CN105664260A - Method for preparing bone tissue engineering three-dimensional porous support based on graphene/silk fibroin - Google Patents

Method for preparing bone tissue engineering three-dimensional porous support based on graphene/silk fibroin Download PDF

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CN105664260A
CN105664260A CN201610097176.2A CN201610097176A CN105664260A CN 105664260 A CN105664260 A CN 105664260A CN 201610097176 A CN201610097176 A CN 201610097176A CN 105664260 A CN105664260 A CN 105664260A
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silk fibroin
graphene
fibroin
solution
prepared
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李明
莫茂松
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Institute of Electrical Engineering of CAS
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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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A61L27/443—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with carbon fillers
    • 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
    • A61L27/36—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
    • 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
    • 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
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Abstract

The invention discloses a method for preparing a bone tissue engineering three-dimensional porous support based on graphene/silk fibroin. According to the preparing method, graphene is introduced to serve as a mechanical reinforcing material, a graphene oxide GO water solution is added into a silk fibroin water solution according to the mass ratio of 0.1-3wt% of GO to silk fibroin, and the three-dimensional porous support is prepared through a salting out method.

Description

Bone tissue engineer method for preparing 3 D porous bracket based on Graphene/fibroin albumen
Technical field
The present invention relates to a kind of bone tissue restoration and the bone tissue engineer method for preparing 3 D porous bracket rebuild, a kind of method being specifically related to bone tissue engineering scaffold utilizing salting out method to prepare Graphene/fibroin albumen three-dimensional porous rack.
Background technology
Along with increasing of social population's aging and athletic injury, various orthopaedic diseases bring puzzlement greatly with painful to patient. Wherein owing to congenital diseases or the such as factor such as wound, infection and tumor resection easily form bigger gap-Cranial defect in bone. Bone defect healing is always up the focus of international research, and the technology such as bone collection (autologous bone transplanting and allogenic bone transplantation), tissue engineering technique, film inductive bone regeneration, gene therapy, growth factor-induced can be adopted to treat Cranial defect.
Tissue engineering technique is a cross discipline comprising seed cell, timbering material, the big key element of somatomedin three. Timbering material is that Growth of Cells provides three dimensional support structure, desirably three-dimensional bone tissue engineering scaffold needs and is beneficial to seed cell in its surface adhesion, propagation and migration, and timbering material need to have biocompatibility, bone conductibility, osteoinductive and certain mechanical property simultaneously.
Research both at home and abroad at present, the natural biologic material for organizational project developed mainly have collagen, chitosan, gelatin, hyaluronic acid, fibroin albumen etc. Fibroin albumen is a kind of natural copolymer extracted from silkworm, is mainly formed (accounting for more than the 95% of total amount) by glycine, alanine, serine and tyrosine. Generally wrap up one layer of sericin (SilkSericin) outside fibroin albumen, there are some researches show that silkworm silk easily causes immunoreation, therefore in use need silkworm silk is carried out degumming process, remove outer layer sericin. As a kind of natural activity albumen, fibroin albumen has the character such as relatively low immunogenicity, good biocompatibility, biodegradability, good mechanical property, cell adhesion and induction bone cell growth can be promoted so that it is the applied research in bio-medical material with field of tissue engineering technology receives more and more attention.
Fibroin albumen is mainly made up of crystalline state and non-crystalline two parts. After the aminoacid being formed as in fibroin albumen of crystal region forms peptide molecule, relatively simple, the little amino acid residue of side chain produces by certain sequential structure formation rule segment, its degree of crystallinity, about 50%～60%, is made up of propylhomoserin, alanine and serine (mol ratio 3:2:1). It is primarily present in amorphous regions with the phenylalanine of larger side base, tyrosine, tryptophan etc. Fibroin albumen has two kinds of conformation SilkI and SilkII.SilkI conformation is mainly random coil structure and class alpha-helix (α-helix-like) conformation produced by intrachain hydrogen bond effect, is a kind of intermediary between alpha-helix and beta sheet; SilkII conformation is then anti-parallel ss-sheet (β-sheet) conformation. SilkI conformational structure is unstable, is very easily changed into SilkII conformation, peptide chain marshalling in this beta sheet structure, has stronger hydrogen bond action and intermolecular force, thus giving the consistency and elasticity that fibroin albumen is higher between chain is disconnected. But, in being applied to the bone tissue restoration process with regeneration, the mechanical property of fibroin albumen three-dimensional porous rack still needs to be improved further, it is therefore desirable to adds good mechanical properties in fibroin albumen, have certain bioactive mechanics reinforcing material.
Graphene is by sp2The two-dimensional layer nano material with cellular crystal lattice structure of hydbridized carbon atoms composition, as a kind of novel nano-material, Graphene has the mechanical property of excellence and good biocompatibility, can be used as the mechanics of Biocomposite material and strengthen phase, to improving the mechanical property of biomaterial. There is scholar to prepare graphite alkene at present and derivant strengthens three-dimensional complex stephanoporate bracket for bone tissue restoration and regeneration, such as porous supports such as Graphene/bioceramic, Graphene/chitosan, GO/ polyvinyl alcohol, Graphene/polycaprolactone, GO/PLGA, GO/ polylactic acid/polyurethane and GO/PLGA/ collagens. Its advantage mainly has: (1) Graphene has bigger specific surface area, it is possible to be fully contacted with matrix material (such as fibroin albumen), it is easy to forms effective netted interconnection architecture in matrix, is conducive to interfacial stress to conduct; (2), when few additive, it is remarkably improved the mechanical properties such as matrix Young's modulus, fracture toughness, yield strength; (3) there is certain biocompatibility, it is possible to produce synergism with fibroin albumen, improve composite biological activity; (4) Graphene can be chemically modified, introduce other active group or material; (5) Graphene has the ability promoting stem cell Osteoblast Differentiation to have research to point out; (6) Graphene has certain antibiotic property, advantageously reduces implant infection rate after abortion operation.
Therefore in fibroin albumen, Graphene is added as mechanics reinforcing material, to improve the mechanical property of support, electrical conductivity and osteogenic activity. Study also in the exploratory stage about the bio-medical of fibroin albumen/Graphene and derivant composite materials thereof at present, correlational study is concentrated mainly on fibroin albumen/Graphene two dimension composite coating/field of film preparation, and domestic and international mistake but without document and patent report utilizes salting out method to prepare Graphene/fibroin albumen three-dimensional porous bone tissue engineering support.
Summary of the invention
It is an object of the invention to the shortcoming overcoming prior art gained silk fibroin bracket poor mechanical property, osteogenic activity is low, it is proposed to the preparation method of a kind of new bone tissue engineer three-dimensional porous rack. The present invention, by introducing Graphene as mechanics reinforcing material, improves the mechanical property for bone tissue restoration and reconstruction silk fibroin bracket and osteogenic activity.
The purpose of the present invention is achieved through the following technical solutions.
1) being shredded by mulberry cocoon (Bombyxmoricocoon), weigh 5g mulberry cocoon, being then placed on 2L concentration is 0.02mol/LNa2CO3Solution boils 30min, removes silkworm silk surface sericin. Use deionized water cleaning and degumming fibroin albumen for several times after boiling end, and naturally dry;
2) by step 1) fibroin albumen prepared is dissolved in 9MLiBr at 60 DEG C and is about 4h, after fibroin albumen is completely dissolved, dialyse 72h with dialyzer, obtain the silk fibroin water solution that mass body fraction is 6-7w/v%, described silk fibroin water solution is left in 4 DEG C of refrigerators stand-by;
3) by step 2) gained 6-7w/v% silk fibroin protein solution is placed in 50ml centrifuge tube, is added in silk fibroin water solution according to GO/ fibroin albumen 0.1～3wt% mass ratio by graphene oxide GO aqueous solution, it is gently mixed uniformly.
4) three-dimensional porous rack is prepared by salting out method, namely using particle diameter for 400～600 μm of sodium chloride as perforating agent, step 3 is added according to 2:1w/v%) the GO/ silk fibroin protein solution prepared, mixture is placed in Ф 65mm culture dish, left at room temperature 48h.
5) until step 4) after the solution prepared becomes gel, it is placed in deionized water and dissolves, remove NaCl perforating agent. After NaCl all dissolves, clean sample 3 times with deionized water, obtain GO/ silk fibroin porous material.
6) step 5 is taken out) the GO/ silk fibroin porous material prepared, utilize the cylindrical three-dimensional porous compound support frame of card punch.
7) by step 6) three-dimensional stephanoporate compound stent prepared is placed in 41mmol/L ascorbic acid solution, and under 95 DEG C of conditions, heat 10min, obtain Graphene/silk fibroin bracket. Then Graphene/silk fibroin bracket is cleaned 3 times with deionized water, 70% alcoholic solution respectively, by described Graphene/silk fibroin bracket submergence in the medium, to treat inoculating cell in Graphene/silk fibroin bracket.
So far, prepared by three-dimensional porous rack of the present invention.
The equipment of Graphene of the present invention/fibroin albumen method for preparing 3 D porous bracket is simple, and easy to operate, technological parameter is easily controllable, and cost is low. Compared with pure silk fibroin bracket, after the addition of Graphene, improve mechanical property and the osteogenic activity of silk fibroin bracket, promoted Graphene in the application in bone tissue engineer field, also promoted the fibroin albumen application prospect in bone defect healing field.
Accompanying drawing explanation
Fig. 1 a is that hMSC cell is inoculated in fibroin albumen and fibroin albumen/rGO compound rest, Osteoblast Differentiation culture medium co-cultures 24h, 672h and 1344h after-poppet macroscopic view optical photograph, Fig. 1 b is that hMSC cell is inoculated in fibroin albumen and fibroin albumen/rGO compound rest, co-cultures the thickness of 24h, 672h and 1344h after-poppet, diameter and volume in Osteoblast Differentiation culture medium;
Fig. 2 hMSC cell is inoculated in fibroin albumen and fibroin albumen/rGO compound rest, co-cultures 24h, 672h and 1344h support inner cell DNA content in Osteoblast Differentiation culture medium;
Fibroin albumen/Graphene the compound rest of Fig. 3 hMSC cell and variable concentrations Graphene balance Young's modulus in a liquid after co-culturing 24h, 672h and 1344h in Osteoblast Differentiation culture medium;
Fibroin albumen/Graphene the compound rest of Fig. 4 hMSC cell and variable concentrations Graphene in Osteoblast Differentiation culture medium, co-culture 672h and 1344h after Ca assay result in cell;
Fibroin albumen/Graphene the compound rest of Fig. 5 ahMSC cell and variable concentrations Graphene in Osteoblast Differentiation culture medium, co-culture 672h and 1344h after BSP expression conditions in cell, the fibroin albumen/Graphene compound rest of Fig. 5 bhMSC cell and variable concentrations Graphene in Osteoblast Differentiation culture medium, co-culture 672h and 1344h after RUNX2 expression conditions in cell.
Detailed description of the invention
The present invention is further illustrated below in conjunction with the drawings and specific embodiments.
Embodiment 1
1) being shredded by mulberry cocoon (Bombyxmoricocoon), weigh 5g mulberry cocoon, being then placed on 2L concentration is 0.02mol/LNa2CO3Solution boils 30min, removes silkworm silk surface sericin.Use deionized water cleaning and degumming fibroin albumen for several times after boiling end, and naturally dry;
2) by step 1) fibroin albumen prepared is dissolved in 9MLiBr at 60 DEG C and is about 4h, after fibroin albumen is completely dissolved, dialyse 72h with dialyzer, obtain the silk fibroin water solution that mass body fraction is 6-7w/v%, described silk fibroin water solution is left in 4 DEG C of refrigerators stand-by;
3) by step 2) gained 6-7w/v% silk fibroin protein solution is placed in 50ml centrifuge tube, is added in silk fibroin water solution according to GO/ fibroin albumen 0.1wt% mass ratio by graphene oxide GO aqueous solution, it is gently mixed uniformly.
4) three-dimensional porous rack is prepared by salting out method, namely using particle diameter for 400～600 μm of sodium chloride as perforating agent, step 3 is added according to 2:1w/v%) the GO/ silk fibroin protein solution prepared, mixture is placed in Ф 65mm culture dish, left at room temperature 48h.
5) until step 4) after the solution prepared becomes gel, it is placed in deionized water and dissolves, remove NaCl perforating agent. After NaCl all dissolves, clean sample 3 times with deionized water, obtain GO/ silk fibroin porous material.
6) step 5 is taken out) the GO/ silk fibroin porous material prepared, utilize diameter to be cut to the cylindrical three-dimensional porous compound support frame of diameter 4mm, thickness 2mm for 4mm card punch.
7) by step 6) three-dimensional stephanoporate compound stent prepared is placed in 41mmol/L ascorbic acid solution, and under 95 DEG C of conditions, heat 10min, obtain Graphene/silk fibroin bracket. Then Graphene/silk fibroin bracket is cleaned 3 times with deionized water, 70% alcoholic solution respectively, by described support submergence in the medium.
8) step 7 is taken out) submergence Graphene/silk fibroin bracket in the medium, in described support, inoculate stem cell, stem cell used is the human mesenchyme's bone marrow stem cell being cultured to forth generation in proliferated culture medium. Proliferated culture medium be containing 10% hyclone, 1% penicillin/streptomycin, 0.1ng/mLbFGF glucose content DMEM culture medium. Take 15 μ L4 × 107Cells/mL cell suspending liquid, drop in the embodiment 3 gained support of Aspirate medium in advance, 20min is stood at 37 DEG C of temperature, afterwards turn over 180 ° of support, drip 8 μ L cell-free medium, at 37 DEG C of temperature, stand 20min, then second time turnover bracket 180 °, drip 8 μ L, repeat this operation about 5～6 times, duration 2h. Then being transferred in 24 orifice plates by the support of inoculated cell, every hole adds 2mL osteogenic, at 37 DEG C/5%CO2Cultivate 4～8 weeks, within every 2～3 days, change a subculture. Osteoblast Differentiation culture medium used is the LG content DMEM culture medium of the L-AA-2-sodium phosphate containing 10% hyclone, 1% Pen .-Strep, 100nM dexamethasone, 10mM sodium β-glycerophosphate and 50 μ g/mL.
The micro structure of Graphene/silk fibroin bracket, DNA content, mechanical property, calcium content and the expression conditions obtained is tested with following instrument or test kit respectively: slide gauge, Picogreen test kit (MolecularProbes, OR), micromechanics tester, Calcium (CPC)Kit test kit (StanbioLaboratory, USA) and RT-PCR analyser.
Graphene/silk fibroin bracket after co-culturing with human mesenchyme's bone marrow stem cell change in size as shown in Figure 1a, after cultivating 672h (week4), support volume shrinks;When in compound rest, rGO content is more than 1%, there is not obvious contraction in support. By measurement bracket thickness and diameter, result as shown in Figure 1 b, rGO content be 1% and 3% compound rest size there is not obvious change, maintain its three-dimensional porous structure preferably.
Human mesenchyme's bone marrow stem cell DNA content in Graphene/silk fibroin bracket as in figure 2 it is shown, with incubation time extend, 1% and 3%rGO silk fibroin bracket inner cell DNA content be gradually increased. After cultivation terminates, 0.5%rGO silk fibroin bracket inner cell DNA content is the highest.
Graphene/silk fibroin bracket is co-culturing after-poppet mechanical property as shown in Figure 3 with human mesenchyme's bone marrow stem cell, when there is no inoculating cell (withoutcells), when rGO content increases to 3%, support Young's modulus significantly improves. After inoculating cell training, sample Young's modulus significantly improves, and along with the increase of rGO content, Young's modulus is gradually increased.
In Graphene/silk fibroin bracket, human mesenchyme's bone marrow stem cell calcium content is as shown in Figure 4, compared with pure silk fibroin bracket, can promote stem cell Osteoblast Differentiation in support after adding rGO, and calcium content dramatically increases.
In Graphene/silk fibroin bracket, in human mesenchyme's bone marrow stem cell, osteogenesis gene expression is as it is shown in figure 5, Fig. 5 a show BSP gene, and Fig. 5 b show RUNX2 gene. After test result indicate that addition Graphene, it is possible to promote stem cell Osteoblast Differentiation in support.
Embodiment 2
1) being shredded by mulberry cocoon (Bombyxmoricocoon), weigh 5g mulberry cocoon, being then placed on 2L concentration is 0.02mol/LNa2CO3Solution boils 30min, removes silkworm silk surface sericin. Use deionized water cleaning and degumming fibroin albumen for several times after boiling end, and naturally dry;
2) by step 1) fibroin albumen prepared is dissolved in 9MLiBr at 60 DEG C and is about 4h, after fibroin albumen is completely dissolved, dialyse 72h with dialyzer, obtain the silk fibroin water solution that mass body fraction is 6-7w/v%, described silk fibroin water solution is left in 4 DEG C of refrigerators stand-by;
3) by step 2) gained 6-7w/v% silk fibroin protein solution is placed in 50ml centrifuge tube, is added in silk fibroin water solution according to GO/ fibroin albumen 0.5wt% mass ratio by graphene oxide GO aqueous solution, it is gently mixed uniformly.
4) three-dimensional porous rack is prepared by salting out method, namely using particle diameter for 400～600 μm of sodium chloride as perforating agent, step 3 is added according to 2:1w/v%) the GO/ silk fibroin protein solution prepared, mixture is placed in Ф 65mm culture dish, left at room temperature 48h.
5) until step 4) after the solution prepared becomes gel, it is placed in deionized water and dissolves, remove NaCl perforating agent. After NaCl all dissolves, clean sample 3 times with deionized water, obtain GO/ silk fibroin porous material.
6) step 5 is taken out) the GO/ silk fibroin porous material prepared, utilize diameter to be cut to the cylindrical three-dimensional porous compound support frame of diameter 4mm, thickness 2mm for 4mm card punch.
7) by step 6) three-dimensional stephanoporate compound stent prepared is placed in 41mmol/L ascorbic acid solution, and under 95 DEG C of conditions, heat 10min, obtain Graphene/silk fibroin bracket. Then described support is cleaned 3 times with deionized water, 70% alcoholic solution respectively, by described Graphene/silk fibroin bracket submergence in the medium, to treat inoculating cell in described support.
Embodiment 3
1) being shredded by mulberry cocoon (Bombyxmoricocoon), weigh 5g mulberry cocoon, being then placed on 2L concentration is 0.02mol/LNa2CO3Solution boils 30min, removes silkworm silk surface sericin.Use deionized water cleaning and degumming fibroin albumen for several times after boiling end, and naturally dry;
2) by step 1) fibroin albumen prepared is dissolved in 9MLiBr at 60 DEG C and is about 4h, after fibroin albumen is completely dissolved, dialyse 72h with dialyzer, obtain the silk fibroin water solution that mass body fraction is 6-7w/v%, described silk fibroin water solution is left in 4 DEG C of refrigerators stand-by;
3) by step 2) gained 6-7w/v% silk fibroin protein solution is placed in 50ml centrifuge tube, is added in silk fibroin water solution according to GO/ fibroin albumen 1wt% mass ratio by graphene oxide GO aqueous solution, it is gently mixed uniformly.
4) three-dimensional porous rack is prepared by salting out method, namely using particle diameter for 400～600 μm of sodium chloride as perforating agent, step 3 is added according to 2:1w/v%) the GO/ silk fibroin protein solution prepared, mixture is placed in Ф 65mm culture dish, left at room temperature 48h.
5) until step 4) after the solution prepared becomes gel, it is placed in deionized water and dissolves, remove NaCl perforating agent. After NaCl all dissolves, clean sample 3 times with deionized water, obtain GO/ silk fibroin porous material.
6) step 5 is taken out) the GO/ silk fibroin porous material prepared, utilize diameter to be cut to the cylindrical three-dimensional porous compound support frame of diameter 4mm, thickness 2mm for 4mm card punch.
7) by step 6) three-dimensional stephanoporate compound stent prepared is placed in 41mmol/L ascorbic acid solution, and under 95 DEG C of conditions, heat 10min, obtain Graphene/silk fibroin bracket. Then described support is cleaned 3 times with deionized water, 70% alcoholic solution respectively, by Graphene/silk fibroin bracket submergence in the medium, to treat inoculating cell in described support.
Embodiment 4
1) being shredded by mulberry cocoon (Bombyxmoricocoon), weigh 5g mulberry cocoon, being then placed on 2L concentration is 0.02mol/LNa2CO3Solution boils 30min, removes silkworm silk surface sericin. Use deionized water cleaning and degumming fibroin albumen for several times after boiling end, and naturally dry;
2) by step 1) fibroin albumen prepared is dissolved in 9MLiBr at 60 DEG C and is about 4h, after fibroin albumen is completely dissolved, dialyse 72h with dialyzer, obtain the silk fibroin water solution that mass body fraction is 6-7w/v%, described silk fibroin water solution is left in 4 DEG C of refrigerators stand-by;
3) by step 2) gained 6-7w/v% silk fibroin protein solution is placed in 50ml centrifuge tube, is added in silk fibroin water solution according to GO/ fibroin albumen 3wt% mass ratio by graphene oxide GO aqueous solution, it is gently mixed uniformly.
4) three-dimensional porous rack is prepared by salting out method, namely using particle diameter for 400～600 μm of sodium chloride as perforating agent, step 3 is added according to 2:1w/v%) the GO/ silk fibroin protein solution prepared, mixture is placed in Ф 65mm culture dish, left at room temperature 48h.
5) until step 4) after the solution prepared becomes gel, it is placed in deionized water and dissolves, remove NaCl perforating agent. After NaCl all dissolves, clean sample 3 times with deionized water, obtain GO/ silk fibroin porous material.
6) step 5 is taken out) the GO/ silk fibroin porous material prepared, utilize diameter to be cut to the cylindrical three-dimensional porous compound support frame of diameter 4mm, thickness 2mm for 4mm card punch.
7) by step 6) three-dimensional stephanoporate compound stent prepared is placed in 41mmol/L ascorbic acid solution, and under 95 DEG C of conditions, heat 10min, obtain Graphene/silk fibroin bracket.Then described support is cleaned 3 times with deionized water, 70% alcoholic solution respectively, by Graphene/silk fibroin bracket submergence in the medium, to treat inoculating cell in described support.

Claims (1)

1. the bone tissue engineer method for preparing 3 D porous bracket based on Graphene/fibroin albumen, it is characterised in that: described preparation method introduces Graphene as mechanics reinforcing material, and step is as follows:
1) being shredded by mulberry cocoon (Bombyxmoricocoon), weigh 5g mulberry cocoon, being placed on 2L concentration is 0.02mol/LNa2CO3Solution boils 30min, removes silkworm silk surface sericin; Use deionized water cleaning and degumming fibroin albumen for several times after boiling end, and naturally dry;
2) by step 1) fibroin albumen prepared is dissolved in 9MLiBr 4h at 60 DEG C, after fibroin albumen dissolves, dialyse 72h with dialyzer, obtain the silk fibroin water solution that mass body fraction is 6-7w/v%, described silk fibroin water solution is left in 4 DEG C of refrigerators stand-by;
3) by step 2) gained 6-7w/v% silk fibroin protein solution is placed in 50ml centrifuge tube, and graphene oxide GO aqueous solution is added in silk fibroin water solution according to GO/ fibroin albumen 0.1～3wt% mass ratio, stirs;
4) three-dimensional porous rack is prepared by salting out method, namely using particle diameter for 400～600 μm of sodium chloride as perforating agent, step 3 is added according to 2:1w/v%) the GO/ silk fibroin protein solution prepared, mixture is placed in Ф 65mm culture dish, left at room temperature 48h;
5) until step 4) after the solution prepared becomes gel, it is placed in deionized water and dissolves, remove NaCl perforating agent; After NaCl all dissolves, clean sample 3 times with deionized water, obtain GO/ silk fibroin porous material;
6) step 5 is taken out) the GO/ silk fibroin porous material prepared, utilize card punch to be cut to cylindrical three-dimensional porous compound support frame;
7) by step 6) three-dimensional stephanoporate compound stent prepared is placed in 41mmol/L ascorbic acid solution, and under 95 DEG C of conditions, heat 10min, obtain Graphene/silk fibroin bracket; Then Graphene/silk fibroin bracket is cleaned 3 times with deionized water, 70% alcoholic solution respectively, by described Graphene/silk fibroin bracket submergence in the medium, to treat inoculating cell in Graphene/silk fibroin bracket.
CN201610097176.2A 2016-02-23 2016-02-23 Method for preparing bone tissue engineering three-dimensional porous support based on graphene/silk fibroin Pending CN105664260A (en)

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CN106400311A (en) * 2016-09-07 2017-02-15 东华大学 Method for preparing composite nanofiber tissue engineering scaffold based on graphene oxide
CN108324998A (en) * 2018-04-19 2018-07-27 四川之江高新材料股份有限公司 The preparation method of polyurethane multichannel endovascular stent and coating liquid used
CN109054047A (en) * 2018-05-24 2018-12-21 华中科技大学同济医学院附属协和医院 A kind of silk gum/graphene oxide composite hydrogel and its preparation method and application
CN111939318A (en) * 2020-08-23 2020-11-17 苏州宣医智慧医疗科技有限公司 Preparation method of reduced graphene oxide silk fibroin composite membrane
CN113087948A (en) * 2021-04-21 2021-07-09 哈尔滨工业大学（深圳） Film and preparation method thereof
CN113499481A (en) * 2021-06-18 2021-10-15 广东省科学院健康医学研究所 Porous scaffold and preparation method and application thereof

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Cited By (8)

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Publication number Priority date Publication date Assignee Title
CN106400312A (en) * 2016-09-07 2017-02-15 东华大学 Method for preparing conductive composite nanofiber nervous tissue engineering scaffold based on graphene
CN106400311A (en) * 2016-09-07 2017-02-15 东华大学 Method for preparing composite nanofiber tissue engineering scaffold based on graphene oxide
CN108324998A (en) * 2018-04-19 2018-07-27 四川之江高新材料股份有限公司 The preparation method of polyurethane multichannel endovascular stent and coating liquid used
CN109054047A (en) * 2018-05-24 2018-12-21 华中科技大学同济医学院附属协和医院 A kind of silk gum/graphene oxide composite hydrogel and its preparation method and application
CN111939318A (en) * 2020-08-23 2020-11-17 苏州宣医智慧医疗科技有限公司 Preparation method of reduced graphene oxide silk fibroin composite membrane
CN113087948A (en) * 2021-04-21 2021-07-09 哈尔滨工业大学（深圳） Film and preparation method thereof
CN113499481A (en) * 2021-06-18 2021-10-15 广东省科学院健康医学研究所 Porous scaffold and preparation method and application thereof
CN113499481B (en) * 2021-06-18 2022-07-19 广东省科学院健康医学研究所 Porous scaffold and preparation method and application thereof

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Application publication date: 20160615