KR102093806B1 - PCL and PEG patch scaffold for tissue regeneration and preparing method thereof - Google Patents

Abstract

The present invention relates to a tissue regeneration support for PCL and PEG patches and a method for manufacturing the same, and more specifically, polycaprolactone is added and stirred after adding polycaprolactone (PCL) and polyethylene glycol (PEG) and an acid to an organic solvent. -Preparing a polyethylene glycol copolymer solution; Preparing an electrospinning solution by adding a growth factor to the solution and then stirring; And connecting the prepared electrospinning solution to a pump, and then operating the electrospinning device to collect nanofibers arranged in a spindle shape on the collector; providing a method of manufacturing a PCL and PEG patch tissue regeneration support.
PCL and PEG patch tissue regeneration support according to the present invention can be usefully used for the treatment of acute and chronic tympanic perforation, in particular, to reduce the proportion of surgery in the treatment method that relies on surgery for the treatment of chronic tympanic perforation and show a high recovery rate of the eardrum You can. In addition, the PCL and PEG patch tissue regeneration support can be applied to regeneration and treatment of other damaged tissues, and it is possible to develop and apply various tissue regeneration supports by simulating the tissue structure of different parts of the human body.

Description

PCL and PEG patch tissue regeneration support and its manufacturing method {PCL and PEG patch scaffold for tissue regeneration and preparing method thereof}

The present invention relates to a PCL and PEG patch tissue regeneration support and a method for manufacturing the same, more specifically, a growth factor release type polycaprolactone (hereinafter referred to as 'PCL') and polyethylene glycol (hereinafter referred to as 'PEG') ') It relates to a patch tissue regeneration support and its manufacturing method.

Chronic otitis media (chronic tympanic perforation) is a disease in which perforation of the eardrum persists, and is one of the most common causes of hearing damage in the otorhinolaryngology area, but most of the therapeutic methods still depend on surgical methods (tympanic regeneration).

The eardrum perforation can be largely divided into acute and chronic. Acute eardrum perforation is likely to regenerate naturally when the size of the perforation is not large, but chronic perforation (chronic otitis media) that is more than 3 months does not heal naturally. If it is old, it can cause complications such as irru, purulent, hearing loss, facial nerve palsy, or brain abscess.

For the treatment of chronic otitis media, a surgical method called tympanic regeneration is generally used, which is expensive, requires more than 1 hour of surgery and complicated procedures such as anesthesia, requires hospitalization, and has a recurrence rate. It has many problems, such as reaching 10% or more.

According to the announcement by the National Statistical Office, the number of patients with otitis media from 2009 to 2013 is about 1.7 million on average, and the cost of medical treatment is also estimated to be about 170 billion won.

Many methods have been studied to overcome the problems of these surgical methods, the most classic of which is the paper patch technique. In other words, by cutting and pasting a paper patch on the perforated area, it is a treatment that plays a role in allowing cells of the perforated eardrum to grow as a paper patch as a support. It is generally known to be effective for acute perforation of small perforation size, but not for chronic perforation at all. That is, it is known that the non-biocompatible material causes inflammation and the success rate is less than 10%.

In addition to the paper patch technique, during the study of applying a patch-type scaffold for the treatment of chronic eardrum perforation, an attempt was made to confirm the therapeutic effect by producing a chitosan patch containing growth factors and applying it to a chronic animal model. . The growth factor was supported on the chitosan patch to have sustained release, and through this, it was confirmed that it showed a high healing rate in the chronic perforated animal model. The epidermal growth factor (hereinafter referred to as 'EGF') supported on the chitosan patch is known as an element that affects the proliferation of stem cells, and the patch carrying this stimulates stem cells remaining in the margins of perforation of the eardrum. Therefore, it has been reported to affect chronic perforation treatment. However, the chitosan patch is not approved by the US Food and Drug Administration, and has a problem in that various attempts to easily apply it to the clinic have not been made.

All cells of the human body are in various environments provided by the extracellular matrix (hereinafter referred to as 'ECM'), and the extracellular matrix is reported to have specific nanostructures. According to a recently reported study, in vitro experiments confirmed that the shape, mobility, as well as function of cells can be controlled by the surface nanostructure of the substrate on which cells adhere and grow. Therefore, even in the case of a tympanic tissue, it is suggested that if an appropriate nano-environment is provided, the shape, mobility, and function of the tympanic stem cells may change in response to the shape or arrangement of the nanostructure.

The easiest way to manufacture nanofibers is the electrospinning method, which is a uniform and continuous method that can produce nanofibers that can exhibit various properties depending on the composition ratio. At this time, in the electrospinning method, the part that acquires the spun nanofibers is called a collector, and the properties of the nanofibers obtained according to the shape of the collector can be controlled. Through active research at home and abroad, it has been found that the properties of cells are controlled even in nanostructures obtained through electrospinning.

However, there is no way to prepare a spindle-shaped tissue regeneration support through electrospinning of a polymer having excellent biodegradability and biocompatibility that can be used for research on drug delivery systems and various medical fields.

Republic of Korea Patent Publication No. 2008-0040516

An object of the present invention is to provide a PCL and PEG patch tissue regeneration support that can be used as a non-surgical treatment method for chronic eardrum perforation that is simple, efficient, and has a low relapse rate.

In order to achieve the above object, the present invention is to prepare a solution by adding polycaprolactone (PCL) and polyethylene glycol (PEG) and an acid to an organic solvent, followed by stirring; Preparing an electrospinning solution by adding a growth factor to the solution and then stirring; And connecting the prepared electrospinning solution to a pump, and then operating the electrospinning device to collect nanofibers arranged in a spindle shape on the collector; providing a method of manufacturing a PCL and PEG patch tissue regeneration support.

In addition, the present invention provides a PCL and PEG patch tissue regeneration support, which is a spindle-shaped nanofiber, including polycaprolactone and polyethylene glycol and growth factors.

In addition, the present invention provides a composition for the prevention or treatment of chronic tympanic perforation, including the PCL and PEG patch tissue regeneration support.

PCL and PEG patch tissue regeneration support according to the present invention can be usefully used for the treatment of acute and chronic tympanic perforation, in particular, to reduce the proportion of surgery in the treatment method that relies on surgery for the treatment of chronic tympanic perforation and show a high recovery rate of the eardrum You can. In addition, the PCL and PEG patch tissue regeneration support can be applied to regeneration and treatment of other damaged tissues, and it is possible to develop and apply various tissue regeneration supports by simulating the tissue structure of different parts of the human body.

Figure 1 is a lower plate design of the collector (a), a drawing in which a cylindrical collector is disposed on the lower plate of the collector (b), a design (c) of a metal pillar with a pointed end to be attached to the lower plate of the collector, and modeling by combining all components A drawing showing an electrospinning collector (d) for producing a spindle pattern patch showing one aspect;
FIG. 2 shows an electron microscope image of the electrospun spindle PCL and PEG patch, a picture (a) of the spindle PCL and PEG patch at 200 magnification, and a picture of the spindle PCL and PEG patch enlarged to 3000 times (b) ), A picture showing a spindle PCL and PEG patch enlarged to 300,000 times (c), and a photograph taken at 300,000 times to analyze the thickness of nanofiber strands by analyzing images (d); And
Figure 3 shows the results of cell proliferation test of tympanic stem cells (TM cells) in spindle PCL and PEG patches prepared using electrospinning, tympanic membrane on the patch prepared according to Example 1 and Comparative Examples 1 to 3 This is a diagram showing the results of stem cell proliferation.

Hereinafter, the tissue regeneration support for PCL and PEG patch according to the present invention and a method for manufacturing the same will be described in more detail.

The inventors of the present invention have found that by using the electrospinning method to control the properties of the nanofibers obtained according to the shape of the collector, the properties of the cells can be controlled even in the nanostructure, especially the growth factors carried by the PCL and PEG patches. The present invention was completed by finding out that it is effective in treating chronic tympanic membrane by excellent adhesion and proliferation of tympanic stem cells on PCL and PEG patch support arranged in a spindle shape.

The present invention comprises the steps of adding a polycaprolactone (PCL) and polyethylene glycol (PEG) and an acid to an organic solvent and stirring to prepare a solution; Preparing an electrospinning solution by adding a growth factor to the solution and then stirring; And connecting the prepared electrospinning solution to a pump, and then operating the electrospinning device to collect nanofibers arranged in a spindle shape on the collector; providing a method of manufacturing a PCL and PEG patch tissue regeneration support.

The polycaprolactone is a polyester-based biodegradable polymer polymerized from caprolactone (CL). Polycaprolactone (PCL) is known as a polymer having excellent biodegradability and biocompatibility. Therefore, it can be used for research on drug delivery systems and various medical fields.

The step of preparing the solution may be stirred at 200 to 500 rpm for 24 to 48 hours after adding polycaprolactone and polyethylene glycol and an acid to the organic solvent, but is not limited thereto.

The concentration of polycaprolactone in the organic solvent may be 100 to 160 mg / ml, but is not limited thereto.

The concentration of polyethylene glycol in the organic solvent may be 40 to 100 mg / ml, but is not limited thereto.

The organic solvent is trifluoroethanol (2,2,2-trifluoroethanol), chloroform, chloroform, dichloromethane, tetrahydrofuran, tetrahydrofuran, dimethylformamide, and dimethylsulfoxide It may be any one selected from the group consisting of, but is not limited thereto.

The acid may be any one selected from the group consisting of acetic acid, formic acid, propionic acid, and lactic acid, but is not limited thereto.

The growth factor is a group consisting of epithelial growth factor (EGF), fibroblast growth factor (hereinafter referred to as 'FGF'), insulin-like growth factor binding protein (hereinafter referred to as 'IGFBP'), and combinations thereof. It may be any one selected from, but is not limited thereto.

The epidermal growth factor (EGF) serves to promote the synthesis of DNA, RNA, protein, hyaluronic acid and fibronectin. The EGF is frequently found in the early stages of the healing of the eardrum, and the growth factor in which the EGF is combined with heparin may play an important role in the healing of the eardrum.

The fibroblast growth factor (FGF) helps the migration of fibroblasts, proliferates and restores the cells.

The insulin-like growth factor binding protein (IGFBP) can prevent cell death and promote growth or differentiation depending on the substrate in the cell.

In the step of preparing the electrospinning solution, growth factors may be added to the solution at 0.16 to 16 μg / ml, but is not limited thereto.

The step of preparing the electrospinning solution may be stirred for 20 to 30 minutes at 0 to 10 ° C after adding growth factors to the solution, but is not limited thereto.

The step of collecting the nanofibers may connect the prepared electrospinning solution to the pump and then release it at a rate of 0.2 to 3.0 μl / hr to operate the electrospinning device, but is not limited thereto.

The nanofiber may have an average thickness of 200 to 800 nm, but is not limited thereto.

The collector may be formed in a cylindrical shape for accommodating the needle inside, but is not limited thereto.

Referring to FIG. 1, the collector serves to collect charged nanofibers, and is composed of a copper pipe having excellent conductivity and a sharp metal needle.

In addition, the present invention provides a PCL and PEG patch tissue regeneration support comprising polycaprolactone and polyethylene glycol as nanofibers arranged in a spindle shape.

The PCL and PEG patch tissue regeneration support may further include growth factors, but is not limited thereto.

The growth factor is a group consisting of epithelial growth factor (EGF), fibroblast growth factor (hereinafter referred to as 'FGF'), insulin-like growth factor binding protein (hereinafter referred to as 'IGFBP'), and combinations thereof. It may be any one selected from, but is not limited thereto.

The PCL and PEG patch tissue regeneration support may include polycaprolactone and growth factors in a weight ratio of (7 × 10 ⁴ to 7 × 10 ⁶ ): 1, but is not limited thereto.

The PCL and PEG patch tissue regeneration support may include polyethylene glycol and growth factors in a weight ratio of (3 × 10 ⁴ to 3 × 10 ⁶ ): 1, but is not limited thereto.

The PCL and PEG patch tissue regeneration support may include polycaprolactone and polyethylene glycol in a weight ratio of (2 to 4): 1, but is not limited thereto.

The growth factor may be any one selected from the group consisting of epithelial growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor binding protein (IGFBP), and combinations thereof, but is not limited thereto.

The nanofiber may have an average thickness of 200 to 800 nm, but is not limited thereto.

In addition, the present invention provides a composition for the prevention or treatment of chronic tympanic perforation, including the PCL and PEG patch tissue regeneration support.

The composition may be a pharmaceutical composition or a health functional food, but is not limited thereto.

The pharmaceutical composition may be formulated and used in the form of oral dosage forms, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., according to a conventional method.

In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc.These solid preparations include at least one excipient such as starch, calcium carbonate, sucrose ( sucrose) or lactose, gelatin, and the like.

Also, lubricants such as magnesium stearate and talc are used in addition to simple excipients. Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, can be included. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.

The amount of the compound used as an active ingredient of the pharmaceutical composition may vary depending on the patient's age, sex, weight, and disease, but is 0.001 to 100 mg / kg, preferably 0.01 to 10 mg / kg once to several times a day. can do.

The dosage of the pharmaceutical composition may be increased or decreased depending on the route of administration, the degree of disease, sex, weight, and age. Therefore, the above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition may be administered to various mammals, such as rats, mice, livestock, and humans. All modes of administration can be envisaged, for example, oral, rectal or intravenous, intramuscular, subcutaneous, bronchosuction, intrauterine dura or intracerebroventricular injection.

In addition, the health functional food may be provided in the form of a powder, granule, tablet, capsule, syrup or beverage, and the health functional food is used together with other food or food additives other than the compound according to the present invention as an active ingredient, It can be suitably used depending on the phosphorus method. The mixing amount of the active ingredient can be appropriately determined according to its purpose of use, for example, prevention, health or therapeutic treatment.

The effective dose of the compound contained in the dietary supplement may be used according to the effective dose of the pharmaceutical composition, but may be below the above range for long-term intake for health and hygiene purposes or for health control purposes. , Since the active ingredient has no problem in terms of safety, it is clear that it can be used in an amount exceeding the above range.

There are no particular restrictions on the type of the dietary supplement, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea , Drinks, alcoholic beverages and vitamin complexes.

Hereinafter, the present invention will be described in more detail by the following examples. However, the present invention is not limited by these examples.

<Example 1>

The distance between the needle end of the 22 gage and the collector was fixed at 10 cm, and a voltage of 18 kV was generated by a voltage generator.

280 mg of polycaprolactone (PCL, MW: 80,000, Sigma Aldrich), and 120 mg of polyethylene glycol (PEG, MW: 3,350, Sigma Aldrich) and acetic acid (Acetic acid) in 2 ml of trifluoroethanol (TFE) , Sigma Aldrich) to prepare a solution. Then, the solution was prepared by stirring at a rate of 300 rpm for 24 to 48 hours so that PCL and PEG were completely dissolved in TFE.

After confirming that PCL and PEG were completely dissolved, 4 μg of EGF was added to the solution. Thereafter, the electrospinning solution was prepared by stirring at 4 ° C. for 20 minutes so that EGF was well mixed with PCL and PEG.

The prepared electrospinning solution was put in a syringe and a syringe pump was connected to spun at a rate of 0.6 μl / hr.

As shown in FIG. 1, a collector composed of a conductive copper pipe and a metal needle with a sharp tip at the center was used to collect in the form of a spindle pattern patch, and conditions were applied to generate an electric field of 1.6 ㎸ / cm. By adjusting and operating the electrospinning device, a spindle-shaped PCL and PEG patch tissue regeneration support was prepared. Hereinafter, the PCL and PEG patch tissue regeneration support prepared according to Example 1 was designated as 'PCL / PEG / EGF-A'.

<Comparative Example 1>

It was under the same conditions as in Example 1, except that a collector generally used for electrospinning was hereinafter referred to as 'PCL / PEG / EGF-R'.

<Comparative Example 2>

It was under the same conditions as in Example 1, except that a collector that is generally used for electrospinning without adding growth factor EGF, was hereinafter referred to as 'PCL / PEG-R'.

<Comparative Example 3>

It was under the same conditions as in Example 1, except that the growth factor, EGF, was not added, and was hereinafter referred to as 'PCL / PEG-A'.

<Experiment 1> Morphological analysis of spindle PCL and PEG patch support for tympanic regeneration

Scanning electron microscopy (hereinafter referred to as 'SEM') using a scanning electron microscope (SUPRA, Carl Zeiss, Germany) to analyze the surface properties of spindle-type PCL and PEG patch supports for tympanic membrane regeneration prepared with different spinning conditions ).

2 (a) to 2 (d) show scanning electron microscope images of spindle PCL and PEG patches obtained from a cylindrical collector for accommodating saliva inside.

Referring to Figure 2 (a), through a picture taken at 200 magnification it was confirmed that each nanofiber strand is facing toward the center.

In addition, FIG. 2 (b) and FIG. 2 (c) are images obtained by randomly enlarging a specific portion of the sample, and it can be confirmed that most of the nanofiber strands extend in a straight line.

In addition, referring to FIG. 2 (d), the thickness of each nanofiber strand did not exceed 300 nm.

<Experimental Example 2> In Vitro test of PCL and PEG nano pattern patch

For in vitro testing, rat fibroblast passage 2 (provided by Ajou University otorhinolaryngology, hereinafter referred to as "TM cell") from a rat eardrum was used. After thawing the frozen TM cells, the medium was prepared by mixing DMEM low glucose, 10% FBS (Fatal Bovine Serum), and 1% penicillin, and cultured for 7-10 hours in a 37 ° C, 5% CO ₂ incubator. After confirming that the culture density of TM cells was confluent, it was separated from the culture plate using trypsin EDTA.

In this case, the experimental group was a PCL and PEG spindle nanopattern patch containing EGF (Example 1, PCL / PEG / EGF-A), a nanopatch that emitted EGF-containing PCL and PEG into a nonwoven fabric (Comparative Example 1, PCL / PEG / EGF-R), a nanopatch in which PCL and PEG containing no EGF were spun into a nonwoven fabric (Comparative Example 2, PCL / PEG-R), and a PCL and PEG spindle nanopattern patch without EGF (comparative) Example 3, PCL / PEG-A) was used to perform the in vitro test.

Then, the four types of patches were prepared in a 96-well plate in a circular shape with a diameter of 6 mm and fixed to each plate. Thereafter, 1 × 10 ³ TM cells per well were aliquoted into 96-well plates.

Thereafter, cell activity was measured by the WST test method at intervals of 1 day, 3 days, and 7 days.

3 is a view showing the results of confirming the proliferation of cells by measuring the activity of cells by culturing tympanic stem cells on a patch for 1 day, 3 days, and 7 days.

Referring to the drawings, PCL and PEG patch containing EGF prepared according to Example 1 and Comparative Example 1 and PCL and PEG patch containing EGF prepared according to Comparative Example 2 and Comparative Example 3, 1 day, On days 3 and 7, it was observed that cell adhesion and proliferation were fast on the patch containing EGF.

However, when comparing the PCL and PEG patches containing EGF prepared according to Example 1 and Comparative Example 1, the tendency of cell adhesion to be excellent in the patch prepared according to Comparative Example 1, but cell proliferation at Day 7 Considering the tendency to be superior in the PCL and PEG patches containing EGF prepared according to Example 1, the fast cell proliferation on PCL and PEG nanopattern patches containing EGF prepared according to Example 1 Was observed.

As described above, although the present invention has been described by a limited number of embodiments and drawings, the present invention is not limited thereto, and the technical spirit of the present invention and the following will be described by those skilled in the art to which the present invention pertains. Of course, various modifications and variations are possible within the scope of the claims to be described.

Claims (20)

Preparing a solution containing polycaprolactone and polyethylene glycol by stirring after adding polycaprolactone (PCL) and polyethylene glycol (PEG) and an acid to the organic solvent;
Preparing a electrospinning solution by stirring after adding growth factors to the solution; And
Connecting the prepared electrospinning solution to a pump, and then operating the electrospinning device to collect nanofibers arranged in a spindle shape in a collector;
Including,
The collector, characterized in that made of a cylindrical shape for accommodating the saliva, PCL and PEG patch method for producing tissue regeneration support. The method according to claim 1,
The step of preparing the solution,
After adding polycaprolactone and polyethylene glycol and an acid to the organic solvent, characterized in that the mixture is stirred at a speed of 200 to 500 rpm for 24 to 48 hours, and a method for preparing a tissue regeneration support for PCL and PEG patches. The method according to claim 1,
The concentration of polycaprolactone in the organic solvent,
A method for producing a tissue regeneration support for PCL and PEG patches, characterized in that it is 100 to 160 mg / ml. The method according to claim 1,
The concentration of polyethylene glycol in the organic solvent,
Characterized in that 40 to 100 mg / ㎖, PCL and PEG patch method for producing tissue regeneration support. The method according to claim 1,
The organic solvent,
In the group consisting of 2,2,2-trifluoroethanol, chloroform, dichloromethane, tetrahydrofuran, dimethylformamide, and dimethylsulfoxide Characterized in that the selected one, PCL and PEG patch method for producing tissue regeneration support. The method according to claim 1,
The acid,
Acetic acid, formic acid, propionic acid, and characterized in that any one selected from the group consisting of lactic acid, PCL and PEG patch method for producing tissue regeneration support. The method according to claim 1,
The growth factor,
Epithelial growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor binding protein (IGFBP), and any one selected from the group consisting of a combination of PCL and PEG patch tissue regeneration support Manufacturing method. The method according to claim 1,
The step of preparing the electrospinning solution,
Method of producing a PCL and PEG patch tissue regeneration support, characterized in that the growth factor is added to the solution at 0.16 to 16 μg / ml. The method according to claim 1,
The step of preparing the electrospinning solution,
After the growth factor is added to the solution, characterized in that it is stirred for 20 to 30 minutes at 0 to 10 ℃, PCL and PEG patch method for producing tissue regeneration support. The method according to claim 1,
The step of collecting the nanofibers,
After the prepared electrospinning solution is connected to a pump, it is released at a rate of 0.2 to 3.0 µl / hr to operate the electrospinning apparatus, and the method for manufacturing a tissue regeneration support for PCL and PEG patches. The method according to claim 1,
The nanofiber,
Method of producing a PCL and PEG patch tissue regeneration support, characterized in that the average thickness is 200 to 800 nm. delete Nanofibers arranged in a spindle shape, comprising polycaprolactone and polyethylene glycol,
PCL and PEG patch tissue regeneration support for the prevention or treatment of chronic tympanic perforation further comprising a growth factor. delete The method according to claim 13,
The PCL and PEG patch tissue regeneration support,
Polycaprolactone and growth factors (7 × 10 ⁴ ~ 7 × 10 ⁶ ), characterized in that it comprises a weight ratio of 1: PCL and PEG patch tissue regeneration support. The method according to claim 13,
The PCL and PEG patch tissue regeneration support,
Polyethylene glycol and growth factors (3 × 10 ⁴ ~ 3 × 10 ⁶ ), characterized in that it comprises a weight ratio of 1: PCL and PEG patch tissue regeneration support. The method according to claim 13,
The PCL and PEG patch tissue regeneration support,
Polycaprolactone and polyethylene glycol (2 ~ 4): characterized in that it comprises a weight ratio of 1, PCL and PEG patch tissue regeneration support. The method according to claim 13,
The growth factor,
PCL and PEG patch tissue regeneration support, characterized in that any one selected from the group consisting of epithelial growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor binding protein (IGFBP), and combinations thereof. The method according to claim 13,
The nanofiber,
PCL and PEG patch tissue regeneration support, characterized in that the average thickness is 200 to 800 nm. A composition for the prevention or treatment of chronic tympanic perforation, comprising the PCL and PEG patch tissue regeneration support according to any one of claims 13 and 15 to 19.

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