thing treats that agarose is cooled down, and takes culture dish lid away, then the groove that a section is arc is left on agarose gel.Can
So that by the depth of the amount adjusting grooves for controlling to add agarose, by changing the cylinder thing of different-diameter, control groove is most
Big width.Above-mentioned support containing cell section is layered in groove, or is placed in groove after curling, add 8-10 mg/mL fibers
Proteinogen and 50-100 IU/mL fibrin ferments, are configured to artificial nerve graft, and this is final finished.
Claims (10)
1. a kind of tissue engineering nerve graft, it is characterised in that the nerve graft is based on slice type Acellularized valve structure
Build and form.
2. a kind of tissue engineering nerve graft according to claim 1, it is characterised in that the nerve graft is by cutting
Chip Acellularized valve and the Geniposide crosslinking cell factor, load autologous stem cells are built-up.
3. a kind of tissue engineering nerve graft according to claim 2, it is characterised in that the autologous stem cells source
In the ecto-mesenchymal stem cell of nose breathing portion mucomembranous cell.
4. a kind of preparation method of tissue engineering nerve graft, it is characterised in that carry out in accordance with the following steps：It is solid with fixer
Fixed tissue, carries out Cell extraction after frozen section, with the Geniposide crosslinking cell factor, prepare on Geniposide crosslinking support；Training
Autologous patient ecto-mesenchymal stem cell is supported, is planted on the support of Geniposide crosslinking, with fibrinogen and fibrin ferment
Bonding carries cell section support and builds nerve graft.
5. a kind of preparation method of tissue engineering nerve graft according to claim 4, it is characterised in that the tissue
Originate is the spinal cord of mammal, skeletal muscle or tendon；The fixer is 4% paraformaldehyde；The frozen section requirement is vertical
Tangential section, slice thickness is 600 μm.
6. the preparation method of a kind of tissue engineering nerve graft according to claim 4, it is characterised in that described to go
Cell processes concrete operations：With 3%（wt.%）Triton X-100,1M NaOH, 10%（wt.%）Dodecyl sodium sulfate
And 4%（wt.%）NaTDC soaks removal section cellular constituent successively.
7. a kind of preparation method of tissue engineering nerve graft according to claim 4, it is characterised in that the capital Buddhist nun
Usual friendship connection cell factor concrete operations be：The Acellularized valve obtained after Cell extraction by volume 1：20 are soaked in
In DMEM/F-12 nutrient solutions, in nutrient solution premix EGF, basic fibroblast growth factor, neurotrophy because
Son and Neurotrophin3, are subsequently added the genipin solution that pre-sterilized phosphate buffer is prepared, in 37 DEG C in cell culture incubator
, there is color reaction in culture 12h, and solution is in navy blue, and section is changed into blue from white.
8. a kind of preparation method of tissue engineering nerve graft according to claim 4, it is characterised in that the patient
Autologous ecto-mesenchymal stem cell derives from nose breathing portion mucomembranous cell.
9. a kind of preparation method of tissue engineering nerve graft according to claim 4, it is characterised in that the fibre
Fibrillarin original content 8-10 mg/mL；The concentration of thrombin 50-100 IU/mL.
10. the preparation method of a kind of tissue engineering nerve graft according to claim 4, it is characterised in that with fiber
Proteinogen and fibrin ferment bonding carry cell section support and build the concrete operations of nerve graft and be：It is arc to prepare containing section
The carrier of groove；Cell section support will be carried to be layered in arc groove, or be placed in groove after curling, add fibrinogen
With fibrin ferment, artificial nerve graft is built.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611027732.5A CN106730034B (en) | 2016-11-22 | 2016-11-22 | Artificial nerve graft constructed based on sliced acellular scaffold and preparation method |
Applications Claiming Priority (1)
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| WO2019100454A1 (en) * | 2017-11-27 | 2019-05-31 | 大连理工大学 | Decellularized porous scaffold for three-dimensional tumor model, and construction method therefor and applications thereof |
| CN109847098A (en) * | 2019-01-22 | 2019-06-07 | 江南大学 | A kind of compound bio timbering material for repairing bone defect |
| CN110507857A (en) * | 2019-08-30 | 2019-11-29 | 江南大学 | A kind of engineered nerve graft and preparation method thereof |
| CN111184912A (en) * | 2019-10-15 | 2020-05-22 | 镇江市中西医结合医院（镇江市第二人民医院） | Genipin modified fibrin gel or microsphere and preparation method and application thereof |
| CN111265717A (en) * | 2020-03-04 | 2020-06-12 | 动之医学技术(上海)有限公司 | Medical cannula and preparation method thereof |
| CN111671975A (en) * | 2020-07-01 | 2020-09-18 | 江南大学 | Composite artificial skin material for repairing skin injury |
| WO2021035679A1 (en) * | 2019-08-30 | 2021-03-04 | 江南大学 | Tissue engineered nerve graft and preparation method therefor |
| CN113430164A (en) * | 2021-05-30 | 2021-09-24 | 华中科技大学同济医学院附属协和医院 | Cell culture system, culture method and application |
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| WO2022138992A1 (en) * | 2020-12-21 | 2022-06-30 | 주식회사 엘앤씨바이오 | Acellular nerve graft material and method for producing same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2019100454A1 (en) * | 2017-11-27 | 2019-05-31 | 大连理工大学 | Decellularized porous scaffold for three-dimensional tumor model, and construction method therefor and applications thereof |
| CN109847098A (en) * | 2019-01-22 | 2019-06-07 | 江南大学 | A kind of compound bio timbering material for repairing bone defect |
| CN110507857A (en) * | 2019-08-30 | 2019-11-29 | 江南大学 | A kind of engineered nerve graft and preparation method thereof |
| CN110507857B (en) * | 2019-08-30 | 2021-01-29 | 江南大学 | Tissue engineering nerve graft and preparation method thereof |
| WO2021035679A1 (en) * | 2019-08-30 | 2021-03-04 | 江南大学 | Tissue engineered nerve graft and preparation method therefor |
| CN111184912A (en) * | 2019-10-15 | 2020-05-22 | 镇江市中西医结合医院（镇江市第二人民医院） | Genipin modified fibrin gel or microsphere and preparation method and application thereof |
| CN111184912B (en) * | 2019-10-15 | 2022-06-28 | 镇江市中西医结合医院（镇江市第二人民医院） | Genipin modified fibrin gel or microsphere and preparation method and application thereof |
| CN111265717A (en) * | 2020-03-04 | 2020-06-12 | 动之医学技术(上海)有限公司 | Medical cannula and preparation method thereof |
| CN111671975A (en) * | 2020-07-01 | 2020-09-18 | 江南大学 | Composite artificial skin material for repairing skin injury |
| CN113430164A (en) * | 2021-05-30 | 2021-09-24 | 华中科技大学同济医学院附属协和医院 | Cell culture system, culture method and application |
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