KR101186004B1 - Method of fabrication of silk nanofibrous membrane with a controllable biodegradability and an implant biomaterial prepared by using the method - Google Patents

Abstract

The present invention relates to a method for controlling the biodegradation rate of silk nanofiber membranes and silk nanofiber membranes prepared therefrom. More specifically, in the manufacturing process of the silk nanofiber membrane, it is to find and provide a biodegradation rate can be adjusted in the process of insolubilizing the spun fiber membrane. According to the present invention, it is possible to improve the biodegradation rate of the silk nanofiber membrane, thereby extending its application in tissue engineering. That is, the present invention provides a biodegradable support prepared from silk nanofiber membranes.

Silk, Nanofiber, Biodegradable, Support, Tissue Engineering, Alcohol, Recrystallization, Insolubilizing, Ethanol, Propanol

Description

Biodegradable silk nanofiber membrane and its preparation method, biodegradable support using the same {Method of fabrication of silk nanofibrous membrane with a controllable biodegradability and an implant biomaterial prepared by using the method}

The present invention relates to a silk nanofiber membrane and a method of manufacturing the same. In particular, the present invention provides a method for producing a silk nanofiber membrane having biodegradable properties in vivo.

Tissue engineering scaffolds are used for the preparation of cell-support complexes cultured on scaffolds in porous structures similar to natural extracellular matrix. Tissue engineering techniques in which tissues are taken from the patient's body, cells are isolated from the tissues, and the cells are cultivated in a scaffold to produce a cell-support complex, which is then transplanted into the human body, is used for tissue regeneration. The key is to prepare a support similar to a living tissue for optimization.

Such scaffolds serve as a framework in which tissue cells adhere to the scaffolds to form tissues with three-dimensional structure. After being implanted in a living body, the scaffold should be non-toxic (biocompatibility). .

Recently, in order to realize excellent cell adhesion in cell culture, a technique of preparing a support having a porous structure includes particle leaching, emulsion freeze-drying, and high pressure gas expansion. And phase separation have been developed.

The particle leaching method is a method of dissolving a polymer having excellent biocompatibility in an organic solvent and then preparing a casting by mixing particles such as salt insoluble in the solution, and then removing the solvent and eluting and removing salt particles using water. . In the particle leaching method, there is a risk of damage to the cells due to the remaining salt salt or its rough shape during the formation of voids.

Emulsification freeze drying method has a limitation in making open cellular pores in the process of forming pores by removing the organic solvent and water by lyophilizing a solution in which a biocompatible polymer is dissolved in an organic solvent and an emulsion of water.

The same applies to the process of putting biocompatible polymers into a mold, applying pressure to make pellets, injecting high-pressure gas into the pellets at a suitable temperature, and then gradually lowering the pressure to release the gas to form voids.

Phase separation is a method of adding a sublimable substance or solvent with different solubility to a solution in which a biocompatible polymer is dissolved in an organic solvent and forming voids by phase separation of the solution according to sublimation or temperature change. There is a problem of being small.

On the other hand, Korean Patent No. 10-0751547 is a method for preparing a support having a porous structure similar to the natural extracellular matrix, a method for producing a support made of silk nanofibers in the form of sponges of three-dimensional structure through the electrospinning fiber spinning solution Presented.

In addition, Korean Patent Publication No. 10-2009-0041271 discloses a method for controlling the pore structure of a nanofiber support for tissue engineering, wherein the nanofiber dispersion solution has a three-dimensional structure from a mixture containing insoluble particles for forming pores and has a porosity of 90. A method of forming a nanofiber scaffold consisting of nanofibers that is more than% is provided.

The silk nanofiber support is a structure similar to a natural extracellular matrix having a three-dimensional structure in which nanofiber protein fibers are entangled and have a large specific surface area and maximize porosity. However, the silk nanofiber support has a disadvantage in that it cannot control the biodegradation rate unlike biodegradable synthetic polymers such as PLGA, although it has many advantages for using as a material for implantation. The limited biodegradability of such silk nanofibers limits the application of silk nanofiber membranes for various applications, particularly for tissue engineering.

The limited biodegradability of silk nanofiber membranes is due to the limitations of the recrystallization methods of regenerated silk proteins that are widely used in the past. Recrystallization (insolubilization) is mainly used to immerse in methanol or ethanol, it is difficult to control the degree of crystallization.

The present invention has been made to solve the above-mentioned problems, to provide a method for controlling biodegradability of silk nanofiber membrane. In addition, the present invention is to provide a silk nanofiber membrane that is easy to control biodegradability. Finally, it is an object of the present invention to provide a biodegradable support that can be used for various purposes in tissue engineering techniques.

The present invention provides a process for preparing a fiber spinning stock solution, spinning the prepared fiber spinning stock while applying a voltage, and insolubilizing the silk fiber after immersing the spun fiber in an alcohol solution. Provided are methods of making a fiber membrane.

In the insolubilization treatment step, the biodegradation rate of the silk nanofiber membrane is controlled by adjusting the type and mixing ratio of the alcohol solution.

Preferably, the alcohol solution may be selected from the group consisting of methanol, ethanol, propanol, butanol and pentanol, one or two or more.

The present invention also provides a silk nanofiber membrane characterized in that the rate of biodegradation is controlled by immersion and recrystallization from the spun fiber into a mixed solution of ethanol and propanol.

The present invention also provides a biodegradable support, which is produced from silk nanofiber membranes.

According to the production method of the present invention as described above it is possible to control the biodegradation rate of the silk nanofiber membrane. Thus, it is possible to produce silk nanofiber membranes with a suitable biodegradation rate depending on the application. Therefore, the silk nanofiber membrane of the present invention can be used in various ways as a support for tissue engineering.

Silk nanofiber membranes are a kind of nonwoven fabric in which irregular fibers are arranged in a fiber of 100 to 400 nm in diameter produced by regenerating silk fibroin (silk protein). The thickness of the silk nanofiber membrane can be freely adjusted, but usually 50-200 um is used. In addition, since the fibers are entangled in structure, they exhibit high porosity of 70-80%. The pore size is approximately 1 um. The crystallized silk nanofiber membranes are brittle in the dry state but flexible in the wet state and easy to handle and deformable.

Silk nanofiber membranes are generally manufactured using electrospinning. The present invention provides a method of controlling the biodegradability of the silk nanofiber membrane in the electrospinning method. Specifically, the manufacturing method of the silk nanofiber membrane is as follows.

1) Preparation of fiber spinning stock

Silk nanofiber membranes are made from silk fibers. Silk fiber is made of thread extracted from silkworm cocoons, and two strands of fibroin are wrapped in a sericin envelope. Fibroin is excellent in biocompatibility and does not affect any surrounding tissues, so it may be manufactured in various forms such as powder, gel, and aqueous solution, and may be used in various foods and medicines. Therefore, in order to use silk fiber, it is preferable to remove sericin of an outer film. After removing sericin, an aqueous solution of silk fibroin is prepared by washing with water, drying and dialysis, and a silk fibroin sponge is prepared by lyophilizing the aqueous solution of silk fibroin. Then, this is dissolved in a solvent to prepare a spinning stock solution of the fiber.

Specifically, in order to prepare a nanofiber stock solution, the cocoon is added to Marseubi soap and sodium carbonate aqueous solution, and treated for 1 hour at a temperature of 100 ° C., followed by repeated washing with distilled water to remove sericin. Next, the cocoons are completely dried, completely dissolved in a calcium chloride / water / ethanol mixed solvent, and dialyzed with a cellulose dialysis membrane to remove salts and ethanol. Finally, the resulting silk fibroin aqueous solution is lyophilized to prepare a sponge. The sponge-like silk fibroin is dissolved in 98% formic acid and impurities and bubbles are removed to prepare a nanofiber stock solution.

2) Preparation of Nanofiber Membrane

Spinning from the fiber stock is done by applying a voltage of 10-20 kV.

Specifically, the nanofiber stock solution is contained in a syringe and discharged at a rate of 0.2-0.5 ml / hr using a delivery pump. At this time, connect the electrode to the syringe needle and apply a constant voltage of the above range to the DC power supply. The formed silk nanofibers collect on the rotating drum surface.

3) Insolubilization of Nanofiber Membrane

Immediately after electrospinning, nanofibers undergo a solubilization process because of their low strength and very susceptibility to moisture. Insolubilization treatment is to immerse and recrystallize spun nanofibers in alcohol. The biodegradability of the nanofiber membrane can be controlled during this insolubilization treatment. In the present invention, the biodegradation rate was measured after recrystallization of nanofibers in various kinds of alcohol solutions. The component change of the silk nanofibers was measured while adjusting the type and mixing ratio of the alcohol solution, and the biodegradation behavior of the prepared silk nanofiber membranes was observed.

Alcohol solution used in the insolubilization treatment process in the present invention is one or two or more selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol Can be used in combination.

According to the type of the alcohol solution, it is possible to produce silk nanofibers having a desirable biodegradation rate suitable for the purpose of using the fibers. Accordingly, the present invention provides a method of controlling the biodegradation rate by the insolubilization process of the silk nanofiber membrane.

Preferably, in the present invention, a mixed alcohol solution of ethanol and propanol is used. The mixing ratio of the mixed alcohol solution is in the range of 0-100% by volume, based on ethanol. The higher the content of ethanol, the higher the content of the beta structure in the nanofibers, and thus the lower the solubility, the slower the biodegradation rate. On the other hand, the use of propanol does not increase the content of the beta structure in the fiber during the insolubilization treatment, which is maintained at about the same level when comparing before and after treatment. However, since the stability to moisture is increased, the strength of the nanofibers is increased and the moisture resistance is improved. On the other hand, the rate of biodegradation appears to be faster when compared to ethanol treatment.

Recrystallization of the silk nanofiber membrane consists of removing the nanofiber membrane from the surface of the drum, which is an integrated device, after the end of the electrospinning, immersing it in an alcohol solution for 1 hour, and drying under pressure to prevent shrinkage.

The present invention will be described in detail through the following examples. However, the present invention should not be construed as being limited thereto.

Example

1) Preparation of Electrospinning Radiation Solution

In a solution of 0.3% and 0.2% of Marseubi soap and sodium carbonate, respectively, the Gajam cocoon was added at a ratio of 1:25 in a bath ratio for 1 hour, and then washed repeatedly with distilled water to remove sericin. Then, the dried cocoons were completely dried, dissolved completely at 85 ° C. in a bath ratio of 1:20 using a calcium chloride / water / ethanol (molar ratio 1: 8: 2) mixed solvent, and dialyzed in distilled water for 3 days using a cellulose dialysis membrane. Salt and ethanol were removed. Silk fibroin aqueous solution prepared in the final process was lyophilized to obtain a sponge.

The nanofiber stock solution was prepared by completely dissolving the prepared silk fibroin sponge in a concentration of 12% in 98% formic acid and removing impurities and bubbles.

2) Electrospinning for Nanofiber Membrane Production

In order to prepare a nanofibrous membrane using electrospinning, the spinning stock solution prepared above was placed in a syringe and discharged at a rate of 0.2-0.5 ml / hr using a delivery pump. An electrode was connected to the syringe needle and a constant voltage of 10 kV (+) was applied with a DC power supply. The formed silk nanofibers were collected on a rotating drum surface of 14 cm in diameter.

3) Insolubilization of Silk Nanofiber Membranes

The mixing ratio of the ethanol (ethyl alcohol: E) / propanol (1-propanol: P) mixed solution used for insolubilization of the electrospun silk nanofibrous membrane (as-spun) was set to E / P = 100/0, 90/10, 80/20, 70/30, 60/40, 50/50, 0/100 were prepared in various ways. Silk nanofiber membranes electrospun in each solution were immersed for 1 hour and then dried under tension.

Next, the beta structure content and biodegradation rate of each silk nanofiber membrane prepared were measured.

4) Beta structure content

It can be seen that silk fibroin has various beta structure contents depending on the mixing ratio of the solution used in the recrystallization process. (FIGS. 2A to 2D) The beta structure of silk fibroin is a very important structural feature in insolubilization. As the beta structure content increases, solubility decreases.

Table 1 below shows the beta structure content of silk fibroin over time according to the mixing ratio of the solution used in the recrystallization process.

The higher the ethanol content of the solution used in the recrystallization treatment, the higher the beta structure content of silk fibroin. On the other hand, when the recrystallization treatment using propanol alone did not show a difference from the beta structure content of the silk fibroin without the recrystallization treatment, it was confirmed that it was sufficiently insoluble due to an increase in stability to moisture.

5) biodegradation rate

Biodegradation behavior by proteinase K of silk nanofiber membranes prepared according to the above recrystallization method was observed. Figure 3 shows the mass change by the proteolytic enzyme of the silk nanofiber membrane with the mixing ratio of the solution used in the recrystallization treatment. 4 is a SEM photograph showing the results of biodegradation on Invitro.

The mass reduction and biodegradation of the silk nanofiber membranes were higher with higher propanol content. That is, the lower the content of the beta structure can be seen that the biodegradation rate increases. Therefore, it can be seen that the biodegradation control by the recrystallization of the present invention is effective.

6) Biodegradation rate (absorption rate) in vivo

Experimental photographs after subdermal insertion of rats by the method of the present invention shows that the absorption rate of the silk membrane in vivo depends on the mixing ratio of the solution used in the silk recrystallization process visually (Fig. 5).

After three weeks, the silk nanofiber membrane was photographed with a scanning electron microscope. Comparing this, it can be seen that the thickness of the fiber is reduced after the passage of tissue insertion (Fig. 6).

It can be seen that the biodegradation is actively occurring in the side portion of the silk nanofiber membrane as shown in Figure 7, and the thickness of the fiber is reduced according to the mixing ratio of the solution used in the recrystallization process.

Figure 1 illustrates the biodegradation rate control process of the radial silk nanofiber membrane of the present invention.

Figures 2a to 2d shows the difference in the beta structure content of the silk nanofibers according to the mixing ratio of the solution used in the recrystallization treatment.

Figure 3 shows the mass change by the proteolytic enzyme of the silk nanofiber membrane with the mixing ratio of the solution used in the recrystallization process.

Figure 4 shows the results of biodegradation on the Invitro of the silk nanofiber membrane according to the mixing ratio of the solution used in the recrystallization process by SEM photograph.

Figure 5 shows the difference in the absorption rate of the silk nanofiber membrane in vivo according to the mixing ratio of the solution used in the visual recrystallization process. (E100: ethanol 100%, EP50: ethanol 50% + propanol 50%, P100: propanol 100%)

6 is a SEM photograph showing the difference in the absorption rate of the silk nanofiber membrane in vivo according to the mixing ratio of the solution used in the recrystallization process.

Figure 7 shows the change in absorbed thickness of silk nanofiber membranes in vivo according to the mixing ratio of the solution used in the recrystallization process with Masson trichrome stain (ethanol 100%, Ethanol 50% + Propanol 50%, respectively) , Propanol 100% data.)

Claims (5)

A process of preparing a fiber spinning stock solution, spinning the prepared fiber spinning stock solution while applying a voltage, and insolubilizing treatment process of immersing the spun fiber in a mixed solution of ethanol and propanol and then recrystallizing it. Method of producing a silk nanofiber membrane, characterized in that to increase the biodegradation rate of the silk nanofiber membrane by increasing the content of propanol in the mixed solution delete delete Silk nanofiber membrane, characterized in that the biodegradation rate produced by the manufacturing method of claim 1 is controlled A biodegradable support, which is prepared from the silk nanofiber membrane of claim 4.

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