KR20180025790A - Cross-linked nanofiber sheet using animal cartilage-derived materials and method for manufacturing the same - Google Patents

Abstract

The present invention relates to a method for manufacturing a crosslinked nanofiber sheet which comprises the following steps: (a) preparing a water-soluble animal cartilage derived tissue by modifying an animal cartilage derived tissue; (b) adding the water-soluble animal cartilage derived tissue to a mixed solvent of a polar organic solvent and an acidic solvent having a pH of 1 to 5, stirring the mixture to prepare a solution, and electrospinning the solution to prepare a nanofiber sheet; and (c) crosslinking the nanofiber sheet. The present invention additionally relates to the crosslinked nanofiber sheet manufactured by the method, and to an anti-adhesion bio-sheet using the same.

Description

[0001] Description [0002] CROSS-LINKED NANOFIBER SHEET USING ANIMAL CARTILAGE-DERIVED MATERIALS AND METHOD FOR MANUFACTURING THE SAME [0003]

The present invention relates to a crosslinked nanofiber sheet using an animal cartilage derived tissue and a method for producing the same.

Cartilage, like other connective tissues, is composed of connective tissue cells and acellular matrix, but unlike native connective tissue, it is a specialized connective tissue containing a rigid but somewhat flexible substrate. Most of the connective tissue cells of the cartilage are composed of one cartilage cell, and these cartilage cells are located in the cartilage cavity in the cartilage matrix.

Cell - free substrates are divided into fibers and intact. Most of the fiber is composed of Ⅱ type collagen (collagen), but some cartilage has abundant elastic fibers. Amorphous consist mainly of protein sugars composed of glycosaminoglycans sulfate. In the cartilaginous matrix, a large number of protein sugar molecules are linked by hyaluronic acid, one of the glycosaminoglycans, to the linking protein to form a macromolecule. These macromolecules are also linked to glue fibers. Protein sugar molecules are attached to glue fibers by the intangible glycoprotein chondronectin.

Other substrates include non-collagen proteins and glycoproteins, which account for 10-15% of the dry weight of the cartilage, which mainly help stabilize the structure of the substrate macromolecules and form organic tissues.

On the other hand, the cellular antigen is recognized by the host during tissue transplantation, which may lead to tissue inflammation or immune rejection. However, since the constituents of cell-free matrix are generally resistant to constituents in allogeneic recipients, various cell-free matrices of tissues including cardiovascular, blood vessels, skin, nerve, bone, tendon, bladder, Many studies have been carried out.

However, to date, there has been no research on a technique for radiating such an animal cartilage-derived tissue as a bio-sheet which can be used as an anti-adhesion bio-sheet without using a synthetic polymer such as polyvinyl alcohol (PVA) alone.

Korean Patent Laid-Open Publication No. 10-2013-0134447 (December 13, 2013)

(A) preparing an aqueous animal cartilage derived tissue by modifying an animal cartilage derived tissue; (b) adding the water-soluble animal cartilage-derived tissue to a mixed solvent of a polar organic solvent and an acidic solvent having a pH of 1 to 5, stirring the mixture to prepare a solution, and spinning the solution to prepare a nanofiber sheet; And (c) crosslinking the nanofiber sheet. The present invention also provides a method for producing a crosslinked nanofiber sheet comprising the step of crosslinking the nanofiber sheet.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

(A) preparing an aqueous animal cartilage derived tissue by modifying an animal cartilage derived tissue; (b) adding the water-soluble animal cartilage-derived tissue to a mixed solvent of a polar organic solvent and an acidic solvent having a pH of 1 to 5, stirring the mixture to prepare a solution, and spinning the solution to prepare a nanofiber sheet; And (c) crosslinking the nanofiber sheet. The present invention also provides a method for producing a crosslinked nanofiber sheet.

The crosslinking may be carried out using a crosslinking solution containing at least one crosslinking agent selected from the group consisting of a carbomide compound, an aldehyde compound, an epoxy compound and a diisocyanate compound.

The crosslinking may be performed by immersing the nanofiber sheet in a crosslinking solution, or ultrasonic jetting or electrospinning the crosslinking solution on the nanofiber sheet.

In one embodiment of the present invention, there is provided a method for producing a cartilage-derived cartilage comprising: (a) modifying an animal cartilage-derived tissue to produce a water-soluble animal cartilage derived tissue; (b-1) introducing a first chemical functional group into the water-soluble animal cartilage-derived tissue, adding it to a polar organic solvent and a mixed solvent of an acidic solvent having a pH of 1 to 5, and then stirring to prepare a first solution; (b-2) introducing a second chemical functional group into the water-soluble animal cartilage-derived tissue, adding it to a polar organic solvent and a mixed solvent of an acidic solvent having a pH of 1 to 5, and then stirring to prepare a second solution; And (c) cross-linking the first solution and the second solution simultaneously with spinning by electrospinning. The present invention also provides a method for producing a crosslinked nanofiber sheet.

(Epoxy group, amine group), (epoxy group, thiol group), (acyl group, amine group), (acyl group, azide group) (Acyl group, thiol group), and (tetrazine, cyclooctene).

The crosslinking may be carried out through a chemical reaction between the first chemical functional group and the second chemical functional group.

The polar organic solvent may be at least one selected from the group consisting of hexafluoroisopropanol (HFIP), hexafluoropropanol (HFP), dimethyl formamide, dimethylsulfoxide, methanol, ethanol, And may be at least one selected from the group consisting of propanol, methylenechloride, acetonitrile, tetra hydrofuran and ethyl acetate.

 The acidic solvent having a pH of 1 to 5 may be at least one selected from the group consisting of 0.1 to 1 M of trifluoroacetic acid, acetic acid and formic acid.

The volume ratio of the polar organic solvent and the acidic solvent having a pH of 1 to 5 may be 5: 1 to 10: 1.

For this solution, the aqueous animal cartilage derived tissue may be from 10% to 20% by weight.

And adding a polar aprotic solvent to the solution.

For this solution, the polar aprotic solvent may be between 5% and 15% by volume.

The radiation may be performed using a syringe having an inner diameter of the injector needle of 18 G to 26 G under conditions of a radiation distance of 5 to 20 cm, a voltage of 20 to 30 kV, and an emission rate of 1 to 20 μl / min.

The animal cartilage derived tissue may be derived from pigs, cows, sheep, horses or cats.

In another embodiment of the present invention, there is provided a crosslinked nanofiber sheet produced by the above method.

As a result of measuring the water contact angle of the crosslinked nanofiber sheet for 5 seconds, an angle of 30 DEG or more can be maintained.

According to another embodiment of the present invention, there is provided an anti-adhesion bio-sheet using the crosslinked nanofiber sheet.

The method for producing a crosslinked nanofiber sheet using an animal cartilage-derived tissue according to the present invention is characterized in that a water-soluble animal cartilage-derived tissue is added to a mixed solvent of a polar organic solvent and an acidic solvent having a pH of 1 to 5 And the mixture is stirred to prepare a solution. The mixed solvent is excellent in volatility and can easily decompose the collagen of the tissue derived from animal cartilage, The animal cartilage-derived tissue itself can enable easy irradiation.

The nanofiber sheet spun by the electrospinning method has properties of biodegradability for bio-application through crosslinking, hydrophobic property, and biocompatibility due to low toxicity. Particularly, since the crosslinked nanofiber sheet can be applied to a living body region where adhesion can take place, the effect of preventing adhesion can be maintained at a constant level over a long term or short term, so that it can be effectively used as a biomaterial sheet, .

FIG. 1 is a view showing a method of producing a nanofiber sheet according to an embodiment of the present invention, a crosslinked nanofiber sheet produced therefrom, and a biomaterial sheet using the same.
FIG. 2A is a view showing a step of manufacturing a nanofiber sheet using the electrospinning method according to Examples 1 to 4, FIG. 2B is a view showing a surface of a nanofiber sheet prepared in Example 1 with a naked eye and a scanning electron microscope It's a picture.
FIGS. 3A to 3D are views showing a method of producing a crosslinked nanofiber sheet according to Examples 2 to 5 and a photograph of a surface of a crosslinked nanofiber sheet in Examples 2 to 5, respectively, by scanning electron microscopy. FIG. The photographs of the surface of the nanofiber sheet prepared in Example 1 were visually observed and the photographs of the surfaces of the nanofiber sheet bridged in Examples 2 to 5 were visually observed.
4 is a photograph showing a comparison of biodegradability for biocompatibility between the nanofiber sheet prepared in Example 1 and the nanofiber sheet crosslinked in Example 2. FIG.
FIG. 5 is a photograph showing a comparison of the water contact angle measured for 5 seconds between the nanofiber sheet prepared in Example 1 and the crosslinked nanofiber sheet in Example 2. FIG.
6 is a photograph showing the result of evaluating the toxicity of the crosslinked nanofiber sheet in Example 2. Fig.
7A and 7B are photographs of a surface of a crosslinked nanofiber sheet in Example 2 observed with a fluorescence microscope and an FE-SEM in order to confirm the results of evaluating the anti-adhesion properties of the crosslinked nanofiber sheet in Example 2. FIG.
8 is a photograph showing an experimental procedure and an experimental result showing the effect of preventing adhesion due to crosslinked nanofibers in Example 2 in a long-term adhesion model rat.

The inventors of the present invention studied a method for producing crosslinked nanofiber sheets by singly spinning tissue derived from animal cartilage without mixing a synthetic polymer such as polyvinyl alcohol (PVA), and found that the water-soluble modified cartilage- It is confirmed that a crosslinked nanofiber sheet excellent in characteristics for application to living bodies can be finally prepared by using a solution prepared by mixing a mixed solvent of a polar organic solvent and a mixed solvent of an acidic solvent having a pH of 1 to 5, .

Hereinafter, the present invention will be described in detail.

Method for producing crosslinked nanofiber sheet (1)

One embodiment of the present invention relates to a method for producing a cartilage-derived animal tissue, comprising the steps of: (a) modifying an animal cartilage-derived tissue to prepare a water-soluble animal cartilage derived tissue; (b) adding the water-soluble animal cartilage-derived tissue to a mixed solvent of a polar organic solvent and an acidic solvent having a pH of 1 to 5, stirring the mixture to prepare a solution, and spinning the solution to prepare a nanofiber sheet; And (c) crosslinking the nanofiber sheet. The present invention also provides a method for producing a crosslinked nanofiber sheet.

That is, the method of producing a crosslinked nanofiber sheet according to an embodiment of the present invention is performed by a two-step reaction in which a water-soluble animal cartilage-derived tissue is radiated by electrospinning and then crosslinked.

First, a method of producing a crosslinked nanofiber sheet according to an embodiment of the present invention includes a step of modifying an animal cartilage-derived tissue to produce a water-soluble animal cartilage-derived tissue (step (a)).

As used herein, the term " nanofiber sheet " refers to a sheet having a mesh structure formed by cross-linking fibers having a nano-sized diameter without chemically cross-linking. The term " crosslinked nanofiber sheet " in the present specification refers to a sheet having a mesh structure formed by crossing each other in a single fiber or chemically crosslinked at a point where the fiber and the fiber intersect.

The term "animal cartilage derived tissue" in this specification refers to an animal derived cartilage tissue, preferably an animal derived cartilage acellular matrix (CAM). Specifically, the animal cartilage derived tissue is a pork, It may be derived from horses or cats, preferably from pigs, but is not limited thereto. The fibrous component constituting the tissue derived from animal cartilage is characterized in that it is mostly composed of collagen.

Specifically, in the present invention, an animal cartilage-derived tissue (for example, a powder form) can be prepared by preparing an animal cartilage-derived tissue (for example, a powder form) by lyophilization and freeze- have.

The lyophilization and freeze-pulverization may be performed at an ultra-low temperature condition using the pretreated animal cartilage-derived tissue, and the ultra-low temperature condition refers to a condition of -50 ° C to -100 ° C and 0.01 mTorr to 10 mTorr. In addition, the freeze pulverization can be carried out using a known pulverizer, and the animal-derived cartilage-derived tissue in the form of powder produced by this method can have a size of 10 mu m to 100 mu m.

Thereafter, the prepared animal cartilage-derived tissue is dissolved in an aqueous solution containing a protease, and then the proteinase is neutralized and then the proteinase is removed by dialysis or the like to produce a water-soluble animal cartilage-derived tissue. Then, freeze-drying can be added at an ultra-low temperature. At this time, an enzyme capable of degrading collagen constituting the tissue derived from animal cartilage can be used as the protease, and it is preferably pepsin, but is not limited thereto.

Next, in the method of producing a crosslinked nanofiber sheet according to an embodiment of the present invention, the water-soluble animal cartilage-derived tissue is added to a mixed solvent of a polar organic solvent and an acidic solvent having a pH of 1 to 5, And then spinning it by electrospinning to produce a nanofiber sheet (step (b)).

The mixed solvent includes a polar organic solvent and an acidic solvent having a pH of 1 to 5. The polar organic solvent is a polarized solvent for performing electrospinning and is highly volatile, can do. Specifically, the polar organic solvent may be at least one selected from the group consisting of hexafluoroisopropanol (HFIP), hexafluoropropanol (HFP), dimethyl formamide, dimethylsulfoxide, methanol, ethanol may be at least one selected from the group consisting of ethanol, propanol, methylenechloride, acetonitrile, tetra hydrofuran and ethyl acetate, and hexafluoroisopropanol (Hexafluoroisopropanol: HFIP), but is not limited thereto. In addition, an acid having a pH of 1 to 5 can easily decompose collagen constituting an animal cartilage-derived tissue by maintaining the pH within the above range. Specifically, the acid having a pH of 1 to 5 is preferably at least one selected from the group consisting of 0.1 to 1 M of trifluoroacetic acid, acetic acid and formic acid, more preferably 0.1 to 1 M of trifluoroacetic acid, It is not limited.

 Specifically, the volume ratio of the polar organic solvent and the acidic solvent having a pH of 1 to 5 in the mixed solvent is preferably 5: 1 to 10: 1, more preferably 7: 1 to 9: 1, It does not. At this time, if the volume ratio of the polar organic solvent is too high, the collagen forming the animal cartilage-derived tissue can not be sufficiently decomposed, the needle of the syringe is clogged and the surface of the produced nanofiber sheet is not smooth. When the volume ratio of the five-phosphoric acid solvent is too large, the strength of the nanofiber sheet produced by decomposing the collagen constituting the animal cartilage-derived tissue is extremely weakened.

For the above solution, the water-soluble animal cartilage-derived tissue is preferably 10 wt% to 20 wt%, more preferably 14 wt% to 16 wt%, but is not limited thereto. If the content of the water-soluble animal cartilage-derived tissues is too low, there is a problem that the viscosity is low and the solvent is not appropriately evaporated when the electrospinning is performed, so that the nanofiber sheet can not be produced. There is a problem that spinning can not be performed by an electric force because of high viscosity.

Optionally, the method may further comprise adding a polar aprotic solvent to the solution.

The polar aprotic solvent may be added to increase the electrospinning efficiency as a well-known solvent. In the polar aprotic solvent, the cation is solvated by O or N having δ-, but since the anion has no δ + band, the solvent is not easily solved and is relatively free. It is called naked anion because it is not bonded by a hard interaction.

Specifically, the polar aprotic solvent is selected from the group consisting of dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), hexamethylphosphoric triamide (DMSO) and ethers A solvent may be used, and it is preferably, but not limited to, dimethyl formamide (DMF).

For this solution, the polar aprotic solvent is preferably 5 vol% to 15 vol%, more preferably 10 vol% to 13 vol%, but is not limited thereto. At this time, when the polar aprotic solvent is less than 5 vol% based on the solution, the electrospinning efficiency can not be sufficiently increased, and when the polar aprotic solvent exceeds 15 vol% There is a problem in that a washing process for removing the washing process must be added.

Thereafter, the solution is irradiated by electrospinning, wherein the electrospinning is carried out without mixing a synthetic polymer such as polyvinyl alcohol (PVA), to produce a nanofiber sheet through the radiation of the animal cartilage derived tissue itself contained in the solution Since the nature of the mixed solvent can be removed at the time of electrospinning, a nanofiber sheet having a mesh structure in which nanofibers cross each other can be manufactured.

The electrospinning can be performed using a known electrospinner, and specifically, a voltage difference is generated between the needle of the syringe and the collector, whereby the mixed solvent of the discharged solution is volatilized and the nanofiber is formed, do.

Specifically, the electrospinning method is performed using a syringe needle having an inner diameter of 18 to 26 G, a spraying distance of 5 to 20 cm between the syringe needle and the collector, a voltage of 20 to 30 kV and a discharge rate of 1 to 20 μl / ≪ / RTI >

Next, a method of producing a crosslinked nanofiber sheet according to an embodiment of the present invention includes a step (c) of crosslinking the nanofiber sheet.

The crosslinking may be performed using a crosslinking solution. Specifically, the crosslinking may be performed by immersing the nanofiber sheet in a crosslinking solution, or ultrasonic jetting or electrospinning the crosslinking solution on the nanofiber sheet. At this time, the crosslinking solution preferably includes at least one crosslinking agent selected from the group consisting of a carbomide compound, an aldehyde compound, an epoxy compound and a diisocyanate compound, but is not limited thereto. The cross-linking agent is preferably 0.1 wt% to 10 wt%, more preferably 0.1 wt% to 5 wt% with respect to the cross-linking solution, but is not limited thereto.

For example, it is possible to use 1-ethyl-3- (3-dimethylaminopropylcarbamido), 1-ethyl-3- (2-morpholinyl-4-ethyl) carbomide or dicyclohexylcarbomide as the carbomide compound Formaldehyde, glutaraldehyde or dextrin aldehyde may be used as the aldehyde-based compound. The aldehyde group of the crosslinking agent can chemically react with the amine group of the collagen, and the amine group of the crosslinking agent can be crosslinked by a chemical reaction with the carboxyl group of the collagen.

In addition to the crosslinking agents listed above, a biocompatible polymer having a carboxyl group at the side chain or terminal may be used. The biocompatible polymer may include polycaprolactone (PCL), and may be at least one selected from the group consisting of copolymerizations of monomers such as glycolide (GA), lactide (LA), lactide, and the like. But is not limited thereto. The aldehyde group of the crosslinking agent can chemically react with the amine group of the collagen, and the carboxyl group of the crosslinking agent can be crosslinked by chemical reaction with the amine group of the collagen.

The nanofiber sheet spun by the electrospinning method has properties of biodegradability for bio-application through crosslinking, hydrophobic property, and biocompatibility due to low toxicity. Particularly, since the crosslinked nanofiber sheet can be applied to a living body region where adhesion can take place, the effect of preventing adhesion can be maintained at a constant level over a long term or short term, so that it can be effectively used as a biomaterial sheet, . On the other hand, the non-crosslinked nanofiber sheet exhibits hydrophilic properties, and its solubility in water is too high, making it difficult to apply it to a living body.

(2) A method for producing a crosslinked nanofiber sheet

Another embodiment of the present invention is a method for producing a cartilage-derived animal tissue, comprising the steps of: (a) modifying an animal cartilage-derived tissue to prepare a water-soluble animal cartilage derived tissue; (b-1) introducing a first chemical functional group into the water-soluble animal cartilage-derived tissue, adding it to a polar organic solvent and a mixed solvent of an acidic solvent having a pH of 1 to 5, and then stirring to prepare a first solution; (b-2) introducing a second chemical functional group into the water-soluble animal cartilage-derived tissue, adding it to a polar organic solvent and a mixed solvent of an acidic solvent having a pH of 1 to 5, and then stirring to prepare a second solution; And (c) cross-linking the first solution and the second solution simultaneously with spinning by electrospinning. The present invention also provides a method for producing a crosslinked nanofiber sheet.

That is, the method for producing a crosslinked nanofiber sheet according to another embodiment of the present invention is performed in a one-step reaction in which a water-soluble animal cartilage-derived tissue is crosslinked simultaneously with spinning by electrospinning.

First, a method for producing a crosslinked nanofiber sheet according to another embodiment of the present invention includes a step of modifying an animal cartilage-derived tissue to prepare a water-soluble animal cartilage-derived tissue (step (a)).

Since details of the step (a) are the same as those described in the method (1) for producing a crosslinked nanofiber sheet, the duplicated description will be omitted.

Next, a method for producing a crosslinked nanofiber sheet according to another embodiment of the present invention comprises introducing a first chemical functional group into the water-soluble animal cartilage-derived tissue, and mixing the solvent with a polar organic solvent and a mixed solvent of an acidic solvent having a pH of 1 to 5 Followed by stirring to prepare a first solution (step (b-1)); And a step of introducing a second chemical functional group into the water-soluble animal cartilage-derived tissue and adding it to a polar organic solvent and a mixed solvent of an acidic solvent having a pH of 1 to 5 and then stirring to prepare a second solution [(b-2) .

The first chemical group and the second chemical group are functional groups introduced for crosslinking. One functional group is introduced at the end of the water-soluble animal cartilage-derived tissue, and the other chemical groups of the water-soluble animal cartilage- And can be crosslinked through a chemical reaction.

Specifically, (the first chemical group and the second chemical group) may be selected from the group consisting of (alkaline-base, azide group), (alkaline-base, thiol group), (epoxy group, amine group), (epoxy group, (Acyl group, thiol group) and (tetrazine, cyclooctene group), but is not limited thereto. Specifically, in order to introduce the first chemical functional group, an amino-PEG4-alkyne (alkaline), an alkyne-PEG5-acid (alkaline), an alkyne-PEG- acrylate, acrylic acid, acryloyl chloride, methyltetrazine-amine (tetrazine), methyltetrazine-PEG4-amine (tetrazine), methyltetrazine-propylamine (tetrazine) (tetrazine), methyltetrazine-PEG-NHS ester (tetrazine), methyltetrazine-PEG4-NHS ester (tetrazine) (tetrazine), methyltetrazine-NHS ester (tetrazine), methyltetrazine-acid (tetrazine), and tetrazine-acid (tetrazine) may be used. In order to introduce the second chemical group, azide-PEG4-amine (Azide group), 3-amino-1-propanethiol (thiol group), 11-mercaptoundecanoic acid (thiol group), aminomethanethiol ), thiol PEG amine (thiol group), ethylene diamine (amine group), PEG diamine (amine group), (S) -3-amino-2- (hydroxymethyl) propionic acid (amine group) Trans cyclooctene-amine (cyclooctene), transcyclooctene-NHS ester (cyclooctene), trans cyclooctene-PEG-NHS ester (cyclooctene), and trans cyclooctene-PEG4-acid (cyclooctene).

In addition, the specific content of the mixed solvent is determined in a mixed solvent of the method (1) for producing a crosslinked nanofiber sheet, and the specific contents of the first solution and the second solution are obtained by a method ), And hence redundant description will be omitted.

Next, a method of producing a crosslinked nanofiber sheet according to another embodiment of the present invention includes a step of mixing the first solution and the second solution, followed by crosslinking the same with electrospinning (step (c)), .

Since the first solution and the second solution contain different chemical functional groups as described above, when the electrospinning is carried out, the crosslinking can be performed simultaneously with the spinning, whereby the physical properties of the crosslinked nanofiber sheet Can be increased.

The crosslinking may be carried out through a chemical reaction between the first chemical functional group and the second chemical functional group.

In addition, the specific contents of the electrospinning method are the same as those described in the method (1) for producing a crosslinked nanofiber sheet, and duplicated description thereof will be omitted.

Crosslinked nanofiber sheet and biomaterial sheet using the same

The present invention provides a crosslinked nanofiber sheet produced by the above method and provides a biomaterial sheet using the crosslinked nanofiber sheet, preferably an anti-adhesion biomaterial sheet.

The nanofiber sheet spun by the electrospinning method has properties of biodegradability for bio-application through crosslinking, hydrophobic property, and biocompatibility due to low toxicity.

In order to evaluate the hydrophobic surface characteristics of the crosslinked nanofiber sheet, it is possible to measure the water contact angle by contacting water with the crosslinked nanofiber sheet. As a result of measuring the water contact angle of the cross-linked nanofiber sheet for 5 seconds, it is possible to maintain the wettability and the hydrophobic surface property by maintaining an angle of 30 DEG or more, preferably 50 DEG or more.

Particularly, since the crosslinked nanofiber sheet can be applied to a living body region where adhesion can take place, the effect of preventing adhesion can be maintained at a constant level over a long term or short term, so that it can be effectively used as a biomaterial sheet, .

The method for producing a crosslinked nanofiber sheet using an animal cartilage-derived tissue according to the present invention is characterized in that a water-soluble animal cartilage-derived tissue is added to a mixed solvent of a polar organic solvent and an acidic solvent having a pH of 1 to 5 And the mixture is stirred to prepare a solution. The mixed solvent is excellent in volatility and can easily decompose the collagen of the tissue derived from animal cartilage, The animal cartilage-derived tissue itself can enable easy irradiation.

The nanofiber sheet spun by the electrospinning method has properties of biodegradability for bio-application through crosslinking, hydrophobic property, and biocompatibility due to low toxicity. Particularly, since the crosslinked nanofiber sheet can be applied to a living body region where adhesion can take place, the effect of preventing adhesion can be maintained at a constant level over a long term or short term, so that it can be effectively used as a biomaterial sheet, .

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  1: Manufacture of Nanofiber Sheet Using Pig Tissue Derived from Pigs

(1) Production of pork cartilage derived powder

After removing the pig knee from the pig cartilage-derived tissues, the pig-cartilage-derived tissues were immersed in 1 × PBS to prevent drying. Separated pig cartilage derived tissues were placed in a beaker and washed with PBS until no blood was visible. PBS was removed, washed once with sterile third order, and sterile third order removed. The washed pig cartilage-derived tissues were lyophilized in a cryogenic freezer at -70 ° C and 5 mTorr for 48 hours. The lyophilized pork cartilage pieces were frozen and ground to prepare animal cartilage derived powder. The pork cartilage-derived powder was added to 1 × PBS and stirred at 4 ° C for 4 hours. The solution was dispensed into a 50-mL tube, centrifuged using a centrifuge, and the supernatant was removed. The cartilage-derived powder was transferred to a beaker, followed by addition of SDS buffer and stirring at 4 ° C for 2 hours. After centrifugation at 3,300 rcf for 10 minutes, the supernatant was removed, sterilized third-order water was added, suspended, and centrifuged. After centrifugation, the supernatant was removed, and the pork cartilage-derived powder was transferred to a beaker, followed by addition of DNase solution and stirring at 100 rpm for 12 hours. The solution was then dispensed into 50 mL tubes and centrifuged at 3,300 rcf for 10 minutes. After removal of the supernatant, sterile third order water was added and the animal cartilage derived powder was suspended and washed. After removing the supernatant, the animal cartilage-derived powder was suspended with a small amount of sterilized third order and lyophilized for 48 hours at -70 ° C and 5 mTorr in a cryogenic freezer.

(2) Preparation of water-soluble pig cartilage-derived powder

25 mL of 800 unit / mL pepsin aqueous solution was added to 1 g of the lyophilized pig cartilage derived powder, and the solution was prepared by stirring. NaOH solution was added to the prepared solution to neutralize the solution to pH 7.4, and the dialyzed membrane was washed with flowing water of 7 to 8 cm, rinsed with sterilized third order, and wetted with sterilized third order water. After fixing one end of the dialysis membrane, the solution neutralized in the dialysis membrane was poured using a funnel. The air inside the dialysis membrane was removed as much as possible, the other end was fixed, and the solution was added to sterilized second order 2 L and stirred at 4 ° C for 24 hours. The dialyzed pepsin aqueous solution was placed in a freeze-dried container and lyophilized at -70 ° C and 0 mTorr for 48 hours in a cryogenic freezer to prepare a water-soluble pig cartilage-derived powder.

(3) Production of Nanofiber Sheet

The water-soluble pig cartilage-derived powder was added to a mixed solvent of hexafluoroisopropanol (HFIP): 0.5 M Trifluoroacetic acid (TFA) = 8: 1 by volume ratio at a ratio of 15:85, and stirred for 48 hours Solution. Then, 11% by volume of dimethylformamide (DMF) was added to the prepared solution. Then, the inner diameter of the syringe needle was 24 G [Stubs Iron Wire Gauge system], and the diameter of wire, metal strap, and metal tube was measured internationally A nanofiber sheet having a mesh structure in which nanofibers cross each other was prepared by using electrospinning (homemade ES-1 (DaeLim Starlet, Siheung, Korea)) using a syringe which is a standardized gauge unit 2A), and its surface was observed through a scanning electron microscope (see FIG. 2B). Specifically, the nanofiber sheet was coated with a plasma-sputtering apparatus (Emitech, K575, Kent, UK) and the surface structure was observed through FE-SEM (JSM-6700F, JEOL, Tokyo, Japan).

Example 2: Crosslinking of Nanofiber Sheet Through Immersion in Crosslinked Solution

A crosslinking solution was prepared by adding 1% by weight of glutaraldehyde to a solvent of hexafluoroisopropanol (HFIP). 40 ml of the cross-linking solution was placed in an 80 ml vial, and the nanofiber sheet prepared in Example 1 was cut into a size of 3 x 3 cm and fully immersed in the cross-linking solution, immediately taken out and crosslinked. The above method was repeated twice to prepare a crosslinked nanofiber sheet, and its surface was observed through a scanning electron microscope and a naked eye (see FIGS. 3A and 3E).

Example 3 Crosslinking of Nanofiber Sheet Through Ultrasonic Injection of Crosslinked Solution

A crosslinking solution was prepared by adding 1% by weight of glutaraldehyde to a solvent of hexafluoroisopropanol (HFIP). The nanofiber sheet prepared in Example 1 was placed under a single-axis ultrasonic atomizer, and the cross-linking solution was put in a syringe and then flowed into a single-axis ultrasonic injector at a flow rate of 0.1 ml / min. Thereafter, the mixture was sprayed at a vibration frequency of 60 Hz for crosslinking. The above method was repeated twice to obtain a crosslinked nanofiber sheet. The surface of the crosslinked nanofiber sheet was observed through a scanning electron microscope and a naked eye (see FIGS. 3B and 3E).

Example 4 Crosslinking of Nanofiber Sheet by Electrospinning of Crosslinked Solution

A crosslinking solution was prepared by adding 1% by weight of glutaraldehyde to a solvent of hexafluoroisopropanol (HFIP). The cross-linked nanofiber sheet was prepared by electrospinning the cross-linking solution with the nanofibers prepared in Example 1 by crosslinking the collector, and the surface thereof was observed through a scanning electron microscope and the naked eye (see FIGS. 3C and 3E ). At this time, the electrospinning was carried out using a syringe having an inner diameter of 24 G of a syringe needle under the conditions of a radiation distance of 5 cm, a voltage of 8 kV, and a release rate of 1 μl / h.

Example 5: Cross-linking of nanofiber sheet through chemical reaction between first and second chemical functional groups

5 mg of transcyclooctene (TC) -PEG-NHS ester was added to the water-soluble pig cartilage-derived powder prepared in Example 1 (2) and PBS was added thereto to prepare a water- The mixture was stirred for 30 minutes and then dialyzed for 72 hours. The mixture was placed in a lyophilized container and lyophilized for 24 hours at -83 ° C and 0 mTorr with a cryogenic freezer to introduce a first chemical functional group into the water-soluble pig cartilage-derived powder . Thereafter, the mixture was added to a mixed solvent of hexafluoroisopropanol (HFIP): 0.5 M Trifluoroacetic acid (TFA) = 9: 1 by weight at a weight ratio of 15:85, and stirred for 48 hours to prepare a first solution .

Meanwhile, water soluble pig cartilage-derived powder solution having a concentration of 10 mg / ml was prepared by adding PBS to the water-soluble pig cartilage-derived powder prepared in Example 1 (2), and 5 mmol of tetrazine (TE) -PEG-NHS ester And the mixture was reacted for 30 minutes with stirring. The mixture was dialyzed for 72 hours and lyophilized in a cryogenic freezer at -83 ° C and 0 mTorr for 24 hours to obtain a second chemical functional group in the water-soluble pig cartilage-derived powder Respectively. Then, the mixture was added to a mixed solvent of hexafluoroisopropanol (HFIP): 0.5 M Trifluoroacetic acid (TFA) = 9: 1 by weight at a weight ratio of 15:85, and stirred for 48 hours to obtain a second solution .

Each of the first solution and the second solution was then subjected to electrospinning (homemade ES-1) using a syringe having an inner diameter of 24 G of a syringe needle under the conditions of a spinning distance of 10 cm, a voltage of 24 kV, (DaeLim Starlet, Siheung, Korea)] to prepare a crosslinked nanofiber sheet. The surface of the crosslinked nanofiber sheet was observed through a scanning electron microscope and a naked eye (see FIGS. 3D and 3E).

Experimental Example

(1) Evaluation of biodegradability for bio-application of nanofiber sheet and crosslinked nanofiber sheet

Water was added to 10 ml of 20 ml vial. The nanofiber sheet prepared in Example 1 and the nanofiber sheet crosslinked in Example 2 were cut into 1 x 1 cm and each was put into water to evaluate biodegradability for biomaterial application.

4 is a photograph showing a comparison of biodegradability for biocompatibility between the nanofiber sheet prepared in Example 1 and the nanofiber sheet crosslinked in Example 2. FIG.

As shown in FIG. 4, the nanofiber sheet prepared in Example 1 was completely dissolved after being immersed in water, but it was confirmed that the nanofiber sheet crosslinked in Example 2 was not completely dissolved even when it was immersed in water. That is, although the non-crosslinked nanofiber sheet has a high solubility in water, it is difficult to apply the crosslinked nanofiber sheet to a living body. However, since the crosslinked nanofiber sheet can be controlled in biodegradability due to crosslinking, it can be applied to a living body. It is confirmed that the sheet has an advantage that it can be utilized as a sheet.

(2) Measurement of water contact angle of nanofiber sheet and crosslinked nanofiber sheet

In order to analyze the surface characteristics of the nanofiber sheet due to crosslinking, the water contact angle was measured for 5 seconds by bringing water into contact with the nanofiber sheet prepared in Example 1 and the nanofiber sheet crosslinked in Example 2.

FIG. 5 is a photograph showing a comparison of the water contact angle measured for 5 seconds between the nanofiber sheet prepared in Example 1 and the crosslinked nanofiber sheet in Example 2. FIG.

As shown in FIG. 5, in the case of the nanofiber sheet prepared in Example 1, the water contact angle was not measured. As a result, the non-crosslinked nanofiber sheet had excellent wettability and hydrophilicity due to high solubility in water . On the other hand, in the case of the crosslinked nanofiber sheet in Example 2, the water contact angle was measured to be about 53.6 DEG, and it was confirmed that the crosslinked nanofiber sheet exhibited hydrophobic properties due to low solubility in water.

(3) Evaluation of toxicity of crosslinked nanofiber sheet

In order to evaluate the toxicity of the nanofiber sheet due to crosslinking, the GFP-HUVECs were cultured on the crosslinked nanofiber sheet in Example 2 for 1, 3, and 7 days, and the toxicity was evaluated by adding MTT solution. The results are shown in Fig.

As shown in FIG. 6, it was confirmed that the GFP-HUVECs were not observed on the surface of the crosslinked nanofiber sheet in Example 2. As a result, the crosslinked nanofiber sheet was not toxic .

(4) Evaluation of anti-adhesion property of crosslinked nanofiber sheet

In order to evaluate the anti-adhesion property of the nanofiber sheet due to crosslinking, GFP-HUVECs were cultured on the crosslinked nanofiber sheet in Example 2 for 1, 3, and 7 days, and the GFP-HUVECs were fixed with 10% formaldehyde And the surface was observed through a fluorescence microscope (Olympus IX51, Tokyo, Japan) and an FE-SEM (JSM-6700F, JEOL, Tokyo, Japan) to confirm the amount of fixed GFP-HUVECs The results are shown in FIGS. 7A and 7B.

As shown in FIGS. 7A and 7B, GFP-HUVECs immobilized on the surface of the crosslinked nanofiber sheet in Example 2 were not observed. The crosslinked nanofiber sheet is excellent in anti-adhesion property in vivo application, It has been confirmed that it has an advantage that it can be advantageously used as a biocompatible seat.

(5) Evaluation of anti-adhesion property of cross-linked nanofiber sheet on tissue by animal experiment

The adhesion of the crosslinked nanofiber sheet to tissues was evaluated by using a long adhesion model rat as an experimental subject. Specifically, 8-week-old Sprague Dawley rats (weight: 230-280 g) in a healthy state were adapted to the laboratory for 3 days and then operated on. The rats were anesthetized and the abdomen was shaved and disinfected with povidone, followed by 3 to 4 cm of the abdominal midline. After the laparotomy, the wound was made 1 × 1 cm in the right peritoneum 1 cm away from the incision, and rubbed to the side of the peritoneal wound to rupture the vaginal opening in the cecum tortoise, causing slight hemorrhage. Thereafter, in Example 2, the crosslinked nanofiber sheet was cut into a size of 2 x 2 cm, and the wound portion was put in contact with the wound portion to cover the entire wound region. The treated rats were fed with sufficient water and food for 7 days, sacrificed, and new incision was made in the left lower abdomen to evaluate the degree of adhesion. The experimental procedure and the results of the experiment are shown in Fig.

As shown in Fig. 8, it was confirmed that the long-adhesion model rats had the effect of preventing adhesion due to the crosslinked nanofibers in Example 2.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (17)

(a) preparing an aqueous animal cartilage derived tissue by modifying an animal cartilage derived tissue;
(b) adding the water-soluble animal cartilage-derived tissue to a mixed solvent of a polar organic solvent and an acidic solvent having a pH of 1 to 5, stirring the mixture to prepare a solution, and spinning the solution to prepare a nanofiber sheet; And
(c) crosslinking the nanofiber sheet.
A method for producing a crosslinked nanofiber sheet.
The method according to claim 1,
The crosslinking is carried out using a crosslinking solution comprising at least one crosslinking agent selected from the group consisting of a carbomide compound, an aldehyde compound, an epoxy compound and a diisocyanate compound
A method for producing a crosslinked nanofiber sheet.
The method according to claim 1,
The crosslinking is carried out by immersing the nanofiber sheet in a crosslinking solution or ultrasonic jetting or electrospinning a crosslinking solution on the nanofiber sheet
A method for producing a crosslinked nanofiber sheet.
(a) preparing an aqueous animal cartilage derived tissue by modifying an animal cartilage derived tissue;
(b-1) introducing a first chemical functional group into the water-soluble animal cartilage-derived tissue, adding it to a polar organic solvent and a mixed solvent of an acidic solvent having a pH of 1 to 5, and then stirring to prepare a first solution;
(b-2) introducing a second chemical functional group into the water-soluble animal cartilage-derived tissue, adding it to a polar organic solvent and a mixed solvent of an acidic solvent having a pH of 1 to 5, and then stirring to prepare a second solution; And
(c) cross-linking the first solution and the second solution after spinning it by electrospinning
A method for producing a crosslinked nanofiber sheet.
5. The method of claim 4,
(Epoxy group, amine group), (epoxy group, thiol group), (acyl group, amine group), (acyl group, azide group) (Acyl group, thiol group), and (tetrazine, cyclooctene).
A method for producing a crosslinked nanofiber sheet.
5. The method of claim 4,
Wherein the crosslinking is carried out through a chemical reaction between the first chemical functional group and the second chemical functional group
A method for producing a crosslinked nanofiber sheet.
The method according to claim 1 or 4,
The polar organic solvent may be at least one selected from the group consisting of hexafluoroisopropanol (HFIP), hexafluoropropanol (HFP), dimethyl formamide, dimethylsulfoxide, methanol, ethanol, At least one member selected from the group consisting of propanol, methylenechloride, acetonitrile, tetra hydrofuran and ethyl acetate,
A method for producing a crosslinked nanofiber sheet.
The method according to claim 1 or 4,
The acidic solvent having a pH of 1 to 5 is preferably at least one selected from the group consisting of 0.1 to 1 M of trifluoroacetic acid, acetic acid and formic acid
A method for producing a crosslinked nanofiber sheet.
The method according to claim 1 or 4,
The volume ratio of the polar organic solvent and the acidic solvent having a pH of 1 to 5 is 5: 1 to 10: 1
A method for producing a crosslinked nanofiber sheet.
The method according to claim 1 or 4,
For the solution, the water-soluble animal cartilage derived tissue is 10 wt% to 20 wt%
A method for producing a crosslinked nanofiber sheet.
The method according to claim 1 or 4,
Further comprising adding a polar aprotic solvent to said solution
A method for producing a crosslinked nanofiber sheet.
12. The method of claim 11,
For this solution, the polar aprotic solvent may comprise from 5 vol% to 15 vol%
A method for producing a crosslinked nanofiber sheet.
The method according to claim 1 or 4,
The radiation is carried out using a syringe having an inner diameter of the injector needle of 18 to 26 G under the conditions of a radiation distance of 5 to 20 cm, a voltage of 20 to 30 kV and a release rate of 1 to 20 μl / min
A method for producing a crosslinked nanofiber sheet.
The method according to claim 1 or 4,
The animal cartilage-derived tissue is derived from pigs, cows, sheep, horses or cats
A method for producing a crosslinked nanofiber sheet.
A crosslinked nanofiber sheet produced by the process according to claims 1 or 4.
16. The method of claim 15,
As a result of measuring the water contact angle of the crosslinked nanofiber sheet for 5 seconds,
Crosslinked nanofiber sheet.
An anti-adhesion bio-sheet using the crosslinked nanofiber sheet according to claim 15.

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