Composition for wound healing or tissue regeneration comprising human placenta-derived genomic DNA fragment and placenta growth factor, and preparation method therefor

Abstract

Provided according to one aspect of the present disclosure is a composition for wound healing or tissue regeneration that comprises a genomic DNA fragment extracted from human placenta-derived mesenchymal stem cells and a placental growth factor and which exhibits excellent tissue regeneration ability. Provided according to another aspect of the present disclosure is a preparation method which enables the efficient, biocompatible, economical, and environmentally friendly production of a composition for wound healing or tissue regeneration having excellent tissue regeneration ability.

Description

Composition for wound treatment or tissue regeneration comprising human placental-derived genomic DNA fragment and placental growth factor, and method for preparing the same

[0001] The present disclosure relates to a composition for wound treatment or tissue regeneration comprising a human placental-derived genomic DNA fragment and a placental growth factor, and a method for preparing the same.

[0002] The skin plays a vital role in protecting the body from external stimuli. A wound is a wound in which the epidermis and dermis, which make up the skin, are damaged by external pressure, such as a cut or tear. Depending on the cause, wounds can be categorized as cuts after cancer surgery, burns, abrasions, lacerations, bedsores, and contusions. If left untreated, these wounds can develop into chronic wounds that do not heal well with standard treatments. This is especially true for diabetic patients, who may experience chronic wounds because they may not heal over time, potentially leading to more serious complications.

[0003] Wound healing is a complex process that involves a complex combination of inflammatory responses, neotissue formation, and re-epithelialization to repair a wound. Wound healing is a highly complex process that requires the simultaneous differentiation and regeneration of various cell types, growth factors, and extracellular matrix, as well as the appropriate coordination of efficient cell proliferation, angiogenesis, skin restoration, and tissue remodeling.

[0004] Various wound healing methods have been known, including suturing, sealants, anti-infective agents, cleansing, debridement, and dressings. Recently, treatments utilizing biomaterials that aid tissue regeneration have emerged. Biomaterials are materials that can be used in vivo, such as artificial organs, drug delivery systems, and tissue engineering scaffolds, and can maintain their function semi-permanently without rejection.

[0005] Polydeoxyribonucleotide (PDRN), an example of a biomaterial used for the treatment of skin damage such as wounds or chronic wounds, is a substance containing a deoxyribonucleotide polymer extracted and processed from the DNA of various plants and animals. PDRN can exert various biological effects such as innate immune regulation, vascular pathology, and hematopoiesis through the A2 purinergic receptor, and can have regenerative effects on joints, musculoskeletal systems, and skin. Furthermore, salmon-derived PDRN is similar to human DNA, and is being used with interest in many hospitals, including orthopedics, pain medicine, and plastic surgery. Recently, it has emerged as a new treatment alternative for degenerative or intractable diseases by utilizing its anti-inflammatory action, growth factor stimulation, and microangiogenesis effects.

[0006] However, since most PDRN is extracted from salmon testes, availability is limited and most are imported. This makes mass production and high-purity purification difficult due to raw material supply issues, and prices are high. Furthermore, the properties and quality of PDRN can vary depending on the salmon species, habitat, and season, making consistent product production difficult. Furthermore, inconsistent biological activity and skin efficacy make it difficult to clearly guarantee product efficacy. Therefore, the development of alternative PDRN biomaterials to address these issues is urgently needed.

[0007] [Prior Art Literature]

[0008] [Patent Document]

[0009] Korean Patent No. 10-0900309 (registered on May 25, 2009)

[0010] According to one aspect of the present disclosure, an economical and effective composition for wound treatment or tissue regeneration can be provided through an extraction and manufacturing process of PDRN that is human-compatible, high-purity, and high-efficiency compared to existing PDRN.

[0011] According to another aspect of the present disclosure, a method for producing a composition for wound treatment or tissue regeneration that is safe and has improved manufacturing efficiency can be provided.

[0012] According to one aspect of the present disclosure, a composition for wound treatment or tissue regeneration is provided, which comprises a genomic DNA fragment extracted from human placenta-derived mesenchymal stem cells isolated and cultured in a GMP for clinical treatment and placental growth factor (PLGF) as active ingredients.

[0013] According to an exemplary aspect of the present disclosure, the size of the genomic DNA fragment may be 100 to 1,000 bp.

[0014] According to an exemplary aspect of the present disclosure, the molecular weight of the genomic DNA fragment may be from 50 to 600 kDa.

[0015] According to an exemplary aspect of the present disclosure, the genomic DNA fragment may be included at a concentration of less than 100 ng / mL.

[0016] According to an exemplary aspect of the present disclosure, the concentration of placental growth factor may be comprised between 0.01 and 0.5 pg / mL.

[0017] According to an exemplary aspect of the present disclosure, the genomic DNA fragment may exhibit an efficacy concentration of 1 to 10 pg per unit area of ​​a wound.

[0018] According to an exemplary aspect of the present disclosure, the wound may include a wound, a burn wound, an abrasion, a laceration, a stab wound, an ulcer, a decubitus, a contusion, hidradenitis suppurativa, inflammatory fibrosis, periodontitis, or a combination thereof, or a wound associated with type 1 or type 2 diabetes.

[0019] According to an exemplary aspect of the present disclosure, the tissue may comprise skin, epidermis, dermis, neovascularization, or skin appendages.

[0020] According to another aspect of the present disclosure, a pharmaceutical composition is provided comprising a genomic DNA fragment extracted from human placenta-derived mesenchymal stem cells and placental growth factor (PLGF) as active ingredients.

[0021] According to another aspect of the present disclosure, a skin regeneration management auxiliary formulation for improving skin elasticity, improving fine wrinkles, whitening skin, improving acne scars, reducing pores or improving skin tone is provided, comprising a genomic DNA fragment extracted from human placenta-derived mesenchymal stem cells and placental growth factor (PLGF) as active ingredients.

[0022] According to another aspect of the present disclosure, a medical device for wound treatment or tissue regeneration is provided, comprising a genomic DNA fragment extracted from human placenta-derived mesenchymal stem cells and placental growth factor (PLGF) as active ingredients.

[0023] According to another aspect of the present disclosure,

[0024] (a) a step of pulverizing human placental-derived mesenchymal stem cells using a dissolution buffer;

[0025] (b) a step of adding isopropanol to the placental-derived mesenchymal stem cell pulverization solution obtained in step (a);

[0026] (c) a step of centrifuging the dissolution buffer to which isopropanol is added in step (b) to separate the supernatant and the precipitate;

[0027] (d) a step of dissolving the precipitate of step (c) with nuclease free water; and

[0028] (e) a step of producing a molecularly reduced genomic DNA fragment by sonicating the dissolved solution in step (d);

[0029] A method for producing a genomic DNA fragment extracted from human placental-derived mesenchymal stem cells is provided.

[0030] According to another exemplary aspect of the present disclosure,

[0031] Between steps (c) and (d), a step of washing the precipitate of step (c) with ethanol to remove impurities may be further included.

[0032] According to another exemplary aspect of the present disclosure,

[0033] (e) Molecular reduction in step

[0034] It can be performed for 20 to 40 minutes under conditions of 50 to 70% amplitude, pulse / rest conditions of 10 to 20 seconds, power of 600 to 800 W, and frequency of 10 to 30 Hz.

[0035] According to another exemplary aspect of the present disclosure, the size of the genomic DNA fragment may be 100 to 1,000 bp.

[0036] According to another exemplary aspect of the present disclosure, the molecular weight of the genomic DNA fragment may be from 50 to 600 kDa.

[0037] According to another exemplary aspect of the present disclosure, the genomic DNA fragment may be included at a concentration of less than 100 ng / mL.

[0038] According to another exemplary aspect of the present disclosure, the concentration of placental growth factor may be comprised between 0.01 and 0.5 pg / mL.

[0039] According to one aspect of the present disclosure, a genomic DNA fragment extracted from human placental-derived mesenchymal stem cells can provide a composition for wound treatment or tissue regeneration that has high biocompatibility, high purity purification and mass production, consistent quality and efficacy, and excellent regenerative effects that allow faster wound recovery.

[0040] According to another aspect of the present disclosure, the manufacturing process is simple, so that time and cost are reduced, which is economical, safe because no toxic substances are used during manufacturing, and environmentally friendly because environmental pollution caused by waste is reduced, making it possible to manufacture a composition for wound treatment or tissue regeneration.

[0041] According to another aspect of the present disclosure, it is possible to manufacture a composition for wound treatment or tissue regeneration with minimal genetic alteration, excellent human compatibility, and no reduction in wound treatment or tissue regeneration effect.

[0042] Figure 1 shows the electrophoresis on an agarose gel after segmenting human placental chorionic valve-derived mesenchymal stem cell genomic DNA using the method of Manufacturing Example 2 (indirect ultrasonic disperser).

[0043] Figure 2 shows the electrophoresis on an agarose gel after fragmenting the human placental chorionic valve-derived mesenchymal stem cell genomic DNA using the method of Comparative Example 1 (ultrasonic grinder).

[0044] Figure 3 shows the result of electrophoresis on an agarose gel after fragmenting human placental chorionic valve-derived mesenchymal stem cell genomic DNA using the method of Comparative Example 2 (ultrasonic grinder).

[0045] Figure 4 shows the electrophoresis on an agarose gel after segmenting the human placental chorionic valve-derived mesenchymal stem cell genomic DNA using the method of Comparative Example 3 (enzyme).

[0046] Figure 5 shows the electrophoresis on an agarose gel after segmenting human placental chorionic valve-derived mesenchymal stem cell genomic DNA using the method of Comparative Example 4 (probe-type ultrasonic segmenter).

[0047] Figure 6 shows the results of a cytotoxicity test using an MTT assay after treating a human placental chorionic valve-derived mesenchymal stem cell genomic DNA fragment to a human keratinocyte cell line, HaCaT, and culturing for 24 hours.

[0048] Figure 7 shows the results of an in vitro wound healing assay of a human placental chorionic valve-derived mesenchymal stem cell genomic DNA fragment.

[0049] Figure 8 shows the results of a wound healing analysis using a human placental chorionic valve-derived mesenchymal stem cell genomic DNA fragment in an in vivo animal wound model.

[0050] Figure 9 shows the results of comparing the degree of wound healing by the difference in the area of ​​granulation tissue (GT) after H&E staining of tissue sections in an animal wound model.

[0051] Figure 10 shows the results of comparing the degree of wound healing by the difference in the area of ​​granulation tissue (GT) after Masson's Trichrome staining of tissue sections in an animal wound model.

[0052] Figure 11 shows the results of comparing the degree of wound healing by the difference in the degree of regeneration progression after staining with PCNA antibody in an animal wound model.

[0053] Hereinafter, the present disclosure will be described in detail. The following description should be understood as describing the present disclosure with specific examples, and the technical concepts of the present disclosure are not limited to the following description. The attached drawings are provided to aid understanding of the present disclosure, but the technical concepts of the present disclosure are not limited to the attached drawings. Furthermore, the thickness and size of each component in the drawings may be exaggerated, omitted, or schematically depicted for convenience of explanation.

[0054] In the description of the structure of the present disclosure described in this specification, positional relationships and directions are based on the drawings attached to this specification, unless otherwise specifically stated.

[0055] In the description of the structure of the present disclosure described herein, descriptions of space or positional relationships refer to the relative positions between components forming the present disclosure. Furthermore, unless otherwise specified, another component may exist in the space between one component and another. For example, when the present disclosure refers to another component being located "above" or "on top of" one component, this includes not only cases where another component is located directly above the one component, but also cases where another component is located between the one component and the other components.

[0056] In this specification, singular expressions may be interpreted to include plural expressions, unless specifically stated otherwise. The expression "includes" in this specification means that the components, parts, operations, features, numbers, etc. described in the description are present, and does not exclude the addition of one or more other components, parts, operations, features, numbers, etc.

[0057] The present disclosure can be fully achieved by the following description. It should be understood that the following description describes preferred embodiments of the present disclosure, and the present disclosure is not necessarily limited thereto. Furthermore, the attached drawings are provided for illustrative purposes only and are not intended to limit the present disclosure. Details regarding individual components can be appropriately understood based on the specific intent of the relevant descriptions described below.

[0058] The present disclosure provides a composition for wound treatment or tissue regeneration comprising a genomic DNA fragment extracted from human placental-derived mesenchymal stem cells and placental growth factor (PLGF) as active ingredients, and a method for preparing the same.

[0059] "Placenta" is a type of reproductive organ that transfers nutrients from the mother to the fetus and waste products from the fetus to the mother during the development of true mammals. In the present disclosure, the placenta may refer to a placenta that is separated and discarded after birth from a healthy mother, but the type is not particularly limited, and may refer to a human placenta that is accompanied by a maternal consent form and a non-infectious test result issued by a medical institution at the time of placenta collection. The placenta may include a chorionic plate membrane.

[0060] "Stem cells" refer to cells that have the ability to differentiate into various types of cells through appropriate environments and stimuli, and have the ability to self-renew.

[0061] "Mesenchymal stem cells (MSC)" refers to a type of stem cell that can be isolated from the chorionic plate of the placenta. Mesenchymal stem cells are multipotent stem cells that can self-renew and differentiate into various lineages, and can be used in the same meaning as mesenchymal progenitor cells. Mesenchymal stem cells can differentiate into bone, fat, cartilage, nerve, muscle, and bone marrow stromal cells depending on the conditions, and thus have various therapeutic effects. Mesenchymal stem cells are a type of adult stem cell and include all mesenchymal stem cells of animal origin, and the animal may mean all animals, including humans, that have developed damaged tissues and can be treated with the mesenchymal stem cells of the present disclosure, and may include mammals such as cows, horses, sheep, and pigs that require treatment for symptoms similar to those of humans, but are not limited thereto. It can be isolated together with hematopoietic stem cells, which are mainly isolated from bone marrow, and unlike hematopoietic stem cells, which have the characteristic of being cultured in a suspended state, it has the characteristic of attaching to culture dishes, etc.

[0062] In the present disclosure, mesenchymal stem cells may refer to mesenchymal stem cells derived from the chorionic valve of the placenta separated from the mother after birth, and various placental-derived stem cells including the same. The method for isolating the stem cells in the present disclosure may be a method for isolating stem cells with high purity disclosed in Korean Patent No. 10-0900309. By utilizing the genomic DNA extracted from the placental-derived mesenchymal stem cells in the present disclosure, mass production of a composition for wound treatment or tissue regeneration may be possible, and by securing highly pure genomic DNA, production of a composition for wound treatment or tissue regeneration with improved quality and consistency may be possible.

[0063] In the present disclosure, the human placental-derived genomic DNA fragment may refer to a state in which genomic DNA extracted from human placental chorionic valve-derived mesenchymal stem cells is fragmented into a certain size and molecular weight range.

[0064] The present disclosure can use genomic DNA fragments of appropriate size that can exhibit anti-fibrosis and tissue regeneration functions from human placental-derived mesenchymal stem cells. The size of the genomic DNA fragment is, for example, 50 to 1,000 bp, 100 to 1,000 bp, 200 to 1,000 bp, 300 to 1,000 bp, 400 to 1,000 bp, 500 to 1,000 bp, 600 to 1,000 bp, 700 to 1,000 bp, 800 to 1,000 bp, 900 to 1,000 bp, 50 to 600 bp, 100 to 600 bp, 200 to 600 bp, 50 to 500 bp, 100 to 500 bp, 200 to 500 bp, 50 to 400 bp, 100 to 400 bp, It may be 200 to 400 bp, 50 to 300 bp, 100 to 300 bp, 150 to 300 bp, 50 to 200 bp, 100 to 200 bp, 150 to 200 bp or 50 to 100 bp, and preferably 100 to 1,000 bp, but is not limited thereto.

[0065] In the present disclosure, the molecular weight of the genomic DNA fragment is, for example, 10 to 700 kDa, 50 to 700 kDa, 100 to 700 kDa, 300 to 700 kDa, 10 to 600 kDa, 50 to 600 kDa, 100 to 600 kDa, 300 to 600 kDa, 10 to 500 kDa, 50 to 500 kDa, 100 to 500 kDa, 250 to 500 kDa, 10 to 400 kDa, 50 to 400 kDa, 100 to 400 kDa, 200 to 400 kDa, 50 to 300 kDa, 100 to 300 kDa, 150 It may be 300 kDa, 50 to 200 kDa, 100 to 200 kDa, 50 to 100 kDa, 60 to 100 kDa, 70 to 100 kDa, 80 to 100 kDa or 90 to 100 kDa, and preferably 50 to 600 kDa, but is not limited thereto.

[0066] "Placental growth factor (PLGF)" is a type of glycoprotein and a subcomponent of the vascular endothelial growth factor (VEGF) family. It refers to a substance produced in the placenta during pregnancy that promotes the growth of new blood vessels and aids fetal growth. Placental growth factor is involved in angiogenesis and vasculogenesis, and plays a major role in embryogenesis. Placental growth factor is found in the endometrium, and is mainly found in chorionic trophoblasts, vascular endothelial cells during heart and angiogenesis, and is mainly formed in uterine trophoblasts during pregnancy. Placental growth factor is involved in various cellular activities, and is involved in the survival, migration, proliferation, metabolism, and activation of not only vascular cells (such as vascular endothelial cells, pericytes, and vascular smooth muscle cells), but also macrophages, dendritic cells, and neurons. In the present disclosure, the placental growth factor may include various natural or synthetic placental growth factors or equivalents thereof, as long as they exhibit the effects according to the present disclosure. The above equivalents include variants, mutations, analogs, or derivatives having the activity of placental growth factors, and may refer to natural or synthetic ones. In the present disclosure, placental growth factors of mammalian origin and form may be used, and human origin and form are preferably used, but are not limited thereto. In the present disclosure, various substances that increase the expression of placental growth factors at the mRNA and / or protein levels and / or increase the activity of placental growth factors may be further included in the composition for wound healing or tissue regeneration.

[0067] In the present disclosure, the term “including as an effective ingredient” may mean including an effective amount capable of exhibiting a wound healing or tissue regeneration effect as a composition for wound healing or tissue regeneration.

[0068] In the present disclosure, the concentration of the genomic DNA fragment extracted from human placental-derived mesenchymal stem cells included in the composition for wound treatment or tissue regeneration is, for example, 0.1 to 100 ng / mL, 0.5 to 100 ng / mL, 1 to 100 ng / mL, 5 to 100 ng / mL, 10 to 100 ng / mL, 50 to 100 ng / mL, 0.1 to 50 ng / mL, 0.5 to 50 ng / mL, 1 to 50 ng / mL, 5 to 50 ng / mL, 10 to 50 ng / mL, 30 to 50 ng / mL, 0.1 to 10 ng / mL, 0.5 to 10 ng / mL, 1 to 10 ng / mL, 5 to 10 ng / mL, 0.1 to 5 It may be ng / mL, 0.5 to 5 ng / mL or 1 to 5 ng / mL, preferably 1 to 100 ng / mL, but is not limited thereto.

[0069] In the present disclosure, the concentration of the placental growth factor included in the composition for wound treatment or tissue regeneration may be, for example, 0.01 to 1 pg / mL, 0.05 to 1 pg / mL, 0.1 to 1 pg / mL, 0.5 to 1 pg / mL, 0.01 to 0.5 pg / mL, 0.05 to 0.5 pg / mL, 0.1 to 0.5 pg / mL, 0.01 to 0.1 pg / mL or 0.05 to 0.1 pg / mL, and preferably 0.01 to 0.5 pg / mL, but is not limited thereto.

[0070] In the present disclosure, “wound” may mean a wound, a burn wound, an abrasion, a laceration, a stab wound, an ulcer, a decubitus, a contusion, hidradenitis suppurativa, inflammatory fibrosis, periodontitis, or a combination thereof, or may mean a wound associated with type 1 or type 2 diabetes.

[0071] In the present disclosure, "tissue" may mean skin, epidermis, dermis, neovascularization, or skin appendages. Accordingly, "tissue regeneration" may mean skin regeneration, epidermal regeneration, dermal regeneration, neovascularization, or skin appendage regeneration.

[0072] In the present disclosure, the "pharmaceutical composition" can be formulated in a suitable form with a pharmaceutically acceptable carrier that is generally used. "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not typically cause allergic reactions or similar reactions such as gastrointestinal upset, dizziness, etc. when administered to humans. Pharmaceutically acceptable carriers can be saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and mixtures of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets, and target organ-specific antibodies or other ligands can be combined with the carrier to specifically act on the target organ. The pharmaceutical composition of the present disclosure may contain, as an active ingredient, a genome fragmented from DNA extracted from placental mesenchymal stem cells through sonication. The pharmaceutical composition of the present disclosure may be formulated in the form of an injection, eye drop, patch, cream, etc., according to conventional pharmaceutical methods. The pharmaceutically acceptable carrier of the present disclosure may include a sterile aqueous solution (e.g., physiological saline solution, etc.), a low concentration of placental growth factor (PLGF, 0.25 pg / mL), and the like, and may include a hydrogel component, if necessary.

[0073] The method of administration of the composition of the present disclosure may apply a known administration method in the relevant technical field, and may be administered parenterally (for example, topically) or orally depending on the intended method, with parenteral administration being preferred, and in the case of parenteral administration, it may be administered in the form of an injection or a patch applied or attached to the skin, and other routes and means are not excluded. The daily dosage may be administered once or several times a day. The dosage range varies greatly depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity, and a dosage known in the relevant technical field may be applied. The dosage of the placental mesenchymal stem cell-derived genome included as an active ingredient in the pharmaceutical composition of the present disclosure may vary depending on the patient's condition, the degree of the disease, and the size of the wound, but may be appropriately selected by a person skilled in the art. The daily dosage of the placental mesenchymal stem cell-derived genome of the present disclosure is, for example, 0.1 to 100 pg / mm 2 , 1 to 100 pg / mm 2 , 5 to 100 pg / mm 2 , 10 to 100 pg / mm 2 , 50 to 100 pg / mm 2 , 0.1 to 50 pg / mm 2 , 1 to 50 pg / mm 2 , 5 to 50 pg / mm 2 , 10 to 50 pg / mm 2 , 20 to 50 pg / mm 2 , 0.1 to 30 pg / mm 2 , 1 to 30 pg / mm 2 , 5 to 30 pg / mm 2 , 10 to 30 pg / mm 2 , 0.1 to 20 pg / mm 2 , 0.5 to 20 pg / mm 2 , 1 to 20 pg / mm 2 , 3 to 20 pg / mm2 , 5 to 20 pg / mm 2 , 10 to 20 pg / mm 2 , 0.1 to 10 pg / mm 2 , 0.5 to 10 pg / mm 2 , 1 to 10 pg / mm 2 , 5 to 10 pg / mm 2 , 0.1 to 5 pg / mm 2 , 0.5 to 5 pg / mm 2 or 1 to 5 pg / mm 2 may be, preferably 1 to 100 pg / mm 2 It may include, but is not limited to.

[0074] In the present disclosure, the genomic DNA fragment and placental growth factor (PLGF) extracted from human placental-derived mesenchymal stem cells exhibit effects of improving skin elasticity, improving fine wrinkles, whitening skin, improving acne scars, reducing pores, and improving skin tone, and thus can be used as a skin regeneration management auxiliary agent.

[0075] In the present disclosure, genomic DNA fragments and placental growth factor (PLGF) extracted from human placental-derived mesenchymal stem cells can be included as active ingredients in medical devices for wound treatment or tissue regeneration.

[0076] The method for producing a genomic DNA fragment extracted from human placental-derived mesenchymal stem cells of the present disclosure is as follows:

[0077] (a) a step of pulverizing human placental-derived mesenchymal stem cells using a dissolution buffer;

[0078] (b) a step of adding isopropanol to the placental-derived mesenchymal stem cell pulverization solution obtained in step (a);

[0079] (c) a step of centrifuging the crushed liquid to which isopropanol has been added in step (b) to separate the supernatant and the precipitate;

[0080] (d) a step of dissolving the precipitate of step (c) with nuclease free water; and

[0081] (e) may include a step of sonicating the solution obtained in step (d) to produce a genomic DNA fragment reduced to an appropriate size,

[0082] Between steps (c) and (d), a step of washing the precipitate of step (c) with ethanol to remove impurities may be further included.

[0083] In the present disclosure, the term "dissolution buffer" is not particularly limited in its type as long as it is known in the art, but preferably means a composition containing 10 mM Tris-Cl pH 8.0, 0.1 M EDTA pH 8.0, and 0.5% SDS.

[0084] The placental-derived mesenchymal stem cells of the above step (a) may refer to various placental-derived stem cells, including mesenchymal stem cells derived from the chorionic valve in the placenta separated from the mother's body after birth.

[0085] The above step (a) can be performed by using stem cells cultured by separating them from the chorionic valve of the placenta, or by thawing cultured stem cells that were frozen and stored, washing them once with phosphate buffered saline (DPBS), and then precipitating them.

[0086] In the above step (a), the placental-derived mesenchymal stem cell pulverization solution may refer to a solution containing human placental-derived mesenchymal stem cells pulverized in a dissolution buffer.

[0087] In the step (a) above, when disrupting cells, a DNA extraction column known in the art including activated carbon may not be used, and thus the unit cost due to column use may be reduced, and the yield of extracted DNA may be improved.

[0088] Typically, after cell disruption, a method is used to increase DNA precipitation efficiency by treating isopropanol with a salt concentration above a certain level to precipitate DNA. However, in the present disclosure, only isopropanol is treated without the addition of additional salt, and the DNA precipitation efficiency is not reduced. Accordingly, since an additional salt removal process is not required in the subsequent process, the manufacturing process, especially the DNA precipitation process, can be simplified, enabling the economical manufacture of a composition for wound healing or tissue regeneration. Furthermore, since isopropanol is used instead of the toxic substance chloroform, environmental pollution due to waste is reduced, enabling eco-friendly manufacturing.

[0089] Since the isopropanol used in the above step (b) has low solubility in DNA, it can precipitate DNA in small amounts. In the present disclosure, the isopropanol added to the pulverization solution may correspond to 0.1 to 1 times the volume of the DNA dissolution solution, and may be 10 to 100% isopropanol.

[0090] In the case of centrifugation in the above step (c), a person skilled in the art can appropriately select and change various conditions such as rotation speed (rpm) and centrifugation time according to the desired work efficiency. In the present disclosure, the centrifugation rotation speed may be, for example, 1,000 to 2,000 rpm, 1,500 to 2,000 rpm, 1,000 to 1,500 rpm, or 1,200 to 1,400 rpm, and preferably 1,000 to 2,000 rpm, but is not limited thereto.

[0091] In the present disclosure, the centrifugation time may be, for example, 1 to 5 minutes, 1 to 4 minutes, 1 to 3 minutes, 1 to 2 minutes, 2 to 5 minutes, 2 to 4 minutes, 2 to 3 minutes, 3 to 5 minutes, 3 to 4 minutes, 4 to 5 minutes, and preferably, 2 to 4 minutes or 3 minutes, but is not limited thereto.

[0092] The method for obtaining a precipitate in the above step (d) can be used without limitation as long as it is a method for obtaining a nucleic acid (DNA fragment polymer) precipitate known in the art, and according to one embodiment of the present disclosure, it can be performed by adding nuclease free water to the precipitate obtained in the above step (d).

[0093] In the present disclosure, nuclease-free water may mean water without nuclease or endonuclease or exonuclease including DNase or RNase, or water without nuclease or endonuclease including DNase or RNase, and may use a commercially available product or a product known in the art, but is not limited thereto. By using nuclease-free water in the present disclosure, contamination by microorganisms, etc. can be minimized or prevented during the manufacture of a composition for wound treatment or tissue regeneration, and by minimizing or preventing DNA degradation during the manufacture process, the yield of DNA finally obtained can be increased. The amount of nuclease-free water used in the present disclosure may be 50 to 100 μL.

[0094] The ultrasonic grinder used for the DNA molecule reduction in step (e) above is not particularly limited in type as long as it is known in the art. However, it may preferably refer to an indirect ultrasonic grinder capable of grinding DNA inside a tube containing DNA without opening the tube. Accordingly, by grinding DNA without opening the tube containing DNA, DNA contamination and loss / damage issues can be minimized or prevented, thereby increasing the yield of the DNA ultimately obtained.

[0095] The DNA molecular reduction in step (e) above may be performed in coolant to maintain the human placental-derived genomic DNA at 4°C. Accordingly, genomic denaturation can be minimized or prevented, thereby ensuring a consistent quality and high-quality efficacy of the resulting DNA.

[0096] The DNA molecule reduction in step (e) above can be performed under conditions of amplitude of, for example, 40 to 80%, 50 to 80%, 60 to 80%, 70 to 80%, 40 to 70%, 50 to 70%, 40 to 60%, 50 to 60%, or 40 to 50%, and preferably, can be performed at an amplitude of 50 to 70% or 60%, but is not limited thereto.

[0097] The DNA molecule reduction in the above step (e) may be performed under pulse / rest conditions of, for example, 5 to 25 seconds, 10 to 25 seconds, 15 to 25 seconds, 20 to 25 seconds, 5 to 20 seconds, 10 to 20 seconds, 15 to 20 seconds, 5 to 15 seconds, 10 to 15 seconds, 5 to 10 seconds, and preferably, but not limited to, 10 to 20 seconds or 15 seconds.

[0098] The molecular reduction in step (e) above can be performed under power conditions of, for example, 500 to 900 W, 600 to 900 W, 700 to 900 W, 800 to 900 W, 500 to 800 W, 600 to 800 W, 700 to 800 W, 500 to 700 W, 600 to 700 W or 500 to 600 W, and preferably, but not limited to, 600 to 800 W or 750 W.

[0099] The DNA molecule reduction in step (e) above can be performed at a frequency condition of, for example, 5 to 40 Hz, 10 to 40 Hz, 20 to 40 Hz, 30 to 40 Hz, 5 to 30 Hz, 10 to 30 Hz, 15 to 30 Hz, 5 to 20 Hz, 10 to 20 Hz or 5 to 10 Hz, and preferably, can be performed at a frequency of 10 to 30 Hz or 20 Hz, but is not limited thereto.

[0100] The DNA molecule reduction in step (e) may be performed for, for example, 15 to 45 minutes, 20 to 45 minutes, 30 to 45 minutes, 15 to 40 minutes, 20 to 40 minutes, 30 to 40 minutes, 15 to 25 minutes, 20 to 25 minutes or 15 to 20 minutes, preferably, 20 to 40 minutes or 30 minutes, but is not limited thereto.

[0101] Between the above steps (c) and (d), in the ethanol washing step that may be further included, the washing may be performed by alcohol treatment, and in the present disclosure, ethanol may be, for example, 50 to 90%, 60 to 80%, 50 to 70%, 50 to 60%, and preferably 60 to 80%, or 70%, but is not limited thereto.

[0102] Hereinafter, the present disclosure will be described in more detail through examples. However, these examples are intended to exemplify the present disclosure, and the scope of the present disclosure is not limited to these examples.

[0103]

[0104] Manufacturing Example 1-1. DNA extraction from placental-derived mesenchymal stem cells

[0105] Placenta-derived Mesenchymal Stem Cells (Universal Placental MSCs, hereinafter referred to as “UNIPla”) manufactured according to Korean Patent Registration No. 10-0900309 were cultured in alpha-MEM medium supplemented with 10% fetal bovine serum (FBS), 1% gentamicin, 25 ng / mL fibroblast growth factor-4 (FGF4), and 1 μg / mL heparin in a CO2 incubator at 37°C for 3 to 4 days. The medium was removed from the medium, and the stem cells were washed with phosphate buffered saline (PBS). Then, the stem cells were detached from the flask by trypsin treatment at 37°C for 2 minutes, and the detached cells were collected using the culture medium and centrifuged at 1,300 rpm for 3 minutes. The supernatant was removed, and 2 mL of culture medium was added to the obtained cell sediment to disperse it, and then the cells were counted using a hemoglobin meter.

[0106] 0.5 to 1 x 10 in one 1.5 mL tube 6 After adding cells, centrifugation was performed at 2,000 rpm for 3 minutes and the supernatant was removed. To remove culture medium components remaining in the cell sediment, the cells were dispersed in 1 mL of phosphate buffer solution, centrifugation was performed at 2,000 rpm for 3 minutes, the supernatant was removed, and the sediment was prepared.

[0107] The cells were well lysed with 500-700 μL of lysis buffer containing 10 mM Tris-Cl pH 8.0, 0.1 M EDTA pH 8.0, and 0.5% SDS to the UNIPla precipitate prepared by the above method, and proteinase K at a concentration of 20 mg / mL was added to reach 250 μg / mL, followed by reaction in a heat block at 37°C for 30 minutes to inactivate DNase, stabilize DNA, and remove histone proteins attached to DNA, enabling pure DNA to be extracted.

[0108] Afterwards, 100% isopropanol corresponding to 0.75 times the volume of the DNA solution was added to the above-mentioned solution (pulverized solution) without adding salt, suspended for 10 seconds, centrifuged at 13,000 rpm for 10 minutes while maintaining 4℃, and the supernatant was removed. 1 mL of 75% ethanol was added to the precipitated DNA, washed, centrifuged at 13,000 rpm for 1 minute while maintaining 4℃, and the supernatant was removed. This process of removing the supernatant was repeated twice. The precipitated DNA was naturally dried for 5 minutes, dissolved in 50-100 μL of nuclease-free water, and the amount of DNA was quantified using a spectrophotometer.

[0109]

[0110] Manufacturing Example 1-2. DNA extraction from frozen placental-derived mesenchymal stem cells.

[0111] In addition to the method according to the above Manufacturing Example 1-1, a frozen UNIPla vial was rapidly thawed in a 37°C water bath for 30 seconds without going through a cell culture step, 3 mL of culture medium was added, mixed well, and centrifuged at 1,300 rpm for 3 minutes. The supernatant was removed, and 2 mL of culture medium was added to the obtained cell sediment to disperse it, and then counted using a hemoglobin meter.

[0112] 0.5 to 1 x 10 in one 1.5 mL tube 6 After adding cells, centrifugation was performed at 2,000 rpm for 3 minutes and the supernatant was removed. To remove culture medium components remaining in the cell sediment, the cells were dispersed in 1 mL of phosphate buffer solution, centrifugation was performed at 2,000 rpm for 3 minutes, the supernatant was removed, and the sediment was prepared.

[0113] The cells were well lysed with 500-700 μL of lysis buffer containing 10 mM Tris-Cl pH 8.0, 0.1 M EDTA pH 8.0, and 0.5% SDS to the UNIPla precipitate prepared by the above method, and proteinase K at a concentration of 20 mg / mL was added to reach 250 μg / mL, followed by reaction in a heat block at 37°C for 30 minutes to inactivate DNase, stabilize DNA, and remove histone proteins attached to DNA, enabling pure DNA to be extracted.

[0114] Afterwards, 100% isopropanol corresponding to 0.75 times the volume of the DNA solution was added to the above-mentioned solution (pulverized solution) without adding salt, suspended for 10 seconds, centrifuged at 13,000 rpm for 10 minutes while maintaining 4℃, and the supernatant was removed. 1 mL of 75% ethanol was added to the precipitated DNA, washed, centrifuged at 13,000 rpm for 1 minute while maintaining 4℃, and the supernatant was removed. This process of removing the supernatant was repeated twice. The precipitated DNA was naturally dried for 5 minutes, dissolved in 50-100 μL of nuclease-free water, and the amount of DNA was quantified using a spectrophotometer.

[0115]

[0116] Manufacturing Example 2. UNIPLa DNA fragment

[0117] The final concentration of the placenta-derived mesenchymal stem cell genomic DNA extracted by the method of Preparation Example 1-1 or Preparation Example 1-2 was 100 μg / mL, and the diluted DNA was dispensed into 0.2 mL tubes (100 μL each). This was placed in an indirect ultrasonic disperser (Qsonica, Q800R3) with the lid closed and treated in cooled water for 20 to 40 minutes under the conditions of an amplitude of 50 to 70%, a pulse / rest of 10 to 20 seconds, a power of 600 to 800 W, and 10 to 30 Hz, thereby obtaining a human placenta-derived mesenchymal stem cell genomic DNA fragment (hereinafter referred to as “UNIPlax”).

[0118] As shown in Fig. 1, DNA was electrophoresed on a 1% agarose gel and visualized under UV light, confirming a genome size of between 100 and 1,000 bp.

[0119]

[0120] Comparative Example 1. UNIPLa DNA fragment

[0121] The final concentration of human placental mesenchymal stem cell genomic DNA extracted by the method of Manufacturing Example 1-1 or Manufacturing Example 1-2 was diluted using nuclease-free water to 72 μg / mL, and 12 μL of this was dispensed into each 1.5 mL tube to prepare.

[0122] As shown in Fig. 2, the DNA was mounted on a water tank-type ultrasonic grinder and treated for 0, 15, 20, 30, and 40 minutes at a power of 40 kHz. Then, the DNA was electrophoresed on a 1% agarose gel and visualized under UV light. As a result, no genome size of 500 bp or less was confirmed.

[0123]

[0124] Comparative Example 2. UNIPLa DNA fragment

[0125] In the case of using an ultrasonic grinder according to the above Comparative Example 1, there is a disadvantage that as the ultrasonic reaction time increases, a lot of heat is generated, causing the temperature of the water tank to rise rapidly. To compensate for this, an ice pack was placed in the water tank and replaced every 10 minutes during the reaction time to maintain the temperature of the water tank below 20℃. In addition, DNA at a concentration of 100 μg / mL was added to a Pyrex tube (glass type) instead of a polypropylene tube and the reaction was performed under 40 kHz conditions for 60 minutes.

[0126] As shown in Fig. 3, DNA was electrophoresed on a 1% agarose gel and visualized under UV light, and no genome size less than 500 bp was identified.

[0127]

[0128] Comparative Example 3. UNIPLa DNA fragment

[0129] In addition to the DNA fragmentation method using ultrasound according to Comparative Example 1 or Comparative Example 2, in order to fragment DNA using an enzyme, 0.5 μL of DNAase I at a concentration of 10 U / μL, 1 μL of 10 x reaction buffer, and 6.5 μL of nuclease-free water were added to 530 ng of DNA, mixed well, and reacted at 37°C for 2 minutes, 10 minutes, and 20 minutes, and then reacted at 75°C for 10 minutes to inactivate the enzyme.

[0130] As shown in Fig. 4, DNA was electrophoresed on a 1% agarose gel and visualized under UV light, and no genome size less than 500 bp was identified.

[0131]

[0132] Comparative Example 4. UNIPLa DNA fragment

[0133] In addition to the method according to Comparative Examples 1 to 3 above, 800 μL of DNA at a concentration of 13.28 μg / mL was placed in a 1.5 mL tube using a probe-type ultrasonic fragmenter, placed on ice, and reacted for 10 seconds at an amplitude of 20, followed by a 10-second rest period, for 20 minutes.

[0134] As shown in Fig. 5, DNA was electrophoresed on a 1% agarose gel and visualized under UV light, and a genome size of between 100 and 500 bp was confirmed. However, it was confirmed that it was not efficient because the DNA sample prepared in the tube was lost due to the intensity of the ultrasound.

[0135]

[0136] Manufacturing Example 3. Manufacturing of a mixed composition of UNIPlax and PLGF

[0137] After measuring the concentration of UNIPlax DNA manufactured according to Manufacturing Example 2 above, 1 / 100 serial dilution and 1 / 10 serial dilution were performed to manufacture a working stock of 800 ng / mL, and an additional 1 / 10 dilution was performed to manufacture a UNIPlax working stock of 80 ng / mL. (The average DNA yield was calculated as 800 μg / mL.)

[0138] Meanwhile, a 5 μg / mL PLGF working stock was prepared by performing 1 / 100 serial dilution three times.

[0139] Based on administering 1.5 μL to 4 sites per experimental mouse, a total of 6 μL of the administration solution was required, so 8 μL of the administration solution was prepared for each dose, taking into account the remaining amount. UNIPlax and PLGF (0.25 pg / mL) prepared according to Manufacturing Example 2 were diluted with distilled water (DW) according to each administration concentration. The composition of each administration solution is as shown in [Table 1].

[0140]

[0141] UNIPlax concentration (ng / mL)UNIPlax (μL)PLGF (μL)DW (μL)Total amount of solution administered (μL)10.1 (80 ng / mL stock)0.4 (5 pg / mL stock)7.5850.5 (80 ng / mL stock)0.4 (5 pg / mL stock)7.18101 (80 ng / mL stock)0.4 (5 pg / mL stock)6.68500.5 (800 ng / mL stock)0.4 (5 pg / mL stock)7.181001 (800 ng / mL stock)0.4 (5 pg / mL stock)6.68

[0142]

[0143] Example 1. MTT assay

[0144] To determine the cytotoxicity of UNIPlax, a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed. First, 2 x 10 of the immortal human keratinocyte cell line HaCaT were seeded in 96 wells. 3 The cells were seeded using a medium containing fetal bovine serum (DMEM / F12 supplemented with 1% penicillin / streptomycin and 10% fetal bovine serum) and cultured for 2 days.

[0145] UNIPlax obtained through the above Manufacturing Example 2 was added to the culture medium at various concentrations (0, 1, 10, 50, 100 ng / mL in 100μL) and cultured for 24, 48, and 72 hours. After the culture time, 20 μL of 5 mg / mL MTT solution was added to each well and left in a CO2 incubator at 37°C for 2 hours. After removing the culture medium, 100 μL of dimethylsulfoxide (DMSO) was added, shaken at room temperature for 3 minutes, and the absorbance was measured at 562 nm. Cell viability (%) was calculated as a percentage by dividing the absorbance of the wells treated with UNIPlax at various concentrations by the absorbance of the wells without UNIPlax and multiplying by 100.

[0146] As shown in Fig. 6, when treated with a concentration of 10 ng / mL of UNIPlax and cultured for 24 hours, cell proliferation was statistically significantly increased compared to the negative control group (excluding UNIPlax). In other concentrations, cell proliferation showed a similar or increasing tendency compared to the negative control group when cultured for 24 or 72 hours, and no difference was confirmed compared to the negative control group when cultured for 48 hours. In other words, it was confirmed that UNIPlax does not exhibit cytotoxicity and can be used safely.

[0147]

[0148] Example 2. In vitro wound healing assay

[0149] To confirm the wound healing effect of UNIPlax, an in vitro wound healing assay was performed.

[0150] Immortalized human keratinocyte cell line HaCaT cells were seeded at 2 x 10 per well in a 6-well plate. 5After seeding and culturing until cell distribution reached 100%, the medium was removed and the cells were damaged by drawing a line down the center of the well with a 1 mL tip. Then, the cells were slowly washed with 1 mL of phosphate buffered solution and replaced with culture medium containing UNIPlax at various concentrations, and the degree of damage was photographed with a microscope camera. After that, each plate was left in a CO2 incubator at 37℃ for 24 hours. After the incubation time was completed, the cells in each well were photographed and the degree of damage recovery according to the treatment with different concentrations of UNIPlax was measured by how much the damaged gap was reduced to compare and analyze the healing effect of UNIPlax.

[0151] As shown in Fig. 7, in the case of the negative control group (no UNIPlax added), the cells recovered to about 40% of the normal cells (undamaged cells), but when treated with UNIPlax at concentrations of 1, 5, and 50 ng / mL, respectively, it was confirmed that the damaged cells recovered to about 76 to 80% of the normal cells. In addition, when treated with UNIPlax at concentrations of 10 and 100 ng / mL, respectively, it was confirmed that the damaged cells recovered to about 52 to 57% of the normal cells. In light of these in vitro test results, it is preferable that UNIPlax is included at a concentration of less than 100 ng / mL, and more specifically, it is preferable that it is included at a concentration of 1 to 10 ng / mL or 1 to 5 ng / mL.

[0152]

[0153] Example 3. In vivo wound healing assay

[0154] In order to examine the tissue regeneration effect of a composition containing UNIPlax and PLGF as active ingredients in an animal wound model, an excisional full-thickness wound with a diameter of 6 mm was induced in experimental mice, and then 6 μL of each of the control group (untreated with saline, UNIPlax, or PLGF), the PLGF 0.25 pg / mL single treatment group, and the mixed solution (PLGF (0.25 pg / mL) and UNIPlax (1, 5, 10, 50, 100 ng / mL)) prepared according to Preparation Example 3 above were applied to the affected area, and the area of ​​the affected area was measured every two days, and on the 9th day, the wound site tissue of each affected area was obtained. The obtained tissue was fixed in paraffin to obtain ultrathin tissue sections. The histological characteristics were compared and analyzed by performing H&E staining and Masson's trichrome staining on the tissue sections.

[0155] As shown in Fig. 8, compared to the control group (Saline) and PLGF only treatment group, it was confirmed that the treatment group treated with PLGF and UNIPlax together showed a faster wound healing rate. As a result of quantitative analysis of the degree of wound healing, the treatment group treated with PLGF and UNIPlax together showed a statistically significant difference in the size of the wound area, which was reduced by approximately 85% compared to the control group or PLGF only treatment group at a concentration of 5 to 50 ng / mL.

[0156] As shown in Fig. 9, on the 9th day after wound induction, wound tissues of experimental mice treated with each experimental group were obtained, and tissue sections were obtained from these and H&E staining was performed. In the PLGF and UNIPlax 5 ng / mL treatment group and the PLGF and UNIPlax 10 ng / mL treatment group, it was confirmed that the area of ​​granulation tissue (GT), which is a newly formed tissue in the dermis during the wound healing process, was significantly reduced by more than 70% compared to the control group. In light of these results, it can be interpreted that the composition containing UNIPlax and PLGF together exhibits a faster wound regeneration rate and thus has excellent tissue regeneration ability.

[0157] As shown in Fig. 10, to confirm the exact site of epidermal formation, staining with Masson's trichrome confirmed a significant decrease in the size of granulation tissue to the same extent in the PLGF and UNIPlax 5 ng / mL treatment group and the PLGF and UNIPlax 10 ng / mL treatment group.

[0158] Meanwhile, wound healing progresses through stages 1 and 2, the coagulation phase, the inflammatory phase, the proliferative phase, and finally the remodeling phase. During the proliferative phase, many cells undergo mitosis in the granulation tissue (GT), and this mitosis can be measured by the expression of the nuclear protein proliferating cell nuclear antigen (PCNA). In other words, a lower number of PCNA-positive cells may indicate faster regeneration.

[0159] As shown in Fig. 11, immunostaining with a PCNA-specific antibody showed that on the 9th day after wound induction, the control group (Saline) and the PLGF-only treatment group showed a significantly higher number of mitoses, whereas the UNIPlax and PLGF treatment group showed a significantly lower number of PCNA-positive cells at concentrations of 1 to 50 ng / mL. In light of these results, it can be interpreted that the granulation tissue in the UNIPlax and PLGF treatment group passed the proliferation phase and entered the remodeling phase in which cell division was suppressed by contact inhibition. In other words, this means that the wound healing rate is faster when UNIPlax and PLGF are treated together.

[0160] The above-described results suggest that when the human placental-derived genomic DNA fragment (UNIPlax) of the present disclosure is treated together with placental growth factor (PLGF), regeneration of skin tissue is actively achieved, resulting in significantly superior wound healing and recovery capabilities.

[0161] The embodiments of the present disclosure have been described above. However, those skilled in the art will appreciate that various modifications to the present disclosure, such as simple design changes, omission of certain components, and simple changes in purpose, can be made within the scope of the technical concepts of the present disclosure as defined in the claims, depending on specific applications of the technology. It is self-evident that such modifications also fall within the scope of the present disclosure.

Claims

1. Contains genomic DNA fragment extracted from human placenta-derived mesenchymal stem cells and placental growth factor (PLGF) as active ingredients. Composition for tissue regeneration.

2. In paragraph 1, The size of the above genomic DNA fragment is 100 to 1,000 bp. Composition for tissue regeneration.

3. In paragraph 1, The molecular weight of the above genomic DNA fragment is 50 to 600 kDa. Composition for tissue regeneration.

4. In paragraph 1, The above genomic DNA fragment is contained at less than 100 ng / mL. Composition for tissue regeneration.

5. In paragraph 1, The above placental growth factor comprises a natural or synthetic placental growth factor, or a modified form, variant, analogue, or derivative thereof. Composition for tissue regeneration.

6. In paragraph 1, The above placental growth factor is included at 0.01 to 0.5 pg / mL. Composition for tissue regeneration.

7. In paragraph 1, The above tissue includes skin, epidermis, dermis, neovascularization or skin appendages. Composition for tissue regeneration.

8. Contains genomic DNA fragment extracted from human placenta-derived mesenchymal stem cells and placental growth factor (PLGF) as active ingredients. Composition for skin care.

9. Contains genomic DNA fragment extracted from human placenta-derived mesenchymal stem cells and placental growth factor (PLGF) as active ingredients. A pharmaceutical composition for wound healing.

10. In paragraph 9, The above wound is one or a combination of two or more selected from the group consisting of wound, burn wound, abrasion, laceration, stab wound, ulcer, decubitus, contusion, hidradenitis suppurativa, inflammatory fibrosis, and periodontitis. A pharmaceutical composition for wound healing.

11. In paragraph 10, The above wound is characterized as a chronic wound. A pharmaceutical composition for wound healing.

12. A pharmaceutical composition comprising any one of claims 9 to 11. Medical devices for wound treatment.

13. A pharmaceutical composition comprising any one of claims 9 to 11. Pharmaceutical preparations for wound healing.

14. In paragraph 13, The above formulation is characterized in that it is in the form of an injection, eye drop, patch, cream, pill, capsule, granule or tablet. Pharmaceutical preparations for wound healing. 15.(a) A step of pulverizing human placental-derived mesenchymal stem cells using a dissolution buffer; (b) a step of adding isopropanol to the placental-derived mesenchymal stem cell pulverization solution obtained in step (a); (c) a step of centrifuging the dissolution buffer to which isopropanol is added in step (b) to separate the supernatant and the precipitate; (d) a step of dissolving the precipitate of step (c) with nuclease free water; and (e) a step of producing a molecularly reduced genomic DNA fragment by sonicating the dissolved solution in step (d); A method for producing a genomic DNA fragment extracted from human placental-derived mesenchymal stem cells.

16. In paragraph 15, The placental-derived mesenchymal stem cells of step (a) above (i) obtaining a chorionic plate membrane from a placenta separated from a mother's womb after birth; (ii) scraping cells inside the chorionic plate membrane obtained in step (i) to collect mesenchymal stem cells; (iii) adding a solution containing trypsin and ethylenediaminetetraacetate to the cells obtained in step (ii) to perform an enzymatic reaction at 20 to 30°C, and adding fetal bovine serum to stop the enzymatic reaction; and (iv) centrifuging the reaction solution obtained in step (iii) to culture the recovered cells in a medium supplemented with fetal bovine serum and antibiotics. Method for producing human placental-derived genomic DNA fragments.

17. In paragraph 15, The placental-derived mesenchymal stem cells of the above step (a) are characterized in that they are cells thawed from a frozen state. A method for producing a genomic DNA fragment extracted from human placental-derived mesenchymal stem cells.

18. In paragraph 15, The volume of isopropanol in the above step (b) is 0.1 to 1 times that of the placental-derived mesenchymal stem cell pulverization solution. A method for producing a genomic DNA fragment extracted from human placental-derived mesenchymal stem cells.

19. In paragraph 15, Before the above step (d), the step of removing impurities by washing the precipitate of the above step (c) with 50 to 90% ethanol is further included. A method for producing a genomic DNA fragment extracted from human placental-derived mesenchymal stem cells.

20. In paragraph 15, The molecular reduction in the above step (e) is performed using an indirect ultrasonic disperser. A method for producing a genomic DNA fragment extracted from human placental-derived mesenchymal stem cells.

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