Composition for alleviating oral injuries, its use, and method of manufacture

Abstract

A composition for alleviating oral injuries, its use, and a method for manufacturing the same are disclosed. The composition includes mitochondria and a biocompatible carrier. The composition can alleviate, repair, improve, or treat oral injuries, and it is expected to be a composition or drug that can alleviate, repair, improve, or treat periodontal disease or oral cancer while having both safety and efficacy.

Description

Technical Field

[0001] The present invention relates to a composition for relieving oral injuries, its use, and a method for manufacturing the same.

Background Art

[0002] Gingival fibroblasts are the main cells of the periodontal tissue. When the periodontal tissue is damaged, gingival fibroblasts regulate various cellular functions such as proliferation, migration, elongation, and adhesion, thereby regulating tissue remodeling and wound healing, maintaining the homeostasis of the periodontal tissue, supporting and stabilizing the teeth. Periodontal disease refers to any disease related to the tissues surrounding and supporting the teeth. Periodontal disease includes gingivitis and periodontitis. The former can be recovered with appropriate oral hygiene and treatment, while the latter is an irreversible condition. If the inflammation of the gums persists, the periodontal tissue may be damaged, and there is a possibility of progression from gingivitis to periodontitis.

[0003] According to research, it has been found that air pollution is also associated with periodontal disease, periodontal abscess, oral submucous fibrosis, leukoplakia, or oral cancer. Air pollutants include carbon monoxide, sulfur oxides, nitrogen oxides, ozone, particulate matter (PM), etc. Particulate matter contains various substances such as ions, metals, ammonium salts, sulfates, nitrates, carbon, organic carbon compounds, silica, etc. The composition varies, and some are water-soluble. Particulate matter is generated not only from artificial pollution but also from natural sources. Recently, evidence has been shown that particulate matter is associated with the occurrence of respiratory diseases, cardiovascular diseases, cerebrovascular diseases, diabetes, and even lung cancer and breast cancer. Furthermore, it has been proven that long-term exposure to high concentrations of particulate matter leads to an increase in periodontal disease and oral cancer.

[0004] Modern people are increasingly exposed to air pollution, and the risks of periodontal disease and oral cancer are on the rise. At the advanced stage of periodontal disease and oral cancer, oral hygiene alone cannot completely relieve the symptoms. Although pain and inflammation can be alleviated with analgesics and anti-inflammatory drugs, the structural changes in the gums remain irreversible. When more than 50% of the periodontal tissue is damaged, severe tooth loss may occur. Therefore, one of the current research goals is to find a way to relieve gum damage before gingivitis progresses to periodontitis and then to oral cancer.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present disclosure provides a new approach for improving oral injuries and opens up a new direction for the treatment of oral injuries. The compositions of the present disclosure are expected to be agents or compositions for alleviating, repairing, improving, or treating periodontal disease, periodontal abscess, oral submucous fibrosis, leukoplakia, or oral cancer.

Means for Solving the Problems

[0006] According to one embodiment of the present disclosure, the use of mitochondria in the manufacture of a composition for alleviating oral injuries is provided.

[0007] According to one embodiment of the present disclosure, the composition comprises mitochondria and a biocompatible carrier.

[0008] According to one embodiment of the present disclosure, a method for manufacturing a composition comprising mitochondria and extracellular vesicles includes culturing cells together with a culture medium in a container, separating the supernatant in the container from the cells attached to the container after culturing, collecting extracellular vesicles from the supernatant, lysing the cells to isolate intracellular mitochondria, and mixing the extracellular vesicles and mitochondria to obtain a composition.

[0009] According to an embodiment of the present disclosure, a composition containing mitochondria can mitigate the damage to gingival fibroblasts caused by particulate matter, thereby reducing the death of gingival fibroblasts. Further, the composition containing mitochondria can reduce the aging of gingival fibroblasts caused by particulate matter. Also, the composition containing mitochondria can reduce the production of reactive oxygen species (ROS) in gingival fibroblasts caused by particulate matter, thereby mitigating further damage to gingival fibroblasts by reactive oxygen species. Furthermore, the composition containing mitochondria can mitigate the damage to mitochondria in gingival fibroblasts caused by particulate matter, thereby improving the mitochondrial function of gingival fibroblasts. Additionally, a composition containing mitochondria and platelet-rich plasma-derived extracellular vesicles, a composition containing mitochondria and stem cell-derived extracellular vesicles, and a composition containing mitochondria and extracellular matrix show a synergistic effect in mitigating, repairing, improving, or treating damage to gingival fibroblasts, significantly reducing the aging or death of gingival fibroblasts caused by particulate matter, further mitigating the production of reactive oxygen species and related damage, and further improving the mitochondrial function of gingival fibroblasts. Therefore, the composition of the embodiment of the present disclosure can achieve the purpose of mitigating, repairing, improving, or treating oral damage, and is expected to be a composition or drug that can mitigate, repair, improve, or treat periodontal disease, periodontal abscess, oral submucous fibrosis, leukoplakia, or oral cancer while having both safety and efficacy.

Brief Description of the Drawings

[0010] The present disclosure will be more fully understood from the detailed description provided below and the accompanying drawings, which are for illustrative purposes only and thus do not limit the present disclosure.

[0011]

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Mode for Carrying Out the Invention

[0012] In the following detailed description, for the purpose of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. According to the description, claims, and drawings disclosed in the specification, those skilled in the art can easily understand the concepts and features of the present invention. The following embodiments further illustrate various aspects of the present invention, but are not intended to limit the scope of the present invention.

[0013] In the present disclosure, features and conditions such as values, numbers, contents, amounts, concentrations, etc. presented as ranges are only for the sake of convenience and brevity. Therefore, a range should be interpreted as encompassing all possible sub-ranges, including integers and non-integers, and individual numbers or values therein. For example, a range of "1.0 to 4.0", "1.0~4.0" or "between 1.0 and 4.0" explicitly discloses all sub-ranges such as 1.0 to 4.0, 1.0 to 3.0, 1.0 to 2.0, 2.0 to 4.0, 2.0 to 3.0, 3.0 to 4.0, etc., and should be understood to include the endpoints of the range. In particular, a sub-range defined by a numerical value or value represented by significant digits 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 0 should be interpreted as encompassing all significant digits of the numerical value or value within the range defined by the endpoints. For example, "1.00 to 2.00" should be interpreted as encompassing all individual values such as 1.00, 1.10, 1.20, 1.30, 1.40, 1.50, 1.60, 1.70, 1.80, 1.90, etc.

[0014] On the premise that the objects and advantages of the present disclosure are achieved, numbers have the precision of significant figures. For example, 10.0 is understood to cover the range from 9.50 to 10.49.

[0015] In one embodiment of the present disclosure, the composition includes mitochondria and a biocompatible carrier. The composition according to one embodiment of the present invention can act on gingival fibroblasts to relieve, repair, improve or treat oral injuries. Oral injuries may include oral diseases, and oral diseases may include periodontal diseases, periodontal abscesses, oral submucous fibrosis, leukoplakia, or oral cancer.

[0016] Mitochondria can be collected from any cell having mitochondria, preferably from mammalian monocytes or stem cells, but not limited thereto. For example, the stem cells may be adipose-derived mesenchymal stem cells, embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, CD34+ stem cells, induced pluripotent stem cells or bone marrow stem cells. In some embodiments, the source of mitochondria depends on the subject to which the composition is administered, and it is preferred that the mitochondria are collected from cells of the same species as the subject to which the composition is administered. For example, when the subject to which the composition is administered is a human, the mitochondria can be collected from human cells, and when the subject to which the composition is administered is a dog, the mitochondria can be collected from dog cells. In some embodiments, the mitochondria can be collected from cells of a species different from the subject to which the composition is administered, or the mitochondria may be exogenous mitochondria obtained from in vitro storage or in vitro culture. In some embodiments, the mitochondria may be used immediately after being taken out, or may be used after in vitro storage or in vitro culture.

[0017] The biocompatible carrier may maintain the activity of mitochondria, encapsulate the mitochondria, facilitate the entry of the mitochondria into cells, and enhance the targeting and specificity of the mitochondria. The biocompatible carrier may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include carriers used in any standard medical product or cosmetic. The biocompatible carrier may be semi-solid or liquid depending on the form of the composition. For example, the biocompatible carrier includes, but is not limited to, water, physiological saline, buffer solution, etc.

[0018] In one embodiment, the concentration of mitochondria in the composition may be from 40 μg / mL to 200 μg / mL. In other embodiments, the concentration of mitochondria in the composition may be from 40 μg / mL to 60 μg / mL. In other embodiments, the concentration of mitochondria in the composition may be from 60 μg / mL to 160 μg / mL. In other embodiments, the concentration of mitochondria in the composition may be from 160 μg / mL to 200 μg / mL. In other embodiments, the concentration of mitochondria in the composition may be at least 60 μg / mL.

[0019] In one embodiment, the effective dosage of mitochondria in the composition may be from 10 μg to 50 μg. In other embodiments, the effective dosage of mitochondria in the composition may be from 10 μg to 15 μg. In other embodiments, the effective dosage of mitochondria in the composition may be from 15 μg to 40 μg. In other embodiments, the effective dosage of mitochondria in the composition may be from 40 μg to 50 μg. In other embodiments, the effective dosage of mitochondria in the composition may be at least 15 μg.

[0020] In one embodiment, the composition may be administered to gingival fibroblasts by oral administration, injection, application, topical application, or infusion.

[0021] In another embodiment, the composition may further comprise an extracellular matrix (ECM). The extracellular matrix is a component synthesized by cells and secreted outside the cells, and includes collagen, elastin, laminin, fibronectin, glycosaminoglycan (GAG), proteoglycan (PG), various growth factors and enzymes, etc. The extracellular matrix has the function of supporting and fixing cells, and provides a site for cell communication and control. The extracellular matrix plays an important role in cell growth and the maintenance of cell structure and function. The extracellular matrix can be commercially obtained by a decellularization process after cell culture or enzymatic treatment to preserve the active components. The decellularization process includes rinsing the cells three times with PBS, removing the rinsing buffer, adding cell lysis buffer and incubating at 37°C for 15 minutes, removing the used cell lysis buffer, adding fresh lysis buffer and incubating at 37°C for 40 to 60 minutes, removing the used cell lysis buffer, washing the cells three to four times with buffer, washing the cells four times with deionized water, then washing with PBS, and finally obtaining the extracellular matrix. The extracellular matrix is treated with a proteolytic enzyme (such as trypsin), and the resulting supernatant is the soluble extracellular matrix (soluble ECM).

[0022] In one embodiment, the concentration of the extracellular matrix in the composition may be from 5 mg / mL to 30 mg / mL. In other embodiments, the concentration of the extracellular matrix in the composition may be from 5 mg / mL to 15 mg / mL. In other embodiments, the concentration of the extracellular matrix in the composition may be from 15 mg / mL to 30 mg / mL. In other embodiments, the concentration of the extracellular matrix in the composition may be 15 mg / mL.

[0023] In one embodiment, the ratio of extracellular matrix to mitochondria in the composition may be from 1 μg:2 μg to 1 μg:32 μg. In other embodiments, the ratio of extracellular matrix to mitochondria in the composition may be from 1 μg:2 μg to 1 μg:5.3 μg. In other embodiments, the ratio of extracellular matrix to mitochondria in the composition may be from 1 μg:15 μg to 1 μg:32 μg. In other embodiments, the ratio of extracellular matrix to mitochondria in the composition may be from 1 μg:4 μg to 1 μg:10.67 μg.

[0024] In other embodiments, the composition may further contain extracellular vesicles. The extracellular vesicles may be derived from platelet-rich plasma (PRP), stem cells, monocytes, fibroblasts, neurons, smooth muscle cells, endothelial cells or epidermal cells. The stem cells may be mesenchymal stem cells (such as adipose-derived mesenchymal stem cells or umbilical cord-derived mesenchymal stem cells), hematopoietic stem cells, neural stem cells, embryonic stem cells, umbilical cord blood stem cells, amniotic stem cells, placental stem cells or induced pluripotent stem cells.

[0025] The method for producing PRP-derived extracellular vesicles will be described in the following embodiments. The PRP-derived extracellular vesicles according to one embodiment of the present disclosure are vesicles having a lipid membrane structure, with a size of about 30 nm to 1000 nm, and encapsulating substances such as nucleic acids, peptides, proteins, and lipids. The PRP-derived extracellular vesicles express platelet-specific surface antigen CD41 and extracellular vesicle-specific surface antigens CD9, CD63, and Alix.

[0026] In one embodiment, the concentration of PRP-derived extracellular vesicles in the composition may be from 0.5 mg / mL to 2.5 mg / mL. In other embodiments, the concentration of PRP-derived extracellular vesicles in the composition may be from 0.5 mg / mL to 1 mg / mL. In other embodiments, the concentration of PRP-derived extracellular vesicles in the composition may be from 1.5 mg / mL to 2.5 mg / mL. In other embodiments, the concentration of PRP-derived extracellular vesicles in the composition may be 1.25 mg / mL.

[0027] In one embodiment, the concentration of PRP-derived extracellular vesicles in the composition may be from 1% (v / v) to 5% (v / v). In other embodiments, the concentration of PRP-derived extracellular vesicles in the composition may be from 1% (v / v) to 2.5% (v / v). In other embodiments, the concentration of PRP-derived extracellular vesicles in the composition may be from 2.5% (v / v) to 5% (v / v). In other embodiments, the concentration of PRP-derived extracellular vesicles in the composition may be 2.5% (v / v).

[0028] In one embodiment, the ratio of PRP-derived extracellular vesicles to mitochondria in the composition may be from 1 mg:64 μg to 1 mg:320 μg. In other embodiments, the ratio of PRP-derived extracellular vesicles to mitochondria in the composition may be from 1 mg:160 μg to 1 mg:320 μg. In other embodiments, the ratio of PRP-derived extracellular vesicles to mitochondria in the composition may be from 1 mg:64 μg to 1 mg:106.6 μg. In other embodiments, the ratio of PRP-derived extracellular vesicles to mitochondria in the composition may be 1 mg:128 μg.

[0029] In one embodiment, the ratio of PRP-derived extracellular vesicles to mitochondria in the composition may be from 1 μL:3.2 μg to 1 μL:16 μg. In other embodiments, the ratio of PRP-derived extracellular vesicles to mitochondria in the composition may be from 1 μL:3.2 μg to 1 μL:6.4 μg. In other embodiments, the ratio of PRP-derived extracellular vesicles to mitochondria in the composition may be from 1 μL:8 μg to 1 μL:16 μg. In other embodiments, the ratio of PRP-derived extracellular vesicles to mitochondria in the composition may be 1 μL:6.4 μg.

[0030] The method for producing stem cell-derived extracellular vesicles will be described in the following embodiments. In the composition according to one embodiment of the present disclosure, the stem cell-derived extracellular vesicles and mitochondria may be derived from the same stem cells. The stem cell-derived extracellular vesicles according to one embodiment of the present disclosure are vesicles having a lipid membrane structure, and their size is about 30 nm to 1000 nm. The vesicles encapsulate substances such as nucleic acids, peptides, proteins, and lipids. Stem cell-derived extracellular vesicles express surface antigens CD40, CD63, CD81, CD9, Alix, Hsp60, Hsp70, and Hsp90.

[0031] In one embodiment, the concentration of stem cell-derived extracellular vesicles in the composition may be from 1 μg / mL to 20 μg / mL. In other embodiments, the concentration of stem cell-derived extracellular vesicles in the composition may be from 1 μg / mL to 5 μg / mL. In other embodiments, the concentration of stem cell-derived extracellular vesicles in the composition may be from 15 μg / mL to 20 μg / mL. In other embodiments, the concentration of stem cell-derived extracellular vesicles in the composition may be 10 μg / mL.

[0032] In an embodiment, to deliver stem cell-derived extracellular vesicles to mitochondria, it is 1 μg:8 μg to 1 μg:160 μg. In another embodiment, transporting stem cell-derived extracellular vesicles to mitochondria is 1 μg:8 μg to 1 μg:10.6 μg. In other embodiments, transporting stem cell-derived extracellular vesicles to mitochondria is 1 μg:32 μg to 1 μg:160 μg. In other embodiments, transporting stem cell-derived extracellular vesicles to mitochondria is 1 μg:16 μg.

[0033] According to another embodiment of the present disclosure, there is provided the use of mitochondria in the manufacture of a composition for alleviating, repairing, improving or treating oral injuries. The composition may be the above composition containing mitochondria. The oral injury may be an injury to gingival fibroblasts. The oral injury may include periodontal disease, periodontal abscess, oral submucous fibrosis, leukoplakia, or oral cancer. The composition may improve the mitochondrial membrane potential and mitochondrial ATP production in the oral cavity. Kill gingival fibroblasts, thereby improving the mitochondrial function of gingival fibroblasts. This composition can reduce the death of gingival fibroblasts, reduce the aging of gingival fibroblasts, reduce the reactive oxygen species generated by gingival fibroblasts, or improve the mitochondrial function of gingival fibroblasts, thereby alleviating, repairing, improving or treating oral injuries.

[0034] According to another embodiment of the present invention, there is provided a method for manufacturing a composition for alleviating oral injuries. This method includes dispersing mitochondria in the extracellular matrix to obtain a composition. Specifically, the mitochondria and the extracellular matrix are sufficiently mixed, for example, by shaking, using a stir bar, pipetting, or magnetic stirring, so that the mitochondria are dispersed in the extracellular matrix to form a composition containing the mitochondria and the extracellular matrix. The mixing time is from 5 minutes to 60 minutes, for example, 15 minutes, 30 minutes, 60 minutes, preferably within 60 minutes. The ratio of the extracellular matrix to mitochondria is from 1 μg:2 μg to 1 μg:32 μg, preferably from 1 μg:4 μg to 1 μg:10.67 μg. After mixing the mitochondria and the extracellular matrix to form a composition, the composition can be administered to the oral injury site.

[0035] According to another embodiment of the present invention, a method for producing a composition containing mitochondria and extracellular vesicles includes culturing cells together with a medium in a container, separating the supernatant in the container from the cells attached to the container after culturing, collecting extracellular vesicles from the supernatant, lysing the cells to isolate intracellular mitochondria, and mixing the extracellular vesicles and mitochondria to obtain a composition. By this method, mitochondria and extracellular vesicles can be simultaneously extracted from the same cells.

[0036] Specifically, according to the type of cells to be cultured, a cell culture medium, a culture container, and a culture method can be selected. After cell culture, the supernatant in the container can be separated from the cells attached to the container by pouring or pipetting. Extracellular vesicles can be collected from the supernatant by tangential flow filtration (TFF). Cells can be collected by enzymatic digestion with trypsin and centrifugation, and intracellular mitochondria can be isolated by lysing the cells by physical grinding or chemical lysis. The obtained extracellular vesicles and the obtained mitochondria are mixed to form a composition. The mixing ratio of extracellular vesicles and mitochondria may be from 1 μg:8 μg to 1 μg:160 μg, for example 1 μg:16 μg. The step of collecting extracellular vesicles and the step of isolating mitochondria may be performed simultaneously or in sequence, and the order is not limited. The cells used in this method may be stem cells such as mesenchymal stem cells (such as adipose-derived mesenchymal stem cells or umbilical cord-derived mesenchymal stem cells), hematopoietic stem cells, neural stem cells, embryonic stem cells, umbilical cord blood stem cells, amniotic stem cells, placental stem cells, or induced pluripotent stem cells.

[0037] The materials used in the experiment are as follows.

[0038] It is obtained from human adipose-derived mesenchymal stem cells (ADSCs). Adipose-derived mesenchymal stem cells express surface markers CD73, CD90, and CD105, and do not express surface markers CD34 and CD45. The stem cell culture medium contains keratinocyte SFM1X solution (Gibco), bovine pituitary extract (BPE, Gibco), and 10% (v / v) FBS (HyClone). First, ADSCs are cultured in a Petri dish to 1.5×10 8 cells, and then washed with Dulbecco's phosphate-buffered saline (DPBS). Next, the DPBS is removed, trypsin for detaching adherent cells from the surface of the dish is added, reacted at 37°C for 3 minutes, and the stem cell medium is added to stop the reaction. Next, the ADSCs are washed out from the dish, dispersed, centrifuged at 600g for 10 minutes, and the supernatant is removed. Next, the remaining ADSCs and 80 mL of IBC-1 buffer (225 mM mannitol, 75 mM sucrose, 0.1 mM EDTA, and 30 mM Tris-HCl pH 7.4) are added to a homogenizer, and the ADSCs are ground with the homogenizer on ice. Next, the ground ADSCs are centrifuged at 600g at 4°C for 5 minutes, and the supernatant is collected. Next, the supernatant is centrifuged at 10000g at 4°C for 10 minutes, and the supernatant is removed. Mitochondria are extracted using a human mitochondrial isolation kit (purchased from Miltenyi Biotec, Germany). Magnetic microbead antibody anti-TOM22 is added to the obtained extract, reacted on ice for 1 hour, and then the mitochondria are purified by magnetic separation. The protein concentration of the purified mitochondria is measured and defined as the weight of the mitochondria.

[0039] The extracellular matrix (ECM) used in the embodiments of the present invention is purchased from Sigma (MaxGelTM ECM, E0282). The extracellular matrix contains human extracellular matrix components such as collagen, laminin, fibronectin, tenascin, elastin, proteoglycan, and glycosaminoglycan. In the following experiments, mitochondria and the extracellular matrix are mixed, the mitochondria are dispersed in the extracellular matrix to form a composition containing mitochondria and the extracellular matrix, and after mixing for 15 minutes, this composition is administered to the experimental cells.

[0040] The PRP-derived extracellular vesicles used in the embodiments of the present disclosure can be produced as follows. Whole blood is collected from the median cubital vein using a 19G butterfly needle, and the first 5 mL of the collected blood is discarded, and about 20 mL of blood is collected. The collected blood is placed in a 50 mL centrifuge tube containing 3.2% (v / v) trisodium citrate. The blood is centrifuged at 2500 g for 15 minutes, and about 5 - 6 mL of the supernatant is collected. This is platelet-rich plasma (PRP). 5 - 6 mL of the obtained PRP is mixed with phosphate-buffered saline (PBS) (without calcium ions and magnesium ions) at a ratio of 1:1, centrifuged at 10000 g at 4 °C for 120 minutes, and the supernatant is removed to obtain 30 to 70 mg of PRP-derived extracellular vesicles. The obtained PRP-derived extracellular vesicles are resuspended in 1 mL of PBS to obtain PRP extracellular vesicles (hereinafter referred to as PRP-EV) at about 50.05 ± 17.66 mg / mL. PRP-derived extracellular vesicles express platelet-specific surface antigen CD41 and extracellular vesicle-specific surface antigens CD9, CD63, and Alix.

[0041] The stem cell-derived extracellular vesicles (MSC-derived extracellular vesicles) used in the embodiments of the present invention can be produced as follows. In the composition containing mitochondria and stem cell-derived extracellular vesicles according to the embodiments of the present invention, the mitochondria and extracellular vesicles are derived from the same stem cells. The stem cells used in the embodiments are adipose-derived mesenchymal stem cells. The culture medium for the stem cells contains keratinocyte SFM 1X solution (Gibco), EGF (Gibco), bovine pituitary extract (BPE, Gibco), N-acetyl-L-cysteine (Sigma), L-ascorbic acid 2-phosphate (magnesium salt hydrate) (Sigma), 10% (v / v) FBS (HyClone). First, the stem cells are cultured until the culture dish is 80% confluent, and then the medium is replaced with fresh medium and cultured for 24 hours. Then, the medium is removed and the cells are washed away with PBS. Then, the washed PBS is removed, fresh medium is added, and the cells are cultured for 48 hours. After culturing, the supernatant in the culture dish and the stem cells attached to the culture dish are separated with a pipette.

[0042] 175 mL of the supernatant is filtered through a 0.22 μm filter. The extracellular vesicles are purified and concentrated from the supernatant using a tangential flow filtration (TFF) system (100 kDam PES filter membrane, D02-E100-05-N) to obtain 35 mL of purified supernatant. The substances contained in the purified supernatant are defined as extracellular vesicles. The stem cells attached to the culture dish are collected by enzymatic digestion with trypsin and pulverized as described in the method for extracting mitochondria to extract and purify mitochondria. By this method, mitochondria and extracellular vesicles can be simultaneously extracted from the same stem cells.

[0043] In the following experiments, human gingival fibroblasts (HGF) are used for the study of oral injuries. The culture medium for human gingival fibroblasts may contain DMEM (Dulbecco's Modified Eagle Medium), 4.5 g / L D-glucose, 110 mg / L sodium pyruvate, 584 mg / L glutamine, 3.7 g / L sodium bicarbonate, 10% (v / v) fetal bovine serum (FBS). Human gingival fibroblasts are subcultured at 3000 - 6000 cells / cm 2It is cultured at 37°C at a density of. Human gingival fibroblasts are cultured until the culture dish is 90% full, then the culture medium is removed and the cells are rinsed with phosphate-buffered saline (PBS). Next, the PBS is removed, 0.25% trypsin is added to the culture dish, incubated at 37°C for 5 minutes, and then fresh culture medium is added to stop the trypsin reaction. Next, the cells are centrifuged at 300g for 5 minutes to remove the supernatant, then fresh culture medium is added to count the cells, and cell subculture is performed according to the experimental requirements.

[0044] In the following experiment, urban particulate matter (purchased from Merck, NIST1648A) is used as a damaging factor for human gingival fibroblasts. Hereinafter, urban particulate matter is referred to as particulate matter or PM. 0.01 g of PM is placed in an Eppendorf and 1 mL of PBS is added. The Eppendorf is sealed with parafilm and shaken for 1 hour using an ultrasonic water bath shaker. After shaking, the solution is used as a stock solution (concentration 10 μg / μL) and stored in a 4°C refrigerator for use in the next experiment.

[0045] In the following experiment, a CCK-8 kit (purchased from Dojindo, CK04) is used to analyze cell viability. WST-8 is the main reagent of the CCK-8 kit. WST-8 has low cytotoxicity, high sensitivity, strong water solubility, and is easy to store. WST-8 reacts with dehydrogenases in living cells and is reduced from pink to orange (formazan dye). The amount of formazan produced is proportional to the number of living cells. Therefore, in cytotoxicity tests and cell proliferation tests, cell viability can be analyzed by measuring the absorbance (OD450nm) due to formazan using a spectrophotometer.

[0046] In the following experiments, the SA-β-galactosidase staining kit (#9860 Senescence β-Galactosidase Staining Kit, purchased from Cell Signaling Technology) was used to evaluate the level of cellular senescence. In senescent cells, senescence-associated β-galactosidase (SA-β-gal) is overexpressed, and SA-β-gal may serve as a biomarker for the level of cellular senescence. Therefore, cellular senescence may be observed through SA-β-gal staining.

[0047] In the following experiments, CM-H2DCFDA (purchased from Invitrogen, C6827) was used to analyze reactive oxygen species (ROS) in cells. CM-H2DCFDA can permeate the cell membrane and react with intracellular ROS to form highly fluorescent products, so it is often used as an indicator of ROS.

[0048] In the following experiments, JC-1 dye (Invitrogen T3168, purchased from Fisher Scientific) was used to analyze the mitochondrial membrane potential. When the mitochondrial function of cells is normal, the mitochondria are polarized and the mitochondrial membrane potential is negatively charged. At this time, the positively charged JC-1 dye accumulates on the mitochondrial membrane to form JC-1 aggregates and emits red fluorescence. When mitochondrial function is damaged, the mitochondria are depolarized and the mitochondrial membrane potential collapses. At this time, the JC-1 dye does not form aggregates, and the JC-1 monomers are distributed in the cell and emit green fluorescence. Therefore, the ratio of JC-1 monomers / JC-1 aggregates (hereinafter referred to as the JC-1 ratio) can be obtained by fluorescence measurement and can be used as an indicator for evaluating mitochondrial function. A high JC-1 monomer / JC-1 aggregate indicates a decrease in the mitochondrial membrane potential and a decrease in the mitochondrial function of the cell.

[0049] In the following experiments, an ATP assay kit (purchased from BioVision, K354-100) is used to analyze the amount of ATP. One of the important functions of mitochondria is to generate ATP via the electron transport system for use in cells. When mitochondria are damaged, their ability to generate ATP is also affected. Therefore, measuring ATP production can indicate the ATP-generating ability of mitochondria and can be used as an indicator to evaluate mitochondrial function.

[0050] Unless otherwise specified, experimental values are shown as mean ± standard deviation and are statistically analyzed by ANOVA test and Tukey's post hoc test.

[0051] Experiment 1: Cytotoxicity of particulate matter (PM) on human gingival fibroblasts

[0052] Human gingival fibroblasts are cultured for 24 hours at a density of 40,000 cells per well in 0.5 mL of culture medium in a 24-well plate with a bottom area of 1.8 cm 2 . Then, after culturing until the cells fill 80% of the well, the culture medium is removed, and the cells are rinsed with 0.5 mL of PBS per well. Next, the rinsed PBS is removed, and fresh DMEM containing 1% FBS (250 μL / well) is added. Then, PM is added to the wells at concentrations of 0, 25, 50, or 100 μg / cm 2 . After culturing the cells with PM at 37°C and 5% CO 2 for 24 hours, cell viability is analyzed using a CCK-8 kit.

[0053] The experimental results are shown in Table 1 and Figure 1. Figure 1 shows the cell viability of human gingival fibroblasts treated with PM compared to the control group. In Figure 1, the control group is cells without PM (PM is 0 μg / cm 2 ), and the symbol "♯" represents a statistically significant difference compared to the control group (♯♯♯ is P < 0.001). From the experimental results, it was found that PM damages human gingival fibroblasts. Also, the degree of damage becomes more severe as the concentration of PM increases.

[0054]

Table 1

[0055] Experiment 2: Aging of human gingival fibroblasts by PM

[0056] The procedure of this experiment is generally the same as that of Experiment 1, and only the differences are described below. PM is added to the wells at a concentration of 0, 25, 50, or 100 μg / cm 2 After culturing the cells with PM at 37 °C, 5% CO 2 for 24 hours, the aging level of the cells is evaluated using the SA-β-gal kit.

[0057] The experimental results are shown in Table 2, Figure 2, and Figure 3. Figure 2 shows the stained images of cell aging of human gingival fibroblasts treated with PM. Figure 3 shows the cell aging level of human gingival fibroblasts treated with PM. In Figure 2 and Figure 3, the control group is cells without PM (PM is 0 μg / cm 2 ), and the symbol "♯" indicates a statistically significant difference compared with the control group (♯♯ is P < 0.01, ♯♯♯ is P < 0.001). From the experimental results, PM induces the aging of human gingival fibroblasts. Also, even when the PM concentration increases, the aging level decreases. The reason is presumably that higher concentrations (50 μg / cm 2 and 100 μg / cm 2 ) of PM cause cell death, resulting in a decrease in the stained cells and thereby a decrease in the aging level.

[0058]

Table 2

[0059] Experiment 3: Production of reactive oxygen species (ROS) in human gingival fibroblasts by PM

[0060] The procedure of this experiment is generally the same as that of Experiment 1, and only the differences will be explained below. Human gingival fibroblasts are cultured for 24 hours, the culture medium is removed, and the cells are rinsed with PBS. Next, the rinsed PBS is removed, and fresh DMEM (250 μL / well) containing 1% FBS and 10 μM CM-H2DCFDA is added, and the reaction is carried out at 37 °C for 45 minutes in the dark. After the reaction, the supernatant in the well is removed, and the cells are rinsed with 0.5 mL PBS per well. Next, the rinsed PBS is removed, and DMEM (250 μL / well) containing fresh 1% FBS is added. Next, PM is added into the well at a concentration of 0, 10, 25, or 50 μg / cm 2 2. The cells are cultured with PM at 37 °C in 5% CO 2 2 in the dark for 24 hours. After cell culture, the supernatant in the well is removed, and the cells are rinsed with 0.5 mL of PBS per well. Next, the rinsed PBS is removed, and 250 μL of RIPA lysis and extraction buffer (purchased from Thermo Scientific, 89900) is added per well to lyse the cells. The obtained solution is collected in a 1.5 mL tube and centrifuged at 300 g for 1 minute. 200 μL of the supernatant is loaded onto a 96-well black plate, and fluorescence is measured at OD485 (excitation) and OD530 (emission) to analyze the amount of ROS.

[0061] The experimental results are shown in Table 3 and Figure 4. Figure 4 shows the comparison of ROS production in PM-treated human gingival fibroblasts with the control group. In Figure 4, the control group is cells without PM (PM is 0 μg / cm 2 2), and the symbols "♯" indicate statistically significant differences compared with the control group (♯ is P < 0.01, ♯♯♯ is P < 0.001). From the experimental results, PM induces the generation of ROS in human gingival fibroblasts. Also, the generated ROS increases as the concentration of PM increases. The generation and increase of ROS further cause oxidative damage to human gingival fibroblasts.

[0062]

Table 3

[0063] Experiment 4: Damage to Mitochondria of Human Gingival Fibroblasts by PM - Membrane Potential Analysis

[0064] The procedure for this experiment is generally the same as that of Experiment 1, and only the differences are explained below. PM was added to the wells at concentrations of 0, 10, 25, and 50 μg / cm 2 , and after culturing the cells at 37°C in 5% CO 2 for 24 hours, the supernatant in the wells was removed, and the cells were washed with 0.5 mL of PBS per well. Then, the washed PBS was removed, and fresh DMEM (250 μL / well) containing 1% FBS and 5 μM JC - 1 was added and reacted at 37°C for 10 minutes. After the reaction, the supernatant in the wells was removed, and the cells were washed twice with 0.5 mL of PBS per well. Then, fresh DMEM (250 μL / well) containing 1% FBS was added. The fluorescence of JC - 1 aggregates was measured at OD520 (excitation) and OD590 (emission), and the fluorescence of JC - 1 monomers was measured at OD490 (excitation) and OD530 (emission), thereby evaluating the mitochondrial membrane potential of human gingival fibroblasts.

[0065] The experimental results are shown in Table 4 and Figure 5. Figure 5 shows the JC - 1 monomer / JC - 1 aggregate ratio (JC - 1 ratio) of mitochondria in PM - treated human gingival fibroblasts. In Figure 5, the control group is cells without PM (PM is 0 μg / cm 2 ), and the symbols "♯" indicate statistically significant differences compared to the control group (♯ is P < 0.01, ♯♯♯ is P < 0.001). From the experimental results, PM increases the JC - 1 monomer / JC - 1 aggregate ratio (JC - 1 ratio) of mitochondria in human gingival fibroblasts, indicating that the mitochondrial membrane is damaged and mitochondrial function is impaired. Also, the degree of mitochondrial damage becomes more severe as the concentration of PM increases.

[0066]

Table 4

[0067] Experiment 5: Damage to Mitochondria of Human Gingival Fibroblasts by PM - ATP Production

[0068] The procedure of this experiment is generally the same as that of Experiment 1, and only the differences will be explained below. Human gingival fibroblasts are cultured in a 10-cm dish (the bottom area of the dish is 60.8 cm 2 ) with 10 mL of DMEM containing 10% FBS at a density of 3.5×10 5 cells for 24 hours. Next, after culturing the cells to 80% of the dish, the medium is removed, and the cells are rinsed with 10 mL of PBS per dish. Next, the rinsed PBS is removed, and fresh DMEM containing 1% FBS (5 mL / dish) is added. Next, PM is added to the dish at a concentration of 0, 10, 25, or 50 μg / cm 2 . After culturing the cells with PM for 24 hours, the mitochondrial ATP production in the cells is analyzed using an ATP assay kit.

[0069] The experimental results are shown in Table 5 and Figure 6. Figure 6 shows the mitochondrial ATP production in PM-treated human gingival fibroblasts. In Figure 6, the control group is cells without PM (PM is 0 μg / cm 2 ), and the symbol "♯" indicates a statistically significant difference compared to the control group (♯♯♯ means P < 0.001). From the experimental results, PM reduces the mitochondrial ATP production in human gingival fibroblasts, indicating that the mitochondrial ATP production ability is impaired and the mitochondrial function is impaired. Furthermore, as the concentration of PM increases, the degree of damage to mitochondria also becomes more severe.

[0070]

Table 5

[0071] Experiment 6: Mitochondria Reduce PM-Induced Cell Death in Human Gingival Fibroblasts

[0072] The procedure of this experiment is generally the same as that of Experiment 1, and only the differences will be explained below. PM is added to the wells at 0 or 50 μg / cm 2Add at the concentration of. Incubate the cells with PM at 37 °C and 5% CO 2 for 6 hours. Then, wash the cells in each well with 0.5 mL of PBS. Next, remove the washed PBS, and add fresh DMEM containing 1% FBS (250 μL / well) and the compositions of each example and comparative example, and incubate at 37 °C and 5% CO 2 for 20 hours. After cell culture, analyze the cell viability using a CCK-8 kit.

[0073] The experimental results of the composition containing extracellular matrix (ECM) and mitochondria are shown in Table 6 and Figure 7. Figure 7 shows the cell viability of human gingival fibroblasts treated with PM and then with the compositions of the examples and comparative examples, compared with the control group. In Figure 7, the control group is cells without PM, mitochondria, and ECM (Control Example 1-1). The symbol "♯" indicates a statistically significant difference (♯♯♯ means P < 0.001) compared with the control group (Control Example 1-1), and the symbol "*" indicates a statistically significant difference (*** means P < 0.001) compared with the comparative example (Comparative Example 1-1). From Control Examples 1-1 to 1-6, when the cells are not damaged, adding only mitochondria or ECM does not decrease the cell viability, but rather slightly increases it. Therefore, it is shown that mitochondria and ECM have no cytotoxicity to human gingival fibroblasts, and it can even be said that the composition containing mitochondria or the composition containing mitochondria and ECM promotes the growth of human gingival fibroblasts. Also, from the examples and comparative examples, when the cells are damaged, it is shown that adding mitochondria improves the cell viability (Examples 1-1 and 1-2). The addition of mitochondria contributes to alleviating, repairing, improving, or treating the damage of human gingival fibroblasts caused by PM, and it is shown that it further reduces the death of human gingival fibroblasts caused by PM. Furthermore, from the examples and comparative examples, when the cells are damaged, adding the composition containing mitochondria and ECM further increases the cell viability (Examples 1-3 and 1-4), and there is a statistically significant difference. This indicates that the addition of the composition containing mitochondria and ECM shows a synergistic effect in alleviating, repairing, improving, or treating the damage to human gingival fibroblasts, and can significantly reduce the death of human gingival fibroblasts caused by PM.

[0074]

Table 6

[0075] The experimental results of the composition containing platelet-rich plasma-derived extracellular vesicles (PRP-EVs) and mitochondria are shown in Table 7 and Figure 8. Figure 8 shows the cell viability of human gingival fibroblasts treated with PM and then with the compositions of the examples and comparative examples, compared with the control group. In Figure 8, the control group is cells that do not contain PM, mitochondria, and PRP-EVs (Control Example 2-1), and the symbol "♯" indicates a statistically significant difference (♯♯♯ means P < 0.001) compared with the control group (Control Example 2-1). From Control Examples 2-1 to 2-3, when the cells are not damaged, adding only mitochondria or PRP-EV does not decrease the cell viability; rather, it slightly increases. Therefore, it is shown that mitochondria and PRP-EV have no cytotoxicity to human gingival fibroblasts, and it can even be said that the composition containing mitochondria promotes the proliferation of human gingival fibroblasts. Also, from the examples and comparative examples, when the cells are damaged, the cell viability can be increased by adding mitochondria (Example 2-1). Therefore, it is shown that the addition of mitochondria contributes to alleviating, repairing, improving, or treating the damage of human gingival fibroblasts caused by PM, and further reducing the death of human gingival fibroblasts caused by PM. Furthermore, from the examples and comparative examples, when the cells are damaged, it can be seen that adding the composition containing mitochondria and PRP-EV further increases the cell viability (Example 2-2). This indicates that the addition of the composition containing mitochondria and PRP-EV exerts a synergistic effect in alleviating, repairing, improving, or treating the damage to human gingival fibroblasts, and can significantly reduce the death of human gingival fibroblasts caused by PM.

[0076]

Table 7

[0077] The experimental results of the composition containing mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) and mitochondria are shown in Table 8 and Figure 9. Figure 9 shows the cell viability of human gingival fibroblasts treated with PM and then with the compositions of the examples and comparative examples, compared with the control group. In Figure 9, the control group is cells that do not contain PM, mitochondria, and MSC-EVs (Control Example 3-1). The symbol "♯" indicates a statistically significant difference (♯♯♯ indicates P < 0.001) compared with the control group (Control Example 3-1), and the symbol "*" indicates a statistically significant difference (*** indicates P < 0.001) compared with the comparative example (Comparative Example 3-1). From Control Examples 3-1 to 3-3, when the cells are not damaged, adding only mitochondria or MSC-EVs does not reduce the cell viability, indicating that mitochondria and MSC-EVs have no cytotoxicity to human gingival fibroblasts. Also, from the examples and comparative examples, when the cells are damaged, adding mitochondria can increase the cell viability (Example 3-1), indicating that the addition of mitochondria contributes to alleviating, repairing, improving, or treating the damage of human gingival fibroblasts caused by PM, and further reducing the death of human gingival fibroblasts caused by PM. Furthermore, from the examples and comparative examples, when the cells are damaged, adding a composition containing mitochondria and MSC-EVs further improves the cell viability (Example 3-2), indicating that the addition of the composition containing mitochondria and MSC-EVs exerts a synergistic effect in alleviating, repairing, improving, or treating the damage to human gingival fibroblasts, and can significantly reduce the death of human gingival fibroblasts caused by PM.

[0078]

Table 8

[0079] Experiment 7: Mitochondria Reduce the Senescence of Human Gingival Fibroblasts Caused by PM

[0080] The procedure of this experiment is generally the same as that of Experiment 1, and only the differences are described below. PM is added to the wells at 0 or 25 μg / cm2 Add at the concentration of. Incubate the cells at 37 °C in 5% CO 2 for 6 hours in PM culture. After that, wash the cells with 0.5 mL of PBS per well. Then, remove the washed PBS and add fresh DMEM (250 μL / well) containing 1% FBS and the compositions of each example and comparative example, and incubate at 37 °C in 5% CO 2 for 20 hours. After cell culture, evaluate the cell senescence level using the SA-β-gal kit.

[0081] The experimental results are shown in Table 9 and Figure 10. Figure 10 shows the cell senescence levels of human gingival fibroblasts treated with the compositions of the examples and comparative examples after PM treatment. In Figure 10, the control group is cells without PM, mitochondria, and ECM (Control Example 4-1). The symbol "♯" indicates a statistically significant difference compared to the control group (Control Example 4-1) (♯♯♯ means P < 0.001), and the symbol "*" indicates a statistically significant difference compared to the comparative example (Comparative Example 4-1) (*** means P < 0.001). From Control Examples 4-1 to 4-6, it can be seen that when the cells are not damaged, adding only mitochondria or ECM does not induce cell senescence. Also, from the examples and comparative examples, when the cells are damaged, adding mitochondria can reduce the cell senescence level (Examples 4-1 and 4-2). Thus, it can be understood that the addition of mitochondria contributes to alleviating, repairing, improving, or treating the damage of human gingival fibroblasts caused by PM and further reducing the senescence of human gingival fibroblasts caused by PM. Furthermore, from the examples and comparative examples, when the cells are damaged, adding a composition containing mitochondria and ECM can further reduce the cell senescence level (Examples 4-3 and 4-4), and there is a statistically significant difference, indicating that the addition of the composition containing mitochondria and ECM shows a synergistic effect in alleviating, repairing, improving, or treating the damage to human gingival fibroblasts and can significantly reduce the senescence of human gingival fibroblasts caused by PM.

[0082]

Table 9

[0083] Experiment 8: Mitochondria Reduce ROS Production in Human Gingival Fibroblasts Induced by PM

[0084] The procedure for this experiment is generally the same as that of Experiment 1, and only the differences are described below. Human gingival fibroblasts are cultured for 24 hours, the culture medium is removed, and the cells are washed with PBS. Then, the rinsed PBS is removed, and fresh DMEM containing 1% FBS and 10 μM CM-H2DCFDA (250 μL / well) is added, and the reaction is carried out at 37 °C for 45 minutes in the dark. After the reaction, the supernatant in the well is removed, the cells are rinsed with 0.5 mL of PBS per well, then the rinsed PBS is removed, and fresh DMEM containing 1% FBS (250 μL / well) is added. Next, PM is added to the well at a concentration of 0 or 50 μg / cm 2 . The cells are cultured with PM at 37 °C, 5% CO 2 for 6 hours, and then the cells are rinsed with 0.5 mL of PBS per well. Next, the rinsed PBS is removed, and fresh DMEM containing 1% FBS (250 μL / well) and the compositions of each example and comparative example are added, and the cells are cultured at 37 °C, 5% CO 2 for 20 hours. After cell culture, the supernatant in the well is removed, and the cells are rinsed with 0.5 mL of PBS per well. Next, the rinsed PBS is removed, and RIPA Lysis and Extraction Buffer (purchased from Thermo Scientific, 89900) is added at 250 μL per well to lyse the cells. The obtained solution is collected in a 1.5 mL tube and centrifuged at 300 g for 1 minute. 200 μL of the supernatant is loaded onto a 96-well black plate, and fluorescence is measured at OD485 (excitation) and OD530 (emission) to analyze the amount of ROS.

[0085] The experimental results of the composition containing extracellular matrix (ECM) and mitochondria are shown in Table 10 and Figure 11. Figure 11 shows the ROS production of human gingival fibroblasts treated with PM and then with the compositions of the examples and comparative examples, compared with the control group. In Figure 11, the control group is cells without PM, mitochondria, and ECM (Control Example 5-1). The symbol "♯" indicates a statistically significant difference compared with the control group (Control Example 5-1) (♯♯♯ indicates P < 0.001), and the symbol "*" indicates a statistically significant difference compared with the comparative example (Comparative Example 5-1) (*** indicates P < 0.001). In Control Examples 5-1 to 5-6, when the cells are not damaged, adding only mitochondria or ECM does not affect ROS production, indicating that mitochondria and ECM do not induce ROS production in human gingival fibroblasts. Also, from the examples and comparative examples, it is shown that when the cells are damaged, adding mitochondria reduces ROS production (Examples 5-1 and 5-2), indicating that the addition of mitochondria contributes to the alleviation, repair, improvement, or treatment of the damage to human gingival fibroblasts caused by PM and further alleviates the damage caused by ROS. Furthermore, from the examples and comparative examples, when the cells are damaged, adding a composition containing mitochondria and ECM can further reduce ROS production compared with adding an equal amount of mitochondria (Examples 5-3 and 5-4), with a statistically significant difference, indicating that the addition of the composition containing mitochondria and ECM shows a synergistic effect in alleviating, repairing, improving, or treating the damage to human gingival fibroblasts and can significantly reduce further damage caused by ROS.

[0086]

Table 10

[0087] The experimental results of the composition containing platelet-rich plasma-derived extracellular vesicles (PRP-EVs) and mitochondria are shown in Table 11 and Figure 12. Figure 12 shows the ROS production of human gingival fibroblasts treated with the compositions of the examples and comparative examples after treatment with PM, compared with the control group. In Figure 12, the control group is cells that do not contain PM, mitochondria, and PRP-EVs (Control Example 6-1). The symbol "♯" indicates a statistically significant difference (♯♯♯ indicates P < 0.001) compared with the control group (Control Example 6-1), and the symbol "*" indicates a statistically significant difference (*** indicates P < 0.001) compared with the comparative example (Comparative Example 6-1). From Control Examples 6-1 to 6-3, when the cells are not damaged, adding only mitochondria or PRP-EVs has no effect on ROS production, indicating that mitochondria and PRP-EVs do not induce ROS production in human gingival fibroblasts. Also, from the examples and comparative examples, when the cells are damaged, adding mitochondria reduces ROS production (Example 6-1), indicating that the addition of mitochondria contributes to the alleviation, repair, improvement, or treatment of the damage to human gingival fibroblasts caused by PM and further alleviates the damage caused by ROS. Furthermore, from the examples and comparative examples, when the cells are damaged, adding a composition containing mitochondria and PRP-EVs can further reduce ROS production compared with adding an equal amount of mitochondria (Example 6-2), with a statistically significant difference, indicating that the addition of the composition containing mitochondria and PRP-EVs shows a synergistic effect in the alleviation, repair, improvement, or treatment of the damage to human gingival fibroblasts and can significantly reduce further damage caused by ROS.

[0088]

Table 11

[0089] The experimental results of the composition containing mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) and mitochondria are shown in Table 12 and Figure 13. Figure 13 shows the ROS production of human gingival fibroblasts treated with the compositions of the examples and comparative examples after treatment with PM, compared with the control group. In Figure 13, the control group is cells that do not contain PM, mitochondria, or MSC-EVs (Control Example 7-1). The symbol "♯" indicates a statistically significant difference (♯♯♯ indicates P < 0.001) compared with the control group (Control Example 7-1), and the symbol "*" indicates a statistically significant difference (*** indicates P < 0.001) compared with the comparative example (Comparative Example 7-1). From Control Examples 7-1 to 7-3, when the cells are not damaged, adding only mitochondria or MSC-EVs has no effect on ROS production, indicating that mitochondria and MSC-EVs do not induce ROS production in human gingival fibroblasts. Also, from the examples and comparative examples, when the cells are damaged, adding mitochondria can reduce ROS production (Example 7-1), indicating that adding mitochondria contributes to alleviating, repairing, improving, or treating the damage to human gingival fibroblasts caused by PM, and further reducing the damage caused by ROS. Furthermore, from the examples and comparative examples, when the cells are damaged, adding a composition containing mitochondria and MSC-EVs can further reduce ROS production compared with adding an equal amount of mitochondria (Example 7-2), with a statistically significant difference, indicating that adding a composition containing mitochondria and MSC-EVs shows a synergistic effect in alleviating, repairing, improving, or treating the damage to human gingival fibroblasts and can significantly reduce further damage caused by ROS.

[0090] [Table 12]

[0091] Experiment 9: Mitochondrial - Membrane Potential Analysis to Alleviate Mitochondrial Damage in Human Gingival Fibroblasts by PM

[0092] The procedure of this experiment is generally the same as that of Experiment 1, and only the differences will be described below. PM is added to the wells at a concentration of 0 or 50 μg / cm 2 . After culturing the cells with PM at 37 °C and 5% CO 2 for 6 hours, the supernatant in the wells is removed, and the cells are washed with 0.5 mL of PBS per well. Then, the washed PBS is removed, and fresh DMEM (250 μL / well) containing 1% FBS and the compositions of each example and comparative example are added, and the reaction is carried out at 37 °C and 5% CO 2 for 20 hours. After cell culture, the supernatant in the wells is removed, and the cells are washed with 0.5 mL of PBS per well. Then, the washed PBS is removed, and the cells are washed with fresh DMEM containing 1% FBS. 5 μM JC-1 (250 μL / well) is added and reacted at 37 °C for 10 minutes. After the reaction, the supernatant in the wells is removed, and the cells are washed twice with 0.5 mL of PBS per well. Next, fresh DMEM (250 μL / well) containing 1% FBS is added. The fluorescence of JC-1 aggregates is measured at OD520 (excitation) and OD590 (emission), and the fluorescence of JC-1 monomers is measured at OD490 (excitation) and OD530 (emission) to evaluate the mitochondrial membrane potential of human gingival fibroblasts.

[0093] The experimental results of the composition containing extracellular matrix (ECM) and mitochondria are shown in Table 13 and Figure 14. Figure 14 shows the ratio of JC-1 monomer / JC-1 aggregate (JC-1 ratio) of mitochondria in human gingival fibroblasts treated with the compositions of the examples and comparative examples after PM treatment. In Figure 14, the control group is cells without PM, mitochondria, and ECM (Control Example 8-1), the symbol "♯" indicates a statistically significant difference compared to the control group (Control Example 8-1) (♯♯♯ means P < 0.001), and the symbol "*" indicates a statistically significant difference compared to the comparative example (Comparative Example 8-1) (*** means P < 0.001). From Control Examples 8-1 to 8-6, when the cells are not damaged, adding only mitochondria or ECM has no effect on the ratio of JC-1 monomer / JC-1 aggregate (JC-1 ratio) of mitochondria in human gingival fibroblasts, indicating that mitochondria and ECM do not have an adverse effect on the mitochondrial function of human gingival fibroblasts. Also, from the examples and comparative examples, when the cells are damaged, it has been confirmed that adding mitochondria reduces the JC-1 ratio (Examples 8-1, 8-2), which indicates that the damage to the mitochondrial membrane of human gingival fibroblasts is improved. Furthermore, adding mitochondria alleviates the damage to the mitochondria of human gingival fibroblasts caused by PM and improves the mitochondrial function of human gingival fibroblasts. Also, from the examples and comparative examples, when the cells are damaged, adding a composition containing mitochondria and ECM can further reduce the JC-1 ratio compared to adding an equal amount of mitochondria (Examples 8-3 and 8-4), and it has been shown to have a statistically significant difference, indicating that the addition of the composition containing mitochondria and ECM exerts a synergistic effect in alleviating, repairing, improving, or treating the damage of human gingival fibroblasts and can further improve the mitochondrial function of human gingival fibroblasts.

[0094] [Table 13]

[0095] The experimental results of the composition containing platelet-rich plasma-derived extracellular vesicles (PRP-EVs) and mitochondria are shown in Table 14 and Figure 15. Figure 15 shows the ratio of JC-1 monomer / JC-1 aggregate (JC-1 ratio) of mitochondria. After treatment with PM, the expression of mitochondrial growth factor receptor (PRP-EV) in human gingival fibroblasts treated with the compositions of the examples and comparative examples was significantly decreased compared with the control group. In Figure 15, the control group is cells without PM, mitochondria, and PRP-EV (Control Example 9-1), the symbol "♯" indicates a statistically significant difference compared with the control group (Control Example 9-1) (♯♯♯ indicates P < 0.001), and the symbol "*" indicates a statistically significant difference compared with the comparative example (Comparative Example 9-1) (*** indicates P < 0.001). In Control Examples 9-1 to 9-3, when the cells were not damaged, adding only mitochondria or RP-EV had no effect on the JC-1 monomer / JC-1 aggregate ratio (JC-1 ratio) of mitochondria in human gingival fibroblasts, indicating that mitochondria and PRP-EV do not have an adverse effect on the mitochondrial function of human gingival fibroblasts. Also, from the examples and comparative examples, when the cells were damaged, it was confirmed that adding mitochondria decreased the JC-1 ratio (Example 9-1), which indicates that the damaged mitochondrial membrane of human gingival fibroblasts was improved. Furthermore, adding mitochondria alleviated the damage to the mitochondria of human gingival fibroblasts caused by PM, indicating that the mitochondrial function of human gingival fibroblasts was improved. Additionally, from the examples and comparative examples, when the cells were damaged, adding a composition containing mitochondria and PRP-EV could further decrease the JC-1 ratio compared with adding an equal amount of mitochondria (Example 9-2), with a statistically significant difference. This shows that the addition of the composition containing mitochondria and PRP-EV exhibits a synergistic effect in alleviating, repairing, improving, or treating the damage of human gingival fibroblasts, and can further improve the mitochondrial function of human gingival fibroblasts.

[0096] [Table 14]

[0097] The experimental results of the composition containing mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) and mitochondria are shown in Table 15 and Figure 16. Figure 16 shows the ratio of JC-1 monomer / JC-1 aggregate (JC-1 ratio) of mitochondria. In Figure 16, the control group is cells without PM, mitochondria, and MSC-EVs (Control Example 10-1). The symbol "♯" indicates a statistically significant difference (♯♯♯ means P < 0.001) compared with the control group (Control Example 10-1), and the symbol "*" indicates a statistically significant difference (*** means P < 0.001) compared with the comparative example (Comparative Example 10-1). In Control Examples 10-1 to 10-3, when the cells were not damaged, adding only mitochondria or MSC-EVs had no effect on the JC-1 monomer / JC-1 aggregate ratio (JC-1 ratio) of mitochondria in human gingival fibroblasts, indicating that mitochondria and MSC-EVs do not have an adverse effect on the mitochondrial function of human gingival fibroblasts. Also, from the examples and comparative examples, when the cells were damaged, it was confirmed that adding mitochondria decreased the JC-1 ratio (Example 10-1), which indicates that the damaged mitochondrial membrane of human gingival fibroblasts was improved. Furthermore, adding mitochondria alleviated the damage of mitochondria in human gingival fibroblasts by PM and improved the mitochondrial function of human gingival fibroblasts. Additionally, from the examples and comparative examples, when the cells were damaged, adding the composition containing mitochondria and MSC-EVs could further decrease the JC-1 ratio compared with adding an equal amount of mitochondria (Example 10-2) and have a statistically significant difference, indicating that the addition of the composition containing mitochondria and MSC-EVs shows a synergistic effect in alleviating, repairing, improving, or treating the damage of human gingival fibroblasts and can further improve the mitochondrial function of human gingival fibroblasts.

[0098]

Table 15

[0099] Experiment 10: Mitochondria that mitigate mitochondrial damage in human gingival fibroblasts by PM-ATP production

[0100] The procedure for this experiment is generally the same as that for Experiment 1, and only the differences will be described below. Human gingival fibroblasts are cultured in a 10-cm dish (the bottom area of the dish is 60.8 cm 2 ) with 10 mL of DMEM containing 10% FBS at a cell density of 3.5×10 5 for 24 hours. Then, after culturing until the cells reach 80% of the dish, the culture medium is removed, and the cells are rinsed with 5 mL of PBS per dish. Next, the rinsed PBS is removed, and fresh DMEM containing 1% FBS (5 mL / dish) is added. Next, PM is added to the dish at a concentration of 0 or 50 μg / cm 2 . The cells are cultured with PM at 37°C and 5% CO 2 for 6 hours, then the supernatant is removed, and the cells are rinsed with PBS. Next, the rinsed PBS is removed, and fresh DMEM containing 1% FBS and the compositions of each example and comparative example are added, and the cells are cultured at 37°C and 5% CO 2 for 20 hours. After cell culture, the ATP production of mitochondria in the cells is analyzed using an ATP assay kit.

[0101] The experimental results are shown in Table 16 and FIG. 17. FIG. 17 shows the mitochondrial ATP production in human gingival fibroblasts treated with the compositions of the examples and comparative examples after treatment with PM. In FIG. 17, the control group is cells without PM, mitochondria, and ECM (Control Example 11-1), and the symbol "♯" indicates a statistically significant difference (♯♯♯ indicates P < 0.001) compared with the control group (Control Example 11-1). In Control Examples 11-1 to 11-6, when the cells are not damaged, adding only mitochondria or ECM has no effect on the mitochondrial ATP production in human gingival fibroblasts, indicating that mitochondria and ECM do not have an adverse effect on the ATP production ability of mitochondria in human gingival fibroblasts. Also, from the examples and comparative examples, when the cells are damaged, adding mitochondria can increase the mitochondrial ATP production in human gingival fibroblasts (Examples 11-1 and 11-2). This indicates that the ATP production ability of damaged mitochondria in human gingival fibroblasts is improved, and further, adding mitochondria alleviates the damage of mitochondria in human gingival fibroblasts caused by PM, indicating that the mitochondrial function of human gingival fibroblasts is improved. Also, from the examples and comparative examples, when the cells are damaged, adding a composition containing mitochondria and ECM can further increase the mitochondrial ATP production in human gingival fibroblasts compared with adding an equal amount of mitochondria (Examples 11-3 and 11-4). This indicates that the addition of a composition containing mitochondria and ECM exerts a synergistic effect in alleviating, repairing, improving, or treating the damage of human gingival fibroblasts and can further improve the mitochondrial function of human gingival fibroblasts.

[0102] [Table 16]

[0103] According to the above experiments and the embodiments of the present disclosure, a composition containing mitochondria can alleviate the damage of gingival fibroblasts by particulate matter, thereby reducing the death of gingival fibroblasts. In addition, a composition containing mitochondria can reduce the aging of gingival fibroblasts caused by particulate matter. Further, a composition containing mitochondria can reduce the production of reactive oxygen species (ROS) in gingival fibroblasts caused by particulate matter, thereby alleviating further damage to gingival fibroblasts by reactive oxygen species. Furthermore, a composition containing mitochondria can alleviate the damage to the mitochondria of gingival fibroblasts by particulate matter, thereby improving the mitochondrial function of gingival fibroblasts. Additionally, a composition containing mitochondria and extracellular vesicles derived from platelet-rich plasma, a composition containing mitochondria and extracellular vesicles derived from stem cells, and a composition containing mitochondria and extracellular matrix show a synergistic effect in alleviating, repairing, improving, or treating damage to gingival fibroblasts, significantly reducing the aging or death of gingival fibroblasts caused by particulate matter, further reducing the production of reactive oxygen species and related damage, and further improving the mitochondrial function of gingival fibroblasts. Therefore, the composition of the embodiments of the present invention can achieve the purpose of alleviating, repairing, improving, or treating oral injuries, and is expected to be a composition or drug that can alleviate, repair, improve, or treat periodontal disease, periodontal abscess, oral submucous fibrosis, leukoplakia, or oral cancer while having both safety and efficacy.

Claims

1. Use of mitochondria in the manufacture of a composition for alleviating oral injuries.

2. The use according to claim 1, wherein the oral injury includes periodontal disease, periodontal abscess, oral submucous fibrosis, leukoplakia or oral cancer.

3. The use according to claim 1, wherein alleviating the oral injury includes reducing the death of gingival fibroblasts.

4. The use according to claim 1, wherein alleviating the oral injury reduces the senescence of gingival fibroblasts.

5. The use according to claim 1, wherein alleviating the oral injury reduces the reactive oxygen species produced by gingival fibroblasts.

6. The use according to claim 1, wherein alleviating the oral injury includes improving the mitochondrial function of gingival fibroblasts.

7. The use according to claim 1, wherein alleviating the oral injury includes improving the mitochondrial membrane potential of gingival fibroblasts or improving the mitochondrial ATP production of gingival fibroblasts.

8. The use according to claim 1, wherein the effective dosage of the mitochondria in the composition is at least 15 μg.

9. The use according to claim 1, wherein the composition further comprises extracellular vesicles or extracellular matrix.

10. The use according to claim 9, wherein the extracellular vesicles are derived from platelet-rich plasma (PRP) or stem cells.

11. The use according to claim 10, wherein the stem cells are mesenchymal stem cells.

12. The use according to claim 10, wherein the mitochondria and the extracellular vesicles are derived from the same stem cells.

13. A composition comprising mitochondria and a biocompatible carrier.

14. The composition according to claim 13, wherein the effective dosage of the mitochondria in the composition is at least 15 μg.

15. The composition according to claim 13, further comprising extracellular vesicles or extracellular matrix.

16. The composition according to claim 15, wherein the extracellular vesicles are derived from platelet-rich plasma (PRP) or stem cells.

17. The composition according to claim 16, wherein the stem cells are mesenchymal stem cells.

18. The composition according to claim 16, wherein the mitochondria and the extracellular vesicles are derived from the same stem cells.

19. Culturing cells in a culture medium in a container, After culturing, separating the supernatant in the container from the cells attached to the container, Collecting extracellular vesicles from the supernatant, Lysing the cells to separate the intracellular mitochondria, Mixing the extracellular vesicles and the mitochondria to obtain a composition. A method for producing a composition comprising mitochondria and extracellular vesicles, including such.

20. The method according to claim 19, wherein the mitochondria and the extracellular vesicles are derived from the same cell.

Images