KR20170060477A

KR20170060477A - Method of manufacturing collagen patch for eardrum regeneration and collagen patch for eardrum regeneration manufactured by the method

Info

Publication number: KR20170060477A
Application number: KR1020150164964A
Authority: KR
Inventors: 박찬흠; 김수현; 이옥주; 주형우; 이정민; 문보미; 박현정; 박예리; 김동욱; 이민채; 정주연; 강길선
Original assignee: 한림대학교 산학협력단
Priority date: 2015-11-24
Filing date: 2015-11-24
Publication date: 2017-06-01
Also published as: KR101778386B1

Abstract

The present invention relates to a method for producing an acid-soluble collagen, which comprises the steps of (1) washing and cutting flippers, (2) extracting acid-soluble collagen from the cleaved tissue subjected to the step (1), (3) (4) dissolving the collagen dried in step (3) in an acidic solution to prepare a collagen solution, (5) freezing the collagen solution obtained in step (4), and (6) ) A method for producing a collagen patch for regenerating eardrum comprising lyophilizing the frozen collagen solution in the step (5) and a collagen patch for regenerating the eardrum produced by the method. According to the present invention, it is possible to shorten the time for regeneration of the eardrum in patients with perforation of the eardrum caused by trauma or chronic diseases, and to induce the restoration boundary to be regenerated without thickening the restored eardrum A collagen patch for eardrum reproduction can be provided.

Description

TECHNICAL FIELD The present invention relates to a collagen patch for regenerating eardrum, and a collagen patch for eardrum regeneration, which is produced by the method.

The present invention relates to a method for producing a collagen patch for regenerating the eardrum and a collagen patch for regenerating the eardrum produced by the method. More particularly, the present invention relates to a collagen patch for regenerating the eardrum, The present invention relates to a method for producing a collagen patch for regenerating the eardrum, which has high physical properties and is effective for perforating the eardrum, and a collagen patch for regenerating the eardrum produced by the method.

Collagen is a major structural protein distributed in the extracellular space of various connective tissues in animals. In addition, collagen is the main component of the connective tissue that forms within the body, and it is the most abundant protein in mammals, accounting for 25% to 35% of the total protein content. Such collagen has a long fiber form and is mainly found in fibrous tissues such as tendons, ligaments, and skin, and also in cornea, cartilage, bone, blood vessels, intestines, intervertebral discs and dentin of teeth.

Collagen proteins are generally composed of two α chains (α ₁ , α ₂ ) and β chains, and are composed of triple helix structures. The amino acid composition of collagen is composed of glycine, proline, alanine, hydroxyproline, glutamic acid and the like. Among them, hydroxyproline Are included in high content. In addition, collagen can be classified into various types according to the structure to be formed, so far 28 types of collagen have been identified. The most common types are collagen I, II, III, IV and V. Type I collagen is the major component of the collagen in the body and is a component of skin, tendons, blood vessels, organs and bones. Type II collagen is the main collagen component of cartilage. Type III collagen is a net structure, It is a major component and is commonly found with type I collagen.

Generally, it is known that as the age of a living body increases, the amount of collagen in the body decreases. This is because as the age increases, the ability to synthesize collagen in the body decreases and the rate of collagen production rapidly decreases. These effects cause aging phenomena such as skin strength and elasticity degradation. It is known that reduction of collagen in the living body affects the regulation of water metabolism in the cells, thereby decreasing the moisture content of the tissues and promoting aging.

For this reason, collagen is known to play a very important role in vivo. With this in mind, research and development of collagen has been proceeding in various fields such as cosmetics, artificial joints, plastic surgery, bone grafting, skin reconstruction surgery, wound healing, and the like.

For example, Patent Document 1 (Japanese Patent Application Laid-Open No. 10-2008-0114798) describes a collagen powder produced by using cowhide and the like, and Patent Document 2 discloses (Japanese Patent Application Laid-Open No. 10-2015-0096566 Discloses a method for producing a cosmetic composition comprising a collagen component derived from a pig skin as a main active ingredient.

Most collagens have been extracted from cattle or pigs and used industrially as described in the prior art, but have problems of limitation due to infectious diseases, religious beliefs or customs due to mad cow disease or foot-and-mouth disease.

On the other hand, the eardrum is located between the ear canal and the middle ear, and is a very small and thin membrane with a width of about 9 × 8 mm and a thickness of about 0.1 mm. The eardrum has a function of amplifying the sound and transmitting it to the ossicle. Such tympanic membrane may cause perforation due to chronic diseases such as otitis media or trauma. In this case, tympanoplasty and eardrum patch surgery can be used as an operative method to promote regeneration of the eardrum.

The above-mentioned tympanoplasty is a surgical operation that helps the eardrum to be regenerated using other soft tissues or autografts at the puncture site. However, soft tissue and autogenous fascia surgery require donor surgery and may cause inflammation and scarring.

The tympanic patch is a method of increasing the success rate of the treatment without complications within a short period of time. It induces the smooth epithelium to be uniformly regenerated along the perineum patch so that the perforation of the tympanic membrane and surrounding patches are in contact with each other. Unlike tympanoplasty, which makes a new tympanic membrane, tympanic patch technique induces natural regeneration of the tympanic membrane by attaching a patch to the perforation of the tympanic membrane, and the regenerated tympanic membrane grows along the patch. However, the conventional eardrum patch uses a paper patch, but has a problem in that efficiency and suitability are poor due to problems such as thickness of regenerated eardrum, regeneration time, and inflammation.

Therefore, in order to overcome these problems, it is required to develop a patch for regenerating the eardrum, which has an optimum physical property suitable for eardrum environment and is effective for perforating the eardrum.

KR 1020080114798 A KR 1020150096566 A

The present invention provides a method for manufacturing a collagen patch for regenerating a eardrum, which has optimal physical properties suitable for the environment of the eardrum by freeze-drying the collagen extracted from the flippers.

The present invention also provides a collagen patch for regenerating the eardrum, which can shorten the time for regenerating the eardrum in patients with perforation of the eardrum caused by trauma or chronic diseases, .

In order to solve the above-mentioned problems, the present invention provides a method for removing collagen, comprising the steps of (1) washing and cutting flippers, (2) extracting acid-soluble collagen from the cleft tissue cut through (1) Soluble collagen extracted in step (2), (4) dissolving the collagen dried in step (3) in an acidic solution to prepare a collagen solution, (5) Freezing the collagen solution, and (6) lyophilizing the frozen collagen solution in the step (5).

The step (2) may include a first collecting step for collecting the supernatant after adding the cleavage tissue to the acidic solution, stirring the supernatant, and centrifuging the supernatant after the first collecting step and collecting the supernatant again And a third collecting step of collecting precipitated collagen after putting the supernatant which has passed through the second collecting step and the second collecting step into ethanol, wherein the acidic solution used is composed of acetic acid, citric acid and formic acid , And the concentration of the acidic solution is preferably 1 to 10% (w / w).

In the step (3), the acid-soluble collagen is dispersed in distilled water and then lyophilized. The lyophilization is performed at a temperature of -40 to 10 ° C and a pressure of 40 to 2000 mTorr for 12 to 72 hours .

The acidic solution used in step (4) may include one or more selected from the group consisting of acetic acid, citric acid, and hydrochloric acid, and the concentration of the acidic solution is preferably 0.01 to 2.0M. In the step (4), the collagen solution may be prepared at a temperature of 2 to 6 ° C, and the concentration of the collagen solution is preferably 0.5 to 5 w / v%. In the step (4), one or more additives selected from the group consisting of chitosan, hyaluronic acid, silk fibroin and gelatin may be further added to the acidic solution to dissolve the acidic solution.

In the step (5), the freezing is preferably performed at -100 to -50 ° C for 12 to 24 hours.

In the step (6), the freeze-drying is preferably performed at a temperature of -40 to 10 ° C and a pressure of 40 to 2000 mTorr for 12 to 72 hours. The collagen patch manufactured through the step (6) may have a sponge-like structure, and the thickness of the collagen patch is preferably 80 to 200 mu m.

The method for preparing a collagen patch for regenerating the eardrum according to the present invention may further comprise a step of crosslinking a collagen patch prepared through the step (6), wherein the crosslinking agent used is genipin, glutaraldehyde (1-ethyl-3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) It can be more than two.

In addition, the present invention provides a collagen patch for regenerating eardrum produced according to the above-described manufacturing method.

According to the method for producing a collagen patch for regenerating the eardrum of the present invention, the acid-soluble collagen is extracted from the flippers and the collagen is formed into a porous sponge form through a lyophilization method. Thus, A collagen patch can be provided.

The collagen patch for regenerating the eardrum produced according to the present invention can shorten the regeneration time of the perforation of the eardrum caused by trauma or chronic diseases and can induce the restoration boundary to be regenerated without thickening the restored eardrum The effect on the perforation of the eardrum is excellent.

In addition, the collagen patch for regenerating the eardrum is excellent in biocompatibility and therefore has a low incidence of inflammation, which is advantageous in use.

1 is a schematic view showing a process of extracting acid-soluble collagen from flippers according to an embodiment.
FIG. 2 is a schematic view showing a process of manufacturing a collagen patch for regenerating eardrum using collagen extracted according to the procedure of FIG.
FIG. 3 shows the results of SDS-PAGE analysis of dried collagen prepared according to the example.
FIG. 4 is a graph comparing the results of SDS-PAGE analysis of the dry collagen prepared according to Examples and Comparative Examples before and after the degradative enzyme reaction experiment.
FIG. 5 is a comparison of gross photographs (A, B) and SEM photographs (C, D) of a collagen patch prepared according to Examples and Comparative Examples.
FIG. 6 is a graph comparing FT-IR analysis results of a follicle collagen patch and a pig collagen patch.
Fig. 7 is a graph comparing the water absorption of the follicle collagen patch (DFC) and the pig collagen patch (PC).
8 is a graph comparing swelling degrees of the follicle collagen patch (DFC) and the pig collagen patch (PC).
FIG. 9 is a graph comparing the porosity of the follicle collagen patch (DFC) and the porcine collagen patch (PC).
FIG. 10 is a graph comparing the analysis results of the cell counting kit-8 (CCK-8) of the follicle collagen patch (DC) and the pig collagen patch (PC).
Fig. 11 shows a comparison of a gill image of a follicle collagen patch and a cigarette paper patch.
FIG. 12 is a comparison of endoscopic photographs after the operation of the collagen, the comparison group, and the control group in which the patch patch technique was performed using the collar patch and paper patches, respectively, and the untreated control group.
FIG. 13 is a graph comparing the healed areas of the experimental group (C), the comparative group (P), and the control group (X) treated with the patches of the follicle collagen and the paper, respectively, .
FIG. 14 is a graph comparing the changes in the cross-section of the eardrum after the operation of the collagen and the comparative group and the untreated control group using the patch collar patch and the paper patch, respectively will be.

The present invention relates to a method for producing a collagen patch for regenerating the eardrum and a collagen patch for regenerating the eardrum produced by the method.

First, a manufacturing method of a collagen patch for regenerating eardrum will be described. The manufacturing method includes (1) washing and cutting a flippers; (2) extracting acid-soluble collagen from the cleaved tissue subjected to the step (1); (3) lyophilizing the acid-soluble collagen extracted in the step (2); (4) dissolving the collagen dried in the step (3) in an acidic solution to prepare a collagen solution; (5) freezing the collagen solution obtained in the step (4); And (6) lyophilizing the frozen collagen solution in the step (5).

1, a process of extracting acid-soluble collagen from flippers is schematically illustrated. FIG. 2 shows a process of preparing a collagen patch for regenerating eardrum using collagen extracted according to the process of FIG. 1, Respectively.

Hereinafter, a method of manufacturing a collagen patch for eardrum reproduction according to an embodiment of the present invention will be described in detail with reference to the accompanying drawings.

(1) Cleaning and cutting step

This step includes washing and cutting the flippers, dipping the flippers in water for 12 to 48 hours to remove the blood and washing in running water, and cutting the washed flippers to a size of 1 mm to 5 mm .

In addition, this step may include removing non-collagen proteins, fats, impurities and the like from the cleaved cut tissue through the above steps. Specifically, the fat may be removed by adding the cut tissue to a basic solution having a concentration of 0.1 to 2.0 M and stirring at 2 to 6 ° C for 12 to 48 hours. Subsequently, the cut tissue from which the fat has been removed is washed in a mixed solution of methanol and chloroform in a weight ratio of 3: 1 to 1: 3, in a mixed solution of any one selected from the group consisting of acetone, ethanol and distilled water Non-collagen proteins and other impurities can be removed. Removal of non-collagen materials through this process can increase the purity of the collagen product, eliminating the cause of odor or decay, thus making the product more stable.

(2) Extraction step of acid-soluble collagen

This step is a step of extracting acid-soluble collagen from the cleaved tissue cut through the step (1), wherein the cleavage tissue is put into an acidic solution and stirred, and then the supernatant is collected, A second collection step of collecting the supernatant after centrifuging the supernatant obtained through the second collection step and a third collecting step of collecting the precipitated collagen after putting the supernatant through the second collection step into ethanol .

The acidic solution used herein may be any organic acid capable of dissolving collagen, and may include any one or more selected from the group consisting of acetic acid, citric acid, and formic acid. Of these, Can be more preferably used. The concentration of the acidic solution is preferably 1 to 10% (w / w).

In the primary collection step, the stirring may be performed at 2 to 6 ° C for 24 to 72 hours, and in the secondary collection step, the centrifugation is performed at a speed of 8,000 to 16,000 rpm at 2 to 6 ° C for 10 to 30 minutes And in the third collecting step, the precipitation may be carried out at 2-6 [deg.] C for 24-72 hours.

In order to completely remove the supernatant remaining in the collagen precipitate after the third collection step, the centrifugal separation is performed at a temperature of 3,000-7,000 rpm at 2-6 ° C for 10-20 minutes. .

As described above, the collagen precipitate in which the supernatant is completely removed is collagen which can be dissolved in acid, that is, acid-soluble collagen. Through this process, the acid-soluble collagen extracted from the connective tissue of lobule is subjected to freeze- It proceeds.

(3) Collagen freeze drying step

This step is a step of lyophilizing acid-soluble collagen extracted from duck-like connective tissue through step (2) to obtain dried collagen. At this time, the acid-soluble collagen may be dispersed in distilled water and then lyophilized. At this time, the freeze-drying may be performed at a temperature of -40 to 10 ° C and a pressure of 40 to 2000 mTorr for 12 to 72 hours. Here, lyophilization is a type of drying method which is obtained by sublimation of freezing the material and lowering the partial pressure of water vapor to make ice directly into steam. At this time, lowering the partial pressure means lowering the pressure to below the triple point of the water. At low pressure, the water in the form of ice does not change into liquid by supplying heat energy but directly sublimates into water vapor. Thus, since the sublimated ice crystals leave space, the dried material has a large number of gaps and is easy to absorb moisture, so that it can be completely re-solution upon re-hydration.

(4) Preparation step of collagen solution

This step is a step of dissolving the collagen dried in the step (3) in an acidic solution to prepare a collagen solution.

The acidic solution may include any organic acid capable of dissolving collagen, and may include any one or more selected from the group consisting of acetic acid, citric acid, and hydrochloric acid. Among them, acetic acid is more preferable Can be used. Here, the acidic solution refers to a solution in which an acid component (acetic acid, citric acid or hydrochloric acid) is dissolved in distilled water, and the concentration of the acidic solution is preferably 0.01 to 2.0M.

The collagen patch manufactured according to the production method of the present invention has a sponge-like structure in which a plurality of pores are present, and is prepared using a collagen dilution solution in which the pH is adjusted using the acid solution. Specifically, by adjusting the pH of the collagen solution to about 2 to 4 by using the acid solution having the above-mentioned concentration range, it is possible to suppress the occurrence of cracks and cracks of the finally obtained collagen patch having the sponge shape, thereby improving the productivity . Particularly, when acetic acid is used as the acidic solution, a collagen patch having a uniform pore diameter can be obtained even if it is frozen and lyophilized without any additional step such as a foaming operation. Therefore, the mechanical property is excellent and cracks and cracks Can be further suppressed.

At this stage, the collagen solution is preferably prepared under a temperature condition of 2 to 6 ° C, and the concentration of the collagen solution thus obtained is preferably 0.5 to 5 w / v%. By adjusting the production temperature and concentration of the collagen solution within the above range, the thickness of the patch for regenerating the eardrum can be adjusted to an appropriate range. Specifically, in consideration of contact with the perforated eardrum, mechanical strength, etc., the thickness of the patch is preferably 80 to 200 mu m.

The collagen solution may further include one or more additives selected from the group consisting of chitosan, hyaluronic acid, silk fibroin, and gelatin. The additive is added to the collagen patch for regenerating the eardrum, which is finally produced, in order to facilitate handling in the dry state. The additive may be added to the collagen solution by being added to the acid solution together with the collagen dried in this step and dissolved therein.

(5) Freezing step of collagen solution

In this step, the collagen solution obtained in the step (4) is frozen. The ice in the collagen solution is frozen through the freezing process to form ice crystals, and the freeze-dried ice crystals are sublimed (dried) in a vacuum state through a freeze-drying step (to be described later) You can get a collagen patch.

As the above freezing method, there can be used a method of rapidly freezing, that is, freezing at a very low temperature (-180 DEG C or less) using liquid nitrogen to minimize the rate at which the collagen solution is frozen to be uniformly frozen. However, And the size of pores that can be formed is restricted. Accordingly, in the present invention, when the collagen solution is frozen, a method of obtaining a uniform pore structure while constantly adjusting the cooling temperature and slowly freezing it to maximize the pore size is used. Considering these points, the freezing in the present invention can be performed at -100 to -50 ° C for 12 to 24 hours.

(6) Freeze-drying step

This step is a step of lyophilizing the frozen collagen solution in the step (5).

The water present in the frozen collagen solution is sublimated into gaseous vapor through the freeze-drying process, and pores are formed at the outlet of the solvent to obtain a light sponge-like collagen patch. Further, since the solvent is removed through freeze-drying, the physical and chemical changes of the collagenous phase are extremely small, and the shrinkage due to drying and the change of shape hardly occur. In addition, collagen natural tissue structure is not destroyed, but exists in a porous state.

Such lyophilization can be carried out by standing at a temperature of -40 to 10 ° C and a pressure of 40 to 2000 mTorr for 12 to 72 hours. The lyophilization conditions greatly affect the quality of the collagen patch base, so careful attention must be paid to its control. Typically, when the lyophilization speed is high, the diameter of the pores formed in the collagen patch base becomes small, so the cooling treatment conditions must be carefully controlled.

By the above-described method, a collagen patch for regenerating the eardrum having a pore-like and flexible sponge-like structure can be produced. Such a collagen patch can be molded into a sheet shape or a shape suitable for the shape of the eardrum for convenience of use.

It is preferable that the thickness of the collagen patch for regenerating the eardrum thus manufactured is 80 to 200 mu m considering the contact between the patch and the puncture eardrum and the mechanical strength of the patch.

The method for preparing a collagen patch for regenerating a eardrum according to the present invention may further comprise a step of crosslinking the collagen patch prepared through the step (6). The cross-linking reaction is carried out in order to give the frozen-dried collagen patch a rigidity capable of maintaining degradation resistance in the body, so that it can serve as a patch for regenerating the eardrum. The crosslinking reaction can be achieved by various methods known in the art, for example, physical or chemical crosslinking methods. The physical crosslinking method includes a dehydrothermal treatment and can be performed, for example, by heating to 90 ° C to 150 ° C under vacuum, followed by holding for 10 to 48 hours. The chemical cross-linking method includes a method using a cross-linking agent, for example, a cross-linking agent such as genipin, glutaraldehyde, glutaraldehyde, One or two or more selected from the group consisting of N-hydroxysuccinimide (NHS), ethyldimethylaminopropyl carbodiimide (EDC), ethyldimethylaminopropyl carbodiimide . &Lt; / RTI >

Meanwhile, the present invention provides a collagen patch for regenerating eardrum produced by the above-described method.

The collagen patch for regenerating the eardrum according to the present invention is used for eardrum patching. The eardrum patch is used for promoting eardrum regeneration in the state where the eardrum is punctured due to trauma or due to inflammation. It is one of the surgical methods. This is the key to improving the success rate of the treatment without complications in a short period of time by inducing the patchy layer to regenerate along the patch by applying patches to contact all margins of the tympanic perforations.

The collagen patch for regenerating the eardrum according to the present invention has a high absorption rate to such a degree that the substance can be transported by diffusion during the period until the generation of a new blood vessel through the patch, and at the same time, . In addition, the collagen patch has a size of pores capable of proliferating and differentiating into the surrounding cells for the formation of a new dermis after application to the perforated margins of the eardrum, and the collagen patch has a new blood vessel And has a connection channel through which blood vessels and substances can be transported between adjacent pores for easy formation, and thus can be suitably used as a patch for regenerating the eardrum.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these embodiments.

Example

&Lt; Preparation of acid-soluble collagen &

A method of producing acid-soluble collagen will be described with reference to FIG.

First, the flippers were immersed in tap water for one day, and the washed flippers were cut into a size of 1 to 5 mm and used as raw materials.

The non-collagen proteins, fats and impurities are removed from the cut tissue of the flippers. For this, the cut tissue is put in 0.5M NaOH solution and the fat is removed by stirring at 4 ° C for 24 hours. The cut tissues from which the fat was removed were sequentially washed with a mixed solution of methanol and chloroform at a volume ratio of 3: 1, acetone, 70% (w / w) ethanol and distilled water to remove noncollagen proteins and impurities Lt; / RTI >

Then, in order to obtain acid-soluble collagen from the cut tissue, the cut tissue was placed in a 5% (w / w) citric acid solution, stirred at 4 ° C for 48 hours, Step. At this time, the citric acid solution was used at a volume ratio of 4 times that of the cut tissue. Subsequently, the supernatant obtained through the primary collection step is centrifuged at 12,000 rpm at 4 DEG C for 15 minutes to collect the supernatant, followed by a secondary collection step. Subsequently, the supernatant obtained through the second collection step is poured into a 100% (w / w) ethanol solution and precipitated at 4 DEG C for 48 hours. Thereafter, the supernatant is removed and the precipitated collagen is recovered, . Subsequently, centrifugation is carried out at 4 ° C for 5 minutes at a speed of 3,500 rpm to completely remove the supernatant remaining in the precipitate after the third collection step. Thus, the process of completely removing the supernatant from the precipitate and extracting acid-soluble collagen from the cuttlefish tissue was completed.

The acid-soluble collagen thus extracted was dispersed in distilled water and lyophilized using a freeze dryer (FDUT-6002; Operon, Korea) until the pressure dropped below 60 mtorr for 48 hours at -37 ° C. Hereinafter referred to as "follicle collagen").

Referring to FIG. 2, a method of manufacturing a collagen patch for regenerating eardrum will be described below.

The dried acid-soluble collagen is dissolved in a 0.5 M acetic acid solution to prepare a 4% (w / v) collagen solution.

The collagen solution is placed in a petri dish and frozen at -80 ° C for 12 hours.

The completely frozen collagen solution was freeze-dried at -37 ° C for 48 hours using a freeze dryer (FDUT-6002; Operon, Korea) until the pressure dropped below 60 mtorr to remove water in the solution, (Hereinafter referred to as "follicle collagen patch").

Comparative Example

A collagen patch (hereinafter referred to as a "pig collagen patch") was prepared in the same manner as in the above example, except that the pig skin collagen supplied from Seewon Cellontec Co., Ltd. was used instead of the flippers.

Assessment Methods

1. Analysis of collagen composition

SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis was performed using 8% gel to examine the composition of duck's foot collagen (DC) prepared according to the above example. Respectively.

As shown in FIG. 3, it was confirmed that two α-chains (α1, α2) having molecular weights of 130 kDa and 110 kDa and one β-chain having a molecular weight of 235 kDa were observed. It is similar to Type I collagen, which constitutes the most part of the collagen in vivo and is a component of skin, tendons, blood vessels, organs and bones. It is known that the composition of collagen is similar to that of Type I collagen .

2. Collagenase enzyme reaction test

In order to confirm the characteristics of collagen reacting only specifically to collagenase, a reaction test for collagenase and collagenase of collagen and pig collagen was carried out. Specifically, each collagen was reacted with collagenase for 1 hour, and subjected to SDS-PAGE analysis using 8% gel. FIG. 4 shows SDS-PAGE analysis results before and after the degradative enzyme reaction of pork collagen and porcine collagen.

4, collagen type I bands appeared normally in the collagenase-treated groups (1 and 2), and collagen type I bands were not observed in the collagenase treated groups (3 and 4) . From this, it can be seen that pork collagen has collagen-specific properties such as collagenase, which is similar to pig collagen.

3. SEM analysis

Sections were observed using a scanning electron microscope (Hitachi, Tokyo, Japan, model: S-3500N) to confirm the structure of the follicle collagen patch prepared according to the above example and the pig collagen patch prepared according to the comparative example Respectively. 5A and 5B each show a gross photograph of a follicle collagen patch and a pig collagen patch, and C and D respectively show SEM photographs of a follicle collagen patch and a pig collagen patch, respectively.

Referring to FIGS. 5C and 5D, it can be seen that both collagen patches have a porous structure that is mutually connected in three dimensions. This patch of porous structure is suitable as a patch for regeneration of the perforation of the eardrum by easy intercellular metabolism and gas exchange during eardrum regeneration.

4. Fourier Transform Infrared Spectroscopy (FT-IR) analysis

Qualitative analysis was performed using an FT-IR measuring device (Perkin Elmer, UK, Model: Frontier) to analyze the structure of the follicle collagen patch and the pig collagen patch manufactured by the above method. Respectively.

Fig. If you look at the FT-IR spectrum shown in Figure 6, the collagen patch, flippers Amide exhibited the peak in the Amide, 1233 cm ^-1 in the Amide, 1555cm ^-1 in the Amide A, 1628cm ^-1 at 3292cm ^-1. Then, the pig collagen patch, Amide exhibited the peak in the Amide, 1237 cm ^-1 in the Amide, 1550cm ^-1 in the Amide A, 1634cm ^-1 at 3316cm ^-1. From the above analysis, it can be confirmed that the follicle collagen patch and the pig collagen patch have similar peak values of Amide A, I, II and III.

5. Assessment of water uptake and swelling ratio

In order to confirm the water absorption and swelling degree of the duck collagen patch (DFC) and the pig collagen patch (PC) manufactured by the above method, each patch was punched into a diameter of 8 mm and immersed in distilled water for 1 hour to obtain water absorption and swelling Respectively. The water absorption degree and swelling degree were calculated by the following equations (1) and (2), respectively, and the results are shown in FIG. 7 and FIG.

Water uptake (%) = (W _s - W _d ) / W _s x 100 (One)

_{Swelling ratio = (W s -W d} ) / W d ... (2)

In the above equations (1) and (2), W _{s represents the} wet weight of the patch, and W _d represents the dry weight of the patch.

Referring to FIG. 7, it can be seen that both the dorsal collagen patch (DFC) and the pig collagen patch (PC) show water absorption of 95% or more. Also, as shown in FIG. 8, the degree of swelling was high in both patches. From these results, it can be seen that the follicle collagen patch (DFC) has excellent hydrophilicity similar to the pig collagen patch (PC).

6. Porosity

Porosity of the follicle collagen patch (DFC) and the porcine collagen patch (PC) prepared by the above method was measured using a liquid displacement process. At this time, ethanol was used as a displacement liquid. A follicle collagen patch and a pig collagen patch were immersed in the same volume of ethanol (V ₁ ) for 10 minutes, respectively. The total volume (V ₂ ) of ethanol in which each patch was immersed was measured. Thereafter, each patch immersed in ethanol was removed, and the volume (V ₃ ) of the residual ethanol was measured. The porosity (P) of each patch was calculated by the following equation, and the results are shown in FIG.

P (%) = [(V ₁ - V ₃ ) / (V ₂ - V ₃ )] × 100

9 shows that porcine collagen patch (PC) and porcupine collagen patch (DFC) exhibit porosity of 88% and 68%, respectively. In the case of a dorsal collagen patch (DFC) Is low.

7. Cytotoxicity test (CCK-8 assay)

Cell Counting Kit-8 (CCK-8) analysis was performed to test the cell viability and toxicity of the follicle collagen patch and the pig collagen patch prepared by the above method. The follicle collagen and porcine collagen patches were prepared to a diameter of 6 mm, sterilized and placed in 96-wells, and NIH 3T3 cells were used. The cells were seeded in each patch for 7 days, and the results of analysis on days 1, 5, and 7 were shown in FIG.

Referring to FIG. 10, it can be seen that the number of cells to be proliferated increases with each period, and it can be seen that each patch has no cytotoxicity. In addition, it can be confirmed that the cell proliferation rate of the follicle collagen patch (DC) is excellent. The data corresponding to Con shown in FIG. 10 are obtained by seeding NIH 3T3 cells with no patches in 96 wells and performing CCK-8 assay. The data were used as criteria for the cell proliferation effect of the collagen patches and pig collagen patches to be.

8. Animal experiments

As a result of the analysis according to the above evaluation methods 3 to 7, the excellent biocompatibility and physical properties of the follicle collagen patch prepared according to the present invention were confirmed, and compared with the pig collagen patch which had been mainly used, the follicle collagen As shown in FIG. From these results, it can be confirmed that the dorsal collagen patch according to the present invention is suitable as a collagen patch for regenerating the eardrum. Therefore, in order to confirm the effect of regenerating the membrane of the collagen patch produced according to the example, a comparative experiment was performed with the cigarette paper used in clinical practice. FIG. 11 shows photographs of a follicle collagen patch (left) and a paper patch (right) manufactured according to the present invention, and the comparative experiment was performed as follows using a rats eardrum perforation model.

Specifically, in order to confirm the eardrum regeneration effect of the paw collagen patch manufactured according to the present invention, an animal experiment was performed in which each patch shown in FIG. 11 was attached to the eardrum perforation model. As experimental animals, experimental rats (SD-Rat, male 8 weeks old) were used and respiration anesthesia was performed using a ventilator. At this time, isoflurane was used as the anesthetic agent, and its capacity was 4% at the initial anesthesia and 2.5% at the maintenance. The end of the disinfected needle was punctured in the eardrum of the experimental rat. Thereafter, a sterilized follicle collagen patch and a paper patch were attached to the puncture site. The rats that had undergone surgery were kept for 14 days, and were photographed with an endoscopic camera once a day.

FIG. 12 shows photographs of the perineal puncture site of rats taken on days 1, 3, 7 and 14 after surgery. Specifically, the control (control) showed that when the patch was not adhered to the perforation site, ) Shows a case where the patch of collagen produced according to the present invention is adhered to the perforation site of the perforation, and a paper of the tobacco paper is adhered to the perforation site of the perforation of the peroneus. As shown in FIG. 12, the naked eye observation showed that the collagen patches were well adhered to the perforation site of the eardrum in comparison with the patches of tobacco paper.

9. Measure healing area of eardrum perforation model

The healing effects of the control group, the experimental group and the comparative group were analyzed using photographs of the perineal puncture sites on the 1st, 3rd, 7th and 14th car shown in FIG. In order to analyze the puncture healing effect according to each case, the area of the puncture site was measured using the Total Imaging Solution software InnerView ^TM Series (ALL RIGHTS RESERVED BY INNERVIEW Co., Ltd.). The healing area of the pericardial perforation measured by the above method is shown in Fig. 13 as a percentage.

13 shows that 16.58% of the control group (X), 44.48% of the control group (C) treated with the follicle collagen patch, and 28.32% of the comparative group (P) treated with the paper patch, Healing effect. This shows that the puncture healing ability of the follicle collagen patch is faster and higher than that of the other groups.

10. Histological Observation of the Eardrum Perforation Model

Epithelial puncture model tissues were removed from experimental rats on the 1st, 3rd, 7th and 14th day after surgery according to the method according to the above evaluation method 8 and histological observation was carried out by hematoxylin & eosin (H & E) Respectively. At this time, the tissues were photographed using a phase contrast microscope (Nikon Eclips 80i, Nikon, Japan), and the results are shown in FIG.

Referring to FIG. 14, it can be seen that the thickness of the reconstructed eardrum (collagen) treated with the paw collagen patch is generally thinner than that of the comparative paper (paper) using the paper patch. The eardrum was composed of three layers of skin layer (outer), intrinsic layer, and mucosal layer (medial). In the experimental group (collagen), the three layers were uniformly formed, It is possible to observe that the composition of the three layers is uneven and thick. These results demonstrate that the follicle collagen patch is more or less attached to the eardrum than the paper patch, and that the effect is more effective in healing the eardrum.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiment, but is to be accorded the widest scope consistent with the appended claims and their equivalents. Should be construed as being included in the scope of the present invention.

Claims

(1) washing and cutting flippers;
(2) extracting acid-soluble collagen from the cleaved tissue subjected to the step (1);
(3) lyophilizing the acid-soluble collagen extracted in the step (2);
(4) dissolving the collagen dried in the step (3) in an acidic solution to prepare a collagen solution;
(5) freezing the collagen solution obtained in the step (4); And
(6) A method for preparing a collagen patch for eardrum, which comprises lyophilizing the frozen collagen solution in the step (5).

The method according to claim 1,
The step (2)
A primary collection step of charging the flint cut tissue into an acidic solution and stirring the same, and collecting the supernatant;
A second collection step of centrifuging the supernatant after the first collection step and collecting the supernatant again; And
And a third collecting step of collecting the precipitated collagen after putting the supernatant passed through the second collecting step into ethanol.

3. The method of claim 2,
Wherein the acidic solution comprises one or more selected from the group consisting of acetic acid, citric acid, and formic acid.

3. The method of claim 2,
Wherein the concentration of the acidic solution is 1 to 10% (w / w).

The method according to claim 1,
Wherein the acid-soluble collagen is dispersed in distilled water and then lyophilized in step (3).

The method according to claim 1,
Wherein the freeze-drying is performed at a temperature of -40 to 10 ° C and a pressure of 40 to 2000 mTorr for 12 to 72 hours.

The method according to claim 1,
Wherein the acidic solution comprises one or more selected from the group consisting of acetic acid, citric acid, and hydrochloric acid in the step (4).

The method according to claim 1,
Wherein the concentration of the acidic solution in step (4) is 0.01 to 2.0M.

The method according to claim 1,
Wherein the collagen solution is prepared at a temperature of 2 to 6 占 폚 in step (4).

The method according to claim 1,
Wherein the concentration of the collagen solution obtained in the step (4) is 0.5 to 5 w / v%.

The method according to claim 1,
The method for producing a collagen patch for regenerating eardrum according to claim 4, wherein, in step (4), one or more additives selected from the group consisting of chitosan, hyaluronic acid, silk fibroin and gelatin are further added and dissolved in the acidic solution.

The method according to claim 1,
Wherein the freezing is performed at -100 to -50 ° C for 12 to 24 hours in the step (5).

The method according to claim 1,
Wherein the collagen patch produced through step (6) has a sponge-like structure.

The method according to claim 1,
Wherein the thickness of the collagen patch manufactured through step (6) is 80 to 200 占 퐉.

The method according to claim 1,
The method for manufacturing a collagen patch for regenerating eardrum according to claim 1, further comprising crosslinking the collagen patch produced through the step (6).

17. The method of claim 16,
The crosslinking can be carried out by using genipin, glutaraldehyde, 1-ethyl-3 (3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide hydroxysuccinimide, NHS) is used as a solvent for the preparation of a collagen patch for regenerating the eardrum.

17. A collagen patch for eardrum reproduction according to any one of claims 1 to 17.