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

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.

What is claims is: 
     
         1 . A preparing method of a sheet for alleviating or improving epidermolysis bullosa, the method comprising:
 (a) obtaining a mesenchymal stem cells-hydrogel-support by attaching mesenchymal stem cells to (i) at least one kind of support selected from the group consisting of biodegradable supports and nondegradable supports; (ii) at least two kinds of nondegradable supports; or (iii) a combination of at least one kind of support and at least one kind of nondegradable support using a hydrogel; and   (b) culturing the mesenchymal stem cells-hydrogel-support obtained in step (a) in a growth medium,   wherein the growth medium is a medium including fetal bovine serum (FBS) and at least one factor selected from the group consisting of a basic fibroblast growth factor (bFGF), an epidermal growth factor (EGF), a transforming growth factor (TGF-beta1), a platelet-derived growth factor (PDGF), a vascular endothelial growth factor (VEGF), a hepatocyte growth factor (HGF) and an insulin-like growth factor (IFG-1), the hydrogel is at least one selected from the group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose and pectin, the biodegradable support is selected from the group consisting of poly-gamma-glutamic acid (PGA), poly lactic acid (PLA), vicryl mesh, human placental membrane, bovine placental membrane, pig collagen, chitin, chitosan, fibronectin and dextran, and the nondegradable support is selected from the group consisting of sterilized nonwoven fabrics, polyethylene terephthalate (PET) films, polyethylene (PE) films, polypropylene (PP) films, polyurethane films, net type polyurethane films, and polyurethane coated with soft silicon on a single surface.   
     
     
         2 . The method of  claim 1 , wherein the sheet contains collagen type VII or laminin-5. 
     
     
         3 . The method of  claim 1 , wherein the fibrin glue includes fibrinogen at a concentration of 0.5 to 45 mg/mL. 
     
     
         4 . The method of  claim 1 , wherein the fibrin glue includes fibrinogen at a concentration of 0.5 to 10 mg/mL. 
     
     
         5 . The method of  claim 1 , further comprising: (c) activating the cells by additionally performing at least one stimulation selected from the group consisting of physical stimulation, hypoxic stimulation, mitogen stimulation, and inflammatory factor stimulation in step (b). 
     
     
         6 . The method of  claim 1 , further comprising:
 (d) adding and freezing the mesenchymal stem cells-hydrogel-support in a freezing preservative containing 1 to 20 w/v % DMSO and 1 to 80 w/v % human serum albumin in step (b),   wherein when thawing after freezing, a survival rate of the mesenchymal stem cells is 90% or more.   
     
     
         7 . The method of  claim 1 , wherein when thawing after freezing for 12 months or more, the survival rate of the mesenchymal stem cells is 90% or more. 
     
     
         8 . The method of  claim 1 , wherein in step (a), 1,000 to 10,000 mesenchymal stem cells per 1 cm 2  of the hydrogel support are applied. 
     
     
         9 . The method of  claim 1 , wherein in step (b), the mesenchymal stem cells are proliferated to 20,000 to 400,000 per 1 cm 2  of the hydrogel support. 
     
     
         10 . The method of  claim 1 , wherein the epidermolysis bullosa is selected from the group consisting of epidermolysis bullosa, acantholysis bullosa, acanthosis bullosa, epidermolysis bullosa acquisita, epidermolysis bullosa hereditaria, epidermolysis bullosa letalis, epidermolysis bullosa tarda, epidermolysis hereditaria tarda, hyperplastic epidermolysis bullosa, keratolysis, localized epidermolysis bullosa, non-scarring epidermolysis bullosa, polydysplastic epidermolysis bullosa, scarring bullosa, simplex epidermolysis bullosa, Weber-Cockayne disease, Dowling-Meara syndrome, Goldscheider's disease, Hallopeau-Siemens disease, Heinrichsbauer syndrome, Herlitz syndrome, and Kobner's disease. 
     
     
         11 . The method of  claim 1 , wherein the mesenchymal stem cells are isolated from any one selected from the group consisting of fat, bone marrow, skin, blood vessels, muscles, brain, blood, placenta, dental pulp and umbilical cord blood. 
     
     
         12 . A method for alleviating or improving epidermolysis bullosa comprising administering a composition comprising:
 mesenchymal stem cells and a hydrogel; and   at least one support selected from the group consisting of a biodegradable support and a nondegradable support; at least two nondegradable supports; or a combination of at least one biodegradable support and nondegradable support as an active ingredient to a subject having epidermolysis bullosa,   wherein the hydrogel is selected from the group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose and pectin, the biodegradable support is selected from the group consisting of poly-gamma-glutamic acid (PGA), poly lactic acid (PLA), vicryl mesh, human placental membrane, bovine placental membrane, pig collagen, chitin, chitosan, fibronectin and dextran, and the nondegradable support is selected from the group consisting of sterilized nonwoven fabrics, polyethylene terephthalate (PET) films, polyethylene (PE) films, polypropylene (PP) films, polyurethane films, net type polyurethane films, and polyurethane coated with soft silicon on a single surface.   
     
     
         13 . The method of  claim 12 , wherein the composition contains collagen type VII or laminin-5. 
     
     
         14 . The method of  claim 12 , wherein the fibrin glue includes fibrinogen at a concentration of 0.5 to 45 mg/mL. 
     
     
         15 . The method of  claim 12 , wherein the fibrin glue includes fibrinogen at a concentration of 0.5 to 10 mg/mL. 
     
     
         16 . The method of  claim 12 , wherein 20,000 to 400,000 mesenchymal stem cells per 1 cm 2  of the hydrogel support are included. 
     
     
         17 . The method of  claim 12 , wherein the epidermolysis bullosa is selected from the group consisting of epidermolysis bullosa, acantholysis bullosa, acanthosis bullosa, epidermolysis bullosa acquisita, epidermolysis bullosa hereditaria, epidermolysis bullosa letalis, epidermolysis bullosa tarda, epidermolysis hereditaria tarda, hyperplastic epidermolysis bullosa, keratolysis, localized epidermolysis bullosa, non-scarring epidermolysis bullosa, polydysplastic epidermolysis bullosa, scarring bullosa, simplex epidermolysis bullosa, Weber-Cockayne disease, Dowling-Meara syndrome, Goldscheider's disease, Hallopeau-Siemens disease, Heinrichsbauer syndrome, Herlitz syndrome, and Kobner's disease. 
     
     
         18 . The method of  claim 12 , when thawing after freezing for 12 months or more, a survival rate of the mesenchymal stem cells is 90% or more in the composition. 
     
     
         19 . The method of  claim 12 , wherein the mesenchymal stem cells are isolated from any one selected from the group consisting of fat, bone marrow, skin, blood vessels, muscles, brain, blood, placenta, dental pulp and umbilical cord blood. 
     
     
         20 . A sheet for alleviating or improving epidermolysis bullosa, containing the composition according to  claim 12  as an active ingredient.