WO2011065672A2

WO2011065672A2 - Method for producing medical dressing using pig skin

Info

Publication number: WO2011065672A2
Application number: PCT/KR2010/007507
Authority: WO
Inventors: 김태운; 김태호
Original assignee: Kim Tae Woon; Kim Tae Ho
Priority date: 2009-11-24
Filing date: 2010-10-29
Publication date: 2011-06-03
Also published as: KR20110057391A; KR101139434B1; WO2011065672A3

Abstract

Disclosed is a method for producing medical dressing made of skin, comprising the following steps: separating, peeling and obtaining the entire thickness of skin from a pig for medical use; dipping the thus-obtained skin into low-concentration glycerol, and spin-culturing the skin using high-concentration glycerol; washing the skin using a sterile saline solution after being cultured in the high-concentration glycerol; spin-culturing the skin in a 0.1 N NaOH solution after washing; neutralizing the skin treated with the NaOH solution, in 0.1 N HCl, and washing the skin using a saline solution; cutting the washed skin, permitting the skin to have a mesh structure, and packing the skin in 20 to 30% glycerol; and placing dry ice around the packed product, and sterilizing the packed product using gamma rays. The skin dressing of the present invention employs a physical treatment method and a chemical treatment method at the same time, thus completely removing contaminants caused by pig hair and immunogens which might remain in the pig skin even after the treatment using the high-concentration glycerol. According to the present invention, the skin which is cultured in high-concentration glycerol is dipped in the low-concentration glycerol during the packing step, freeze-stored and sterilized using gamma rays, thus preventing deformation of the skin, which might occur in the event of gamma ray sterilization performed in high-concentration glycerol, and also preventing discoloration of glycerol.

Description

Manufacturing method of dressing material using pig skin

The present invention relates to a method for producing a dressing material using a pig skin sterilized gamma rays within a range that is maintained stably without changing the original structure of the skin tissue.

Self-healing function in living organisms loses its function when a certain level of damage occurs, and thus requires additional treatment. In particular, in case of skin damage, such as burns, trauma, surgery or chronic ulcers, a skin transplantation process is required. In the case of burns, which are the most common causes of skin damage, the first-degree burns with damaged epidermal layers rapidly spread the apocrine gland, eccrine gland, sebaceous gland, and epidermal cells around the hair root to restore the damaged area. In the case of second degree burns in which a part of the dermis is damaged, the wound is recovered in a similar manner to the first degree burns, but apocrine gland, eccrine gland, sebaceous gland, and many parts of the hair root are damaged and only a part of them remain, resulting in slow recovery and infection. Increases the risk. In third degree burns that damage the epidermis, dermis and subcutaneous layer, all parts of the apocrine, eccrine and hair roots are damaged and the epidermal cells proliferate only at the borderline with healthy skin, which significantly reduces the recovery rate. do,

Currently used skin transplantation treatment is a self-transplantation method that removes the healthy skin tissue of the body and transplants it to the traumatic area. It takes a lot, and the economic burden is also very large. In addition, when there is not enough healthy body parts such as severe burn patients, autograft cannot be applied or transplantation should be performed several times. Recently, a reticular graft method for processing healthy skin into reticular grafts and implanting them in a wider area has been carried out, but there are problems such as scarring and shrinkage in the graft site. In addition, when allogeneic skin is used, a large cryogenic freezer is required for the storage because it must be stored by cryogenic freezing, and there is a problem that structural damage of the skin tissue occurs due to the cryogenic storage. In addition, when using the stored skin, there was a problem that preparation work such as thawing and washing before use is necessary.

In addition, when the skin is stored and used, sterilization is required. In most cases, ethylene oxide is used as a method of sterilizing the skin. However, the ethylene oxide may modify the biological properties of the skin to be transplanted, and there is a problem of generating harmful toxic residues such as ethylene oxide, ethylene chlorohydrin, and ethylene glycerol. And there was a problem that the heat or chemical treatment can not be applied to the soft tissue by the sterilization method. In addition, gamma-ray sterilization of high concentration of glycerol may cause crosslinking of collagen matrix or breakdown of connective tissue of polymers, and these processes are difficult to control, and thus, there is a problem that the matrix structure of the skin can be hardened or worsened and thus cannot be used.

In addition, the sterilization and long-term storage of bacteria by simple glycerol culture, which has been developed in the past, is a technology that can be used only for humans, and there is a problem in that it is insufficient in securing safety for disease transmission for application to tissues derived from heterologous animals.

An object of the present invention is to prepare a safe biological dressing material by gamma-ray sterilization using low concentration glycerol culture. The present invention is intended to use as a dressing material for the treatment of skin trauma patients by collecting skin tissue from pigs selected for medical purposes (pig that passed the hematological and histological examination for the common pain disease).

In addition, in order to secure the safety of skin tissue derived from heterologous animals, using a low concentration glycerol culture method and frozen gamma-ray sterilization method using dry ice to prepare a biological dressing material that is cheaper than the existing biological dressing material and the supply is easy.

In another aspect, the present invention aims to remove the immunogenic substances with the removal of hair, hair follicles, hair roots of the pig skin as a raw material in the manufacturing process.

In addition, by immersing the skin cultured in a high concentration of glycerol in a low concentration of glycerol in the packaging step and frozen storage to perform gamma-ray sterilization to prevent changes in the original structure of the skin tissue to prevent discoloration of glycerol.

Method for preparing a skin dressing agent of the present invention is to remove the entire skin layer from the medical pig in order to solve the above problems and peeling; the step of immersing the collected skin in a low concentration of glycerol and stirring culture with a high concentration of glycerol; Washing the skin with sterile physiological saline after culturing the high concentration of glycerol; stirring and culturing the skin with 0.1 N NaOH solution after the washing; neutralizing the skin treated with the NaOH solution in 0.1 N HCl and washing with physiological saline ; Cutting the washed skin is completed and made into a network structure and then put in 20 to 30% glycerol and packaging; and Gamma-ray sterilization of the packaged product in the dry ice load.

The present invention uses medical pig skin. Pig's skin is so thick and densely distributed that it can not be compared to human skin, so it is possible to use both physical shaving and chemical treatment at the same time to completely remove pollutants and immunogens caused by hair. Removed. In particular, the use of 0.1 N NaOH solution can completely eliminate immunogens that may remain in hair as well as high concentrations of glycerol treatment.

The glycerol serves to prevent contamination by the skin microorganisms and bacteria to be transplanted. In addition, the glycerol enables the storage of biological dressings without the need for rapid freezing facilities. The glycerol may further use an antibiotic.

Preferably, the gamma rays of the gamma irradiation step is characterized in that irradiated with a 20 to 30kGy dose. Most preferably irradiated with gamma rays of 25 kGy dose. The irradiation not only inhibits the activity of enzymes involved in autolysis to inhibit the degradation of the skin at low temperatures, but also kills bacteria and viruses that may remain after glycerol treatment. The irradiated skin is preferably sealed in an aluminum pouch in order to minimize moisture absorption. The sensitivity of gamma rays decreases in the order of pests, E. coli, spore-forming bacteria, spore-forming bacteria, spores and viruses. The pest belongs to Hwaranggok moths, cinnabar beetle, powder mites, cockroaches, beetles, weevil pests, such as low grain pests, and the gamma radiation dose is killed at 0.5 to 3 kGy. The E. coli group includes E. coli, Salmonella, typhoid fever, Listeria, dysentery, pneumococcal (Legionella), etc., the gamma radiation dose is killed at 1 to 5 kGy. The spore-forming bacteria and spore-forming bacteria belong to Vibrio bacteria, Pseudomonas aeruginosa, Anthrax, Bacillus subtilis, Staphylococcus aureus, Streptococcus, and various fungi, but the gamma-irradiation is killed at 5 to 10 kGy. The spores belong to bacterial spores, Endotoxin, Exotoxin, fungal poison (Mycotoxin, Aflatoxin), botulinum poison, etc., the gamma radiation dose is killed at 10 to 20 kGy. The virus belongs to foot-and-mouth disease, ebola, and various viruses, and is killed when the gamma-irradiation dose is 30 kGy or more. The dressing material safely kills bacteria that may remain despite glycerol treatment by irradiating gamma rays with a 20-30 kGy dose. In gamma ray sterilization, gamma rays are characterized in that 20 ~ 30 kGy, which is a range without degeneration of the skin tissue to serve as a dressing material.

Preferably, the glycerol treatment step, collecting and washing the skin of the mammal to a thickness of 0.2 ~ 2mm and then transporting the skin in a sterile 45-55% primary glycerol solution; Shaking culture of the skin contained in the primary glycerol at 37 ° C. for 1.5-2.5 hours in sterile 70-80% secondary glycerol solution; And refrigeration of the shake cultured skin in sterile 80 to 90% tertiary glycerol solution for 2 weeks at 4 ℃. The reason for treating the glycerol while gradually increasing the concentration is to remove the water contained in the skin to be transplanted step by step. This is because if the treatment of high concentration of glycerol immediately may damage the skin tissue by osmotic pressure is to process step by step from low concentration. In this step, a high concentration of glycerol is used to kill cells through the high concentration of glycerol and to kill bacteria and viruses that may be included in the skin to be transplanted.

In addition, the conditions of stirring culture with glycerol is characterized in that the culture for 3 to 12 hours at room temperature of 20 ~ 25 ℃.

On the other hand, when the gamma ray sterilization is stored in a low concentration of glycerol of 20 to 30%, the packaged product is sterilized by keeping the frozen state below -70 ℃ with dry ice, thereby preventing degeneration of the skin tissue. Safe dressing materials can be produced.

The skin dressing of the present invention solves a problem when using the skin of a medical pig whose hair is thick and densely distributed so as to be incomparable with human skin by simultaneously using a physical and chemical treatment method to treat pollutants caused by hair and a high concentration of glycerol. There is also an effect that completely eliminates the immunogen that can remain.

In addition, there is no structural damage to the skin tissue as it is processed at room temperature, and by irradiation with gamma rays, it prevents the activity of enzymes related to autolysis, inhibits the decomposition of the skin at low temperatures, and removes bacteria or viruses that may remain after glycerol treatment. It is safe and useful because it does not cause secondary infection during skin graft and no residual toxic substances remain.

In addition, the skin dressing of the present invention can prevent the deformation of the skin caused by the gamma-ray sterilization in the existing high-concentration glycerol by sterilizing gamma-ray sterilization by placing the skin cultured in a high concentration of glycerol in a low concentration of glycerol in the packaging step and frozen. It is also effective in preventing discoloration of glycerol.

In the above description of the preferred embodiment of the present invention, those skilled in the art will be able to perform various modifications without departing from the gist of the present invention, and such modifications are within the protection scope of the present invention. The protection scope of the present invention should be construed as described in the claims.

1 is a photograph of gamma-ray sterilized skin tissue cultured in high concentration glycerol,

2 is a photograph of gamma-ray sterile skin tissue stored in low concentration glycerol,

3 is a photograph of H & E tissue staining before removing the immunogen by treatment with 0.1N NaOH,

Figure 4 is a H & E tissue staining picture after removing the immunogen by treatment with 0.1N NaOH,

5 is an immunohistochemical staining photo before 0.1N NaOH treatment,

Figure 6 is a picture of immunohistochemical staining after cell removal by treatment with NaOH,

7 is a photo of the gamma-ray sterilization by loading the finished product with dry ice,

8 is a photograph showing the skin washed with glycerol and irradiated with gamma rays,

9 is a diagram showing the electron microscope at 100 magnification by irradiating the skin with gamma rays of different degrees and staining with H & E stain (hematoxylin and eosin stain).

10 is a diagram showing the elastin fibers at 100 times magnification of the elastin fibers after irradiating the skin with gamma rays having different degrees.

FIG. 11 is a diagram showing collagen at 100-fold magnification after irradiating the skin with gamma rays having different degrees.

Hereinafter will be described in detail step by step for the manufacturing method of the skin dressing material according to the present invention.

1. 의료용 돼지로부터 피부 채취1. Skin Collection from Medical Pigs

Medical pigs that passed hematological and histological examinations, such as acquired common infectious diseases, were sacrificed using drugs, and then the entire skin layer was separated after disinfecting the skin with antiseptic drugs.

2. 박피2. Peel

Skin tissue including the separated epidermal and dermal layers was peeled with a thickness of 0.2 ~ 0.8 mm using a medical peeler.

3. 글리세롤 배양3. Glycerol Culture

The collected skin was stored in a glycerol solution containing antibiotics and antibacterial agents at room temperature and maintained at 50% concentration for 1 day. The skin tissue stored in 50% glycerol solution was visually inspected to confirm that there was no problem with the skin tissue and solution, and transferred to a glycerol solution containing antibiotics and antibacterial agents at room temperature and maintained at 75% concentration for 2 hours. Incubated at 37 ℃ temperature, and transferred to a glycerol solution to maintain a concentration of 85% again and incubated again under the same conditions for 3 to 12 hours.

It was confirmed that the water contained in the skin tissue is gradually removed by the treatment of the glycerol. The glycerol treated skin is transferred back to fresh 85% glycerol and stored for 2 weeks at 4 ° C. refrigeration to kill cells through high concentration of glycerol and to kill bacteria and viruses that may be contained in the skin. It was. After 2 weeks, the refrigerated skin was washed twice with physiological saline for 10 minutes in a shaker.

4. 멸균생리식염수 세척4. Sterile physiological saline washing

After incubation with 85% high concentration of glycerol, the skin was washed three times for 10 minutes with 0.9% sterile saline solution.

5. 0.1N NaOH 처리5. 0.1N NaOH Treatment

The skin washed with physiological saline was stirred and incubated for 24 hours using 0.1N NaOH solution to remove hair, hair follicles, hair roots and other immunogenic substances from the skin. That is, due to the nature of the pig skin by chemical treatment using 0.1N NaOH not only to remove the skin of the pig skin, there is an effect that can completely remove the immunogen that may remain in the general high concentration glycerol treatment. 3 and 4 are H & E histostained photographs before and after 0.1 N NaOH treatment, it can be seen that the immunogen is removed by NaOH treatment. 5 and 6 show the immunohistochemical staining after removal of the cells by 0.1N NaOH treatment and 0.1N NaOH treatment, respectively.

6. 0.1N HCl 처리6. 0.1N HCl treatment

The 0.1N NaOH-treated skin was neutralized in 0.1N HCl for 10 minutes and washed with sterile saline for 30 minutes.

7. 피부재단 및 그물망 절단기(mesher)로 제품 제작 및 포장7. Production and packing of products with skin foundation and mesh cutter

Cut the skin after washing with physiological saline to the standard and make a network structure using a mesh cutter. In low concentration of glycerol it was packaged in individual packaging. If the glycerol concentration is less than 20% during storage, there is a problem of tissue damage due to crystal formation. If the glycerol concentration is more than 30%, skin deformation occurs during sterilization of gamma rays, so the finished product should be stored in 20 to 30% glycerol. do.

8. 감마선 멸균8. Gamma Ray Sterilization

Skin cultured in a high concentration of glycerol was stored in a low concentration of 20% glycerol in the packaging step, and frozen in the presence of dry ice at -78 ℃ was sterilized using gamma rays (60-Co gamma irradiation).

1 is a photograph of skin tissue sterilized by gamma rays while being stored in a high concentration of glycerol, which causes deformation of skin tissue and discoloration of glycerol. On the other hand, as shown in Figure 2 stored in a low concentration of glycerol of 20% and gamma sterilized skin tissue in the frozen state of -78 ℃ did not change the skin tissue or discoloration of glycerol.

That is, since it is stored in a low concentration of glycerol and gamma-ray sterilization in a frozen state of less than -70 ℃, it is possible to prevent the deformation (hardening) of the skin and discoloration of glycerol generated by gamma-ray sterilization in a high concentration of glycerol.

As shown in Table 1, the optimum gamma irradiation dose for the preparation of the skin dressing material of the present invention was confirmed within the range of 20 to 30 kGy.

Table 1

Confirmation of Gamma Ray Optimal Dose

Radiation dose (kGy) Sterilization temperature Glycerol concentration	15		20		25		30		35		40
	Room temperature	-70	Room temperature	-70	Room temperature	-70	Room temperature	-70	Room temperature	-70	Room temperature	-70
	85	20	85	20	85	20	85	20	85	20	85	20
Normal spore-forming bacteria and spore-forming bacteria	Almost killed		Death		Death		Death		Death		Death
Microstructure of keratinocytes (fear formation)	Fear Formation11%	No change	18%	10%	19%	15%	25%	18%	41%	27%	46%	35%
Microstructure of Fibroblasts (Adipose Expansion)	Private body expansion 28%	17%	28%	18%	29%	20%	35%	20%	37%	20%	Private somatic cell destruction	25%
Vascular endothelial cells	No damage	No damage	No damage	No damage	No damage	No damage	Private body expansion 15%	No damage	Private body expansion 23%	No damage	Private body expansion 28%	Private body expansion 15%
Collagen Microstructure	No damage	No damage	No damage	No damage	No damage	No damage	No damage	No damage	No damage	No damage	No damage	No damage

As shown in Table 1, when the gamma-irradiation dose is 20 to 30 kGy, the skin tissue of the dressing material can be stably maintained without changing the original structure of the tissue.

Hereinafter, it was examined through experiments whether the skin tissue damage caused by the gamma irradiation.

* 감마선 조사에 따른 진피의 미세구조의 손상여부* Damage to the microstructure of the dermis following gamma irradiation

To determine how gamma rays affect skin tissue, the skin was taken from a normal abdominal skin of a 35-year-old woman with the dermis layer down to 24.5 × 24.5㎠A control group and an experimental group were prepared by placing the plate on an assay plate of size and cutting it into a size of 4 × 12 to 5 × 8 cm 2 to be rectangular.

The experimental group was irradiated with a gamma ray dose of 15, 20, 25, 30, 35, and 40 KGy to the skin prepared by the method described above and then observed the microstructure of the skin by electron microscopy. Referring to FIG. 8, it can be seen that there is no change in appearance by irradiating gamma rays to the skin treated with glycerol.

Referring to FIG. 9, the skin was irradiated with gamma rays having different degrees, and then stained with H & E stain (hematoxylin and eosin stain) to determine whether the structure of the skin was damaged. The collagen matrix is observed. Looking at the dermal structure of the skin irradiated with gamma rays and the control group without irradiating gamma rays, it was observed that the dermal structure was not destroyed or damaged. Despite the high dose, the dermal structure was not significantly different from the control group. .

* 감마선 조사에 따른 엘라스틴 섬유의 미세구조의 손상여부* Microstructure of Elastin Fibers Damaged by Gamma Irradiation

The experimental group irradiated gamma rays to the skin prepared by the above-described method at 15, 20, 25, 30, 35, and 40 KGy, respectively, and then observed the microstructure of the skin by electron microscopy.

Referring to FIG. 10, the skin was irradiated with gamma rays having different degrees, and then elastin fibers were observed with an electron microscope at a magnification of 100 to determine whether the structure of the skin was damaged. The elastin fibers did not have a difference between the irradiated skin and the irradiated skin, and even when irradiated with a high degree of gamma rays, the structure of the elastin fibers did not change, indicating that the gamma rays did not affect the elastin fibers.

* 감마선 조사에 따른 콜라겐의 미세구조의 손상여부* Damage of collagen microstructure by gamma irradiation

To determine how gamma rays affect skin tissue, the skin was taken from a normal abdominal skin of a 35-year-old woman with the dermis layer down to 24.5 × 24.5㎠The control plate and the experimental group were prepared by placing the plate on an assay plate of size and cutting it into a size of 4 × 12 to 5 × 8 cm 2 so as to be rectangular.

The experimental group irradiated gamma rays to the prepared skin by gamma rays at 15, 25, and 35 KGy, respectively, and observed the microstructure of the skin by electron microscopy.

Referring to FIG. 11, the skin was irradiated with gamma rays of different degrees to determine whether collagen was damaged by gamma irradiation, and then the collagen was observed under an electron microscope. It was observed that there was no structural difference among the tissues irradiated with gamma by irradiation dose compared to the tissues without irradiated gamma. In addition, it was confirmed that the cells are affected by the irradiation dose of gamma rays above 30KGy, it was confirmed that the epidermis (epidermis) drop due to some autolysis of itself.

In addition, it was confirmed that the microstructure of keratinocytes, the microstructure of fibroblasts, the microstructure of hematopoietic endothelial cells, and the microstructure of gelatinous fibers were not affected by gamma irradiation.

Claims

Separating and peeling the entire skin layer from the medical pig;

Immersing the collected skin in glycerol at a concentration of 45 to 55% and then stirring and culturing in glycerol at a concentration of 70 to 90%;

Washing the skin with sterile physiological saline after culturing the high concentration of glycerol;

Stirring and culturing the skin in 0.1 N NaOH solution after the washing;

Neutralizing the skin treated with the NaOH solution in 0.1 N HCl and washing with physiological saline;

Cutting the completed skin and making a network structure and then packing the glycerol at a concentration of 20 to 30%; and

Method for producing a skin dressing material comprising the step of gamma-ray sterilization of the packaged product in a frozen state.
The method of claim 1,

The gamma ray is a method for producing a skin dressing material of 20 ~ 30 kGy.
The method of claim 1,

Method for producing a skin dressing material, characterized in that the culture for 3 to 12 hours at room temperature of 20 ~ 25 ℃ stirred culture with glycerol.
The method of claim 1,

The skin tissue packaged during gamma-ray sterilization is a method of manufacturing a skin dressing material, characterized in that it maintains a frozen state below -70 ℃ with dry ice.