CN110075353A - A kind of preparation method of natural spider silk bio-ink - Google Patents
A kind of preparation method of natural spider silk bio-ink Download PDFInfo
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- CN110075353A CN110075353A CN201910371849.2A CN201910371849A CN110075353A CN 110075353 A CN110075353 A CN 110075353A CN 201910371849 A CN201910371849 A CN 201910371849A CN 110075353 A CN110075353 A CN 110075353A
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- spider silk
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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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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Transplantation (AREA)
- Dermatology (AREA)
- Medicinal Chemistry (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Dispersion Chemistry (AREA)
- Materials For Medical Uses (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to a kind of preparation methods of natural spider silk bio-ink, by the way that natural spider silk is carried out degumming process with sodium hydroxide solution, after freeze-drying, formic acid solution is added to dissolve under heating water bath, then spider's thread protein bio-ink is made in the gelatinous precipitate that repeatedly centrifugation obtains, and obtains tissue engineering bracket for 3D printing molding.Preparation method of the present invention is simple, obtained spider's thread protein bio-ink has good rheological characteristic, cooperate certain printing strategy that can prepare 3D printing spider's thread protein tissue engineering bracket, with good cell compatibility, it is expected to become ideal tissue engineering bracket, is able to satisfy the individual requirement of medical implant instantly.
Description
Technical field
The invention belongs to tissue engineering bracket field, in particular to a kind of preparation method of natural spider silk bio-ink.
Background technique
The combination of 3D printing technique and organizational project is able to solve many traditional medicines and is difficult even insurmountable difficulty
Topic.For example, traditional medicine can obtain the 3-dimensional image data of patient, the 3D solid of more visual pattern cannot be but provided
Model, especially complicated lesion.In addition to this, 3D printing will push personalized and precision medical treatment fast-developing.It is a
Property and precision medical treatment be the following medical treatment development important directions, and the development of the personalized precisely medical treatment of the power-assisted of 3D printing at present
It is mainly reflected in the personalized customization for human tissue engineering support and bone tissue etc..Therefore, 3D printing organizational project
Bracket has important research significance and application prospect on promoting the personalized precisely development of medical treatment.
When 3D printing at present is used for external medical assistance, including medical model, the medical instrument, rehabilitation used outside type-script
Accessory, artificial limb etc., the requirement to Biocompatibility are lower, it is only necessary to which material is capable of providing suitable mechanical strength and can beat
Print performance.When preparing 3D printing tissue engineering bracket, then require material that there should be good biocompatibility.
And this natural biologic material of spider silk, it is formed with its unique material, structure and good biocompatibility have
Hoping becomes ideal tissue engineering bracket material.Research shows that spider silk is by the high-intensitive miracle with high ductibility perfect combination,
There is indivisible contact with exquisite hierarchical microarchitecture unique inside spider silk.Spider silk is that a kind of native protein is fine
Dimension, spider's thread protein are then composed of the polypeptide chain that various amino acid form by certain way.Analysis shows spider silk fibroin
Amino acid form based on glycine and alanine, the two accounts for about the 70% of spider's thread protein amino acid content.Spider silk it is strong
Degree, elasticity and flexibility are all fine, and impact resistance is strong.In addition to this, resistance to low temperature is excellent, is still able to maintain at -40 DEG C
Good elasticity.Since the main constituents of spider silk are protein, thus there is biodegradability.
The research of domestic and international rarely seen natural spider's thread protein 3D printing, current report relate generally to gene recombination spider's thread protein
3D printing.As Thomas professor Scheibel of Germany is extracted recombinant spider silk proteins eADF4 using technique for gene engineering
(C16) and it is made to obtain bio-ink in conjunction with rgd peptide, the cell loaded in 7 days after printing activity still with higher, but
It is that it has uncontrollable risk.Simultaneously as the machine-shaping of natural spider's thread protein is difficult, the development of spider's thread industry is hindered.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of preparation method of natural spider silk bio-ink, this method
Natural spider silk is filled up in 3D printing using upper blank, manufactured 3D printing spider's thread protein tissue engineering bracket has good
Cell compatibility and mechanical property, be expected to become ideal tissue engineering bracket, be able to satisfy the individual character of medical implant instantly
Change and requires.
The present invention provides a kind of preparation methods of natural spider silk bio-ink, comprising:
(1) it in the natural spider silk merging sodium hydroxide solution after cleaning, boils, hot water injection, freeze-drying；
(2) the freeze-drying spider silk that step (1) obtains is mixed, heating reaction with formic acid solution, abandons bottom after low-speed centrifugal
Portion's impurity, then collected after high speed centrifugation and obtain natural spider silk bio-ink.
The amount ratio of natural spider silk and sodium hydroxide solution in the step (1) is 1g:200-400mL.
The mass fraction of sodium hydroxide solution in the step (1) is 0.5%-2.0%.
The temperature of hot water in the step (1) is 60-90 DEG C.
Freeze-drying in the step (1) specifically: be placed in -80 DEG C of refrigerator freezing 8-12h, then be placed in 3- in freeze drier
5 days.
The use temperature of the freeze drier is -100 to -80 DEG C, air pressure 5-10pa.
The mass fraction of formic acid solution in the step (2) is 80-100%.
The amount ratio of freeze-drying spider silk and formic acid solution in the step (2) is 1g:40-70mL.
Heating reaction temperature in the step (2) is 45-65 DEG C, and the heating reaction time is 8-12h.
The technological parameter of low-speed centrifugal in the step (2) is centrifugal force 1800-4500 × g, 5-20 DEG C of temperature, is centrifuged
Time 5-15min；Ultracentrifugal technological parameter be centrifugal force 7500-17500 × g, 5-20 DEG C of temperature, centrifugation time 5-
15min。
Natural spider silk bio-ink obtained in the step (2) is used to prepare 3D printing spider's thread protein organizational project branch
Frame.
The technological parameter of the 3D printing is 0.15-0.30MPa, and print temperature is 5-15 DEG C, uses syringe needle internal diameter 0.25-
0.41mm, syringe internal diameter 22.35mm.
Beneficial effect
(1) preparation method of the present invention is easy to operate, convenient feasible；The use of the natural spider's thread is raw material, avoids genetic engineering
Technology bring non-controllable risk；
(2) spider's thread bio-ink prepared by the present invention has good rheological characteristic, has the function of shear shinning；
(3) present invention has spider's thread bio-ink by the spider's thread protein tissue engineering bracket that 3D printing is prepared good
Good cell compatibility is expected to become ideal tissue engineering bracket, is able to satisfy the personalization of medical implant instantly, discloses
Potential application of the spider's thread protein biomaterial in personalized implantation extends the natural spider's thread again at the same time and leads in organizational project
Use direction in domain has more extensive application prospect.
Detailed description of the invention
Fig. 1 is the product photo in preparation process of the invention；
Fig. 2 is to print SEM observation of the obtained bracket at 215 times (A) with 10000 times (B) to scheme；
Fig. 3 left figure is bio-ink frequency and storage modulus, the relational graph of loss modulus under 1% strained condition；Right figure
For under 1Hz frequency condition, bio-ink shear strain and storage modulus, the relational graph of loss modulus；
Fig. 4 is to be planted in bracket and culture 1,3,5 on slide (control) with rat bone marrow mesenchymal stem cells (BMSCs)
It, and the result figure dyed with calcein.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Agents useful for same or instrument in the preparation and 3D printing technique of the natural spider silk bio-ink that embodiment provides
It is bought by market, the raw material used are the Jingzhao chilobrachys spider spider's thread, are provided by Hainan Zhu Wang pharmaceutcal corporation, Ltd.
Embodiment 1
(1) 5g natural spider silk is cleaned, is placed in 0.5% sodium hydroxide solution of 2000ml, is boiled in electromagnetic cooker
20min is repeated 3 times, then with 60 DEG C of hot water injection；
(2) it after the complete spider silk of hot water injection is cooled to room temperature, is placed in -80 DEG C of refrigerators and freezes 12h, then be placed in freezing
3 days in drying machine, degumming spider silk is obtained；
(3) 2.0g degumming spider silk and the formic acid solution of 100ml 98% is taken to mix, in 60 DEG C of heating water baths, magnetic agitation
8h；
(4) reacted mixed system is placed in supercentrifuge and centrifugal force is set for 1914 × g, room temperature is centrifuged
5min collects its supernatant, removes impurities at bottom；
(5) supernatant collected, which is again placed in supercentrifuge, is arranged centrifugal force as 11963 × g, and room temperature is centrifuged 5min,
It collects bottom gelatinous precipitate and bio-ink is made；
(6) ink is placed in printing syringe (syringe internal diameter 22.35mm) and is pushed away at 10 DEG C with supercharging air (0.3MPa)
Into 3D printing (syringe needle model: 24G) is carried out, the bracket printed is placed in deionized water again impregnates 4h, then is freeze-dried acquisition
3D printing spider's thread protein tissue engineering bracket.
As shown in Figure 2, the bracket of printing has certain porosity, appropriate cell growth.
From the figure 3, it may be seen that the spider's thread protein bio-ink of preparation has the function of shear shinning, meet the spy of 3D printing ink
Point.
As shown in Figure 4, the BMSCs cell proliferative conditions of plantation are good, illustrate that the spider's thread protein bio-ink of preparation has
Certain biocompatibility.
Claims (10)
1. a kind of preparation method of natural spider silk bio-ink, comprising:
(1) it in the natural spider silk merging sodium hydroxide solution after cleaning, boils, hot water injection, freeze-drying；
(2) the freeze-drying spider silk that step (1) obtains is mixed with formic acid solution, heating reaction, it is miscellaneous to abandon bottom after low-speed centrifugal
It is collected after matter, then high speed centrifugation and obtains natural spider silk bio-ink.
2. a kind of preparation method of natural spider silk bio-ink according to claim 1, it is characterised in that: the step
(1) amount ratio of natural spider silk and sodium hydroxide solution in is 1g:200-400mL.
3. a kind of preparation method of natural spider silk bio-ink according to claim 1, it is characterised in that: the step
(1) mass fraction of the sodium hydroxide solution in is 0.5%-2.0%.
4. a kind of preparation method of natural spider silk bio-ink according to claim 1, it is characterised in that: the step
(1) temperature of the hot water in is 60-90 DEG C.
5. a kind of preparation method of natural spider silk bio-ink according to claim 1, it is characterised in that: the step
(1) freeze-drying in specifically: be placed in -80 DEG C of refrigerator freezing 8-12h, then be placed in freeze drier 3-5 days.
6. a kind of preparation method of natural spider silk bio-ink according to claim 1, it is characterised in that: the step
(2) amount ratio of freeze-drying spider silk and formic acid solution in is 1g:40-70mL.
7. a kind of preparation method of natural spider silk bio-ink according to claim 1, it is characterised in that: the step
(2) the heating reaction temperature in is 45-65 DEG C, and the heating reaction time is 8-12h.
8. a kind of preparation method of natural spider silk bio-ink according to claim 1, it is characterised in that: the step
(2) technological parameter of the low-speed centrifugal in be centrifugal force 1800-4500 × g, 5-20 DEG C of temperature, centrifugation time 5-15min；At a high speed
The technological parameter of centrifugation be centrifugal force 7500-17500 × g, 5-20 DEG C of temperature, centrifugation time 5-15min.
9. a kind of preparation method of natural spider silk bio-ink according to claim 1, it is characterised in that: the step
(2) natural spider silk bio-ink obtained in is used to prepare 3D printing spider's thread protein tissue engineering bracket.
10. a kind of preparation method of natural spider silk bio-ink according to claim 9, it is characterised in that: the 3D
The technological parameter of printing is 0.15-0.30MPa, and print temperature is 5-15 DEG C, using syringe needle internal diameter 0.25-0.41mm, in syringe
Diameter 22.35mm.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910371849.2A CN110075353B (en) | 2019-05-06 | 2019-05-06 | Preparation method of natural spider silk biological ink |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910371849.2A CN110075353B (en) | 2019-05-06 | 2019-05-06 | Preparation method of natural spider silk biological ink |
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| CN110075353A true CN110075353A (en) | 2019-08-02 |
| CN110075353B CN110075353B (en) | 2021-10-26 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115227871A (en) * | 2022-08-10 | 2022-10-25 | 北京积水潭医院 | Silk fibroin biomaterial ink and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107029289A (en) * | 2017-03-31 | 2017-08-11 | 芜湖扬展新材料科技服务有限公司 | A kind of spider silk fibroin tissue renovation material |
-
2019
- 2019-05-06 CN CN201910371849.2A patent/CN110075353B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107029289A (en) * | 2017-03-31 | 2017-08-11 | 芜湖扬展新材料科技服务有限公司 | A kind of spider silk fibroin tissue renovation material |
Non-Patent Citations (4)
| Title |
|---|
| SHIKHA CHAWLA ET AL: "Silk-Based Bioinks for 3D Bioprinting", 《ADVANCED HEALTHCARE MATERIALS》 * |
| 刁均艳: "虎纹捕鸟蛛丝的结构与性能及其静电纺再生加工", 《中国硕士学位论文库》 * |
| 李晓春: "《针织物染整工艺学》", 30 October 2005, 中国纺织出版社 * |
| 段久芳: "《天然高分子材料》", 31 March 2016, 华中科技大学 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115227871A (en) * | 2022-08-10 | 2022-10-25 | 北京积水潭医院 | Silk fibroin biomaterial ink and preparation method and application thereof |
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