CN114699556A - Preparation method of silk fibroin repair patch and silk fibroin repair patch - Google Patents
Preparation method of silk fibroin repair patch and silk fibroin repair patch Download PDFInfo
- Publication number
- CN114699556A CN114699556A CN202210366624.XA CN202210366624A CN114699556A CN 114699556 A CN114699556 A CN 114699556A CN 202210366624 A CN202210366624 A CN 202210366624A CN 114699556 A CN114699556 A CN 114699556A
- Authority
- CN
- China
- Prior art keywords
- silk fibroin
- formic acid
- repair patch
- preparation
- acid solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010022355 Fibroins Proteins 0.000 title claims abstract description 200
- 230000008439 repair process Effects 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 124
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 62
- 235000019253 formic acid Nutrition 0.000 claims abstract description 62
- 239000000835 fiber Substances 0.000 claims abstract description 41
- 239000012528 membrane Substances 0.000 claims abstract description 41
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 27
- 238000001035 drying Methods 0.000 claims abstract description 26
- 238000010041 electrostatic spinning Methods 0.000 claims abstract description 21
- 238000002791 soaking Methods 0.000 claims abstract description 19
- 239000002994 raw material Substances 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 77
- 238000000502 dialysis Methods 0.000 claims description 37
- 238000001523 electrospinning Methods 0.000 claims description 28
- 239000006228 supernatant Substances 0.000 claims description 25
- 241000255789 Bombyx mori Species 0.000 claims description 23
- 238000004140 cleaning Methods 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 19
- 239000012265 solid product Substances 0.000 claims description 18
- 238000009987 spinning Methods 0.000 claims description 18
- AMXOYNBUYSYVKV-UHFFFAOYSA-M lithium bromide Chemical compound [Li+].[Br-] AMXOYNBUYSYVKV-UHFFFAOYSA-M 0.000 claims description 16
- 238000005119 centrifugation Methods 0.000 claims description 15
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 14
- 239000012498 ultrapure water Substances 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 12
- 238000004108 freeze drying Methods 0.000 claims description 7
- 230000001476 alcoholic effect Effects 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 238000009826 distribution Methods 0.000 abstract description 9
- 230000010261 cell growth Effects 0.000 abstract description 6
- 239000011148 porous material Substances 0.000 abstract description 6
- 238000004132 cross linking Methods 0.000 abstract 1
- 238000004925 denaturation Methods 0.000 abstract 1
- 230000036425 denaturation Effects 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 13
- 230000017423 tissue regeneration Effects 0.000 description 11
- 229910000029 sodium carbonate Inorganic materials 0.000 description 8
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Substances [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 8
- 238000000034 method Methods 0.000 description 7
- 239000000463 material Substances 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 238000009835 boiling Methods 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 238000010438 heat treatment Methods 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- 206010061218 Inflammation Diseases 0.000 description 3
- 239000007857 degradation product Substances 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 229920002994 synthetic fiber Polymers 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 108010013296 Sericins Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 230000005859 cell recognition Effects 0.000 description 1
- 239000011365 complex material Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000010981 drying operation Methods 0.000 description 1
- 210000001951 dura mater Anatomy 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000005661 hydrophobic surface Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 239000002121 nanofiber Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000002980 postoperative effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000037816 tissue injury Diseases 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000009827 uniform distribution Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- 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
-
- 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
-
- 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
- A61L27/58—Materials at least partially resorbable by the body
-
- D—TEXTILES; PAPER
- D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
- D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
- D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
- D04H1/70—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres
- D04H1/72—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged
- D04H1/728—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged by electro-spinning
-
- 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
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
Landscapes
- Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Dermatology (AREA)
- Medicinal Chemistry (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Transplantation (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Textile Engineering (AREA)
- Artificial Filaments (AREA)
- Nonwoven Fabrics (AREA)
Abstract
The invention discloses a preparation method of a silk fibroin repair patch and the silk fibroin repair patch prepared by the preparation method. The preparation method of the silk fibroin repair patch comprises the following steps: providing a formic acid solution of silk fibroin; performing electrostatic spinning by taking the formic acid solution of the silk fibroin as a raw material to obtain a silk fibroin fiber membrane; and soaking the silk fibroin fibrous membrane in a methanol solution, fully soaking the silk fibroin fibrous membrane until the silk fibroin fibrous membrane is subjected to crosslinking denaturation, taking out the silk fibroin fibrous membrane and drying to obtain the silk fibroin repairing patch. The data in the test example part of the specification show that the silk fibroin repairing patch prepared by the preparation method of the silk fibroin repairing patch has uniform fiber diameter and uniform fiber distribution, and pores exist among the fibers, so that the adhesion growth of cells can be met, and the cells can grow into the silk fibroin repairing patch.
Description
Technical Field
The invention relates to the technical field of tissue repair materials, in particular to a silk fibroin repair patch preparation method and a silk fibroin repair patch prepared by the silk fibroin repair patch preparation method.
Background
The regeneration and repair of human tissue injury is receiving more and more attention, and along with the progress of medical technology and the requirement of patients on high-quality life, the tissue repair patch is widely applied to surgical operations as a novel medical implantation instrument. The tissue repair patch not only can play a role in supporting the tissue mechanics, but also has good biocompatibility and a hole structure, can meet the requirements of cell adhesion, growth and proliferation, repairs defective tissues, accelerates postoperative recovery, and is widely recognized and accepted in the clinical field of tissue repair.
At present, two types of products of animal origin and artificial synthetic materials are mainly used as common tissue repair patches in clinic. However, the animal-derived tissue repair patch has the defects of limited material sources, complex material sources, high standard production difficulty, difficulty in controlling stability and degradation rate, and risks of viral infection, inflammatory reaction and the like. The tissue repair patch prepared from the artificial synthetic material has a hydrophobic surface, lacks cell recognition sites, and degradation products easily cause inflammatory reaction, so that the tissue repair patch is not favorable for adhesion and growth of cells, and the synthetic material has larger difference with a human tissue structure, so that the tissue repair patch is not favorable for repairing soft tissues.
The silk fibroin repair patch prepared by taking silk as a raw material has good human body affinity, pores exist among silk fibroin fibers, the adhesion growth of cells can be met, and the silk fibroin repair patch is a good tissue repair material,
however, the silk fibroin repairing patch with uniformly distributed fibers is difficult to prepare by the traditional preparation method of the silk fibroin repairing patch.
Disclosure of Invention
Therefore, the silk fibroin repair patch prepared by the method has uniform fiber distribution.
In addition, the silk fibroin repairing patch prepared by the preparation method for preparing the silk fibroin repairing patch is also necessary to be provided.
A preparation method of a silk fibroin repair patch comprises the following steps:
providing a formic acid solution of silk fibroin, wherein the mass concentration of the formic acid solution of silk fibroin is 6% -15%;
performing electrostatic spinning by using the formic acid solution of the silk fibroin as a raw material to obtain a silk fibroin fibrous membrane, wherein the electrostatic spinning voltage is 15V-30V, the spinning distance is 10 cm-30 cm, the spinning speed is 0.5 mL/h-2 mL/h, the rotating speed of a roller is 100 rpm-3000 rpm, the humidity in an electrospinning box is 35% -45%, and the temperature in the electrospinning box is 18-30 ℃; and
and soaking the silk fibroin fibrous membrane in an alcoholic solution, fully soaking the silk fibroin fibrous membrane until the silk fibroin fibrous membrane is crosslinked and denatured, taking out the silk fibroin fibrous membrane and drying the silk fibroin fibrous membrane to obtain the silk fibroin repairing patch.
In one embodiment, the formic acid solution of silk fibroin has a mass concentration of 10%;
the electrostatic spinning voltage is 24V, the spinning distance is 12cm, the spinning speed is 0.8mL/h, the rotating speed of the roller is 1000rpm, the humidity in the electrospinning box is 35%, and the temperature in the electrospinning box is 24 ℃.
In one embodiment, the mass concentration of the methanol solution is 60% to 100%, and the mass concentration of the ethanol solution is 60% to 100%.
In one embodiment, the formic acid solution of silk fibroin is prepared by:
extracting silk fibroin from silkworm cocoons;
and preparing the silk fibroin into a formic acid solution of the silk fibroin.
In one embodiment, the operation of extracting silk fibroin from silkworm cocoons is: and (3) taking the silkworm cocoons, and sequentially carrying out primary cleaning, degumming treatment, secondary cleaning and drying treatment to obtain the silk fibroin.
In one embodiment, the formulating the silk fibroin into the formic acid solution of the silk fibroin is performed by:
according to the solid-liquid ratio of 0.5-2: 4, dissolving the silk fibroin into CaCl with the mass concentration of 2-30 percent2And then naturally drying in the formic acid solution to obtain a dry film, cleaning and drying the dry film, and dissolving the dry film into formic acid to obtain the formic acid solution of the silk fibroin.
In one embodiment, the formulating the silk fibroin into the formic acid solution of the silk fibroin is performed by:
according to the solid-liquid ratio of 0.5-2: 4, dissolving the silk fibroin into a LiBr solution with the concentration of 8-10.6 mol/L to obtain a mixed solution, transferring the mixed solution into a dialysis bag with the molecular weight cutoff of 3500-100000, then placing the dialysis bag into ultrapure water for dialysis, centrifuging the liquid in the dialysis bag after the dialysis is finished, retaining the supernatant, drying the supernatant to obtain a solid product, and dissolving the solid product into formic acid to obtain the formic acid solution of the silk fibroin.
In one embodiment, in the operation of centrifuging the liquid in the dialysis bag, the centrifugation speed is 5000 rpm-10000 rpm, the centrifugation temperature is 0-8 ℃, and the centrifugation time is 10 min-30 min;
the operation of drying the supernatant is as follows: pre-cooling the supernatant at the temperature of-20 ℃ to 0 ℃, and then freeze-drying the supernatant for 12 to 36 hours at the temperature of-20 ℃ to-80 ℃.
The silk fibroin repairing patch is prepared by the preparation method of the silk fibroin repairing patch.
In one embodiment, the silk fibroin repair patch has a thickness of 0.5mm to 3 mm.
The data in the test example part of the specification show that the silk fibroin repairing patch prepared by the preparation method of the silk fibroin repairing patch has uniform fiber diameter and uniform fiber distribution, and pores exist among the fibers, so that the adhesion growth of cells can be met, and the cells can grow into the silk fibroin repairing patch.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Wherein:
fig. 1 is a flowchart of a method for preparing a silk fibroin repair patch according to an embodiment.
Fig. 2 is a photomicrograph of the silk fibroin repair patch obtained in example 1.
Fig. 3 is a photomicrograph of the silk fibroin repair patch obtained in example 1.
Fig. 4 is a photomicrograph of the silk fibroin repair patch obtained in comparative example 1.
Fig. 5 is a photomicrograph of the silk fibroin repair patch obtained in comparative example 2.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The preparation method of the silk fibroin repair patch shown in fig. 1 comprises the following steps:
s10, providing a formic acid solution of silk fibroin, wherein the mass concentration of the formic acid solution of silk fibroin is 6% -15%.
In the embodiment, formic acid is used as a solvent of the silk fibroin, and the formic acid is a volatile substance, so that the finally prepared silk fibroin repairing patch is ensured to contain no toxic substances.
In the invention, the quality concentration control of the formic acid solution of the silk fibroin is very important, when the concentration is too low, microspheres and beads are easy to appear, the performance of the silk fibroin repair patch is influenced, and when the concentration is too high, a needle head is easy to block, and the silk fibroin repair patch cannot be electrospun.
More preferably, the concentration of the formic acid solution of silk fibroin is 10% by mass.
Preferably, in this embodiment, the formic acid solution of silk fibroin is prepared by: extracting silk fibroin from silkworm cocoons; the silk fibroin is prepared into formic acid solution of the silk fibroin.
Specifically, the operation of extracting silk fibroin from silkworm cocoons may be: taking silkworm cocoons, and sequentially carrying out primary cleaning, degumming treatment, secondary cleaning and drying treatment to obtain silk fibroin.
Further, the operation of the primary cleaning and degumming treatment may be: weighing silkworm cocoon, cutting into pieces, cleaning with ultrapure water for three times, removing impurities, and preparing 0.02M Na2CO3Heating the solution to boil, adding the silk cocoon pieces to the boiled Na2CO3Boiling the solution for 30min, and repeating for 3 times.
Further, the secondary cleaning is as follows: soaking silkworm cocoon in ultrapure water of 60 deg.C, stirring, cleaning for 30min, and repeating for 3 times to remove sericin.
Further, the drying operation may be: placing the silk fibroin in an oven at 40 ℃ overnight, and drying to obtain the silk fibroin.
In other embodiments, finished silk fibroin can also be purchased directly.
Specifically, the preparation of the silk fibroin into the formic acid solution of the silk fibroin is performed as follows: according to the solid-liquid ratio of 0.5-2: 4 (preferably 1: 4), dissolving the silk fibroin into CaCl with the mass concentration of 2-30% (preferably 5%) (2Then naturally drying in the formic acid solution to obtain a dry film, cleaning the dry film, drying, dissolving the dry film in formic acid,obtaining the formic acid solution of the silk fibroin.
It should be noted that before dissolving the dry film into formic acid, the dry film needs to be cut into pieces, so as to ensure the dry film to be fully dissolved, and avoid the phenomenon that the needle is blocked in the subsequent electrospinning operation because the dry film is not completely dissolved to form flocculent silk fibroin.
Specifically, the operation of formulating silk fibroin into a formic acid solution of silk fibroin can also be: according to the solid-liquid ratio of 0.5-2: 4 (preferably 1: 4), dissolving silk fibroin into a LiBr solution with the concentration of 8-10.6 mol/L (preferably 9.3mol/L) to obtain a mixed solution, transferring the mixed solution into a dialysis bag with the molecular weight cutoff of 3500-100000 (preferably 8000), then putting the dialysis bag into ultrapure water for dialysis, centrifuging the liquid in the dialysis bag after the dialysis is finished, retaining the supernatant, drying the supernatant to obtain a solid product, and dissolving the solid product into formic acid to obtain a formic acid solution of the silk fibroin.
In the invention, LiBr is removed by dialysis, the LiBr removal effect is better, in addition, dialysis bags with different specifications are selected, the molecular weight of silk fibroin in a product is controlled according to the interception amount of the dialysis bags, and the performance of the silk fibroin fibrous membrane can be better regulated and controlled (for example, the silk fibroin fibrous membrane is less prone to degradation when the molecular weight is higher).
It should be noted that before the solid product is dissolved in the formic acid, the solid product needs to be sheared to ensure that the solid product is fully dissolved as much as possible, so as to avoid the phenomenon that the needle is blocked in the subsequent electrospinning operation because flocculent silk fibroin is formed due to incomplete dissolution of the solid product.
In one embodiment, the liquid in the dialysis bag is centrifuged at 5000-10000 rpm, 0-8 deg.C, and 10-30 min.
Preferably, the operation of drying the supernatant is: precooling the supernatant at-20-0 ℃, and then freeze-drying for 12-36 h at-20-80 ℃.
In the invention, the supernatant is freeze-dried, so that on one hand, the supernatant can be fully dried, and on the other hand, the solid product obtained after freeze-drying is more easily dissolved in formic acid, thereby avoiding the phenomenon that the needle head is blocked in the subsequent electrospinning operation because the solid product is incompletely dissolved to form flocculent silk fibroin.
S20, performing electrostatic spinning by taking a formic acid solution of silk fibroin as a raw material to obtain the silk fibroin fiber membrane.
Specifically, the voltage of electrostatic spinning is 15V-30V, the spinning distance is 10 cm-30 cm, the spinning speed is 0.5 mL/h-2 mL/h, the rotating speed of a roller is 100 rpm-3000 rpm, the humidity in an electrospinning box is 35% -45%, and the temperature in the electrospinning box is 18-30 ℃.
In the electrostatic spinning operation, the control of humidity and temperature is crucial, and specifically, the humidity and temperature affect the volatilization speed of formic acid, thereby further affecting the fiber diameter size and fiber distribution inside the silk fibroin repair patch finally obtained.
In the invention, the humidity in the electrospinning box is controlled to be 35-45% and the temperature in the electrospinning box is controlled to be 18-30 ℃ in the electrostatic spinning process, so that the uniform diameter and the uniform distribution of fibers in the finally obtained silk fibroin repairing patch are ensured.
It should be noted that the selection of the drum rotation speed is an important link in the electrospinning process, and if the rotation speed is too high, the fiber distribution tends to be isotropic, and the difference of the mechanical properties is large.
In the invention, the silk fibroin fibrous membrane with the same mechanical properties in each direction is obtained by selecting a proper rotating speed.
Particularly preferably, the electrospinning voltage is 24V, the spinning distance is 12cm, the spinning speed is 0.8mL/h, the drum rotation speed is 1000rpm, the humidity in the electrospinning chamber is 35%, and the temperature in the electrospinning chamber is 24 ℃.
S30, soaking the silk fibroin fibrous membrane in an alcohol solution, fully soaking the silk fibroin fibrous membrane until the silk fibroin fibrous membrane is crosslinked and denatured, taking out the silk fibroin fibrous membrane and drying the silk fibroin fibrous membrane to obtain the silk fibroin repairing patch.
In the present embodiment, the alcohol solution may be a methanol solution or an ethanol solution.
Generally, the mass concentration of the methanol solution is 60% to 100% (preferably 100%), and the mass concentration of the ethanol solution is 60% to 100%.
The soaking time can be set according to the thickness of the actual silk fibroin fiber membrane. Generally, the soaking time is 6min to 20min (preferably 10 min).
The data in the test example part of the specification show that the silk fibroin repairing patch prepared by the preparation method of the silk fibroin repairing patch has uniform fiber diameter and uniform fiber distribution, and pores exist among the fibers, so that the adhesion growth of cells can be met, and the cells can grow into the silk fibroin repairing patch.
The preparation method of the silk fibroin repair patch has the following advantages: 1) by electrostatic spinning of pure fibroin, the raw material source is single, the material is easy to obtain, the operation is simple, and the production cost is low; 2) the low-toxicity solvent is used for electrospinning, has good biocompatibility, is non-toxic and has no environmental pollution; 3) the silk fibroin repair patch is degradable, the sizes of pores on the surface of the membrane and the fiber diameter can be realized according to the adjustment of electrostatic spinning parameters, the adjustability of the degradation of the silk fibroin membrane is further realized, different silk fibroin molecular weights can be selected through dialysis, the adjustability of the degradation of the silk fibroin membrane is realized, and pure fibroin and degradation products thereof are easier to be absorbed by surrounding tissues compared with degradation products of polymers, are not easy to generate inflammatory reaction, and can be used as an absorbable tissue repair patch; 4) the process conditions are as follows: the temperature and the humidity regulate and control the fiber forming performance in the electrostatic spinning process, so that the nanofiber membrane with uniform fiber distribution is obtained and the mechanical properties of all directions are the same.
The invention also discloses the silk fibroin repair patch of an embodiment, which is prepared by the preparation method of the silk fibroin repair patch.
Preferably, the thickness of the silk fibroin repairing patch is 0.5 mm-3 mm.
The silk fibroin repairing patch of the invention can be used as an excellent tissue repairing material for breast reconstruction, artificial skin, dura mater, oral cavity repairing membrane and the like.
The following are specific examples.
Example 1
Weighing Bombyx mori cocoon (Chuan silkworm No. 26), cutting into pieces, washing with ultrapure water for three times, removing impurities, and preparing 0.02M Na2CO3Heating the solution to boil, adding the silk cocoon pieces to the boiled Na2CO3Boiling the solution for 30min, and repeating for 3 times. And then soaking the silkworm cocoons in ultrapure water at 60 ℃, stirring and cleaning, cleaning for 30min, repeating for 3 times, finally placing the silkworm cocoons in an oven at 40 ℃ overnight, and drying to obtain the silk fibroin.
According to the solid-liquid ratio of 1: 4, dissolving silk fibroin into a LiBr solution with the concentration of 9.3mol/L to obtain a mixed solution, transferring the mixed solution into a dialysis bag with the molecular weight cutoff of 8000, then putting the dialysis bag into ultrapure water for dialysis, centrifuging liquid in the dialysis bag after the dialysis is finished, retaining supernatant, precooling the supernatant at-20 ℃, freeze-drying the supernatant for 24 hours at-40 ℃ to obtain a solid product, and dissolving the solid product into formic acid to obtain a formic acid solution of the silk fibroin. Wherein the centrifugation speed is 9000rpm, the centrifugation temperature is 4 ℃, the centrifugation time is 20min, and the mass concentration of the formic acid solution of the silk fibroin is 10%.
And (3) performing electrostatic spinning by taking a formic acid solution of silk fibroin as a raw material to obtain the silk fibroin fiber membrane with the thickness of 1 mm. Wherein the electrostatic spinning voltage is 24V, the spinning distance is 12cm, the spinning speed is 0.8mL/h, the roller rotating speed is 1000rpm, the humidity in the electrospinning box is 35%, and the temperature in the electrospinning box is 24 ℃.
And soaking the silk fibroin fibrous membrane in methanol for 10min until the silk fibroin fibrous membrane is crosslinked and denatured, taking out and drying to obtain the silk fibroin repairing patch.
Example 2
Weighing Bombyx mori cocoon (Chuan silkworm No. 26), cutting into pieces, washing with ultrapure water for three times, removing impurities, and preparing 0.02M Na2CO3Heating the solution to boil, adding the silk cocoon pieces to the boiled Na2CO3Boiling the solution for 30min, and repeating for 3 times. Then soaking silkworm cocoon in ultra-pure water at 60 deg.CStirring and cleaning in water, cleaning for 30min, repeating for 3 times, finally placing the silkworm cocoon in an oven at 40 ℃ overnight, and drying to obtain the silk fibroin.
According to the solid-liquid ratio of 1: 4, dissolving silk fibroin into a LiBr solution with the concentration of 9.3mol/L to obtain a mixed solution, transferring the mixed solution into a dialysis bag with the molecular weight cutoff of 8000, then putting the dialysis bag into ultrapure water for dialysis, centrifuging liquid in the dialysis bag after the dialysis is finished, retaining supernatant, precooling the supernatant at-20 ℃, freeze-drying the supernatant for 24 hours at-40 ℃ to obtain a solid product, and dissolving the solid product into formic acid to obtain a formic acid solution of the silk fibroin. Wherein the centrifugation speed is 9000rpm, the centrifugation temperature is 4 ℃, the centrifugation time is 20min, and the mass concentration of the formic acid solution of the silk fibroin is 6%.
And (3) performing electrostatic spinning by taking a formic acid solution of silk fibroin as a raw material to obtain the silk fibroin fiber membrane with the thickness of 1 mm. Wherein the electrostatic spinning voltage is 24V, the spinning distance is 12cm, the spinning speed is 0.8mL/h, the roller rotating speed is 1000rpm, the humidity in the electrospinning box is 40%, and the temperature in the electrospinning box is 22 ℃.
And soaking the silk fibroin fibrous membrane in a methanol solution with the mass concentration of 80%, soaking for 10min until the silk fibroin fibrous membrane is crosslinked and denatured, taking out and drying to obtain the silk fibroin repairing patch.
Example 3
Weighing Bombyx mori cocoon (Chuan silkworm No. 26), cutting into pieces, washing with ultrapure water for three times, removing impurities, and preparing 0.02M Na2CO3Heating the solution to boil, adding the silk cocoon pieces to the boiled Na2CO3Boiling the solution for 30min, and repeating for 3 times. And then soaking the silkworm cocoons in ultrapure water at 60 ℃, stirring and cleaning, cleaning for 30min, repeating for 3 times, finally placing the silkworm cocoons in an oven at 40 ℃ overnight, and drying to obtain the silk fibroin.
According to the solid-liquid ratio of 1: 4, dissolving silk fibroin into a LiBr solution with the concentration of 9.3mol/L to obtain a mixed solution, transferring the mixed solution into a dialysis bag with the molecular weight cutoff of 8000, then putting the dialysis bag into ultrapure water for dialysis, centrifuging liquid in the dialysis bag after the dialysis is finished, retaining supernatant, precooling the supernatant at-20 ℃, freeze-drying the supernatant for 24 hours at-40 ℃ to obtain a solid product, and dissolving the solid product into formic acid to obtain a formic acid solution of the silk fibroin. Wherein the centrifugation speed is 9000rpm, the centrifugation temperature is 4 ℃, the centrifugation time is 20min, and the mass concentration of the formic acid solution of the silk fibroin is 15%.
And (3) performing electrostatic spinning by taking a formic acid solution of silk fibroin as a raw material to obtain the silk fibroin fiber membrane with the thickness of 1 mm. Wherein the electrostatic spinning voltage is 24V, the spinning distance is 12cm, the spinning speed is 0.8mL/h, the roller rotating speed is 1000rpm, the humidity in the electrospinning box is 45%, and the temperature in the electrospinning box is 20 ℃.
And soaking the silk fibroin fibrous membrane in a methanol solution with the mass concentration of 60%, soaking for 10min until the silk fibroin fibrous membrane is crosslinked and denatured, taking out and drying to obtain the silk fibroin repairing patch.
Comparative example 1
Comparative example 1 is substantially identical to example 1 except that in comparative example 1, the formic acid solution of silk fibroin as an electrospinning raw material has a mass concentration of 4%.
Comparative example 2
Comparative example 2 is substantially identical to example 1 except that in comparative example 2, humidity control is not performed as the electrospinning process.
Test example
Morphological observation
The silk fibroin repair patches obtained in example 1, comparative example 1, and comparative example 2 were subjected to microscopic observation, respectively, and fig. 2, fig. 3, fig. 4, and fig. 5.
As can be seen from fig. 2 and fig. 3, the silk fibroin repair patch obtained in example 1 has a nano silk fibroin fiber structure, the fiber diameter size distribution is uniform, the fiber distribution is uniform, pores exist among the fibers, the cell adhesion growth can be met, and the cells can grow and extend into the patch.
As can be seen from the combination of FIG. 4, the silk fibroin repair patch obtained in the comparative example 1 has a nanometer silk fibroin fiber structure, the fiber diameters are different, and microspheres and beads appear on the fibers, so that the performance of the silk fibroin repair patch is influenced.
As can be seen from fig. 5, the silk fibroin repair patch obtained in comparative example 2 has a nano silk fibroin fiber structure, and a small amount of liquid drops also appear on the fiber.
In addition, in comparative example 2, when the silk fibroin repair patch is prepared, the viscosity of the silk fibroin fiber film obtained after electrostatic spinning is high due to the slow volatilization speed of formic acid, and the silk fibroin fiber film is not easy to be uncovered from a roller receiving device.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the claims. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Claims (10)
1. A preparation method of the silk fibroin repairing patch is characterized by comprising the following steps:
providing a formic acid solution of silk fibroin, wherein the mass concentration of the formic acid solution of silk fibroin is 6% -15%;
performing electrostatic spinning by using the formic acid solution of the silk fibroin as a raw material to obtain a silk fibroin fibrous membrane, wherein the electrostatic spinning voltage is 15V-30V, the spinning distance is 10 cm-30 cm, the spinning speed is 0.5 mL/h-2 mL/h, the rotating speed of a roller is 100 rpm-3000 rpm, the humidity in an electrospinning box is 35% -45%, and the temperature in the electrospinning box is 18-30 ℃; and
and soaking the silk fibroin fiber membrane in an alcoholic solution, fully soaking the silk fibroin fiber membrane until the silk fibroin fiber membrane is crosslinked and denatured, taking out the silk fibroin fiber membrane and drying the silk fibroin fiber membrane to obtain the silk fibroin repairing patch, wherein the alcoholic solution is a methanol solution or an ethanol solution.
2. The preparation method of the silk fibroin repair patch according to claim 1, wherein the mass concentration of the formic acid solution of the silk fibroin is 10%;
the electrostatic spinning voltage is 24V, the spinning distance is 12cm, the spinning speed is 0.8mL/h, the drum rotation speed is 1000rpm, the humidity in the electrospinning box is 35%, and the temperature in the electrospinning box is 24 ℃.
3. The preparation method of the silk fibroin repair patch according to claim 1, wherein the mass concentration of the methanol solution is 60% -100%, and the mass concentration of the ethanol solution is 60% -100%.
4. The preparation method of the silk fibroin repair patch according to any one of claims 1-3, wherein the formic acid solution of silk fibroin is prepared by:
extracting silk fibroin from silkworm cocoons;
and preparing the silk fibroin into a formic acid solution of the silk fibroin.
5. The preparation method of the silk fibroin repair patch according to claim 4, wherein the operation of extracting silk fibroin from silkworm cocoons is: and (3) taking the silkworm cocoons, and sequentially carrying out primary cleaning, degumming treatment, secondary cleaning and drying treatment to obtain the silk fibroin.
6. The preparation method of the silk fibroin repair patch of claim 4, wherein the formulation of the silk fibroin into the formic acid solution of the silk fibroin is performed by:
according to the solid-liquid ratio of 0.5-2: 4, dissolving the silk fibroin into CaCl with the mass concentration of 2-30 percent2And then naturally drying in the formic acid solution to obtain a dry film, cleaning and drying the dry film, and dissolving the dry film into formic acid to obtain the formic acid solution of the silk fibroin.
7. The preparation method of the silk fibroin repair patch according to claim 4, wherein the formulating of the silk fibroin into the formic acid solution of the silk fibroin is:
according to the solid-liquid ratio of 0.5-2: 4, dissolving the silk fibroin into a LiBr solution with the concentration of 8-10.6 mol/L to obtain a mixed solution, transferring the mixed solution into a dialysis bag with the molecular weight cutoff of 3500-100000, then placing the dialysis bag into ultrapure water for dialysis, centrifuging the liquid in the dialysis bag after the dialysis is finished, retaining the supernatant, drying the supernatant to obtain a solid product, and dissolving the solid product into formic acid to obtain the formic acid solution of the silk fibroin.
8. The preparation method of the silk fibroin repair patch according to claim 7, wherein in the operation of centrifuging the liquid in the dialysis bag, the centrifugation speed is 5000-10000 rpm, the centrifugation temperature is 0-8 ℃, and the centrifugation time is 10-30 min;
the operation of drying the supernatant is as follows: pre-cooling the supernatant at the temperature of-20 ℃ to 0 ℃, and then freeze-drying the supernatant for 12 to 36 hours at the temperature of-20 ℃ to-80 ℃.
9. The silk fibroin repair patch is characterized by being prepared by the preparation method of the silk fibroin repair patch as claimed in any one of claims 1-8.
10. The silk fibroin repair patch of claim 9, wherein the silk fibroin repair patch has a thickness of 0.5mm to 3 mm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210366624.XA CN114699556A (en) | 2022-04-08 | 2022-04-08 | Preparation method of silk fibroin repair patch and silk fibroin repair patch |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210366624.XA CN114699556A (en) | 2022-04-08 | 2022-04-08 | Preparation method of silk fibroin repair patch and silk fibroin repair patch |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114699556A true CN114699556A (en) | 2022-07-05 |
Family
ID=82173807
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210366624.XA Pending CN114699556A (en) | 2022-04-08 | 2022-04-08 | Preparation method of silk fibroin repair patch and silk fibroin repair patch |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114699556A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115463257A (en) * | 2022-09-13 | 2022-12-13 | 娜斯嘉生物科技(嘉兴)有限公司 | Composite enhanced sclera patch and preparation method thereof |
| CN116392636A (en) * | 2023-03-29 | 2023-07-07 | 深圳高性能医疗器械国家研究院有限公司 | Silk repairing patch and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103341209A (en) * | 2013-07-08 | 2013-10-09 | 苏州大学 | Silk fibroin nanofiber membrane and preparation method thereof |
| CN110453378A (en) * | 2019-07-03 | 2019-11-15 | 上海大学 | A kind of sulfonic acid based quantum dot/fibroin albumen composite nano-fiber membrane and its preparation method and application |
| CN110903374A (en) * | 2019-12-20 | 2020-03-24 | 重庆医科大学 | Silk fibroin extraction method, composite skin scaffold based on silk fibroin/usnic acid and preparation method of composite skin scaffold |
| JP2022001669A (en) * | 2018-07-19 | 2022-01-06 | Ｓｐｉｂｅｒ株式会社 | Method of manufacturing protein fiber |
| CN114249982A (en) * | 2022-01-25 | 2022-03-29 | 武汉纺织大学 | Preparation method and application of high-strength high-modulus silk material |
-
2022
- 2022-04-08 CN CN202210366624.XA patent/CN114699556A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103341209A (en) * | 2013-07-08 | 2013-10-09 | 苏州大学 | Silk fibroin nanofiber membrane and preparation method thereof |
| JP2022001669A (en) * | 2018-07-19 | 2022-01-06 | Ｓｐｉｂｅｒ株式会社 | Method of manufacturing protein fiber |
| CN110453378A (en) * | 2019-07-03 | 2019-11-15 | 上海大学 | A kind of sulfonic acid based quantum dot/fibroin albumen composite nano-fiber membrane and its preparation method and application |
| CN110903374A (en) * | 2019-12-20 | 2020-03-24 | 重庆医科大学 | Silk fibroin extraction method, composite skin scaffold based on silk fibroin/usnic acid and preparation method of composite skin scaffold |
| CN114249982A (en) * | 2022-01-25 | 2022-03-29 | 武汉纺织大学 | Preparation method and application of high-strength high-modulus silk material |
Non-Patent Citations (1)
| Title |
|---|
| 杨鸣波等, 中国轻工业出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115463257A (en) * | 2022-09-13 | 2022-12-13 | 娜斯嘉生物科技(嘉兴)有限公司 | Composite enhanced sclera patch and preparation method thereof |
| CN115463257B (en) * | 2022-09-13 | 2023-12-29 | 娜斯嘉生物科技(嘉兴)有限公司 | Composite reinforced scleral patch and preparation method thereof |
| CN116392636A (en) * | 2023-03-29 | 2023-07-07 | 深圳高性能医疗器械国家研究院有限公司 | Silk repairing patch and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106075598B (en) | Photo-crosslinked sericin hydrogel and preparation method and application thereof | |
| CN114699556A (en) | Preparation method of silk fibroin repair patch and silk fibroin repair patch | |
| CN103341209B (en) | Silk fibroin nanofiber membrane and preparation method thereof | |
| US20060095137A1 (en) | Nanofibrous nonwoven membrane of silk fibroin for guided bone tissue regeneration and manufacturing method thereof | |
| CN109893680B (en) | Repair fiber membrane, repair sleeve pipe and preparation method and application thereof | |
| EP1436346A1 (en) | Method for the preparation of silk fibroin hydrogels | |
| CN106310380A (en) | Nano-fibrosis silk fibroin gel and preparation method thereof | |
| Li et al. | A native sericin wound dressing spun directly from silkworms enhances wound healing | |
| CN108815578B (en) | Artificial biological endocranium and preparation method thereof | |
| CN113046917B (en) | Electrostatic spinning collagen membrane and preparation method thereof | |
| CN106267336B (en) | A kind of bone renovating material and preparation method thereof | |
| Sah et al. | Preparation, characterization and in vitro study of biocompatible fibroin hydrogel | |
| CN111235662A (en) | Collagen long fiber with natural structure and preparation method and application thereof | |
| CN110615913B (en) | Silk protein porous sponge and preparation method thereof | |
| CN114539564A (en) | Preparation method of rapid-gelling silk fibroin-based strong hydrogel | |
| CN115679695B (en) | Hybrid fiber for promoting tissue regeneration, method for producing the same, and fiber structure using the same | |
| Vojoudi et al. | A novel inexpensive method for preparation of silk nanofibers from cocoons | |
| CN117982737A (en) | Silk protein anti-adhesion repair film and preparation method and application thereof | |
| CN115121195B (en) | alginate-GelMA fiber microsphere, preparation method and application of alginate-GelMA fiber microsphere as cell carrier | |
| CN117180506B (en) | Bracket for treating infectious bone defect and preparation method and application thereof | |
| CN116392636A (en) | Silk repairing patch and preparation method thereof | |
| CN114949348B (en) | alginate-hDPSCs-loaded GelMA fiber microsphere and preparation method thereof | |
| CN117942426B (en) | Multi-stage porous material with oriented pore structure, and preparation method and application thereof | |
| CN116510078A (en) | Jellyfish collagen solution and nanofiber membrane for in-situ skin repair | |
| CN115197450A (en) | Photo-crosslinking chitin whisker composite liquid crystal gel material and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220705 |