KR20230015191A - A method for increasing thickness of acellular dermal matrix - Google Patents

Abstract

The present invention relates to a method for increasing the thickness of an acellular dermal graft material. By using the method according to one aspect, the thickness of the allogeneic dermis can be increased to be similar to the skin thickness of a site to be transplanted, and at the same time, a more flexible and resilient tissue can be produced. Therefore, the method can induce smooth skin tissue regeneration by engraftment without separation of the tissue at a transplant site. The method for increasing the thickness of an acellular dermal graft material comprises a step of immersing the cell-removed dermis layer in a reaction vessel containing a hydrogen peroxide solution.

Description

A method for increasing the thickness of an acellular dermal layer graft material {A method for increasing thickness of acellular dermal matrix}

The present invention relates to a method for increasing the thickness of an acellular dermal layer graft material.

Skin is an organ that covers the surface of the body, and is composed of epidermis, dermis, and subcutaneous tissue, and has accessory organs such as sebaceous glands, sweat glands, hair, pubic hair muscles, and nails. It protects the body from the external environment and at the same time is in charge ^of various physiological functions such as temperature control function, sensory function, and vitamin D synthesis function.

Currently, autologous transplantation is mainly used as a method for regenerating damaged skin tissue. New trauma occurs in the healthy area from which the skin is removed, increasing the patient's pain, requiring a lot of time for complete recovery, and economic burden. big. In addition, when there are not enough healthy body parts left, such as severe burn patients, there is a problem that autologous transplantation cannot be applied or transplantation must be performed several times. In order to solve the above problems, allotransplantation using the skin of another person, xenotransplantation using the skin of another animal such as pigs, etc. have been attempted (Korean Patent Publication No. 10-2018-0028229). The skin graft material is manufactured in the form of an acellular dermal matrix (ADM) that maintains the three-dimensional structure of the dermal layer as it is by removing cells in the epidermis and dermis that cause immune reactions from donated human skin. Such acellular dermis provides a skeleton necessary for influx of fibroblasts, generation of nerves, regeneration of blood vessels, etc., to skin tissue damaged by trauma without immune rejection.

On the other hand, since the thickness of the skin is different depending on the site, thick skin is required depending on the site where transplantation is required, but there are cases in which the donated skin does not satisfy the required thickness. Therefore, there is a need for a method capable of effectively increasing the thickness of the skin graft material.

One aspect provides a method for increasing the thickness of a cell-free dermal layer transplant material comprising: immersing the dermal layer from which cells are removed in a reaction vessel containing a hydrogen peroxide solution.

Another aspect provides an acellular dermal layer transplant material prepared by the above method.

One aspect provides a method for increasing the thickness of a cell-free dermal layer graft material comprising the steps of immersing the dermal layer from which cells are removed in a reaction vessel containing a hydrogen peroxide solution.

The term "dermal layer from which cells are removed" can be used interchangeably with the term "acellular dermal layer", and is skin tissue in a state in which cells in the epidermis and dermis that cause an immune response in skin tissue isolated from an individual are removed, It may mean an acellular dermal matrix (ADM) that provides a three-dimensional structural support for influx of fibroblasts, generation of nerves, and regeneration of blood vessels. The above “cell-free” or “cell-free” means that cells capable of causing immune rejection are “substantially removed” and “substantially does not contain” cells, and “substantially” is compared with the raw material. Thus, it may mean a state in which 90% or more of the cells are removed, for example, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

The dermal layer from which the cells are removed is the step of removing the epidermal layer from the skin tissue separated from the subject; And removing the cells of the dermal layer from the skin tissue from which the epidermal layer was removed. Therefore, the method may include, prior to the immersion step, removing the epidermal layer from the skin tissue separated from the subject; and removing cells of the dermal layer from the skin tissue from which the epidermal layer is removed. Removal of the epidermal layer and removal of cells in the dermal layer may be performed according to various methods known in the art.

In one embodiment, the epidermal layer may be removed by separating from the dermal layer using a proteolytic enzyme, an ionic solution, or a combination thereof. In the case of using an enzyme, if the concentration used is low or the treatment time is too short, the separation is not performed well, and if the concentration is high or the treatment time is too long, it may cause damage to cells or tissues. The proteolytic enzyme may be neutral proteolytic enzymes such as dispase, thermolysin, and trypsin. The dermal and epidermal layers can be separated by treatment with dispase at a concentration of 1.0 units/ml at 37°C for 60 to 120 minutes. Alternatively, treatment with 200 μg/ml of termolysin at 37° C. for 30 minutes can separate the dermal and epidermal layers. When an ionic solution is used, the degree of separation may vary depending on conditions such as ionic strength, treatment time, and treatment temperature. The ionic solution may be used alone or in combination with a sodium chloride solution, an ethylenediaminetetraacetic acid (EDTA) solution, or the like. The dermal layer and the epidermal layer can be separated by treatment at 37°C for 14 to 32 hours with 1M or more sodium chloride solution. In a sodium chloride solution of 1M or more, since bacteria or fungi cannot proliferate, the risk of microbial contamination can be reduced. Alternatively, the dermal layer and the epidermal layer may be separated by treatment with a 20mM ethylenediaminetetraacetic acid (EDTA) solution at 37° C. for 14 to 32 hours. When EDTA is used, tissue damage caused by proteases can be reduced because EDTA acts as a protease inhibitor. Therefore, treatment by adding 1 to 10 mM EDTA to a 1 M sodium chloride solution minimizes microbial contamination or tissue damage caused by enzymes, and separates the dermal and epidermal layers.

In one embodiment, the step of removing the cells of the dermal layer may be to remove by treatment with a surfactant, ultrasonic treatment, or a combination thereof. Surfactants include ionic surfactants such as sodium dodecyl sulfate (SDS), Triton X-100, Tween 20, Tween 40, Tween 60, Tween 80, Nonidet P-10 (NP-10), Nonidet P-40 (NP-40) and the like can be used, and their treatment concentration and treatment time vary depending on each surfactant. For example, SDS is treated for 30 to 120 minutes at a concentration of 0.2 to 1% (w/v) at room temperature, and Tween 20 is treated at a concentration of 0.1 to 2.0% (w/v) at room temperature for 30 to 120 minutes. It is treated for 180 minutes, and in the case of Triton X-100 or Nonidetfi-40 solution, cells can be removed by treatment at a concentration of 0.2 to 2% (w / v) at 22 to 37 ° C. for 30 to 180 minutes. In the case of using ultrasonic waves, cells may be removed by irradiating ultrasonic waves of 10 to 100 kHz for 5 to 60 minutes. Alternatively, cells can be removed without tissue damage even when the surfactant and ultrasound are used in combination. Since most of the immune response is induced by membrane proteins present in the cell membrane, when the cells of the dermal layer are removed by the above method, the cause of the immune response is removed, and tissue detachment due to immune rejection after transplantation is eliminated. may not occur.

In one embodiment, the subject may be a mammal, for example, a human, cow, pig, guinea pig, or dog. Specifically, the isolated skin tissue may be allogeneic skin isolated from a human cadaver. there is.

A method of increasing the thickness of the acellular dermal layer graft material is described in detail as follows.

As shown in FIG. 1, the method may include immersing the dermal layer from which cells are removed in a reaction vessel containing a hydrogen peroxide solution.

In one embodiment, the hydrogen peroxide solution may have a concentration of 5.0 to 30.0% (w/v). The concentration may vary depending on the desired skin thickness within the above range, for example, the concentration of the hydrogen peroxide solution is 5.0 to 30.0% (w / v), 5.0 to 25.0% (w / v), 5.0 to 20.0 % (w/v), 5.0 to 15.0% (w/v), 5.0 to 10.0% (w/v), 10.0 to 30.0% (w/v), 10.0 to 25.0% (w/v), 10.0 to 20.0 % (w/v), 10.0 to 15.0% (w/v), 15.0 to 30.0% (w/v), 15.0 to 25.0% (w/v), or 15.0 to 20.0% (w/v). . If the concentration of hydrogen peroxide is too low outside the above range, the thickness may not be increased well, and if the concentration is too high, even if the thickness is increased, the density of the tissue may decrease and become stiff overall, or the thickness may increase Even if it is made, there is damage and disintegration of the tissue by hydrogen peroxide, so fluff is severely generated, and thus the mechanical strength may be weakened. The immersing step may be performed at room temperature, for example, at 25 to 40 °C.

Then, as shown in FIG. 1 , the method may further include applying a vacuum condition to the reaction vessel. As confirmed in one embodiment, when reacting by performing more vacuum treatment than reacting by treating only hydrogen peroxide, it is possible to manufacture a skin graft material having a more flexible and resilient tissue while achieving the desired thickness increase effect. there is. In one embodiment, the acellular dermal layer transplant material having an increased thickness may be used for tissue reconstruction such as a breast that requires soft and elastic tissue.

In one embodiment. The applying of the vacuum condition may include applying a pressure condition of 1 to 950 mTorr to the reaction vessel. In other words, the step of applying the vacuum condition is to increase the thickness by maintaining the pressure of the gas inside the reaction vessel at 1 to 950 mTorr to react the cell-removed dermal layer and the hydrogen peroxide solution under a vacuum condition for 30 minutes to 24 hours it could be For example, the pressure condition is 1 to 900 mTorr, 1 to 700 mTorr, 1 to 500 mTorr, 1 to 300 mTorr, 1 to 100 mTorr, 1 to 50 mTorr, 1 to 30 mTorr, 1 to 10 mTorr, 5 to 10 mTorr 900 mTorr, 5 to 700 mTorr, 5 to 500 mTorr, 5 to 300 mTorr, 5 to 100 mTorr, 5 to 50 mTorr, 5 to 30 mTorr, 5 to 10 mTorr, 10 to 900 mTorr, 10 to 700 mTorr, 10 to 700 mTorr 500 mTorr, 10 to 300 mTorr, 10 to 100 mTorr, 10 to 50 mTorr, or 10 to 30 mTorr. If the pressure is too high out of the above range, the effect of improving the mechanical strength weakened by the increase in thickness may not appear, and if the pressure is too low, the dermal layer tissue due to the pressure may be densified or damaged or structural deformation may occur. Further, for example, the reaction time under the vacuum condition is 30 minutes to 24 hours, 30 minutes to 18 hours, 30 minutes to 12 hours, 30 minutes to 6 hours, 30 minutes to 3 hours, 30 minutes to 2 hours, 30 minutes to 1 hour, 1 hour to 24 hours, 1 hour to 18 hours, 1 hour to 12 hours, 1 hour to 6 hours, 1 hour to 3 hours, 1 hour to 2 hours, 2 hours to 24 hours, 2 hours to 18 hours, 2 hours to 12 hours, 2 hours to 6 hours, 2 hours to 3 hours, 3 hours to 24 hours, 3 hours to 18 hours, 3 hours to 12 hours, 3 hours to 6 hours, 6 hours to 24 hours hours, 6 to 18 hours, 6 to 12 hours, 12 to 24 hours, or 12 to 18 hours. If the reaction time is too short outside the above range, the thickness increase may not be achieved well, and if the reaction time is too long, even if the thickness is increased, the density of the tissue may decrease and the overall stiffness may be exhibited, or the thickness increase is made Even if it loses, there is damage and disintegration of the tissue due to hydrogen peroxide, so fluff is severely generated, and thus the mechanical strength may be weakened.

Thereafter, the method may further include, as shown in FIG. 2 , after applying the vacuum condition, irradiating the dermal layer with electron beams.

Specifically, after the step of applying the vacuum condition, after removing the residual hydrogen peroxide solution by treating a washing solution such as PBS, the dermal layer having an increased thickness is immersed in a storage solution to irradiate with an electron beam (E-beam). . The storage solution may be sterile water, physiological saline, or a mixture thereof, and any known storage solution used in the art may be used.

In one embodiment, the irradiation dose of the electron beam may be 1 to 30 kGy. For example, the irradiation dose of the electron beam is 1 to 30 kGy, 5 to 30 kGy, 10 to 30 kGy, 15 to 30 kGy, 20 to 30 kGy, 25 to 30 kGy, 1 to 25 kGy, 5 to 25 kGy, 10 to 25 kGy, 15 to 25 kGy, 20 to 25 kGy, 1 to 20 kGy, 5 to 20 kGy, 10 to 20 kGy, 15 to 20 kGy, 1 to 15 kGy, 5 to 15 kGy, 10 to 15 kGy, 1 to 10 kGy, or 5 to 10 kGy. If the irradiation dose is too low outside the above range, the mechanical strength weakened by the increase in thickness may not improve, or the increased thickness may decrease over time, and if the irradiation dose is too high outside the above range, the secondary The compactness of the structure may increase, the tendency to stratify may increase, the protein bundle may be deformed by thermal deformation to exhibit an overall stiff aspect, and the thickness increased by the previous process may be reduced.

As shown in FIG. 3, the method according to one aspect may further include forming a plurality of holes on the surface of the dermal layer from which the cells are removed, prior to the immersion step. The plurality of holes may be formed using fine needles. For example, a plurality of fine-sized holes are formed by stabbing the upper and lower surfaces of the dermal layer from which cells are removed 1 to 10 times using a roller to which fine needles are attached. It may be formed uniformly. As a plurality of fine holes are formed on the surface of the dermal layer from which the cells are removed, hydrogen peroxide penetrates well in the subsequent hydrogen peroxide immersion process, and the thickness increase effect may be improved by about three times. Accordingly, when very thick skin is required at the site to be transplanted, the step of forming the plurality of holes may be further added. The plurality of holes may be formed on the upper and/or lower surface of the dermal layer from which cells are removed, but may be formed on both the upper and lower surfaces, rather than on any one surface, in order to uniformly increase the thickness.

Another aspect provides a method for manufacturing an acellular dermal layer graft material comprising the step of immersing the dermal layer from which cells are removed in a reaction vessel containing a hydrogen peroxide solution. The method may further include applying a vacuum condition to the reaction vessel after the immersion step. In addition, the method may further include forming a plurality of holes on the surface of the dermal layer from which cells are removed, prior to the immersion step. In addition, the method may further include, after applying a vacuum condition, irradiating electron beams to the dermal layer. In the above method, the dermal layer from which cells are removed, hydrogen peroxide solution, immersion, vacuum conditions, electron beam irradiation, and formation of a plurality of holes are the same as described above. The method may further include a step of applying a vacuum condition and/or an electron beam irradiation step, so that a skin graft material having a more flexible and resilient tissue may be manufactured while achieving a desired thickness increase effect. In one embodiment, the method may be to prepare an acellular dermal layer graft material for tissue reconstruction such as breast.

Another aspect provides an acellular dermal layer transplant material prepared by the above method. The acellular dermal layer graft material prepared by the above method does not cause immune rejection in transplant patients and can form a thickness similar to that of the defective skin tissue, so it engrafts without tissue detachment, inflow of fibroblasts, generation of nerves, and blood vessels. It is possible to provide a skeleton on which regeneration and the like can be smoothly performed.

The method according to one aspect can increase the thickness of the allogeneic dermis similar to the thickness of the skin of the site to be transplanted while maintaining mechanical strength, and can induce smooth skin tissue regeneration by engraftment without separation of the tissue at the transplant site.

1 is a flow chart of a method of increasing the thickness of an acellular dermal layer graft material according to an embodiment.
2 is a flow chart of a method of increasing the thickness of an acellular dermal layer graft material according to an embodiment.
3 is a flow chart of a method of increasing the thickness of an acellular dermal layer graft material according to an embodiment.
Figure 4 shows the tensile force and maximum force of Example 1 in which hydrogen peroxide treatment and vacuum treatment were performed on the acellular dermal layer, Comparative Example 1 in which hydrogen peroxide treatment and vacuum treatment were not performed, and Comparative Example 2 in which hydrogen peroxide treatment was performed , and the result of comparing the elongation force.
5 shows Example 3 in which electron beam irradiation (5 kGy, 15 kGy) was performed after performing hydrogen peroxide treatment and vacuum treatment without spike treatment on the acellular dermal layer, and only hydrogen peroxide treatment and vacuum treatment without spike treatment. It is the result of comparing the tensile force, maximum force, and elongation force after storage for 4 weeks in the storage solution with respect to Example 1 before electron beam irradiation and Comparative Example 1 before thickness increase treatment.
6 shows the change in thickness during storage in storage solution for 4 weeks by increasing the thickness by performing a hydrogen peroxide treatment step and a vacuum treatment step on the acellular dermal layer in the electron beam untreated group, the electron beam 5 kGy irradiation group, and the electron beam 15 kGy irradiation group. is the result of comparison.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of acellular dermal layer transplant material subjected to hydrogen peroxide treatment and vacuum treatment (BC-H ₂ O ₂ (vacuum))

The skin of the cadaver obtained from the tissue bank was transported so that it could be maintained in a frozen state. The transported skin tissue was taken out and cut into appropriate sizes with the dermis layer facing down. After washing the skin tissue, it was treated with sterile distilled water containing 1M NaCl + 10mM EDTA. The epidermal layer was lightly held using tweezers, then peeled off from the dermal layer to remove the epidermal layer, and washed to remove the remaining ionic solution. Then, 40 ml of Dulbecco's phosphate buffered saline (Sigma Chem. Co., USA) was added, and the mixture was gently shaken at room temperature at a speed of 30 ± 5 rpm for 5 minutes. Subsequently, the phosphate buffer solution was removed, and 40 ml of Dulbecco's phosphate buffer solution to which 1% (w/v) Tween 20 was added was added again, followed by gentle shaking at room temperature at a speed of 30±5 rpm for 120 minutes. Subsequently, an acellular dermal layer for transplantation was prepared by washing with sterile distilled water containing 1 mM EDTA and gentamicin.

The prepared acellular dermal layer was immersed in a 30% (w/v) hydrogen peroxide solution at room temperature and treated under vacuum conditions (900 mTorr) for about 1 hour, followed by PBS washing.

Example 2. Preparation of an acellular dermal layer transplant material subjected to hydrogen peroxide treatment and vacuum treatment after Spike pretreatment (BC-spike + H ₂ O ₂ (vacuum))

An acellular dermal layer was prepared in the same manner as in Example 1, except that a spike treatment step was further performed prior to the immersion in the hydrogen peroxide solution in Example 1. Specifically, using a roller attached with a fine needle (hereinafter referred to as 'spike treatment'), poke the upper and lower surfaces of the acellular dermis layer 5 times each to make small holes, and then the acellular dermis layer with a plurality of holes is formed. After being immersed in a 30% (w/v) hydrogen peroxide solution at room temperature and treated in a vacuum condition (900 mTorr) for about 1 hour, PBS washing was performed.

Example 3. Preparation of acellular dermal layer transplant material subjected to hydrogen peroxide treatment, vacuum treatment, and electron beam irradiation (BC-H ₂ O ₂ (vacuum) + electron beam irradiation (5, 15 kGy))

An acellular dermal layer was prepared in the same manner as in Example 1, except that an electron beam irradiation step was further performed in Example 1. Specifically, the electron beam irradiation step was carried out by requesting Seoul Radiation Service Co., Ltd., entering the electron beam irradiation facility along the automatic conveyor, setting the target electron beam dose to 5 kGy and 15 kGy, respectively, and cross-section irradiation of the electron beam in a vertical downward direction was carried out. The dose of the electron beam was confirmed through a separate dosimetry system.

Example 4. Production of acellular dermal layer transplant material subjected to hydrogen peroxide treatment, vacuum treatment, and electron beam irradiation after Spike pretreatment (BC-spike + H ₂ O ₂ (vacuum) + electron beam irradiation (5, 15 kGy))

An acellular dermal layer was prepared in the same manner as in Example 2, except that an electron beam irradiation step was further performed in Example 2. Specifically, the electron beam irradiation step was carried out by requesting Seoul Radiation Service Co., Ltd., entering the electron beam irradiation facility along the automatic conveyor, setting the target electron beam dose to 5 kGy and 15 kGy, respectively, and cross-section irradiation of the electron beam in a vertical downward direction was carried out. The dose of the electron beam was confirmed through a separate dosimetry system.

Comparative Example 1. Preparation of acellular dermal layer graft material (BC)

An acellular dermal layer was prepared in the same manner as in Example 1 except for omitting the step of immersing the acellular dermal layer in a hydrogen peroxide solution and treating it under vacuum conditions in Example 1.

Comparative Example 2. Preparation of acellular dermal layer transplant material treated with hydrogen peroxide (BC-H ₂ O ₂ )

Except for omitting the vacuum treatment step in Example 1, the acellular dermal layer was immersed in a 30% (w / v) hydrogen peroxide solution at room temperature for about 1 hour, and then washed with PBS. An acellular dermal layer was prepared in the same manner as in Example 1.

Experimental Example 1: Confirmation of the effect of hydrogen peroxide treatment and vacuum treatment

1.1. Confirmation of thickness increase effect by hydrogen peroxide treatment and vacuum treatment

In order to confirm the effect of increasing the thickness of the acellular dermal layer by hydrogen peroxide treatment and vacuum treatment, the difference in thickness of the acellular dermal layer before and after hydrogen peroxide treatment and vacuum treatment was evaluated. Table 1 below shows the thickness difference between Comparative Example 2 in which only hydrogen peroxide treatment (30.0% (w/v), 1 hour) and Example 1 in which hydrogen peroxide (30.0% (w/v), 1 hour) and vacuum treatment were performed This is the result of comparative evaluation with Comparative Example 1, which is an untreated control group.

As a result, as shown in Table 1 below, it was confirmed that the thickness of the acellular dermal layer of Example 1 treated with hydrogen peroxide and vacuum was increased by about 0.47 mm on average compared to the untreated control group. This means that the thickness of the acellular dermal layer can be significantly increased through the hydrogen peroxide treatment and the vacuum treatment process.

division
(Unit: mm) original thickness
(Comparative Example 1) H ₂ O ₂ Thickness after treatment
(Comparative Example 2) H ₂ O ₂ + Thickness after vacuum treatment
(Example 1) Thickness difference before/after Average difference in thickness before/after One 2.82 3.80 - 0.98 0.98 2 2.86 3.83 - 0.97 3 2.76 3.75 - 0.99 4 2.77 - 3.18 0.41 0.47 5 2.75 - 3.14 0.39 6 2.70 - 3.30 0.60

1.2. Confirmation of thickness increase effect by spike pretreatment

In order to confirm whether the spike pretreatment on the acellular dermis layer before the thickness increase process has an effect on the thickness increase, the acellular dermal layer of Example 1 in which hydrogen peroxide treatment and vacuum treatment were performed without spike pretreatment and after spike pretreatment For the acellular dermal layer of Example 2 subjected to hydrogen peroxide treatment and vacuum treatment, the degree of thickness increase was compared with that of Comparative Example 1, which was an untreated control group. The following [Table 2] shows the results of confirming the degree of thickness increase compared to Comparative Example 1, which is an untreated control, in Example 1 without spike treatment and Example 2 with spike treatment.

As a result, as shown in Table 2 below, in the non-spike treatment group, the thickness increased by about 0.462 mm compared to Comparative Example 1, but in the spike treatment group, it was confirmed that the thickness increased by about 1.183 mm compared to Comparative Example 1. This means that the effect of increasing the thickness by performing the hydrogen peroxide treatment and vacuum treatment step can be increased by about 3 times on average by performing the spike pretreatment step.

division In the case of Spike (○)
Thickness difference before/after augmentation
(Example 2) For Spike (X)
Thickness difference before/after augmentation
(Example 1) One 2.04 mm 0.40 mm 2 1.33mm 0.45 mm 3 0.88 mm 0.20 mm 4 1.38mm 0.37 mm 5 0.80 mm 0.89 mm 6 0.67 mm 0.46 mm Average 1.183 mm 0.462 mm

1.3. Confirmation of the improvement effect of physical property deterioration due to vacuum treatment

Since the mechanical properties of the acellular dermal layer can be deteriorated according to the structural transformation that increases the thickness of the acellular dermis layer, in order to confirm whether this reduction in physical properties can be improved by performing the vacuum treatment step, Example 1 and Comparative Example 1, And mechanical properties were evaluated for Comparative Example 2.

Specifically, each tissue sample was cut into a size of 1 × 5 cm, fixed to both jigs of a universal test machine, and the distance between the grips on both sides of the specimen was adjusted to 3 cm, and then cross-head speed 50 mm / min Tensile force, maximum force, and elongation force were measured by stretching at a rate of .

Figure 4 is a result of comparing the tensile force, maximum force, and elongation force of Example 1 in which a hydrogen peroxide treatment step and a vacuum treatment step were performed on the acellular dermal layer, Comparative Example 2 in which only the hydrogen peroxide treatment step was performed, and Comparative Example 1, which is an untreated control group am.

As a result, as shown in FIG. 4, in the case of Comparative Example 2, as the thickness is increased by the hydrogen peroxide treatment, the tensile force and the maximum force, which are physically and structurally enduring forces, decrease, and the elongation force increases in inverse proportion to this, thereby exhibiting high adhesion and covering power. confirmed that there is On the other hand, in the case of Example 1 in which hydrogen peroxide and vacuum treatment were performed, tensile force and maximum force values were higher than those of Comparative Example 2, so the physical structural strength reduced by the thickness increase can be improved by vacuum treatment confirmed. In addition, it was found that the increased tensile force due to the thickness increase was reduced by the vacuum treatment, but it was confirmed that the tensile force was still higher than that of Comparative Example 1. The above results show that this process of performing hydrogen peroxide and vacuum treatment can produce a more flexible and resilient tissue while achieving the desired thickness increase effect rather than treating only hydrogen peroxide, so that a skin graft material with excellent marketability can be manufactured. In particular, In the case of tissues such as breasts, soft and resilient tissues are required, which means that they can be usefully used in manufacturing skin graft materials for breast reconstruction.

Experimental Example 2: Confirmation of the effect of electron beam irradiation

2.1. Confirmation of the effect of improving the degradation of physical properties by electron beam irradiation

In order to confirm whether the decrease in physical properties due to the increase in thickness can be further improved by electron beam irradiation, the mechanical properties before and after electron beam irradiation were compared and evaluated in Examples 1 and 3, which are spike untreated groups, and Examples 2 and 4, which are spike treated groups. Mechanical property evaluation was performed in the same manner as in Experimental Example 1.3.

Table 3 below shows Example 1 in which only the hydrogen peroxide treatment step (30% (w / v), 1 hour) and the vacuum treatment step were performed in the spike untreated group, and electron beam irradiation (5 kGy, 15 kGy) was further performed This is a result of comparative evaluation of the mechanical properties of Example 3. In addition, Table 4 below shows Example 2 in which only the hydrogen peroxide treatment step (30% (w / v), 1 hour) and the vacuum treatment step were performed in the spike treatment group, and electron beam irradiation (5 kGy, 15 kGy) was further added thereto. This is the result of comparative evaluation of the mechanical properties of Example 4 performed.

Example 1
(before electron beam irradiation) division Tensile Strength (MPa) Load at Maximum Load (N) Percentage Total Elongation at Maximum Force 9.05 130.12 173.18 8.35 128.34 155.64 8.14 156.24 173.56 Average 8.51 138.23 167.46 STDEV 0.48 15.62 10.24 Example 3 (after electron beam irradiation (5 kGy)) division Tensile Strength (MPa) Load at Maximum Load (N) Percentage Total Elongation at Maximum Force 9.24 145.91 146.79 8.17 130.93 163.28 9.15 157.25 151.42 Average 8.85 144.70 153.83 STDEV 0.59 13.20 8.51 Example 3 (after electron beam irradiation (15 kGy)) division Tensile Strength (MPa) Load at Maximum Load (N) Percentage Total Elongation at Maximum Force 10.56 210.72 125.66 13.87 225.69 149.61 11.23 208.31 134.52 Average 11.89 214.91 136.60 STDEV 1.75 9.42 12.11

Example 2
(before electron beam irradiation) division Tensile Strength (MPa) Load at Maximum Load (N) Percentage Total Elongation at Maximum Force 8.02 134.22 172.32 8.11 137.56 168.34 7.92 120.44 178.33 Average 8.02 130.74 173.00 STDEV 0.10 9.08 5.03 Example 4 (after electron beam irradiation (5 kGy)) division Tensile Strength (MPa) Load at Maximum Load (N) Percentage Total Elongation at Maximum Force 10.38 206.85 148.92 14.27 246.33 172.34 12.65 233.46 156.28 Average 12.43 228.88 159.18 STDEV 1.95 20.13 11.98 Example 4 (after electron beam irradiation (15 kGy)) division Tensile Strength (MPa) Load at Maximum Load (N) Percentage Total Elongation at Maximum Force 10.97 223.00 136.94 17.13 315.20 178.32 12.57 275.38 118.55 Average 13.56 271.19 144.60 STDEV 3.20 46.24 30.61

As a result, as shown in Tables 3 and 4 above, it was confirmed that the tensile force and maximum force values increased before and after electron beam irradiation in both the spike-treated group and the spike-untreated group, indicating that the reduced physical and structural strength due to the increase in thickness It was confirmed that it could be further improved by electron beam irradiation. In addition, it was confirmed that the increased elongation due to the thickness increase decreased before and after the electron beam irradiation, but the elongation value was still higher than that of the original material. This tendency was more evident in the 15 kGy irradiation condition. The above results show that this process, in which electron beam irradiation is performed more than hydrogen peroxide and vacuum treatment alone, achieves the desired thickness increase effect, while making a more flexible and resilient tissue, so that a skin graft material having excellent marketability can be manufactured, In particular, in the case of a tissue such as a breast, soft and elastic tissue is required, so it means that it can be usefully used for manufacturing a skin graft material for breast reconstruction.

In addition, in order to confirm whether the effect of improving the decrease in physical properties due to electron beam irradiation can be maintained for a long period of time, the mechanical properties according to the electron beam dose after being stored in the storage solution for 4 weeks were compared and evaluated with that of Comparative Example 1, which is an untreated group.

Figure 5 is Example 1 in which only the hydrogen peroxide treatment step (30% (w / v), 1 hour) and the vacuum treatment step were performed in the spike untreated group, an example in which electron beam irradiation (5 kGy, 15 kGy) was further performed These are the results of comparing the tensile force, maximum force, and elongation force after storage for 4 weeks in the storage solution for Example 3 and Comparative Example 1, which is an untreated group.

As a result, as shown in FIG. 5, it was confirmed that the maximum force and extension force except for the tensile force were maintained at levels similar to those of Comparative Example 1 before the thickness increase treatment under the irradiation condition of 15 kGy after 4 weeks in the storage solution. This means that, in particular, under the condition of 15 kGy irradiation, the crosslinking of the collagen layer is more dense and stratified, thereby forming favorable conditions for maintaining the structure, so that the effect of improving the decrease in physical properties due to electron beam irradiation can be maintained for a long period of time.

2.2. Confirmation of thickness maintenance effect by electron beam irradiation

In order to confirm whether the thickness increase effect by hydrogen peroxide treatment and vacuum treatment can be maintained by electron beam irradiation, the thickness increase degree confirmed in [Table 2] and the thickness increase degree after further treatment of electron beam irradiation are compared to change the thickness observed. The following [Table 5] shows the degree of thickness increase compared to Comparative Example 1 of Examples 1 and 2 after thickness increase, the degree of thickness increase compared to Comparative Example 1 of Examples 3 and 4 irradiated with electron beams after thickness increase, before and after electron beam irradiation This is the result of confirming the degree of thickness reduction.

As a result, as shown in Table 5 below, although the thickness decreased slightly compared to before irradiation according to the electron beam irradiation, it was confirmed that the thickness decrease was smaller than that of 5 kGy under the 15 kGy irradiation condition. This means that it is possible to minimize the reduction of the increased thickness by the electron beam irradiation under the condition of 15 kGy when the electron beam is irradiated to enhance the mechanical properties.

division Spike (○) Spike (X) Thickness difference before electron beam irradiation
(Example 2) Thickness difference after electron beam irradiation (Example 4) Degree of reduction before/after electron beam Thickness difference before electron beam irradiation
(Example 1) Thickness difference after electron beam irradiation (Example 3) Degree of reduction before/after electron beam 5 kGy One 2.04 mm 1.75mm -0.29 mm 0.40 mm 0.29 mm -0.11 mm 2 1.33mm 1.13mm -0.20 mm 0.45 mm 0.37 mm -0.08 mm 3 0.88 mm 0.80 mm -0.08 mm 0.20 mm -0.20 mm -0.40 mm Average -0.19 mm - 0.20 mm 15 kGy 4 1.38mm 1.07mm -0.31 mm 0.37 mm 0.30 mm -0.07 mm 5 0.80 mm 0.70 mm -0.10 mm 0.89 mm 0.76 mm -0.13 mm 6 0.67 mm 0.56mm -0.11 mm 0.46 mm 0.39 mm -0.07 mm Average -0.17 mm -0.09 mm overall average 1.183 mm 1.00 mm -0.18 mm 0.462 mm 0.32 mm -0.14 mm

In addition, in order to check whether the effect of increasing the thickness can be maintained for a long period of time by irradiating the electron beam, the change in thickness after 4 weeks of storage in the storage solution was compared according to the dose of the electron beam. Figure 6 shows the change in thickness during storage for 4 weeks in a storage solution by increasing the thickness by performing a hydrogen peroxide treatment step and a vacuum treatment step on the acellular dermal layer in the electron beam untreated group, the electron beam 5 kGy irradiation group, and the electron beam 15 kGy irradiation group. is the result of comparison.

As a result, as shown in FIG. 6, in the case of the electron beam untreated group, the thickness change after 4 weeks in the storage solution state was relatively large, but in the case of the electron beam treatment group (5 kGy, 15 kGy), it increased even after 4 weeks. It was found that the increased thickness was maintained, and in particular, the increased thickness was best maintained even after 4 weeks under the irradiation condition of 15 kGy, and it was confirmed that some degree of thickness increase additionally appeared even in the storage solution state beyond the maintained state. This means that the effect of increasing the thickness by hydrogen peroxide and vacuum treatment can be maintained and improved for a longer period of time by irradiation with electron beams.

Claims (11)

A method for increasing the thickness of an acellular dermal layer graft material comprising: immersing the dermal layer from which cells are removed in a reaction vessel containing a hydrogen peroxide solution. The method according to claim 1, wherein the method further comprises applying a vacuum condition to the reaction vessel. The method according to claim 2, wherein the step of applying the vacuum condition is to apply a pressure condition of 1 to 950 mTorr to the reaction vessel. The method of claim 1, wherein the hydrogen peroxide solution has a concentration of 5.0 to 30.0% (w/v). The method according to claim 1, wherein the method further comprises forming a plurality of holes on the surface of the dermal layer from which the cells are removed, prior to the immersion step. The method according to claim 2, wherein the method further comprises, after applying the vacuum condition, irradiating the dermal layer with electron beams. The method according to claim 6, wherein the irradiation dose of the electron beam is 1 to 30 kGy. The method according to claim 1, wherein the method, prior to the immersion step, the step of removing the epidermal layer from the skin tissue separated from the subject; and removing cells of the dermal layer from the skin tissue from which the epidermal layer is removed. The method according to claim 8, wherein the epidermal layer is removed by separating it from the dermal layer using a proteolytic enzyme, an ionic solution, or a combination thereof. The method according to claim 8, wherein the step of removing the cells of the dermal layer is to remove by treatment with a surfactant, ultrasonic treatment, or a combination thereof. An acellular dermal layer graft material prepared by the method of any one of claims 1 to 10.

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