Bio-ink composition for dermal regeneration sheet, method for producing customized dermal regeneration sheet using same, and customized dermal regeneration sheet produced by said production method
阅读说明：本技术 真皮再生片用生物墨水组合物、利用其的定制型真皮再生片的制造方法以及利用所述制造方法制造的定制型真皮再生片 (Bio-ink composition for dermal regeneration sheet, method for producing customized dermal regeneration sheet using same, and customized dermal regeneration sheet produced by said production method ) 是由 柳石焕 于 2018-10-19 设计创作，主要内容包括：本发明涉及一种真皮再生片用生物墨水组合物(adipose tissue derived stromal vascular fraction)以及利用其的定制型真皮再生片的制造方法,所述真皮再生片用生物墨水组合物包括：包含脂肪组织源性基质血管组分、细胞外基质及纤维蛋白原的第一液；以及包含凝血酶的第二液。(The present invention relates to a bio-ink composition for a regenerated dermis (induced structural vacuum fraction) comprising: a first fluid comprising adipose tissue-derived stromal vascular fraction, extracellular matrix, and fibrinogen; and a second liquid comprising thrombin.)
1. A bio-ink composition for dermal regeneration patch, comprising:
a first fluid comprising adipose tissue-derived stromal vascular fraction, extracellular matrix, and fibrinogen; and
a second liquid comprising thrombin.
2. The bio-ink composition for dermal renewal sheet according to claim 1,
the concentration of the adipose tissue-derived stromal vascular fraction in the first liquid is 105Per ml to 107One per ml.
3. The bio-ink composition for dermal renewal sheet according to claim 1,
the extracellular matrix content in the first liquid is 20 wt% to 60 wt%.
4. The bio-ink composition for dermal renewal sheet according to claim 1,
the extracellular matrix is a particle of 5 to 100 μm in diameter.
5. The bio-ink composition for dermal renewal sheet according to claim 1,
the concentration of fibrinogen in the first liquid is 4 mg/ml to 50 mg/ml.
6. The bio-ink composition for dermal renewal sheet according to claim 1,
the concentration of thrombin in the second liquid is 30IU/ml to 250 IU/ml.
7. The bio-ink composition for dermal renewal sheet according to claim 1,
the adipose tissue-derived stromal vascular fraction and the extracellular matrix are separately extracted from autologous adipose tissue.
8. The bio-ink composition for dermal renewal sheet according to claim 1,
the first liquid further comprises aprotinin.
9. A method of manufacturing a customized dermal renewal sheet, comprising:
a) acquiring three-dimensional data of a dermal region of a defect using a 3D scanner;
b) a step of forming a first layer corresponding to the shape of the acquired three-dimensional data by using a first liquid containing an adipose tissue-derived stromal vascular fraction, an extracellular matrix, and fibrinogen;
c) a step of forming a second layer by applying a second liquid containing thrombin onto the first layer; and
d) and a step of forming a dermal regeneration sheet by reacting the first layer and the second layer.
10. The method of manufacturing a customized dermal renewal sheet according to claim 9,
further comprising: a1) a step of manufacturing a mold corresponding to the shape of the acquired three-dimensional data by using a 3D printer;
in step b), the first liquid is applied inside the mould.
11. The method of manufacturing a customized dermal renewal sheet according to claim 9,
the step b) and the step c) are alternately and repeatedly executed for more than 2 times.
12. The method of manufacturing a customized dermal renewal sheet according to claim 9,
step d) is completed in less than 10 minutes.
13. The method of manufacturing a customized dermal renewal sheet according to claim 1,
steps b) and c) are performed using inkjet printing or 3D printing.
14. A customized dermal renewal sheet manufactured by the manufacturing method according to claim 9.
Technical Field
The present specification claims the benefit of the application date of korean patent application No. 10-2018-0012220, filed from korean patent office at 31.1.2018, the entire contents of which are incorporated herein by reference.
The present invention relates to a bio-ink composition for a dermal regeneration sheet, a method for manufacturing a customized dermal regeneration sheet using the same, and a customized dermal regeneration sheet manufactured by the manufacturing method.
Background
The present description advocates the benefit of the application date of korean patent application No. 10-2018-0012220, filed in the korean patent office on 31.1.2018, the contents of which are all included in the present description.
The present invention relates to a bio-ink composition for a dermal regeneration sheet, a method for manufacturing a customized dermal regeneration sheet using the same, and a customized dermal regeneration sheet manufactured by the manufacturing method.
Background of the invention
The skin, as the largest organ covering the entire surface of the human body, performs functions of preventing loss of body fluid, blocking invasion of harmful substances and microorganisms from the outside, protecting our body from physical irritation, radiation, ultraviolet rays, and the like. The skin has many appendages such as hair follicles, hairs, sweat glands, and sebaceous glands, and is an important comprehensive organ that performs many different functions in addition to the function of a protective membrane. The skin is roughly divided into epidermis (epidermis), dermis (dermis) and hypodermis (hypodermis).
The dermis, which is a layer located below the epidermis, is a layer that provides a matrix to support various structures such as blood vessels, nerves, etc., and is composed of collagen, elastic fibers, and an extracellular matrix (extracellular matrix). The dermis is composed of a papillary layer (papillary layer) rich in fibroblasts (fibroplast) and having capillary blood vessels in the upper layer and a reticular connective tissue (connective tissue) rich in thick collagen fibers.
In skin tissues, a part of the tissues may be damaged by burns, trauma, skin diseases, and the like, and in this case, autograft (autograft) of transplanting the skin tissues of the patient, allograft (homograft, allograft) of transplanting the skin tissues of another person, and xenograft (hetereograft, xenograft) of transplanting the skin of an animal are used for healing the damaged tissues or for reshaping the damaged tissues. Among these methods, autografting is most desirable, but with the difficulty: when the treatment site is wide, the site where the tissue can be secured is limited, and a new wound site is left at the skin-removing site. The allografts serve to help the cells in the periphery of the wound to move and heal, rather than to help the permanent implant.
Artificial skin (skin equivalent or reconstructed skin) is a three-dimensional reconstructed skin using skin cells and collagen, elastin, and the like as skin constituent substances, and is called a biomimetic skin (skin equivalent or reconstructed skin) because it is composed of living fibroblasts and keratinocytes and exhibits morphological and physiological properties similar to those of actual skin. Artificial skin was developed in 1980, and is intended to treat severe burn patients who need skin transplantation but cannot be transplanted only on their own skin due to excessive burn degree or too large affected part, and is currently applied to various fields such as skin physiological research, skin irritation evaluation, skin efficacy evaluation and the like through continuous improvement and research. However, the conventional artificial skin has a problem in that the dermis gradually shrinks during tissue culture in terms of the production method, and the entire shape of the artificial skin is deformed when the dermis shrinks severely.
The original cell therapeutic agent adopts a method of transplanting autologous cells or transplanting allogeneic cells. Transplantation of allogeneic cells, although having a paracrine (paracrine) effect, has a problem in that they are eventually lost by the body as foreign bodies (bodies). In addition, when autologous cells are transplanted by an injection method, if the cells do not have an environment (e.g., a scaffold) necessary for growth, the probability of disappearance is high, and it is difficult to see the effect of the injected cells.
Further, autografting, in which a part of the autologous tissue is subjected to proliferation culture, has problems in that there is a possibility that scars will occur in the donor area, that the donor area will be insufficient when the graft site is large, and the like; the problems of the allografts and the xenotransplants are immune rejection or inflammation. Thus, it is true that there is a need to develop a method for replacing or regenerating damaged skin with a minimum of side effects as described above.
[ Prior art documents ]
[ patent document ]
Korean granted patent gazette: 10-1710615
Disclosure of Invention
Technical problem to be solved by the invention
The present invention aims to provide a bio-ink composition for a dermal regeneration sheet, which can minimize graft rejection and can be used for manufacturing a customized dermal regeneration sheet for a patient, a method for manufacturing a customized dermal regeneration sheet using the same, and a dermal regeneration sheet.
Means for solving the problems
In one embodiment of the present invention, there is provided a bio-ink composition for a dermal regeneration sheet, including: a first fluid comprising adipose tissue-derived stromal vascular fraction, extracellular matrix, and fibrinogen; and a second liquid comprising thrombin.
Another embodiment of the present invention provides a method for manufacturing a customized dermal regeneration sheet, including: a) acquiring three-dimensional data of a dermal region of a defect using a 3D scanner; b) a step of forming a first layer corresponding to the shape of the acquired three-dimensional data by using a first liquid containing an adipose tissue-derived stromal vascular fraction, an extracellular matrix, and fibrinogen; c) a step of forming a second layer by applying a second liquid containing thrombin onto the first layer; and d) a step of forming a dermal renewal sheet by reacting the first layer and the second layer.
In another embodiment of the present invention, a customized dermal renewal sheet manufactured by the manufacturing method is provided.
ADVANTAGEOUS EFFECTS OF INVENTION
According to the bio-ink composition for a dermal regeneration sheet and the method for manufacturing a customized dermal regeneration sheet using the same of the present invention, there is an advantage in that a dermal regeneration sheet suitable for a damaged skin tissue of a patient can be manufactured. Specifically, the method for manufacturing a customized dermal regeneration sheet according to the present invention can manufacture a dermal regeneration sheet having a shape conforming to an affected part by 3D scanning of the exact shape of the affected part.
The dermal regeneration sheet according to the present invention has advantages in that it can be transplanted without immune rejection, and can be produced in a short time, so that healthy cells can be transplanted into an affected part.
Drawings
Fig. 1 illustrates a process of preparing a first liquid and a second liquid, respectively, for manufacturing a customized dermal regeneration sheet according to the present invention.
Fig. 2 shows photographs for confirming the cell viability of the customized dermal renewal sheet according to experimental example 1.
Fig. 3 shows a photograph of the removed skin in the animal experiment for carrying out experimental example 2.
Fig. 4 shows the customized dermal renewal sheets of the control group and the examples manufactured according to experimental example 2.
Fig. 5 shows a process of transplanting the customized dermal renewal sheet according to experimental example 2.
FIG. 6 shows the result of the trichrome staining of the masson pine in the animal experiment of Experimental example 2.
FIG. 7 is the result of the animal experiment according to Experimental example 2, whether or not the graft site was vascularized, observed using CD31 staining.
Detailed Description
In the present specification, when a member is referred to as being "on" another member, it does not merely refer to the case where the member is in contact with another member, but includes the case where another member exists between the two members.
In the present specification, when a part "includes" a certain component, unless specifically stated to the contrary, it does not mean that other component is excluded, but means that other component may be included.
The present invention will be described in detail below.
In one embodiment of the present invention, there is provided a bio-ink composition for a dermal regeneration sheet, including: a first liquid comprising an adipose tissue derived stromal vascular fraction, an extracellular matrix, and fibrinogen; and a second liquid comprising thrombin.
The bio-ink composition for a dermal regeneration sheet according to the present invention is a two-component type, and the first liquid and the second liquid are sequentially applied and then react with each other to form the dermal regeneration sheet. Specifically, the thrombin in the second liquid and the fibrinogen in the first liquid can react to form a fibrin network, which can serve to sufficiently immobilize the adipose tissue-derived stromal vascular fraction and extracellular matrix.
The adipose tissue-derived stromal vascular fraction comprises adipose tissue-derived stem cells. Preferably, the adipose tissue-derived stromal vascular fraction may be substantially free of cells other than adipose tissue-derived stem cells (e.g., adipocytes, red blood cells, and other stromal cells, etc.) and extracellular matrix material, and more preferably, may be completely free of cells and extracellular matrix material.
The adipose tissue-derived stromal vascular fraction may be extracted from adipose tissue of a homogeneous or heterogeneous animal. Preferably, the adipose tissue-derived stromal vascular fraction may be extracted from autologous adipose tissue. More specifically, the adipose tissue-derived stromal vascular fraction may be extracted using adipose tissue of a subject patient or animal. The adipose tissue-derived stromal vascular fraction may be obtained by extracting adipose tissue-derived Stromal Vascular Fraction (SVF) and extracellular matrix (ECM) and the like from adipose tissue in the form of micro-clusters (micro or nano SVF/ECM clusters), or may be separately isolated and used as needed. For example, the extract can be extracted from adipose tissue using a Lipocell kit from Tiss' you. However, the adipose tissue-derived stromal vascular fraction may be obtained by a method known in the art, an extraction kit, or the like.
According to one embodiment of the invention, the adipose tissue-derived stromal vascular fraction may be present in the first liquid at a concentration of 105Per ml to 107One per ml. When the concentration of the adipose tissue-derived stromal vascular fraction is within the range, the fabricated customized dermal renewal sheet is capable of effectively performing differentiation into dermal cells at the transplantation site.
The effect is improved as the content of the adipose tissue-derived stromal vascular fraction is higher, whereas the effect by extracellular matrix, fibrinogen, and the like is hardly exhibited when the content is too high. Therefore, the adipose tissue-derived stromal vascular fraction may be present in an amount that is the balance of the amount of other constituents present in the first liquid.
The adipose tissue-derived stromal vascular fraction can be used in combination with an adipose tissue-derived extracellular matrix to promote differentiation into dermal cells. Specifically, the adipose tissue-derived extracellular matrix has biochemical factors required for cell growth and differentiation necessary for healing an affected wound, and can provide a physical environment in which the adipose tissue-derived matrix vascular component can be fixed after being differentiated into dermal cells.
According to one embodiment of the invention, the extracellular matrix can be extracted from adipose tissue (or cells) of a homogeneous or heterogeneous animal. In addition, the extracellular matrix may be extracted from fibrotic tissue (or cells) of the same or a different animal. In particular, the extracellular matrix may be extracted from autologous adipose tissue (or cells), or autologous fibrotic tissue (or cells). More specifically, the extracellular matrix may be extracted using adipocytes of a surgical subject patient or animal.
According to one embodiment of the invention, the extracellular matrix may be decellularized.
According to one embodiment of the invention, the extracellular matrix may be particles with a diameter of 5 μm to 100 μm. Specifically, the extracellular matrix may be physically decellularized and pulverized into particles for use. Further, the extracellular matrix can be obtained by a method known in the art.
The fibrin matrix obtained by the reaction of the fibrinogen of the first liquid and the thrombin of the second liquid can function to immobilize the adipose tissue-derived matrix vascular fraction and the extracellular matrix.
According to one embodiment of the invention, the extracellular matrix content in the first liquid may be between 20 wt% and 60 wt%. Specifically, the content of the extracellular matrix in the first liquid may be 20 wt% to 50 wt%. When the content of the extracellular matrix is within the above range, the survival rate of cells that differentiate into dermal cells is increased, and regeneration of a defective dermal site can be effectively achieved. When the content of the extracellular matrix is too high, the contents of fibrinogen and thrombin used for manufacturing the dermal renewal sheet are relatively reduced, and thus there is a problem that the form of the manufactured dermal renewal sheet is difficult to maintain. In addition, when the content of the extracellular matrix is too small, the contents of fibrinogen and thrombin used for manufacturing the dermal regeneration sheet are too high, so that the manufactured dermal regeneration sheet is too firm and the effect of the extracellular matrix is hardly expected. Therefore, it is preferable to adjust the content of the extracellular matrix to the above range, thereby improving the survival rate of cells that differentiate into dermal cells and further facilitating the shape maintenance of the dermal renewal sheet.
According to an embodiment of the present invention, the concentration of fibrinogen in the first liquid may be from 4 mg/ml to 50 mg/ml. The concentration of fibrinogen in the first liquid may be from 5 mg/ml to 40 mg/ml, or from 7 mg/ml to 30 mg/ml.
According to one embodiment of the invention, the concentration of thrombin in the second liquid may be between 30IU/ml and 250 IU/ml. Further, the concentration of thrombin in the second liquid may be from 40IU/ml to 250IU/ml, from 40IU/ml to 100IU/ml, or from 45IU/ml to 80 IU/ml.
When the concentrations of the fibrinogen and the thrombin are within the ranges, it is possible to ensure an appropriate curing speed and to maintain a uniform distribution of the adipose tissue-derived stromal vascular fraction. Thus, the cell distribution in the manufactured customized dermal regeneration sheet can be maintained uniform, and the cell differentiation ability can be effectively realized after the implantation of the customized dermal regeneration sheet. In addition, when the concentrations of the fibrinogen and the thrombin are within the above ranges, there is an advantage in that the form of the produced dermal renewal sheet can be favorably maintained and the appropriate hardness can be maintained, so that the transplantation can be suitable for the affected part.
According to one embodiment of the invention, the first liquid may also comprise aprotinin. The aprotinin (aprotinin), which is an inhibitor of proteases secreted by the pancreas, is a polypeptide consisting of 58 amino acids in total. It is known that it is mainly extracted from bovine lung and stops fibrin decomposition in blood, thereby playing a role of hemostasis.
According to one embodiment of the invention, the first solution may comprise 900 to 1,100 units of said aprotinin per 1ml of first solution, in particular 1000 units of kininogen Inactivator.
According to one embodiment of the invention, the second liquid may be thrombin dispersed in a calcium chloride solution. Specifically, the second liquid may contain 40IU to 250IU of thrombin per 1ml, and 5 mg to 6.5 mg of calcium chloride.
The solvent of the first liquid and the second liquid may be water, and may specifically be normal saline. In addition, the fibrinogen in the first liquid and the thrombin in the second liquid may be purchased from commercial fibrin glue kits.
The bio-ink composition for the dermal regeneration sheet has an advantage in that the dermal regeneration sheet can be manufactured on site using a 3D printer at a surgical site because the first liquid and the second liquid complete a reaction within 10 minutes, preferably within 5 minutes.
The present invention uses fibrin glue consisting of fibrinogen and fibrin as a binder, which can ensure higher viscosity than hyaluronic acid binder or collagen binder, so that the customized dermal renewal sheet has excellent adhesive force with the affected part, thereby maintaining high strength.
Another embodiment of the present invention provides a method for manufacturing a customized dermal regeneration sheet, including: a) acquiring three-dimensional data of a dermal region of a defect using a 3D scanner; b) a step of forming a first layer corresponding to the shape of the acquired three-dimensional data by using a first liquid containing an adipose tissue-derived stromal vascular fraction, an extracellular matrix, and fibrinogen; c) a step of forming a second layer by applying a second liquid containing thrombin onto the first layer; and d) a step of forming a dermal renewal sheet by reacting the first layer and the second layer.
Furthermore, according to an implementation state of the present invention, the method may further include: a1) a step of manufacturing a mold corresponding to the shape of the acquired three-dimensional data by using a 3D printer; in step b), the first liquid may be applied inside the mold.
Step a) may utilize 3D scanner equipment or 3D printing equipment known in the art. Further, step a1) may utilize 3D printing equipment known in the industry. The die can fix the three-dimensional shape when the first liquid and the second liquid are applied. The mold may be removed after the custom dermal renewal sheet is formed. The mold may be formed using biocompatible polymers generally used in the industry.
According to one embodiment of the invention, steps b) and c) can be carried out by means of inkjet printing or 3D printing. Specifically, in steps b) and c), a printing apparatus having 2 or more nozzles known in the art may be used, and the nozzles may discharge the first liquid and the second liquid to form a three-dimensional shape. Further, when the first layer is formed, a 3D printer or an ink jet printer may be used to prevent cell clustering in the customized dermal regeneration sheet by providing a uniform cell distribution.
According to an embodiment of the present invention, the steps b) and c) may be performed repeatedly for more than 2 times. Specifically, when a large-sized dermal regeneration sheet is to be formed, steps b) and c) may be alternately performed to be stacked in the same manner as "first layer/second layer/first layer/second layer", and then solidified to form the dermal regeneration sheet.
According to an embodiment of the invention, step d) can be performed within 10 minutes, preferably within 5 minutes. Specifically, in the step d), the first layer and the second layer may react for 3 to 7 minutes to form the customized dermal regeneration sheet.
In the method of manufacturing the customized dermal renewal sheet according to the present invention, the adipose tissue-derived stromal vascular fraction and extracellular matrix may be extracted from the adipose tissue of a patient or an animal for use, without an additional culturing step. After the first solution is produced using the same, the customized dermal renewal sheet can be produced in a short time and transplanted to the affected part, and thus the cell activity can be maximized.
In addition, the method for manufacturing the customized dermal renewal sheet can manufacture the dermal renewal sheet having the same shape as the affected part, and thus can minimize the gap between the transplantation site and the dermal renewal sheet, and can restore the natural state after recovery.
In another embodiment of the present invention, a customized dermal renewal sheet manufactured by the manufacturing method is provided.
As described above, the customized dermal regeneration sheet can be produced into a shape suitable for the affected part in a short time by using the adipose tissues of a patient or an animal, and then transplanted. The dermal regeneration sheet can be transplanted to an affected part, and the affected part is protected by a dressing so as to recover damaged skin. After the customized dermal regeneration sheet is transplanted, the adipose tissue-derived matrix vascular component is differentiated into dermal cells, and the differentiated dermal cells are fixed in the adipose tissue-derived extracellular matrix and the fibrin matrix, so that damaged skin can be recovered.
Hereinafter, the present invention will be described in detail with reference to examples. However, the embodiments according to the present invention may be modified into other various forms, and the scope of the present invention is not to be construed as being limited to the embodiments described below. The embodiments of the present description are provided to more fully describe the invention to those of ordinary skill in the art.
Adipose tissue-derived Stromal Vascular Fraction (SVF)
Adipose tissue-derived Stromal Vascular Fraction (SVF) cells were obtained by the following procedure.
1. In a sterile operating room, liposuction was performed under the control of a doctor, about 60cc of adipose tissue was extracted, and 0.075% collagenase (collagenase) was added thereto in the same amount, and the mixture was incubated at 37 ℃ for 30 minutes by shaking at 230 rpm.
2. After the reaction as described above, the reaction mixture was centrifuged at a speed of 420g [ Relative Centrifugal Force (RCF) ] for 10 minutes at 25 ℃ to separate the reaction mixture into 3 layers, i.e., an SVF pellet layer, a collagenase layer and an oil layer.
3. The supernatant except the SVF pellet layer was removed, and after the SVF pellet layer was resuspended in Phosphate Buffered Saline (PBS), the fibrous mass and residual impurities of the resuspended SVF layer were removed using a 100 μm nylon filter (Cell filter).
4. The results of counting nucleated cells using a cell counter after resuspending the filtered SVF-containing solution, centrifuging 3 times, removing the pellet to leave the lowest pellet, were confirmed: SVFs of 0.26X 106 to 2.2X 106 per 1ml were obtained.
Extracellular matrix (ECM)
Liposuction is performed in a sterile operating room under the direction of a doctor, and after the adipose tissue is extracted, it is mixed into physiological saline. The ECM was mechanically separated from the adipose tissue using a homogenizer at 12,000rpm to 20,000rpm, and then centrifuged. The ECM was finally obtained after repeating the process of re-centrifuging the centrifuged precipitate 3 to 5 times.
Experimental example 1 in vitro experiment for confirming cell survival ability in an Engineered dermal regeneration patch
Fig. 1 shows a process of preparing a first liquid and a second liquid separately for manufacturing a customized dermal regeneration sheet of the present invention. Specifically, the obtained SVF and ECM were mixed with a mixing syringe (mix syring), and then mixed with 1ml of aprotinin solution mixed with 4.5 mg/ml fibrinogen with the mixing syringe (mix syring) to prepare a first solution. At this time, the ECM content in the first liquid was adjusted to 10 wt% or 20 wt% as shown in fig. 2.
Further, a thrombin-dispersed calcium chloride solution was prepared in the same volume as the first liquid phase prepared. At this time, the concentration of thrombin in the second liquid was adjusted to 7.8IU/ml to 250IU/ml as shown in FIG. 2.
The prepared first liquid was applied to form a first layer, and the prepared second liquid was applied to the first layer to produce a custom-made dermal renewal sheet.
Fig. 2 shows photographs for confirming the cell viability of the customized dermal renewal sheet according to experimental example 1. Specifically, fig. 2 shows that the cell viability was confirmed by Confocal microscopy [ Confocal microscopics (leica, TCS SP5) ] after staining the customized dermal regeneration sheets with green fluorescence calcein-AM (calcein AM) for confirming living cells and staining with red fluorescence ethidium homomodimer-1 (ethidium bromide dimer 1, EthD-1) for confirming the loss of plasma membrane. From FIG. 2, it was confirmed that when the content of ECM was 20 wt%, the interaction (interaction) between SVF and ECM was increased, and the cell growth was active, and that when the thrombin concentration was 50IU/ml or more, the cell growth was more active.
Experimental example 2 animal experiment for confirming efficacy of the manufactured dermal renewal sheet (in vivo experiment)
In order to verify the efficacy of the dermis renewal sheet according to the invention, after the skin of a 4-month-old pig is removed at a size of 2.5 cm × 2.5 cm, the SVF and ECM are obtained from the adipose tissue of the pig as described above. Fig. 3 shows a photograph of the removed skin in the animal experiment used to present experimental example 2.
Further, the obtained SVF and ECM were mixed by a mixing syringe (mix syring), and then mixed with 1ml of aprotinin solution mixed with 9 mg/ml fibrinogen by the mixing syringe (mix syring) to prepare a first solution. At this time, the ECM content in the first solution was 20 wt%, and the concentration of SVF was 6.25X 106/ml. In addition, a second liquid containing thrombin at a concentration of 50IU/ml was prepared.
Then, the removed skin area was scanned by a 3D scanner, and then the prepared first liquid and second liquid were applied in this order using a 3D Bio 3D printer (INVIVO, ROKIT), followed by curing for 5 minutes, thereby producing a custom-made dermal regeneration sheet. The thus-produced dermal regeneration sheet was transplanted to a removed skin region and then subjected to dressing treatment, and the degree of recovery of the skin was observed for 14 days.
As a control group, a custom-made dermal renewal sheet was produced in the same manner without ECM in the first liquid, and was transplanted to a removed skin area and then subjected to dressing treatment, and the degree of recovery of the skin was observed for 14 days.
Fig. 4 shows the customized dermal renewal sheets of the control group and the examples manufactured according to experimental example 2.
Fig. 5 shows a process of transplanting the customized dermal renewal sheet according to experimental example 2. Specifically, fig. 5 shows photographs of the transplantation of the customized dermal renewal sheets according to the control group (SVF only) and the example (SVF + ECM), showing the passage from the transplantation day (day zero) to 14 days later.
FIG. 6 shows the result of the trichrome staining of the masson pine in the animal experiment of Experimental example 2. Specifically, fig. 6 shows masson trichrome staining and observation after 14 days after the removal of the skin, for a non-damaged skin (normal) area, a skin-removed area (defect only), and an area in which a customized dermal renewal sheet (SVF + ECM) according to the example was transplanted in the skin-removed area, respectively. From fig. 6, it can be confirmed that, when left to stand for 14 days after removal of the skin (only the defect), the collagen complex is formed at a very low concentration in the dermal layer. In contrast, when the customized dermal regeneration sheet (SVF + ECM) according to the example was transplanted, it was confirmed that the dermal layer was stained sharply blue, the collagen complex was formed at a high density in the dermal layer, and the epidermal layer was stained sharply red, and it was confirmed that keratin, cytoplasm, etc., which are components of the epidermal layer, were formed.
FIG. 7 is the result of the animal experiment according to Experimental example 2, whether or not the graft site was vascularized, observed using CD31 staining. Specifically, fig. 7 shows CD31 staining and observation of whether or not blood vessels are regenerated after 14 days from the removal of the skin, respectively, for a non-damaged skin (normal) area, a skin-removed area (defect only), and an area where a customized dermal regeneration patch (SVF + ECM) according to the embodiment is transplanted in the skin-removed area. From fig. 7, it can be confirmed that when left to stand for 14 days after removal of the skin (only the defect), the blood vessel region stained brown appears sporadically and narrowly. In contrast, when the customized dermal regeneration sheets (SVF + ECM) according to the example were transplanted, it was confirmed that a large, numerous brown-stained angiogenesis region existed.
From the experimental results of experimental example 2, it was found that when the customized dermal regeneration sheet according to the present invention is transplanted to the skin, the regeneration of the skin can be promoted, and further, the angiogenesis in the skin can be promoted, so that the skin can be restored to be similar to the original skin.