WO2019221470A1 - Collagen gel strength controller, method for manufacturing artificial skin by using same, and artificial skin - Google Patents

Abstract

Provided according to an embodiment are: a collagen gel strength controller comprising a structural unit represented by a particular chemical formula; a method for manufacturing artificial skin by using same; and artificial skin comprising same.

Description

Collagen gel strength modifier, artificial skin preparation method and artificial skin using the same

The present disclosure relates to a collagen gel strength modifier for artificial skin, a method for preparing artificial skin using the same, and an artificial skin manufactured according to the method.

The skin is an organ that covers the outside of the body and consists of three layers from the outside: the epidermis, the dermis, and the subcutaneous fat layer. The epidermis is mostly composed of keratinocytes of stratified squamous epithelium. The dermis, which consists of matrix proteins such as collagen fibers and elastic fibers, is located beneath the epidermis, and the dermis contains blood vessels, nerves, and sweat glands. Subcutaneous fat layer is composed of fat cells. The skin maintains its shape by interacting with various cells and components as described above, and exhibits various functions such as body temperature control and a barrier to the external environment.

Artificial skin is a three-dimensional reconstruction of the skin using skin cells and skin constituents, such as collagen and elastin, and consists of living fibroblasts and keratinocytes. It is also called a skin equivalent or reconstructed skin because of its functional properties. Artificial skin is a polymer composite that exhibits similar physical properties to skin, elasticity, strength, and material permeability. The difference is that artificial skin does not exhibit life phenomena such as skin. Artificial skin is used not only for replacing (permanent engraftment) or regenerating (temporary coating type) of damaged skin such as burns and trauma, but also in various areas such as skin physiology research, skin irritation evaluation and skin efficacy evaluation. .

The dermal layer constituting our skin contains collagen gel, and the strength of the dermal layer varies depending on the strength of the collagen gel. The strength of the skin is changing due to various diseases or aging. If a new artificial skin can be produced that simulates the strength of the skin, it can be used as a personalized artificial skin and used as an aging research platform. It can dramatically improve the problem of skin elasticity due to aging.

Modifications of the artificial skin model to date rely mainly on cell biological methods of altering cells or adding or removing specific substances. To this end, there are no techniques that can change the physical characteristics of artificial skin models such as tissue strength, so there are limitations in terms of various expansion of artificial skin models. In one embodiment, the artificial skin is made by using a polymer including a specific structural unit, which can directly adjust the strength of the collagen gel. Thus, a new skin simulation model can be manufactured by adjusting the strength of the artificial skin model.

According to one embodiment, it provides a collagen gel strength modifier comprising a structural unit represented by the formula (1).

[Formula 1]

In Chemical Formula 1,

R ¹ is a hydrogen atom or a substituted or unsubstituted C1 to C20 alkyl group,

L ¹ is a substituted or unsubstituted C1 to C20 alkylene group,

m and n are each independently an integer of 1 to 10000.

The structural unit represented by Formula 1 may satisfy Equation 1 below.

[Equation 1]

0 <n / (n + m) x 100 ≤ 5

The collagen gel strength modifier may further include calcium ions.

The collagen gel strength modifier may have a weight average molecular weight of 10,000 g / mol to 1,000,000 g / mol.

According to another embodiment, a step of preparing a dermal replica layer by mixing fibroblasts, collagen and the collagen gel strength modifier according to any one of claims 1 to 4; Applying keratinocytes on the dermis replicating layer; And it provides an artificial skin manufacturing method comprising the step of culturing.

The step of applying keratinocytes on the dermis replicating layer may be a step of placing keratinocytes on the dermis replicating layer and culturing for two days.

The culturing step may be performed while exposed to air.

Another embodiment provides an artificial skin comprising the collagen gel strength modifier.

Another embodiment provides an artificial skin manufactured according to the artificial skin manufacturing method.

Collagen gel strength modifier according to one embodiment includes a polymer comprising a specific structural unit, the polymer is added to the collagen solution, it can be variously adjusted to the desired level of collagen gel constituting the dermal layer of artificial skin. .

Figure 1 is a schematic diagram showing the structure of the collagen gel strength modifier according to one embodiment.

2 is a flow chart showing a method for manufacturing artificial skin according to an embodiment.

3 and 4 are each independently NMR data of the compound according to Comparative Example 1 and Example 1.

5 is a view showing a strength control mechanism to adjust the collagen gel strength according to one embodiment.

6 and 7 are independently pyrene fluorescence data of the compounds according to Example 1 and Example 2, respectively.

8 and 9 are schematic diagrams showing the structures of the compounds according to Example 1 and Example 2, respectively independently.

10 is a graph showing the collagen gel strength according to the content of the collagen gel strength modifier according to one embodiment.

11 is a graph showing collagen gel strength according to the type of compound used as a collagen gel strength modifier.

12 is a scanning electron microscope (SEM) photograph of the internal structure of the dermal layer according to the content of the collagen gel strength modifier (scale bar = 5㎛).

Figure 13 is a graph showing the change in fibroblast behavior according to the addition of the collagen gel strength modifier according to one embodiment.

14 is a photograph of the dermal and epidermal layers of an artificial skin model prepared by adding a collagen gel strength modifier according to one embodiment.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily practice. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention.

As used herein, the term "artificial skin" refers to a three-dimensional reconstitution of skin using skin cells and collagen, which is a skin component, and a polymer composite exhibiting structural and functional characteristics similar to those of actual skin. Ramen is the broadest meaning to include all.

Unless stated otherwise in the specification, "alkyl group" means a C1 to C20 alkyl group, "alkenyl group" means a C2 to C20 alkenyl group, and "cycloalkenyl group" means a C3 to C20 cycloalkenyl group , "Heterocycloalkenyl group" means a C3 to C20 heterocycloalkenyl group, "aryl group" means a C6 to C20 aryl group, "arylalkyl group" means a C6 to C20 arylalkyl group, "alkylene group" Means C1 to C20 alkylene group, "arylene group" means C6 to C20 arylene group, "alkyl arylene group" means C6 to C20 alkylarylene group, and "heteroarylene group" means C3 to C20 hetero It means an arylene group and a "alkoxy group" means a C1-C20 alkoxylene group.

Unless stated otherwise in the present specification, "substituted" means that at least one hydrogen atom is a halogen atom (F, Cl, Br, I), hydroxy group, C1 to C20 alkoxy group, nitro group, cyano group, amine group, imino group , Azido groups, amidino groups, hydrazino groups, hydrazono groups, carbonyl groups, carbamyl groups, thiol groups, ester groups, ether groups, carboxyl groups or salts thereof, sulfonic acid groups or salts thereof, phosphoric acid or salts thereof, C1 to C20 alkyl group, C2 to C20 alkenyl group, C2 to C20 alkynyl group, C6 to C20 aryl group, C3 to C20 cycloalkyl group, C3 to C20 cycloalkenyl group, C3 to C20 cycloalkynyl group, C2 to C20 heterocycloalkyl group, It means substituted with a substituent of a C2 to C20 heterocycloalkenyl group, a C2 to C20 heterocycloalkynyl group, a C3 to C20 heteroaryl group or a combination thereof.

In addition, unless otherwise specified in the present specification, "hetero" means that at least one hetero atom of N, O, S, and P is included in a chemical formula.

Also, unless stated otherwise in the present specification, "(meth) acrylate" means that both "acrylate" and "methacrylate" are possible, and "(meth) acrylic acid" means "acrylic acid" and "methacrylic acid". "Both mean it's possible.

Unless otherwise stated herein, "combination" means mixed or copolymerized.

Unless otherwise defined in the chemical formulas herein, when a chemical bond is not drawn at a position where a chemical bond is to be drawn, it means that a hydrogen atom is bonded at the position.

When a portion of a layer, film, region, plate, or the like is said to be "on top" of another part, this includes not only the case where the other part is "right over" but also another part in the middle. On the contrary, when a part is "just above" another part, there is no other part in the middle.

Hereinafter, a collagen gel strength modifier according to one embodiment will be described.

Collagen gel strength modifier according to one embodiment is represented by the formula (1).

[Formula 1]

In Chemical Formula 1,

R ¹ is a hydrogen atom or a substituted or unsubstituted C1 to C20 alkyl group,

L ¹ is a substituted or unsubstituted C1 to C20 alkylene group,

m and n are each independently an integer of 1 to 10000.

The collagen gel strength modifier represented by the formula (1) includes both m mole number of repeating units and n mole number of repeating units, the n mole number of repeating units, unlike m mole number of repeating units, including the grafted acrylate group , Michael addition reaction occurs between the amino group of the collagen fiber and the grafted acrylate group, the collagen gel strength modifier represented by the formula (1) can be attached to the collagen fiber, thereby easily controlling the strength of the collagen gel .

For example, the structural unit represented by Formula 1 may satisfy Equation 1 below.

[Equation 1]

0 <n / (n + m) x 100 ≤ 5

Collagen gel strength modifier according to one embodiment is a kind of graft copolymer comprising a structural unit represented by the formula (1), depending on how the acrylate group is grafted to the backbone, the desired effect, that is, desired Whether the level of intensity control is possible may vary. That is, the degree of grafting of the main chain of the acrylate group, which is a grafting group (Degree of Substitution, DS) is important, and when the structural unit represented by the formula (1) (more than 0 mol%) has a grafting degree of 5 mol% or less It is possible to control the strength to a desired level, and when the grafting exceeds 5 mol%, self-assembly occurs (self-assembly formation), which leads to a decrease in strength control. Equation 1 is an equation for displaying the graft degree. That is, the grafting degree means the number of repeating units of n moles per 100 polymer units composed only of the structural unit represented by the formula (1).

In general, the higher the number of acrylate groups grafted to the main chain in order to increase the Michael addition rate with collagen fibers may be expected to be excellent in strength control, but the grafting degree of the pyrene of the strength modifier of 5 mol% or less (pyrene) fluorescence data of Figure 6 I ₃ / I ₁ value is almost constant, but Fig. 7 of the pyrene fluorescence data of the strength modifier grafting exceeds 5 mol% I ₃ / I ₁ value It can be seen that the sigmoidal form increases. This is because the microenvironmental polarity of the pyrene molecule is reduced, and the more acrylate groups are included, the more evidence that the formation of the self-assembly is started in the aqueous solution.

In other words, when the degree of grafting increases because the acrylate group has a weak hydrophobicity, since the structural unit represented by Formula 1 self-assembles in an aqueous solution, the acrylate group to be reacted with collagen fibers and Michael is a self-assembly. It is trapped inside, and the Michael addition reaction rate with collagen fiber falls, and the strength control power is inevitably reduced. On the other hand, when the grafting degree is lower than 5 mol%, there are relatively more sites capable of ionic cross-linking due to the addition of calcium ions, which will be described later, so that the physical ion crosslinking ability is improved, and ultimately the strength Control is bound to be excellent.

8 and 9 are both schematic diagrams showing structural units represented by Chemical Formula 1, and FIG. 8 shows a case in which the grafting degree is 5 mol% or less, but the grafting degree is more than 5 mol%. 9 shows that the self-assembly is formed.

The collagen gel strength modifier may further include calcium ions. The calcium ions may be ion-bonded with the main chain of Chemical Formula 1, thereby assisting further binding of collagen fibers, thereby helping to control the strength of the collagen gel.

From FIG. 5, the collagen gel strength modifier including the structural unit represented by Formula 1 is introduced into the collagen fiber to control the strength of the collagen gel, and additionally calcium ions are added thereto, whereby the collagen gel is obtained through ion crosslinking. It can be seen that the control of the intensity is easier. In particular, calcium ions can give an excellent effect in controlling the strength of the dermal layer in the artificial skin through the ion cross-linking in the manufacture of artificial skin.

The collagen gel strength modifier may have a weight average molecular weight of 10,000 g / mol to 1,000,000 g / mol. If the molecular weight of the collagen gel strength regulator is less than 10,000 g / mol cross-linking is difficult, and if more than 1,000,000 g / mol may cause problems of viscosity increase and solubility.

Meanwhile, the collagen gel strength modifier is used as an additive, but does not adversely affect fibroblast behavior. That is, the collagen gel strength modulator according to one embodiment is cell friendly.

Another embodiment is to prepare a dermal replica layer by mixing fibroblasts, collagen and the collagen gel strength modifier; Applying keratinocytes on the dermis replicating layer; And it provides an artificial skin manufacturing method comprising the step of culturing.

Referring to FIG. 2, the collagen fibers are bonded to each other through a Michael addition reaction between an amino group in the collagen fiber and an acrylate group, which is a grafting group in the collagen gel strength modifier, by the collagen gel strength modifier in the collagen fiber. Due to the calcium ions added during the air exposure culture after keratinocyte seeding, due to the ionic binding between the main chain and the calcium ions constituting the collagen gel strength regulator, additional binding between collagen fibers occurs, It can be confirmed that artificial skin is produced.

The dermis is a layer of skin below the epidermis consisting of connective tissue that buffers the body and protects it from stress and strain. The dermis is tightly connected to the epidermis through the basement membrane. The dermis supplies the substrate that supports the various appendages, such as the blood vessels and nerves inherent in the structure.

The dermal mimetic layer is formed using a mixture of fibroblasts and collagen from the viewpoint of human correlation with the actual skin. The dermis replicating layer may be composed of one layer or two or more layers, and may include, for example, a first dermal layer containing collagen and a second dermal layer containing fibroblasts, collagen and fibroblasts. It may consist of a single dermal simulating layer containing. In this case, the dermis shrinkage phenomenon of the artificial skin can be further alleviated.

The first dermal layer may contain collagen, and extracellular matrix constituting the dermis such as elastin, chitosan, glycosaminoglycans (GAGs), and hyaluronic acid (HA). extracellular matrix).

The second dermal layer may be located on top of the first dermal layer and contains fibroblasts, elastin, chitosan, glycosaminoglycans (GAGs), hyaluronic acid (HA) It may further contain an extracellular matrix constituting the dermis, such as).

The single dermal mimetic layer may contain collagen and fibroblasts, wherein the collagen and fibroblasts are as described above.

The collagen used may be collagen of bovine origin, from a let tail or from fish, or of any other origin of natural collagen or collagen produced by genetic engineering, which may contract in the presence of fibroblasts.

The thickness of the dermis replica layer is generally at least 0.05 cm, in particular approximately 0.05 cm to 2 cm, but can be increased or decreased as long as it does not impair the advantageous properties of the artificial skin according to the invention.

After preparing the dermal mimetic layer using a mixture of the fibroblasts and collagen, it may be cultured, for example, for about 5 to 9 days, about 6 to about 8 days, or about 7 days.

Once the dermis mimetic layer is formed, the keratinocytes are applied thereon and then cultured. That is, applying the keratinocytes on the dermis replicating layer may be a step of placing keratinocytes on the dermis replicating layer and culturing for two days.

The keratinocytes make up about 80% to 90% of the epidermal cells and are formed through mitosis in the basal layer, the deepest layer of the epidermal layer, and then up to the skin surface.

The step of culturing after applying the keratinocytes is a first step for forming an epidermal replica layer of artificial skin, wherein the keratinocytes used herein may be, for example, keratinocytes derived from humans. The keratinocytes may be any commercially available keratinocytes, and keratinocytes derived from other cells as well as keratinocytes cultured after being isolated or separated directly from humans may be used. Commercially available human keratinocytes include NHEK-Neo, Pooled (Neonatal Normal Human Epidermal Keratinocytes, Pooled: Catalog No. 00192906, Tissue Acquisition No. P867, Whites), NHEK-Neo (Tab. No. 00192907) available from Lonza. Acquisition number: 20647, white), NHEK-Adult (model number 00192627, organization acquisition number: 21155, white), NHEK-Neo (model number 00192907, organization acquisition number: 18080, black). And HEKn (Human Epidermal keratinocytes, neonatal: product number C0015C, tissue acquisition number 1781129, white) provided by Thermo Fisher, HEKn (product number C0015C, tissue acquisition number 1803827, black). In order to maintain the propagation properties of keratinocytes by attaching to the base membrane, the dish is preferably coated with 0.1% to 0.2% gelatin or 0.1 mg / ml to 0.2 mg / ml collagen type IV. The cells may be cultured in a 35 ° C. to 37 ° C., 5% to 10% CO ₂ incubator and passaged at a density of about 70% to 80%.

The keratinocytes may be applied on the dermis replicating layer by, for example, seeding, and the culture period may be, for example, 1 day to 4 days, 1 day to 3 days, or 1 day to 2 days, but is not limited thereto. .

After applying and culturing the keratinocytes, the cells are cultured in the epidermal layer-forming medium.

Herein, "epidermal layering medium" means a medium for forming an epidermal layer in artificial skin, which may be used in any vessel known in the art. For example, the epidermal layering medium may include bovine pituitary extract (BPE), human epidermal growth factor (hEGF), bovine insulin (bovine insulin), hydrocortisone (Hydrocortisone), and gentamicin and cancer cells. It may be a medium containing erysine-B (GA-1000 (Gentamicin, Amphotericin-B)). In addition, the medium may further contain epinephrine (Epinephrine) and transferrin (Transferrin) in the component. Commercially available examples of such media include CnT-3D-PR (CellnTEC).

The epidermal layer forming medium may be installed to contact the lower surface of the dermis replicating layer, and the opposite side of the side where the epidermal layer forming medium is installed may be exposed to air. Cultivation in such epidermal layering medium may be performed for, for example, 3 to 20 days, 4 to 18 days, 5 to 15 days, or 7 to 14 days, but is not limited thereto.

By following the above steps, an artificial skin structure in which the epidermal replica layer is formed on the dermis replica layer can be obtained.

When the artificial skin is manufactured according to the above-described method, not only an artificial skin model substantially similar to that found in human skin can be obtained, but the strength of the artificial skin can be controlled by the collagen gel strength modifier to a desired level. Can be.

That is, without using any conventional cross-linking agent or regulator, it is possible to implement a desired level of strength control, artificial skin model that does not adversely affect the behavior of fibroblasts.

According to another embodiment, it provides an artificial skin comprising the collagen gel strength modifier described above.

 According to another embodiment, it provides an artificial skin manufactured according to the above-described manufacturing method.

Through the following examples will be described in more detail the embodiment of the present invention. However, the following examples are merely for illustrative purposes and do not limit the scope of the present invention.

Collagen Gel Strength Modulator Synthesis

Example 1

Alginic acid sodium salt (Sigma) was 1.0% (w / w) in 0.1 M 2- (N-morpholino) ethanesulfonic acid (MES, Sigma) buffer at pH 6.4. v), and the mixture was sufficiently dissolved at 30 to 40 ° C while stirring. When completely dissolved, 1-hydroxybenzotriazole (HOBt, Sigma), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (1-ethyl-3- (3) -dimethylaminopropyl) carbodiimide, EDC, Sigma) and 2-aminoethyl methacrylate (2-aminoethyl methacrylate, AEMA, Sigma) were added slowly and reacted with stirring for 18 hours. After the reaction, the reaction solution was dialyzed with distilled water for 3 days, and then lyophilized to obtain a molecule represented by the following Chemical Formula E-1 in which methacrylate was grafted. The synthesized molecules were dissolved in D ₂ O and analyzed by ¹ H-NMR (Avance II, Bruker Biospin) at 400 MHz, and the results are shown in FIG. 4. The degree of grafting (DS) of the synthetic molecule was calculated (5 mol%) by measuring unreacted COOH free carboxylate groups by titration with sodium hydroxide (NaOH, Sigma).

[Formula E-1]

Example 2

The molecule | numerator represented by the said Formula (E-1) was obtained like Example 1 except having set the grafting degree to 7 mol%.

Comparative Example 1

Alginic Acid Used in Example 1

Evaluation 1: structural analysis

¹ H-NMR spectra at D ₂ O of the molecules according to Example 1 and Comparative Example 1 are shown in FIGS. 3 and 4.

Unlike FIG. 3 (Comparative Example 1), it can be seen that FIG. 4 (Example 1) has several different peaks, which may enable the strength control of the collagen gel.

Evaluation 2: Changes According to Graft Degree

Pyrene (fluorescence) analysis of the synthetic molecules according to Example 1 and Example 2, the results are shown in Figures 5 and 6.

5 and 6, in the case of Example 1 having a low degree of grafting, the value of I ₃ / I ₁ is almost constant, but in Example 2 having a high degree of grafting, the value of I ₃ / I ₁ increases in the sigmodal form. It can be seen that this is because the microenvironmental polarity of the pyrene molecule is reduced, in the case of Example 2 containing a large number of acrylate groups, it can be inferred that the self-assembly was formed in the aqueous solution. That is, unlike Example 1, in the case of Example 2, it can be inferred that the acrylate group will be contained in the self-assembly, and will not react with the collagen fiber and Michael.

Evaluation 3: Change in Strength of Collagen Gel

(1) Artificial skin Dermal dermis was prepared by adding a synthesized molecule (Example 1) to confirm the effect of controlling the dermis strength. The dermal mixture contains collagen solution (Type I, 6 mg / ml, Advaced BioMatrix), P buffer (NaOH, NaHCO ₃ , HEPES in distilled water) and R buffer (Dulbecco's Modified Eagle Medium powder, Ham's F-12 Nutrient Mxiture, Antibiotic- Antimycotic in DI water), 5N NaOH solution was prepared in a ratio of 8: 1: 1: 0.05. Synthetic molecules (Example 1) were prepared in phosphate buffer saline (PBS) at a concentration of 3% and added to the dermal mixture at various concentrations. Human dermal fibroblasts (neonatal, Invitrogen) were treated with low serum growth supplement (LSGS, 50x, Gibco) and 1% penicillin-streptomycin (P / S, 100X, Biowest) in media 106 (Gibco) in a 75 cm ² flask. The culture was added to the culture medium at 37 ℃, 5% CO ₂ environment. Cultured dermal fibroblasts were added to the dermal mixture so as to encapsulate 5 × 10 ⁴ cells per well. The dermal mixture was put into 500 μL each 48well plate gelled for 1 hour in a 37 ℃ incubator to prepare artificial dermal dermis. After 48 hours of preparation, cell stability and Michael addition reaction between collagen and acrylate of the synthetic molecule (Example 1) were induced. In order to induce gelation of the synthetic molecules, the culture medium was added with 5 mM calcium ions, and then cultured by replacing with an existing culture medium after 12 hours, and the results are shown in FIG. 10.

As shown in FIG. 10, it can be seen that the strength of the collagen gel is increased when the synthetic molecules are added and when the content of the synthetic molecules is increased, rather than when the synthetic molecules are not added. That is, it can be seen that the collagen gel strength modifier according to one embodiment can control the strength of the collagen gel.

(2) In order to measure the strength of artificial skin dermis into which a synthetic molecule (Example 1) was introduced, artificial skin dermis without cells was prepared. After the same culture process, the strength of artificial skin dermis prepared on the 6th day of manufacture was performed using a universal testing machine (UTM, DrTech). Load cell guide was installed to measure the strength without removing the prepared artificial dermis from the 48 well plate. The setting value of the universal testing machine was the same as the measuring distance test rate of 10%, the test speed of 0.5 mm / min, and the load range of 1.0 kgf. In compressive strength measurement, the unit area was set to 7 mm, the size of the tip of the load cell guide. The results are shown in FIG.

11, when the acrylate group is not grafted, it can be seen that there is no effect of increasing the collagen gel strength.

(3) Crosslinking degree and internal content of collagen gel when the synthetic molecules (Example 1) were not used, and when the content of the synthetic molecules (Example 1) was used differently (0.6 mg, 1.2 mg, 2.4 mg) Photographs were taken to confirm the structural changes, and the results are shown in FIG. 12. (The internal microstructure of artificial skin dermis into which synthetic molecules were introduced was observed using a scanning electron microscope (FE-Scanning electron microscope, SIGMA, Carl Ziess). The artificial skin dermis to be observed was lyophilized and platinum coated. )

It can be seen from FIG. 12 that the use of synthetic molecules (Example 1) is more thicker than the case where no synthetic molecules are used, and the collagen fibers become thicker as the amount of the synthetic molecules is increased, resulting in a denser internal structure.

Evaluation 4: Whether Fibroblasts Changed with Synthetic Molecules

MTT assay was performed to confirm the cell viability of dermal fibroblasts in artificial skin dermis to which a synthetic molecule (Example 1) was added. 3- (4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-tetrazolium bromide, sigma) was prepared in PBS at a concentration of 5 mg / ml and then added to the dermal dermal culture 1:10. After 4 hours, the cultures were removed and 500 μL lysis buffer (20% Sodium dodecyl sulfate in N, N Dimethylformamid / DI water (1/1), adjusted to pH 4.7 with 1N HCl) was added and formazan in artificial dermis for 12 hours. Melted. Thereafter, 100 μL of the culture solution was extracted from each well to measure the concentration of formazan, and placed in a 96well plate, and the absorbance (Optical density, OD) was measured at a wavelength of 570 nm. WST-1 and MTT assays were performed on Days 1, 3, 5 and 7 after artificial dermal dermal preparation. The measurement results are shown in FIG. 13.

13, it can be seen that the synthetic molecule (Example 1) does not adversely affect fibroblast behavior (cell friendly). That is, it can be confirmed that the synthetic molecule (Example 1) can be used for cell experiments such as artificial skin preparation.

Artificial skin production

Example 3

(1) Step 1: Preparation of dermal simulating layer

After mixing fibroblasts (NDFn, ThermoFisher), collagen (Nutragen, Advanced Biomatrix) and synthetic molecules (Example 1) to prepare a dermal replica layer, M106 medium containing LSGS (ThermoFisher) and 100μg / ml ascorbic acid (TermoFisher) was incubated for 1 week. The concentration of the mixed synthetic molecules (Example 1) was fixed at 1.2 mg.

(2) step 2: application and culture of keratinocytes

After the first step, keratinocytes (HEKn, Thermo Fisher) were put on the dermis replicating layer and incubated for 2 days.

(3) step 3: culturing in a first medium

After the second step, the lower part of the dermal mimetic was installed to reach the first medium (CnT-3D-PR, CellnTEC), and the epidermal layer was cultured for 8 days of air exposure exposing to air.

A microscopic cross-sectional photograph of the artificial skin model of Example 3 is shown in FIG. 14.

Evaluation 5: Cross Section Observation of Artificial Skin Model

The cross section of the artificial skin model obtained in Example 3 is observed under a microscope.

14 is a photomicrograph of the artificial skin obtained in Example 3 after staining with H & E tissue.

Referring to FIG. 14, in the artificial skin model to which the synthetic molecule (Example 1) is added, growth of normal dermal layer and epidermal layer may be confirmed.

Although preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to the scope of the invention.

Claims (9)

1. Collagen gel strength modifier comprising a structural unit represented by the formula (1):

   [Formula 1]

   

   In Chemical Formula 1,

   R ¹ is a hydrogen atom or a substituted or unsubstituted C1 to C20 alkyl group,

   L ¹ is a substituted or unsubstituted C1 to C20 alkylene group,

   m and n are each independently an integer of 1 to 10000.
2. In claim 1,

   The structural unit represented by Formula 1 is a collagen gel strength modifier that satisfies the following formula (1).

   [Equation 1]

   0 <n / (n + m) x 100 ≤ 5
3. In claim 1,

   The collagen gel strength modifier further comprises a calcium ion collagen gel strength modifier.
4. In claim 1,

   The collagen gel strength regulator is a collagen gel strength regulator having a weight average molecular weight of 10,000 g / mol to 1,000,000 g / mol.
5. Preparing a dermal replica layer by mixing fibroblasts, collagen and the collagen gel strength modifier according to any one of claims 1 to 4;

   Applying keratinocytes on the dermis replicating layer; And

   Incubation step

   Artificial skin manufacturing method comprising a.
6. In claim 5,

   The step of applying keratinocytes on the dermis replicating layer is a step of placing the keratinocytes on the dermis replicating layer and culturing for two days.
7. In claim 5,

   The culturing step is an artificial skin manufacturing method that proceeds in a state exposed to air.
8. Artificial skin comprising the collagen gel strength modifier according to any one of claims 1 to 4.
9. Artificial skin prepared according to the method according to any one of claims 5 to 7.

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