KR101406637B1 - Method of fabricating silk fibroin plate and the silk fibroin plate fabricated thereby - Google Patents

Abstract

The present invention relates to a method of producing a silk plate and a silk plate produced thereby, which comprises a step of producing a silk fibroin aqueous solution and a bacterial cellulose thin film, and a step of pouring a silk fibroin aqueous solution into the bacterial cellulose thin film and drying.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to a method for producing a silk plate,

The present invention relates to a process for producing a bone fixing silk plate using silk fibroin and a silk plate produced thereby.

Fractures with bone dislocation cause functional and cosmetic problems. In contrast, fractured bone fragments should be placed in place and fixed to restore function and appearance. Traditionally, metal plates and screws were used to fix bone fragments. However, metal fixation systems can dissolve toxic metal ions (eg, cobalt, nickel, vanadium, etc.) into the body. Furthermore, it may inhibit bone growth in ovine, induce osteopenia, and cause inflammatory and allergic reactions.

In order to solve the disadvantages of the metal fixing system, an absorbent fixing system has been developed and used for over 20 years. Polylactic acid (PLA) was first developed and used as a suture, a sheet and a plate. Polyglycolic acid (PGA), poly-L-lactic acid, poly-D-lactic acid and poly acid is also used. However, the conventional absorbent plates have a disadvantage in that stability and stability of the decomposition period are uncertain and foreign matter reaction is caused. Inflammatory reactions have been reported to delay absorption in patients treated with PLA or PGA plates. In addition, post-operative infection may occur, which may cause the screw to loosen and wounds causing the plate to be exposed, requiring the removal of the plate early. Conventional absorptive plates are very expensive, and technically difficult to fix fractured pieces in segmental fractures.

However, silk fibroin is a typical natural polymer material extracted from silkworms and has been used for medical materials such as textile materials and sutures for a long time. It has excellent biocompatibility, good permeability of oxygen and vapor, and biodegradability, and has been attracting attention as various biomedical materials. The silk matrix supports the adhesion, growth and differentiation of bone marrow stromal stromal cells and is known to improve bone regeneration. In addition, since silk fibroin is excellent in strength and durability, it can provide sufficient stability during bone growth.

However, silk fibroin is fragile and fragile and has the disadvantage that it is difficult to make it to a certain thickness. Therefore, in order to use silk fibroin as an absorbent plate, it should be able to be made to a certain thickness without breaking, and to promote bone regeneration.

It is an object of the present invention to provide a method of manufacturing a silk plate and a silk plate which can be made to have a predetermined thickness with appropriate tensile strength and promote bone regeneration.

(B) a step of pouring the silk fibroin aqueous solution into the bacterial cellulose thin film and drying the silk fibroin aqueous solution; and (c) preparing a silk fibroin aqueous solution and a bacterial cellulose thin film. ≪ / RTI >

Here, the step of producing the aqueous solution of silk fibroin comprises a first step of removing the sericin protein and a disintegrant from the silkworm cocoon, a second step of dissolving and dissolving the resultant of the first step in a salt solution, and a step of dissolving the resultant of the second step in distilled water To the dialysis solution.

Here, the step of producing the bacterial cellulose thin film is a step of cultivating the activated bacteria and the culture liquid in a culture dish and culturing the result, the result of the first step is immersed in sodium hydroxide solution and purified, followed by washing with demineralized water to neutralize Homogenizing the resultant product in the second step and the second step, and then filtering and sieving using a sieve.

In the manufacturing method of the silk plate, in the step (b), it is preferable that the silk fibroin aqueous solution is poured into the bacterial cellulose thin film, dried at room temperature and then dried in a vacuum oven.

In order to achieve the above object, the present invention provides a silk plate produced by the above-described method for producing a silk plate.

As described above, according to the present invention, since the bacterial cellulose is added to silk fibroin, it is possible to provide an absorbent silk plate capable of promoting bone regeneration with appropriate tensile strength and thickness. Further, according to the present invention, an absorbent silk plate can be produced by an easy and simple method, which is advantageous in terms of manufacturing cost and process.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photograph of a naked eye of a silk plate produced by a method of producing a silk plate according to an embodiment of the present invention, and SEM photographs of 100 times and 250 times.
2 is a photograph of an animal model for an experiment of bone regeneration of a silk plate produced by a method of producing a silk plate according to an embodiment of the present invention.
FIG. 3 is a photograph of the naked eye of 1, 2, 4 and 8 weeks of the bone regeneration experimental animal using the silk plate produced by the silk plate manufacturing method according to the embodiment of the present invention.
4 is a 2D and 3D CT photograph of a control group and a silk plate fixed group for confirming the bone regeneration effect of the silk plate produced by the silk plate manufacturing method according to the embodiment of the present invention.
FIG. 5 is a photograph of a control group and a silk-plate fixed group photographed at a magnification of 200 and stained for the control group and the fixed group in FIG.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, it should be understood that the following embodiments are provided so that those skilled in the art will be able to fully understand the present invention, and that various modifications may be made without departing from the scope of the present invention. It is not.

The method for producing a silk plate according to the present invention includes a step of producing a silk fibroin aqueous solution and a bacterial cellulose thin film, and a step of pouring a silk fibroin aqueous solution into the bacterial cellulose thin film followed by drying. By adding the bacterial cellulose to the silk fibroin solution, the tensile strength can be increased and a uniform thickness can be produced.

Here, the step of producing the aqueous solution of silk fibroin comprises a first step of removing the sericin protein and a disintegrant from the silkworm cocoon, a second step of dissolving and dissolving the resultant of the first step in a salt solution, and a step of dissolving the resultant of the second step in distilled water .

The process for producing a bacterial cellulose thin film is a process for producing a bacterial cellulosic thin film, which comprises a first step of culturing the activated bacteria and a culture solution in a culture dish and culturing the resultant product, a step of immersing the resultant product in a sodium hydroxide solution for purification, Homogenizing the resultant product in the second step and the second step, and then filtering and drying the resultant using a sieve.

Furthermore, in the step of pouring the aqueous solution of silk fibroin into the bacterial cellulose thin film and drying it, a method may be used in which the aqueous solution of silk fibroin is poured into the bacterial cellulose thin film, dried at room temperature and then dried in a vacuum oven.

According to this manufacturing method, an absorbent silk plate having appropriate tensile strength and thickness can be produced. The silk plate according to the present invention promotes bone regeneration and can replace the conventional absorbent plate.

Hereinafter, one embodiment of a method for producing a silk plate according to the present invention will be described.

1) Preparation of aqueous solution of silk fibroin

First, sericin proteins and impurities are removed from Bombyx mori. To this end, cocoon cocoons are added to a basic aqueous solution such as sodium carbonate, heated, and then washed with distilled water. Through this process, silk fibroin can be obtained, and then silk fibroin is dissolved in a salt solution such as calcium chloride.

It is then dialyzed against distilled water to remove ionic components from the silk fibroin solution. The dialysis time is preferably 36 to 72 hours. Thus, silk fibroin usable in vivo is extracted.

2) Manufacture of bacterial cellulose thin film

The bacterial cellulose thin film can be synthesized from "A. xylinum" or the like. To do this, the activated bacteria and culture are injected into a culture dish and cultured. Pre-culture is to maximize the activity of the bacteria to obtain maximum production. For example, 50 μL of activated bacteria and 10 mL of culture can be injected into a culture dish and incubated at 30 ° C for 10 days.

The thus prepared bacterial cellulose thin film was purified by immersing it in a sodium hydroxide solution at room temperature for 48 hours, and then repeatedly washed with deionized water to neutralize it. After homogenization using an Ultra-Turrax T25, the mixture is sieved using a sieve having a pore size of about 200 nm and dried in a 60 ° C vacuum oven for 24 hours.

3) Fabrication of silk fibroin plate

A 10 mL silk fibroin aqueous solution was poured into the bacterial cellulose thin film, dried at room temperature for 2 days, and then dried in a vacuum oven. Because the bacterial cellulose membrane has nanostructures, it penetrates the silk fibroin between these nanostructures and strengthens the overall structure.

silk  Plate The tensile strength  Measure

The thickness of the prepared silk plate was 0.3 mm, and it had elasticity. The tensile strength was 68.28 ± 6.71 MPa, the elongation at break was 5.84 ± 1.65%, the tensile modulus, Was measured at 722.56 ± 56.15 MPa.

silk  Shooting plate

In order to confirm the structure of the prepared silk plate, scanning electron microscope was taken at 100 times and 250 times. 1 is a photograph (A, B, C) of a silk plate produced by a method of producing a silk plate according to an embodiment of the present invention and SEM photographs (D, E) of 100 times and 250 times. Referring to FIG. 1B, the thickness of the silk plate is 0.3 mm. Referring to FIG. 1C, it can be seen that the silk plate has elasticity. In addition, it can be seen from the photographs D and E of FIG. 1 that the silk plate has a very dense structure without voids.

silk  Evaluation of plates

1) Effect of bone regeneration

In order to confirm the possibility of the biocompatibility and absorptive plate of the silk plate manufactured in the above example, an experiment was conducted to confirm the bone regeneration process of the osteolysis without osteosynthesis.

2 is a photograph of an animal model for an experiment of bone regeneration of a silk plate produced by a method of producing a silk plate according to an embodiment of the present invention. Rats were used as experimental animals. Two-mm-sized fracture pieces were removed from both ganglia and covered with silk plates on one side and no additional control was performed on the other side.

At 1, 2, 4, and 8 weeks after osteolysis, changes of bone loss site were observed by microscopic observation and micro CT was taken. FIG. 3 is a photograph of the naked eye of 1, 2, 4 and 8 weeks of the bone regeneration experimental animal using the silk plate produced by the silk plate manufacturing method according to the embodiment of the present invention. As shown in the photograph of FIG. 3, there was no complication such as inflammation, abscess or necrosis caused by silk plate in the visual examination, and degree of adhesion between the silk plate and surrounding tissue increased with time.

4 is a 2D and 3D CT photograph of a control group and a silk plate fixed group for confirming the bone regeneration effect of the silk plate produced by the silk plate manufacturing method according to the embodiment of the present invention. In Fig. 4, the left side is the 2D and 3D CT photographs of the control group, and the right side is the 2D and 3D CT photographs using the silk plate. In the control group, bone regeneration was not observed at all for 8 weeks, but bone formation was observed at 1 week in the group to which the silk plate was fixed, osseous ossification was observed at 4 weeks, and the bone was completely reformed after 8 weeks .

2) Activation of oocyte

Histological examinations were performed to confirm the activation of the osteoblast during the bone regeneration period of the silk plate. For this purpose, 1, 2, 4, and 8 weeks old silk plates and fractured pieces were stained with hematoxylin and Eosin and fixed at 200x magnification. FIG. 5 is a photograph of a control group and a silk-plate fixed group photographed with Hematoxylin and Eosin staining at 200 magnifications for the control group and the fixed group of FIG.

From Fig. 5, when the silk plate was fixed, new bone was formed at the cross section of the fracture with activation of the osteocytes, and it was confirmed that the bone was completely healed after 8 weeks.

From the above results, it can be confirmed that the silk plate of the present invention is excellent in bone regeneration effect and activates osteoblast cells without any adverse side effects. Therefore, the silk plate of the present invention can be utilized as an absorbent plate.

Claims (5)

(a) preparing a silk fibroin aqueous solution and a bacterial cellulose thin film;
(b) pouring the silk fibroin aqueous solution into the bacterial cellulose thin film and drying the silk fibroin aqueous solution. The method for producing a silk fibroin aqueous solution according to claim 1,
A first step of removing the sericin protein and the impurity from the cocoon;
A second step of putting the resultant of the first step in a salt solution and dissolving it;
And a step of dialyzing the result of the second step with distilled water. The method according to claim 1, wherein the step of preparing the bacterial cellulose thin film comprises:
A first step of culturing the activated bacteria and the culture solution by injecting them into a culture dish;
A second step of immersing the resultant of the first step in a sodium hydroxide solution to purify and then neutralizing the resultant by washing with deionized water;
A step of homogenizing the result of the second step, followed by a step of filtering and drying the resultant using a sieve. The method according to claim 1, wherein the step (b)
Wherein the silk fibroin aqueous solution is poured into the bacterial cellulose thin film, dried at room temperature, and then dried in a vacuum oven. A silk plate produced by a process for producing a silk plate according to any one of claims 1 to 4.

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