JP2015027448A - Bioconjugate device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a bio-conjugate device with a base of amnion, having improved strength.SOLUTION: The bio-conjugate device includes a laminate of amnion and silk fiber of unrefined raw silk subjected to semi-melting treatment, with use of solubilized fibroin.

Description

  The present invention relates to a medical material used as a bioconjugate device, and more particularly to a bioconjugate device in which amnion and silk fiber are combined.

Amniotic membrane is a tough biological membrane composed of collagen and elastic fibers, and has anti-inflammatory action and epithelialization promoting effect, so it is used for wound treatment and the like.
The group of Toshio Nikaido, who is also the inventor of the present applicant, has applied the amniotic membrane drying treatment method (patent document 1) capable of long-term storage of the amniotic membrane (patent document 1) and the application of the dried amnion to medical materials (patent document 2) Has proposed.
However, when used as a patch during cornea perforation in the ophthalmologic region, there is a concern of falling off from the affected area due to collagenase or the like, and enhancement of the amniotic membrane strength during prep formation in glaucoma is desired.
Further, it has been desired to improve the strength of the amniotic membrane when used as a cerebral dural patch extensively in the brain surgery region, or when used as a patch when the eardrum is damaged in the otolaryngology region.
On the other hand, silk has long been used for surgical sutures as a medical material.
In recent years, attempts have been made to use silk or silk protein as a scaffold for regenerating a living tissue.
For example, scouring silk to remove sericin and the like, and using it for the formation of a tissue-supporting prosthetic device for implantation (Patent Document 3), obtaining a complex of silk protein and collagen (Patent Document 4), There is one that obtains a complex of salt-shrinked silk and chitosan (Patent Document 5).
Therefore, the present inventors tried to bond the amniotic membrane and silk fiber, but they were dissociated from each other simply by being laminated, and sufficient performance could not be obtained.
After that, the present invention has been achieved by further improving.

Japanese Patent No. 4977345 Japanese Patent No. 5092119 JP 2011-147790 A JP-A-11-228837 JP 2004-131647 A

  An object of the present invention is to provide a bioconjugate device based on amniotic membrane with improved strength.

The bioconjugate device according to the present invention is characterized by laminating amniotic membrane and silk fiber of unrefined raw silk that has been semi-dissolved using solubilized fibroin.
Here, the raw silk silk fibers that have not been refined are used for the following reason.
A silk thread made from a silkworm cocoon and pulled out has a cross-sectional structure in which two fibroin fibers are surrounded by sericin, and has a unique gloss and is called raw silk.
In contrast, raw silk is scoured with an alkaline liquid such as soap, lye, soda water, and the silk thread from which sericin has been removed is referred to as kneaded yarn.
The present invention is characterized in that this unrefined raw silk is used after being semi-dissolved.
Here, the semi-dissolution treatment refers to a state in which a part of sericin surrounding the fibroin fiber of raw silk is dissolved and weakly refined so that sericin still remains.
Therefore, it means that sericin has been scoured to the extent that it remains, and is not meant to be limited to half scouring.
The chemical solution is preferably a calcium salt solution, and examples of the calcium salt used for semi-dissolution include calcium nitrate and calcium chloride, with calcium chloride being preferred.
The density | concentration of a calcium salt should just be 40 to 60% (weight%).
Moreover, heating should just boil calcium salt aqueous solution, and processing time should just be 5 to 10 minutes.
The semi-dissolution treatment may be performed by adding a monohydric alcohol such as methanol or ethanol.
The addition of alcohol is preferably performed after the aqueous calcium salt solution has boiled.
The half dissolution treatment time including addition of alcohol is 5 to 30 minutes.
Further, the solubilized fibroin refers to a fibroin fiber obtained by scouring raw silk and dissolved in a gel form.
Examples of chemical solutions used for solubilization include solutions of copper ethylenediamine, lithium bromide, and calcium chloride.
For example, it can be obtained by putting a scoured silk thread in a 30-50 mass% calcium chloride aqueous solution, heating to gel, removing neutral salts by dialysis or the like, and removing impurities by filtration or the like.
In addition, you may perform the neutralization process etc. before dialysis.

Although details will be described later, it is insufficient to improve the strength of the amniotic membrane only by impregnating the dried amniotic membrane with the solubilized fibroin.
In addition, as a trial, a raw silk silk fabric was semi-dissolved and laminated on a dry amnion, but it did not adhere sufficiently.
Therefore, in the present invention, when the amniotic membrane and the semi-dissolved raw silk fiber are laminated using solubilized fibroin, the adhesion between the amniotic membrane and the semi-dissolved silk fiber is excellent, and the strength is greatly improved. .
Examples of the method of using solubilized fibroin for the integration include an example in which silk fibers of raw silk that has been semi-dissolved after impregnating the solubilized fibroin into the amniotic membrane are superimposed.

There are no restrictions on the shape of the silk fiber in terms of improving the strength with the silk fiber of the raw silk that has been semi-dissolved in the amniotic membrane. However, from the viewpoint of easy handling, the silk fiber is a plain weave of warp and weft alternately A plain woven fabric composed of a structure is preferred.
Examples of thin, lightly transparent plain fabrics include silk organdy and picture silk used mainly for painting Japanese paintings.
Moreover, it is easy to handle the amniotic membrane used for laminating with the semi-dissolved silk fiber by using a dried and dried amniotic membrane.
As the dried amniotic membrane, those obtained by drying human amniotic membrane or horse amniotic membrane using the method described in Patent Document 1 are preferable.
The dried amniotic membrane may be fixed with glutaraldehyde.

  A bioconjugate device in which a half-dissolved raw silk fiber and an amniotic membrane impregnated with solubilized fibroin are laminated has a significantly improved strength compared to the amniotic membrane alone, and can provide a novel medical material.

The schematic of the drying apparatus used for drying amnion etc. is shown.

  Hereinafter, although a reference example, a manufacture example, a test example, etc. explain the present invention, the present invention is not limited to these.

(Reference Example 1: Drying method of amniotic membrane and bioconjugate device)
The amnion and bioconjugate device used in the present invention were dried using the drying apparatus shown in FIG.
In this drying apparatus, a magnetron having an output of 1.5 KW was used as the microwave irradiation apparatus 30.
Moreover, the temperature setting of the far-infrared heater 14 was set to 50 ° C., and the far-infrared ray was continuously irradiated on the amniotic membrane from the start to the end of drying.
Furthermore, when the amniotic membrane was not placed in the treatment tank 10, the maximum pressure reduction achieved by the vacuum pump 18 was set to 0.4 kPa.

When drying the amniotic membrane etc. with the drying apparatus shown in FIG. 1, the amniotic membrane etc. (for example, raw amnion 50 g) is spread without wrinkles on the cooking paper spread so as not to be wrinkled. Was placed on a tray.
Furthermore, after placing this tray on the turntable 12 in the processing tank 10, the turntable 12 was rotated.
The turntable 12 was continuously rotated by a motor 16 from the start to the end of drying.

The far-infrared heater 14 was turned on, the vacuum pump 18 was driven, the electromagnetic valve 20 was opened, and a pressure reducing operation for reducing the pressure inside the processing tank 10 was started.
Since the decompression speed has decreased for a while after the start of decompression, when the maximum decompression ultimate pressure reaches 0.90 kPa, the vacuum pump 18 is stopped and the solenoid valve 20 is closed and the solenoid valve 26 is opened. A return pressure operation for introducing air in which bacteria were filtered into the treatment tank 10 was started, and the pressure in the treatment tank 10 was restored to 4.53 kPa.

  Simultaneously with the start of the decompression operation, the magnetron as the microwave irradiation device 30 was turned on, and a heating operation was performed to irradiate the amniotic membrane on the rotary table 12 with microwaves.

After performing the heating operation with the far infrared heater 14 and the magnetron for 3 minutes, the magnetron was turned off, and the decompression operation was resumed while the far infrared heater 14 was turned on.
After reducing the pressure in the processing tank 10 to 0.62 kPa by the restarted pressure reducing operation, the pressure reducing operation for returning the pressure in the processing tank 10 to 4.63 kPa, and the heating operation for 3 minutes by the far infrared heater 14 and the magnetron And gave.
This decompression operation, heating operation, and decompression operation were performed a total of 6 times to finish drying the amniotic membrane.
The end of this drying was judged by the maximum pressure reduction ultimate pressure in the treatment tank 10 by the fifth decompression operation and the maximum pressure reduction ultimate pressure when no amniotic membrane was placed in the treatment tank 10.
That is, the maximum reduced pressure attainment pressure of the sixth decompression operation reached 0.40 kPa and became equal to the maximum reduced pressure attainment pressure when no amniotic membrane was placed in the treatment tank 10, so it was determined that the drying was finished.

  The dried amniotic membrane after drying was dehydrated and dried to 1 g with respect to 50 g of raw amniotic membrane placed in the treatment tank 10, and sealed and stored in a sterilized pack containing a desiccant.

(Reference Example 2: Production of solubilized fibroin)
Salt (0.2%) electrolyzed alkaline water (pH 9.0 to 10.5) was added by 50 times the weight of silk fiber, and 70 ° C. in an extractor made by Toyo High Pressure Co., Ltd. (TFS-2L; 100 MPa). Scouring to remove sericin for 1 hour was performed.
2.5 g of the fibroin fiber thus obtained was put into 50 ml of calcium chloride: water (50:50 w / w) and heated.
After boiling, 50 ml of ethanol was added.
Thereafter, the mixture was cooled, dialyzed with a dialysis tube (cellulose tube), then placed in a seamless cellulose tube and treated in running water for 5 days to obtain a gelled product (solubilized fibroin A) in which fibroin was solubilized. .
Moreover, after cooling and neutralizing to pH 7.0, a gelled product (solubilized fibroin B) was obtained by dialysis treatment in running water for 1 day.

(Reference Example 3: Production of semi-dissolved organdy)
Organdy 2.5 g consisting of silk plain fabric of raw silk is added to 100 ml of a mixed aqueous solution of calcium chloride: pure water (50:50 (w / w)), and heated and boiled.
Next, 30 ml of ethanol is gradually added with a Komagome pipette.
Next, the semi-dissolved organdy is washed with running water (around 15 ° C.) for 1 hour.

(Production Example 1: Production of laminate A of solubilized fibroin A-impregnated dry amnion and semi-dissolved organdy)
The dried amniotic membrane obtained in Reference Example 1 was treated with glutaraldehyde (immersed in a 0.1% glutaraldehyde aqueous solution for 30 minutes).
The treated product was washed with physiological saline and dried by the method of Reference Example 1.
Next, it was immersed in solubilized fibroin A for several seconds at room temperature, and organdy that had been semi-dissolved in Reference Example 3 was attached.
Further, glutaraldehyde treatment / washing was performed, and the mixture was again dried by the method of Reference Example 1 to obtain a laminate A of dried amnion and semi-dissolved organdy.

(Production Example 2: Production of laminate B of solubilized fibroin B-impregnated dry amniotic membrane and semi-dissolved organdy) The dry amniotic membrane obtained in Reference Example 1 was treated with physiological saline for 30 minutes without being treated with glutaraldehyde this time. It was immersed and dried by the method of Reference Example 1.
Subsequently, the organdy which was immersed in solubilized fibroin B for several seconds at room temperature and semi-dissolved in Reference Example 3 was attached.

Samples for comparative evaluation of strength were prepared as follows.
Sample 1: The dried amniotic membrane obtained in Reference Example 1 was immersed in a 0.1% glutaraldehyde aqueous solution for 30 minutes, washed with physiological saline, and then dried again by the method of Reference Example 1.
Sample 2: The bioconjugate device according to the present invention obtained in Production Example 1.
Sample 3: The dried amniotic membrane obtained by the method of Reference Example 1 was immersed in the solubilized fibroin A obtained in Reference Example 2 and impregnated.
Sample 4: The semi-dissolved organdy obtained in Reference Example 3 was immersed in a 0.1% glutaraldehyde aqueous solution for 30 minutes, washed with physiological saline, and dried in Reference Example 1.
Sample 5: The dried amniotic membrane obtained in Reference Example 1 was moistened with physiological saline.
Sample 6: Bioconjugate device according to the present invention obtained in Production Example 2.

(Test Example 1: Tensile test)
The strength was measured with a tensile strength meter TENSION / UTM- (KK) -500 (manufactured by Toyo Baldwin).
<Method>
Samples 1 to 5 were cut into a size of 20 mm × 20 mm and immersed in phosphate buffered saline for 5 minutes or longer.
Next, a sample immersed in phosphate buffered saline was attached to a pattern paper filter paper.
The sample was fixed to the air jaw together with the pattern paper filter paper, and the pattern paper filter paper that was not sandwiched between the air jaws was removed.
The tensile strength until the sample broke was measured.
The test conditions were a tensile start distance of 10 mm, a width of 20 mm, and a tensile speed of 10 mm / min.

The test results are shown in Table 1.
<Result>

  From the results in Table 1, it can be seen that the strength of the bioconjugate device according to the present invention of Sample 2 is improved compared to Sample 1 of amniotic membrane alone and Organdy sample 4 subjected to semi-dissolution treatment.

(Test Example 2: Tensile test)
Tensile test is performed in the same manner as in Test Example 1 using the sample 5 cut into the same size of 10 mm × 10 mm for comparison with the sample 5 ′ cut into the size of 10 mm × 10 mm. Went.
As a result, Sample 5 ′ had a strength of 579 (gf) and Sample 6 had a strength of 670 (gf).
This result also shows that the strength of the bioconjugate device according to the present invention has been improved.

  The bioconjugate device according to the present invention is useful as a medical material for new treatments such as development of a brain dura mater that requires strength, a tympanic membrane that requires strength, and an artificial trachea / artificial esophagus.

DESCRIPTION OF SYMBOLS 10 Treatment tank 12 Rotary table 14 Far-infrared heater 16 Motor 18 Vacuum pump 20, 26 Solenoid valve 22 Pressure-reduction piping 24 Filter 28 Pressure-reduction piping 30 Microwave irradiation apparatus

Claims (4)

  A bioconjugate device, wherein amniotic membrane and non-refined raw silk silk fibers are laminated using solubilized fibroin.   The bioconjugate device according to claim 1, wherein the silk fiber is a raw silk fabric.   3. The bioconjugate device according to claim 2, wherein the raw silk fabric is a plain fabric.   The bioconjugate device according to any one of claims 1 to 3, wherein the silk fiber semi-dissolution treatment uses a calcium salt solution.

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