CN105330881B - A kind of gentle and a large amount of methods for preparing mineralising fibroin protein film - Google Patents
A kind of gentle and a large amount of methods for preparing mineralising fibroin protein film Download PDFInfo
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- CN105330881B CN105330881B CN201510787482.4A CN201510787482A CN105330881B CN 105330881 B CN105330881 B CN 105330881B CN 201510787482 A CN201510787482 A CN 201510787482A CN 105330881 B CN105330881 B CN 105330881B
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Abstract
The present invention provides a kind of gentle and a large amount of methods for preparing mineralising fibroin protein films, including the preparation of fibroin protein film, the enzyme solutions pretreatment of fibroin protein film, phosphatoptosis, calcium deposition obtain the fibroin protein film of surface permineralization.Prepared mineralising fibroin protein film can be used for bone renovating material, such as repair of cartilage, periosteum for repairing, ligament reparation.This method needs not move through repeated multiple times immersion, and preparation process is simple；Preparation condition is gentle, and most of preparation process all carries out at 37 DEG C；Obtained mineralising area is big and measures more, morphology controllable.
Description
Technical field
The present invention relates to the preparation method of mineralising fibroin protein film, and in particular to a kind of gentle and a large amount of preparation mineralising fibroins
The method of protein film.This method belongs to technical field of biological material, and prepared material can be widely used for bone tissue reparation.
Background technology
Fibroin material is due to good biocompatibility, machinability, mechanical property, in technical field of biological material
In receive much attention, the application especially in bone renovating material is very extensive.The important performance of bone renovating material is exactly after being implanted into
Synostosis can be realized with bone tissue, due to being rich in organic substance collagen and inorganic substances hydroxyapatite in bone tissue.It is single
Calcification is slow in vivo for silk fibroin material, and in order to improve the feature, the mineralising of fibroin material is largely paid close attention to.
The patent of application number 201010528523.5 discloses original position and prepares organic silk-inorganic phosphate hydrogen calcium from crystallisation
The method of composite material, mainly passes through Na2HPO4Solution and CaCl2Solution soaks multiple cycles repeatedly, is finally formed in situ
Inorganic phosphate hydrogen calcium mineralized layer.The patent of Application No. 201210283703.0 discloses a kind of tussah silk for promoting cell growth
The mineralization method of element, is first prepared for fibroin protein film, then by repeatedly in Na2HPO4Solution and CaCl2Soaked in solution
The method of bubble, has been prepared the fibroin membrane of mineralising.The patent of Application No. 201210411395 discloses a kind of by three-dimensional silk
Fibroin stent carry out mineralising method, be specifically first calcium chloride ethanol solution, absolute ethyl alcohol, aqueous dibasic potassium phosphate solution,
Soaked in deionized water；After stent repeatedly is soaked 5 ~ 30min in calcium chloride ethanol solution afterwards, then use deionized water
3 ~ 10min is soaked, makes three-dimensional silk fibroin stent pre-calcification；Finally the three-dimensional silk fibroin stent of pre-calcification part is soaked in
In bionical calcium ion buffer solution, constant temperature incubation 4 ~ 8 days at 37 DEG C, obtain the three-dimensional silk fibroin stent of mineralising.Application No.
Stearylamine is added in linoleic acid and alcohol mixed solution by 201510406788 patent, and stirring adds Ca to being completely dissolved
(NO3)2After aqueous solution stratification, Na is added3PO4Aqueous solution stirs；Silk fiber is added, solution is submerged silk fibroin
Dimension, high-temperature heating；Cleaning removes uncombined component, realizes the mineralising of silk fiber.The obtained mineralized fiber of above method is equal
It is to be obtained by the immersion or reaction of two kinds of salting liquids, and shows only to observe by the obtained fibroin material of immersion process
To a small amount of mineralized material.Only substantial amounts of hydroxyapatite nano rod has been observed in the patent of Application No. 201510406788
Formation, but its preparation condition harshness, it is necessary to be carried out at 100 DEG C.Due to protein mutability at high temperature, especially for
The fibroin material of carrying active medicine, easily makes drug inactivation under high temperature.Therefore need to invent a kind of reaction condition gentle
Method can prepare the more fibroin albumen mineralising films of mineralising amount.
The content of the invention
For overcome the deficiencies in the prior art, the present invention provides a kind of gentle and a large amount of sides for preparing mineralising fibroin protein film
Method, prepared mineralising fibroin protein film can be used for bone renovating material, such as repair of cartilage, periosteum for repairing, ligament reparation.
For purpose as realization, the present invention is achieved through the following technical solutions：
A kind of gentle and a large amount of methods for preparing mineralising fibroin protein film, it is characterised in that include the following steps：
（1）The preparation of fibroin protein film：Mass fraction is that the silk fibroin water solution of 3%-30% is cast at 37-70 DEG C
Film forming；
（2）The enzyme solutions pretreatment of fibroin protein film：Fibroin protein film is soaked in the protease that concentration is 1-6U/mL
When 1-48 is small in XIV solution, it is placed in 37 DEG C of shaking table, it is 50-200 revolutions per minute to control rotating speed, then takes out and is delayed with phosphate
Rush solution cleaning（The PBS of 0.01mol/L）, air drying；
（3）Phosphatoptosis：It is 0.05- that fibroin protein film through albumen enzyme pretreatment, which is soaked in phosphorus acid ion concentration,
In the PBS solution of 0.2mol/L, be subsequently placed in stationary incubation at 37 DEG C, after 3 days taking-up cleaned with distilled water, air drying obtains
Phosphate mineralising fibroin protein film；
（4）Calcium deposition；By phosphate mineralising fibroin protein film be soaked in concentration be 0.2-0.5mol/L calcium chloride or
In calcium nitrate solution 24 it is small when, obtain the fibroin protein film of surface permineralization.
The fibroin protein film is the composite membrane based on fibroin albumen, such as fibroin albumen and polysaccharide, albumen, synthesis high score
One or more composite membranes in son, cellulose etc..
The fibroin protein film for carry medicine fibroin protein film, contained medicine for growth factor, antibiotic, antitumor drug,
Treat the one or more in medicine for treating osteoporosis etc..
Advantage of the invention is that：（1）This method needs not move through repeated multiple times immersion, and preparation process is simple；（2）The party
Method prepares mild condition, and most of preparation process all carries out at 37 DEG C；（3）Obtained mineralising area is big and measures more, pattern
It is controllable.
Brief description of the drawings
Attached drawing 1 is the stereoscan photograph of mineralising fibroin protein film obtained by embodiment 1；
Attached drawing 2 is the stereoscan photograph of mineralising fibroin protein film obtained by embodiment 2.
Embodiment
Technical scheme is further described below by way of specific embodiment.Following embodiment is to this
The further explanation of invention, and do not limit the scope of the invention.
Embodiment 1：
（1）The preparation of fibroin protein film：The silk fibroin water solution that mass fraction is 5% casting film at 37 DEG C；
（2）The enzyme solutions pretreatment of fibroin protein film：Fibroin protein film is soaked in the protease XIV that concentration is 1U/mL
In solution 24 it is small when, be placed in 37 DEG C of shaking table, it is 100 revolutions per minute to control rotating speed, is then taken out clear with phosphate buffer solution
Wash（The PBS of 0.01mol/L）, air drying；
（3）Phosphatoptosis：Fibroin protein film through albumen enzyme pretreatment is soaked in phosphorus acid ion concentration as 0.1
In the PBS solution of mol/L, be subsequently placed in stationary incubation at 37 DEG C, after 3 days taking-up cleaned with distilled water, air drying obtains phosphorus
Hydrochlorate mineralising fibroin protein film.
（4）Calcium deposition；Phosphate mineralising fibroin protein film is soaked in the calcium chloride or nitric acid that concentration is 0.2mol/L
In calcium solution 24 it is small when, obtain mineralising fibroin protein film.
Embodiment 2：
（1）The preparation of fibroin protein film：The silk fibroin water solution that mass fraction is 15% casting film at 60 DEG C；
（2）The enzyme solutions pretreatment of fibroin protein film：Fibroin protein film is soaked in the protease XIV that concentration is 2U/mL
In solution 24 it is small when, be placed in 37 DEG C of shaking table, it is 100 revolutions per minute to control rotating speed, is then taken out clear with phosphate buffer solution
Wash（The PBS of 0.01 mol/L）, air drying；
（3）Phosphatoptosis：Fibroin protein film through albumen enzyme pretreatment is soaked in phosphorus acid ion concentration as 0.1
In the PBS solution of mol/L, be subsequently placed in stationary incubation at 37 DEG C, after 3 days taking-up cleaned with distilled water, air drying obtains phosphorus
Hydrochlorate mineralising fibroin protein film.
（4）Calcium deposition；Phosphate mineralising fibroin protein film is soaked in the calcium chloride or nitric acid that concentration is 0.2 mol/L
In calcium solution 24 it is small when, obtain mineralising fibroin protein film.
Embodiment 3：
（1）The preparation of fibroin protein film：The silk fibroin water solution that mass fraction is 30% casting film at 37 DEG C；
（2）The enzyme solutions pretreatment of fibroin protein film：Fibroin protein film is soaked in the protease XIV that concentration is 6U/mL
In solution 2 it is small when, be placed in 37 DEG C of shaking table, it is 200 revolutions per minute to control rotating speed, then takes out and is cleaned with phosphate buffer solution
（The PBS of 0.01mol/L）, air drying；
（3）Phosphatoptosis：Fibroin protein film through albumen enzyme pretreatment is soaked in phosphorus acid ion concentration as 0.05
In the PBS solution of mol/L, be subsequently placed in stationary incubation at 37 DEG C, after 3 days taking-up cleaned with distilled water, air drying obtains phosphorus
Hydrochlorate mineralising fibroin protein film.
（4）Calcium deposition；Phosphate mineralising fibroin protein film is soaked in the calcium chloride or nitric acid that concentration is 0.2mol/L
In calcium solution 24 it is small when, obtain mineralising fibroin protein film.
Embodiment 4：
（1）The preparation of silk fibroin composite membrane：Mass fraction is 5% silk fibroin water solution, by fibroin mass percent
For 10% add gelatin, after stirring evenly at 37 DEG C casting film,；
（2）The enzyme solutions pretreatment of fibroin albumen/synthesized thin film：Fibroin albumen/synthesized thin film is soaked in concentration
For in the protease XIV solution of 1U/mL 24 it is small when, be placed in 37 DEG C of shaking table, it is 100 revolutions per minute to control rotating speed, is then taken out
Cleaned with phosphate buffer solution（The PBS of 0.01mol/L）, air drying；
（3）Phosphatoptosis：Fibroin albumen/synthesized thin film through albumen enzyme pretreatment is soaked in phosphorus acid ion concentration
In PBS solution for 0.1 mol/L, be subsequently placed in stationary incubation at 37 DEG C, after 3 days taking-up cleaned with distilled water, air drying
Obtain phosphate mineralising fibroin protein film.
（4）Calcium deposition；Phosphate mineralising fibroin albumen/synthesized thin film is soaked in the chlorination that concentration is 0.2mol/L
In calcium or calcium nitrate solution 24 it is small when, obtain mineralising fibroin protein film.
Embodiment 5：
（1）The preparation of silk fibroin composite membrane：Mass fraction is 5% silk fibroin water solution, by fibroin mass percent
Hyaluronic acid, the casting film at 37 DEG C are added for 5%；
（2）The enzyme solutions pretreatment of fibroin albumen/hyaluronic acid membrane：Fibroin albumen/hyaluronic acid membrane is dense in being soaked in
Spend in the protease XIV solution for 3U/mL 12 it is small when, be placed in 37 DEG C of shaking table, it is 50 revolutions per minute to control rotating speed, is then taken out
Cleaned with phosphate buffer solution（The PBS of 0.01mol/L）, air drying；
（3）Phosphatoptosis：Fibroin albumen/hyaluronic acid membrane through albumen enzyme pretreatment is soaked in phosphorus acid ion concentration
In PBS solution for 0.2 mol/L, be subsequently placed in stationary incubation at 37 DEG C, after 3 days taking-up cleaned with distilled water, air drying
Obtain phosphate mineralising fibroin albumen/hyaluronic acid membrane.
（4）Calcium deposition；Phosphate mineralising fibroin albumen/hyaluronic acid membrane is soaked in the chlorine that concentration is 0.2 mol/L
Change in calcium or calcium nitrate solution 24 it is small when, obtain mineralising fibroin albumen/hyaluronic acid membrane.
Claims (4)
1. a kind of gentle and a large amount of methods for preparing mineralising fibroin protein film, it is characterised in that include the following steps：
（1）The preparation of fibroin protein film：Mass fraction is that the silk fibroin water solution of 3%-30% is cast at 37-70 DEG C
Film；
（2）The enzyme solutions pretreatment of fibroin protein film：It is molten that fibroin protein film is soaked in the protease XIV that concentration is 1-6U/mL
When 1-48 is small in liquid, it is placed in 37 DEG C of shaking table, it is 50-200 revolutions per minute to control rotating speed, then takes out the PBS with 0.01mol/L
Phosphate buffer solution is cleaned, air drying；
（3）Phosphatoptosis：It is 0.05- that fibroin protein film through albumen enzyme pretreatment, which is soaked in phosphorus acid ion concentration,
In the PBS solution of 0.2mol/L, be subsequently placed in stationary incubation at 37 DEG C, after 3 days taking-up cleaned with distilled water, air drying obtains
Phosphate mineralising fibroin protein film；
（4）Calcium deposition：Phosphate mineralising fibroin protein film is soaked in the calcium chloride or nitric acid that concentration is 0.2-0.5mol/L
In calcium solution 24 it is small when, obtain the fibroin protein film of surface permineralization.
2. method that is a kind of gentle as claimed in claim 1 and largely preparing mineralising fibroin protein film, it is characterised in that described
Fibroin protein film is the composite membrane based on fibroin albumen, fibroin albumen and the one or more in polysaccharide, albumen, synthesis macromolecule
Composite membrane.
3. method that is a kind of gentle as claimed in claim 2 and largely preparing mineralising fibroin protein film, it is characterised in that described
Polysaccharide is cellulose.
4. method that is a kind of gentle as claimed in claim 1 and largely preparing mineralising fibroin protein film, it is characterised in that the silk
For fibroin film to carry medicine fibroin protein film, contained medicine is growth factor, antibiotic, antitumor drug, treatment osteoporosis drug
One or more in thing.
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| CN201510787482.4A CN105330881B (en) | 2015-11-17 | 2015-11-17 | A kind of gentle and a large amount of methods for preparing mineralising fibroin protein film |
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| CN201510787482.4A CN105330881B (en) | 2015-11-17 | 2015-11-17 | A kind of gentle and a large amount of methods for preparing mineralising fibroin protein film |
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| CN105330881B true CN105330881B (en) | 2018-05-11 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107050511A (en) * | 2017-04-11 | 2017-08-18 | 江南大学 | A kind of biological enzyme fibroin albumen/calcium phosphate composite materials preparation method |
| CN107233619A (en) * | 2017-07-14 | 2017-10-10 | 中国人民解放军第四军医大学 | A kind of Porous titanium bone implant material of functionalization and preparation method thereof |
| CN115581801B (en) * | 2022-09-29 | 2024-05-17 | 苏州大学 | Calcium phosphate mineralized silk micro-nano fiber film and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1562386A (en) * | 2004-03-19 | 2005-01-12 | 清华大学 | Mineralized fibroin material and preparation method |
| CN103421201A (en) * | 2012-05-14 | 2013-12-04 | 江南大学 | Preparation method for antibacterial silk fibroin membrane by biological enzyme method |
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2015
- 2015-11-17 CN CN201510787482.4A patent/CN105330881B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1562386A (en) * | 2004-03-19 | 2005-01-12 | 清华大学 | Mineralized fibroin material and preparation method |
| CN103421201A (en) * | 2012-05-14 | 2013-12-04 | 江南大学 | Preparation method for antibacterial silk fibroin membrane by biological enzyme method |
Non-Patent Citations (3)
| Title |
|---|
| A Moldable Putty Containing Silk Fibroin Yolk Shell Particles for Improved Hemostasis and Bone Repair;Kushagra Saran, et al.;《Adv. Healthcare Mater.》;20141009;第4卷;第432-445页 * |
| Enzymatic grafting of chitosan onto Bombyx mori silk fibroin: kinetic and IR vibrational studies;Sandra Sampaio, et al.;《Journal of Biotechnology》;20051231;第116卷;第21-33页 * |
| Enzymatic Mineralization of Silk Scaffolds;Sangram K. Samal, et al.;《Macromol. Biosci.》;20140307;第14卷;第991-1003页 * |
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