KR101655333B1 - Surface-modified Silk Fibroin Implant for Biological Film and Preparation Method Thereof - Google Patents
Surface-modified Silk Fibroin Implant for Biological Film and Preparation Method Thereof Download PDFInfo
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- KR101655333B1 KR101655333B1 KR1020150036953A KR20150036953A KR101655333B1 KR 101655333 B1 KR101655333 B1 KR 101655333B1 KR 1020150036953 A KR1020150036953 A KR 1020150036953A KR 20150036953 A KR20150036953 A KR 20150036953A KR 101655333 B1 KR101655333 B1 KR 101655333B1
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
- C08J7/12—Chemical modification
- C08J7/123—Treatment by wave energy or particle radiation
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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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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
- C08J5/18—Manufacture of films or sheets
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2389/00—Characterised by the use of proteins; Derivatives thereof
Abstract
Description
The present invention relates to a surface-modified biocompatible silk fibroin film and a process for producing the same, and more particularly, to a process for producing a film by using silk fibroin When the tissue engineering biofilm corneal endothelial layer suitable for living body transplantation is prepared and applied by increasing the cell adhesion and proliferation efficiency, the prepared tissue biomaterial corneal endothelial layer maintains transparency and the cultured cells are single The corneal endothelial cells are required to be transplanted into the corneal endothelium, and the corneal endothelial cells necessary for transplantation are sufficiently cultured to exhibit excellent functions as corneal endothelial cells. Therefore, in the future, Lt; RTI ID = 0.0 > biocompatible < / RTI > silk fibroin film.
Tissue engineering has been suggested as an ideal way to overcome limitations due to the lack of tissue or organ that can adequately combine cells, factors, and environments to replace or replace damaged and destroyed tissues. Tissue engineering is to attach specific cells separated and cultured from a patient to an implant made of a biocompatible and biodegradable material and to organize them by biochemical stimulation using a bioactive factor or physical stimulation using a bioreactor.
Technologically manufactured artificial organs using this method are similar to the biomedical tissue of our body, and thus there are many possibilities that autogenous tissue is a substitute for plants. As described above, tissue engineering is defined by three factors, cell, stimulant, and support. In the present invention, the present invention focuses on improving the surface properties of a conventional support and the relationship between the support and cells having an improved surface , And especially the biological characteristics of the cells cultured on the support with improved surface properties were greatly increased. Since silk has been used as a suture for many centuries, it can be a good biomaterial in tissue engineering applications. Silk derived from Bombyx mori (B.mori) is composed of two proteins, fibroin and sericin .
At this time, the fibroin is a protein which occupies 75% of silk cocoon and is composed of glycine, alanine, serine, etc. up to about 90% of insoluble protein. Fibroin also does not cause an immune response during in vivo transplantation, can control the rate of degradation, has excellent mechanical strength and good permeability to oxygen and water.
In addition, silk fibroin supports the adhesion and growth of human limbal stem cells and fibroblasts, and is used in a variety of applications including incising agents, enzyme immobilization membranes, cell culture media, artificial skin, and soft contact lenses , Two-dimensional silk fibroin film has been reported to provide good biocompatibility by many researchers.
In addition, sericin constitutes the remaining 25% of silk, but it is used after refining to remove sericin, as it causes immune and allergic reactions.
The purpose of surface treatment through plasma is to increase the low surface energy of various materials such as plastic, metal, glass, paper and so on to activate the surface and increase the hydrophilicity and adhesion. In addition, since surface treatment occurs only on the surface, it enhances the surface's ability to apply cells without affecting the original properties of the various materials applied.
The corneal endothelial cells do not proliferate in the body, they stop at the G1 phase of the cell cycle, and the function of the corneal endothelial cells gradually decreases with age. Loss of corneal endothelial cells varies from external causes such as trauma, infection, and contact lens use to genetic internal causes. However, the proliferation of the corneal endothelial cells in the body is limited by the ability to recover from damage and loss of cells, leading to blindness. Therefore, transplantation has been regarded as a therapeutic method in order to restore damaged cells and is recognized as the sole treatment until now. However, there are limitations in the number of corneas donated globally that can not adequately meet the number of patients requiring transplant surgery. Therefore, the necessity of artificial cornea manufactured by tissue engineering is urgently required.
On the other hand, the above-mentioned silk fibroin is used for tissue engineering by dissolving purified silk fibroin using an organic solvent after lyophilization and then casting the film, and the surface of the silk fibroin film thus produced is hydrophobic, Has limitations on cell adhesion and proliferation.
In the conventional silk fibroin solution, the powder obtained by freeze-drying the purified silk fibroin was dissolved in an organic solvent. When the cells were inoculated on the silk fibroin film produced using the powder, the cell adhesion rate and the growth rate were low. However, corneal endothelial cells do not proliferate after birth, and when they are severely damaged, they cause visual loss and visual loss. Therefore, it is imperative to obtain a sufficient number of donated corneas for transplantation, Do.
In addition, even if cells are obtained from a patient for a transplant operation, since the number of cells is very small, it is necessary to provide an efficient environment in order to secure sufficient cell numbers for transplantation.
It should be understood that the foregoing description of the background art is merely for the purpose of promoting an understanding of the background of the present invention and is not to be construed as adhering to the prior art already known to those skilled in the art.
Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to improve the low cell adhesion rate and proliferation rate of a silk fibroin which has been used as a bio-
More specifically, the present invention relates to a method for producing a silk fibroin film, which has a limited ability to adhere and propagate cells of a conventional organic solvent-derived silk fibroin film by improving the surface characteristics, thereby producing an efficient surface, The tissue engineering bioprosthetic corneal endothelial layer, which exhibits optimal functional manifestation, has been shown to have no significant effect on transparency and transmittance as a corneal transplantation application, as well as being selected from the group consisting of corneal epithelium, retina, The present invention provides a tissue-engineered biocompatible material excellent in application and transplantation of tissue cells to be treated.
As described above, the present invention is a conventional technique for improving the disadvantages of silk fibroin, which can improve the surface characteristics of existing silk fibroin film to sufficiently satisfy the number of deficient cells, and ultimately, As a solution to the problem.
Accordingly, an object of the present invention is to improve the cell adhesion rate and the growth rate after transplantation by preparing silk fibroin, which is a conventional biocompatible material, as a film and modifying the surface thereof.
Another object of the present invention is to provide a novel silk fibroin film whose surface properties of the silk fibroin film have been surface-modified through plasma treatment.
It is a further object of the present invention to provide a tissue engineered bioartificial corneal endothelial layer to replace a deficient donor cornea using the surface modified silk fibroin film.
It is still another object of the present invention to provide a surface-modified silk fibroin film as described above and a method of manufacturing an artificial corneal endothelial layer therefrom.
In order to solve the above problems, the present invention provides a surface-modified biocompatible silk fibroin film having a surface of a film made of silk fibroin treated with plasma and having a transparent property.
The present invention also relates to a process for purifying silk fibroin from silkworm or silk fiber; Preparing a silk fibroin film with purified silk fibroin; And subjecting the surface of the silk fibroin film to plasma treatment. The present invention also provides a method for producing a biocompatible silk fibroin film.
In addition, the present invention provides a tissue engineering biofilm endothelial layer produced by culturing corneal endothelial cells on a plasma-modified biocompatible silk fibroin film.
The present invention also relates to a method for producing a silk fibroin film, comprising the steps of: preparing a silk fibroin film from silk fibroin purified from cocoon or silk fiber; Subjecting the surface of the silk fibroin film to plasma treatment; Isolating and culturing corneal endothelial cells; Treating the surface of the surface treated silk fibroin film with a corneal endothelial cell isolated and cultured to produce a tissue engineering bio-corneal endothelial layer.
Features and advantages of the biocompatible silk fibroin film according to the present invention and the tissue engineering artificial corneal endothelial cell layer using the same are summarized as follows:
(i) The present invention can artificially provide a tissue-engineered bioartificial corneal endothelial layer for transplantation which is insufficient in transplantation conditions using biomaterials and corneal endothelial cells.
(Ii) In addition, the base membrane of the manufactured tissue engineering bio-artificial corneal endothelial layer according to the present invention is a plasma-treated silk fibroin film having excellent biocompatibility and can significantly improve cell adhesion rate and proliferation rate.
(Iii) The silt fibroin film having improved surface properties according to the plasma treatment according to the present invention is used when there is a need to rapidly proliferate a shortage of the number of deficient cells in a short time, and when a variety of tissue implants It has an effect that can be used for surgery.
(Iv) In addition, the plasma treatment according to the present invention has an improved surface
The tissue engineering bioartificial corneal endothelial layer produced by applying corneal endothelial cells to silk fibroin film has the effect of suggesting the possibility of replacing the deficient donor cornea.
FIG. 1 is a schematic diagram of a functional group generated when a plasma is treated on a surface of a silk fibroin according to a preferred embodiment of the present invention.
FIGS. 2A and 2B show the results of confirming the dry state, the wet state, and the transparency after cell seeding of the silk fibroin film surface-treated with the plasma produced according to the present invention.
FIGS. 3A and 3B are AFM results of the plasma treated silk fibroin film produced according to the present invention. FIG.
4 shows the results of measuring the thickness of the treated silk fibroin film according to the presence or absence of the plasma treatment produced according to the present invention and the conditions.
FIG. 5 is a result of measuring the contact angle to confirm the hydrophilicity of the surface-modified biocompatible silk fibroin film surface according to the present invention.
6A and 6B are graphs comparing the initial adhesion rate (30 minutes after sowing) and the growth rate (culturing for 5 days) of cells seeded on the surface-modified biocompatible silk fibroin film according to the present invention.
FIGS. 7A and 7B show the results of confirming the expression of mRNAs of cultured cells after seeding the surface-modified biocompatible silk fibroin film according to the present invention.
FIGS. 8A, 8B, and 8C are the results of observing functional expression of cells cultured on the surface-modified biocompatible silk fibroin film according to the present invention through immunofluorescence staining.
Hereinafter, the present invention will be described in more detail as an embodiment.
According to a preferred embodiment of the present invention, a silk fibroin solution is made into a film and the plasma is treated so that the surface property of the silk fibroin film is more hydrophilic than that of the existing silk fibroin film surface, Film, in which a corneal endothelial cell is cultured to provide a bioartificial corneal endothelial layer that forms a monolayer.
The composite grafts prepared using conventional silk fibroin have been produced by casting method and have a low cell attachment rate and proliferation rate when seeding the cells because the surface characteristics are hydrophobic. Because human corneal endothelial cells have limited proliferative capacity in the body, external donors are needed for patients requiring recovery. In contrast, the present invention aims to develop a tissue engineering biofilm endothelial layer which improves the hydrophobic characteristics of the surface of a conventional silk fibroin film and effectively induces cell proliferation.
As a result, according to the present invention, when producing a silk fibroin solution, the silk fibroin film can be overcome the characteristic limit of the surface of the silk fibroin film by processing the silk fibroin film after preparing the film by preparing the silkworm cocoons or the silk fiber.
In addition, according to the present invention, it was confirmed that cell characteristics were maintained by seeding cells on the surface of the surface-modified biocompatible silk fibroin film, and it was confirmed that the physical properties were not changed by the plasma treatment.
Therefore, plasma-treated silk fibroin film according to the present invention can serve as a good basement membrane for the tissue engineering bio-corneal endothelial layer production, which can solve the conventional lack of donor cornea.
Particularly, according to the present invention, a new proposal of improving the surface characteristics of the silk fibroin film by treating the plasma can be used to cultivate cells on a support having improved surface characteristics to obtain a sufficient number of cells for transplantation, It is possible to produce a tissue-engineered artificial cornea that can be substituted.
According to a preferred embodiment of the present invention, the silk fibroin may be purified from cocoon or silk fiber.
According to a preferred embodiment, the silk fibroin, for example, dissolve the silk was dried after the cocoon or Silk boiled put it became a Na 2 CO 3 solution was transparent, the lithium bromide (LiBr) solution, a few days, preferably from about Dialyzed and filtered for 3 days to obtain purified silk fibroin.
According to a preferred embodiment of the present invention, the transparency does not change even when plasma is treated on the film made of the silk fibroin solution obtained as described above.
According to a preferred embodiment of the present invention, the plasma treatment is carried out such that the ratio of nitrogen to oxygen on the surface of the silk fibroin film is in the range of 8: 1 to 1: 1, preferably 3: 6: 1, The surface treated silk fibroin film can be produced by treating the plasma under a vacuum condition. If the nitrogen ratio is too high, the hydrophilicity of the surface increases greatly, and thus the wetting and swelling of the support occurs excessively, so that there is a problem in the combination with the tissue due to the swelling of the support after transplantation. There is a problem that the capital is reduced. In particular, according to a preferred embodiment of the present invention, it is very important to treat the plasma within the ratio that the concentration of oxygen does not exceed the content of at least 10% and not more than 35% by volume in the plasma surface treatment. If these conditions are not maintained, the amount of change in hydrophilic water that greatly affects the interaction with the cell during the modification of the surface is increased, and depending on the degree of wetting and swelling of the support, There is a concern that there will be a gap in.
According to a preferred embodiment of the present invention, when the surface of the silk fibroin film is treated with a plasma, it is preferable to treat the plasma for 100 seconds, 30 seconds, The surface treatment can be carried out by a treatment method.
According to a preferred embodiment of the present invention, the surface of the silk fibroin film surface-modified by the plasma is more hydrophilic than the surface of the non-plasma-treated silk fibroin film. Preferably, the hydrophilic property may be increased by 5-80% relative to the film before the plasma treatment.
According to a preferred embodiment of the present invention, the surface-modified biocompatible silk fibroin film produced by the present invention has hydrophilicity, physico-chemical properties such as transparency, surface roughness and surface energy necessary for cell adhesion and proliferation, . ≪ / RTI >
According to a preferred embodiment of the present invention, the biocompatible silk fibroin film surface-modified by the plasma treatment as described above is applied to at least one tissue cell or stem cell selected from the group consisting of corneal epithelium, corneal endothelium, retina, And it is possible to attach and propagate.
According to a preferred embodiment of the present invention, the surface-modified biocompatible silk fibroin film produced by the present invention is more favorable for adhesion and proliferation of cells, It may have a silk fibroin surface property with increased roughness than before plasma treatment.
According to a preferred embodiment of the present invention, the surface-modified biocompatible silk fibroin film produced according to the present invention is a preferred example: (i) preparing a silk fibroin film; (Ii) treating the film with silk fibroin purified from cocoon or silk fiber; (Iii) isolating and culturing corneal endothelial cells; And (iv) seeding the corneal endothelial cells isolated and cultured in step (iii) on the film produced in step (ii) to produce a tissue engineering biofilm endothelial layer; In the form of a plasma-treated silk fibroin film-corneal endothelial cell layer.
According to a preferred embodiment of the present invention, after the step (iii), at least one tissue cell or stem cell selected from the group consisting of the corneal epithelium, the corneal endothelium, the retina, the eardrum, and the oral cavity is applied to the plasma treated silk fibroin film And may further include a step of seeding and adhering and propagating.
According to the present invention, as described above, a silk fibroin film is produced by preparing a film with a solution of silk fibroin, treating the plasma to provide an improved surface, and cultivating and culturing the isolated corneal endothelial cells To provide a tissue engineering biofilm endothelial layer.
According to a preferred embodiment of the present invention, the corneal endothelial cells of rabbits which can be easily accessed and easily separated and cultured can be used for producing the tissue engineering biofilm endothelial layer. However, since the corneal endothelial cells of rats, pigs, dogs, cows, humans and the like can be isolated and cultured as well as rabbit corneal endothelial cells for producing the corneal endothelial layer, they are not necessarily limited thereto.
According to another preferred embodiment of the present invention,
(I) purifying silk fibroin;
(Ii) preparing and drying the purified silk fibroin as a film;
(Iii) subjecting the produced silk fibroin film to plasma treatment;
(Iv) separating the corneal endothelial cells;
(V) seeding the isolated corneal endothelial cells into a plasma-treated silk fibroin film; And
(Vi) culturing corneal endothelial cells on a plasma-treated silk fibroin film to produce a tissue engineering bio-artificial corneal endothelial layer;
To produce a tissue-engineered bioartificial corneal endothelial cell layer.
According to a preferred embodiment of the present invention, the method of the present invention further comprises, in the step (iv), one or more tissue cells or stem cells selected from the group consisting of corneal epithelium, retina, eardrum, oral cavity and blood vessel .
The manufacturing method of the present invention will be described in detail as a more specific embodiment in each step as follows:
(I) purifying silk fibroin
In the present invention, silk which is prepared by boiling silkworm cocoons or silk fiber in a sodium carbonate (Na 2 CO 3 ) solution and drying it is dissolved again in lithium bromide (LiBr) solution, and then a dialysis membrane having a cut- Dialyzed and filtered. The prepared aqueous solution of silk fibroin is frozen at -80 占 폚 for 24 hours to 72 hours and then dried for 48 hours to 96 hours to prepare a solid purified silk fibroin.
In addition, the silk fibroin can be extracted and purified from a silk worm, a spider and the like, preferably a Silk worm, particularly a Bombyx mori is recommended.
(Ii) preparing purified silk fibroin as a solution
In the present invention, the purified solid silk fibroin is dissolved in an organic solvent at a concentration of 10-200 mg / mL to prepare a silk fibroin solution. Preferably, a concentration of 50-150 mg / mL is prepared, most preferably a concentration of 100 mg / mL.
The solvent is not limited as long as it can dissolve the purified solid silk fibroin, and preferably hexafluoroisopropanol or formic acid can be used, and hexafluoroisopropanol is more preferably used.
(Iii) casting the silk fibroin solution to produce a film
The silk fibroin solution prepared in the step (ii) is cast in a glass dish of 10 to 100 mm in diameter, preferably in a glass dish of 30 to 70 mm, more preferably in a glass dish of 50 mm, And dried for 240 hours. Drying is preferably performed for 48 to 192 hours, more preferably for 120 hours at a temperature range of 10 to 35 DEG C, preferably at a temperature range of 15 to 30 DEG C, more preferably at a temperature range of 20 to 25 DEG C Dry.
(iv) crystallizing the dried film
50 to 100% of methanol is poured into the film prepared in the step (iii) and crystallized at room temperature. Preferably 70 to 100% of methanol is used, more preferably 70 to 100% of methanol is used. The above-mentioned methanol may be crystallized by using a mixed solution of ethanol and ethanol / methanol.
The conditions of time are preferably crystallized for 1 to 100 minutes, more preferably for 10 to 80 minutes, most preferably for 72 hours. The uncrystallized film occupies a larger proportion of the α-helix structure than the formed β-sheet, making it impossible to completely provide the film for a minimum time to cultivate the cell, and it is difficult to manufacture and handle the film.
(V) drying the crystallized film to obtain a transparent silk fibroin film
After crystallizing the film produced in the step (iv), the film is washed three times or more with the third distilled water and dried. The room temperature condition for drying is preferably dried at a temperature range of 10 to 35 占 폚, preferably at a temperature range of 15 to 30 占 폚, more preferably at a temperature range of 20 to 25 占 폚, . Before the plasma treatment, it is prepared by sterilizing with ethylene oxide gas. However, it can be sterilized by using gamma ray, alcohol, chloroform, etc., and the process itself may be optional since the plasma itself has a sterilizing function at the same time.
(Vi) plasma treatment of the prepared film
Plasma is treated on the surface of the produced film. According to a preferred embodiment of the present invention, the plasma treatment is performed under a low-temperature plasma treatment condition in which the volume ratio of nitrogen and oxygen is used in the range of 8: 1 to 1: 1, preferably 6: 1 to 2: 1, Is used in a ratio of 5: 1 to 3: 1, most preferably 4: 1.
The plasma treated on the surface of the film thus produced is reduced in efficiency of improvement by the plasma treatment during storage for a long period of time in the air, and is preferably treated with the third distilled water immediately after the plasma treatment.
(Ⅶ) Separation of corneal endothelial cells
In step (vi), the corneal endothelial cells were separated from the rabbits to inoculate the corneal endothelial cells to the plasma treated silk fibroin film. The corneal endothelial layer was separated from the rabbit cornea with desde membranes and chemically separated by treatment with collagenase. The isolated corneal endothelial cells were cultured in an incubator at 37 ° C and 5% CO 2 using Endothelial growth medium-2 (Lonza, USA) as a growth medium to obtain
(Ⅷ) Step of seeding isolated corneal endothelial cells
The corneal endothelial cells prepared in step (i) are seeded with plasma-treated silk fibroin film at a cell density of 10-2000 cells / mm 2 per unit area of the corneal endothelial cells. In order to confirm the initial adhesion, seeding is preferably performed at 50 - 1500 cells / mm 2 , more preferably 100 - 1000 cells / mm 2 . Most preferably 500 cells / mm < 2 > and cultured in serum free medium for 30 minutes. In order to confirm the growth rate, seeding is preferably performed at 100 cells / mm 2 and cultured in an endothelial growth medium-2 (Lonza, USA). The culture conditions are preferably 10 to 10 days at 20 to 40 ° C and 1 to 10% carbon dioxide concentration. It is preferable to observe the growth rate by culturing for 5 days at a cell density of 100 cells / mm 2 desirable. Culturable cells can be cultured in tissue cells such as corneal epithelial cells, skin epithelial cells, fibroblasts, vascular endothelial cells, and stem cells.
According to the present invention, the corneal endothelial layer produced by the process according to the present invention as described above can be applied not only to the cornea but also to the implantation site requiring the support layer-cell relationship.
In particular, the tissue-engineered corneal endothelial layer produced according to the present invention maintains transparency, and the cultured cells form a monolayer, and the corneal endothelial cells necessary for transplantation are sufficient. Therefore, it is very useful for the preparation of a tissue engineering biomaterial corneal endothelial layer for transplantation which can replace the donated cornea in the future.
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.
Example One
(I) Production of silk fibroin film
The purified silk fibroin (SF) solution was prepared by dissolving silk cocoon in 0.02N sodium carbonate (Na 2 CO 3 ) solution, boiling and drying the dried silk again in 9.3M lithium bromide (LiBr) solution, Dialyzed with a dialysis membrane having a molecular weight cut-off of 3500 for 3 days, filtered, and lyophilized. 100 mg of silk fibroin obtained by freeze-drying was dissolved in 1 mL of hexafluoroisopropanol to prepare a 0.1% silk fibroin solution. The prepared solution was poured into a 60 mm glass dish and dried for 72 hours.
(Ii) crystallizing the dried film
The dried silk fibroin film was treated with 100% methanol for 30 minutes to crystallize the film, separated from the glass dish, and re-dried at room temperature for 48 hours.
(Iii) treating the produced silk fibroin film with plasma
In order to modify the surface characteristics, the silk fibroin film was treated with a plasma in a vacuum state at a ratio of N 2 : O 2 of 4: 1. The treatment conditions were as follows. The processing power was fixed at 100 W and the processing time was set to 30 seconds, 1 minute, 5 minutes and 10 minutes. The processing time was fixed to 5 minutes, and the power was 25 W, 50 W, 100 W, The surface of the silk fibroin film was modified according to the difference in time and processing power. Since the surface modification and the sterilization are possible in the plasma treatment, the support is not separately sterilized.
(Vi) Sowing corneal endothelial cells
The corneal endothelial cells isolated from rabbits were seeded at the density of 500 cells / mm 2 for initial adhesion and cultured in serum-free medium for 30 minutes. At this time, seeded cells were cultured at 37 ° C under a carbon dioxide concentration of 5%. The cells were seeded at a density of 100 cells / mm 2 and cultured in endothelial growth medium-2 (Lonza, USA) for 5 days. The seeded cells were cultured at 37 ° C, 5%.
Comparative Example One
The silk fibroin film prepared in the step (ii) was prepared in the same manner as in Example 1 except for the plasma treatment. Specifically, the method for producing the silk fibroin solution used in Comparative Example 1 is as follows. Silk cocoon was added to a 0.02N sodium carbonate (Na 2 CO 3 ) solution and boiled and dried. The dried silk was again dissolved in 9.3 M lithium bromide (LiBr) solution, and water was changed every 12 hours. , Dialyzed and dialyzed, and dialyzed. The dialyzed silk aqueous solution, which had been filtered with a gauze filter, was then poured into a plastic dish for 24 hours at -80 ° C., followed by lyophilization until complete drying. The resulting solution was dissolved in hexafluoroisopropanol The film was prepared using the dissolved solution and the plasma was not treated.
The results of the same tests as those of Examples 1 to 6 were compared with those of Examples 1 to 9, respectively.
Experimental Example One: plasma Treated Silk fibroin Film FTIR Measure
The FTIR was measured to confirm the functional groups formed on the surface of the plasma-treated silk fibroin film prepared in Example 1. The results are shown in FIG. The silk fibroin film showed that the amide I and II groups appeared regardless of the plasma treatment, confirming that the characteristic of the material silk fibroin was maintained. Silk fibroin film also suggests addition of peaks such as 3500-3700 cm -1 and 2100-2400 cm -1 by the treated plasma in addition to hydrogen, amine, hydroxyl, and imidazole residues on the surface. These results support that the surface properties have been modified.
Experimental Example 2: Confirmation of transparency - Visual observation and Optical density Measure
The transparency of the plasma-treated silk fibroin film prepared in Example 1 and the silk fibroin film prepared in Comparative Example 1 were visually observed and optical density was measured and shown in FIGS. 2 (a) and 2 (b). (A), it was confirmed that the films were very transparent. When the optical density was measured in the wavelength range of 380 nm to 780 nm, it was observed that there was no difference in transparency between (b) and (b). It was found that the plasma processing time and processing power did not affect the transparency of the silk fibroin film.
Experimental Example 3: surface of film FESEM and AFM result
Each film was completely dried to measure changes in the surface of the plasma treated film. The film was coated with osmium and the surface was observed with a field emission scanning electron microscope (FESEM, SUPRA 40VP, Carl Zeiss, Germany), and a silk fibroin film treated at 100 W for 5 minutes (Atomic Force Microscope, AFM, Multimode-8, Bruker, USA). The results are shown in Figs. 3 (a) and 3 (b). As a result of FESEM observation, it was confirmed that the surface roughness was increased as the plasma treatment time was increased, than that of the silk fibroin surface which was not treated with surface treatment. Among the silk fibroin films treated at 100 W for 300 seconds, Respectively. Therefore, based on the FESEM results, the surface of the silk fibroin film treated with 300s / 100w condition was measured through the AFM, and the plasma was treated at 300s / 100W with respect to the surface of the silk fibroin film not treated with plasma It was confirmed that the surface curvature of the silk fibroin film was larger and evenly distributed.
Experimental Example 4: plasma Treated Silk fibroin Film Thickness Measurement
Each film was completely dried to measure the thickness of the film. The film was coated with osmium and observed with a Field Emission Scanning Electron Microscope (FE-SEM, SUPRA 40VP, Carl Zeiss, Germany). The observation results are shown in FIG.
As shown in FIG. 4, the films produced in Example 1 and Comparative Example 1 had a thickness in the range of 39 to 45 μm (1.9 ± 3.0 μm), and the plasma treatment had no effect on the thickness difference of the films None).
Experimental Example 5: Hydrophilicity measurement result of surface
The contact angle was measured in order to confirm that the hydrophilicity of the surface of the film was changed by the plasma treatment and is shown in Fig. Although the film of silk fibroin without plasma had an initial contact angle of 68 degrees, the hydrophilicity increased as the plasma treatment time increased, and the hydrophilicity increased as the plasma treatment power increased.
Experimental Example 6: " plasma process Silk fibroin film- Corneal endothelial cells "Characterization of Endothelial Layer of Human Artificial Cornea
Experimental Example 6-1: Rabbit corneal endothelial cell seeding
In order to confirm the relationship between the plasma treated silk fibroin film and the cells, first-generation corneal endothelial cells isolated from rabbits were inoculated at 100 cell density (100 cells / mm 2 ) per unit area and cultured for 5 days at 37 ° C, %, Respectively, and the relationship with the cells was confirmed.
Experimental Example 6-2: Measurement of initial adhesion degree and growth rate
The corneal endothelial cells transplanted in Experimental Example 6-1 were cultured on the films of Example 1 and Comparative Example 1 in order to evaluate the initial adhesion rate and proliferation rate of the seeded cells in the surface modified silk fibroin film by the
Experimental Example 6-3: Measurement of mRNA expression level by RT-PCR
In order to evaluate the expression of specific mRNA markers of cells seeded in the surface-modified silk fibroin film by the plasma treatment, the corneal endothelial cells transplanted in Experimental Example 6-1 were cultured on the films of Example 1 and Comparative Example 1 The expression of the extracted mRNAs is shown in Fig. 7 (a), bands were obtained by electrophoresis of the cultured corneal endothelial cell-specific mRNAs. In Fig. 7 (b), the expression ratio of collagen type VIII, which has the largest difference, actin in the presence of the inhibitor. The mRNA expression of mRAN in the corneal endothelial cells cultured in the plasma treated group was found to be different in the mRNA expression in the plasma treated group. Especially, in case of the collagen type VIII gene, the plasma was not treated when the plasma was treated We observed that there was a significant difference in the incidence of corneal endothelial cells cultured on untreated films.
Experimental Example 6-4: Morphology and monolayer formation of corneal endothelial cells of rabbit
The adhesion and cell morphology of the corneal endothelial cells of rabbits were observed by field emission scanning electron microscopy and are shown in Fig. 8 (a) shows that the seeded cells proliferated at 300 s / 100 w and maintained morphological specificity of the corneal endothelial cells on the surface of the treated silk fibroin film on the 5th day, and Fig. 8 (b) The cultured corneal endothelial cells were confirmed to form a monolayer on the surface of the film under the same condition and to be well maintained by observing the nucleus through immunofluorescence staining. Thus, the plasma-treated silk fibroin film according to the present invention is biocompatible and can be used as a biological artificial corneal endothelial layer for regenerating the corneal endothelium by observing that the morphological characteristic of the polygonal cell, which is a morphological characteristic of the corneal endothelial cell, It can be suggested that
EXPERIMENTAL EXAMPLE 6-5: Expression of functional protein in corneal endothelial cells by immunofluorescence staining
Anti-ZO-1 and anti-Na + / K + -ATPase, which are specific functional proteins of cells seeded / cultured on silk fibroin surface treated with 300s / 100w condition among the surface modified silk fibroin films by plasma treatment Was confirmed by immunofluorescence staining and shown in Fig. 8 (c). There was no quantitative difference between anti-ZO-1 and anti-Na + / K + -ATPase expressed on corneal endothelial cells cultured on silk fibroin surface treated with plasma treated silk fibroin film and 300s / 100w condition However, it was confirmed that it functions as a corneal endothelial cell layer by observing that it is well expressed.
The plasma treated silk fibroin film of the present invention is transparent and has a surface property modified to be hydrophilic favorable to cell adhesion and superior in ability to interact with cells It can be confirmed that it is suitable as an implant capable of transplanting various tissue cells including corneal endothelial cells.
That is, the present invention provides a structural environment for the attachment, proliferation, and cell transfer of graft cells by improving the adhesion, growth and functional expression of cells by modifying the surface characteristics of conventional silk fibroin film, It can function as a basolateral membrane. In addition, the plasma treatment has no significant effect on the thickness and transparency of the silk fibroin support and is advantageous for adhering and propagating tissue cells selected from the group consisting of corneal epithelium, corneal endothelium, retina, eardrum and oral cavity.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (14)
Preparing a silk fibroin film with a solution of purified silk fibroin in hexafluoro isopropanol;
Subjecting the surface of the silk fibroin film to plasma treatment;
, ≪ / RTI &
The plasma treatment may be performed by plasma treating the surface of the silk fibroin film at a volume ratio of nitrogen to oxygen of 3 to 6: 1,
Wherein the plasma treatment is performed at 100 W for 5 minutes. ≪ RTI ID = 0.0 > 11. < / RTI >
Preparing a tissue engineering biofilm endothelial layer by seeding the corneal endothelial cells on the surface-modified biocompatible silk fibroin film;
≪ / RTI > wherein the thickness of the corneal endothelial layer is greater than the thickness of the corneal endothelial layer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116492505A (en) * | 2023-05-11 | 2023-07-28 | 山东第一医科大学附属眼科医院(山东省眼科医院) | Artificial cornea endothelial transplanting sheet and application thereof |
| WO2023195785A1 (en) * | 2022-04-07 | 2023-10-12 | 대한민국(농촌진흥청장) | Method for generating information code using fluorescent silk and information code generated thereby |
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Non-Patent Citations (2)
| Title |
|---|
| Improvement of early cell adhesion on Thai silk fibroin surface(2013.07.)* * |
| 생체적합 재료를 이용한 인간각막재생 연구(2014.02.)* * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023195785A1 (en) * | 2022-04-07 | 2023-10-12 | 대한민국(농촌진흥청장) | Method for generating information code using fluorescent silk and information code generated thereby |
| CN116492505A (en) * | 2023-05-11 | 2023-07-28 | 山东第一医科大学附属眼科医院(山东省眼科医院) | Artificial cornea endothelial transplanting sheet and application thereof |
| CN116492505B (en) * | 2023-05-11 | 2023-12-08 | 山东第一医科大学附属眼科医院(山东省眼科医院) | Artificial cornea endothelial transplanting sheet and application thereof |
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