CN105879113A - Method for preparing three-dimensional cell scaffolds on basis of silk fibroins - Google Patents
Method for preparing three-dimensional cell scaffolds on basis of silk fibroins Download PDFInfo
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- CN105879113A CN105879113A CN201610438113.9A CN201610438113A CN105879113A CN 105879113 A CN105879113 A CN 105879113A CN 201610438113 A CN201610438113 A CN 201610438113A CN 105879113 A CN105879113 A CN 105879113A
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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
- A61L27/56—Porous materials, e.g. foams or sponges
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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
- A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
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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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- 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
- A61L2430/22—Materials or treatment for tissue regeneration for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus
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Abstract
The invention discloses a method for preparing three-dimensional cell scaffolds on the basis of silk fibroins. Each three-dimensional cell scaffold is of a silk scaffold network structure with internal uniform holes. The method includes preparing mono-dispersion micro-spheres with uniform sizes by the aid of micro-fluidic devices; preparing templates for protein structures by the aid of self-assembly functions of the mono-dispersion micro-spheres; filling the templates with silk fibroin solution; cooling and drying the silk fibroin solution and removing the templates to obtain the network-shaped silk fibroin three-dimensional cell scaffolds with the internal uniform holes. The method for constructing the three-dimensional cell scaffolds has the advantages that the method is easy to implement and low in cost, and the three-dimensional cell scaffolds prepared by the aid of the method are excellent in mechanical performance, biocompatibility and biodegradability.
Description
Technical field
The invention belongs to three-dimensional cell timbering material studying technological domain, be specifically related to a kind of three-dimensional based on fibroin albumen
Cytoskeletal preparation method.
Background technology
Regenerating, replace, maintain or repairing the function aspects of damaged tissues, finding and develop suitable biology is tissue work
The most promising method of one in journey, and in various problems involved among these, cytoskeletal research then has weight
The meaning wanted.
Cytoskeletal structure and character are studied under material science and biomedical engineering background widely,
Support is required to include: (1) is used for preparing cytoskeletal material must have bio-compatible row and biodegradability,
Positive reaction is there is simultaneously for the cell together with inoculation；(2) this cytoskeleton should include the network structure of cavity, and
After can form interconnective system in three-dimensional；(3) this cytoskeleton should have certain mechanical performance to adapt to spy
Fixed application, is included in the application requirement generating the aspects such as cartilage, bone, artificial blood vessel.
In order to obtain an excellent three-dimensional cell support, researchers propose a lot of methods, including lyophilization,
High Temperature High Pressure, separated, electrostatic spinning etc..But most method has certain limitation, such as electrostatic spinning to obtain
Material be difficult to expand on real three-dimensional rack, other method also has size irregular, cavity heterogeneity, poor connectivity
Deng shortcomings.
Cytoskeletal aperture and structural relation to cell adhesion, migrate and be distributed, and nutrient substance and metabolism are produced
The exchange of thing, so it has very important significance for the cultivation of cell.Diameter and knot currently for support cavity
Structure, researchers are made that substantial amounts of effort and trial, but the interconnection between the homogeneity of cavity and cavity is the most urgently
Difficult point to be solved.Based on this problem, counter opal structure is considered as a kind of preferably model, because counter opal structure
There is the most consistent cavity, and inside has fairly regular three-dimensional net structure.Although having had some based on anti-egg
The three-dimensional cell support of white stone structure is developed, but the material that they are used is not the most to have biodegradability
, which limits its application in clinic.
Silkworm silk is one of native protein of being utilized the earliest, and human use's silkworm silk is as the history existing 5,000 of fabric
For many years. silkworm silk makes it in new and high technologies such as biological medicines because of mechanical property and the good biocompatibility of its excellence in recent years
Field is increasingly subject to pay attention to, and silkworm silk is made up of two kinds of albumen, and internal layer is fibroin albumen, and outer layer is coated with by sericin.Wherein silk
Fibroin content is about 70%-80%.Fibroin albumen contains 18 kinds of aminoacid, wherein glycine, alanine, serine content about
Account for 87%, how with-Gly-Ala-Gly-Ala-serine-polypeptide form exist.Fibroin albumen has two
The charged property of property, the premium properties such as nontoxic, good human body affinity, biological degradability and biocompatibility, can make
Standby become variform, such as cellular, membranaceous and tubulose etc., is a kind of to develop the material that medical material is more satisfactory.
As biological medicine material, its biological degradability is critically important performance indications.Such as, in organizational project
Timbering material, does not require nothing more than good biocompatibility, and the longest speed being more desirable to its degradation speed and tissue matches, Fang Keda
To good repairing effect.Therefore, how can control fibroin material degradation speed in vivo is that many researcheres close
The problem of the heart.Owing to natural silk still retains the mechanical property of more than 50% in implantation human body after 60 d, according to usual people
Definition to " degradable " material, silkworm silk is not belonging to biodegradable material.But research subsequently proves, silkworm silk is as one
Protein, is by some enzymatic degradation, and also finally can be absorbed in implanting human body, the most required time ratio one
As degradation material in meaning to grow.Affect a lot of because have of material degradation performance, such as the structure of material, form, degraded
The process degraded, mechanism, influence factor etc. are only had sufficiently understanding, can be only achieved effectively by condition, physiochemical environment etc.
Control material degradation behavior and the purpose of degradation speed.
Summary of the invention
Present invention aim at providing the preparation method of a kind of three-dimensional cell support based on fibroin albumen, solve at present
Technology be difficult to the network structure three-dimensional cell support with consistent diameter cavity, meet cytoskeleton at biology simultaneously
Requirement in terms of the compatibility and biodegradability.
In order to solve these problems of the prior art, present invention provide the technical scheme that
The preparation method of a kind of three-dimensional cell support based on fibroin albumen, described three-dimensional cell support is that one is internal containing all
The fibroin albumen network structure of one hole;It is characterized in that said method comprising the steps of:
(1) extraction of silk fibroin protein solution:
Raw Bombyx bombycis is carried out, utilize saltout, dialyse, the method such as concentration is extracted from 3% to 15% variable concentrations from raw Bombyx bombycis
The silk fibroin water solution of gradient.
(2) preparation process of micron size microsphere:
Select suitable material respectively as the continuous phase during micro-fluidic and discontinuous phase, utilize micro fluidic device (Fig. 1 a
Shown in) prepare the mono-dispersion microballoon of size tunable.The mono-dispersion microballoon of preparation is carried out and collects.
(3) template of mono-dispersion microballoon is prepared and replicates:
Utilize chemical vapour deposition technique, by monodispersed microsphere natural subsidence, preparation in the steps such as slow evaporating solution (2)
Mono-dispersion microballoon be self-assembled into opal structural template, then the silk fibroin protein solution extracted in step (1) is filled into albumen
In the template of stone structure, solidified finally by the mode such as ultrasound gel or lyophilization.
(4) collection of three-dimensional cell support processes:
Fibroin albumen after solidification is collected and cleaned, removes the mono-dispersion microballoon in removing template, and ultimately form internal containing communicating
The network-like three dimensional structure support of cavity.
Preferably, in described method step (2), micro fluidic device discontinuous phase includes silicon dioxide, polystyrene, gathers oneself
One or more material in lactone, cyanoacrylate, polyvinyl alcohol, benzene, dichloromethane.
Preferably, in described method step (2), micro fluidic device continuous phase includes methyl-silicone oil, hexadecane, polyethylene
One or more material in alcohol.
Preferably, described method step (4) go the remover of mono-dispersion microballoon in removing template to be selected from Fluohydric acid., hydroxide
Sodium, acetone, normal hexane or dichloromethane.
Preferably, in described method step (1) silk fibroin protein solution concentration between 3% ~ 15%.
Preferably, in described method in step (2) monodisperse particles particle diameter between 30 μm-300 μm.
Three-dimensional cell support of the present invention is compared to other materials, and advantage is will have consistent size in inside
Cavity, the diameter of cavity is controlled between 30-300 μm, the cultivation of suitable different types of cell.Simultaneously cavity between mutually
Connection forms network-like structure, it is possible to contributes to cell adhesion in incubation, migration, nutrient substance and metabolism and produces
The transport of thing.
Three-dimensional cell support of the present invention is based on fibroin material, it is possible to have good mechanical performance, is answering
It is able to maintain that certain stability with in organism, and there is biocompatibility and the biodegradability of excellence, real
By property widely.Its concrete preparation method comprises the following steps:
1. the extraction of silk fibroin protein solution:
Raw Bombyx bombycis is carried out, removes sericin by heated and boiled.Then the salt utilizing LiBr (0.9-1.1 mol/L) is molten
Fibroin is dissolved by liquid, load in bag filter dialyse in deionized water, the solution obtained afterwards of dialysing needs the removal of impurity,
Then the silk fibroin water solution of the variable concentrations from 3% to 15% it is condensed into according to different needs.
2. the preparation process of micron size microsphere:
Select suitable material respectively as the continuous phase during micro-fluidic and discontinuous phase, continuous phase and noncontinuity respectively
It is made up of oil phase or aqueous phase.So in micro fluidic device, different dispersion phases are to form shearing force crossing, and continuous phase is by non-
Continuous phase cuts into the emulsion of size uniformity.Utilize the emulsion of size uniformity of self-control micro-fluidic chip and device through overcuring
Can form microsphere, be calculated by accurate, emulsion diameter after being solidified into microsphere can control as required in 30-300 μm
Between monodisperse particles.The mono-dispersion microballoon of preparation is carried out and collects.Wherein said discontinuous phase includes two
One or more in silicon oxide, polystyrene, polycaprolactone, cyanoacrylate, polyvinyl alcohol, benzene, dichloromethane
Material；Described continuous phase includes one or more the material in methyl-silicone oil, hexadecane, polyvinyl alcohol.
3. the template of mono-dispersion microballoon is prepared and replicates:
Utilize chemical vapour deposition technique etc. by step 2. in the mono-dispersion microballoon of preparation be self-assembled into opal structural template, then will
The step 1. middle silk fibroin protein solution extracted is filled in the template of opal structural, finally by ultrasound gel or lyophilization
Etc. mode, fibroin albumen is shaped.
4. the collection of three-dimensional cell support processes:
Fibroin albumen after shaping is collected and is cleaned, and removes the mono-dispersion microballoon in removing template, and ultimately forms internal containing communicating
The network-like three dimensional structure support of cavity.Go in removing template the remover of mono-dispersion microballoon selected from Fluohydric acid., sodium hydroxide, third
Ketone, normal hexane or dichloromethane.
Accompanying drawing explanation
Fig. 1. preparation method schematic diagram (a) of a kind of three-dimensional cell support based on fibroin albumen and pictorial diagram (b): 1)
Utilize micro fluidic device to prepare and touch an edition microsphere, 2) microsphere is self-assembly of opal structural, 3) fibroin albumen is filled into egg
In white stone structure, 4) microsphere touched in version is removed, obtain counter opal structure three-dimensional cell support.
Specific implementation method
Below in conjunction with specific embodiment, such scheme is described further.Should be understood that these embodiments are for this is described
Bright and be not limited to limit the scope of the present invention.The implementation condition used in embodiment can be done into one according to the condition of concrete producer
Successive step, not marked implementation condition is usually the condition in normal experiment.
Embodiment 1 one kinds three-dimensional cell based on fibroin albumen support preparation method
(1) preparation of Monodispersed polystyrene latex:
We prepare micron order polystyrene (PS, Sigma) emulsion micro fluidic device (shown in Fig. 1 a) internal phase select be
PS/ benzene/dichloroethanes, mass volume ratio be 0.8 g:10 mL:10 mL. foreign minister be polyvinyl alcohol (PVA, Sigma)
Aqueous solution, mass volume ratio is 5%.Utilize stable flow rate of liquid (interior to flow velocity 0.5 mL/h) and shearing force (export-oriented flow velocity
5 mL/h), the oil phase of PS is cut into the oil-in-water list emulsion structure that diameter is consistent by the aqueous phase of PVA, and then emulsion passes through collecting pipe
Enter in the collecting pit filling PVA aqueous phase.As surfactant, the existence of PVA can effectively prevent in the process collected
The phenomenon that middle emulsion mutually merges；
(2) solidification of polystyrene emulsion:
Prepared emulsion droplets is transferred on the flask of Rotary Evaporators, from the beginning of room temperature, within every 5 minutes, raises 5 DEG C, until 60
DEG C, it is 40 rpm that rotary speed controls；Temperature arrives after 60 DEG C, programming rate reduce to every 30 minutes 2 DEG C, stop rising after 70 DEG C
Temperature；After temperature is more than 64 DEG C, rotating speed increases to 80rpm；Stop heating up and check whether solidification in latter 1 hour.PS micron ball by solidification
Repeatedly clean with ultra-pure water, 60 DEG C of oven dryings；
(3) replicate and remove template microsphere and obtain cytoskeleton
Polystyrene microsphere is placed in bottom centrifuge tube, is dried in 60 DEG C of baking ovens after adding pure water, can be bottom centrifuge tube
Obtain regularly arranged polystyrene moulding.Silk fibroin protein solution (15%) it is added thereto and carries out lyophilization, i.e. being become
Cytoskeleton after shape, finally uses acetone to remove polystyrene；
Described silk fibroin protein solution is prepared by following steps:
Raw Bombyx bombycis is carried out, removes sericin by heated and boiled.Then the salt utilizing LiBr (0.9-1.1 mol/L) is molten
Fibroin is dissolved by liquid, load in bag filter dialyse in deionized water, the solution obtained afterwards of dialysing needs the removal of impurity,
Then the silk fibroin water solution of the variable concentrations from 3% to 15% it is condensed into according to different needs.
Embodiment 2 one kinds three-dimensional cell based on fibroin albumen support preparation method
(1) preparation of single dispersing polycaprolactone (PCL) emulsion
What we prepared the micro fluidic device internal phase selection of micron order PCL emulsion is PCL/ dichloromethane, and the mass fraction of PCL is
5%wt.Foreign minister is the aqueous solution of PVA, and mass volume ratio is 5%.Utilize stable flow rate of liquid (interior to flow velocity 0.8 mL/h) and
Shearing force (export-oriented flow velocity 8 mL/h), the oil phase of PCL is cut into the oil-in-water list emulsion structure that diameter is consistent by the aqueous phase of PVA, so
Rear emulsion is entered by collecting pipe in the collecting pit filling PVA aqueous phase.As surfactant, the existence of PVA can be effective
Prevent the phenomenon that emulsion mutually merges during collecting；
(2) solidification of PCL emulsion droplets
Use 100ml to fill the blue mouth bottle of PVA solution PCL is collected, then in fume hood and keep 30 DEG C of constant temperature to add
Heat overnight, enables dichloromethane fully to volatilize, and is finally cleaned with a large amount of ultra-pure waters by the PCL solution of solidification, i.e. can get PCL
Microsphere；
(3) replicate and remove template microsphere and obtain cytoskeleton
Polycaprolactone microballoon sphere is placed in bottom centrifuge tube, with 80rpm shaken overnight on shaking table after addition pure water, along with moisture
Volatilization can obtain regularly arranged PC template bottom centrifuge tube, then it adds in 50 DEG C of baking ovens 1h, goes out
Polycaprolactone template is made to be fixed up while removing moisture.Finally silk fibroin protein solution it is added thereto and carries out freezing dry
Dry, after i.e. being shaped cytoskeleton, finally uses dichloromethane to remove polycaprolactone, then allows dichloromethane in fume hood
Alkane fully volatilizees and obtains desired cytoskeleton.
Claims (5)
1. the preparation method of a three-dimensional cell support based on fibroin albumen, it is characterised in that described method is real according to the following steps
Existing:
(1) extraction of silk fibroin protein solution:
Being carried out raw Bombyx bombycis, utilization is saltoutd, dialyses, is concentrated, and extracts the fibroin albumen from 3wt%-15wt% from raw Bombyx bombycis
Aqueous solution；
(2) preparation process of micron size microsphere:
Micro fluidic device, discontinuous phase and continuous phase is utilized to prepare the mono-dispersion microballoon of size tunable, micro-to the single dispersing of preparation
Ball is carried out and collects；
(3) template of mono-dispersion microballoon is prepared and replicates:
Utilize chemical vapour deposition technique that the mono-dispersion microballoon of preparation in step (2) is self-assembled into opal structural template, then will
The silk fibroin protein solution extracted in step (1) is filled in the template of opal structural, finally by ultrasound gel or freezing dry
Dry solidified；
(4) collection of three-dimensional cell support processes:
Fibroin albumen after solidification is collected and cleaned, utilizes remover to remove the mono-dispersion microballoon in removing template, and ultimately form
Internal containing the network-like three-dimensional cell support communicating cavity.
Method the most according to claim 1, it is characterised in that in described method step (2) discontinuous phase be silicon dioxide,
One or more material in polystyrene, polycaprolactone, cyanoacrylate, polyvinyl alcohol, benzene, dichloromethane.
Method the most according to claim 1, it is characterised in that in described method step (2), micro fluidic device continuous phase is first
One or more material in base silicone oil, hexadecane, polyvinyl alcohol.
4. according to according to the method described in claim 1, it is characterised in that described method step removes mono-dispersion microballoon in removing template in (4)
Remover be Fluohydric acid., sodium hydroxide, acetone, normal hexane or dichloromethane.
Method the most according to claim 1, it is characterised in that in described method, in step (2), monodisperse particles particle diameter exists
Between 30 μm-300 μm.
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Cited By (7)
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| CN106309383A (en) * | 2016-11-08 | 2017-01-11 | 东南大学 | Drug microcarrier based on egg white and preparation method thereof |
| CN107865979A (en) * | 2017-09-06 | 2018-04-03 | 北京航空航天大学 | A kind of three-dimensional manometer fibrous framework based on microflow control technique and electrostatic spinning technique and preparation method thereof |
| CN109856300A (en) * | 2018-11-22 | 2019-06-07 | 天津大学 | A kind of preparation method of silica inverse opal hydrogel photonic crystal microballoon |
| CN113144275A (en) * | 2020-01-22 | 2021-07-23 | 鸡西代悦科技有限公司 | Hydrogel adhesive and preparation method and application thereof |
| CN113244460A (en) * | 2021-04-29 | 2021-08-13 | 南开大学 | Oriented microchannel bracket for promoting tissue regeneration and preparation method thereof |
| CN114306737A (en) * | 2021-11-30 | 2022-04-12 | 厦门大学 | Silk fibroin-based porous microsphere and preparation method thereof |
| CN115501396A (en) * | 2022-09-13 | 2022-12-23 | 四川大学 | Degradable tissue scaffold and preparation method and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106309383A (en) * | 2016-11-08 | 2017-01-11 | 东南大学 | Drug microcarrier based on egg white and preparation method thereof |
| CN107865979A (en) * | 2017-09-06 | 2018-04-03 | 北京航空航天大学 | A kind of three-dimensional manometer fibrous framework based on microflow control technique and electrostatic spinning technique and preparation method thereof |
| CN109856300A (en) * | 2018-11-22 | 2019-06-07 | 天津大学 | A kind of preparation method of silica inverse opal hydrogel photonic crystal microballoon |
| CN113144275A (en) * | 2020-01-22 | 2021-07-23 | 鸡西代悦科技有限公司 | Hydrogel adhesive and preparation method and application thereof |
| CN113244460A (en) * | 2021-04-29 | 2021-08-13 | 南开大学 | Oriented microchannel bracket for promoting tissue regeneration and preparation method thereof |
| CN114306737A (en) * | 2021-11-30 | 2022-04-12 | 厦门大学 | Silk fibroin-based porous microsphere and preparation method thereof |
| CN115501396A (en) * | 2022-09-13 | 2022-12-23 | 四川大学 | Degradable tissue scaffold and preparation method and application thereof |
| CN115501396B (en) * | 2022-09-13 | 2024-03-22 | 长春达康园医疗器械有限公司 | Degradable tissue scaffold and preparation method and application thereof |
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Application publication date: 20160824 |