CN110201225A - 3D printing fibroin/gelatin bracket and preparation method thereof for repair of cartilage - Google Patents
3D printing fibroin/gelatin bracket and preparation method thereof for repair of cartilage Download PDFInfo
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- CN110201225A CN110201225A CN201910370206.6A CN201910370206A CN110201225A CN 110201225 A CN110201225 A CN 110201225A CN 201910370206 A CN201910370206 A CN 201910370206A CN 110201225 A CN110201225 A CN 110201225A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/222—Gelatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y10/00—Processes of additive manufacturing
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y80/00—Products made by additive manufacturing
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
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Abstract
3D printing fibroin/gelatin bracket and preparation method thereof that the invention discloses a kind of for repair of cartilage, the bracket is using fibroin albumen, tyrasamine piece-root grafting branch modified gelatin, horseradish peroxidase as slurry, porous aquagel bracket is constructed using 3D printing technique, enzyme crosslinking is realized by hydrogen peroxide dipping, further utilizes alcoholic solution processing enhancing mechanical property.Bracket of the present invention combines the excellent characteristics of gelatin and fibroin, has good biocompatibility, mechanical property and compression anti-fatigue performance, can be applied to regenerative agent of cartilaginous tissue reparation.
Description
Technical field
The present invention relates to the technical fields of bio-medical material, refer in particular to a kind of 3D printing silk for repair of cartilage
Element/gelatin bracket and preparation method thereof.
Background technique
Once cartilaginous tissue defect is difficult to realize selfreparing, although clinically there are many technologies to be applied to cartilage defect
Treatment, but various therapies all Shortcomings.The research and development of tissue engineering bone/cartilage are that the reparation of cartilage defect brings new think of
Road.The degradation rate and mechanical property that cartilage support material need to have good biocompatibility, match with regenerative agent of cartilaginous tissue
The characteristics such as energy；Cartilage tissue engineering rack is built with there are many modes, compared to some conventional stent molding modes, 3D printing
The structure of bracket can be precisely controlled.Hydrogel has structure similar with human body soft tissue and water content, in recent years 3D
The research of printing hydrogel also attracts attention, and also achieves many achievements.
3D printing hydrogel has material certain requirement, realizes that 3D printing can obtain fidelity using gelatin Thermo-sensitive
Spend preferable three-dimensional structure.Using gelatin as cartilage repair material, it is advantageous that it is reported that glutinous for cell recognition on strand
Attached site, have good biocompatibility, but gelatin there is also poor mechanical property, degradation time in vivo is short the defects of.Phase
Instead, fibroin albumen is as a kind of natural polymer extracted from silk, have preferable mechanical property and it is opposite can
The degradation rate of control, but do not have cell attachment sites on natural fibroin albumen strand, be unfavorable for cell migration on it,
Adherency and growth.The two, which is combined building 3D printing bracket, may be implemented the mutual supplement with each other's advantages of two kinds of materials.
Horseradish peroxidase enzyme catalytic crosslinking is a kind of quick, mild crosslinking method.Gelatin can be by strand
Upper modification tyrasamine root, to realize enzyme crosslinking；And natural fibroin albumen strand inherently has the junket for enzyme-catalyzed cross-linking
Histidine residue.The two can be subjected to covalent cross-linking by enzyme crosslinking reaction, to realize the mutual supplement with each other's advantages of the two.In addition, enzyme is handed over
Silk fibroin hydrogel afterwards, then the mechanical property of hydrogel can be significantly increased after alcoholic solution processing causes conformation transition.
Summary of the invention
The purpose of the present invention is to overcome the shortcomings of the existing technology with it is insufficient, propose a kind of 3D for repair of cartilage and beat
Fibroin/gelatin bracket and preparation method thereof is printed, which combines the excellent characteristics of gelatin and fibroin, has good biological compatible
Property, mechanical property and compression anti-fatigue performance, can be applied to regenerative agent of cartilaginous tissue reparation.
To achieve the above object, technical solution provided by the present invention are as follows: 3D printing fibroin/gelatin for repair of cartilage
Bracket, the bracket utilize 3D printing technique using fibroin albumen, tyrasamine piece-root grafting branch modified gelatin, horseradish peroxidase as slurry
Porous aquagel bracket is constructed, enzyme crosslinking is realized by hydrogen peroxide dipping, further utilizes alcoholic solution processing enhancing mechanical property.
3D printing fibroin/gelatin bracket preparation method for repair of cartilage, comprising the following steps:
1) prepared by printed material
1.1) preparation of tyrasamine piece-root grafting branch modified gelatin: configuration 500ml concentration is 50mM morpholino b acid buffer, is added
10g gelatin powder is stirred at 50 DEG C, is dissolved sufficiently, 5g Hydrphenacetamine Hydrochloride is added, sufficiently, solution is cooled to room for stirring, dissolution
Wen Hou sequentially adds carboxyl activator n-hydroxysuccinimide and 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide salt
Carboxyl in acid salt activating gelatin chains, reacts 12h at room temperature；It is 10000- that reaction product, which is packed into molecular cut off,
It in 12000 bag filter, dialyses 4 days under deionized water environment, finally removes moisture using freeze drier, obtain platinum sponge
Shape modified product stores in moisture-resistant cabinet, spare；
1.2) preparation of silk fibroin protein solution: 4g boiled silk is added in beaker, 9.3mol/l lithium bromide is then added
Solution 20ml；Place the beaker heating for dissolving boiled silk 4h in 60 DEG C of water-baths；After completely dissolution, solution is transferred to retention
In the bag filter that molecular weight is 3500, dialyses 3 days under deionized water environment, change 2-3 water daily；After dialysis, it will dialyse
Solution in bag is centrifuged twice in centrifuge, removes insoluble impurity；After centrifugation, clarification silk fibroin protein solution, silk are obtained
The concentration of fibroin solution is obtained by specific gravity in oven dry method, is placed in 4 DEG C of refrigerators and is saved backup, and is used in three weeks；
2) configuration of slurry is printed
Modified gelatin addition is diluted in the silk fibroin protein solution of normal concentration, about 2h is dissolved at 50 DEG C, it is sufficiently mixed
It is even；Horseradish peroxidase is added after Gelatin；It is transferred in barrel after mixing, it is spare；Wherein, gelatin concentration is
15wt%, fibroin albumen concentration are 0~5w/v%, horseradish peroxidase concentration is 30~240Units/ml；
3) printing and post-processing of hydrogel scaffold
Barrel equipped with printing slurry in step 2) is transferred in 3D printer, by barrel temperature and printing deposition platform
After temperature is set, barrel is allowed to be kept the temperature；After the parameter for setting internal stent and external structure, pressure is squeezed out by debugging
Start to print after power, draft speed；After printing, the structure that printing is obtained impregnates hydrogen peroxide solution, causes fibroin albumen
Enzyme-catalyzed cross-linking between modified gelatin；The hydrogel scaffold after enzyme crosslinking is finally further handled with alcoholic solution, promotes fibroin
Protein conformation transformation, to enhance the mechanical property of bracket；After treatment, cleans that remove remaining alcohol three times molten with deionized water
Liquid obtains a kind of 3D printing fibroin albumen/gelatin hydrogel bracket.
In step 1.1), the carboxyl activator n-hydroxysuccinimide and 1- (3- dimethylamino-propyl) -3- second
Base carbodiimide hydrochloride dosage is respectively as follows: 0.37g, 0.11g.
In step 3), the barrel temperature is set as 28~30 DEG C, and platform temperature is 4~8 DEG C.
In step 3), the shape and size of the bracket outer structure can be designed according to actual needs；It is described
Internal stent structure is the fiber filament that spacing is 0.4~1mm, and angle is 90 ° between every two layers of fiber filament, and thickness is 180 μm~400
μm。
In step 3), the extrusion pressure is 1.5~4bar；The draft speed is 10~20mm/s.
In step 3), the hydrogen peroxide concentration is 5~10mM, and soaking time is 30~60min.
In step 3), the alcoholic solution is 70~90w/v% methanol or ethanol water, and soaking time is 6~10h.
Compared with prior art, the present invention have the following advantages that with the utility model has the advantages that
1, material therefor of the present invention is two kinds of natural reproducible macromolecules of gelatin and fibroin albumen, from a wealth of sources easily to obtain
It takes, there is good biocompatibility.
2,3D printing fibroin/gelatin bracket prepared by the present invention has the pore structure of internal connection, is conducive to cell and exists
The growth and proliferation of internal stent.
3,3D printing fibroin prepared by the present invention/gelatin bracket compression modulus with higher and compression fatigue resistance,
It is adapted to the stress condition of natural cartilage tissue.
4, preparation process proposed by the present invention is simple and easy, and building that can be efficient, stable is repaired for cartilage tissue engineered
Multiple three-dimensional porous rack.
Detailed description of the invention
Fig. 1 is bracket 3D printing preparation process schematic diagram.
Fig. 2 is the scanning electron microscope (SEM) photograph after the bracket freeze-drying prepared in Examples 1 to 5.
Fig. 3 is that the bracket for preparing is after being inoculated with the adipose-derived mescenchymal stem cell of people in Examples 1 to 5, with CCK-8 method table
Levy the histogram of cell proliferative condition.
Fig. 4 is the compression modulus histogram of the bracket prepared in Examples 1 to 5.
Fig. 5 is that the bracket of Examples 1 to 4 preparation unloads the load-deformation curve in compression verification in linear CYCLIC LOADING.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is further elaborated, but embodiments of the present invention are not limited to this.
Embodiment 1
1) prepared by printed material
1.1) preparation of tyrasamine piece-root grafting branch modified gelatin: configuration 500ml concentration is 50mM morpholino b acid buffer, is added
10g gelatin powder is stirred at 50 DEG C, is dissolved sufficiently.5g Hydrphenacetamine Hydrochloride is added, stirring, dissolution are abundant；Solution is cooled to room
Wen Hou sequentially adds carboxyl activator n-hydroxysuccinimide and 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide salt
Hydrochlorate 0.37g/0.11g reacts 12h with the carboxyl on active gelatin strand at room temperature；Reaction product is packed into retention molecule
Amount is to dialyse 4 days under deionized water environment in the bag filter of 10000-12000, finally removes moisture using freeze drier, obtains
To white sponge modified product, stored in moisture-resistant cabinet, it is spare.
1.2) preparation of silk fibroin protein solution: 4g boiled silk is added in beaker, 9.3mol/l lithium bromide is then added
Solution 20ml；Place the beaker heating for dissolving boiled silk 4h in 60 DEG C of water-baths；After completely dissolution, solution is transferred to retention
In the bag filter that molecular weight is 3500, is dialysed 2 days with ion water environment, change 2-3 water daily；After dialysis, by bag filter
In solution be centrifuged in centrifuge twice, remove insoluble impurity；After centrifugation, clarification silk fibroin protein solution, fibroin are obtained
The concentration of protein solution is obtained by specific gravity in oven dry method, and silk fibroin protein solution is diluted to 2.5w/v% with deionized water.
2) configuration of slurry is printed
It is that the modified gelatin that modified gelatin to concentration is 15w/v% is added in 2.5w/v% silk fibroin protein solution in concentration,
About 2h is dissolved at 50 DEG C, is mixed well；Horseradish peroxidase is added after Gelatin, makes its concentration 60Units/
ml；It is transferred in barrel after mixing.
3) printing and post-processing of hydrogel scaffold
Barrel equipped with printing slurry in step 2) is transferred in 3D printer, barrel temperature is set as 29 DEG C, is beaten
Print deposition platform temperature is set as 4 DEG C, and barrel is allowed to keep the temperature 2h；Bracket outer pattern is set as the cuboid of 10*10*4mm, it is internal
Structure are as follows: fiber filament spacing is 0.6mm, and the angle between every two layers of fiber filament is 90 °；Extrusion pressure is set as 2bar, drawing-off speed
Degree starts to print after being 15mm/s；After printing, the structure that printing is obtained impregnates hydrogen peroxide solution 30min, causes fibroin
Enzyme-catalyzed cross-linking between albumen and modified gelatin is cleaned three times with deionized water, obtains the SF2.5GT15 hydrogel branch of 3D printing
Frame.
Embodiment 2
1) prepared by printed material
1.1) preparation of tyrasamine piece-root grafting branch modified gelatin: configuration 500ml concentration is 50mM morpholino b acid buffer, is added
10g gelatin powder is stirred at 50 DEG C, is dissolved sufficiently.5g Hydrphenacetamine Hydrochloride is added, stirring, dissolution are abundant；Solution is cooled to room
Wen Hou sequentially adds carboxyl activator n-hydroxysuccinimide and 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide salt
Hydrochlorate 0.37g/0.11g reacts 12h with the carboxyl on active gelatin strand at room temperature；Reaction product is packed into retention molecule
Amount is to dialyse 4 days under deionized water environment in the bag filter of 10000-12000, finally removes moisture using freeze drier, obtains
To white sponge modified product, stored in moisture-resistant cabinet, it is spare.
1.2) preparation of silk fibroin protein solution: 4g boiled silk is added in beaker, 9.3mol/l lithium bromide is then added
Solution 20ml；Place the beaker heating for dissolving boiled silk 4h in 60 DEG C of water-baths；After completely dissolution, solution is transferred to retention
In the bag filter that molecular weight is 3500, is dialysed 2 days with ionized water, change 2-3 water daily；It, will be in bag filter after dialysis
Solution is centrifuged twice in centrifuge, removes insoluble impurity；After centrifugation, clarification silk fibroin protein solution, fibroin albumen are obtained
The concentration of solution is obtained by specific gravity in oven dry method, and silk fibroin protein solution is diluted to 2.5w/v% with deionized water.
2) configuration of slurry is printed
It is that addition modified gelatin to concentration is 15w/v% in 2.5w/v% silk fibroin protein solution in concentration, it is molten at 50 DEG C
Terminate an agreement 2h, mixes well；Horseradish peroxidase is added after Gelatin, makes its concentration 60Units/ml；Turn after mixing
It moves in barrel.
3) printing and post-processing of hydrogel scaffold
Barrel equipped with printing slurry in step 2) is transferred in 3D printer, barrel temperature is set as 29 DEG C, is beaten
Print deposition platform temperature is set as 4 DEG C, and barrel is allowed to keep the temperature 2h；Bracket outer pattern is set as the cuboid of 10*10*4mm, it is internal
Structure are as follows: fiber filament spacing is 0.6mm, and the angle between every two layers of fiber filament is 90 °, sets extrusion pressure as 2.5bar, drawing-off
Speed starts to print after being 15mm/s；After printing, the structure that printing is obtained impregnates hydrogen peroxide solution 30min, causes silk
Enzyme-catalyzed cross-linking between fibroin and modified gelatin, finally further with the water after 75wt% methanol solution immersion treatment enzyme crosslinking
Gel stent 10h；After treatment is cleaned three times with deionized water, obtains the SF2.5GT15/ alcohol processing hydrogel of 3D printing
Bracket.
Embodiment 3
1) prepared by printed material
1.1) preparation of tyrasamine piece-root grafting branch modified gelatin: configuration 500ml concentration is 50mM morpholino b acid buffer, is added
10g gelatin powder is stirred at 50 DEG C, is dissolved sufficiently.5g Hydrphenacetamine Hydrochloride is added, stirring, dissolution are abundant；Solution is cooled to room
Wen Hou sequentially adds carboxyl activator n-hydroxysuccinimide and 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide salt
Hydrochlorate 0.37g/0.11g reacts 12h with the carboxyl on active gelatin strand at room temperature；Reaction product is packed into retention molecule
Amount is to dialyse 4 days under deionized water environment in the bag filter of 10000-12000, finally removes moisture using freeze drier, obtains
To white sponge modified product, stored in moisture-resistant cabinet, it is spare.
1.2) preparation of silk fibroin protein solution: 4g boiled silk is added in beaker, 9.3mol/l lithium bromide is then added
Solution 20ml；Place the beaker heating for dissolving boiled silk 4h in 60 DEG C of water-baths；After completely dissolution, solution is transferred to retention
In the bag filter that molecular weight is 3500, dialyses 2 days under deionized water environment, change 2-3 water daily；After dialysis, it will dialyse
Solution in bag is centrifuged twice in centrifuge, removes insoluble impurity；After centrifugation, clarification silk fibroin protein solution, silk are obtained
The concentration of fibroin solution is obtained by specific gravity in oven dry method, and silk fibroin protein solution is diluted to 5w/v% with deionized water.
2) configuration of slurry is printed:
It is that addition modified gelatin to concentration is 15w/v% in 5w/v% silk fibroin protein solution in concentration, is dissolved at 50 DEG C
About 2h is mixed well；Horseradish peroxidase is added after Gelatin, makes its concentration 60U/ml；Material is transferred to after mixing
In cylinder.
3) printing and post-processing of hydrogel scaffold
Barrel equipped with printing slurry in step 2) is transferred in 3D printer, barrel temperature is set as 30 DEG C, is beaten
Print deposition platform temperature is set as 4 DEG C, and barrel is allowed to keep the temperature 2h；Set bracket outer pattern are as follows: the cuboid of 10*10*4mm, it is interior
Portion's structure are as follows: fiber filament spacing is 0.6mm, and the angle between every two layers of fiber filament is 90 °, sets extrusion pressure as 3bar, drawing-off
Speed starts to print after being 15mm/s；After printing, the structure that printing is obtained impregnates hydrogen peroxide solution 30min, causes silk
Enzyme-catalyzed cross-linking between fibroin and modified gelatin is cleaned three times with deionized water, obtains the SF5GT15 hydrogel branch of 3D printing
Frame.
Embodiment 4
1) prepared by printed material
1.1) preparation of tyrasamine piece-root grafting branch modified gelatin: configuration 500ml concentration is 50mM morpholino b acid buffer, is added
10g gelatin powder is stirred at 50 DEG C, is dissolved sufficiently.5g Hydrphenacetamine Hydrochloride is added, stirring, dissolution are abundant；Solution is cooled to room
Wen Hou sequentially adds carboxyl activator n-hydroxysuccinimide and 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide salt
Hydrochlorate 0.37g/0.11g reacts 12h with the carboxyl on active gelatin strand at room temperature；Reaction product is packed into retention molecule
Amount is to dialyse 4 days under deionized water environment in the bag filter of 10000-12000, finally removes moisture using freeze drier, obtains
To white sponge modified product, stored in moisture-resistant cabinet, it is spare.
1.2) preparation of silk fibroin protein solution: 4g boiled silk is added in beaker, 9.3mol/l lithium bromide is then added
Solution 20ml；Place the beaker heating for dissolving boiled silk 4h in 60 DEG C of water-baths；After completely dissolution, solution is transferred to retention
In the bag filter that molecular weight is 3500, dialyses 3 days under deionized water environment, change 2-3 water daily；After dialysis, it will dialyse
Solution in bag is centrifuged twice in centrifuge, removes insoluble impurity；After centrifugation, clarification silk fibroin protein solution, silk are obtained
The concentration of fibroin solution is obtained by specific gravity in oven dry method, and silk fibroin protein solution is diluted to 5w/v% with deionized water.
2) configuration of slurry is printed
It is that addition modified gelatin to concentration is 15w/v% in 5w/v% silk fibroin protein solution in concentration, is dissolved at 50 DEG C
About 2h is mixed well；Horseradish peroxidase is added after Gelatin, makes its concentration 60Units/ml；It is shifted after mixing
Into barrel
3) printing and post-processing of hydrogel scaffold
Barrel equipped with printing slurry in step 2) is transferred in 3D printer, barrel temperature is set as 30 DEG C, is beaten
Print deposition platform temperature is set as 4 DEG C, and barrel is allowed to keep the temperature 2h；Set bracket outer pattern are as follows: the cuboid of 10*10*4mm, it is interior
Portion's structure are as follows: fiber filament spacing is 0.6mm, and angle is 90 ° between every two layers of fiber filament, sets extrusion pressure as 3bar, drawing-off speed
Degree starts to print after being 15mm/s；After printing, the structure that printing is obtained impregnates hydrogen peroxide solution 30min, causes fibroin
Enzyme-catalyzed cross-linking between albumen and modified gelatin；Finally further with the water-setting after 75wt% methanol solution immersion treatment enzyme crosslinking
Glue bracket 10h；After treatment cleans three times with deionized water and removes remaining alcoholic solution, obtains the SF5GT15/ of 3D printing
Alcohol handles hydrogel scaffold.
Embodiment 5 (comparative example)
1) prepared by printed material
1.1) preparation of tyrasamine piece-root grafting branch modified gelatin: configuration 500ml concentration is 50mM morpholino b acid buffer, is added
10g gelatin powder is stirred at 50 DEG C, is dissolved sufficiently.5g Hydrphenacetamine Hydrochloride is added, stirring, dissolution are abundant；Solution is cooled to room
Wen Hou sequentially adds carboxyl activator n-hydroxysuccinimide and 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide salt
Hydrochlorate 0.37g/0.11g reacts 12h with the carboxyl on active gelatin strand at room temperature；Reaction product is packed into retention molecule
Amount is to dialyse 4 days under deionized water environment in the bag filter of 10000-12000, finally removes moisture using freeze drier, obtains
To white sponge modified product, stored in moisture-resistant cabinet, it is spare.
2) configuration of slurry is printed
Modified gelatin is added in deionized water, makes its concentration 15w/v%, about 2h is dissolved at 50 DEG C, is mixed well；
Horseradish peroxidase is added after Gelatin, makes its concentration 60U/ml；It is transferred in barrel after mixing.
3) printing and post-processing of hydrogel scaffold
Barrel equipped with printing slurry in step 2) is transferred in 3D printer, barrel temperature is set as 28 DEG C, is beaten
Print deposition platform temperature is set as 4 DEG C, and barrel is allowed to keep the temperature 2h；Set bracket outer pattern are as follows: the cuboid branch of 10*10*4mm
Frame, internal structure are as follows: fiber filament spacing is 0.6mm, and angle is 90 ° between every two layers of fiber filament, by debugging extrusion pressure, drawing-off
Start to print after speed；After printing, the obtained structure of printing is impregnated into hydrogen peroxide solution 30min, cause fibroin albumen and
Enzyme-catalyzed cross-linking between modified gelatin；Finally further with the hydrogel scaffold after 75wt% methanol solution immersion treatment enzyme crosslinking
10h；After treatment cleans three times with deionized water and removes remaining alcoholic solution, obtains the pure gelatin hydrogel bracket of 3D printing.
In conclusion the present invention is mainly printing slurry with horseradish peroxidase, modified gelatin and fibroin albumen, pass through
Temperature control 3D printing constructs porous aquagel bracket, causes bracket using hydrogen peroxide dipping and is crosslinked, then further at alcoholic solution
Reason causes Conformation Transition of Silk Fibroin, improves material physicochemical property, obtains a kind of bracket suitable for regenerative agent of cartilaginous tissue.Referring to
Shown in Fig. 1 to Fig. 5, it is followed successively by bracket 3D printing preparation process schematic diagram；Sweeping after the bracket freeze-drying prepared in Examples 1 to 5
Retouch electron microscope；The bracket prepared in Examples 1 to 5 is characterized thin after being inoculated with the adipose-derived mescenchymal stem cell of people with CCK-8 method
The histogram of born of the same parents' proliferative conditions；The compression modulus histogram of the bracket prepared in Examples 1 to 5；The branch of Examples 1 to 4 preparation
Load-deformation curve of the frame in linear CYCLIC LOADING unloading compression verification.
But embodiment of the present invention are not limited by the above embodiments, other any without departing from spirit of the invention
Essence and made changes, modifications, substitutions, combinations, simplifications under principle, are equivalent substitute mode, are included in the present invention
Protection scope within.
Claims (8)
1. being used for 3D printing fibroin/gelatin bracket of repair of cartilage, it is characterised in that: the bracket is with fibroin albumen, tyrasamine piece-root grafting
Branch modified gelatin, horseradish peroxidase are slurry, construct porous aquagel bracket using 3D printing technique, pass through dioxygen water logging
Bubble realizes enzyme crosslinking, further utilizes alcoholic solution processing enhancing mechanical property.
2. 3D printing fibroin/gelatin bracket preparation method described in claim 1 for repair of cartilage, which is characterized in that
The following steps are included:
1) prepared by printed material
1.1) preparation of tyrasamine piece-root grafting branch modified gelatin: configuration 500ml concentration is 50mM morpholino b acid buffer, and 10g is added
Gelatin powder is stirred at 50 DEG C, is dissolved sufficiently, 5g Hydrphenacetamine Hydrochloride is added, sufficiently, solution is cooled to room temperature for stirring, dissolution
Afterwards, carboxyl activator n-hydroxysuccinimide and 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide hydrochloride are sequentially added
Carboxyl on salt active gelatin strand, reacts 12h at room temperature；It is 10000-12000 that reaction product, which is packed into molecular cut off,
Bag filter in, dialyse 4 days under deionized water environment, finally using freeze drier remove moisture, obtain white sponge and change
Property product stores in moisture-resistant cabinet, spare；
1.2) preparation of silk fibroin protein solution: 4g boiled silk is added in beaker, 9.3mol/l lithium-bromide solution is then added
20ml；Place the beaker heating for dissolving boiled silk 4h in 60 DEG C of water-baths；After completely dissolution, solution is transferred to retention molecule
In the bag filter that amount is 3500, dialyses 3 days under deionized water environment, change 2-3 water daily；It, will be in bag filter after dialysis
Solution be centrifuged in centrifuge twice, remove insoluble impurity；After centrifugation, clarification silk fibroin protein solution, fibroin egg are obtained
The concentration of white solution is obtained by specific gravity in oven dry method, is placed in 4 DEG C of refrigerators and is saved backup, and is used in three weeks；
2) configuration of slurry is printed
Modified gelatin addition is diluted in the silk fibroin protein solution of normal concentration, about 2h is dissolved at 50 DEG C, is mixed well；To
Horseradish peroxidase is added after Gelatin；It is transferred in barrel after mixing, it is spare；Wherein, gelatin concentration 15wt%, silk
Fibroin concentration is 0~5w/v%, horseradish peroxidase concentration is 30~240Units/ml；
3) printing and post-processing of hydrogel scaffold
Barrel equipped with printing slurry in step 2) is transferred in 3D printer, by barrel temperature and printing deposition platform temperature
After setting, barrel is allowed to be kept the temperature；After the parameter for setting internal stent and external structure, by debugging extrusion pressure, lead
Start to print after stretching speed；After printing, the structure that printing is obtained impregnates hydrogen peroxide solution, causes fibroin albumen and modification
Enzyme-catalyzed cross-linking between gelatin；The hydrogel scaffold after enzyme crosslinking is finally further handled with alcoholic solution, promotes fibroin albumen structure
As transformation, to enhance the mechanical property of bracket；After treatment cleans three times with deionized water and removes remaining alcoholic solution, obtains
To a kind of 3D printing fibroin albumen/gelatin hydrogel bracket.
3. 3D printing fibroin/gelatin bracket preparation method according to claim 2 for repair of cartilage, feature exist
In: in step 1.1), the carboxyl activator n-hydroxysuccinimide and 1- (3- dimethylamino-propyl) -3- ethyl carbon two
Inferior amine salt hydrochlorate dosage is respectively as follows: 0.37g, 0.11g.
4. 3D printing fibroin/gelatin bracket preparation method according to claim 2 for repair of cartilage, feature exist
In: in step 3), the barrel temperature is set as 28~30 DEG C, and platform temperature is 4~8 DEG C.
5. 3D printing fibroin/gelatin bracket preparation method according to claim 2 for repair of cartilage, feature exist
In: in step 3), the shape and size of the bracket outer structure can be designed according to actual needs；In the bracket
Portion's structure is the fiber filament that spacing is 0.4~1mm, and angle is 90 ° between every two layers of fiber filament, and thickness is 180 μm~400 μm.
6. 3D printing fibroin/gelatin bracket preparation method according to claim 2 for repair of cartilage, feature exist
In: in step 3), the extrusion pressure is 1.5~4bar；The draft speed is 10~20mm/s.
7. 3D printing fibroin/gelatin bracket preparation method according to claim 2 for repair of cartilage, feature exist
In: in step 3), the hydrogen peroxide concentration is 5~10mM, and soaking time is 30~60min.
8. 3D printing fibroin/gelatin bracket preparation method according to claim 2 for repair of cartilage, feature exist
In: in step 3), the alcoholic solution is 70~90w/v% methanol or ethanol water, and soaking time is 6~10h.
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| CN110639060A (en) * | 2019-11-04 | 2020-01-03 | 西安工程大学 | 3D bio-printing silk fibroin-based tissue engineering scaffold and preparation method and application thereof |
| CN112972765A (en) * | 2021-02-22 | 2021-06-18 | 苏州大学 | Silk fibroin 3D printing biological ink and application thereof |
| CN113030219A (en) * | 2021-05-24 | 2021-06-25 | 广州新诚生物科技有限公司 | Continuous glucose monitoring sensor and application thereof |
| CN113350577A (en) * | 2021-06-15 | 2021-09-07 | 华南理工大学 | 3D printing composite hydrogel support, preparation method thereof and sterile freeze-drying support |
| CN114533953A (en) * | 2022-01-24 | 2022-05-27 | 华南理工大学 | Nanoparticle composite hydrogel nerve conduit and preparation method thereof |
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Application publication date: 20190906 |