Decellularized placenta-derived amniotic membrane-based hydrogel using supercritical fluid and organic solvent system, use thereof, and method for preparing same

Abstract

The present invention relates to a decellularized placenta-derived amniotic membrane-based hydrogel, a use thereof, and a method for preparing same, the hydrogel being characterized by comprising a decellularized placenta-derived amniotic membrane, wherein the content of collagen in the decellularized placenta-derived amniotic membrane is 9 µg / mg to 15 µg / mg, and the content of glycosaminoglycans (GAGs) is 0.3 µg / mg to 1.0 µg / mg.

Description

Decellularized placental-derived amniotic membrane-based hydrogel using a supercritical fluid and organic solvent system, its use, and its preparation method

[0001] The present invention relates to a decellularized placental-derived amniotic membrane-based hydrogel using a supercritical fluid and organic solvent system, its use, and a method for producing the same.

[0002] Amniotic tissue, the inner lining of the placenta, is physiologically safe, contains few immunogens, and possesses favorable mechanical properties (permeability, stability, elasticity, flexibility, and absorbability) and favorable cell adhesion properties. Furthermore, amniotic tissue exhibits anti-inflammatory, anti-fibrotic, anti-cytotoxic, and analgesic effects, as well as tissue growth factor, cytokine, and stem cell properties. Accordingly, amniotic tissue is widely studied and utilized in various tissue regeneration fields, including corneal regeneration, chronic wound healing, reproductive organ reconstruction, bone regeneration, microvascular reconstruction, nerve regeneration, and oral regeneration. In particular, amniotic tissue exhibits excellent skin regeneration properties, which can help control inflammation and promote wound healing.

[0003] Meanwhile, wound dressings rely on their own regenerative capacity to provide a moist environment to the injured area. While tissue-derived bioactive ingredients that can be included in these wound dressings can promote regeneration at the injured site, they pose the risk of an accompanying immune response. Therefore, while various processes for decellularizing tissue exist, they often result in significant removal of bioactive ingredients compared to the native tissue.

[0004] Therefore, in order to utilize amniotic tissue, which is rich in physiologically active components, as a wound dressing, research on a decellularization process to minimize immune response is necessary.

[0005] The present invention provides a decellularized placental-derived amniotic membrane-based hydrogel, characterized in that the content of collagen in the decellularized placental-derived amniotic membrane is 9 μg / mg to 15 μg / mg, and the content of glycosaminoglycans (GAGs) is 0.3 μg / mg to 1.0 μg / mg.

[0006] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0007] The present invention provides a decellularized placental-derived amniotic membrane-based hydrogel, characterized in that the content of collagen in the decellularized placental-derived amniotic membrane is 9 μg / mg to 15 μg / mg, and the content of glycosaminoglycans (GAGs) is 0.3 μg / mg to 1.0 μg / mg.

[0008] The content of DNA in the amniotic membrane derived from the decellularized placenta may be 500 ng / mg or less.

[0009] The above decellularized placental-derived amniotic membrane may be decellularized amniotic tissue isolated from an animal or human placenta.

[0010] The above decellularized placental-derived amniotic membrane expressed Decorin, interferon-α (IFN-α), interferon-γ (IFN-γ), interleukin-1α (IL-1α), interleukin-1 family member 4 (IL-1F4), interleukin-1 family member 5 (IL-1F5), interleukin-8 (IL-8), interleukin-13 (IL-13), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-28B (IL-28B), interleukin-32α (IL-32α), CC motif chemokine ligand 1 (CCL1 / I-309), CC motif chemokine ligand 2 (CCL2 / MCP-1), CC motif chemokine ligand 3 (CCL3 / MIP-1α), CC motif chemokine ligand It may include one or more inflammation-related factors selected from the group consisting of CXC motif chemokine ligand 4 (CCL4 / MIP-1β), C-X-C motif chemokine ligand 1 (CXCL1 / GROα), C-X-C motif chemokine ligand 9 (CXCL9 / MIG), C-X-C motif chemokine ligand 10 (CXCL10 / IP-10), and tumor necrosis factor-α (TNF-α).

[0011] The above decellularized placental-derived amniotic membrane may contain one or more vascular regeneration-related factors selected from the group consisting of platelet-derived growth factor-AB (PDGF-AB), platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), tissue metalloproteinase inhibitor-1 (TIMP-1), tissue metalloproteinase inhibitor-2 (TIMP-2), angiogenin, thrombospondin-1, platelet factor 4 (PF4), Serpin E1, amphiregulin, coaulation factor 3, insulin-like growth factor binding protein-1 (IGFBP-1), pentraxin 3 (PTX-3), and urokinase-type plasminogen activator (μPA).

[0012] The weight ratio of the decellularized placental-derived amniotic membrane in the hydrogel may be 0.1 (w / v)% to 10 (w / v)%.

[0013] The hydrogel may have a storage modulus of 100 Pa to 1000 Pa and a loss modulus of 50 Pa to 500 Pa at 10 Hz.

[0014] In one embodiment of the present invention, a composition for wound treatment or tissue regeneration comprising the decellularized placental-derived amniotic membrane-based hydrogel is provided.

[0015] In another embodiment of the present invention, a method for producing a decellularized placental-derived amniotic membrane-based hydrogel is provided, comprising the steps of: (a) preparing placental-derived amniotic tissue and then decellularizing it using a supercritical fluid and organic solvent system; (b) washing the decellularized tissue and then freeze-drying it; (c) dissolving the freeze-dried tissue in a pepsin and acidic solution and then neutralizing it; and (d) gelling the neutralized tissue solution to produce a hydrogel including decellularized placental-derived amniotic membrane, wherein in step (d), the content of collagen in the decellularized placental-derived amniotic membrane is 9 μg / mg to 15 μg / mg and the content of glycosaminoglycans (GAGs) is 0.3 μg / mg to 1.0 μg / mg.

[0016] In the above step (a), the supercritical fluid and organic solvent system can be set to react for 2 to 20 hours under conditions of a pressure of 300 to 400 bar and a temperature of 30°C to 40°C.

[0017] In the above step (b), washing can be performed for 2 to 100 hours with a DNase-containing PBS buffer.

[0018] In the above step (d), a separate gel may not be mixed during gelation.

[0019] The hydrogel according to the present invention comprises a decellularized placental-derived amniotic membrane, and can be manufactured through a decellularization process and a gelation process using a supercritical fluid and an organic solvent system. Accordingly, the collagen content in the decellularized placental-derived amniotic membrane is 9 μg / mg to 15 μg / mg, and the glycosaminoglycan (GAG) content is 0.3 μg / mg to 1.0 μg / mg, while minimizing the content of DNA remaining in the decellularized placental-derived amniotic membrane. In addition, the hydrogel according to the present invention has excellent viscoelastic properties due to its porous structure, and has the advantages of excellent anti-inflammatory effects, vascular regeneration effects, and skin regeneration effects.

[0020] Therefore, the hydrogel according to the present invention can be usefully utilized for wound treatment or tissue regeneration while minimizing immune response.

[0021] Figures 1(a) and (b) schematically illustrate a method for manufacturing a hydrogel through a gelation process following a decellularization process using a supercritical fluid and organic solvent system targeting placental-derived amniotic membrane tissue.

[0022] Figure 2(a) shows the results of observing the histological images before freeze-drying after decellularization using a supercritical fluid and organic solvent system for amniotic tissue isolated from porcine placenta, and Figures 2(b) and (c) show the results of analyzing the components of DNA, collagen, and glycosaminoglycans (GAGs) after decellularization using a supercritical fluid and organic solvent system, freeze-drying, and milling for amniotic tissue isolated from porcine placenta. In addition, Figure 2(d) shows the results of analyzing the expression of factors related to inflammation or vascular regeneration before and after the decellularization process using a supercritical fluid and organic solvent system for amniotic tissue isolated from porcine placenta.

[0023] Figure 3(a) shows the results of histological observations of amniotic tissue isolated from bovine placenta before and after lyophilization using a supercritical fluid and organic solvent system, and Figure 3(b) shows the results of analyzing the components of DNA, collagen, and glycosaminoglycans (GAGs) in amniotic tissue isolated from bovine placenta after decellularization using a supercritical fluid and organic solvent system, lyophilization, and milling. In addition, Figure 3(c) shows the results of analyzing the expression of inflammatory factors in amniotic tissue isolated from bovine placenta before and after the decellularization process using a supercritical fluid and organic solvent system.

[0024] Figure 4(a) shows the results of observing the histological images before freeze-drying after decellularization using a supercritical fluid and organic solvent system for amniotic tissue isolated from human placenta, and Figure 3(b) shows the results of analyzing the components of DNA, collagen, and glycosaminoglycans (GAGs) after decellularization, freeze-drying, and milling using a supercritical fluid and organic solvent system for amniotic tissue isolated from human placenta. In addition, Figure 3(c) shows the results of analyzing the expression of factors related to vascular regeneration or inflammation before and after the decellularization process using a supercritical fluid and organic solvent system for amniotic tissue isolated from human placenta.

[0025] Figure 5 shows the results of evaluating the structure and properties of a decellularized porcine placenta-derived amniotic membrane-based hydrogel using a supercritical fluid and organic solvent system.

[0026] Figure 6 shows the results of evaluating the angiogenic effect of a decellularized porcine placental-derived amniotic membrane-based hydrogel using a supercritical fluid and organic solvent system.

[0027] Figure 7(a) shows the results of visual and histological evaluations of the anti-inflammatory, hair follicle regeneration, or skin regeneration effects of a decellularized porcine placenta-derived amniotic membrane-based hydrogel using a supercritical fluid and organic solvent system in vivo, and Figure 7(b) shows the results of analyzing the expression of factors related to inflammation or skin regeneration of a decellularized porcine placenta-derived amniotic membrane-based hydrogel using a supercritical fluid and organic solvent system in vivo.

[0028] The present inventors applied a supercritical fluid and organic solvent system to the decellularization process to minimize the immune response while preserving the physiologically active components in the production of a hydrogel through a gelation process after a decellularization process targeting placental-derived amniotic tissue, and confirmed the effect thereof, thereby completing the present invention.

[0029]

[0030] Hereinafter, the present invention will be described in detail.

[0031]

[0032] Decellularized placental-derived amniotic membrane-based hydrogel and its use

[0033]

[0034] The present invention provides a decellularized placental-derived amniotic membrane-based hydrogel, characterized in that the content of collagen in the decellularized placental-derived amniotic membrane is 9 μg / mg to 15 μg / mg, and the content of glycosaminoglycans (GAGs) is 0.3 μg / mg to 1.0 μg / mg.

[0035]

[0036] The hydrogel according to the present invention is characterized by comprising a decellularized placental-derived amniotic membrane.

[0037] The above decellularized placental-derived amniotic membrane can be manufactured by separating amniotic tissue from the placenta and then performing a decellularization process using a supercritical fluid and organic solvent system.

[0038] First, the decellularized placenta-derived amniotic membrane is amniotic tissue isolated from an animal or human placenta, which is decellularized. The placenta may be an animal placenta or a human placenta, and in particular, in the case of an animal placenta, it may be a pig placenta or a bovine placenta, but is not limited thereto.

[0039] In addition, the separated amniotic tissue is in the form of a thin membrane, and unlike other tissues, it has the advantage of being directly applicable to a decellularization process using a supercritical fluid and organic solvent system without separate chopping.

[0040] The decellularization process using the above supercritical fluid and organic solvent system is performed by adding an organic solvent to a high-pressure reactor and then injecting the supercritical fluid. Specifically, the reaction may be performed under conditions of a pressure of 300 to 400 bar and a temperature of 30°C to 40°C for 2 to 20 hours. At this time, the organic solvent may be an alcohol-based solvent, and is preferably an alcohol-based solvent having 1 to 4 carbon atoms (particularly, ethanol), but is not limited thereto. In addition, the supercritical fluid may be a known compressed gas, and is preferably, but not limited to, carbon dioxide, dichlorotrifluoroethane, difluoromethane, difluoroethane, trifluoroethane, tetrafluoroethane, pentafluoroethane, heptafluoropropane, hexafluoropropane, pentafluoropropane, sulfur hexafluoride, perfluorocyclobutane, dichlorofluoroethane, chlorodifluoroethane, chlorofluoromethane, dimethyl ether, nitrogen dioxide, propane, butane, etc. (especially, carbon dioxide).

[0041] The content of collagen in the decellularized placenta-derived amniotic membrane may be 9 μg / mg to 15 μg / mg (preferably, 9 μg / mg to 12 μg / mg), and the content of glycosaminoglycans (GAGs) may be 0.3 μg / mg to 1.0 μg / mg.

[0042] Additionally, the content of DNA remaining in the decellularized placental amniotic membrane can be minimized. More specifically, the content of DNA in the decellularized placental amniotic membrane can be 500 ng / mg or less, preferably 50 ng / mg or less, but is not limited thereto. This minimizes the immune response.

[0043] This preservation of the physiologically active components in the amniotic membrane derived from the decellularized placenta while minimizing the immune response is due to the decellularization process using a supercritical fluid and organic solvent system, and thus has excellent anti-inflammatory, vascular regeneration, and skin regeneration effects.

[0044] Specifically, the decellularized placenta-derived amniotic membrane expresses Decorin, interferon-α (IFN-α), interferon-γ (IFN-γ), interleukin-1α (IL-1α), interleukin-1 family member 4 (IL-1F4), interleukin-1 family member 5 (IL-1F5), interleukin-8 (IL-8), interleukin-13 (IL-13), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-28B (IL-28B), interleukin-32α (IL-32α), CC motif chemokine ligand 1 (CCL1 / I-309), CC motif chemokine ligand 2 (CCL2 / MCP-1), CC motif chemokine ligand 3 (CCL3 / MIP-1α), CC motif chemokine It may include one or more inflammation-related factors selected from the group consisting of ligand 4 (CCL4 / MIP-1β), C-X-C motif chemokine ligand 1 (CXCL1 / GROα), C-X-C motif chemokine ligand 9 (CXCL9 / MIG), C-X-C motif chemokine ligand 10 (CXCL10 / IP-10), and tumor necrosis factor-α (TNF-α), and decorin, interleukin-1 family member 4 (IL-1F4), interleukin-1 family member 5 (IL-1F5), interleukin-13 (IL-13), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-28B (IL-28B), interleukin-32α (IL-32α), C-C motif chemokine ligand It is preferable to include, but is not limited to, one or more inflammation-related factors selected from the group consisting of CCL1 / I-309, C-C motif chemokine ligand 2 (CCL2 / MCP-1), C-C motif chemokine ligand 3 (CCL3 / MIP-1α), C-C motif chemokine ligand 4 (CCL4 / MIP-1β), C-X-C motif chemokine ligand 1 (CXCL1 / GROα), and C-X-C motif chemokine ligand 9 (CXCL9 / MIG). Accordingly, it is possible to suppress the immune response and provide a suitable microenvironment for tissue repair.

[0045] The above decellularized placental-derived amniotic membrane may contain one or more vascular regeneration-related factors selected from the group consisting of platelet-derived growth factor-AB (PDGF-AB), platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), tissue metalloproteinase inhibitor-1 (TIMP-1), tissue metalloproteinase inhibitor-2 (TIMP-2), angiogenin, thrombospondin-1, platelet factor 4 (PF4), Serpin E1, amphiregulin, coaulation factor 3, insulin-like growth factor binding protein-1 (IGFBP-1), pentraxin 3 (PTX-3), and urokinase-type plasminogen activator (μPA), and platelet-derived growth factor-AB (PDGF-AB). It is preferable to include, but is not limited to, one or more vascular regeneration-related factors selected from the group consisting of platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), tissue metalloproteinase inhibitor-1 (TIMP-1), angiogenin, thrombospondin-1, platelet factor 4 (PF4), Serpin E1, amphiregulin, coaulation factor 3, insulin-like growth factor binding protein-1 (IGFBP-1), and pentraxin 3 (PTX-3). Therefore, there is an advantage that it can be utilized as a tissue regeneration material.

[0046]

[0047] The weight ratio of the decellularized placental-derived amniotic membrane in the hydrogel may be 0.1 (w / v)% to 10 (w / v)%, preferably 1 (w / v)% to 5 (w / v)%, but is not limited thereto. As a result, excellent viscoelastic properties can be achieved.

[0048] More specifically, by maintaining the above weight ratio while maintaining a high content of collagen in the decellularized placenta-derived amniotic membrane, the hydrogel may have a storage modulus of 100 Pa to 1,000 Pa (preferably, 100 Pa to 500 Pa) and a loss modulus of 50 Pa to 500 Pa (preferably, 50 Pa to 200 Pa) at 10 Hz.

[0049]

[0050] In addition, the present invention provides a composition for wound treatment or tissue regeneration comprising the decellularized placental-derived amniotic membrane-based hydrogel.

[0051] The composition for wound treatment or tissue regeneration according to the present invention is characterized by including the decellularized placenta-derived amniotic membrane-based hydrogel. Since the "decellularized placenta-derived amniotic membrane-based hydrogel" has been described above, a redundant description thereof will be omitted.

[0052] In this specification, “wound” may be used in a broad sense including wound, burn wound, abrasion, laceration, stab wound, ulcer, etc., and “tissue” may be used in a broad sense including angiogenesis, epidermis, dermis, skin appendages, nerves, tendons, ligaments, muscles, etc.

[0053]

[0054] Alternatively, the present invention provides a use for a composition for wound healing or tissue regeneration comprising the decellularized placental-derived amniotic membrane-based hydrogel.

[0055] Alternatively, the present invention provides a wound treatment or tissue regeneration method comprising applying the decellularized placental-derived amniotic membrane-based hydrogel to a subject. Here, the term "subject" refers to a subject in need of treatment or improvement of a disease, and more specifically, a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, or cow.

[0056]

[0057] Method for manufacturing a hydrogel based on decellularized placental amniotic membrane

[0058]

[0059] The present invention provides a method for producing a decellularized placental-derived amniotic membrane-based hydrogel, comprising the steps of: (a) preparing placental-derived amniotic membrane tissue and then decellularizing it using a supercritical fluid and organic solvent system; (b) washing the decellularized tissue and then freeze-drying it; (c) dissolving the freeze-dried tissue in a pepsin and acidic solution and then neutralizing it; and (d) gelling the neutralized tissue solution to produce a hydrogel including decellularized placental-derived amniotic membrane, wherein the content of collagen in the decellularized placental-derived amniotic membrane in the step (d) is 9 μg / mg to 15 μg / mg and the content of glycosaminoglycans (GAGs) is 0.3 μg / mg to 1.0 μg / mg.

[0060]

[0061] The method for manufacturing a decellularized placental-derived amniotic membrane-based hydrogel according to the present invention comprises the step [step (a)] of preparing placental-derived amniotic membrane tissue and then decellularizing it using a supercritical fluid and organic solvent system.

[0062] The above placental-derived amniotic tissue may be isolated from an animal placenta or a human placenta, and is in the form of a thin membrane, and thus, unlike other tissues, has the advantage of being directly applicable to a decellularization process using a supercritical fluid and organic solvent system without separate chopping.

[0063] The decellularization process using the above supercritical fluid and organic solvent system is performed by adding an organic solvent to a high-pressure reactor and then injecting the supercritical fluid. Specifically, the reaction may be performed under conditions of a pressure of 300 to 400 bar and a temperature of 30°C to 40°C for 2 to 20 hours. At this time, the organic solvent may be an alcohol-based solvent, and is preferably an alcohol-based solvent having 1 to 4 carbon atoms (particularly, ethanol), but is not limited thereto. In addition, the supercritical fluid may be a known compressed gas, and is preferably, but not limited to, carbon dioxide, dichlorotrifluoroethane, difluoromethane, difluoroethane, trifluoroethane, tetrafluoroethane, pentafluoroethane, heptafluoropropane, hexafluoropropane, pentafluoropropane, sulfur hexafluoride, perfluorocyclobutane, dichlorofluoroethane, chlorodifluoroethane, chlorofluoromethane, dimethyl ether, nitrogen dioxide, propane, butane, etc. (especially, carbon dioxide).

[0064]

[0065] The method for manufacturing a decellularized placental-derived amniotic membrane-based hydrogel according to the present invention includes a step [step (b)] of washing the decellularized tissue and then freeze-drying it.

[0066] The washing can be performed with a DNase-containing PBS buffer for 2 to 100 hours (preferably, 2 to 4 days). This washing time can be shortened as the decellularized tissue has a thin membrane shape. After the washing, an additional washing can be performed with distilled water for 2 to 100 hours (preferably, 2 to 3 days).

[0067]

[0068] The method for producing a decellularized placental-derived amniotic membrane-based hydrogel according to the present invention includes a step [step (c)] of dissolving the freeze-dried tissue in pepsin and an acidic solution and then neutralizing it.

[0069] The above acidic solution may be a solution that can be adjusted to a pH of 2.5 to 4.5, and preferably includes at least one selected from the group consisting of acetic acid, hydrochloric acid, paratoluenesulfonic acid, and maleic acid, and more preferably includes acetic acid, but is not limited thereto. The neutralization may be performed using a basic solution, and the basic solution may be a solution that can be adjusted to a pH of 5.5 to 7.8 (preferably, pH 6.5 to 7.5), and preferably includes at least one selected from the group consisting of sodium hydroxide, sodium carbonate, sodium bicarbonate, disodium hydrogen phosphate, calcium bicarbonate, calcium hydroxide, calcium nitrate, calcium chloride, calcium cyanate, potassium hydroxide, ammonium hydroxide, and sodium acetate, and more preferably sodium hydroxide, but is not limited thereto.

[0070]

[0071] The method for producing a decellularized placental-derived amniotic membrane-based hydrogel according to the present invention includes a step [step (d)] of producing a hydrogel including a decellularized placental-derived amniotic membrane by gelling the neutralized tissue solution.

[0072] The above gelation can be performed at a temperature of 30°C to 40°C for 10 minutes to 1 hour. Since the neutralized tissue solution has a high collagen content, a separate gel may not be mixed during the gelation.

[0073] Accordingly, the content of collagen in the decellularized placenta-derived amniotic membrane is characterized by being 9 μg / mg to 15 μg / mg, and the content of glycosaminoglycans (GAGs) is 0.3 μg / mg to 1.0 μg / mg. In addition, the content of DNA remaining in the decellularized placenta-derived amniotic membrane may be minimized. In addition, since the "decellularized placenta-derived amniotic membrane-based hydrogel" has been described above, a redundant description will be omitted.

[0074]

[0075] As reviewed above, the hydrogel according to the present invention comprises a decellularized placental-derived amniotic membrane, and can be manufactured through a decellularization process and a gelation process using a supercritical fluid and an organic solvent system. Accordingly, the collagen content in the decellularized placental-derived amniotic membrane is 9 μg / mg to 15 μg / mg, and the glycosaminoglycan (GAG) content is 0.3 μg / mg to 1.0 μg / mg, while minimizing the content of DNA remaining in the decellularized placental-derived amniotic membrane. In addition, the hydrogel according to the present invention has excellent viscoelastic properties due to its porous structure, and has the advantages of excellent anti-inflammatory effects, vascular regeneration effects, and skin regeneration effects.

[0076] Therefore, the hydrogel according to the present invention can be usefully utilized for wound treatment or tissue regeneration while minimizing immune response.

[0077]

[0078] [Example]

[0079] Example 1: Preparation of a decellularized porcine placental amniotic membrane-based hydrogel using a supercritical fluid and organic solvent system.

[0080] (Step 1: Decellularization Process) Amniotic tissue was isolated from the porcine placenta, washed with PBS, and then pretreated with 100% ethanol for 24 hours. Then, a high-pressure reactor (Ilshinautoclave, Korea) was prepared as a supercritical fluid and organic solvent system, and 100% ethanol was added as an organic solvent to the high-pressure reactor, and carbon dioxide was injected as a supercritical fluid. Meanwhile, the system was set to react for 6 hours under the conditions of a pressure of 300-350 bar and a temperature of 37°C to perform decellularization. Thereafter, it was washed with a PBS buffer containing 0.3% DNase for 2 hours, and further washed with distilled water (DW) for 72 hours, followed by lyophilization and milling (see Fig. 1(a)).

[0081] (Step 2: Gelation Process) The lyophilized tissue that had been ground (milled) was dissolved in 3 mg / ml pepsin (Sigma Aldrich, P7125, USA) containing 0.5 M acetic acid (Sigma Aldrich, USA) at 4°C for 48 hours, and then neutralized using 5 N NaOH (Sigma Aldrich). The neutralized tissue solution was then gelated in a 37°C incubator for 30 minutes to produce hydrogels (1% dPAM, 2% dPAM, 3% dPAM) containing 1-3 (w / v)% of decellularized placental-derived amniotic membrane (see Fig. 1(b)).

[0082]

[0083] Example 2: Preparation of decellularized bovine placental amniotic membrane-based hydrogel using a supercritical fluid and organic solvent system.

[0084] A decellularized bovine placental-derived amniotic membrane-based hydrogel was prepared in the same manner as in Example 1, except that amniotic membrane tissue was isolated from the bovine placenta.

[0085]

[0086] Example 3: Preparation of a decellularized human placental amniotic membrane-based hydrogel using a supercritical fluid and organic solvent system.

[0087] A decellularized human placental-derived amniotic membrane-based hydrogel was prepared in the same manner as in Example 1, except that amniotic membrane tissue was isolated from the human placenta.

[0088]

[0089] Comparative Example 1: Preparation of a Decellularized Porcine Placenta-Derived Amniotic Membrane-Based Hydrogel Using a Supercritical Fluid-Only System

[0090] Decellularized porcine placental-derived amniotic membrane-based hydrogels were prepared using a supercritical fluid-only system in the same manner as in Example 1, except that the addition of 100% ethanol as an organic solvent to the high-pressure reactor was excluded.

[0091]

[0092] Experimental Example 1: Histological observation and component analysis of decellularized porcine placental-derived amniotic membrane.

[0093] When using amniotic tissue isolated from pig placenta, first, histological photographs were observed before freeze-drying (dPAM) after decellularization in Example 1. As shown in Fig. 2(a), unlike when decellularization was omitted (PAM), cell nuclei were effectively removed after decellularization, and collagen, an ECM component, was confirmed to remain well.

[0094] Next, the components of DNA, collagen, and glycosaminoglycans (GAGs) were analyzed after decellularization, freeze-drying, and milling (dPAM) in Example 1. As shown in Fig. 2(b), unlike when decellularization was omitted (PAM), DNA was significantly reduced due to decellularization, but collagen and glycosaminoglycans (GAGs), which are effective components for skin regeneration, were confirmed to remain significantly.

[0095] In particular, in Example 1, the DNA components were comparatively analyzed after decellularization, freeze-drying, and milling (dPAM (supercritical fluid-organic solvent)). As shown in Fig. 2(c), unlike the case where decellularization was omitted (PAM), it was confirmed that decellularization using a supercritical fluid-organic solvent resulted in a significant reduction in DNA, with only 50 ng / mg or less remaining (about 5% of the case where decellularization was omitted (PAM)). However, in Comparative Example 1, after decellularization, freeze-drying, and milling (dPAM (supercritical fluid)), DNA remained in excess of 500 ng / mg (about 30% of the case where decellularization was omitted (PAM)), so it can be seen that the decellularization effect is minimal.

[0096] Next, the expression of factors related to inflammation and vascular regeneration was analyzed before and after decellularization of a hydrogel (2% dPAM) containing 2 (w / v)% of the decellularized porcine placental-derived amniotic membrane manufactured in Example 1. As shown in Fig. 2(d), even after the decellularization according to Example 1, it was confirmed that a significant amount of Decorin and interleukin-28B (IL-28B) as inflammatory factors remained. Accordingly, this suggests that it can suppress immune responses and provide a suitable microenvironment for tissue repair. In addition, even after the decellularization according to Example 1, it was confirmed that a certain level or more of factors related to vascular regeneration, such as platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), and tissue metalloproteinase inhibitor-2 (TIMP-2), remained.

[0097]

[0098] Experimental Example 2: Histological observation and component analysis of decellularized bovine placental-derived amniotic membrane.

[0099] When using amniotic tissue isolated from bovine placenta, histological photographs were first observed before freeze-drying (dBAM) after decellularization in Example 2.

[0100] As shown in Fig. 3(a), unlike the case where decellularization was omitted (BAM) in the histological photograph, it was confirmed that cell nuclei were effectively removed after decellularization, and collagen, an ECM component, remained well.

[0101] Next, in Example 2, the components of DNA, collagen, and glycosaminoglycans (GAGs) were analyzed after decellularization, freeze-drying, and milling (dBAM).

[0102] As shown in Fig. 3(b), unlike the case where decellularization was omitted (BAM), DNA was significantly reduced due to decellularization, but collagen and glycosaminoglycans (GAGs), which are effective components for skin regeneration, were confirmed to remain significantly.

[0103] Next, the expression of inflammatory factors was analyzed before and after decellularization of a hydrogel (2% dBAM) containing 2 (w / v)% of the decellularized bovine placental-derived amniotic membrane prepared in Example 2.

[0104] As shown in Fig. 3(c), even after decellularization according to Example 2, it was confirmed that a significant amount of inflammation-related factors, such as interleukin-1 family member 5 (IL-1F5), interleukin-13 (IL-13), interleukin-21 (IL-21), and C-X-C motif chemokine ligand 9 (CXCL9 / MIG), remained. Therefore, this suggests that it can suppress immune responses and provide a suitable microenvironment for tissue repair.

[0105]

[0106] Experimental Example 3: Histological observation and component analysis of decellularized human placental-derived amniotic membrane.

[0107] When using amniotic tissue isolated from human placenta, histological photographs were first observed before freeze-drying (dAM) after decellularization in Example 3.

[0108] As shown in Fig. 4(a), unlike the case where decellularization was omitted (AM) in the histological photograph, it was confirmed that cell nuclei were effectively removed after decellularization, and collagen, an ECM component, remained well.

[0109] Next, in Example 3, the components of DNA, collagen, and glycosaminoglycans (GAGs) were analyzed after decellularization, freeze-drying, and milling (dAM).

[0110] As shown in Fig. 4(b), unlike when decellularization was omitted (AM), DNA was significantly reduced due to decellularization, but collagen and glycosaminoglycans (GAGs), which are effective components for skin regeneration, were confirmed to remain significantly.

[0111] Next, the expression of vascular regeneration or inflammation-related factors was analyzed before and after decellularization of a hydrogel (2% dBAM) containing 2 (w / v)% of the decellularized human placental-derived amniotic membrane prepared in Example 3.

[0112] As shown in Fig. 4(c), even after decellularization according to Example 3, it was confirmed that factors related to vascular regeneration, such as platelet-derived growth factor-AB / BB (PDGF-AB / PDGF-BB), angiogenin, vascular endothelial growth factor (VEGF), and thrombospondin-1, were maintained at a significant level or higher. In addition, platelet factor 4 (PF4), Serpin E1, and tissue metalloproteinase inhibitor-1 (TIMP-1) were also confirmed to remain significantly, thus indicating an advantage in that they can be utilized as a tissue regeneration material. In addition, amphiregulin, coagulation factor 3, insulin-like growth factor binding protein-1 (IGFBP-1), pentraxin 3 (PTX-3), and urokinase-type plasminogen activator (μPA) were also confirmed to remain at a certain level or higher.

[0113] In addition, it was confirmed that even after decellularization according to Example 3, inflammation-related factors such as interleukin-13 (IL-13), C-C motif chemokine ligand 1 (CCL1 / I-309), interleukin-1 family member 4 (IL-1F4), and interleukin-32α (IL-32α) still remain significantly. In addition, C-C motif chemokine ligand 2 (CCL2 / MCP-1), C-C motif chemokine ligand 3 (CCL3 / MIP-1α), C-C motif chemokine ligand 4 (CCL4 / MIP-1β), and C-X-C motif chemokine ligand 1 (CXCL1 / GROα) also remain significantly, and interleukin-8 (IL-8) was also confirmed to remain at a certain level or higher, suggesting that it can suppress immune responses and provide a suitable microenvironment for tissue repair.

[0114]

[0115] Experimental Example 4: Structural and Physical Properties of Decellularized Porcine Placenta-Derived Amniotic Membrane-Based Hydrogels

[0116] The structure and properties of a hydrogel (2% dPAM) containing 2 (w / v)% of the decellularized porcine placental-derived amniotic membrane prepared in Example 1 were evaluated.

[0117] As shown in Fig. 5, the hydrogel (2% dPAM) according to Example 1 was confirmed to have a porous structure. In addition, as a result of evaluating the storage modulus and loss modulus using a rheometer, the hydrogel (2% dPAM) according to Example 1 had a storage modulus of about 200 Pa and a loss modulus of about 100 Pa at 10 Hz, which are both higher values ​​than collagen, and can be seen to have excellent viscoelastic properties.

[0118]

[0119] Experimental Example 5: Evaluation of the Angiogenic Effect of Decellularized Porcine Placenta-Derived Amniotic Membrane-Based Hydrogel

[0120] The angiogenic effect of the hydrogel (2% dPAM) containing 2 (w / v)% of the decellularized porcine placental-derived amniotic membrane prepared in Example 1 was evaluated through a tube formation assay using HUVEC cells.

[0121] As shown in Fig. 6, in the case of the hydrogel (2% dPAM) according to Example 1, it was confirmed that the most junctions, segments, and meshes were formed when HUVEC cells formed tubes. Since all of these values ​​were maintained at a higher level than collagen, it can be seen that the angiogenic effect is excellent.

[0122]

[0123] Experimental Example 6: Evaluation of the anti-inflammatory or skin regeneration effects of a decellularized porcine placental-derived amniotic membrane-based hydrogel (in vivo)

[0124] UVB energy (500 mJ / cm) was applied to experimental mice 2 ) for inflammatory skin disease (2×2 cm) 2 ), a hydrogel (2% dPAM) containing 2 (w / v)% collagen or decellularized porcine placental-derived amniotic membrane prepared in Example 1 was applied to the affected area, and tissues were obtained on the 5th, 7th, and 14th days.

[0125] Afterwards, the degree of wound closure was visually observed for the obtained tissue. Furthermore, the obtained tissue was fixed in paraffin to obtain ultrathin tissue sections, which were then stained with H&E and MT for histological analysis.

[0126] As shown in Fig. 7(a), when the hydrogel (2% dPAM) according to Example 1 was applied, it was visually confirmed that hair was growing as inflammation disappeared on the 14th day. In addition, as a result of histological characteristic analysis, when the hydrogel (2% dPAM) according to Example 1 was applied, the number of neutrophils was the lowest on the 5th day compared to collagen, and it was confirmed that hair follicles, which are one of the important skin appendages in wound healing, were formed on the 14th day. In addition, when the hydrogel (2% dPAM) according to Example 1 was applied, it was confirmed that it had a skin regeneration effect as the epidermal layer and dermal layer were well distinguished on the 14th day.

[0127] Afterwards, the expression of factors related to inflammation or skin regeneration was confirmed through immunofluorescence staining of the obtained tissue.

[0128] As shown in Fig. 7(b), according to immunofluorescence staining of the tissue on the 5th day, when the hydrogel (2% dPAM) according to Example 1 was applied, the expression of myeloperoxidase, a neutrophil marker, was confirmed to be the lowest, and the expression of CD206, an anti-inflammatory macrophage marker, was confirmed to be the highest. Therefore, it can be seen that the hydrogel (2% dPAM) according to Example 1 can promote regeneration by alleviating the inflammatory response in the early inflammatory state compared to collagen.

[0129] Meanwhile, according to immunofluorescence staining of the tissue on the 14th day, when the hydrogel (2% dPAM) according to Example 1 was applied, the expression of involucrin, an epidermal regeneration marker, and CD31, an angiogenesis marker, were confirmed to be the greatest, confirming the skin regeneration effect.

[0130]

[0131] The foregoing description of the present invention is for illustrative purposes only. Those skilled in the art will readily appreciate that modifications to other specific embodiments can be made without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. In a hydrogel containing decellularized placental-derived amniotic membrane, A decellularized placenta-derived amniotic membrane-based hydrogel, characterized in that the content of collagen in the decellularized placenta-derived amniotic membrane is 9 μg / mg to 15 μg / mg, and the content of glycosaminoglycans (GAGs) is 0.3 μg / mg to 1.0 μg / mg.

2. In paragraph 1, A decellularized placental-derived amniotic membrane-based hydrogel, characterized in that the content of DNA in the decellularized placental-derived amniotic membrane is 500 ng / mg or less.

3. In paragraph 1, The above decellularized placental-derived amniotic membrane is a decellularized placental-derived amniotic membrane-based hydrogel characterized by decellularizing amniotic membrane tissue isolated from an animal or human placenta.

4. In paragraph 1, The above decellularized placental-derived amniotic membrane expressed Decorin, interferon-α (IFN-α), interferon-γ (IFN-γ), interleukin-1α (IL-1α), interleukin-1 family member 4 (IL-1F4), interleukin-1 family member 5 (IL-1F5), interleukin-8 (IL-8), interleukin-13 (IL-13), interleukin-18 (IL-18), interleukin-21 (IL-21), interleukin-28B (IL-28B), interleukin-32α (IL-32α), CC motif chemokine ligand 1 (CCL1 / I-309), CC motif chemokine ligand 2 (CCL2 / MCP-1), CC motif chemokine ligand 3 (CCL3 / MIP-1α), CC motif chemokine ligand A decellularized placental-derived amniotic membrane-based hydrogel characterized by comprising one or more inflammation-related factors selected from the group consisting of CXCL4 / MIP-1β, C-X-C motif chemokine ligand 1 (CXCL1 / GROα), C-X-C motif chemokine ligand 9 (CXCL9 / MIG), C-X-C motif chemokine ligand 10 (CXCL10 / IP-10), and tumor necrosis factor-α (TNF-α).

5. In paragraph 1, A decellularized placental-derived amniotic membrane-based hydrogel characterized in that the decellularized placental-derived amniotic membrane comprises at least one vascular regeneration-related factor selected from the group consisting of platelet-derived growth factor-AB (PDGF-AB), platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), tissue metalloproteinase inhibitor-1 (TIMP-1), tissue metalloproteinase inhibitor-2 (TIMP-2), angiogenin, thrombospondin-1, platelet factor 4 (PF4), Serpin E1, amphiregulin, coaulation factor 3, insulin-like growth factor binding protein-1 (IGFBP-1), pentraxin 3 (PTX-3), and urokinase-type plasminogen activator (μPA).

6. In paragraph 1, A decellularized placental-derived amniotic membrane-based hydrogel, characterized in that the weight ratio of the decellularized placental-derived amniotic membrane in the hydrogel is 0.1 (w / v)% to 10 (w / v)%.

7. In paragraph 1, A decellularized placental-derived amniotic membrane-based hydrogel, characterized in that the hydrogel has a storage modulus of 100 Pa to 1,000 Pa and a loss modulus of 50 Pa to 500 Pa at 10 Hz.

8. A composition for wound treatment or tissue regeneration comprising a decellularized placental-derived amniotic membrane-based hydrogel according to any one of claims 1 to 7. 9.(a) A step of preparing placental-derived amniotic tissue and then decellularizing it using a supercritical fluid and organic solvent system; (b) a step of washing the decellularized tissue and then freeze-drying it; (c) a step of dissolving the freeze-dried tissue in pepsin and an acid solution and then neutralizing it; and (d) a step of gelling the neutralized tissue solution to prepare a hydrogel containing decellularized placental-derived amniotic membrane, A method for producing a decellularized placenta-derived amniotic membrane-based hydrogel, characterized in that the content of collagen in the decellularized placenta-derived amniotic membrane in the step (d) is 9 μg / mg to 15 μg / mg, and the content of glycosaminoglycans (GAGs) is 0.3 μg / mg to 1.0 μg / mg.

10. In paragraph 9, A method for producing a decellularized placenta-derived amniotic membrane-based hydrogel, characterized in that in the step (a), the supercritical fluid and organic solvent system are set to react for 2 to 20 hours under conditions of a pressure of 300 to 400 bar and a temperature of 30°C to 40°C.

11. In paragraph 9, A method for producing a decellularized placental-derived amniotic membrane-based hydrogel, characterized in that washing in the step (b) is performed for 2 to 100 hours with a DNase-containing PBS buffer.

12. In paragraph 9, A method for producing a decellularized placenta-derived amniotic membrane-based hydrogel, characterized in that a separate gel is not mixed during gelation in the above step (d).

Images