KR20130134447A - New nanofiber membrane and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a manufacturing method of a nanofiber membrane, comprising; a step of improving animal cartilage powder made of animal cartilage structures into presenting water solubility and a step of producing a nanofiber membrane by mixing the improved animal cartilage powder with PVA(polyvinyl alcohol), a synthesized polymer through electric radiation method. The present invention is suitable for producing a hydrogel product or a film type product that cannot be produced with the conventional animal cartilage powder because the animal cartilage powder is improved into an easily soluble material in neutral liquid like a culture medium or a PBS solution. [Reference numerals] (AA) Diameter (nm)

Description

New nanofiber membrane and manufacturing method thereof

The present invention is to modify the animal cartilage powder prepared by using animal cartilage tissue so that it can be easily dissolved in a neutral aqueous solution to mix the hydrogel of the cartilage powder obtained and the synthetic polymer PVA (poly vinyl alcohol) in an appropriate ratio and The present invention relates to a nanofiber membrane prepared by spinning in fibrous form by a spinning method and a method of manufacturing the same.

Cartilage, like other connective tissue, is composed of connective tissue cells and extracellular matrix, but unlike connective tissue, it is a special connective tissue containing a rigid and somewhat flexible substrate. The connective tissue cells of the cartilage are mostly composed of one kind of chondrocyte, and these chondrocytes are located in the cartilage lumen inside the cartilage substrate.

Extracellular matrix is divided into fibers and ground substance. The fiber component is mostly composed of type II collagen (type II collagen, type II collagen), but some cartilage is rich in elastic fibers (elastic fiber). Intangibles are mainly composed of proteoglycans, which consist mainly of sulfated glycosaminoglycans. In cartilage substrate, a large number of protein sugar molecules are linked to hyaluronic acid, one of glycosaminoglycans, by a link protein to form macromolecules. These macromolecules are also combined with collagen fibers. Protein molecules are attached to glial fibers by chondronectin, an intangible glycoprotein.

Other substrates, such as noncollagenous protein and glycoprotein, account for 10-15% of the dry weight of cartilage, which mainly helps to stabilize the structure of the matrix macromolecules and form organic tissues. give.

On the other hand, since the cell antigen is recognized by the host at the time of tissue transplantation, it may cause an inflammatory reaction or immune rejection of the tissue. However, since extracellular matrix components are generally resistant to allogeneic receptors, various extracellular substrates in tissues including cardiovascular, blood vessels, skin, nerves, bones, tendons, bladder, liver, etc. Much research has been conducted regarding the application of.

Conventional techniques known to date include decellularized in natural tissue and the method of using the extracellular matrix of the existing tissue as it is, and the method of using cartilage powder (US Pat. No. 4,656,137). However, these prior arts have limitations in size, porosity, shape, structure, etc. in the use of the extracellular matrix, and thus have limitations in application to commercial and therapeutic purposes.

In view of such a problem, the Republic of Korea Patent Publication No. 10-2010-0041027, after collecting the cartilage of the animal, "powderization" and "use the particle extraction method" porosity without restrictions in physical properties, porosity, form, structure and size 3 The dimensional support was produced. Animal cartilage powder prepared from cartilage tissue having the above-described characteristics can be used more efficiently by removing cells, nuclei, and the like and reducing rejection of components through decellularization.

Animal cartilage powder disclosed in the Republic of Korea Patent Publication No. 10-2010-0041027 is used for the production of cartilage regeneration is used for the production of porous three-dimensional support. In addition, the animal cartilage powder contains a component suitable for cartilage regeneration and various growth factors, etc., mainly composed of type 2 collagen, and has excellent biocompatibility and biodegradability. 0041027.

However, even though the technology using the animal cartilage powder of the Republic of Korea Patent Publication No. 10-2010-0041027 is excellent technology, as in the other technical fields, continuous improvement of the technology is required in the technical field to which the present invention belongs. For example, the conventional animal cartilage powder is not easily dissolved in a neutral aqueous solution, so there is a limit to manufacturing it in the form of a hydrogel, and thus has a disadvantage in that its application range is limited. That is, the conventional animal cartilage powder is not dissolved in a neutral aqueous solution such as distilled water or PBS solution, so that various applications as a biomaterial are difficult, and in particular, there is a limitation in that it is impossible to manufacture various biomaterials in the form of nanofibers formed by spinning in fibrous form.

Accordingly, the present inventors have determined that if animal cartilage powder is modified into a material that is easily dissolved in a neutral aqueous solution such as a culture solution or a PBS solution, it is possible to make a hydrogel or film-type product that cannot be manufactured with conventional animal cartilage powder. As a result, a method of dissolving animal cartilage powder in an acid solution in which pepsin is dissolved and neutralizing the acid solution was devised.

In addition, the present inventors have neutralized the animal cartilage powder by optimizing the appropriate acid solution, the concentration and the treatment time necessary for modifying the animal cartilage powder in consideration of the difference in the concentration and treatment time of the acid solution used according to the substance to be dissolved. It has been successful to modify it so that it can be easily dissolved in an aqueous solution.

On the other hand, the electrospinning method is one of the most widely used method of making nanofibers, but in order to spin the modified animal cartilage powder solution into the fibrous form by the electrospinning method, a smooth solvent volatilization should be made during the electrospinning, Condensation reaction of the polymer occurs to enable a smooth fibrous formation. Therefore, the present inventors also consider the above problems and introduce a process for mixing synthetic polyvinyl alcohol (PVA) as a synthetic animal cartilage powder solution as described above to prepare a new nanofibrous membrane capable of producing a nanofibrous membrane by electrospinning method. The method was completed.

US Patent Publication No. 4,656,137 (1987.04.07) Republic of Korea Patent Publication No. 10-2010-0041027 (2010.04.22) United States Patent Application Publication No. 2007/0248638 A1 (2007.10.25) United States Patent Application Publication No. 2008/0124374 A1 (2008.05.29)

The present invention is to modify the animal cartilage powder prepared by using animal cartilage tissue so that it can be easily dissolved in a neutral aqueous solution to mix the hydrogel of the cartilage powder obtained and the synthetic polymer PVA (poly vinyl alcohol) in an appropriate ratio and It is an object of the present invention to provide a nanofiber membrane prepared by spinning in a fibrous manner by a spinning method and a method of manufacturing the same.

Furthermore, an object of the present invention is to produce a nanofiber membrane having physicochemical properties suitable for use as a biofilm such as an anti-adhesion membrane, using the manufacturing method as described above.

In order to achieve the above object, the present invention comprises the steps of modifying the animal cartilage powder produced by using animal cartilage tissue in water soluble, and mixing the modified animal cartilage powder with synthetic polymer PVA (poly vinyl alcohol) in an appropriate ratio It provides a method for producing a nanofiber membrane comprising the step of producing a nanofiber membrane by the electrospinning method.

Nanofibrous membrane production method of an embodiment of the present invention, (a) preparing an acidic aqueous solution containing the proteolytic enzyme, and (b) animal cartilage powder prepared using the animal cartilage tissue in the prepared acidic aqueous solution Dissolving; (c) neutralizing the acidic aqueous solution with a neutral aqueous solution; (d) removing the remaining proteolytic enzymes to obtain an aqueous cartilage powder modified with water solubility; Preparing a solution in which the modified animal cartilage powder is mixed with polyvinyl alcohol (PVA), and (f) spinning the solution prepared in step (e) by electrospinning to produce a nanofiber membrane.

In the method of manufacturing a nanofiber membrane of an embodiment of the present invention, the proteolytic enzyme may be pepsin, but the present invention is not limited thereto, and any enzyme may be used as long as it is an enzyme capable of degrading collagen constituting cartilage. Of course.

In the method of manufacturing a nanofiber membrane of an embodiment of the present invention, the removal of the proteolytic enzyme may be made by dialysis.

In the method of manufacturing a nanofiber membrane of an embodiment of the present invention, the step of obtaining the water-soluble modified animal cartilage powder may include a step of lyophilization and lyophilization. The water soluble modified cartilage powder may be modified in hydrogel form.

In the method for producing a nanofibrous membrane of an embodiment of the present invention, the mixing ratio of the water soluble modified animal cartilage powder and polyvinyl alcohol (PVA) is 0.5: 1 to 2: 1 by weight. In this case, the polyvinyl alcohol (PVA) is preferably prepared with a solution of PVA dissolved in HFP (Hexafluoro-propanol), which is a highly volatile organic solvent, and then mixed with the water-modified animal cartilage powder.

The method of manufacturing a nanofiber membrane according to an embodiment of the present invention may further include crosslinking the nanofiber membrane. Crosslinking of the nanofiber membrane may be performed by putting the nanofiber membrane in a solution of diluting glutaraldehyde in methanol.

In a preferred embodiment of the method for producing a nanofiber membrane of the present invention, the animal cartilage tissue may be cartilage tissue derived from a spinal cord animal such as pigs, cows, sheep, horses, dogs or cats. More preferably the animal cartilage powder is pig cartilage powder. However, the present invention is, of course, not limited to, including various animal cartilage powders obtained from the prior art for pulverizing animal cartilage. That is, in the present invention, the animal cartilage powder can be used as long as it is a powder obtained by pulverizing the animal cartilage and decellularizing before or after the animal cartilage, or simultaneously with the powdering.

Nanofiber membrane of an embodiment of the present invention has a mesh structure formed by crossing the nanofibers having a diameter of nano units with each other, the composition ratio of animal cartilage powder and PVA (poly vinyl alcohol) is 0.5: 1 ~ 2: 1 by weight ratio It is characterized by.

Nanofiber membrane of an embodiment of the present invention is characterized in that it is used as a biofilm. The biofilm may be an anti-adhesion film.

The present invention can be modified to a material that is easily dissolved in a neutral aqueous solution, such as culture medium or PBS solution, so that the animal cartilage powder is suitable for producing a product in the form of a hydrogel or film that can not be produced with conventional animal cartilage powder.

In addition, the present invention can be modified so that the animal cartilage powder can be easily dissolved in a neutral aqueous solution by optimizing the appropriate acid solution and its concentration and processing time necessary for modifying the animal cartilage powder, so that the modified animal cartilage powder solution By introducing a process of mixing the synthetic polymer PVA (poly vinyl alcohol) in an appropriate ratio, it is possible to make a fiber-like spinning by the electrospinning method has the advantage that the production of nanofiber membrane.

Furthermore, the present invention has the advantage of using a manufacturing method as described above, to produce a nanofiber membrane having a physicochemical property suitable for use as a biofilm such as anti-adhesion film. That is, the nanofibrous membrane prepared according to the production method of the present invention has a high affinity with the cells, thereby high cell engraftment rate, high cell proliferation rate can shorten the process of tissue formation of the cell.

1 is a graph showing the results of measuring the chemical structures of natural cartilage, PCP and PCP-WS using the ATR-IR spectrum.
2 is a view schematically showing an electrospinning apparatus used to manufacture a PCP nanofiber membrane of an embodiment of the present invention.
3 is a scanning electron showing the shape and diameter of the nanofibers prepared by fixing the ratio of PVA to 1 and then adjusting the ratio of PCP-WS to 0 to 2 to prepare a blend solution with various mixing ratios and electrospinning them. Photomicrograph.
4 is a graph showing the results of measuring the tensile load before and after crosslinking of the blended nanofiber membrane prepared from the PCP-WS / PVA blend.
5 is a graph showing the cell adhesion rate of chondrocytes and fibroblasts in the PCP-WS nanofibrous membrane according to the mixing ratio with PVA.
FIG. 6 is a graph showing the cell proliferation rate of fibroblasts over time in the PCP-WS nanofibrous membrane according to the mixing ratio with PVA.
7 is a graph showing the cell proliferation rate of chondrocytes over time in the PCP-WS nanofibrous membrane according to the mixing ratio with PVA.

Hereinafter, the present invention will be described in detail according to embodiments which do not limit the present invention. It should be understood that the following embodiments of the present invention are only for embodying the present invention and do not limit or limit the scope of the present invention. Therefore, what can be easily inferred by the expert in the technical field to which this invention belongs from the detailed description and the Example of this invention is interpreted as belonging to the scope of the present invention. The references cited in the present invention are incorporated herein by reference.

Reference Example 1 Preparation of Porcine Cartilage Powder

Pig cartilage powder (abbreviated as "PCP"), which is an example of animal cartilage powder, is a pig according to the production method described in Korean Patent Publication No. 10-2010-0041027 "Method for Producing Porous Three-dimensional Support Using Animal Tissue Powder". After cartilage was separated and frozen and pulverized, it was prepared by removing the chondrocytes and gene components present in the pig cartilage powder through decellularization.

Example 1 PCP Modification and Characterization of Modified PCP

Proteolytic enzyme pepsin (pepsin) was dissolved in 0.5M hydrochloric acid to prepare a 0.1% pepsin solution, and the solution was added to the PCP prepared according to Reference Example 1 and stirred at 100 to 200 rpm at room temperature for 24 hours.

After dissolving, the acidic PCP solution was neutralized with NaOH solution to pH 7.4, followed by dialysis to remove the remaining pepsin. In dialysis, the PCP solution was stirred at 100-200 rpm at 4 ° C. for 24 hours, and ultrapure water was replaced at intervals of 6-12 hours.

Finally, the dialysis-complete PCP solution was lyophilized and then lyophilized to prepare a water-soluble PCP (hereinafter, abbreviated as "PCP-WS").

1-1. Determination of the Main Components of PCP-WS

In order to measure collagen content, 1 mL of 4N hydrochloric acid aqueous solution was added per 1 mg of PCP-WS prepared in Example 1, and hydrolyzed at 120 ° C. for 4 hours, followed by centrifugation at 12,000 rpm for 10 minutes. . The assay method was a hydroxy proline assay (Hydroxy proline assay). The hydroxyproline assay deoxidizes hydroxyproline, which is specifically present in collagen, by oxidative reaction and then develops 4- (N, N-dimethylamino) benzaldehyde as a coloring reagent [4- (N, N-dimethylamino) benzaldehyde]. Formation of the quinoid compound by the addition of this, the colorimetric method using the absorption spectrum is changed according to the concentration of hydroxy proline (hydroxy proline).

GAG (Glycosaminoglycan) content was quantified using a commercially available Glycosaminoglycan assay kit. The analysis method of the kit is a colorimetric method using a specific compound between the sulfated GAG anionic polymer and DBM blue cationic dye to form a complex, wherein the absorption spectrum is changed according to the concentration of sulfated GAG to be.

As a result of the component test for natural cartilage and PCP-WS as described above, the content of collagen in natural cartilage was about 44%, the content of GAG was about 20%, and in the case of PCP-WS The content was about 38% and the content of GAG was about 12%, similar to natural cartilage (Table 1).

Comparison of Main Component Contents of Natural Cartilage and PCP-WS ingredient content Collagen GAG Natural cartilage 44.31 ± 1.0 19.93 ± 0.1 PCP-WS 38.62 ± 0.9 11.54 ± 0.1

1-2. Chemical Structure Analysis of PCP-WS

1 is a view showing the results of measuring the chemical structure of natural cartilage, PCP and PCP-WS using the ATR-IR spectrum.

The natural cartilage peak appears at 3300cm ^-1 of an amine group (Amine group) as shown in Figure 1, and PCP were equally found in the PCP-WS, the peak of the amide I, amide II and amide III, 1630, respectively cm ^-1 , 1520 cm ^-1 and 1220 cm ^-1 .

In addition, the absorption of each peak (absorbance) showed a tendency to decrease with the progress of the modification from natural cartilage to PCP-WS. These results indicate that amide bonds are produced by the production of PCP from natural cartilage and by the enzymatic digestion of PCP on acidic aqueous solution and the modification of PCP-WS into collagen in the extracellular matrix to be modified into low molecular weight collagen. This decrease was judged as a result. In addition, in the case of the phosphate peak (Phosphate peak), it was confirmed that the intensity of the natural cartilage was detected at a much higher level than PCP and PCP-WS at 1040cm ^-1 . On the other hand, as a result of measuring the chemical structures of natural cartilage, PCP and PCP-WS, it was confirmed that the modified PCP-WS also maintains the same chemical structure as natural cartilage.

1-3. Solubility of PCP-WS

As a result of checking the solubility of the modified PCP-WS in the neutral aqueous solution, PCP-WS showed excellent dissolution characteristics in the neutral aqueous solution (see Table 2 below). This is because the molecular structure of α-helix is dissociated in acidic environment, but in neutral environment, collagen, which has the property of coagulation through self-assembly mechanism, is dissociated into acidic aqueous solution and treated with decomposing enzyme again to lower-molecular weight protein. It is the result of modification to an extracellular matrix containing low molecular weight collagen that can be dissolved in a neutral environment by conversion to a chain.

Solubility of PCP-WS in Neutral Aqueous Solution PCP Neutral Aqueous Concentration PBS Tertiary distilled water Blank 100 100 One% 39.36 14.69 5% 1.68 2.28

In addition, low molecular weight collagen can be dissociated smoothly in a buffer having a certain level or more of ionic strength, so that a solution can be prepared. The PCP-WS of the present invention also contains an extracellular matrix-based biomaterial containing a large amount of collagen. As a result, it showed better solubility than tertiary distilled water in PBS solution with ion concentration of 55 mM.

Example 2 Fabrication and Characterization of PCP-WS Nanofiber Membranes

Electrospinning using the electrospinning machine (nano NC, es robot) as shown in Figure 2, the voltage 25kV, the distance between the needle and the collector 10cm, the speed of the syringe pump 1ml / hr, temperature 20 ~ 30 ℃, humidity 30 It progressed on conditions below%.

The nanofibrous membrane is formed by stacking the nano-sized fibers and stacking them on the collector as the voltage difference between the needle and the collector of the syringe is generated and the solvent of the discharged polymer solution is volatilized.

However, when performing nanofiber production using only the PCP-WS solution, a problem arises in that the removal of ultrapure water, which is a water-soluble solvent, is not smooth. Therefore, in the nanofibrous membrane manufacturing method of the present invention, a blend solution of PCP-WS solution and PVA (polyvinyl alcohol), which is a biocompatible polymer, was prepared and spun onto a fiber to prepare nanofibers. The PVA used in the present embodiment enables the electrospinning of the modified PCP-WS solution, and has excellent biocompatibility as the synthetic biopolymer and excellent physical properties of the manufactured nanofibers.

2-1. Appearance Analysis of Nanofiber Membrane According to Blending Ratio of Blend Solution

The blend solution was prepared by mixing PCP-WS dissolved in tertiary distilled water and a solution of PVA dissolved in Hexafluoro-propanol (HFP), which is a highly volatile organic solvent, by weight ratio.

After fixing the ratio of PVA to 1, the ratio of PCP-WS was adjusted to 0 to 2 to prepare a blend solution at various mixing ratios, and then electrospun to prepare a nanofiber membrane. And, using a scanning electron microscope (scanning electron microscope) was confirmed the shape and diameter of the nanofibers produced for each mixing ratio (see Figure 3). As a result, as shown in FIG. 3, when the weight ratio of PCP-WS: PVA is 0.5: 1 to 2: 1, a uniform nanofiber was formed and the diameter of the nanofiber was also good.

2-2. Evaluation of Biodegradability of Nanofiber Membrane According to the Mixing Ratio of Blend Solution

In order to evaluate the biodegradability of the PCP-WS nanofibrous membrane prepared according to step 2-1, after measuring the dry weight of the square nanofibrous membrane cut to a size of 1.5 × 1.5 cm and put the sample in PBS solution 37 The mixture was stirred at 150 rpm for 24 hours. As a result of the experiment, it was confirmed that all nanofiber membranes were completely decomposed as shown in Table 3 below.

Biodegradability Evaluation of PVA Nanofiber Membranes and Nanofiber Membranes Prepared from PCP-WS / PVA Blend Solution PVA PCP-WS 0.5
: PVA 1 PCP-WS 1
: PVA 1 PCP-WS 2
: PVA 1 Weight before decomposition (mg) 12 12 12 13 Weight after decomposition (mg) Disassembled

2-3. Tensile strength measurement after chemical crosslinking to strengthen the properties of nanofiber membrane

Nanofibers prepared from PVA and PCP-WS / PVA blends are composed of water-soluble polymers, which are unstable in aqueous solutions such as PBS solution and cell culture solution. To this end, first, dilute glutaraldehyde in methanol to prepare a 10% glutaraldehyde solution, and then add nanofiber membranes to the solution and stir at 100 to 200 rpm for 4 hours at 37 ° C to crosslink the nanofibers.

In order to confirm the physical properties of the nanofibrous membrane before and after crosslinking, the tensile strength of the nanofibrous membrane was measured before and after crosslinking. Tensile strength specimens were prepared by cutting a nanofibrous membrane into rectangles of 1 × 3 cm size, and the thicknesses were measured using a universal measuring instrument (Tinius Olsen, H5KT).

As shown in FIG. 4, the tensile load before and after the crosslinking was measured, and in the case of the blended nanofiber membrane prepared from the PCP-WS / PVA blend, the tensile load after the crosslinking was increased. However, when the mixing ratio of PCP-WS and PVA was 2: 1, and the content of PCP-WS was higher, the physical properties of the specimen before crosslinking could not be measured.

Example 3 Evaluation of Cell Affinity of Nanofiber Membranes of the Present Invention

3-1. Chondrocyte Culture Isolated from Porcine Cartilage Tissue

The knee joints of the Yorkshire pigs within 2 weeks of age were taken, and the knee tube insulated bone tissues were removed from the tibia and recovered. The cartilage flakes were chopped to about 1-2 mm. To separate chondrocytes from cartilage tissue, 20 mL of 0.1% collagenase (Collagenase type II, Washington, USA) solution per 1 g of cartilage was added and placed in a stirrer in a cell incubator at 37 ° C., 5% CO _2. It stirred for 18 hours at 100-200 rpm in conditions.

After 18 hours of 0.1% collagenase treatment, only the chondrocytes were filtered with a cell filter, centrifuged (1500-2000 rpm, 5 min), and the supernatant was removed to separate only the chondrocytes. The isolated porcine chondrocytes were seeded at a concentration of 1 × 10 ⁴ cells in a 100Φ culture dish. During cell culture, medium [DMEM + 20% NCS (Newborn Calf Serum) + 1% antibiotic (penicillin- streptomycin solution) + 0.05% ascorbic acid] was exchanged three times a week for two to three days. .

3-2. Comparison of Cell Adhesion Rate in PCP-WS Nanofiber Membranes According to Mixing Ratio with PVA

In order to evaluate the cell adhesion rate, the PCP-WS nanofibrous membrane prepared according to the mixing ratio with PVA by the method of Example 2 was first cut to a size of 1.2 × 1.2 cm and placed on a 24 well culture dish. In the experimental group, the chondrocytes isolated in step 3-1 were seeded at a concentration of 1 × 10 ³ cells, and in the control group, the fibroblasts (fibroblast, HFF-1) purchased from ATTC were seeded.

After incubation for 6 hours in a cell incubator at 37 ° C., 5% CO ₂ , washed with PBS and fresh medium was added. Then, CCK-8 kit (Cell Counting Kit-8, DOJINDO, Japan) was added and cultured in a cell incubator at 37 ° C. and 5% CO ₂ conditions for another 4 hours, and 200 μm of each medium was dispensed into 96 wells. Next, absorbance at 450 nm was measured using a microplate reader (iMark, Biorad, Japan). The results are shown in FIG. 5 and both the experimental group (chondrocytes) and the control group (fibroblasts) showed good cell adhesion rates to the nanofibrous membrane.

3-3. Comparison of Cell Proliferation Rate in PCP-WS Nanofibrous Membranes According to the Mixing Ratio with PVA

Chondrocytes and fibroblasts were seeded at a concentration of 1 × 10 ³ on the PCP-WS nanofibrous membrane in the same manner as described above in 3-2. Then, the cells were cultured in a cell incubator at 37 ° C. and 5% CO ₂ , and the proliferation rate of the cells was measured on days 1, 3, and 5 after the cultivation (see FIGS. 6 and 7).

To evaluate the cell proliferation rate, CCK-8 kit (Cell Counting Kit-8, DOJINDO, Japan) was added to the medium cultured for 1 day, 3 days and 5 days, and the cells were treated at 37 ° C. and 5% CO ₂ for 4 hours. After culturing in an incubator, each medium was dispensed into 96 wells at 200 μm, and then absorbance was measured at 450 nm using a microplate reader (iMark, Biorad, Japan).

The results are shown in Figures 6 and 7, it can be seen that the higher the content of PCP-WS, the more proliferation rate of the fibroblasts as well as the chondrocytes. In conclusion, it was confirmed that physical properties and cell affinity were improved when the nanofiber membrane was prepared by mixing the hydrogel dissolved in tertiary distilled water with the modified water soluble PCP in an appropriate ratio.

Therefore, the nanofibrous membrane prepared according to the manufacturing method of the present invention has a high affinity with the cells, thereby high cell engraftment rate, high cell proliferation rate, and can shorten the process of cell tissue formation. That is, the nanofibrous membrane prepared according to the manufacturing method of the present invention has a physicochemical property suitable for use as a biofilm such as an anti-adhesion membrane.

In addition, since the nanofibrous membrane of the present invention is manufactured by mixing a natural biomaterial and a synthetic synthetic polymer for medical use, the nanofibrous membrane is less toxic and has biodegradability, so that it is absorbed in vivo and does not remain as a foreign substance. Therefore, the nanofiber membrane of the present invention may be applied as an anti-adhesion membrane by satisfying the requirements of the anti-adhesion agent in the form of a film in response to the recent development of anti-adhesion products based on biomaterials.

Although the present invention has been described in accordance with the above-described embodiments, the present invention is not limited to the disclosed embodiments. Those skilled in the art will appreciate that modifications can be made in accordance with the spirit of the invention, and such modifications also fall within the scope of the invention.

Claims (12)

(a) preparing an acidic aqueous solution containing proteolytic enzymes,
(b) dissolving the animal cartilage powder prepared by using animal cartilage tissue in the prepared acidic aqueous solution;
(c) neutralizing the acidic aqueous solution with a neutral aqueous solution;
(d) removing the remaining protease and obtaining a water soluble modified animal cartilage powder,
(e) preparing a solution in which the water-soluble modified animal cartilage powder is mixed with polyvinyl alcohol (PVA),
(f) manufacturing a nanofiber membrane comprising the step of producing a nanofiber membrane by spinning the solution prepared in step (e) by electrospinning. The method of claim 1,
Obtaining the water-modified animal cartilage powder is a method of producing a nanofibrous membrane, characterized in that it comprises a step of lyophilization and lyophilization. The method of claim 1,
The method for producing a nanofibrous membrane, characterized in that the water-soluble modified animal cartilage powder is modified in a hydrogel form. 4. The method according to any one of claims 1 to 3,
The mixing ratio of the water-soluble modified animal cartilage powder and polyvinyl alcohol (PVA) is 0.5: 1 to 2: 1 by weight ratio. 4. The method according to any one of claims 1 to 3,
The polyvinyl alcohol (PVA) is prepared as a solution of PVA dissolved in HFP (Hexafluoro-propanol) and then mixed with the aqueous cartilage powder modified with water solubility. 4. The method according to any one of claims 1 to 3,
Method for producing a nanofiber membrane further comprising the step of crosslinking the nanofiber membrane. The method according to claim 6,
The step of crosslinking the nanofiber membrane is a method of producing a nanofiber membrane, characterized in that the nanofiber membrane is put in a solution diluted with glutaraldehyde (glutaraldehyde) in methanol. 4. The method according to any one of claims 1 to 3,
The animal cartilage tissue is a method of producing a nanofibrous membrane, characterized in that the cartilage tissue derived from pigs, cows, sheep, horses, dogs or cats. 9. The method of claim 8,
The animal cartilage powder is a method for producing a nanofibrous membrane, characterized in that the pig cartilage powder. In the nanofiber membrane,
Nanofiber membrane, characterized in that the nanofibers having a diameter of the nano unit has a mesh structure formed while crossing each other, the composition ratio of animal cartilage powder and polyvinyl alcohol (PVA) is 0.5: 1 ~ 2: 1 by weight ratio. The method of claim 10,
Nanofiber membrane, characterized in that used as a biological membrane. 12. The method of claim 11,
The biofilm is a nanofiber membrane, characterized in that the anti-adhesion film.

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