CN107432952A - Three-dimensional grapheme-collagen composite support and its preparation method and application - Google Patents

Three-dimensional grapheme-collagen composite support and its preparation method and application Download PDF

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Publication number
CN107432952A
CN107432952A CN201610353589.2A CN201610353589A CN107432952A CN 107432952 A CN107432952 A CN 107432952A CN 201610353589 A CN201610353589 A CN 201610353589A CN 107432952 A CN107432952 A CN 107432952A
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dimensional grapheme
dimensional
collagen composite
support
composite support
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CN107432952B (en
Inventor
马勤勤
程国胜
杨令延
姜自云
肖淼
张栋
马迅
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Suzhou Institute of Nano Tech and Nano Bionics of CAS
University of Shanghai for Science and Technology
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Suzhou Institute of Nano Tech and Nano Bionics of CAS
University of Shanghai for Science and Technology
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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
    • A61L27/02—Inorganic materials
    • A61L27/08—Carbon ; Graphite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • 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
    • 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
    • A61L27/56—Porous materials, e.g. foams or sponges
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/0007—Electro-spinning
    • D01D5/0061—Electro-spinning characterised by the electro-spinning apparatus
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/0007—Electro-spinning
    • D01D5/0061—Electro-spinning characterised by the electro-spinning apparatus
    • D01D5/0076—Electro-spinning characterised by the electro-spinning apparatus characterised by the collecting device, e.g. drum, wheel, endless belt, plate or grid
    • D01D5/0084—Coating by electro-spinning, i.e. the electro-spun fibres are not removed from the collecting device but remain integral with it, e.g. coating of prostheses
    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
    • D04H1/04—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres from fleeces or layers composed of fibres having existing or potential cohesive properties, e.g. natural fibres, prestretched or fibrillated artificial fibres
    • D04H1/30—Collagen
    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
    • D04H1/70—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres
    • D04H1/72—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged
    • D04H1/728—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged by electro-spinning
    • 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
    • A61L2300/10—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing inorganic materials
    • A61L2300/108—Elemental carbon, e.g. charcoal
    • 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
    • A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
    • A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
    • 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

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  • Health & Medical Sciences (AREA)
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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Animal Behavior & Ethology (AREA)
  • Dermatology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
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  • Textile Engineering (AREA)
  • Mechanical Engineering (AREA)
  • Dispersion Chemistry (AREA)
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  • Inorganic Chemistry (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Materials For Medical Uses (AREA)

Abstract

The invention discloses a kind of three-dimensional grapheme collagen composite support and its preparation method and application.The compound rest includes：Three-dimensional grapheme support, and, at least it is incorporated into the collagenous fibres of the three-dimensional grapheme rack surface；The three-dimensional grapheme collagen composite support has three-dimensional porous structure, and the aperture of its contained hole is to be several to some tens of pm, and porosity is more than 98%.Further, the collagenous fibres are incorporated into three-dimensional grapheme rack surface by physical adsorption way.The manufacture craft of three-dimensional grapheme collagen composite support of the present invention is simple, cost is low, obtained three-dimensional grapheme collagen composite support has ideal support aperture, maintain the porous three dimensional structure of three-dimensional grapheme, have the good characteristic of three-dimensional grapheme and collagen concurrently, when applied to culture cell, there is very high cell loading rate, propagation and the differentiation of NSC, and the formation of neutral net can also be effectively facilitated.

Description

Three-dimensional grapheme-collagen composite support and its preparation method and application
Technical field
Present invention relates particularly to a kind of three-dimensional grapheme-collagen composite support and its preparation method and application, belong to stem cell tissue engineering field.
Background technology
Tissue engineering material is the support and template of regeneration, is that seed cell is seeded in the biomaterial for coming gentrify function of organization in biological support surface.Core of the biologic bracket material as organizational project, the adhesion of cell is not only supported, while propagation to seed cell and differentiation have facilitation.Three-dimensional grapheme has been widely used in the research of stem cell support due to its excellent mechanics, electric property and chemical property.Three-dimensional grapheme has the ability of adsorbed proteins and small-molecule substance, and it can be promoted to have sufficient exchange between the secretion of adjacent cell or cell, and cell is adsorbed onto into the surface of material and influences propagation and the differentiation of cell.Collagen is the skeleton for forming extracellular matrix, is extracellular most important fibrin, and the support of attachment is provided for cell.
NSC is a kind of cell with more differentiation potentials and self-renewal capacity.The regeneration potential of NSC is limited, and in many cases, adult central nervous system can not be regenerated to improve or repair its function after suffering from pathology wound or disease.Because nerve to occur is restricted in vivo, NSC has been demonstrated to separate from Adult Mammals brain, and is used in vitro study.The in vitro culture of NSC functions not only as studying neurogenetic good model, while also can be as the excellent source of human stem cell of potential cell therapy.Many studies have shown that, there are NSC great potentiality to be used to treat many cental system neurogenic diseases as seed cell in recent years.
Although NSC turns into one of most novel and most rich promising cell therapy instrument for treating various pathological tissues and neomorph, there are still many problems to hamper extensive and quick application of the NSC in regenerative medicine field, and the Stem Cell Activity being such as adhered on biomaterial is poor, differentiation efficiency is low and cell function is restricted.
Graphene has form of excellent biocompatibility, various two and three dimensions etc. as cell culturing rack material, it has also become promotes and regulation and control stem cell is divided into specific cells pedigree（Neuron, Gegenbaur's cell, spongiocyte etc.）One of candidate material, then the cell loading rate of existing three-dimensional grapheme material is relatively low, it is difficult to meets the needs of practical application.
The content of the invention
It is a primary object of the present invention to provide a kind of three-dimensional grapheme-collagen composite support and its preparation method and application, to overcome deficiency of the prior art.
In order to reach foregoing invention purpose, the technical solution adopted by the present invention includes：
The embodiments of the invention provide a kind of three-dimensional grapheme-collagen composite support, it includes：Three-dimensional grapheme support, and, at least it is incorporated into the collagenous fibres of the three-dimensional grapheme rack surface；The three-dimensional grapheme-collagen composite support has three-dimensional porous structure, and the aperture of hole contained by the three-dimensional porous structure is more than 1 μm less than 100 μm, and porosity is more than 98%, and the diameter of the collagenous fibres is more than 10nm but less than 10 μm.
The embodiment of the present invention additionally provides a kind of preparation method of three-dimensional grapheme-collagen composite support, and it includes：
Three-dimensional grapheme support is provided, and the three-dimensional grapheme branch is placed in the high-voltage electrostatic field that intensity is 5kV~10kV；
And, collagenous fibres are made at least to be incorporated into three-dimensional grapheme rack surface under electrostatic field, form three-dimensional grapheme-collagen composite support, the three-dimensional grapheme-collagen composite support has three-dimensional porous structure, the aperture of hole contained by the three-dimensional porous structure is more than 1 μm and is less than 100 μm, porosity is more than 98%, and the diameter of the collagenous fibres is more than 10nm but less than 10 μm.
The embodiment of the present invention additionally provides the three-dimensional grapheme-collagen composite support in cultivating the purposes in cell.Wherein, the cell includes supporting any one in NSC, neural progenitor cell, mescenchymal stem cell, fibroblast or two or more combinations.
Compared with prior art, the present invention is by using method of electrostatic spinning by collagenous fibres and three-dimensional grapheme Material cladding, obtain the compound support frame material with good biocompatibility and mechanical performance, its cell loading rate significantly improves than existing three-dimensional grapheme material, propagation and differentiation and the formation of neutral net of NSC can be effectively facilitated, there is wide application prospect.
Brief description of the drawings
In order to illustrate more clearly of architectural feature of the present invention and technical essential, the present invention is described in detail with reference to the accompanying drawings and detailed description.
Fig. 1 a- Fig. 1 b are the scanning electron microscope diagram pieces of three-dimensional grapheme and three-dimensional grapheme-collagen composite support in an exemplary embodiments of the invention；
Fig. 2 is immunostaining fluorescence picture when being broken up 15 days using three-dimensional grapheme and three-dimensional grapheme-collagen composite support culture of neural stem cells neural in the exemplary embodiments of the present invention；
Fig. 3 is that the typical case of the present invention implements to determine the MTT pictures of cell viability when being and using three-dimensional grapheme and three-dimensional grapheme-collagen composite support culture of neural stem cells neural breeding 3 days in an exemplary embodiments of the invention.
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, the embodiment of the present invention is described in detail below.The example of these preferred embodiments is illustrated in the accompanying drawings.What the embodiments of the present invention described shown in accompanying drawing and with reference to the accompanying drawings were merely exemplary, and the present invention is not limited to these embodiments.
Here, it should also be noted that, in order to avoid having obscured the present invention because of unnecessary details, the structure and/or processing step closely related with the solution of the present invention are illustrate only in the accompanying drawings, and eliminate the other details little with relation of the present invention.
In view of the deficiencies in the prior art, inventor is through numerous studies and practice, it is found surprisingly that, when by three-dimensional grapheme material and collagenous fibres, especially through electrostatic spinning technique by collagenous fibres and three-dimensional grapheme Material cladding, the compound rest of formation shows the cell loading rate of far superior to existing three-dimensional grapheme material when applied to cell culture.Based on this discovery, inventor is able to propose technical scheme.
The one side of the embodiment of the present invention provides a kind of three-dimensional grapheme-collagen composite support, and it includes：Three-dimensional grapheme support, and, at least it is incorporated into the collagenous fibres m of the three-dimensional grapheme rack surface；The three-dimensional grapheme-collagen composite support has three-dimensional porous structure, and the aperture of hole contained by the three-dimensional porous structure is more than 1 μm less than 100 μm, and porosity is more than 98%, and the diameter of the collagenous fibres is more than 10nm, but less than 10 μm.
Further, the collagenous fibres pass through non-chemical bonding pattern（Physical adsorption way）It is incorporated into three-dimensional grapheme rack surface.
More preferable, the collagenous fibres are formed and are attached on three-dimensional grapheme support by electrostatic spinning process.
The other side of the embodiment of the present invention additionally provides a kind of preparation method of three-dimensional grapheme-collagen composite support, and it includes：Three-dimensional grapheme support is connected with collagenous fibres, three-dimensional grapheme-collagen composite support is formed after coupling agent treatment.
Further, the preparation method can include：
Three-dimensional grapheme support is provided, and the three-dimensional grapheme branch is placed in high-voltage electrostatic field, its electric-field intensity is about the kV of 5kV~10；
And, collagenous fibres are made at least to be incorporated into three-dimensional grapheme rack surface under electrostatic field, form three-dimensional grapheme-collagen composite support, the three-dimensional grapheme-collagen composite support has three-dimensional porous structure, the aperture of hole contained by the three-dimensional porous structure is more than 1 μm and is less than 100 μm, porosity is more than 98%, and the diameter of the collagenous fibres is more than 10nm, but less than 10 μm.
More preferable, described preparation method can include：
The three-dimensional grapheme branch is placed in high-voltage electrostatic field,
And the collagen solution that concentration is 4wt%~8wt% is spurted into the high-voltage electrostatic field with injection apparatus, and the collagenous fibres to be formed is attached to the three-dimensional grapheme rack surface as reception device.
More preferable, the distance between nozzle and three-dimensional grapheme support of the injection apparatus are 9cm~20cm.
More preferable, the speed of the injection apparatus injection collagen solution is 0.1~1ml/h.
More preferable, the electrostatic spinning time used in the preparation method is is less than or equal to 2 minutes more than 0.
In some more specific embodiment, the preparation method includes：Collagen solution is sprayed by the first-class injection apparatus of syringe needle, nanometer or micron-sized collagenous fibres-electrostatic spinning technique are formed off field in high-pressure electrostatic.
More preferable, described preparation method may also include：Collagenous fibres is attached to three-dimensional grapheme rack surface and after forming three-dimensional grapheme-collagen composite materials, three-dimensional grapheme-the collagen composite materials are placed in coupling agent solution again, after filling crosslinking point reaction, the three-dimensional grapheme-collagen composite support is made.Handled by aforementioned coupling agents, the water absorption rate and expansion rate that can make collagen reduce, and improve the structural stability of collagen, are allowed to be not susceptible to degrade.
More preferable, the coupling agent solution is about 1 comprising volume ratio:1 n-hydroxysuccinimide and 1-（3- dimethylamino-propyls）- 3- ethyl carbodiimides.
More preferable, the coupling agent solution includes the n-hydroxysuccinimide that concentration is 50nM.
More preferable, described preparation method includes：Three-dimensional grapheme-the collagen composite materials are placed in coupling agent solution 0~4 DEG C（It is preferred that 4 DEG C or so）Overnight, the three-dimensional grapheme-collagen composite support is made in reaction, cleans, sterilizes afterwards.
In some more specific embodiment, described preparation method can include：The three-dimensional grapheme-collagen composite support is cleaned more than 2 times using deionized water, then is soaked in 75% ethanol and sterilizes.
The three-dimensional grapheme used in the present invention can be prepared using mode known to industry, such as can be synthesized by chemical vapour deposition technique.
The manufacture craft of three-dimensional grapheme of the present invention-collagen composite support is simple, cost is low, obtained three-dimensional grapheme-collagen composite support has ideal support aperture, higher porosity and high-specific surface area, maintain the porous three dimensional structure of three-dimensional grapheme, have the good characteristic of three-dimensional grapheme and collagen concurrently, such as have good biocompatibility and excellent mechanical property concurrently, being capable of more preferable analog cell epimatrix, good growing environment is provided for cell, therefore when applied to culture cell, with very high cell loading rate, propagation and differentiation and the formation of neutral net of NSC can also be effectively facilitated, it can apply in stem cell body transplant, treat the fields such as the damage of nervous centralis.
Thus, the embodiment of the present invention additionally provides the three-dimensional grapheme-collagen composite support in cultivating the purposes in cell.
Further, the cell includes supporting any one in NSC, neural progenitor cell, mescenchymal stem cell, fibroblast or two or more combinations, and not limited to this.
For example, in some application schemes, Culture of neural stem cells can be inoculated in the compound rest, study the support to nerve stem cell proliferation and the influence of differentiation, the NSC comes from the hippocampus position of mouse in newborn 24 hours.
Below in conjunction with some embodiments and accompanying drawing the technical solution of the present invention is further explained explanation.
Embodiment 1 ：
Three-dimensional grapheme prepared by chemical vapour deposition technique（Pattern refers to Fig. 1 a）It is placed in the high-voltage electrostatic field that intensity is 5kV~10kV, collagen solution of the concentration as 4wt%~8wt% is sprayed to three-dimensional grapheme surface according to 0.1~1ml/h speed using syringe needle, and the distance between syringe needle and three-dimensional grapheme is set to be 9cm~20cm, so as to form collagenous fibres by electrostatic spinning technique and be allowed to be incorporated on three-dimensional grapheme, it is 1 to immerse volume ratio afterwards:1, concentration be 50nM n-hydroxysuccinimide and 1-（3- dimethylamino-propyls）The mixing coupling agent solution of -3- ethyl carbodiimides, three-dimensional grapheme-collagen composite support is formed in 4 DEG C of overnight cross-linking reactions, the compound rest is cleaned 2 times with deionized water afterwards, and absolute ethyl alcohol is replaced once, naturally dry, form three-dimensional grapheme-collagen composite support（Pattern see Fig. 1 b）, the compound rest is placed in culture dish, and fixed using silica gel, after silica dehydrator, soaked overnight in 75% ethanol is placed in, is dried in fume hood.
The compound rest is modified 40 minutes and 6 hours in 37 DEG C of priorities with poly-ornithine and laminin solution, is cleaned 2 times with phosphate buffer afterwards, is inoculated with the NSC of suitable concentration（P2-P6）In the culture dish, using differential medium（DMEM/12 basal mediums, 10% hyclone, 5 nM retinoic acids）It is incubated 15 days, detects neural stem cell differentiating situation.
As shown in Fig. 2 through GFAP antibody mediated immunity fluorescent stainings, it has been observed that compared with three-dimensional grapheme support, three-dimensional grapheme-collagen composite support can effectively facilitate the neural stem cell differentiating formation for astroglia and neutral net.
Embodiment 2 ：
With reference to embodiment 1, three-dimensional grapheme prepared by chemical vapour deposition technique, it is placed in 5kV~10kV high-voltage electrostatic field, collagenous fibres is formed by electrostatic spinning technique and are allowed to be incorporated on three-dimensional grapheme support, it is 1 to immerse volume ratio afterwards:1 concentration is 50nM n-hydroxysuccinimide and 1-（3- dimethylamino-propyls）The coupling agent solution of -3- ethyl carbodiimides mixing, three-dimensional grapheme-collagen composite support is formed in 4 DEG C of overnight cross-linking reactions, thereafter the compound rest is cleaned 2 times with deionized water, absolute ethyl alcohol is replaced once, naturally dry, form three-dimensional grapheme-collagen composite support, the compound rest is placed in culture dish, and fixed using silica gel, after silica dehydrator, soaked overnight in 75% ethanol is placed in, is dried in fume hood.
The compound rest is modified 40 minutes and 6 hours in 37 DEG C of priorities with poly-ornithine and laminin solution, phosphate buffer cleans 2 times, is inoculated with the NSC of suitable concentration（P2-P6）In the culture dish, using proliferated culture medium（DMEM/12 basal mediums, 2%B27,20 ng/ml EGF, 20 ng/ml FGF）After being incubated 3 days, the effect of nerve stem cell proliferation is observed.
As shown in figure 3, MTT experiment result is shown, compared to three-dimensional grapheme group, three-dimensional grapheme-collagen composite support group is obviously promoted the propagation of NSC.
It should be understood that; the technical concept and architectural feature of above-mentioned embodiment only to illustrate the invention; purpose is the stakeholder for being familiar with technique according to this can implement; but above said content is not intended to limit protection scope of the present invention; any equivalent change or modification that every Spirit Essence according to the present invention is made, all should fall under the scope of the present invention.

Claims (11)

  1. A kind of 1. three-dimensional grapheme-collagen composite support, it is characterised in that including：Three-dimensional grapheme support, and, at least it is incorporated into the collagenous fibres of the three-dimensional grapheme rack surface；The three-dimensional grapheme-collagen composite support has three-dimensional porous structure, and the aperture of hole contained by the three-dimensional porous structure is more than 1 μm less than 100 μm, and porosity is more than 98%, and the diameter of the collagenous fibres is more than 10nm but less than 10 μm.
  2. 2. three-dimensional grapheme according to claim 1-collagen composite support, it is characterised in that：The collagenous fibres are incorporated into three-dimensional grapheme rack surface by physical adsorption way.
  3. 3. three-dimensional grapheme according to claim 1 or 2-collagen composite support, it is characterised in that：The collagenous fibres are to form and be attached to three-dimensional grapheme support by electrostatic spinning process.
  4. A kind of 4. preparation method of three-dimensional grapheme-collagen composite support, it is characterised in that including：
    Three-dimensional grapheme support is provided, and the three-dimensional grapheme branch is placed in high-voltage electrostatic field of the intensity for the kV of 5kV~10；
    And, collagenous fibres are made at least to be incorporated into three-dimensional grapheme rack surface under electrostatic field, form three-dimensional grapheme-collagen composite support, the three-dimensional grapheme-collagen composite support has three-dimensional porous structure, the aperture of hole contained by the three-dimensional porous structure is more than 1 μm and is less than 100 μm, porosity is more than 98%, and the diameter of the collagenous fibres is more than 10nm but less than 10 μm.
  5. 5. preparation method according to claim 4, it is characterised in that including：
    The three-dimensional grapheme branch is placed in high-voltage electrostatic field,
    And the collagen solution that concentration is 4~8wt% is spurted into the high-voltage electrostatic field with injection apparatus, and the collagenous fibres to be formed is attached to the three-dimensional grapheme rack surface as reception device.
  6. 6. preparation method according to claim 5, it is characterised in that：The distance between nozzle and three-dimensional grapheme support of the injection apparatus are 9cm~20cm；And/or the speed of the injection apparatus injection collagen solution is 0.1-1ml/h；And/or the electrostatic spinning time used is is less than or equal to 2 minutes more than 0.
  7. 7. preparation method according to claim 4, it is characterised in that also include：Collagenous fibres is attached to three-dimensional grapheme rack surface and after forming three-dimensional grapheme-collagen composite materials, three-dimensional grapheme-the collagen composite materials are placed in coupling agent solution again, after full cross-linked reaction, the three-dimensional grapheme-collagen composite support is made.
  8. 8. preparation method according to claim 7, it is characterised in that：It is 1 that the coupling agent solution, which includes volume ratio,:1 n-hydroxysuccinimide and 1-（3- dimethylamino-propyls）- 3- ethyl carbodiimides；And/or the coupling agent solution includes the n-hydroxysuccinimide that concentration is 50nM.
  9. 9. preparation method according to claim 7, it is characterised in that including：Three-dimensional grapheme-the collagen composite materials are placed in 0~4 DEG C of reaction in coupling agent solution and overnight, the three-dimensional grapheme-collagen composite support are made, cleans, sterilize afterwards.
  10. 10. three-dimensional grapheme any one of claim 1-3-collagen composite support is in cultivating the purposes in cell.
  11. 11. purposes according to claim 10, it is characterised in that：The cell includes supporting any one in NSC, neural progenitor cell, mescenchymal stem cell, fibroblast or two or more combinations.
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CN108619580A (en) * 2018-06-19 2018-10-09 佛山皖阳生物科技有限公司 A kind of preparation method of hydrophilic coating coronary artery bracket material
CN109847105A (en) * 2019-01-10 2019-06-07 东华大学 A kind of new type nerve catheter holder and its preparation method and application
CN110452397A (en) * 2019-08-19 2019-11-15 中国科学院苏州纳米技术与纳米仿生研究所 Three-dimensional graphene foam/natural polysaccharide based aquagel compound rest and its preparation method
CN110452397B (en) * 2019-08-19 2022-07-29 中国科学院苏州纳米技术与纳米仿生研究所 Three-dimensional graphene foam/natural polysaccharide-based hydrogel composite scaffold and preparation method thereof
CN111028983A (en) * 2019-12-16 2020-04-17 天新福（北京）医疗器材股份有限公司 Conductive composite material and preparation method and application thereof
CN111028983B (en) * 2019-12-16 2021-07-30 天新福（北京）医疗器材股份有限公司 Conductive composite material and preparation method and application thereof
CN111944750A (en) * 2020-08-20 2020-11-17 中国科学院苏州纳米技术与纳米仿生研究所 Three-dimensional annular cell scaffold responding to wireless electrical stimulation and preparation method and application thereof
CN111944750B (en) * 2020-08-20 2024-01-12 中国科学院苏州纳米技术与纳米仿生研究所 Three-dimensional annular cell scaffold with radio stimulation response and preparation method and application thereof
CN114904047A (en) * 2021-02-07 2022-08-16 上海大学 Three-dimensional graphene/extracellular matrix composite scaffold and preparation method and application thereof

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