Cell for treating burns and scalds and method for treating burns and scalds

Abstract

The present invention addresses the problem of effectively promoting granulation proliferation in dermal defects or subcutaneous tissue defects of severe burns and scalds. As a solution, a cell for treating burns and scalds, which is used in the treatment of burns and scalds, is derived from a placenta of an animal including a human, and promotes healing.

Description

Technical Field

[0001] The present invention relates to cells for repairing the periphery of defective dermal tissue (including subcutaneous tissue) in the treatment of burns and scalds and a burns and scalds treatment method. Background Art

[0002] The severity of burns is determined by the area and depth. Regarding the depth, it is divided into I to III degrees according to the color of the surface. II degree burns are burns that reach deep into the dermis, while III degree burns are a state where the entire skin is damaged.

[0003] Burns of degree II covering more than 30% of the body surface area or burns of degree III covering more than 10% of the body surface area are considered severe and require intensive treatment at an emergency center.

[0004] Third degree burns in the field of emergency surgery damage not only the skin but also the subcutaneous tissue, nerves and blood vessels, so surgical treatment is essential. In addition, the risk of infection is increased, so conventional wound covering materials are not suitable. The damaged area due to burns should be removed as soon as possible, and the formation of granulation tissue as a transplant bed should be observed, and skin transplantation should be performed.

[0005] However, the following problems often occur during transplantation: the indispensable wound bed preparation (good granulation) is not fully performed, so the transplanted skin is difficult to survive, inducing sepsis, etc., worsening the condition and leading to death.

[0006] According to non-patent document 1 ("Burns and Scalds Diagnosis and Treatment Guidelines (Revised 2nd Edition) 2015" published by the Japanese Society of Burns and Scalds), reports on wound covering materials are for II degree burns and scalds, and there is no positive evidence for the use of wound covering materials on III degree burns and scalds. It should be noted that the wound covering materials used in burns and scalds are roughly divided into foam materials, fiber materials, and colloid materials, and are used according to their shape and absorption of exudate. All of them are aimed at forming good granulation tissue.

[0007] It should be noted that the amniotic membrane is a strong biological membrane composed of collagen and elastic fibers, and fresh amniotic membrane is reported to be a useful covering material for trauma and burns (Non-Patent Document 2).

[0008] However, since it is not immediately available when needed, and its storage and handling are complicated, its use in actual clinical applications is limited. In contrast, dried amniotic membrane (extremely dried human dried amniotic membrane: hereinafter referred to as HD-AM) produced by a specific drying process has been reported (Patent Document 1, Non-Patent Document 3). There are also reports on granulation formation using HD-AM (Patent Document 2).

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: Japanese Patent Application Publication No. 2007-54015

[0012] Patent Document 2: Japanese Patent Application Publication No. 2021-020868

[0013] Patent Document 3: International Publication No. 2013 / 077428

[0014] Non-patent literature

[0015] Non-patent literature 1: Burn and Scald Diagnosis and Treatment Guideline, Revised 2nd Edition, 2015, Japan Society for Burn and Scald Diseases

[0016] Non-patent document 2: Gruss, JS & Jirsch, DW Human amniotic membrane: A versatile wound dressing. Can. Med. Assoc. J. 118, 1237-1246 (1978).

[0017] Non-patent document 3: Okabe, M. et al. Hyperdry human amniotic membrane is useful material for tissue engineering: Physical, morphological properties, and safety as the new biological material. J. Biomed. Mater. Res. - Part A 102, 862-870 (2014). Summary of the invention

[0018] Problem that the invention aims to solve

[0019] As mentioned above, in the past, there was no effective method to prevent infection and promote good granulation formation for the treatment of third-degree burns and scalds. The current situation was that the patient's vitality and immunity could only be relied upon.

[0020] In the treatment of third-degree burns, it is necessary to allow granulation tissue to proliferate as quickly as possible, but there is no effective method.

[0021] Therefore, early skin transplantation to form good granulation tissue and avoid infection can help prolong the life of patients with severe burns and scalds.

[0022] The inventors prepared burn model animals with burn depths of II to III degrees, and confirmed from the perspectives of histology, immunochemistry, and molecular biology that placenta-derived cells can effectively promote granulation proliferation in the dermis defect or subcutaneous tissue defect of the burn model animals, which led to the present invention.

[0023] Solutions for solving problems

[0024] The cells for treating burns and scalds according to the present invention are characterized in that they are cells derived from the placenta of animals including humans and are used in the treatment of burns and scalds, and the cells promote healing.

[0025] By adopting this structure, especially when used as cells for regeneration of dermal defect or subcutaneous tissue of patients with severe burns and scalds, the secretion of physiologically active substances such as inflammatory cytokines and growth factors at the wound site of severe burns and scalds is promoted, and the infection caused by foreign bodies from the outside is resisted by the inflammatory cytokines, and the secretion of IL-6, IFN-γ, IL-10, COX2 (PGE2) and the like are promoted, and the wound healing of severe burns and scalds is promoted through these positive effects. In addition, safe regenerative medicine with few ethical issues and few immune rejection reactions caused by allogeneic transplantation can be realized.

[0026] In addition, the invention may be characterized in that the invention comprises amniotic mesenchymal stem cells.

[0027] According to this structure, the cells have high differentiation and proliferation abilities, and promote wound healing of severe burns and scalds.

[0028] In addition, the invention may be characterized in that it is used for the regeneration of burn tissue and the promotion of granulation formation in the treatment of burns and scalds.

[0029] In addition, the cells may be characterized in that they are cells having tissue regeneration for burns and scalds and antibacterial and anti-inflammatory effects.

[0030] In addition, the invention may be characterized in that the solution is contained in a solvent so as to be added dropwise to a burn or scald wound.

[0031] According to this configuration, placenta-derived cells can be easily placed in the burn wound.

[0032] In addition, it may be characterized in that the above-mentioned solvent has high viscosity.

[0033] According to this configuration, it is possible to prevent placenta-derived cells from being lost from the burn wound.

[0034] In addition, the burns and scalds may be characterized in that the burns and scalds are II degree and / or III degree burns and scalds.

[0035] According to this structure, it is particularly used in the regeneration of dermal defects or subcutaneous tissues in patients with severe burns and scalds, and the secretion of physiologically active substances such as inflammatory cytokines and growth factors in the wound site of severe burns and scalds is promoted. The inflammatory cytokines are used to resist infection caused by foreign bodies from the outside, and the secretion of IL-6, IFN-γ, IL-10, COX2 (PGE2) and the like are promoted. Through these positive effects, the healing of severe burns and scalds wounds is promoted.

[0036] In addition, the present invention may be characterized in that the cells can be used together with a covering material for burns and scalds and / or a regenerative scaffold material for burns and scalds, or can be used alone.

[0037] In addition, the characteristic may be that the above-mentioned covering material for burns and scalds and / or the above-mentioned regenerative scaffold material for burns and scalds is a dried amniotic membrane obtained by drying the fresh amniotic membrane that wraps the fetus of an animal including a human, and the dried amniotic membrane is dried so that it can be preserved in the atmosphere, and when it is immersed in water or a buffer solution and rehydrated, the epithelial cells, basement membrane and connective tissue that constitute the fresh amniotic membrane are maintained.

[0038] With this configuration, the dried amniotic membrane can function as a scaffold for cells, induce growth factors and cell migration chemokines, and promote local anti-inflammation and tissue regeneration by regulating differentiation into M2 macrophages.

[0039] The method for treating burns and scalds according to the present invention is characterized in that healing is promoted by placing cells derived from the placenta of animals including humans at the burn and scald wound site.

[0040] According to this method, when used as cells for regeneration of dermal defects or subcutaneous tissues in patients with severe burns and scalds, the secretion of physiologically active substances such as inflammatory cytokines and growth factors at the wound site of severe burns and scalds is promoted, and the infection caused by foreign bodies from the outside is resisted by the inflammatory cytokines, and the secretion of IL-6, IFN-γ, IL-10, COX2 (PGE2) and the like are promoted, and the wound healing of severe burns and scalds is promoted through these positive effects. In addition, safe regenerative medicine with few ethical issues and few immune rejection reactions caused by allogeneic transplantation can be achieved.

[0041] In addition, the present invention may be characterized in that the placenta-derived cells include amniotic mesenchymal stem cells.

[0042] According to this structure, the cells have high differentiation and proliferation abilities, and promote wound healing of severe burns and scalds.

[0043] In addition, the method may be characterized in that the placenta-derived cells are mixed in a solvent and the mixture is dripped onto the burn wound.

[0044] In addition, the present invention may be characterized in that 100 or more of the placenta-derived cells are contained in 50 μL of the solvent.

[0045] In addition, the present invention may be characterized in that 300,000 of the placenta-derived cells are contained in 50 μL of the solvent.

[0046] In addition, it may be characterized in that the above-mentioned solvent has high viscosity.

[0047] According to this method, it is possible to prevent the solvent containing placenta-derived cells from leaking out from the burn wound.

[0048] In addition, the characteristic may be that cells derived from the placenta of animals including humans are arranged at the burn and scald wound site, and a dry amniotic membrane is arranged from above. The dry amniotic membrane is obtained by drying the fresh amniotic membrane that wraps the fetus of an animal including humans. The dry amniotic membrane is dried so that it can be preserved in the atmosphere, and the epithelial cells, basement membrane and connective tissue that constitute the fresh amniotic membrane are maintained when it is immersed in water or buffer solution and rehydrated.

[0049] According to this method, the dried amniotic membrane can function as a scaffold for cells, induce growth factors and cell migration chemokines, and promote local anti-inflammation and tissue regeneration by regulating differentiation into M2 macrophages.

[0050] In addition, the burns and scalds may be characterized in that the burns and scalds are II degree and / or III degree burns and scalds.

[0051] According to this method, it is particularly used in the regeneration of dermal defects or subcutaneous tissues in patients with severe burns and scalds, and the secretion of physiologically active substances such as inflammatory cytokines and growth factors in the wound site of severe burns and scalds is promoted. The inflammatory cytokines are used to resist infection caused by foreign bodies from the outside, and the secretion of IL-6, IFN-γ, IL-10, COX2 (PGE2) and the like are promoted. Through these positive effects, the healing of severe burns and scalds wounds is promoted.

[0052] Effects of the Invention

[0053] The cells for treating burns and scalds and the burns and scalds treatment method according to the present invention can promote the healing of severe burns and scalds. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 : Figure 1 A to Figure 1 C is a diagram illustrating the experimental mouse model and the application of HD-AM. Figure 1 A and Figure 1 B is a diagram showing the preparation of burn sites on experimental mice. Figure 1C is a graph showing suitability as an experimental model for third-degree burns.

[0055] Figure 2 : Figure 2 Figures illustrating the use of HD-AM and placenta-derived cells (control group: schematic diagram of mice without HD-AM and placenta-derived cells, cell group: schematic diagram of mice using placenta-derived cells, HD-AM group: schematic diagram of mice using HD-AM, HD-AM / cell group: schematic diagram of mice using HD-AM and placenta-derived cells).

[0056] Figure 3 : Figure 3 A and Figure 3 B is a schematic diagram of the method for measuring granulation tissue thickness. Figure 3 A is a diagram obtained by observing over time a tissue stained by the AZAN staining method which selectively stains connective tissue after a burn or scald. Figure 3 B is a schematic diagram showing a method for measuring the thickness of granulation tissue formed at a treatment site after a burn or scald occurs.

[0057] Figure 4 : Figure 4 The table shows the primers used in quantitative reverse transcription polymerase chain reaction (qRT-PCR).

[0058] Figure 5 : Figure 5 A and Figure 5 B is a graph comparing microscopic photographs of granulation tissue formed over time by post-wounding treatment and the average thickness of granulation tissue. Figure 5 A is a microscope photo of the control group, cell group, HD-AM group, and HD-AM / cell group at 1, 4, and 7 days after treatment (POD1, POD4, and POD7). The thickness of the granulation tissue is indicated by a black arrow. Figure 5 B is a graph comparing the average values ​​of the thickness of granulation tissue.

[0059] Figure 6 : Figure 6 A to Figure 6 C is a diagram illustrating the formation of blood vessels in the granulation tissue. Figure 6 A is a photograph showing the state of angiogenesis in POD4. Figure 6 B is a photograph showing the state of angiogenesis in POD7. Figure 6 C is a graph showing the comparison of the measurement sites of blood vessels formed in the granulation tissue and the averages of the measurement values.

[0060] Figure 7 : Figure 7 A to Figure 7D is a graph comparing inflammatory and anti-inflammatory cytokines using quantitative RT-PCR. Figure 7 A is a graph showing the mRNA expression of cytokines associated with inflammation. Figure 7 B is a graph and a photograph showing the expression of inflammatory cytokine IL-6 mRNA and the distribution of IL-6. Figure 7 C is a graph and a photograph showing the expression of mRNA of a Type II macrophage marker (CD163) associated with anti-inflammation and the distribution of Type II macrophages. Figure 7 D is a graph and a photograph showing the expression of inflammatory cytokine IL-10 mRNA and the distribution of IL-10.

[0061] Figure 8 : Figure 8 A diagram showing a drying device for producing dried amniotic membrane (HD-AM). DETAILED DESCRIPTION

[0062] (Placenta-derived cells)

[0063] First, placenta-derived cells will be described. In the following experiments, placenta-derived cells are amniotic mesenchymal cells and amniotic mesenchymal stem cells, but are not limited to these.

[0064] The amniotic membrane is an extraembryonic tissue composed of epithelial cells derived from the ectoderm and mesenchymal cells derived from the mesoderm, and contains a cell population with pluripotent stem cell characteristics. The amniotic membrane is discarded as excretion after birth, so there are few ethical issues in using it as a biomaterial. In addition, the amniotic membrane also has special properties in immunology and has low immunogenicity, so the immune rejection reaction caused by allogeneic transplantation is also relatively mild.

[0065] Placenta-derived cells are collected from the amniotic membrane of mammals including humans. The amniotic membrane is composed of epithelial cells and mesenchymal cells. When collecting a cell population of mesenchymal cells from the amniotic membrane, the epithelial cells can be removed from the amniotic membrane and separated. The collection of the cell population of mesenchymal cells can be carried out according to the method described in Japanese Patent Application Laid-Open No. 2003-231639, for example.

[0066] It should be noted that human amniotic membrane can be collected from pregnant women with informed consent, for example, by cesarean section.

[0067] The collected amniotic mesenchymal cell population contains cells with different proliferation capabilities, life spans, and properties. Therefore, when the mesenchymal cell population is maintained under conventional culture conditions, the epithelial cells that begin to adhere and proliferate at the beginning of the culture occupy most of the culture surface, and the amniotic mesenchymal stem cells that begin to proliferate later cannot proliferate and are squeezed out, resulting in the inability to be isolated.

[0068] Therefore, in order to prepare a cell population of amniotic mesenchymal stem cells from the collected cell population of amniotic mesenchymal cells, it can be carried out according to the method described in International Publication No. 2013 / 077428, for example.

[0069] The cell population of amnion mesenchymal stem cells includes cells having a spindle-shaped morphology and has a high proliferation capacity, and is preferably capable of 50 or more cell divisions (population doublings).

[0070] When placenta-derived cells are used to treat burns or scalds, for example, each 50 μL of solvent contains more than 100 placenta-derived cells, and preferably each 50 μL of solvent contains 300,000 placenta-derived cells. By dripping a solution containing placenta-derived cells to the burn or scald wound, the placenta-derived cells can be arranged at the burn or scald site.

[0071] In addition, when a solution containing cells derived from the placenta is added to the burn wound, in order to avoid the loss of the solution containing cells derived from the placenta, a wound covering material such as a plaster can be attached to the burn wound after the solution containing cells derived from the placenta is added to the burn wound. Wherein, in the case of III degree burns, conventional wound covering materials cannot be applied sometimes, so the dried amniotic membrane (HD-AM) described later can be used as a covering material to avoid the loss of the solution containing cells derived from the placenta. It should be noted that by making the cells derived from the placenta contained in a solvent with high viscosity to the extent that the fluidity is not lost, the solution containing cells derived from the placenta can also be avoided from being lost from the burn wound. When the cells derived from the placenta are contained in a solvent with high viscosity, the higher the viscosity, the fewer the number of cells contained per unit solvent.

[0072] Furthermore, when placenta-derived cells are used for the treatment of burns and scalds, the placenta-derived cells may be used alone or in combination with dried amniotic membrane (HD-AM) described below.

[0073] (Dried amniotic membrane)

[0074] The dried amniotic membrane produced by a specific drying process (extreme drying) is, for example, the dried amniotic membrane described in Patent Document 1. That is, the fresh amniotic membrane placed in the treatment tank is continuously heated by an infrared heater provided in the treatment tank, and during the decompression operation of making the treatment tank in a decompressed state and the re-pressurization operation of slightly raising the pressure in the treatment tank in a decompressed state toward the atmospheric pressure side, the fresh amniotic membrane is irradiated with microwaves from a microwave generator provided in the treatment tank, thereby drying while imparting energy to the water molecules present in the amniotic membrane. The dried amniotic membrane (HD-AM) produced by repeating this process multiple times has its cell / tissue structure preserved, although the amniotic cells themselves are inactivated. Specifically, the dried amniotic membrane (HD-AM) is dried so that it can be stored in the atmosphere, and the epithelial cells, basement membrane, and connective tissue constituting the fresh amniotic membrane are preserved when it is rehydrated by immersing in water or a buffer solution.

[0075] (Combination use of placenta-derived cells and dried amniotic membrane)

[0076] Third degree burns in the field of emergency surgery require surgical treatment because they damage not only the skin but also the subcutaneous tissue, nerves, and blood vessels. Conventional wound covering materials are not suitable because of the increased risk of infection. The damaged area due to burns should be removed as soon as possible, and the formation of granulation tissue as a transplant bed should be observed, and skin transplantation should be performed.

[0077] However, the following problems often occur during transplantation: the indispensable wound bed preparation (good granulation) is not fully performed, so the transplanted skin is difficult to survive, inducing sepsis, etc., worsening the condition and leading to death.

[0078] Therefore, in the present embodiment, experiments were conducted on the formation of good granulation tissue using placenta-derived cells alone or in combination with HD-AM.

[0079] Furthermore, by placing HD-AM as a covering material and scaffold material in the wound of severe burns and scalds, the appearance of inflammatory effector cells including type I macrophages and a transient increase in inflammatory cytokines promotes infection defense and angiogenesis. Then, with the reduction of these and the appearance of type II macrophages, anti-inflammatory cytokines increase, thereby promoting positive effects such as high-quality granulation formation.

[0080] In this embodiment, when HD-AM is used as a covering material and a support material, for example, it can be formed into a suitable shape using scissors or the like according to the wound site such as burns or severe burns that cannot use conventional wound covering materials. In addition, drainage holes for discharging exudates from the body can be made on the covered HD-AM.

[0081] (Using animals to make burn models)

[0082] Figure 1 A to Figure 1 C and Figure 2 Shown are diagrams illustrating the use of experimental model mice and HD-AM.

[0083] The mice were placed in a supine position under anesthesia and their limbs were fixed. Then, the back skin was shaved and depilatory cream was used to remove hair. Then, a tube ( Figure 1 A) Expose the 10 mm area of ​​the back to hot water at 90 degrees for 10 seconds ( Figure 1 A), with a size of φ = 10 mm in the center of the back ( Figure 1 B) Formation of III degree burn wounds.

[0084] Figure 1 C shows an image of the third-degree burn experimental model. After being treated with hot water at 90 degrees for 10 seconds, the exposed skin immediately turned pale, and all layers of the skin necrotized and fell off on the 7th day. Based on this result, it is judged that this experimental model is appropriate as a third-degree burn model, and it is used as a third-degree burn model for studying the effects of burn treatment such as granulation formation.

[0085] It should be noted that the handling of experimental animals was carried out in accordance with the guidelines of the National Institutes of Health and was approved by the Animal Experiment Committee of the University of Toyama. In addition, the experiments were carried out in accordance with the guidelines of the Animal Experiment Committee of the University of Toyama.

[0086] This method can not only adjust the depth of burns and scalds by changing the temperature of the hot water and the exposure time of the hot water, but also change the injured area of ​​burns and scalds by changing the diameter of the tube used. Therefore, for burns and scalds that were previously made using expensive equipment, the required burn and scald models can be simply and stably made using cheap materials.

[0087] (Treatment of burns using HD-AM and / or placenta-derived cells)

[0088] Figure 2 This figure shows an explanatory diagram of using HD-AM and / or placenta-derived cells in the treatment of burns and scalds. In the following description, an example of using amniotic mesenchymal stem cells as placenta-derived cells is described.

[0089] In the experiment, the following four groups were prepared: a group using cells derived from the placenta (cell group), a group using HD-AM (HD-AM group), a group using HD-AM and cells derived from the placenta (HD-AM / cell group), and a group that did not use any of the above as a control group (control group).

[0090] A total of 72 mice were used in order to evaluate the results on postoperative day 1 (POD 1), postoperative day 4 (POD 4), and postoperative day 7 (POD 7) using 6 mice per group.

[0091] In the control group, the burn wound (Wall of exposed bowel) of the mouse model was covered with a polyurethane foam covering material (Tegaderm (registered trademark) Diamond transparent film (registered trademark), 3M Deutschand GmbH Health Care Business, Germany) and then wrapped with a stainless steel mesh (0.06 mmφ, 150 mesh).

[0092] In the cell group, 50 μL of a solution containing 300,000 placenta-derived cells was dripped onto the burn wound (Wall of exposed bowel) of the mouse model, and then covered with a polyurethane foam covering material (Tegaderm (registered trademark) Diamond transparent film (registered trademark), 3M Deutschand GmbH Health Care Business, Germany), and then wrapped with a stainless steel mesh (0.06 mmφ, 150 mesh).

[0093] In the HD-AM group, HD-AM was placed on the burn wound (Wall of exposed bowel) of the mouse model, covered with a polyurethane foam covering material (Tegaderm (registered trademark) Diamond transparent film (registered trademark), 3M Deutschand GmbH Health Care Business, Germany), and then wrapped with a stainless steel mesh (0.06 mmφ, 150 mesh).

[0094] In the HD-AM / cell group, 50 μL of a solution containing 300,000 placenta-derived cells was dripped onto the burn wound (Wall of exposed bowel) of the mouse model, and HD-AM was placed on it, which was then covered with a polyurethane foam covering material (Tegaderm (registered trademark) Diamond transparent film (registered trademark), 3M Deutschand GmbH Health Care Business, Germany), and then wrapped with a stainless steel mesh (0.06 mmφ, 150 mesh).

[0095] It should be noted that the solvent containing the placenta-derived cells can be physiological saline or phosphate-buffered saline (PBS).

[0096] Furthermore, in order to prevent placenta-derived cells from being lost from the burn wound, placenta-derived cells can be contained in a cell culture medium or a jelly-like medium. A jelly-like medium for cell culture can be, for example, agar. Jelly-like refers to a highly viscous fluid that has not lost fluidity. Viscosity can be imparted to saline or phosphate-buffered saline (PBS).

[0097] It should be noted that the stainless steel mesh is used to prevent the HD-AM, wound covering materials, etc. from being detached due to the movement of the mouse.

[0098] (Histological staining and immunohistochemical staining)

[0099] For histological observation, the burn wounds were collected from each mouse on POD 1, POD 4, and POD 7 and embedded in paraffin. Thin sections cut from paraffin blocks were stained with hematoxylin-eosin (H&E) and AZAN staining. In addition, immunohistochemical staining for CD31, αSMA, IL-6, CD163, and IL-10 was performed. The stained tissues were photographed using a Leica (registered trademark) DMRBE microscope (Leica, Wetzlar, Germany) and a DP73 system (Olympus, Tokyo, Japan).

[0100] (Measurement of granulation tissue (regeneration area) at the treated area of ​​burns and scalds)

[0101] Figure 3 A and Figure 3 B is a schematic diagram showing a method for measuring the thickness of granulation tissue. Figure 3 A is a diagram obtained by observing the tissue after being stained by the AZAN staining method for selectively staining connective tissue after burns and scalds occurred over time. The tissue immediately below the HD-AM is set as tissue 1, the upper layer of the collagen layer is set as tissue 2, and the lower layer of the collagen layer is set as tissue 3.

[0102] Figure 3 B is a schematic diagram showing the measurement site of the thickness of granulation tissue formed at the wound site by treatment after burns and scalds occur.

[0103] It should be noted that Figure 3 In A, the newly constructed granulation tissue in tissue 3 was measured in contrast to tissues 1, 2, and 3. Figure 3 The schematic diagram of B shows that tissue 3 is located on the upper layer of adipose tissue.

[0104] AZAN staining allows for easy visualization of newly constructed granulation tissue, distinguishable from collagen fibers and fibrin. The thickness of granulation tissue containing collagen fibers was measured using the Olympus (registered trademark) CellSens (registered trademark) imaging program (version 1.7; Olympus, Tokyo, Japan).

[0105] On the 4th day after treatment (POD4), a layer of collagen membrane was observed between the subcutaneous tissue and the muscular layer of the surrounding normal tissue, regardless of whether HD-AM was added. In order to observe the behavior of infiltrating cells and granulation formation using this membrane as a boundary, Figure 3 A determines each area.

[0106] like Figure 3 B. Observe the changes in these areas over time. The measurement sites are set as the epithelial defect stump a, the central part b of the epithelial defect stump, and the midpoint c between a and b. Measure the thickness of the granulation tissue formed on the tissues 3 at a, b, and c, and take the average value as the thickness of the granulation tissue.

[0107] (Quantitative reverse transcription polymerase chain reaction (qRT-PCR))

[0108] Figure 4 The sequences of primers used in quantitative reverse transcription polymerase chain reaction (qRT-PCR) are shown.

[0109] In order to extract mRNA from the granulation tissue of the wound caused by burns, the target site is selectively collected from each sample. During collection, the granulation tissue is cut in the same way for any subject so that they are as consistent as possible in anatomy to avoid being affected by differences in the collection site and range. Total mRNA was extracted from the tissue using Isogen II (Nippon Gene Co. LTD., Tokyo, Japan) according to the manufacturer's instructions. A portion of the mRNA, i.e. 3 μg, was treated with deoxyribonuclease I (DNase I, Sigma-Aldrich, Inc., Tokyo, Japan) at room temperature for 15 minutes. Using ReverTra Ace qPCR RT Kit (Toyobo Co., Ltd., Osaka, Japan), 500 ng of DNase I was used to treat RNA to synthesize cDNA. Gene expression was quantified by real-time RT-PCR analysis using the Mx3000P quantitative polymerase chain reaction (qPCR) system (Stratagene; Agilent Technologies Japan Ltd., Japan) and Brilliant SYBR Green QRT-PCR Mix (Stratagene; Agilent Technologies Japan Ltd., Japan).

[0110] On POD 1, POD 4, and POD 7, mRNA was extracted from 6 mice / group, and single measurements were performed on inducible nitric oxide synthase (iNOS) produced by type I macrophages, CD163 as an indicator of type II macrophages, inflammatory cytokines IL-6, IFN-γ, and COX-2 that induces PGE2 involved in angiogenesis.

[0111] In order to clarify that the changes in cytokines are postoperative changes, tissues were collected immediately after the six model mice were created, and the mRNA expression levels of each primer immediately after surgery were investigated as POD 0. The expression levels of each mRNA were corrected using GAPDH as an internal control, and relative comparisons were performed based on the average value of the control group on POD 7.

[0112] (Evaluation of the thickness of granulation tissue at the treatment site of burns and scalds)

[0113] Figure 5 A and Figure 5 B shows representative microscopic photographs of the wound during treatment and a comparative graph of the measured average thickness of granulation tissue.

[0114] Figure 5A is a representative microscopic photograph of the control group, cell group, HD-AM group, and HD-AM / cell group at 1, 4, and 7 days after treatment (POD1, POD4, and POD7). The thickness of the granulation tissue at the wound site of each group is indicated by the black arrow.

[0115] Figure 5 B is a graph comparing the average values ​​of the measured granulation tissue.

[0116] Granulation tissue was hardly observed in all groups at POD1.

[0117] On POD 4, no difference was found in the thickness of granulation tissue between the treatment groups.

[0118] On POD7, the granulation tissue of the HD-AM / cell group showed significantly thicker granulation tissue than that of the other groups (n=5, *p<0.05, **p<0.01).

[0119] (Vascularization within the formed granulation tissue)

[0120] Figure 6 A to Figure 6 C shows a photograph illustrating the vascularization within the granulation tissue.

[0121] Figure 6 A shows the angiogenesis status of each group except the control group on POD 4. In general, CD31-positive cells and α-SMA-positive cells were observed in the granulation tissue.

[0122] In the cell group and HD-AM / cell group, images were observed in which CD31-positive cells constituted the lumen of blood vessels and α-SMA-positive cells surrounded them.

[0123] In the HD-AM group, although CD31-positive cells were present, no vascular lumen was observed.

[0124] Figure 6 B shows the angiogenesis status of POD7 in each group except the control group.

[0125] In the HD-AM group, CD31-positive cells and α-SMA-positive cells were observed to aggregate and form blood vessels within the granulation tissue.

[0126] In the cell group and HD-AM / cell group, the number of these cells decreased and the images were observed to run in the form of complete blood vessels. In the cell group and HD-AM / cell group, blood vessel formation was observed in deeper layers than in the HD-AM group.

[0127] Figure 6C shows a comparison of blood vessel formation in granulation tissue. The left photograph shows an example of granulation tissue magnified with a 20x objective lens, and the inner circumference length of blood vessels formed by CD31-positive cells in one field of view was measured in each group of POD4 and POD7. The measurement results are shown in the graph on the right.

[0128] Observing the graph of POD4, at POD4, the blood vessel length of the control group was extremely short, close to 0, while the blood vessel length of the cell group, HD-AM group, and HD-AM / cell group increased significantly. The length of the cell group was about 1500μm, the length of the HD-AM group was about 1100μm, and the length of the HD-AM / cell group was about 2500μm.

[0129] On POD 7, the cell group had a significantly increased length of blood vessels, reaching about 5700 μm. In the cell group and HD-AM / cell group, the number of blood vessels was small, but blood vessels were observed to run over a large area (from the surface to the deep layer).

[0130] (Comparison of expression patterns of mRNA related to type I and type II macrophage markers and mRNA expression patterns of inflammatory cytokines and anti-inflammatory cytokines during the healing process)

[0131] Figure 7 A to Figure 7 D shows the mRNA expression of cell markers and cytokines related to inflammation.

[0132] In the cell group, HD-AM group, and HD-AM / cell group at POD4, the expression level of iNOS (type I macrophages) tended to increase, and a trend of HD-AM / cell group > cell group > HD-AM group was observed. At POD7, a trend of decreased expression of iNOS (type I macrophages) was observed in all groups.

[0133] Regarding the expression of COX-2 (PGE2), all groups tended to increase their expression at POD4. At POD7, except for the control group, their expression tended to decrease. Thus, COX2, which is present upstream in the generation of PGE2 that causes angiogenesis, tended to increase at POD4 and decrease in all groups at POD7, which supports the results of immunohistochemistry (CD31 and αSMA).

[0134] Regarding the expression level of the inflammatory cytokine IFN-γ, the expression levels in the cell group and the HD-AM / cell group tended to be higher than those in the control group and the HD-AM group on POD 4. On POD 7, the expression levels in all groups tended to decrease.

[0135] Figure 7B shows the expression of inflammatory cytokine IL-6 mRNA and the distribution of IL-6.

[0136] Regarding the expression level of IL-6 mRNA, in all groups, there was a tendency that the expression increased on POD4 and decreased on POD7 compared to POD1.

[0137] The results of immunohistochemical staining (POD4, cell group) showed that IL-6 positive cells were diffusely present in the granulation tissue.

[0138] Figure 7 C shows the expression pattern of type II macrophage marker-related mRNA (CD163) and the distribution of CD163-positive cells.

[0139] Regarding the expression level of CD163 (Type II Mφ), it was not expressed much on POD 1, but tended to increase in all groups on POD 4. On POD 7, the expression tended to increase in the HD-AM group.

[0140] In addition, the granulation tissue stained by immunohistochemistry is shown on the right (POD7, HD-AM / cell group), and CD163-positive cells were clearly observed in the newly formed granulation tissue on POD7.

[0141] Figure 7 D shows the expression of inflammatory cytokine IL-10 mRNA and the distribution of IL-10.

[0142] Regarding the expression level of IL-10 mRNA, on POD7, the expression level in the HD-AM / cell group was significantly higher than that in the control group and the HD-AM group.

[0143] The above experimental results indicate that, for severe burns, namely, third-degree burns, the use of placenta-derived cells alone or in combination with HD-AM promotes the formation of granulation tissue rich in blood vessels in the subcutaneous tissue.

[0144] (Manufacture of HD-AM)

[0145] use Figure 8 The drying device shown dries fresh amniotic membrane under the conditions of the following vacuum, far infrared ray and microwave irradiation devices to produce dried amniotic membrane (HD-AM).

[0146] The fresh amniotic membrane placed on the rotating table 12 in the processing tank 10 is continuously heated by the far-infrared heater 14 provided in the processing tank 10, and is irradiated with microwaves from the microwave irradiation device 30 provided in the processing tank during the decompression operation of bringing the processing tank 10 into a decompressed state and the re-pressurization operation of slightly raising the decompressed processing tank 10 toward the atmospheric pressure side, thereby applying energy to the water molecules in the amniotic membrane while drying it. This process is repeated several times to obtain the dried amniotic membrane while maintaining the cell / tissue structure, which has not only improved preservability but also easy handling.

[0147] · Drying tank heating: 50℃, FIR: 50℃, closing valve: 37%, maximum pressure 0.34kPa, maximum pressure 0.33kPa when running

[0148] Drying method

[0149] (1) Decompression 180 seconds

[0150] (2) Repressurization for 30 seconds (close valve opening 37%), microwave irradiation 0.1kw-180 seconds (continuous repressurization)

[0151] (3) Decompression 180 seconds

[0152] (4) Then repeat (2) and (3)

[0153] (5) Confirm that the pressure reaches 0.30 to 0.35 kPa after the 180-second decompression in (3), and manually complete the drying.

[0154] Return to atmospheric pressure and end.

[0155] Industrial Applicability

[0156] Cells derived from the placenta of animals including humans are used as regenerative medical materials, especially in the treatment of severe burns and scalds. In addition, by using dried amniotic membrane as a medical device in combination with cells derived from the placenta, it can be used to cover the dermal defect or subcutaneous tissue of patients with severe burns and scalds with cells derived from the placenta, and to perform tissue regeneration of skin transplantation (with good granulation formation accompanied by angiogenesis), thereby achieving the necessary skin transplantation for third-degree burns and scalds as soon as possible. Furthermore, it is usually used for the purpose of preventing infection of the burn wound, which can improve the patient's survival rate and treatment effect (no need to worry about keloids).

Claims

1. A cell for treating burns and scalds, characterized in that: It is a cell derived from the placenta of animals including humans, which is used in the treatment of burns and scalds and promotes healing.

2. The cell for treating burns and scalds according to claim 1, characterized in that: It contains amniotic mesenchymal stem cells.

3. The cell for treating burns and scalds according to claim 1, characterized in that: In the treatment of burns and scalds, it is used for tissue regeneration of burns and scalds and promotes granulation formation.

4. The cell for treating burns and scalds according to claim 1, characterized in that: These cells have antibacterial and anti-inflammatory effects and are useful for tissue regeneration in burns and scalds.

5. The cell for treating burns and scalds according to claim 1, characterized in that: It is contained in a solvent in order to be added dropwise to the burn wound.

6. The cell for treating burns and scalds according to claim 5, characterized in that: The solvent has high viscosity.

7. The cell for treating burns and scalds according to claim 1, wherein: Burns and scalds are II and / or III degree burns and scalds.

8. The cell for treating burns and scalds according to claim 1, characterized in that: These cells can be used together with a covering material for burns and scalds and / or a regenerative scaffold material for burns and scalds, or can be used alone.

9. The cell for treating burns and scalds according to claim 8, characterized in that: The covering material for burns and scalds and / or the regenerative scaffold material for burns and scalds is a dried amniotic membrane obtained by drying the fresh amniotic membrane that wraps the fetus of an animal including a human. The dried amniotic membrane is dried so that it can be stored in the atmosphere, and the epithelial cells, basement membrane and connective tissue that constitute the fresh amniotic membrane are maintained when it is immersed in water or a buffer solution and rehydrated.

10. A method for treating burns and scalds, characterized in that: Healing is promoted by placing cells derived from the placenta of animals including humans at burn wounds.

11. The method for treating burns and scalds according to claim 10, characterized in that: The placenta-derived cells include amniotic mesenchymal stem cells.

12. The method for treating burns and scalds according to claim 10, characterized in that: The placenta-derived cells are mixed in a solvent and added dropwise to the burn wound.

13. The method for treating burns and scalds according to claim 12, characterized in that: More than 100 placenta-derived cells are contained in 50 μL of the solvent.

14. The method for treating burns and scalds according to claim 13, characterized in that: 300,000 of the placenta-derived cells were contained in 50 μL of solvent.

15. The method for treating burns and scalds according to claim 12, characterized in that: The solvent has high viscosity.

16. The method for treating burns and scalds according to claim 10, characterized in that: Cells derived from the placenta of animals including humans are placed on a burn or scald wound, and a dried amniotic membrane is placed thereon. The dried amniotic membrane is obtained by drying fresh amniotic membrane that wraps a fetus of an animal including humans. The dried amniotic membrane is dried so that it can be stored in the atmosphere, and when it is rehydrated by being immersed in water or a buffer solution, the epithelial cells, basement membrane and connective tissue that constitute the fresh amniotic membrane are maintained.

17. The method for treating burns and scalds according to claim 10, wherein: Burns and scalds are II and / or III degree burns and scalds.

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