Patent Application
20200354686
The present application claims priority based on Korean Patent Application No. 10-2019-0053748, filed on May 8, 2019, the entire content of which is incorporated herein for all purposes by this reference.
The present invention relates to an artificial tissue including an artificial papillary layer, an artificial skin, and a method of manufacturing the same.
In the field of tissue engineering and regenerative medicine, there is urgent need to replace technologies dependent upon animal experimentation or to fabricate implantable artificial tissues by manufacturing artificial tissues that closely mimic human physiology by mimicking micro-environments inside the human body using cells and cell-friendly biomaterials.
Many research groups have already worked on manufacturing artificial tissues that mimic the human body using biomaterials and multiple cells, and the resultant tissues may be used for implantation, cosmetics, and drug testing. In conventional techniques, artificial tissues having a layer structure have been manufactured by simply stacking materials and cells, but have a limitation in that they cannot mimic the interlayer microstructure of real human tissue.
In the case of human skin, a dermo-epidermal junction is present between the dermal layer and the epidermal layer, and the uneven structure under the epidermis due to the protruding dermal papillary layer of the dermo-epidermal junction also affects the function of actual skin tissue.
Therefore, there is the need for a method of manufacturing a tissue capable of more closely simulating human skin functions by mimicking the uneven structure of the dermo-epidermal junction.
Accordingly, an objective of the present invention is to provide an artificial tissue capable of overcoming the structural limitation of the flat dermal layer of existing artificial tissue.
Another objective of the present invention is to provide a method of manufacturing an artificial tissue including an artificial papillary layer by performing cell arrangement, rather than a general process of performing surface patterning using a hydrogel alone.
Still another objective of the present invention is to provide an artificial skin capable of more closely simulating the function of real human skin by manufacturing the artificial skin including the artificial tissue and a method of manufacturing the same.
An aspect of the present invention provides an artificial tissue, including an extracellular matrix layer including a first extracellular matrix and an artificial papillary layer formed on the extracellular matrix layer and including a papilla or papillae, each papilla including a cell and a second extracellular matrix.
Also, the second extracellular matrix may be induced by the cell and contracted.
Also, the cell may include an extracellular matrix receptor, the second extracellular matrix may include at least one selected from the group consisting of a protein and a polysaccharide, and an adhesion adhering the extracellular matrix receptor to at least one selected from the group consisting of a protein and a polysaccharide may be formed. Also, the adhesion may be formed by at least one selected from the group consisting of van der Waals attraction, electrostatic attraction, ionic bonding, hydrogen bonding, and covalent bonding.
Also, the extracellular matrix receptor may include at least one selected from the group consisting of integrin, discoidin domain receptor, glycoprotein VI, immunoglobulin-like cell adhesion molecule, selectin, syndecan, dystrophin glycoprotein complex, and hyaladherin.
Also, the cell may include at least one selected from the group consisting of a fibroblast, a myoblast, a nerve cell, and a vascular endothelial cell.
Also, the fibroblast may include at least one selected from the group consisting of a mammalian fibroblast, an avian fibroblast, a reptile fibroblast, an amphibian fibroblast, and a fish fibroblast.
Also, the artificial papillary layer may have a shape corresponding to a pattern.
Also, the pattern may include at least one selected from the group consisting of a straight-line shape, a curved shape, a polygonal shape, a circular shape, an oval shape, an arc shape, a fan shape and combinations thereof.
Also, each of the extracellular matrix layer and the artificial papillary layer may be a hydrogel.
Also, each of the first extracellular matrix and the second extracellular matrix may independently include at least one selected from the group consisting of collagen, gelatin, nanocellulose, fibrinogen, hyaluronic acid, gelatin methacrylate (GelMA), and elastin.
Another aspect of the present invention provides an artificial skin, including an extracellular matrix layer including a first extracellular matrix, an artificial papillary layer formed on the extracellular matrix layer and including a papilla or papillae, and an artificial epidermal layer formed on the artificial papillary layer and including a keratinocyte, each papilla including a cell and a second extracellular matrix.
Also, the artificial epidermal layer may further include at least one selected from the group consisting of an epidermal cell and a melanocyte.
Still another aspect of the present invention provides a method of manufacturing an artificial tissue, including (a) preparing a mixed solution including a first extracellular matrix in a vessel, (b) forming a bioink pattern by discharging bioink including a cell and a second extracellular matrix on the mixed solution, and (c) culturing the mixed solution and the bioink pattern.
Also, in step (c), each of the first extracellular matrix and the second extracellular matrix may independently be cured by at least one selected from the group consisting of van der Waals attraction, hydrogen bonding, ionic bonding, and covalent bonding.
Also, the curing may occur due to a decrease in hydrogen bonding with a water molecule independently of each of the first extracellular matrix and the second extracellular matrix owing to an elevation in temperature, and due to an increase in self-aggregation owing to van der Waals attraction.
Also, the artificial tissue may include an extracellular matrix layer including the first extracellular matrix, and an artificial papillary layer formed on the extracellular matrix layer and including a papilla or papillae, each papilla including the cell and the second extracellular matrix.
Also, the cell may include an extracellular matrix receptor, the second extracellular matrix may include at least one selected from the group consisting of a protein and a polysaccharide, and the papilla may be formed to protrude by contracting and compacting all or part of the second extracellular matrix due to formation of an adhesion adhering the extracellular matrix receptor to at least one selected from the group consisting of a protein and a polysaccharide. Also, the bioink pattern may be formed through any one process selected from the group consisting of micro-extrusion printing, inkjet printing, laser printing, valve-type printing, spray printing, micro-stamping, and masking.
Yet another aspect of the present invention provides a method of manufacturing an artificial skin, including (a′) preparing a mixed solution including a first extracellular matrix in a vessel, (b′) forming a bioink pattern by discharging bioink including a cell and a second extracellular matrix on the mixed solution, (c′) culturing the mixed solution and the bioink pattern, and (d′) printing and culturing a keratinocyte solution including a keratinocyte on the mixed solution and the bioink pattern, which are cultured.
The artificial tissue according to the present invention includes an artificial papillary layer including a papilla or papillae, thereby overcoming the structural limitation of the flat epidermis of existing artificial tissue.
In addition, the method of manufacturing the artificial tissue according to the present invention is capable of manufacturing an artificial tissue having excellent ability to mimic human physiology using artificial arrangement of cells rather than a general process of forming a surface pattern using a hydrogel alone, and moreover, can be utilized to produce all kinds of tissues and organs having a fine uneven structure in the human body.
In addition, the present invention is capable of applying various patterns when forming the artificial papillary layer, thus inducing an artificial papillary layer in various shapes related to the patterns, thereby providing artificial skin with a controllable state or degree of aging.
Since these drawings are for reference in describing exemplary embodiments of the present invention, the technical spirit of the present invention should not be construed as being limited to the accompanying drawings, in which:
Hereinafter, exemplary embodiments of the present invention are described in detail with reference to the appended drawings so as to be easily performed by a person having ordinary skill in the art.
However, the following description does not limit the present invention to specific embodiments, and in the description of the present invention, detailed descriptions of related known techniques incorporated herein will be omitted when the same may make the gist of the present invention unclear.
The terms herein are used to explain specific embodiments, and are not intended to limit the present invention. Unless otherwise stated, a singular expression includes a plural expression. In the present application, the terms “comprise”, “include” or “have” are used to designate the presence of features, numbers, steps, operations, elements, parts, or combinations thereof described in the specification, and should be understood as not excluding the presence or additional possible presence of one or more different features, numbers, steps, operations, elements, parts, or combinations thereof.
As used herein, the terms “first”, “second”, etc. may be used to describe various elements, but these elements are not to be limited by these terms. These terms are only used to distinguish one element from another. For example, a first element may be termed a second element, and similarly, a second element may be termed a first element, without departing from the scope of the present invention.
Further, it will be understood that when an element is referred to as being “formed” or “stacked” on another element, it can be formed or stacked so as to be directly attached to all surfaces or to one surface of the other element, or intervening elements may be present therebetween.
Hereinafter, a detailed description will be given of an artificial tissue including an artificial papillary layer, an artificial skin and a method of manufacturing the same according to the present invention, which is set forth to illustrate but is not to be construed as limiting the present invention, and the present invention is defined only by the accompanying claims.
The present invention pertains to an artificial tissue, including an extracellular matrix layer including a first extracellular matrix and an artificial papillary layer formed on the extracellular matrix layer and including a papilla or papillae, each papilla including a cell and a second extracellular matrix.
Also, the second extracellular matrix may be induced by the cell and contracted.
Also, the cell may include an extracellular matrix receptor, the second extracellular matrix may include at least one selected from the group consisting of a protein and a polysaccharide, and an adhesion adhering the extracellular matrix receptor to at least one selected from the group consisting of a protein and a polysaccharide may be formed.
The papilla or papillae may be formed by contracting the second extracellular matrix due to traction force of the cell caused by the extracellular matrix receptor in the adhesion.
Also, the adhesion may be formed by at least one selected from the group consisting of van der Waals attraction, electrostatic attraction, ionic bonding, hydrogen bonding, and covalent bonding.
Also, the extracellular matrix receptor may include at least one selected from the group consisting of integrin, discoidin domain receptor, glycoprotein VI, immunoglobulin-like cell adhesion molecule, selectin, syndecan, dystrophin glycoprotein complex, and hyaladherin.
Also, the cell may include at least one selected from the group consisting of a fibroblast, a myoblast, a nerve cell, and a vascular endothelial cell, and preferably includes a fibroblast.
Also, the fibroblast may include at least one selected from the group consisting of a mammalian fibroblast, an avian fibroblast, a reptile fibroblast, an amphibian fibroblast, and a fish fibroblast, and preferably includes a mammalian fibroblast. Most preferably, a human dermal fibroblast is used.
Also, the artificial papillary layer may have a shape corresponding to a pattern.
Also, the pattern may include at least one selected from the group consisting of a straight-line shape, a curved shape, a polygonal shape, a circular shape, an oval shape, an arc shape, a fan shape and combinations thereof.
Also, each of the extracellular matrix layer and the artificial papillary layer may be a hydrogel.
Also, each of the first extracellular matrix and the second extracellular matrix may independently include at least one selected from the group consisting of collagen, gelatin, nanocellulose, fibrinogen, hyaluronic acid, gelatin methacrylate (GelMA) and elastin, and preferably includes collagen.
The present invention pertains to an artificial epidermis, including an extracellular matrix layer including a first extracellular matrix, an artificial papillary layer formed on the extracellular matrix layer and including a papilla or papillae, and an artificial epidermal layer formed on the artificial papillary layer and including a keratinocyte, each papilla including a cell and a second extracellular matrix.
Also, the artificial epidermal layer may further include at least one selected from the group consisting of an epidermal cell and a melanocyte.
With reference to
Also, in step (c), each of the first extracellular matrix and the second extracellular matrix may independently be cured by at least one selected from the group consisting of van der Waals attraction, hydrogen bonding, ionic bonding, and covalent bonding.
Here, the curing may occur due to a decrease in hydrogen bonding with a water molecule independently of each of the first extracellular matrix and the second extracellular matrix owing to an elevation in temperature, and due to an increase in self-aggregation owing to van der Waals attraction.
Also, the artificial tissue may include an extracellular matrix layer including the first extracellular matrix and an artificial papillary layer formed on the extracellular matrix layer and including a papilla or papillae, each papilla including the cell and the second extracellular matrix.
Also, the cell may include an extracellular matrix receptor, the second extracellular matrix may include at least one selected from the group consisting of a protein and a polysaccharide, and the papilla may be formed to protrude by contracting and compacting all or part of the second extracellular matrix due to formation of an adhesion adhering the extracellular matrix receptor to at least one selected from the group consisting of a protein and a polysaccharide.
Also, the bioink pattern may be formed through any one process selected from the group consisting of micro-extrusion printing, inkjet printing, laser printing, valve-type printing, spray printing, micro-stamping, and masking.
With referenced to
The present invention pertains to a method of manufacturing an artificial skin, including (a′) preparing a mixed solution including a first extracellular matrix in a vessel, (b′) forming a bioink pattern by discharging bioink including a cell and a second extracellular matrix on the mixed solution, (c′) culturing the mixed solution and the bioink pattern, and (d′) printing and culturing a keratinocyte solution including a keratinocyte on the mixed solution and the bioink pattern, which are cultured.
The keratinocyte solution may further include at least one selected from the group consisting of an epidermal cell and a melanocyte.
A better understanding of the present invention will be given through the following examples, which are merely set forth to illustrate the present invention but are not to be construed as limiting the scope of the present invention.
Lyophilized Type-I pig skin extract collagen was dissolved in 0.1 v/v % acetic acid, added with a nutrient solution containing Dulbecco's Modified Eagle Medium (DMEM), Ham's F-12, and penicillin/streptomycin, uniformly mixed, added with a mixed solution of 0.05 M NaOH, NaHCO3 and HEPES, and titrated to a neutral pH, thus preparing a mixed solution. Here, the volume ratio of NaOH to NaHCO3 to HEPES was 8:1:1.
The mixed solution was added with human dermal fibroblasts at a concentration of 4.0×106 cells/ml, thereby manufacturing bioink.
Bioink was manufactured in the same manner as in Preparation Example 1, with the exception that human dermal fibroblasts were not added, in lieu of addition of human dermal fibroblasts at a concentration of 4.0×106 cells/ml.
HEKn cells were harvested using Accutase® (Innovative Cell Technologies, USA) and resuspended in an epidermal cell culture medium to afford a keratinocyte solution having a final cell concentration of 1.2×107 cells/ml.
With reference to
The bioink prepared in Preparation Example 1 was discharged on the mixed solution, thus forming a circular bioink pattern having a size of 0.5 mm.
The mixed solution and the bioink pattern were placed in a CO2 cell incubator at 37° C. and cured at an elevated temperature. After 2 hr, a cell culture solution was added on the mixed solution and the bioink pattern, the solution was replaced every two days, and the tissue was matured for a predetermined period of time (1-2 weeks), thus manufacturing an artificial tissue including papillae having a width of 0.3 mm and a height of 50.4 μm.
An artificial tissue was manufactured in the same manner as in Example 1, with the exception that papillae having a width of 0.6 mm and a height of 85.3 μm were formed by forming a bioink pattern having a size of 1.0 mm, in lieu of formation of the papillae having a width of 0.3 mm and a height of 50.4 μm by forming the bioink pattern having a size of 0.5 mm.
An artificial tissue was manufactured in the same manner as in Example 1, with the exception that papillae having a width of 1.0 mm and a height of 94.1 μm were formed by forming a bioink pattern having a size of 2.0 mm, in lieu of formation of the papillae having a width of 0.3 mm and a height of 50.4 μm by forming the bioink pattern having a size of 0.5 mm.
With reference to
The bioink prepared in Preparation Example 1 was discharged on the mixed solution, thus forming a circular bioink pattern having a size of 0.5 mm.
The mixed solution and the bioink pattern were placed in a CO2 cell incubator at 37° C. and cured at an elevated temperature. After 2 hr, a cell culture solution was added on the mixed solution and the bioink pattern, the solution was replaced every two days, and the tissue was matured for a predetermined period of time (1-2 weeks).
Thereafter, the keratinocyte solution prepared in Preparation Example 3 was printed and cultured on the mixed solution and the bioink pattern, thereby manufacturing an artificial skin.
An artificial tissue was manufactured in the same manner as in Example 1, with the exception that the bioink prepared in Preparation Example 1 was not discharged, in lieu of discharging the bioink prepared in Preparation Example 1 on the mixed solution.
An artificial tissue was manufactured in the same manner as in Example 1, with the exception that the bioink prepared in Preparation Example 2 was used, in lieu of using the bioink prepared in Preparation Example 1.
With reference to
Moreover, the height of the formed papillae was increased with an increase in the size of the bioink pattern. When the bioink pattern was formed to a size of 0.5, 1.0 and 2.0 mm in Examples 1, 2 and 3, the formed papillae had a width of 0.3 mm, 0.6 mm and 1.0 mm and a height of 50.4 μm, 85.3 μm and 94.1 lam, respectively.
Therefore, it can be confirmed that the collagen structure was horizontally contracted but vertically raised relative to the size of the printed bioink pattern.
The scope of the invention is defined by the claims below rather than the aforementioned detailed description, and all changes or modified forms that are capable of being derived from the meaning, range, and equivalent concepts of the appended claims should be construed as being included in the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0053748 | May 2019 | KR | national |
