Mesenchymal stem cells-hydrogel-biodegradable or mesenchymal stem cells-hydrogel-nondegradable support composition for alleviating or improving epidermolysis bullosa

a technology of mesenchymal stem cells and hydrogel, which is applied in the field of composition and a sheet for alleviating or improving epidermolysis bullosa, can solve the problems of no known technique for a dressing agent to exhibit skin regeneration effects without, and no method for the full recovery of the disease is available, so as to improve epidermolysis bullosa, improve skin reproduction and re-epithelization ability, and significantly increase survival time

Inactive Publication Date: 2018-05-03
ANTEROGEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0016]According to the present invention, the composition or sheet for alleviating or improving epidermolysis bullosa, containing the mesenchymal stem cells-hydrogel-biodegradable support or nondegradable support increases the expression of specifically, collagen type VII and laminin-5 to express a clinically significant effect when being applied to the wounded part of epidermolysis bullosa.
[0017]Further, in the composition or sheet containing high-active mesenchymal stem cells, the high-active mesenchymal stem cells may be applied to a wounded part of a patient with epidermolysis bullosa as it is without isolation (selection) using proteases, and in the culturing, an extracellular matrix such as collagen, laminin, fibronectin, and elastin secreted from the mesenchymal stem cells is wholly present on the hydrogel to have an advantageous effect that skin reproduction and re-epithelization abilities are significantly excellent when the composition or sheet is applied to the wounded part of the patient with epidermolysis bullosa as compared with conventional therapeutic agents.
[0018]More specifically, the mesenchymal stem cells-hydrogel-support according to the present invention keep a form of fibroblasts even in a serum-free medium, and the cells survive 90% or more after 1 week elapses and thus the survival time is significantly increased as compared with a conventional stem cell therapeutic agent. Also, when thawing after freezing, the shape and the strength of the sheet are kept as it is and the cells in the sheet also survive 95% or more and long-term freezing at −80° C. is enabled without damage of the cells as a main active ingredient, and even when 1 year or more elapses when long-term freezing, the cells survive 95% or more when thawing. Further, various growth factors and cytokines promoting cell proliferation and angiogenesis are continuously secreted, various types of extracellular matrixes containing collagen type VII and laminin-5 are secreted in large amount, and the secreted extracellular matrixes are left in the hydrogel and applied to the wounded part to provide various substrates, thereby facilitating treatment of epidermolysis bullosa. Also, even if the inflammation occurs in the wounded part without causing the immune response, a secretion amount of TNF-α which is secreted by immune cells in large amount to increase immunoreactivity is significantly reduced to alleviate the inflammation, thereby helping in healing blisters and inflammation in patients with epidermolysis bullosa. Specifically, the expression of collagen type VII is significantly increased, thereby promoting tissue reproduction and re-epithelization.

Problems solved by technology

Up to now, a method of modifying keratin mixtures produced in the skin, methods such as bone marrow transplantation, and the like have been studied, but there is no method for the full recovery of the disease available yet.
However, there are no known techniques for a dressing agent exhibits skin regeneration effects without irritation when the dressing agent is applied to a wounded site of epidermolysis bullosa patients.

Method used

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  • Mesenchymal stem cells-hydrogel-biodegradable or mesenchymal stem cells-hydrogel-nondegradable support composition for alleviating or improving epidermolysis bullosa
  • Mesenchymal stem cells-hydrogel-biodegradable or mesenchymal stem cells-hydrogel-nondegradable support composition for alleviating or improving epidermolysis bullosa
  • Mesenchymal stem cells-hydrogel-biodegradable or mesenchymal stem cells-hydrogel-nondegradable support composition for alleviating or improving epidermolysis bullosa

Examples

Experimental program
Comparison scheme
Effect test

example 1

Culturing Human Adipose-Derived Mesenchymal Stem Cells

[0086]Adipose tissue may be usually obtained by liposuction, but is not limited thereto. Adipose-derived mesenchymal stem cells were isolated from the adipose tissue obtained by liposuction as follows: To remove blood, the adipose tissue was washed, and then added with the same volume of collagenase solution as the adipose tissue thereto, and reacted at 37° C. in a water bath.

[0087]After the centrifugation, the fat layer as a supernatant was removed, and the collagenase solution as the lower layer was carefully isolated without shaking, suspended in a substrate medium, and then centrifuged at 20° C. and 1500 rpm for 5 minutes. At this time, the lower layer served as the stroma-vascular fraction, and the supernatant was removed. The stroma-vascular fraction was suspended in the substrate medium, inoculated into a culture vessel, and cultured in a 5% CO2 incubator at 37° C. for 24 hours.

[0088]After the removal of the culture medium...

example 2

tion of Concentration of Fibrin Glue as Hydrogel

[0089]Lyophilized thrombin was added to 1 mL of a calcium chloride solution to be 400 to 600 I.U. Alternatively, the frozen thrombin was thawed and adjusted to the same concentration, and then used. The lyophilized thrombin was added with 1 mL of an aprotinin solution or thawed to prepare a undiluted solution and then the undiluted solution was diluted stepwise to 1:5, 1:10, 1:20, and 1:40. The cells subcultured two times or more in Example 1 were collected and suspended, mixed with thrombin at a ratio (v / v) of 40 to 50:1, and then mixed with fibrigen diluted stepwise at 1:1 to form a fibrin gel. When the gel was completely hardened, the cells were added with a culture medium containing 10% FBS and 1 ng / mL bFGF and cultured in a 5% CO2 incubator at 37° C. for 5 days.

[0090]On the 2nd and 5th day of the culture, the cell-fibrin gel mixture was taken and made into thin slices, stained with 10 μg / mL of acridine orange / ethidium bromide (AO / ...

example 3

on of Human Adipose-Derived Mesenchymal Stem Cells-Hydrogel-Biodegradable or Nondegradable Support Sheet

[0093]The mesenchymal stem cells subcultured two times or more in Example 1 were collected and suspended in the growth medium. Based on the results of Example 2, thrombin was added to the cell suspension to be a final 8 to 15 I.U.

[0094]The fibrinogen at a concentration of about 3 to 6.5 mg / mL was applied evenly on vicryl mesh or bovine placental membrane as a biodegradable support having a square shape of about 5×5 cm or gauze, polyurethane coated with soft silicon on a single surface, and a PET film as a nondegradable support, as a support. Thereafter, the cell suspension containing thrombin was applied to the support to have about 5,000 cells per cm2, and then the cell-fibrin gel was uniformly formed and attached to the support. When the fibrin gel was completely hardened, the cells were added with a growth medium and cultured at 37° C. in a 5% CO2 incubator for 3 to 7 days.

[009...

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Abstract

Provided are a composition and a sheet, including a mesenchymal stem cells-hydrogel-biodegradable support or a mesenchymal stem cells-hydrogel-nondegradable support and a preparing method thereof. More specifically, in the sheet including a mesenchymal stem cells-hydrogel-biodegradable support or a mesenchymal stem cells-hydrogel-nondegradable support according to the present invention, the high-active mesenchymal stem cells may be applied to a wounded part of a patient with epidermolysis bullosa as it is without isolation using proteases, and in the culturing, an extracellular matrix such as collagen, laminin, fibronectin, and elastin secreted from the mesenchymal stem cells is wholly present on the hydrogel to have an advantageous effect that skin reproduction and re-epithelization abilities are significantly excellent as compared with conventional dressing agents used for epidermolysis bullosa.

Description

REFERENCE TO RELATED APPLICATIONS[0001]This is a Continuation-In-Part (CIP) application of PCT / KR2017 / 003982 filed Apr. 12, 2017 which claims priority to KR Application 10-2016-0044514 filed Apr. 12, 2016, the disclosure of which being entirely incorporated herein as reference.TECHNICAL FIELD[0002]The present invention relates to a composition and a sheet for alleviating or improving epidermolysis bullosa, containing high-active living mesenchymal stem cells which are attached and cultured to a biodegradable or nondegradable support by suspending the cultured mesenchymal stem cells in a hydrogel, and a preparing method thereof. Specifically, the present invention is to provide a composition, a sheet, and a preparing method thereof capable of expressing a clinically significant effect when applied to a wounded part of epidermolysis bullosa by increasing expression of collagen type VII and laminin-5. Therefore, the composition or sheet may be usefully used for alleviating or improving...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61L27/38C12N5/0775A61K38/39A61K47/30A61L27/52C12N5/071C12N5/00A61P17/00A61L27/54
CPCA61L27/3813C12N5/0665A61K38/39A61K47/30A61L27/52C12N5/0625C12N5/0062C12N5/0068A61P17/00A61L27/54A61L2300/20A61L2300/64A61K9/70A61K9/0014A61K9/7007A61K9/7015A61K47/32A61K47/36A61K47/38C12N5/0662C12N2501/10C12N2533/50C12N2533/30C12N2533/70C12N2533/90A61L27/3834
Inventor LEE, SUNG-KOOKIM, MIHYUNG
Owner ANTEROGEN
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