KR102098691B1 - Hydrogels with controlled mechanical strengths and method for preparing thereof - Google Patents

Abstract

The present invention relates to a hydrogel for artificial skin having a controlled mechanical strength and a method for manufacturing the same, and more specifically, a hydrogel that exhibits porosity by continuously crosslinking collagen and monomers can be controlled by controlling the concentration of monomers. , As it is confirmed that the mechanical properties of the hydrogel adjusted as such are very important cell physiological regulatory factors for growth, proliferation and activity, the collagen gel in which the collagen and monomers of the present invention are continuously crosslinked is provided as artificial skin of various strengths according to age. Can be.

Description

Hydrogels with controlled mechanical strength and method for preparing thereof

The present invention provides a hydrogel for artificial skin with controlled mechanical strength and a method for manufacturing the same.

The skin is the largest organ in the human body and protects the human body from surrounding toxic substances and microorganisms and prevents moisture evaporation. In general, the skin tissue of the human body is largely divided into three parts, the outermost layer of the skin and the dermis layer below it, and thus the subcutaneous tissue. Of these, the epidermal layer is composed of epithelial cells differentiated into several layers from the basement membrane that allows the epidermal layer and the dermal layer to tightly bind, and melanocytes and immune cells, and the dermal layer below the epidermal layer mainly consists of fibroblasts and these. It consists of several extracellular matrices secreted by cells.

Wounded or damaged skin is regenerated from a variety of sources, such as xenografts, allografts, and autografts. However, due to the antigenicity and the limited area of the crab, such a skin substitute is not easy to achieve the purpose of skin regeneration, and thus a histological approach to skin tissue regeneration is required.

The decisive factor in tissue engineering is the preparation of structures, and in order to produce ideal structures that induce skin regeneration, biomaterials must exhibit excellent biocompatibility, appropriate microstructure, adjustable biodegradability and appropriate mechanical properties.

Cellulose derivatives, carrageenan, polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, silicone, alginate, and the like are widely used synthetic and natural polymers for skin tissue regeneration. Collagen and the like. In particular, collagen is the most promising structural material in tissue engineering, and is used in tissue engineering due to its excellent biocompatibility and biodegradability as well as improving the mechanical properties of the structure using various crosslinking agents.

However, like most natural biomaterials such as alginate and silk fibroin, collagen has low processability at room temperature and is manufactured by a direct rapid-prototyping process due to its very high hydrophilicity, resulting in a precisely controlled void structure. There is a problem that it is difficult to obtain a structure.

In addition, the conventional artificial dermal structure exhibits instability, making it difficult to control physical properties, and it is impossible to manufacture a mimetic body due to aging of human skin.

Republic of Korea Patent No. 10-2011-0032381 (2011.03.30. Published)

The present invention provides a hydrogel that exhibits porosity by continuously crosslinking collagen and monomers to provide a three-dimensional collagen structure with precisely controlled strength, and hydrogel for artificial skin whose mechanical strength is controlled by supporting dermal cells in the hydrogel. Want to provide

The present invention provides a hydrogel for artificial skin with controlled mechanical strength, characterized in that collagen and monomers are continuously crosslinked to form a hydrogel having porous pores, and the dermal cells are supported in the hydrogel.

The present invention comprises the steps of preparing a collagen gel by incubating a mixed solution consisting of a collagen solution and a buffer solution (first step); Applying and incubating a mixed solution composed of a monomer, a crosslinking agent, and an initiator on the collagen gel prepared in the first step (second step); Preparing a hydrogel in which collagen and a monomer are continuously crosslinked by irradiating ultraviolet rays to the incubated collagen gel in the second step (step 3); Lyophilizing the hydrogel prepared in the third step (step 4); And applying a dermal cell solution to the lyophilized hydrogel and incubating it (step 5) to provide a method for manufacturing a hydrogel for artificial skin with controlled mechanical strength.

In addition, the present invention provides an artificial skin comprising the hydrogel for artificial skin.

According to the present invention, the hydrogel exhibiting porosity by continuously crosslinking collagen and monomers can be controlled in mechanical strength by controlling the concentration of the monomers, and the mechanical properties of the hydrogels thus controlled are very good for cells to grow, proliferate, and activity. As it is confirmed that it is an important cell physiological regulation factor, the collagen gel in which the collagen and the monomer of the present invention are continuously crosslinked can be provided as artificial skins of various strengths according to age.

1 is a schematic diagram showing the manufacturing process of skin dermal cell-supported collagen gel through continuous crosslinking.
2 is a SEM (Scanning electron microscopic) image confirming the pores formed in the continuously cross-linked collagen gel (scale bars = 1.0 μM).
3 is a result of confirming the change in the elastic modulus of the acrylamide (acrylamide, AAm) hydrogel according to the concentration change of the monomer and initiator.
4 is a result of confirming the change in the elastic modulus of the hydrogel-based collagen gel through continuous crosslinking.
5 is a result confirming the cell metabolic activity according to the elastic modulus of the collagen gel.
6 is a result of confirming the glycosaminoglycan (GAG) synthesis and secretion levels according to the gel strength.

Hereinafter, the present invention will be described in more detail.

In order to control the strength of the collagen gel, conventionally, a short cross-linker having a divalent or polyhydric aldehyde was used, but the aldehyde molecular group remaining in the gel that does not participate in collagen crosslinking is toxic to cells. As problems were identified, the present inventors developed a hydrogel-based collagen gel in which biomaterials collagen and monomeric acrylamide were crosslinked while researching a method for more safely controlling the strength of the collagen gel in the human body or cells. The present invention has been completed.

The present invention can provide a hydrogel for artificial skin with controlled mechanical strength, characterized in that collagen and monomers are continuously crosslinked to form a hydrogel having porous pores, and the dermal cells are supported in the hydrogel.

The hydrogel may include 0.1 to 1.0 parts by weight of collagen and 3 to 10 parts by weight of monomer with respect to 100 parts by weight in total.

More specifically, with respect to 100 parts by weight of the hydrogel, 0.6 parts by weight of collagen and 3 to 10 parts by weight of the monomer are included, and the mechanical strength of the hydrogel can be controlled by the concentration of the monomer.

When the monomer is included below the range of the content, there may be a problem that does not form a hydrogel, and when included above the range of the content, the mechanical strength increases, which causes problems with the growth, proliferation and activity of dermal cells. Can be caused.

The monomer may be selected from the group consisting of acrylamide, N-isopropylacrylamide, acrylic acid and poly (ethylene glycol) diacrylate, It is not limited.

The dermal cells may be supported on a hydrogel at a concentration of 1 × 10 ⁴ to 1 × 10 ⁶ cells / mL, but are not limited thereto.

The present invention comprises the steps of preparing a collagen gel by incubating a mixed solution consisting of a collagen solution and a buffer solution (first step); Applying and incubating a mixed solution composed of a monomer, a crosslinking agent, and an initiator on the collagen gel prepared in the first step (second step); Preparing a hydrogel in which collagen and a monomer are continuously crosslinked by irradiating ultraviolet rays to the incubated collagen gel of the second step (step 3); Lyophilizing the hydrogel prepared in the third step (step 4); And applying a dermal cell solution to the lyophilized hydrogel and incubating it (step 5) to provide a method for manufacturing a hydrogel for artificial skin with controlled mechanical strength.

The monomer is contained in 3 to 10 parts by weight based on 100 parts by weight of the collagen gel, and the mechanical strength of the hydrogel can be controlled by the concentration of the monomer.

The monomer may be selected from the group consisting of acrylamide, N-isopropylacrylamide, acrylic acid and poly (ethylene glycol) diacrylate.

The crosslinking agent may be selected from the group consisting of methylenebisacrylamide (N, N'-methylenebisacrylamide), polyethylene glycol diacrylate, and methacrylic alginate.

In addition, the mixed solution of the third step may further include an initiator.

The initiator may be Irgacure-2959, but is not limited thereto.

In the third step, a hydrogel in which collagen and monomers are continuously crosslinked can be prepared by photopolymerizing a monomer in collagen by irradiating 360 to 379 nm ultraviolet rays on a collagen gel coated with a mixed solution composed of a monomer and a crosslinking agent.

The step of freeze-drying the hydrogel of the fourth step may be -120 to -125 ° C, and freeze-drying the hydrogel under vacuum.

The dermal cells of the fifth step may be applied to a lyophilized hydrogel at a concentration of 1 × 10 ⁴ to 1 × 10 ⁶ cells / mL.

In addition, the present invention can provide an artificial skin comprising the hydrogel for artificial skin.

Hereinafter, examples will be described in detail to help understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Continuous crosslinked  Collagen gel production and characterization

One. Continuous crosslinked  Collagen gel production

A certain amount of RES buffer solution (0.26M NaHCO ₃ (sodiumbicarbonate), 0.2M 4- (2-hydroxyethyl) in a collagen solution (collagen type I, 6.1 mg / ml, Advanced BioMatrix Inc., USA) at a low temperature of 4 ° C ) -1-piperazineethanesulfonic acid (HEPES) and 0.04M NaOH) were added and incubated at 37 ° C. for 4 hours to prepare a pure collagen gel.

To prepare a hydrogel-based collagen gel, acrylamide (acrylamide, AAm, Sigma, USA) as a monomer, methylenebisacrylamide (N, N'-methylenebisacrylamide, MBA, Sigma) as a crosslinking agent, and Irgacure-2959 (initiator) Sigma) was used.

First, a certain amount of collagen solution and RES buffer solution were introduced into a quartz plate on which two spacers having a thickness of 1.0 mm were placed, covered with another quartz plate, and incubated at 37 ° C for 30 minutes to prepare a collagen gel. Thereafter, a mixture of AAm and MBA was applied to the collagen gel and covered with a quartz plate to allow sufficient penetration through the collagen fibers for 3 minutes. At this time, the concentration of the crosslinking agent MBA was fixed at 0.1% (w / v) and the concentration of AAm was variously adjusted to 5 to 10% (w / v).

Finally, 365 nm UV (UV lamp, Vilber Lourmat, France) was irradiated for 10 minutes to photopolymerize AAm monomers inside the collagen gel, thereby preparing a hydrogel-based collagen gel by a continuous crosslinking method. The gel thus prepared was standardized to 8 mm in diameter and 1 mm in thickness to be used for physical property analysis and cell loading studies.

The collagen gel prepared through the continuous crosslinking process was freeze-dried at -121 ° C and vacuum conditions to introduce porous pores inside the collagen as shown in FIG. 2, and the dried gel was sterilized with UV for 10 minutes.

2. Continuous crosslinked  Collagen Gel  Strength check

In order to confirm the physical properties of collagen gels prepared using various concentrations of monomers as in Example 1, the Young's modulus ( E , elastic modulus) of each collagen gel was calculated as the ratio of stress to strain and measured for each sample. The average value was confirmed.

As a result, as shown in FIGS. 3 and 4, as the AAm concentration introduced through continuous crosslinking to the collagen gel increased, it was confirmed that collagen gels having various strengths from 1.5 kPa to 3.0 kPa were prepared.

From the above results, it was confirmed that the collagen gel continuously crosslinked through the monomer concentration control can be provided as a skin structure exhibiting various strengths.

< Example  2> Dermal cells Supported  Collagen gel production and characterization

One. Dermal cells Supported  Collagen gel production

The dried collagen gel introduced with porous pores prepared as in Example 1 absorbs the dermal cell solution rapidly and swells at the same time, so that the dermal cells can be encapsulated inside the gel. First, 2% (v / v) low serum growth supplement (LSGS) and 1% (v / v) penicillin-strepto in M106 (Gibco) culture in response to dermal fibroblasts (human dermal fibroblasts, HDFs, Gibco, USA) Mycin (Penicillin-Streptomycin, P / S, Biowest, France) was added and cultured at 37 ° C and 5% CO ₂ . The cells cultured in this way were applied to the dried gel at a concentration of 1.0 × 10 ⁵ cells per ml of the collagen gel solution (100 μL) to rapidly swell the collagen gel for 20 minutes while supporting the cells inside the gel. The collagen gel encapsulating the cells was washed twice with PBS (phosphate-buffered saline, Biowest) and incubated in 37 ° C. and 5% CO ₂ conditions for 2 weeks in M106 culture solution to which LSGS and P / S were added.

2. Dermal cells Supported  Check the level of cellular metabolism in collagen gel

After 2 weeks of incubation, the level of cell metabolism in each collagen gel was standardized based on the level of cell metabolism in the collagen gel showing 1.5 kPa.

As a result, as shown in FIG. 5, as it was confirmed that the cell metabolic activity was reduced by about 2 times in the gel of 3.0 kPa compared to 1.5 kPa, it was confirmed that the level of cellular metabolism decreased as the gel strength increased.

3. Dermal cells Supported  In collagen gel Glycosamino glucan (GAG) Check synthesis level

The synthesis and secretion levels of glycosaminoglycan (GAG) of cells according to gel strength were confirmed inside the collagen gel.

In order to confirm the level of GAGs synthesized and secreted by cells inside collagen gels of various strengths, each collagen gel was stained with alcian blue, and the degree of staining was analyzed with ImageJ and compared with the reference GAG concentration. , Relative GAG expression level according to gel strength was normalized to the number of cells in each gel.

As a result, as shown in FIG. 5B, in a pure collagen gel having a strength of 1.5 kPa, about 8.1 μg / ml of GAG was secreted per 5 × 10 ⁴ cells, but in a collagen gel of 3.0 kPa whose intensity increased twice, 5 × 10 ⁴ It was confirmed that the amount of GAG secretion was increased to about 17.3 μg / ml per cell.

Compared to the cell proliferation results of FIG. 4 identified above, it was confirmed that cell growth and proliferation were decreased in the gel with increased strength, but GAG synthesis and secretion were activated.

From the above results, it was confirmed that the mechanical properties of the collagen gel are very important cell physiological regulation factors for growth, proliferation and activity, and through this, the continuously crosslinked collagen gel of the present invention can be provided as artificial skin having various strengths. Yes, it was confirmed.

< Comparative example  1> Collagen solution, buffer solution and Hydrogel  Using mixed solution Simultaneous crosslinked Check the properties of collagen gel

The collagen gel formation was induced by incubating the mixed solution of the collagen solution, the buffer solution, and the monomer solution at 37 ° C for 30 minutes.

Then, 365nm UV was irradiated to photopolymerize the AAm monomer to prepare a hydrogel-based collagen gel that was cross-linked.

After lyophilization in the same manner as in Example 2, dermal cell loading and culture were performed.

As a result, collagen fibers were not formed well in collagen gels using collagen solution, buffer solution, and hydrogel mixed solution, which can control mechanical strength, but adhesion between dermal cells and extracellular matrix is not well achieved. May limit cell growth, proliferation and activity.

The specific parts of the present invention have been described in detail above, and it is obvious to those skilled in the art that this specific technology is only a preferred embodiment, whereby the scope of the present invention is not limited. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

Preparing a collagen gel by incubating a mixed solution consisting of a collagen solution and a buffer solution (first step);
Applying a mixed solution consisting of a monomer and a crosslinking agent to the collagen gel prepared in the first step and incubating it (second step);
Preparing a hydrogel in which collagen and a monomer are crosslinked by irradiating ultraviolet rays to the incubated collagen gel in the second step (step 3);
Preparing a hydrogel having porous pores by freeze-drying the hydrogel prepared in the third step at -120 to -125 ° C in a vacuum condition (step 4); And applying the dermal cell solution to the lyophilized hydrogel, followed by incubation, whereby the dermal cells are loaded into the hydrogel (step 5), wherein the monomer is 3 to 10 weight per 100 parts by weight of the collagen gel. Hydrogel for artificial skin with mechanical strength controlled, characterized in that the mechanical strength of the hydrogel is controlled by the concentration of monomers included as a part. The method according to claim 1, The hydrogel is a hydrogel for artificial skin, characterized in that it contains 0.1 to 1.0 parts by weight of collagen and 3 to 10 parts by weight of monomer, based on 100 parts by weight in total. delete The method according to claim 1, The monomer is selected from the group consisting of acrylamide (acrylamide), N- isopropylacrylamide (N-isopropylacrylamide), acrylic acid (acrylic acid) and polyethylene glycol diacrylate (Poly (ethylene glycol) diacrylate) Hydrogel for artificial skin, characterized in that. The method according to claim 1, wherein the dermal cell is a hydrogel for artificial skin, characterized in that supported on a hydrogel at a concentration of 1 × 10 ⁴ to 1 × 10 ⁶ cells / mL. Preparing a collagen gel by incubating a mixed solution consisting of a collagen solution and a buffer solution (first step);
Applying a mixed solution consisting of a monomer and a crosslinking agent to the collagen gel prepared in the first step and incubating it (second step);
Preparing a hydrogel in which collagen and a monomer are crosslinked by irradiating ultraviolet rays to the incubated collagen gel in the second step (step 3);
Lyophilizing the hydrogel prepared in the third step at -120 to -125 ° C under vacuum conditions (fourth step); And applying a dermal cell solution to the lyophilized hydrogel, followed by incubation (step 5), wherein the monomer is included in 3 to 10 parts by weight with respect to 100 parts by weight of the collagen gel, depending on the concentration of the monomer. Method for manufacturing a hydrogel for artificial skin with controlled mechanical strength, characterized in that the mechanical strength of the hydrogel is controlled. delete The method according to claim 6, The monomer is selected from the group consisting of acrylamide (acrylamide), N-isopropylacrylamide (N-isopropylacrylamide), acrylic acid (acrylic acid) and polyethylene glycol diacrylate (Poly (ethylene glycol) diacrylate) Method of manufacturing a hydrogel for artificial skin with controlled mechanical strength, characterized in that. The method according to claim 6, wherein the third step is to produce a hydrogel in which collagen and a monomer are crosslinked by photopolymerizing a monomer in collagen by irradiating 360 to 379 nm ultraviolet rays on a collagen gel coated with a mixed solution consisting of a monomer and a crosslinking agent. Method for manufacturing a hydrogel for artificial skin with controlled mechanical strength. The method according to claim 6, The mixed solution of the third step is a mechanical strength controlled artificial skin hydrogel manufacturing method characterized in that it further comprises an initiator. delete The method according to claim 6, wherein the dermal cells of the fifth step is 1 × 10 ⁴ to 1 × 10 ⁶ cells / mL concentration of mechanical strength controlled artificial skin hydrogel production characterized in that applied to the lyophilized hydrogel Way. Artificial skin comprising a hydrogel for artificial skin according to claim 1.

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