Composition for preparing acellular allogeneic dermis, and method for preparing acellular allogeneic dermis by using same

Abstract

According to a composition for preparing acellular allogeneic dermis, and a method for preparing acellular allogeneic dermis by using same, of the present invention, epidermal removal, cell removal, fat removal, and hair root removal from allogeneic skin tissue can be simultaneously performed, and loss of growth factors and collagen present in skin tissue, that can occur during preparation of acellular allogeneic dermis, can be minimized.

Description

Composition for producing acellular allogeneic dermis and method for producing acellular allogeneic dermis using the same

[0001] The present invention relates to a composition for producing acellular allogeneic dermis and a method for producing acellular allogeneic dermis using the same.

[0002] Skin tissue is composed of the epidermis, dermis, and subcutaneous tissue, and the dermis layer below the epidermis layer is mainly composed of fibroblasts and extracellular matrix such as collagen secreted by fibroblasts.

[0003] The skin, formed from the above skin tissue, is the largest organ covering the entire surface of the human body and performs the important function of preventing the loss of body fluids and the inflow of harmful substances and microorganisms from the outside, and protecting our body from physical and chemical stimuli.

[0004] Meanwhile, in cases where the skin tissue is severely damaged due to burns, trauma, ulcers, bedsores, etc., it is necessary to regenerate it, and for this purpose, autologous transplantation, which is a method that can minimize immune rejection reactions, is mainly used.

[0005] However, in the case of autologous transplantation, there is a problem that the patient's pain may increase due to the surgical burden of having to remove the patient's own skin tissue, and there is a problem that it may be dangerous if the patient's health is not good. Therefore, in order to overcome the problems of the autologous transplantation method, many studies are being conducted recently on methods of using processed skin substitutes.

[0006] Skin substitutes can be classified into human-derived allogeneic dermis, animal-derived xenogeneic dermis, and synthetic ones. Allogeneic dermis-derived skin substitutes (acellular allogeneic dermis) are relatively expensive, but they have the advantage of not causing immune rejection or inflammatory reactions after transplantation, and are therefore considered the optimal skin substitute.

[0007] Acellular Dermal Matrix is ​​a product that selectively removes only the cellular antigens that are the target of the immune response while maintaining various structural proteins and components without damaging the three-dimensional structure of the dermal layer of the skin tissue provided by cadaveric skin from donors. It has important characteristics such as very low antigenicity, rapid blood vessel formation ability, and stability as a dermal layer, so it can be used in areas where skin tissue is severely damaged due to burns, trauma, or other diseases.

[0008] Specifically, acellular allogeneic dermis can be transplanted onto a skin defect, and fibroblasts and blood vessels from the patient's wound surface grow into the three-dimensional network-like structure of acellular allogeneic dermis, serving as a support to form a new dermis. This not only reduces scarring but also creates an immediate physiological closed wound, preventing external infection. In addition, the basement membrane between the dermis and epidermis is preserved, helping epidermal regeneration and engraftment, and since new dermal tissue is formed quickly, the patient's own skin graft can be performed simultaneously, thereby shortening the hospital stay.

[0009] However, the characteristics of the acellular allogeneic dermis can be greatly influenced by the final components of the processed acellular allogeneic dermis, and how the donated cadaveric skin tissue is handled or processed can have a significant difference in the treatment effect for the patient.

[0010] Accordingly, the inventor of the present invention conducted research to prevent changes in the components or properties of skin tissue that may occur during the multi-step processing of cadaver skin tissue, as well as to minimize the loss of effective ingredients that may affect the survival rate, such as growth factors contained in skin tissue, and as a result, simplified the method for producing acellular allogeneic dermis and derived a composition for producing acellular allogeneic dermis in which specific effective ingredients are combined to minimize the loss of effective ingredients, and a method for producing the same, which were embodied in the present invention.

[0011] [Prior Art Literature]

[0012] [Patent Document]

[0013] (Patent Document 1) KR 10-1107022 B1

[0014] The purpose of the present invention is to provide a composition for producing acellular allogeneic dermis and a method for producing acellular allogeneic dermis using the same.

[0015] Another object of the present invention is to provide a composition for producing an acellular allogeneic dermis capable of simultaneously performing epidermal removal, cell removal, fat removal, and hair root removal on homologous skin tissue due to the effect of mixing each effective ingredient, and a method for producing an acellular allogeneic dermis using the same.

[0016] Another object of the present invention is to provide a composition for producing acellular allogeneic dermis, which can minimize the loss of growth factors and collagen present in skin tissue that may occur during the process of producing acellular allogeneic dermis, and also maintain excellent mechanical strength, and a method for producing acellular allogeneic dermis using the same.

[0017] In order to achieve the above purpose, a composition for producing an acellular allogeneic dermis according to one embodiment of the present invention comprises an epidermal separating agent, a nonionic surfactant, sodium deoxycholate and a thioglycolate compound, and can simultaneously perform epidermal removal, cell removal, fat removal and hair root removal on allogeneic skin tissue.

[0018] The above-mentioned epidermal separator is selected from the group consisting of sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA), and mixtures thereof.

[0019] The above nonionic surfactant is selected from the group consisting of Triton X-100, Tween 20, Tween 40, Tween 60, Tween 80, Nonidetpy-10 (NP-10), Nonidetpy-40 (NP-40) and mixtures thereof.

[0020] The pH range of the above composition is 6.5 to 7.5.

[0021] The above composition has an excellent effect of maintaining collagen and growth factors in acellular homologous dermis.

[0022] According to another embodiment of the present invention, a method for producing an acellular allogeneic dermis comprises: 1) a first washing step of washing allogeneic skin tissue in distilled water; 2) a step of treating the allogeneic skin tissue that has undergone the first washing step with a composition of the present invention; and 3) a second washing step of treating the allogeneic skin tissue that has undergone the treatment step with phosphate buffered saline (PBS).

[0023] According to another embodiment of the present invention, an acellular homologous dermis is manufactured by the above manufacturing method.

[0024] According to the composition for producing acellular allogeneic dermis of the present invention and the method for producing acellular allogeneic dermis using the same, not only can epidermal removal, cell removal, fat removal, and hair root removal of allogeneic skin tissue be performed simultaneously, but also the loss of growth factors and collagen present in skin tissue that may occur during the process of producing acellular allogeneic dermis can be minimized.

[0025] Figure 1 shows the results of confirming the simultaneous removal effect on the epidermis, cells, fat, and hair roots of a composition for producing acellular homologous dermis according to one embodiment of the present invention.

[0026] Figure 2 shows the results of confirming the simultaneous removal effect on the epidermis, cells, fat, and hair roots of a composition for producing acellular homologous dermis according to one embodiment of the present invention.

[0027] The present invention relates to a composition for producing an acellular allogeneic dermis, which comprises an epidermal separating agent, a nonionic surfactant, sodium deoxycholate and a thioglycolate compound, and which can simultaneously perform epidermal removal, cell removal, fat removal and hair root removal on allogeneic skin tissue.

[0028] Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement them. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[0029] The term "acellular dermal layer" or "acellular allogeneic dermis" in the specification of the present invention may refer to an acellular dermal matrix (ADM) that is skin tissue in which cells in the epidermis or dermis that may cause an immune response in a patient after transplantation are substantially removed from skin tissue separated from an individual, and can provide a three-dimensional structural support for the influx of fibroblasts, nerve generation, and blood vessel regeneration after transplantation. The term "substantially removed" as described above means "substantially not including" because it is substantially removed, and "substantially" may refer to a state in which cells, etc. are removed by 90% or more, for example, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, compared to the raw material. Meanwhile, the acellular allogeneic dermis of the present invention is processed using an allogeneic skin tissue derived from a human body.

[0030] Early studies on the processing and preparation of acellular allograft dermis employed various methods, including trypsin treatment, repeated freeze-thawing, and prolonged incubation to allow intradermal enzymes to function. However, the acellular allograft dermis produced using these methods harbored excessive antigenicity, raising concerns about immune rejection. Consequently, research and development have focused on developing processing compositions and methods for efficiently processing acellular allograft dermis.

[0031] Meanwhile, a brief look at the processing and manufacturing process of acellular allogeneic dermis involves conducting a raw material test on the received cadaver skin, washing it with a protective solution to protect the tissue, and then applying an epidermal stripping solution to remove the epidermis, which is the antigen target of the immune response. Afterwards, any remaining epidermal stripping solution is washed with saline solution, and then the cells within the dermis, another factor that causes immune rejection, are treated with a detergent. Afterwards, any remaining hair roots and lichen planus are removed, and then freeze-dried for preservation and storage.

[0032] Specifically, prior art KR 10-1523878 B1 discloses a step of removing epidermal tissue by treating skin tissue with a neutral proteolytic enzyme, and then a step of removing cells of the dermal layer using a surfactant. However, when using a proteolytic enzyme in the epidermal removal process, there is a disadvantage in that the concentration and reaction time must be finely adjusted depending on the tissue sample, and there is also the inconvenience of having to perform de-epidermization and decellularization in separate processes using separate reagents.

[0033] Meanwhile, prior art 10-2450461 B1 discloses a technique capable of simultaneously performing de-epidermization, de-fat removal, and decellularization, including a step of treating skin tissue with a hypotonic solution containing an ionic surfactant and then washing it with an isotonic solution. This partially solves the problems of the existing multi-step process in that the de-epidermization and decellularization processes are simultaneously performed in a single process. However, not only does the process of removing hair roots on the skin surface still have the inconvenience of having to be performed as a separate step, but there is also a problem that the ionic surfactant used may dissociate and may inhibit the survival rate by adsorbing and remaining on proteins in the dermis.

[0034] Accordingly, the inventors of the present invention recognized the problems existing in the above-mentioned prior technologies and developed a one-step composition for producing acellular homologous dermis, which can simultaneously perform the effects of existing de-epidermization, decellularization, and defatting, as well as the effect of hair root removal, in a single process, and can minimize the loss of growth factors and collagen, which are effective ingredients in the dermis, and also maintain excellent mechanical strength.

[0035] A composition for producing an acellular allogeneic dermis according to one embodiment of the present invention comprises an epidermal separating agent, a nonionic surfactant, sodium deoxycholate, and a thioglycolate compound, and can simultaneously perform epidermal removal, cell removal, fat removal, and hair root removal on allogeneic skin tissue.

[0036] That is, as mentioned above, the purpose of the present invention is to provide a composition for producing an acellular allogeneic dermis designed to simultaneously perform epidermal removal, cell removal, fat removal and hair root removal in one process for allogeneic skin tissue, and the composition can additionally exhibit a hair root removal effect in addition to de-epidermalization, decellularization and defatting by the effect exhibited by mixing the effective ingredients, namely, an epidermal separating agent, a nonionic surfactant, sodium deoxycholate and a thioglycolate compound.

[0037] In order to remove hair follicles during the processing of homologous dermal tissue, a separate step of manually pulling them out using forceps was typically performed, resulting in inconvenience. Therefore, the present invention is characterized by simultaneously achieving decellularization, defatting, and de-epidermization effects, as well as hair follicle removal, in a single step of processing the composition, without the need for a separate hair follicle removal step using forceps.

[0038] Specifically, the thioglycolate compound is an effective ingredient included for the purpose of not only cutting hair by cutting the sulfur (S) bond of keratin present in the hair or hair root, but also removing the hair root. The thioglycolate compound may include at least one of thioglycolic acid, calcium thioglycolate, sodium thioglycolate, and potassium thioglycolate, and may preferably be thioglycolic acid.

[0039] That is, the composition of the present invention has the advantage of simultaneously exhibiting the effects of de-epidermization, de-cellularization, and de-fat removal as well as hair root removal in a single process by including a thioglycolate compound as an active ingredient.

[0040] Meanwhile, deoxycholic acid, a substance primarily extracted from the intestines of livestock, is a component of bile acids and is known to produce various organic and molecular compounds. Furthermore, deoxycholic acid can play a role in removing accumulated fat from the body or skin by destroying intracellular granular structures.

[0041] Accordingly, the present invention is characterized in that it can simultaneously dissolve skin cell membranes and exert the effects of decomposing and removing fat cells by including sodium deoxycholate, a salt of the above-described deoxycholic acid, as an active ingredient.

[0042] Meanwhile, the composition of the present invention may additionally include a pH regulator. The pH regulator is an effective ingredient for controlling the pH of the composition of the present invention within a certain range, and may be, for example, NaOH. However, the present invention is not limited thereto, and is defined as including all substances that can be applied by a person skilled in the art to control the pH of the composition itself.

[0043] The above-mentioned epidermal separator is selected from the group consisting of sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA), and mixtures thereof.

[0044] In the past, in order to remove the epidermal layer from an allogeneic dermis, a proteolytic enzyme was used to separate the epidermal layer from the dermal layer and remove it. Specifically, dispase, thermolysin, trypsin, etc. were used as the proteolytic enzyme, and for example, a method was used to separate the dermal layer and the epidermal layer by treating with 1.0 units / ㎖ of dispase at 37°C for 60 to 120 minutes or treating with 200 μg / ㎖ of thermolysin at 37°C for 30 minutes.

[0045] However, in the case of the method using the above protein-decomposing enzyme, if the concentration used is low or the treatment time is too short, the epidermal layer is not separated well, and if the concentration is too high or the treatment time is too long, damage to cells or tissues may occur, so there was a problem that the treatment concentration and time had to be adjusted differently for each sample.

[0046] Accordingly, the present invention is characterized in that it applies an epidermal layer separating agent selected from the group consisting of sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA), and a mixture thereof to remove the epidermal layer.

[0047] Specifically, the epidermal layer separating agent may include sodium chloride and ethylenediaminetetraacetic acid.

[0048] For example, it contains a 1 M to 1.5 M sodium chloride solution and 10 mM to 20 mM ethylenediaminetetraacetic acid (EDTA). Since bacteria and mold cannot grow in a sodium chloride solution of 1.5 M or more, the risk of microbial contamination can be reduced. In addition, since the EDTA acts as a protease inhibitor, it can suppress the decomposition of effective proteins, thereby reducing tissue damage.

[0049] Therefore, when sodium chloride and EDTA are mixed and used, the dermal and epidermal layers can be separated while minimizing microbial contamination and tissue damage during the processing. Meanwhile, it is preferable that the sodium chloride and EDTA be mixed in a weight ratio of 1:1 to 2:1, which maximizes the effect of separating and removing the epidermal layer.

[0050] The above nonionic surfactant is selected from the group consisting of Triton X-100, Tween 20, Tween 40, Tween 60, Tween 80, Nonidetpy-10 (NP-10), Nonidetpy-40 (NP-40) and mixtures thereof.

[0051] Specifically, the nonionic surfactant is an effective ingredient that plays a role in removing cells of the dermal layer, and the present invention is characterized by using the nonionic surfactant that does not have an ionic nature of a cationic or anionic.

[0052] Meanwhile, among the prior art techniques that disclose a method for producing acellular homogeneous dermis, a technique is disclosed for treating acellular skin tissue in a storage solution containing an ionic surfactant such as SDS to simultaneously perform de-epidermization, de-fat removal, and decellularization.

[0053] However, when using an ionic surfactant as a surfactant, the ionic surfactant is dissociated during the treatment process with skin tissue and is adsorbed to proteins in the dermis. In this case, even if a washing step is performed, some of the adsorbed surfactant components remain, which can not only inhibit the survival rate following transplantation, but also have the problem of not being able to simultaneously exert the effect of removing hair roots.

[0054] Accordingly, the present invention has the advantage of not only maintaining an excellent survival rate by minimizing the surfactant that may remain on the processed acellular homogeneous dermis by including a nonionic surfactant instead of an ionic surfactant as an effective ingredient for removing cells from the dermis layer, but also simultaneously exhibiting an excellent hair root removal effect.

[0055] The pH range of the above composition is 6.5 to 7.5.

[0056] In order for the composition of the present invention to be applied to homogeneous skin tissue and exhibit excellent epidermal removal, cell removal, fat removal, and hair root removal effects, it is desirable to maintain a certain level of pH, and in particular, when pH 7.5 is maintained, it has the characteristic of being able to exhibit all of the above effects excellently.

[0057] The above composition has an excellent effect of maintaining collagen and growth factors in acellular homologous dermis.

[0058] Typically, reagents applied at each stage during the processing of acellular dermal allografts can cause loss of intracellular collagen or growth factors or structural deformation. In these cases, even when the processed acellular dermal allograft is transplanted to a defect site, nerve formation and vascular regeneration may not be smoothly induced, potentially reducing the survival rate.

[0059] Accordingly, the present invention provides a one-step treatment composition in which specific effective ingredients are combined, thereby minimizing the loss of collagen and growth factors that may occur during a multi-step process, and also having the characteristic of maintaining excellent mechanical strength.

[0060] Preferably, the composition of the present invention may include 50 to 70 parts by weight of a nonionic surfactant, 50 to 70 parts by weight of sodium deoxycholate, and 30 to 50 parts by weight of a thioglycolate compound, per 100 parts by weight of an epidermal separator.

[0061] When the composition of the present invention is mixed in the above weight range, it has the advantage of maximizing the simultaneous removal effect of the epidermis, cells, fat, and hair roots due to the synergistic effect of mixing each active ingredient, and also has the advantage of maximizing the collagen and growth factor maintenance effect and mechanical strength maintenance effect of the acellular homologous dermis.

[0062] However, if the weight exceeds or falls below the above weight range, the effect may be minimal.

[0063] More preferably, the composition of the present invention may further include a compound represented by the following chemical formula 1 and a compound represented by the following chemical formula 2:

[0064] [Chemical Formula 1]

[0065]

[0066] [Chemical Formula 2]

[0067]

[0068] Specifically, when a nonionic surfactant is used as an effective ingredient of the composition of the present invention, the problem of reduced survival rate due to the dissociation characteristics of the ionic surfactant can be overcome as mentioned above, but compared to the ionic surfactant, it can destroy the microstructure of the extracellular matrix of skin tissue and cause loss of growth factors.

[0069] Accordingly, the present invention is characterized in that it includes compounds represented by the chemical formulas 1 and 2 as active ingredients to overcome the problems of the nonionic surfactant.

[0070] Specifically, when the compounds represented by the above chemical formulas 1 and 2 are included, a protective effect can be exerted on the surface of skin tissue and the microstructure of the extracellular matrix due to the action of a specific functional group included in each compound, thereby minimizing the loss of growth factors and overcoming the disadvantages of using nonionic surfactants.

[0071] Furthermore, when the compounds represented by the above chemical formulas 1 and 2 are included as active ingredients, each compound activates the thioglycolic acid, thereby maximizing the hair removal and root removal effects desired by the present invention. Furthermore, the mechanical strength of the processed acellular homologous dermis can also be excellently maintained.

[0072] More preferably, the compounds represented by the above chemical formulas 1 and 2 may include 5 to 10 parts by weight of the compound represented by the above chemical formula 1 and 5 to 10 parts by weight of the compound represented by the above chemical formula 2, per 100 parts by weight of the epidermal separator.

[0073] When the compounds represented by the additionally included chemical formulas 1 and 2 are mixed in the above-mentioned specific weight range, the synergistic effect of mixing each compound can further maximize the epidermal removal, cell removal, and fat removal effects, and the protective effect on the surface of skin tissue and the microstructure of the extracellular matrix can be maximized, thereby further enhancing the collagen and growth factor maintenance effect. Furthermore, there is an advantage in that the hair root removal effect can also be maximized by further activating thioglycolic acid.

[0074] According to another embodiment of the present invention, a method for producing an acellular allogeneic dermis comprises: 1) a first washing step of washing allogeneic skin tissue in distilled water; 2) a step of treating the allogeneic skin tissue that has undergone the first washing step with a composition of the present invention; and 3) a second washing step of treating the allogeneic skin tissue that has undergone the treatment step with phosphate buffered saline (PBS).

[0075] Step 1) above is the primary washing step of the homologous skin tissue, which involves removing a protective solution and foreign substances that protect the tissue. Specifically, the homologous skin tissue and distilled water are placed inside a shaking incubator and operated for washing. Washing is preferably performed at a temperature of 20 to 27°C for 10 to 15 hours.

[0076] The above step 2) is a step of treating the first-washed homologous skin tissue with the composition according to the present invention. Specifically, it is a step of immersing the first-washed homologous skin tissue in the composition of the present invention, and it is preferable to immerse it for 10 to 15 hours at a temperature of 35 to 40°C.

[0077] In the case of step 2) above, each effective ingredient included in the composition of the present invention acts on homologous skin tissue, thereby simultaneously performing epidermal removal, cell removal, fat removal, and hair root removal. That is, when treating homologous skin tissue using the composition of the present invention, there is a characteristic that the de-epidermalization, decellularization, fat removal, and hair root removal steps, which were previously performed in multiple steps, can be performed in a single step.

[0078] Meanwhile, the step 3) above is a step of secondarily washing the homogeneous skin tissue processed through the immersion treatment step with a buffer, and specifically, by secondarily washing using phosphate buffered saline (PBS) at a temperature of 20 to 27°C for 10 to 15 hours, the composition of the present invention and other impurities remaining after the immersion treatment step can be removed.

[0079] More preferably, the method for producing an acellular allogeneic dermis of the present invention may include, after the first washing step of step 1), a step of 2-1) immersing the allogeneic skin tissue that has undergone the pre-washing step with the composition of the present invention; 2-2) low-temperature treating the allogeneic skin tissue that has undergone the immersion treatment step at a temperature of 5 to 10°C for 1 to 2 hours; and 3) a step of secondarily washing the allogeneic skin tissue that has undergone the low-temperature treatment step with phosphate buffered saline (PBS).

[0080] Specifically, when the low-temperature treatment step of step 2-2) above is additionally performed, the composition remaining after the immersion step can act more effectively on the tissue, thereby maximizing the effect of simultaneous removal of the epidermis, cells, fat, and hair roots as desired in the present invention.

[0081] Meanwhile, the method for manufacturing the above-mentioned acellular homologous dermis can proceed with an additional process after step 3).

[0082] Specifically, 4) a step of finely adjusting the thickness of the skin tissue that has gone through the secondary washing step and flattening it using a thickness-control device; 5) a step of removing the swarf on the surface of the skin tissue by treating it with 3% H2O2 at a temperature of 20 to 27°C for 10 to 15 hours; 6) a step of removing the remaining fat by treating the skin tissue from which the swarf has been removed with a mixed solution of EtOH and IPA at a temperature of 20 to 27°C for 10 to 15 hours; and 7) a step of preserving the acellular allogeneic dermis from which the remaining fat has been removed using any one of freezing, freeze-drying, and hydration, and an additional washing step using distilled water may be further included between each of the above steps.

[0083] According to another embodiment of the present invention, an acellular homologous dermis is manufactured by the above manufacturing method.

[0084]

[0085] [Manufacturing example]

[0086] Preparation of a composition for producing acellular homologous dermis

[0087] The composition of the present invention was prepared by mixing and stirring a skin separating agent containing 1M NaCl and 0.1% EDTA in a 1:1 weight ratio, a nonionic surfactant (0.25% Triton X-100), 0.25% sodium deoxycholate, 0.05 to 0.5% thioglycolic acid, a compound represented by the following chemical formula 1, and a compound represented by the following chemical formula 2 in the weight ranges shown in Table 1 below. Meanwhile, each of the above materials was purchased from Tokyo Chemical and used:

[0088] [Chemical Formula 1]

[0089]

[0090] [Chemical Formula 2]

[0091]

[0092] PZ1PZ2PZ3PZ4PZ5PZ6PZ7PZ8PZ9PZ10Epidermal Separator100100100100100100100100100100100Nonionic Surfactant45506070756060606060Sodium Deoxycholate45506070756060606060Thioglycolic Acid25304050554040404040Chemical Formula 1-----357.51012Chemical Formula 2-----357.51012

[0093] (Unit: parts by weight)

[0094] Preparation of acellular allogeneic dermis

[0095] Example 1

[0096] After purchasing cadaveric skin tissues from EURO skin bank, Allosource, and CTS, tissues larger than 1 mm were selected and used.

[0097] 1) The skin tissue was first washed using distilled water at 20 to 27°C for 10 to 12 hours through a shaking incubator. 2) Thereafter, the skin tissue that had completed the first washing was immersed in the PZ1 composition at 35 to 38°C for 10 to 12 hours. 3) Thereafter, the skin tissue processed through the immersion treatment step was secondarily washed with PBS buffer at 20 to 27°C for 10 hours to remove impurities, thereby producing the acellular allogeneic dermis of the present invention.

[0098] Examples 2 to 10

[0099] Acellular homologous dermis of Examples 2 to 10 were prepared in the same manner as in Example 1, except that the PZ1 composition of Example 1 was replaced with PZ2 to PZ10, respectively.

[0100] Example 11

[0101] 1) The skin tissue was first washed using distilled water at 20 to 27°C for 10 to 12 hours through a shaking incubator. 2-1) Afterwards, the skin tissue that had completed the first washing was immersed in the PZ8 composition at 35 to 38°C for 10 to 12 hours. 2-2) Afterwards, the skin tissue that had completed the immersion treatment step was taken out and low-temperature treated at 5 to 10°C for 1.5 hours. 3) Afterwards, the skin tissue processed through the low-temperature treatment step was secondarily washed with PBS buffer at 20 to 27°C for 10 hours, and impurities were removed to prepare the acellular allogeneic dermis of Example 11.

[0102] Comparative Example 1

[0103] An acellular homogeneous dermis of Comparative Example 1 was prepared in the same manner as in Example 3, except that 0.25% Triton X-100 was replaced with 0.25% SDS, an ionic surfactant, in the combination of the PZ3 composition used in Example 3.

[0104] Comparative Example 2

[0105] An acellular homogeneous dermis of Comparative Example 2 was prepared in the same manner as in Example 8, except that 0.25% Triton X-100 was replaced with 0.25% SDS, an ionic surfactant, in the combination of the PZ8 composition used in Example 8.

[0106]

[0107] [Experimental Example]

[0108] Simultaneous removal of epidermis, cells, fat and hair roots confirmed

[0109] The decellularization, de-epidermization, de-fat and hair root removal effects of the human-derived acellular homogeneous dermal matrix of Example 3 above were confirmed.

[0110] Specifically, the effects on decellularization, de-epidermization, and defatting are evaluated through histological examination using H&E staining, and the method is specifically as follows.

[0111] (1) Paraffin blocks were cut into 4 μm thick sections and dried to produce paraffin sections.

[0112] (2) For the deparaffinization process, the sample was reacted three times in xylene for 5 minutes each, three times in 100% ethanol for 2 minutes each, once in 90% ethanol for 1 minute each, once in 80% ethanol for 1 minute each, and once in 70% ethanol for 1 minute each, and then washed in running water for 10 minutes.

[0113] (3) After reacting with hematoxylin staining solution for 10 minutes, washed in running water for 3 minutes, reacted with eosin staining solution for 10 minutes, and washed in running water until no eosin staining solution came out. After reacting with 70% ethanol 10 times for 1 second, 80% ethanol 10 times for 1 second, 90% ethanol 10 times for 1 second, 100% ethanol 2 times for 1 minute, and xylene 3 times for 3 minutes, it was mounted with mounting solution.

[0114] (4) Afterwards, the skin tissue was photographed using an optical microscope (Olympus BX51, H&E staining) and a scanning electron microscope (Hitachi S-4700, Japan).

[0115] The results are as shown in Fig. 1.

[0116] Referring to FIG. 1, in the case of Example 3, which is a skin tissue treated with the PZ3 composition of the present invention (FIG. 1(a)), it can be confirmed that the epidermis was peeled off and not only the cells in the dermis were removed, but also the fat was removed, compared to the control group (normal skin, FIG. 1(b)).

[0117] Meanwhile, the results of the root removal effect of Example 3 can be confirmed through Fig. 2, which is a photograph of the acellular homologous dermis manufactured by the manufacturing method of Example 3.

[0118] According to the above drawing 2, it can be confirmed that the hair root of the manufactured acellular homogeneous dermis has been removed, and thus, it can be confirmed that when the composition of the present invention is used, not only the de-epidermization, decellularization, and defatting effects but also the hair root removal effect can be simultaneously exhibited in a single step of processing the composition without having to go through a separate hair root removal step using a forcep.

[0119] Accordingly, it was confirmed that when producing an acellular homologous dermis using the composition of the present invention, the effect of simultaneously removing the epidermis, cells, fat, and hair roots of skin tissue can be achieved in a one-step process through the mixing action of the effective ingredients in the composition.

[0120] Meanwhile, using the same method as the method for confirming the effect of removing the epidermis, cells, fat and hair roots for Example 3, the above effects were compared and tested for Examples 1, 2, 4 to 11 and Comparative Examples 1 and 2.

[0121] For a comparative experiment with the effects according to Example 3, the epidermal removal, cell removal, fat removal, and hair root removal effects of Example 3 were each set to an index of 5, and the results of the remaining examples and comparative examples were evaluated with an index of 1 to 10, and the results are shown in Table 2 below.

[0122] The higher the index number, the better the effect of epidermal removal, cell removal, fat removal, and hair root removal, and the lower the number, the worse the effect.

[0123] Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Example 9 Example 10 Example 11 Comparative Example 1 Comparative Example 2 Epidermal removal effect 34553799971023 Cell removal effect 35544699971012 Fat removal effect 3554379987922 Hair root removal effect 3554389101081011

[0124] (Unit: Index)

[0125] Referring to the results in Table 2 above, it can be confirmed that Examples 1 to 11 of the present invention have a simultaneous removal effect of the epidermis, cells, fat, and hair roots that is equal to or greater than that of Example 3.

[0126] Meanwhile, in the case of Examples 6 to 10, it can be confirmed that the effect is further maximized, and in particular, in the case of Examples 7 to 9, an even better effect can be confirmed. This corresponds to the effect resulting from the mixing of the compounds represented by Chemical Formulas 1 and 2.

[0127] Furthermore, in Example 11, the best simultaneous removal activity of epidermis, cells, fat and hair roots was confirmed.

[0128] However, in the case of Comparative Examples 1 and 2, it was confirmed that the epidermal removal, cell removal, and fat removal effects were somewhat lower than in the examples, and in particular, the hair root removal effect was confirmed to be extremely low. This is because the ionic surfactant dissociated and was absorbed into the dermis, thereby reducing the hair root removal activity.

[0129] Confirmation of the effect of maintaining the content of growth factor bFGF

[0130] When processing acellular allogeneic dermis, growth factors necessary for tissue regeneration after transplantation may also be lost. Therefore, it was confirmed by comparing and evaluating the content of basic fibroblast growth factor (bFGF), one of the representative growth factors, whether the tissues of Examples 1 to 11 and Comparative Examples 1 and 2 effectively maintained factors useful for tissue regeneration in the dermal layer.

[0131] Specifically, the acellular homogenous dermal layer samples of Examples 1 to 11 and Comparative Examples 1 and 2 were finely chopped, 100 μl of Tissue Extraction Buffer was added per 10 mg, and extracted using a homogenizer at 4°C for 1 hour. After extraction, the samples were centrifuged at 12,000 rpm at 4°C for 20 minutes, and the supernatant was recovered and quantified using a bFGF ELISA kit at UV 450 nm. The results are shown in Table 3 below.

[0132] Confirmed effect of maintaining collagen content

[0133] When processing acellular allogeneic dermis, the extracellular matrix necessary for providing a skeleton after transplantation may also be lost. Therefore, in order to confirm whether the tissues of Examples 1 to 11 and Comparative Examples 1 and 2 effectively maintain the extracellular matrix of the dermal layer, the collagen content of each acellular allogeneic dermis layer was comparatively evaluated.

[0134] Specifically, a certain amount of acellular homologous dermal layer samples from Examples 1 to 11 and Comparative Examples 1 and 2 were taken, and PITC labeling was performed using the PICO tag method. The PITC-labeled samples were dissolved in 400 μl of buffer, and 10 μl of the samples were loaded onto HPLC. Among the amino acid quantitative values, the hydroxyproline value was used to quantify collagen according to the following Equation 1, and the results are shown in Table 3 below:

[0135] [Formula 1]

[0136]

[0137] Confirmation of mechanical strength maintenance effect

[0138] During the processing of acellular allogeneic dermis, the collagen structure may be damaged, resulting in a decrease in mechanical properties. To confirm whether the dermis layers according to the examples and comparative examples effectively maintain mechanical properties, the tensile strength of the acellular allogeneic dermis layers manufactured in examples 1 to 11 and comparative examples 1 and 2 was compared and evaluated.

[0139] Specifically, the acellular homogeneous dermal layer samples manufactured in Examples 1 to 11 and Comparative Examples 1 and 2 were cut to a length of 10×50 mm and prepared. After positioning the distance between the grips of a universal test machine (UTM) to 30 mm, the sample was fixed to the equipment and tensioned at a crosshead speed of 50 mm / min. The tensile strength was determined using the value calculated as the force value reaching the breaking point, and the results are shown in Table 3 below.

[0140] Control group (normal) skin) Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Example 9 Example 10 Example 11 Comparative Example 1 Comparative Example 2 b FGF amount (wt%) 100 6 2.46 9.47 0.16 8.16 0.17 5.48 0.18 0.48 1.17 4.6 8 5.45 5.45 1.4 Collagen amount (wt%) 100 6 6.47 2.57 4.57 4.0 6 5.47 9.58 2.48 4.58 3.18 0.18 8.45 0.44 9.5 Tensile strength (Mpa) 30.8 15.9 19.5 18.119 115.5 21.4 26.9 27.5 25.5 20.43 0.41 0.0 10.9

[0141] Referring to Table 3 above, in the case of Examples 1 to 11, it was confirmed that the content of growth factors and collagen was maintained to a certain extent even after going through the processing process, and the tensile strength was also confirmed to be above a certain level.

[0142] Specifically, for Examples 6 to 10, it was confirmed that the effects were excellent when additional compound combinations were additionally included, and in particular, the effects were maximized for Examples 7 to 9. Meanwhile, Example 11, which additionally included a low-temperature treatment process, showed the best effects.

[0143] On the other hand, in the case of Comparative Examples 1 and 2, it was confirmed that growth factors and collagen were lost significantly during the processing compared to the examples of the present invention, and the tensile strength was also confirmed to be reduced.

[0144]

[0145] Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention.

[0146] The present invention relates to a composition for producing acellular allogeneic dermis and a method for producing acellular allogeneic dermis using the same.

Claims

1. Contains a skin separating agent, a nonionic surfactant, sodium deoxycholate and thioglycolate compounds. It can perform epidermal removal, cell removal, fat removal and hair root removal simultaneously on homologous skin tissue. A composition for preparing an acellular homogeneous dermis.

2. In paragraph 1, The above-mentioned epidermal separator is selected from the group consisting of sodium chloride (NaCl), ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. A composition for preparing an acellular homogeneous dermis.

3. In paragraph 1, The above nonionic surfactant is selected from the group consisting of Triton X-100, Tween 20, Tween 40, Tween 60, Tween 80, Nonidetpy-10 (NP-10), Nonidetpy-40 (NP-40) and mixtures thereof. A composition for preparing an acellular homogeneous dermis.

4. In paragraph 1, The pH range of the above composition is 6.5 to 7.

5. A composition for preparing an acellular homogeneous dermis.

5. In paragraph 1, The above composition has an excellent effect of maintaining collagen and growth factors in acellular homogeneous dermis. A composition for preparing an acellular homogeneous dermis. 6.1) Primary washing step of washing homogeneous skin tissue in distilled water; 2) a step of treating homogeneous skin tissue that has undergone the first washing step with a composition according to claim 1; and 3) A step of washing the homogeneous skin tissue that has undergone the above processing step a second time with phosphate buffered saline (PBS). Method for producing acellular homogeneous dermis.

7. Manufactured by the method for manufacturing acellular homogeneous dermis according to Article 6 Acellular allogeneic dermis.

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