Combination of amniotic mesenchymal stem cells and dried amniotic membrane

Abstract

To provide effective promotion of granulation growth in dermal or subcutaneous tissue defects of severe burns.SOLUTION: Provided are cells derived from the placenta of animals, including humans, the cells being used to treat burns and promote healing.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to repairing the periphery of the dermis tissue (including subcutaneous tissue) damaged in the treatment of burns. Combination of amniotic mesenchymal stem cells and dried amniotic membrane It relates thereto.

Background Art

[0002] The severity of a burn is determined by the area and depth. The depth is classified into degrees I to III from the surface color tone. A second-degree burn is a burn that reaches the dermis, and a third-degree burn is a state in which all the skin is damaged. A burn with a second-degree depth of 30% or more of the body surface area or a third-degree depth of 10% or more of the body surface area is severe and requires intensive treatment at an emergency center. In the field of emergency surgery, third-degree burns require surgical treatment because not only the skin but also the underlying tissues, nerves, and blood vessels are damaged. In addition, since the risk of infection and the like increases, general wound dressings are not applicable. The damaged area due to the burn is removed as soon as possible, and skin grafting is performed after observing the formation of granulation as a graft bed. However, since the wound bed preparation (good granulation formation) essential at the time of transplantation is not sufficiently performed, the transplanted skin is difficult to engraft, often causing problems such as deterioration of the disease condition and death due to inducing sepsis.

[0003] According to Non-Patent Document 1 ("Burn Treatment Guidelines [Revised 2nd Edition], 2015" published by the Japan Burn Society), reports on wound dressings are for second-degree burns, and there is no positive evidence for using a wound dressing for third-degree burn wounds. The wound dressings used for burns are roughly classified into foam materials, fiber materials, and colloid materials, and are used appropriately according to the shape and absorbency of exudate. All are aimed at good granulation formation.

[0004] Amnion is a tough biological membrane composed of collagen and elastic fibers, and fresh amnion has been reported as a useful covering material for trauma and burns (Non-Patent Document 2). However, its use in actual clinical practice has been limited due to its unavailability when needed and its complicated storage and handling. In response to this, dried amniotic membrane produced by a specific drying process (Hyperdry Human Dried Amniotic Membrane: hereinafter HD-AM) has been reported (Patent Document 1, Non-Patent Document 3). There are also reports on granulation tissue formation using HD-AM (Patent Document 2). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2007-54015 [Patent Document 2] Japanese Patent Publication No. 2021-020868 [Patent Document 3] International Publication No. 2013 / 077428 [Non-patent literature]

[0006] [Non-Patent Document 1] Burn Treatment Guidelines, Revised 2nd Edition, 2015, The Japanese Society for Burn Injuries [Non-Patent Document 2] Gruss, JS & Jirsch, DW Human amniotic membrane: A versatile wound dressing. Can. Med. Assoc. J. 118, 1237-1246 (1978). [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). [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] As mentioned above, there are currently no treatment methods for third-degree burns that prevent infection or promote healthy granulation tissue formation, and treatment must rely on the patient's vitality and immune system. While it is necessary to promote granulation tissue formation early in the treatment of third-degree burns, there is currently no effective treatment. Therefore, early formation of healthy granulation tissue and early skin grafting without infection are effective in prolonging the life of severely burned patients.

[0008] The inventors created burn model animals with burn depths of second to third degree, and histologically, immunochemically, and molecularly confirmed that placenta-derived cells can effectively promote granulation tissue formation in dermal or subcutaneous tissue defects in these burn model animals, which led to the invention of this invention. [Means for solving the problem]

[0009] According to the present invention The combination of amniotic mesenchymal stem cells and dried amniotic membrane, for use in the treatment of burns, comprises amniotic mesenchymal stem cells, 300,000 of which are dropped onto the burn wound site in a quantity of 50 μL of solvent per drop, and dried amniotic membrane placed on top of the dropped amniotic mesenchymal stem cells, wherein the dried amniotic membrane is dried so as to be stored in the atmosphere, and retains the epithelial cells, basement membrane, and connective tissue that constitute the living amniotic membrane when rehydrated by immersion in water or buffer solution. By adopting this configuration, especially when used as cells for regenerating dermal defects or subcutaneous tissue in severe burn patients, it promotes the secretion of physiologically active substances such as inflammatory cytokines and growth factors at the wound site of severe burns. These inflammatory cytokines protect against infection by foreign substances from the outside, and through active effects such as promoting the secretion of IL-6, IFN-γ, IL-10, and COX2 (PGE2), it promotes wound healing in severe burns. Furthermore, it enables safe regenerative medicine with fewer ethical issues and fewer immune rejection reactions from allogeneic transplantation. Furthermore, this configuration possesses high differentiation and proliferative capabilities, promoting wound healing in severe burns. Furthermore, this configuration allows the dried amniotic membrane to function as a cell scaffold, induce growth factors and chemokines for cell migration, and promote local anti-inflammatory effects and tissue regeneration by regulating differentiation into M2 macrophages. [Effects of the Invention]

[0026] According to the cells for burn treatment and the burn treatment method of the present invention, healing in severe burn wound sites can be promoted.

Brief Description of Drawings

[0027] [Figure 1] It is a diagram explaining the experimental mouse model and the application of HD-AM. (A) and (B) are diagrams showing the creation of burn sites in experimental mice. (C) is a diagram showing the validity as a third-degree burn experimental model. [Figure 2] It is a diagram explaining the application of HD-AM and placenta-derived cells (Control group: schematic diagram of a mouse not using HD-AM and placenta-derived cells, Cell group: schematic diagram of a mouse using placenta-derived cells, HD-AM group: schematic diagram of a mouse using HD-AM, HD-AM / Cell group: schematic diagram of a mouse using HD-AM and placenta-derived cells). [Figure 3] It is a schematic diagram of a method for measuring the thickness of granulation tissue. (A) is a diagram showing the tissue stained by the azan staining method that selectively stains connective tissue over time after burn injury. (B) is a schematic diagram showing a method for measuring the thickness of the formed granulation after treatment after burn injury. [Figure 4] It is a table showing the primers used in quantitative reverse transcription polymerase chain reaction (q RT-PCR). [Figure 5] It is a graph comparing microscopic photographs of time-dependent granulation formation and the average thickness of granulation tissue in the treatment after injury. (A) Microscopic photographs of the Control group, Cell group, HD-AM group, and HD-AM / Cell group on the 1st, 4th, and 7th days (POD1, POD4, POD7) after treatment. The thickness of the granulation tissue is indicated by black arrows. (B) is a graph comparing the average values of the thickness of the granulation tissue. [Figure 6] It is a diagram explaining angiogenesis in granulation tissue. (A) is a photograph showing the state of angiogenesis on POD4. (B) is a photograph showing the state of angiogenesis on POD7. (C) is a graph comparing the measurement sites and the average of the measured values of the blood vessels formed in the granulation tissue. <00s0089> [Figure 7]Graph comparing inflammation and anti-inflammatory cytokines using quantitative RT-PCR. (A) Graph showing the mRNA expression of cytokines involved in inflammation. (B) Graph and photograph showing the mRNA expression and distribution of the inflammatory cytokine IL-6. (C) Graph and photograph showing the mRNA expression and distribution of the anti-inflammatory Type II macrophage marker (CD163). (D) Graph and photograph showing the mRNA expression and distribution of the inflammatory cytokine IL-10. [Figure 8] Figure showing a drying device for creating dried amnion (HD-AM).

Mode for Carrying Out the Invention

[0028] (Placenta-derived cells) First, placenta-derived cells will be described. Placenta-derived cells are amniotic mesenchymal cells and amniotic mesenchymal stem cells in the following experiments, but are not limited thereto. The amnion is an extraembryonic tissue composed of epithelial cells derived from the ectoderm and mesenchymal cells derived from the mesoderm, and contains a cell group having characteristics as pluripotent stem cells. Since the amnion is discarded as excrement after childbirth, there are few ethical problems in using it as a biological material. In addition, the amnion has special immunological properties and low immunogenicity, so the immune rejection reaction due to allogeneic transplantation is relatively mild.

[0029] Placenta-derived cells are collected from the amnion of mammals including humans. The amnion is composed of epithelial cells and mesenchymal cells. To collect a cell population of mesenchymal cells from the amnion, epithelial cells can be removed from the amnion and a separation operation can be performed. The collection of the cell population of mesenchymal cells can be carried out, for example, according to the method described in JP-A-2003-231639. Note that human amnion can be collected by cesarean section from a pregnant woman who has obtained informed consent, for example.

[0030] The collected amniotic mesenchymal stem cell population contains cells with various proliferative abilities, lifespans, and properties. Therefore, when a mesenchymal cell population is maintained under general culture conditions, epithelial-like cells that adhere and begin to proliferate early in the culture process occupy most of the culture surface. As a result, amniotic mesenchymal stem cells that begin to proliferate later are unable to proliferate, are eliminated, and cannot be isolated. Therefore, a cell population of amniotic mesenchymal stem cells can be prepared from a cell population of collected amniotic mesenchymal cells, for example, by following the method described in International Publication No. 2013 / 077428. The amniotic mesenchymal stem cell population includes cells with a spindle-shaped morphology and possesses high proliferative capacity, preferably capable of 50 or more cell divisions (population doublings).

[0031] When using placenta-derived cells for burn treatment, for example, by containing 100 or more placenta-derived cells per 50 μL of solvent, preferably 300,000 placenta-derived cells per 50 μL of solvent, the placenta-derived cells can be placed on the burn site by dropping the solution containing the placenta-derived cells onto the burn wound. Furthermore, when applying a solution containing placental cells to a burn wound, it is advisable to apply a wound dressing such as an adhesive bandage over the solution to prevent it from flowing out. However, in the case of third-degree burns, general wound dressings may not be applicable, so dried amniotic membrane (HD-AM), described later, can be used as a dressing to prevent the solution containing placental cells from flowing out. By encapsulating the placental cells in a solvent with high viscosity without losing fluidity, the solution containing the placental cells can be prevented from flowing out of the burn wound. When encapsulating placental cells in a highly viscous solvent, the higher the viscosity, the fewer cells may be encapsulated per unit of solvent. Furthermore, when using placental-derived cells to treat burns, they may be used alone or in combination with dried amniotic membrane (HD-AM), as described later.

[0032] (dried amnion) Dried amniotic membrane produced by a specific drying process (hyperdrying) is, for example, the dried amniotic membrane described in Patent Document 1. Specifically, the raw amniotic membrane placed in a processing tank is continuously heated by an infrared heater installed in the processing tank, and during a depressurization operation to reduce the pressure inside the processing tank, and a restoration operation to slightly raise the pressure inside the depressurized processing tank to atmospheric pressure, microwaves are also irradiated onto the raw amniotic membrane from a microwave generator installed in the processing tank to add energy to the water molecules present in the amniotic membrane while drying. Dried amniotic membrane (HD-AM) produced by repeating this process multiple times has its amniotic membrane cells themselves inactivated, but its cellular and tissue structure is preserved. Specifically, the dried amniotic membrane (HD-AM) is dried so that it can be stored in the atmosphere, and the amniotic membrane rehydrated by immersion in water or buffer solution retains the epithelial cells, basement membrane, and connective tissue that constitute the raw amniotic membrane.

[0033] (Combination of placental-derived cells and dried amniotic membrane) Third-degree burns in emergency surgery require surgical treatment because they damage not only the skin but also the underlying tissues, nerves, and blood vessels. General wound dressings are unsuitable due to the increased risk of infection. The burned area should be removed as quickly as possible, and skin grafting should be performed after observing the formation of granulation tissue to serve as a graft site. However, because woundbed preparation (good granulation tissue formation), which is essential during transplantation, is not adequately performed, the transplanted skin has difficulty taking root, often leading to problems such as sepsis, worsening of the patient's condition, and death. Therefore, in this embodiment, experiments were conducted on the formation of good granulation tissue using placental-derived cells alone or in combination with HD-AM. Furthermore, by placing HD-AM as a dressing and scaffolding material on the wound of a severe burn, the appearance of inflammatory effect cells, including Type I macrophages, and a transient increase in inflammatory cytokines promote infection defense and angiogenesis. Subsequently, as these cells decrease and Type II macrophages appear, the levels of anti-inflammatory cytokines increase, thereby promoting active effects such as the formation of high-quality granulation tissue.

[0034] In this embodiment, when using HD-AM as a covering material and scaffolding material, for example, it is sufficient to mold it into an appropriate shape with scissors or the like to fit wound sites such as burns or severe burns where general wound dressings cannot be used. Furthermore, it is preferable to create drainage holes in the covered HD-AM to allow exudate and other fluids to be discharged from the body.

[0035] (Creating burn models in animals) Figures 1 and 2 illustrate the experimental model mouse and the application of HD-AM. The mice were anesthetized and placed in a prone position, and their limbs were fixed in place. Next, the skin on their backs was shaved and the hair was removed with depilatory cream. Then, using a tube (Figure 1A), a 10 mm area on the back was exposed to hot water at 90 degrees Celsius for 10 seconds, creating a third-degree burn wound in the center of the back (Figure 1B) with a diameter of 10 mm. Figure 1C shows that in the third-degree burn experimental model, after treatment with hot water at 90 degrees Celsius for 10 seconds, the exposed skin immediately became pale, and by the 7th day, the entire skin layer had necrotized and fallen off. Based on these results, this experimental model was judged to be valid as a third-degree burn model and was used as a third-degree burn model to investigate the effects of burn treatment, such as granulation tissue formation. Furthermore, the handling of experimental animals was carried out in accordance with the guidelines of the National Institutes of Health, with permission obtained from the Animal Experimentation Committee of Toyama University. The experiments were also conducted in accordance with the guidelines of the Animal Experimentation Committee of Toyama University.

[0036] This method allows for the adjustment of burn depth by varying the temperature of the hot water introduced and the exposure time, as well as the change in the burn area by changing the diameter of the tube used. This makes it possible to easily and reliably create burn models as needed using inexpensive materials, whereas previously expensive equipment was required.

[0037] (Burn treatment using HD-AM and / or placental-derived cells) Figure 2 shows an explanatory diagram of the use of HD-AM and / or placental-derived cells in burn treatment. The following explanation describes an example in which amniotic mesenchymal stem cells were used as placenta-derived cells. In the experiment, four groups were created: a group using placental-derived cells (Cell group), a group using HD-AM (HD-AM group), a group using HD-AM and placental-derived cells (HD-AM / Cell group), and a control group using neither (Control group). Each group consisted of 6 mice, and they were evaluated at 1 day post-surgery (POD 1), 4 days post-surgery (POD 4), and 7 days post-surgery (POD 7), for a total of 72 mice used.

[0038] In the control group, the burn wounds (Wall of exposed bowel) of the mouse models were covered with a polyurethane foam covering (Tegaderm® Diamond transparent film®, 3M Deutschiand GmbH Health Care Business, Germany), and then further covered with stainless steel mesh (0.06 mmφ, 150 mesh).

[0039] In the cell group, 50 μL of a solution containing 300,000 placenta-derived cells was dropped onto a burn wound (Wall of Exposed Bowel) in a mouse model. This was then covered with a polyurethane foam covering (Tegaderm® Diamond transparent film®, 3M Deutschiand GmbH Health Care Business, Germany), and further covered with a stainless steel mesh (0.06 mmφ, 150 mesh).

[0040] In the HD-AM group, HD-AM was placed on the burn wound (Wall of Exposed Bowel) of a mouse model, covered with a polyurethane foam covering (Tegaderm® Diamond transparent film®, 3M Deutschiand GmbH Health Care Business, Germany), and then covered with stainless steel mesh (0.06 mmφ, 150 mesh).

[0041] In the HD-AM / Cell group, 50 μL of a solution containing 300,000 placenta-derived cells was dropped onto a burn wound (Wall of Exposed Bowel) in a mouse model. HD-AM was then placed on top of the cells, covered with a polyurethane foam covering (Tegaderm® Diamond transparent film®, 3M Deutschiand GmbH Health Care Business, Germany), and further covered with a stainless steel mesh (0.06 mmφ, 150 mesh).

[0042] Furthermore, the solvent used to contain the placenta-derived cells can be physiological saline (saline) or phosphate-buffered saline (PBS). Furthermore, to prevent placental-derived cells from flowing out of the burn wound, it is preferable to contain the placental-derived cells in a cell culture medium or a gel-like medium. A gel-like medium for cell culture can be, for example, agar. A gel-like medium refers to a highly viscous fluid that has not lost its fluidity. Viscosity may be added to physiological saline (saline) or phosphate-buffered saline (PBS).

[0043] The stainless steel mesh is used to cover HD-AM, wound dressings, etc., to prevent them from shifting due to the mouse's movements.

[0044] (Histological and immunohistochemical staining) For histological observation, burn wounds were collected from mice in POD 1, POD 4, and POD 7 and embedded in paraffin. Sections obtained from the paraffin blocks were stained with hematoxylin-eosin (H&E) and Azan staining. Immunohistochemical staining for CD31, αSMA, IL-6, CD163, and IL-10 was also performed. The stained tissues were imaged using a Leica® DMRBE microscope (Leica, Wetzlar, Germany) and a DP73 system (Olympus, Tokyo, Japan).

[0045] (Measurement of granulation tissue (regenerated tissue) in burn treatment sites) Figure 3 shows a schematic diagram of a method for measuring the thickness of granulation tissue. Figure 3A shows the tissue stained with Azan staining, which selectively stains connective tissue, after burn injury, observed over time. The tissue directly beneath HD-AM is designated as tissue 1, the layer above the collagen layer as tissue 2, and the layer below the collagen layer as tissue 3. Figure 3B is a schematic diagram showing the site where the thickness of granulation tissue formed at the wound site is measured during post-burn treatment. In Figure 3A, tissues 1, 2, and 3 were distinguished, and the granulation tissue newly formed in tissue 3 was measured. The schematic diagram in Figure 3B shows that tissue 3 is located above the adipose tissue.

[0046] Azan staining distinguished collagen fibers from fibrin, facilitating the observation of newly formed granulation tissue. The thickness of the granulation tissue containing collagen fibers was measured using the Olympus® CellSens® imaging program (version 1.7; Olympus, Tokyo, Japan). On day 4 post-treatment (POD4), regardless of whether HD-AM was attached, a layer of collagen membrane was observed emerging from between the subcutaneous tissue and muscle layer of the surrounding normal tissue. Since the movement of infiltrating cells and granulation tissue formation were observed along this membrane, each region was determined as shown in Figure 3A. As shown in Figure 3B, changes in these regions were observed over time. Measurements were taken at three points: the epithelial defect stump (a), the central part of the epithelial defect stump (b), and the midpoint between a and b (c). The thickness of the granulation tissue formed in tissue 3 at these three points (a, b, and c) was measured, and the average value was defined as the thickness of the granulation tissue.

[0047] (Quantitative reverse transcription polymerase chain reaction (qRT-PCR)) Figure 4 shows the primer sequences used in quantitative reverse transcription polymerase chain reaction (qRT-PCR). To extract mRNA from granulation tissue in burn wounds, target sites were selectively collected from each sample. During collection, granulation tissue was dissected similarly from each sample, ensuring anatomical uniformity to minimize differences in collection site and extent. 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 this mRNA (3 μg) was treated with deoxyribonuclease I (DNase I, Sigma-Aldrich, Inc., Tokyo, Japan) for 15 minutes at room temperature. cDNA was synthesized using 500 ng of DNase I-treated RNA with a Rever Tra Ace qPCR RT Kit (Toyobo Co., Ltd., Osaka, Japan). Gene expression was quantified using the Mx3000P quantitative polymerase chain reaction (qPCR) system (Stratagene; Agilent Technologies Japan Ltd, Japan) and real-time RT-PCR analysis with Brilliant SYBR Green QRT-PCR Mix (Stratagene; Agilent Technologies Japan Ltd, Japan). mRNA was extracted from 6 mice / group in POD1, POD4, and POD7, and single-stage measurements were performed for inducible nitric oxide synthase (iNOS) produced by Type I macrophages, CD163 (an indicator of Type II macrophages), inflammatory cytokines IL-6 and IFN-γ, and COX-2, which induces PGE2 involved in angiogenesis. To clearly demonstrate that cytokine fluctuations were postoperative changes, tissue samples were collected immediately after creating six model mice, and mRNA expression levels of each primer were examined immediately after surgery (POD0). Each mRNA expression level was corrected for GAPDH as an internal control, and relative comparisons were made using the mean value of the Control group (POD7) as the baseline.

[0048] (Evaluating the thickness of granulation tissue at the burn treatment site) Figure 5 shows a graph comparing typical microscopic images taken during post-injury treatment with the average thickness of granulation tissue measured. Figure 5A shows representative micrographs of the Control, Cell, HD-AM, and HD-AM / Cell groups at 1, 4, and 7 days post-treatment (POD1, POD4, and POD7). The thickness of granulation tissue at the wound site in each group is indicated by the black arrows. Figure 5B is a graph comparing the average values ​​of the measured granulation tissue. In POD1, almost no granulation tissue was observed in any of the groups. In POD4, no difference in granulation tissue thickness was observed between the treatment groups. In POD7, the HD-AM / Cell group showed significantly thicker granulation tissue compared to the other groups (n=5, * p<0.05, ** p<0.01).

[0049] (Angiogenesis within formed granulation tissue) Figure 6 shows a photograph illustrating angiogenesis within granulation tissue. Figure 6A shows the state of angiogenesis in POD4 in each group other than the control group. Overall, CD31-positive cells and α-SMA-positive cells were observed in the granulation tissue. In the Cell group and HD-AM / Cell group, CD31-positive cells formed the vascular lumen, surrounded by α-SMA-positive cells. In the HD-AM group, CD31-positive cells were present, but vascular lumens were not observed.

[0050] Figure 6B shows the angiogenesis status of POD7 in each group other than the control group. In the HD-AM group, CD31-positive and α-SMA-positive cells aggregated, and vascular formation within the granulation tissue was observed. In the Cell group and HD-AM / Cell group, the number of these cells decreased, and images of complete blood vessels were observed. Deeper angiogenesis was observed in the Cell group and HD-AM / Cell group compared to the HD-AM group.

[0051] Figure 6C shows a comparison of angiogenesis within granulation tissue. The image on the left 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 present in a single field of view was measured in each group under POD4 and POD7. The results of the measurements are shown in the graph on the right. Looking at the POD4 graph, the length of blood vessels in the control group was extremely short, close to zero, but the length of blood vessels increased significantly in the Cell group, HD-AM group, and HD-AM / Cell group. The length was approximately 1500 μm in the Cell group, approximately 1100 μm in the HD-AM group, and approximately 2500 μm in the HD-AM / Cell group. In POD7, the Cell group showed a significant increase in vascular length, reaching approximately 5700 μm. In both the Cell group and the HD-AM / Cell group, although the number of blood vessels was small, vascular pathways were observed over a wide area (from the surface to the deep layers).

[0052] (Comparison of the expression patterns of Type I and Type II macrophage marker-related mRNAs and the expression patterns of inflammatory and anti-inflammatory cytokine mRNAs during the healing process) Figure 7 shows the expression of mRNA for markers and cytokines in cells involved in inflammation. iNOS (Type I macrophage) expression tended to increase in the cell, HD-AM, and HD-AM / cell groups in POD4, with a trend of HD-AM / Cell group > Cell group > HD-AM group. In POD7, iNOS (Type I macrophage) expression tended to decrease in all groups. COX-2 (PGE2) expression levels tended to increase in all groups during POD4. In POD7, expression levels tended to decrease in all groups except the control group. Thus, COX-2, which is upstream in the PGE2 production process that triggers angiogenesis, tended to increase in POD4 and decrease in all groups during POD7, supporting the results of immunohistochemistry (CD31 and αSMA). In POD4, the expression level of the inflammatory cytokine IFN-γ tended to be higher in the cell group and HD-AM / cell group compared to the control group and HD-AM group. In POD7, expression tended to be lower in all groups.

[0053] Figure 7B shows the mRNA expression and distribution of the inflammatory cytokine IL-6. IL-6 mRNA expression levels tended to increase in POD4 compared to POD1 in all groups, and decrease in POD7. Immunohistochemical staining results (POD4, cell group) showed that IL-6-positive cells were diffusely present within the granulation tissue.

[0054] Figure 7C shows the expression pattern of Type II macrophage marker-related mRNA (CD163) and the distribution of CD163-positive cells. CD163 (Type II Mφ) expression was almost nonexistent in POD1, but tended to increase in all groups in POD4. In POD7, expression tended to increase in the HD-AM group. The image on the right shows immunohistochemically stained granulation tissue (POD7, HD-AM / cell group), which reveals that CD163-positive cells were prominently observed in the newly formed granulation tissue of POD7.

[0055] Figure 7D shows the mRNA expression and distribution of the inflammatory cytokine IL-10. In POD7, IL-10 mRNA expression levels were significantly higher in the HD-AM / cell group compared to the control group and the HD-AM group.

[0056] The experimental results described above demonstrate that, in the case of severe third-degree burns, the use of placental-derived cells alone or in combination with HD-AM promotes the formation of vascularized granulation tissue in the subcutaneous tissue.

[0057] (Manufacturing of HD-AM) Using the drying apparatus shown in Figure 8, dried amniotic membrane (HD-AM) was produced by drying the raw amniotic membrane under the following conditions: vacuum, far-infrared radiation, and microwave irradiation. The raw amniotic membrane, placed on a rotating table 12 in the processing tank 10, is continuously heated by a far-infrared heater 14 installed in the processing tank 10. During the depressurization operation, which reduces the pressure inside the processing tank 10, and the restoration operation, which slightly increases the pressure inside the depressurized processing tank 10 to atmospheric pressure, microwaves are also irradiated onto the raw amniotic membrane from a microwave irradiation device 30 installed in the processing tank to add energy to the water molecules present in the amniotic membrane and dry it. By repeating this process multiple times, the amniotic membrane is dried while preserving its cell and tissue structure, resulting in improved shelf life and easier handling.

[0058] Drying chamber heating: 50°C, FIR: 50°C, Stop valve: 37%, Maximum achievable pressure: 0.34kPa, Maximum achievable pressure during dry operation: 0.33kPa • Drying method (1) Depressurize for 180 seconds (2) Restoration pressure 30 sec (stop valve opening 37%) Microwave input 0.1kW-180sec (pressure restoration continues) (3) Depressurize for 180 seconds (4) Repeat (2) and (3) thereafter. (5) To complete the drying process, check the pressure reached after 180 seconds of depressurization as in (3) (0.30~0.35 kPa) and perform the process manually. The process is completed after restoring pressure to atmospheric pressure. [Industrial applicability]

[0059] Placental cells derived from animals, including humans, are used as regenerative medicine materials, particularly in the treatment of severe burns. Furthermore, by using dried amniotic membrane as a medical device in combination with placental cells, it is possible to cover dermal defects or subcutaneous tissue in severely burned patients to which placental cells have been placed. It can also be used for tissue regeneration for skin grafting (good granulation tissue formation accompanied by angiogenesis), enabling early skin grafting, which is essential for third-degree burns and other severe burns. In addition, by using it to prevent infection of burn wounds, it can improve patient survival rates and treatment effectiveness (eliminating concerns about siloid formation). [Explanation of Symbols]

[0060] 10 Processing tanks 12 Rotating Table 14 Far-infrared heater 16 motors 18 Vacuum pump 20 Solenoid valves 22 Pressure Reducing Piping 24 filters 28. Pressure restoration piping 30 Microwave irradiation device

Claims

[Claim 1] A combination of amniotic mesenchymal stem cells and dried amniotic membrane for use in the treatment of burns, The amniotic mesenchymal stem cells, 300,000 of which are dropped onto the burn wound site in 50 μL of solvent per drop, The dried amniotic membrane is placed on top of the amniotic mesenchymal stem cells that have been dropped onto it, The combination of amniotic mesenchymal stem cells and dried amniotic membrane is characterized in that the dried amniotic membrane is dried so that it can be stored in the atmosphere, and when rehydrated by immersion in water or a buffer solution, the epithelial cells, basement membrane, and connective tissue that constitute the living amniotic membrane are retained.

Images