Extracellular vesicles for supporting cell repair, manufacturing method thereof, and preparation thereof

Abstract

To provide silicate-induced extracellular vesicles formed by inducing secretion in stem cells using silicate, which aid in cell repair.SOLUTION: Extracellular vesicles that support cell repair of the present invention are formed by inducing secretion in stem cells using silicate, contribute to the expression of indicator factors such as anti-inflammation, tissue regeneration, and cell repair, and promote cell growth in wound tissue and a wound repair process. The method for producing the extracellular vesicles that support cell repair of the present invention includes the steps of taking 1 g of calcium silicate, adding it to 10 to 100 ml of a medium, soaking it for at least 24 hours, and collecting the clear liquid to prepare a silicate medium; cultivating the stem cells in the silicate medium; and isolating the extracellular vesicles secreted by the stem cells.SELECTED DRAWING: Figure 3A

Description

[Technical field]

[0001] The present invention relates to extracellular vesicles, in particular extracellular vesicles that aid in cellular repair, methods for their production and formulations thereof. [Background technology]

[0002] There are many factors that can cause poor wound healing, ranging from improper wound cleaning and care to the patient's physical condition, such as old age, malnutrition, diabetes, autoimmune diseases, cancer, or bad habits such as smoking or drinking, all of which are closely related to how quickly the wound heals.

[0003] In addition to surgical procedures, the use of antibiotics, and wound dressing, newer treatment methods include the topical application of human growth factors, negative pressure therapy, hyperbaric oxygen therapy, and biotechnology skin grafts, which are not only cost prohibitive, but also have limited ability to accelerate wound healing. Therefore, the development of technologies to help promote wound healing remains an urgent research goal in related fields. Summary of the Invention [Problem to be solved by the invention]

[0004] To develop a technology that helps promote wound healing, the present invention provides extracellular vesicles that support cell repair, which are silicate-induced extracellular vesicles formed by inducing stem cells to secrete with silicate. [Means for solving the problem]

[0005] In the present invention, the step of producing the silicate-derived extracellular vesicles comprises: Taking 1g of calcium silicate, adding it to 10-100ml of culture medium, soaking it for at least 24 hours, collecting the clear liquid to remove calcium element, thus preparing a silicate culture medium; Culturing the stem cells in the silicate medium; isolating silicate-derived extracellular vesicles secreted by the stem cells; Includes.

[0006] In the present invention, the medium includes a medium that is mesenchymal stem cell medium (MSCM) or Dulbecco's Modified Eagle Medium (DMEM).

[0007] The present invention further provides a formulation for supporting cell repair comprising silicate-induced extracellular vesicles (CSEVs) formed by inducing secretion from stem cells with silicate.

[0008] The present invention includes a hydrogel for retaining said silicate-derived extracellular vesicles (CSEVs).

[0009] The ratio of the silicate-derived extracellular vesicles (CSEVs) in the hydrogel is 10 9 ~10 14 pieces / mL. Effect of the Invention

[0010] The silicate-induced extracellular vesicles (CSEV) provided by the present invention have improved surface area, total protein amount and membrane-permeable protein content compared to ordinary extracellular vesicles, which helps to improve the affinity of the silicate-induced extracellular vesicles (CSEV)-receptor cells to undergo endocytosis. Cell and animal experiments have demonstrated that the silicate-induced extracellular vesicles (CSEV) easily enter cells through endocytosis, contributing to the expression of indicator factors such as anti-inflammation, tissue regeneration and cell repair, and promoting cell growth in wound tissue and the wound repair process. [Brief description of the drawings]

[0011] [Figure 1] FIG. 2 is a graph showing the change in calcium ion and silicon ion content in the silicate medium provided by the present invention. [Diagram 2]FIG. 1 is a diagram showing cell activity detection after stem cell culture provided by the present invention. [Figure 3A] FIG. 1 is a transmission electron microscope image of the extracellular vesicles provided by the present invention. [Figure 3B] FIG. 1 shows the analysis and detection of nanoparticle tracking of extracellular vesicles provided by the present invention. [Figure 3C] This is a diagram showing potential detection on the membrane surface of extracellular vesicles provided by the present invention. [Figure 3D] FIG. 1 shows detection of protein expression in extracellular vesicles provided by the present invention. [Figure 3E] This is an analysis diagram of the expression ratios of extracellular vesicle-permeable proteins CD9, CD63 and CD81 provided by the present invention. [Figure 3F] This is an analysis diagram of the expression ratios of extracellular vesicle-permeable proteins CD9, CD63 and CD81 provided by the present invention. [Figure 3G] This is an analysis diagram of the expression ratios of extracellular vesicle-permeable proteins CD9, CD63 and CD81 provided by the present invention. [Figure 3H] FIG. 1 is an image of the expression level of extracellular vesicle proteins provided by the present invention. [Figure 4A] FIG. 1 is a diagram showing cell activity detection in a diabetic cell model provided by the present invention. [Figure 4B] FIG. 1 shows detection of intracellular reactive oxygen free radicals in the diabetic cell model provided by the present invention. [Figure 4C] FIG. 2 is a fluorescent staining image of cells in the diabetic cell model provided by the present invention. [Figure 4D] FIG. 1 is a graph showing the amount of fluorescence expression in cells of the diabetic cell model provided by the present invention. [Figure 5A] FIG. 1 shows a diagram of detection of cell activity after extracellular vesicle reaction in a diabetic cell model provided by the present invention. [Figure 5B] FIG. 1 shows the detection of intracellular reactive oxygen free radicals after extracellular vesicle reaction in the diabetic cell model provided by the present invention. [Figure 5C] FIG. 1 is an image of wound healing after extracellular vesicle reaction in a diabetic cell model provided by the present invention. [Figure 5D]FIG. 1 shows the cell migration rate after extracellular vesicle reaction in the diabetic cell model provided by the present invention. [Figure 6] FIG. 1 is an illustration of the hydrogel provided by the present invention carrying extracellular vesicles and cell cultures. [Figure 7] FIG. 1 shows the cell activity detection diagram after culturing the diabetic cell model provided by the present invention with extracellular vesicles supported on hydrogel. [Figure 8] FIG. 1 is a graph showing the detection of the expression level of indicator factors after culturing the diabetic cell model provided by the present invention. [Figure 9A] FIG. 1 is an analysis of miRNA expression levels after culturing a diabetic cell model provided by the present invention. [Figure 9B] FIG. 1 is an analysis of miRNA expression levels after culturing a diabetic cell model provided by the present invention. [Figure 9C] FIG. 1 is an analysis of miRNA expression levels after culturing a diabetic cell model provided by the present invention. [Figure 10] FIG. 1 is a stained photograph of a wound repair section of a diabetic animal model provided by the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] As used herein, "extracellular vesicles (EVs)" refer to bilayer lipid vesicles secreted by cells into the extracellular space. Extracellular vesicles include exosomes and microvesicles, and as a new method of intercellular communication, they have become a new research field in physiology, pathology, nanoscience, etc. in recent years. Extracellular vesicles are highly heterogeneous vesicle-like bodies secreted by cells, and their contents include proteins, mRNA / miRNA, DNA, lipids, etc., and they are classified based on their biological origin, release route, size, entry and function, and purification methods are distinguished according to particle size.

[0013] The present invention will be described below in combination with the specification, drawings and specific examples, but the examples do not impose any formal limitations on the present invention. Unless otherwise specified, the reagents, methods and devices used in the present invention are conventional reagents, methods and devices in the technical field.

[0014] The present invention uses silicate to induce the secretion of silicate-induced extracellular vesicles (CSEV) from stem cells, where the stem cells are preferably human stem cells, which selectively include, but are not limited to, human adipose-derived stem cells (ADSCs), bone marrow-derived stem cells (BMSCs), dental pulp stem cells (DPSCs), Wharton's jelly MSCs (WJMSC), etc.

[0015] In this embodiment, as described below, human adipose-derived stem cells (ADSCs) are used as source cells for secretion of silicate-induced extracellular vesicles. The human stem cells are cultured by adding a silicic acid medium containing a silicic acid component, and the human adipose-derived stem cells (ADSCs) are induced to secrete the extracellular vesicles, and the extracellular vesicles secreted after induction by the silicate medium are further defined herein as the silicate-induced extracellular vesicles (CSEVs).

[0016] Here, the method of culturing the human adipose stem cells (ADSCs) is not particularly limited, and any method capable of culturing the human adipose stem cells (ADSCs) to obtain the silicic acid-induced cells (CSEV) is within the scope of the present invention.

[0017] Here, the silicate is a silicic acid compound (Si x O y ). The silicate compound further includes one or more metals or hydrogen. In this embodiment, the silicate is calcium silicate.

[0018] Here, the medium used to culture the human adipose stem cells by adding the silicate is not limited, and may be mesenchymal stem cell medium (MSCM, ScienCell Research Laboratories), Dulbecco's Modified Eagle Medium (DMEM), or other media for culturing common cells, all of which are within the scope of the present invention.

[0019] Further, the step of culturing the silicate-induced extracellular vesicles (CSEVs) includes:

[0020] In step S1-1, the silicate medium is prepared by taking 1 g of calcium silicate, adding it to 10 to 100 ml of mesenchymal stem cell medium (MSCM), soaking it for at least 24 hours, and collecting the clear liquid to prepare the silicate medium.

[0021] Here, the clear liquid can be collected by filtration, centrifugation, or the like.

[0022] Here, in the step of soaking for at least 24 hours, a temperature environment or a shaking table can be simultaneously provided to promote the reaction of calcium silicate in the mesenchymal stem cell culture medium.

[0023] Referring to Figure 1, further use inductively coupled plasma mass spectrometry (ICP-MS) technology to analyze the change in calcium ion and silicon ion content in the mesenchymal stem cell medium when calcium silicate is added. On day 0, the release of calcium ion is obvious because the calcium silicate is added to the mesenchymal stem cell medium, and after the calcium silicate reacts in the mesenchymal stem cell medium for 24 hours to prepare the silicate medium, the calcium ion and silicon ion content are clearly measured by the preparation of the silicate medium, which proves the effectiveness of the reaction of calcium silicate in the mesenchymal stem cell medium.

[0024] In step S1-2, the human adipose stem cells (ADSCs) are cultured in the silicate medium and cultured in a cell culture incubator containing 5% CO2 at 37°C.

[0025] Here, the human adipose stem cells (ADSCs) can be cultured in a T175 culture bottle filled with the mesenchymal stem cell medium, and the concentration of the silicate medium in the mesenchymal stem cell medium is 5 to 50%.

[0026] Referring to Figure 2, the human adipose stem cells (ADSCs) cultured in the silicate medium are further defined as a cell culture example. As a comparative example of cell culture, the human adipose stem cells (ADSCs) are cultured in the mesenchymal stem cell medium. By detecting the cell proliferation effect through cell activity, the human adipose stem cells (ADSCs) cultured in the silicate medium have a significantly larger cell number than the comparative cell culture example from the third day, and it can be inferred that the human adipose stem cells (ADSCs) have a cell proliferation effect after being cultured in the silicate medium.

[0027] Here, the cell activity can be analyzed for cell viability and proliferation effect by detecting fluorescence or absorbance using MTT assay, PrestroBlue assay, Alamar Blue assay, etc. In this embodiment, cell activity detection is achieved using PrestroBlue assay.

[0028] In step S1-3, the silicate-induced extracellular vesicles (CSEV) are separated. The cultured human adipose stem cells (ADSCs) are collected together with the silicate medium, centrifuged at 1500 g for 5 minutes, and the first supernatant liquid formed is collected. Next, the first supernatant liquid is filtered under a pore size of 0.22 microns (μm), then placed in a concentrated centrifuge tube (Amicon(R) Ultra-15 Centrifugal Filter Unit, Merck), and centrifuged under a centrifugation condition of 5000 gravitational acceleration (g) for 15 minutes, and the silicate-induced extracellular vesicles (CSEV) remain in the second supernatant liquid formed. Next, the second supernatant liquid is collected, and the silicate-induced extracellular vesicles (CSEV), which have low solubility, are precipitated and separated out from the second supernatant liquid using the property that high molecular weight polyethylene glycol can bind to hydrophobic proteins and lipid molecules in competition with released water molecules according to the principle of polymer precipitation. Finally, the mixture is centrifuged at 1500 g for 30 to 40 minutes to remove the supernatant, and the mixture is redissolved in phosphate buffered saline (PBS) and precipitated to obtain highly pure silicate-derived extracellular vesicles (CSEVs).

[0029] Experiment 1: Analysis of the properties of the silicate-induced extracellular vesicles (CSEVs) The present invention further compares and analyzes the protein expression levels, electrical properties of the membrane surface and particle size of the collected silicate-induced extracellular vesicles (CSEVs) and normal extracellular vesicles (EVs) obtained after culture in a comparative example, and identifies the external surface characteristics of the silicate-induced extracellular vesicles (CSEVs) and the physiological data of each cell.

[0030] In FIG. 3A, using a transmission electron microscope (TEM), it can be observed that there is no significant difference in particle size between the normal extracellular vesicles (EVs) and the silicate-derived extracellular vesicles (CSEVs), confirming that the surface features of the two are similar.

[0031] In Figure 3B, nanoparticle tracking analysis (NTA) can be used to confirm whether the particle size of the normal extracellular vesicles (EVs) and the silicate-induced extracellular vesicles (CSEVs) is similar, and further detect whether the concentration of the normal extracellular vesicles (EVs) and the silicate-induced extracellular vesicles (CSEVs) is similar, demonstrating that both the normal extracellular vesicles (EVs) and the silicate-induced extracellular vesicles (CSEVs) belong to the extracellular vesicles.

[0032] In FIG. 3C, the present invention measures the membrane surface potential of the normal extracellular vesicles (EVs) and the silicate-induced extracellular vesicles (CSEVs). Comparing the results, it can be seen that the membrane surface potential of the silicate-induced extracellular vesicles (CSEVs) is lower than -20mV, preferably between -25 and -30mV, and the degree of the negative value is much greater than that of the normal extracellular vesicles (EVs).

[0033] In Figures 3D-3H, the present invention further analyzes the total protein content and expression levels of multiple extracellular vesicle marker proteins in the silicate-induced extracellular vesicles (CSEVs) by Western blot, where the marker proteins include transmembrane proteins CD9, CD63, and CD81, and intramembrane proteins Alix, TSG101, and HSP70.

[0034] From these results, the total protein content of the silicate-induced extracellular vesicles (CSEV) was 4 μg / 10 9 It can be seen that this is higher than that of normal extracellular vesicles (EVs).

[0035] It is noteworthy that more than 40% of the silicate-induced extracellular vesicles (CSEVs) expressed the transmembrane proteins CD9, CD63, and CD81, which play important roles in specific binding, endocytosis, and drug delivery (Figures 3E-G).

[0036] From the above detection results, it can be seen that the silicate-induced extracellular vesicles (CSEV) secreted by human adipose stem cells (ADSCs) after induction with the silicate culture medium have significantly improved surface potential, total protein amount and transmembrane protein content, which is helpful in improving the affinity of the silicate-induced extracellular vesicles (CSEV) recipient cells for endocytosis.

[0037] To confirm whether the silicate-derived extracellular vesicles (CSEVs) have an effect on cell proliferation, repair, anti-inflammation, etc., the present invention further targets a diabetic cell model that is commonly used in the study of wound healing, cell proliferation and repair.

[0038] Here, the construction of the diabetic cell model was based on the experimental steps described in the literature, and human dermal fibroblasts (HDF) were cultured in a high glucose medium (High Glucose DMEM, HDMEM) to induce the human dermal fibroblasts (HDF) to simulate the physiological characteristics of cells in diabetic patients. Here, the glucose concentration in the high glucose medium (High Glucose DMEM, HDMEM) was between 13.5 and 25 mM.

[0039] As shown in Figures 4A and 4B, the proliferation effect of the human dermal fibroblasts decreases with increasing glucose and number of days in the high glucose medium. Figure 4B shows that the human dermal fibroblast group (HG) cultured in the high glucose medium (High Glucose DMEM, HDMEM) with a glucose concentration of 25 mM and the human dermal fibroblast group (NG) cultured in a general medium were measured for reactive oxygen species (ROS) concentration in the cells, which is used as an index of inflammatory response. It can be seen that the reactive oxygen free radical activity of the human dermal fibroblast group (HG) cultured in the high glucose medium (High Glucose DMEM, HDMEM) is significantly higher than that of the human dermal fibroblast group (NG) cultured in a general medium. The above-mentioned reduction in cell proliferation effect and the enhancement of intracellular reactive oxygen species (ROS) free radical activity can be regarded as the main physiological indicators of the diabetic cell model, and the present invention will explain the subsequent experiments using the diabetic cell model cultured in a high glucose medium (High Glucose DMEM, HDMEM) with a glucose concentration of 25 mM.

[0040] Experiment 1: affinity test of diabetic cell model and said silicate-induced extracellular vesicles. Diabetic cell model is treated with normal extracellular vesicles and said silicate-induced extracellular vesicles respectively, and the endocytosis effect of diabetic cell model on said normal extracellular vesicles or said silicate-induced extracellular vesicles is analyzed when treated for 24 hours and 48 hours respectively. Here, the group treated with said normal extracellular vesicles is the control group (EV), and the group treated with said silicate-induced extracellular vesicles is the experimental group (CSEV).

[0041] In Figure 4C, the intracellular area of ​​the diabetic cell model can be defined by red fluorescent-labeled actin (F-actin), the normal extracellular vesicles and / or the silicate-induced extracellular vesicles are labeled with green fluorescence, and blue fluorescence is used to label the cell nucleus. The results show that the green fluorescence representing the normal extracellular vesicles and / or the silicate-induced extracellular vesicles is distributed within the intracellular area defined by the red fluorescence, demonstrating that the normal extracellular vesicles (EVs) and / or the silicate-induced extracellular vesicles (CSEVs) have indeed entered the diabetic cell model by endocytosis.

[0042] The reaction between the experimental group and the silicate-induced extracellular vesicles was further observed up to 24 hours, and the fluorescence performance of the silicate-induced extracellular vesicles in the diabetic cell model was significantly higher than that of the control group, and the difference in the green fluorescence performance between the control group (EV) and the experimental group (CSEV) was even more significant at 48 hours. The fluorescence brightness of the green fluorescence was detected and shown numerically in Figure 4D, allowing a clear comparison of the difference in the content of the silicate-induced extracellular vesicles between the experimental group (CSEV) and the control group (EV) after 48 hours.

[0043] Furthermore, the effect of cell proliferation after the diabetic cell model is co-cultured with the silicate-induced extracellular vesicles is confirmed. In FIG. 5A, the cell viability detection shows that the number of cells in the diabetic cell model co-cultured with the silicate-induced extracellular vesicles (experimental group CSEV) is significantly higher than that in the first control group (Ctl) without any treatment and the second control group (EV) co-cultured with the normal extracellular vesicles from the third day. Here, a significant difference is generated from the seventh day of co-culture onwards. From this, it can be seen that the diabetic cell model generates a cell proliferation effect after co-culture treatment with the silicate-induced extracellular vesicles (CSEV).

[0044] In Fig. 5B, the intracellular reactive oxygen species (ROS) free radical concentration was measured for the first control group (Ctl), the second control group (EV) and the experimental group (CSEV) as an index of inflammatory response. The results show that the intracellular reactive oxygen free radical performance in the experimental group (CSEV) after the diabetic cell model was co-cultured with the silicate-induced extracellular vesicles was significantly lower than that of the first control group (Ctl) and the second control group (EV).

[0045] FIG. 5C shows a wound healing assay performed on the first control group (Ctl), the second control group (EV) and the experimental group (CSEV). Scratches were created on the cell monolayer of each group, and cell images were taken under a microscope at 0 and 24 hours, respectively, to confirm the effect of cell migration and to evaluate the wound healing. From this result, it can be seen that the cells are uniformly distributed in the scratch area in the cell image taken 24 hours after scratching the experimental group (CSEV). Even after converting the data into cell migration rate based on the cell coverage rate per unit area of ​​the scratch (FIG. 5D), it can be seen that the effect of cell migration after the diabetic cell model was co-cultured with the silicate-induced extracellular vesicles was significantly superior to that of the first control group (Ctl) and the second control group (EV).

[0046] Through the above experimental results, it can be preliminarily confirmed that the silicate-derived extracellular vesicles can promote cell proliferation, cell repair, and reduce the production of anti-inflammatory factors.

[0047] Furthermore, the present invention further provides a formulation containing said silicate-derived extracellular vesicles (CSEV), using a hydrogel as a carrier for carrying said silicate-derived extracellular vesicles (CSEV).

[0048] Here, the hydrogel may have a three-dimensional mesh structure formed by cross-linking a material such as collagen, gelatin, hyaluronic acid, or chitosan.

[0049] Here, the crosslinking may be physical or chemical. The physical crosslinking can be achieved by the principles of van der Waals force, ionic attraction, polymer chain entanglement, etc., while the chemical crosslinking can be achieved by the principle of covalent bond. In this embodiment, the hydrogel is formed of collagen, as described in the subsequent embodiments.

[0050] Here, the method of loading the silicate-derived extracellular vesicles on the hydrogel is not limited, but in this embodiment, 5×10 9 The silicate-derived extracellular vesicles were mixed with 1 ml of sterile hydrogel to form a mixture, and 50 μL of the mixture was placed in a culture dish and reacted at 37° C. for 30 minutes to carry out the gelation reaction.

[0051] Here, by further adding a photosensitizer, the mixture can undergo a gelling reaction by photocuring technology.

[0052] In experiment 2, the release effect after the hydrogel carries the normal extracellular vesicles and the silicate-induced extracellular vesicles is compared. The normal extracellular vesicles and the silicate-induced extracellular vesicles are gelled with the hydrogel, respectively, and then added to PBS for culture. After culturing for 0, 1, 2, 3, 5, 7, 10, and 14 hours, respectively, the liquid is extracted into a culture dish, and the number of the extracellular vesicles in the liquid is measured by NTA to estimate the amount of the extracellular vesicles released. It can be confirmed that the silicate-induced extracellular vesicles secreted by silicate do not affect the release rate in the hydrogel.

[0053] In experiment 3, the effects on the proliferation of the diabetic cell model after the hydrogel is loaded with the normal extracellular vesicles and the silicate-induced extracellular vesicles are compared. Referring to FIG. 6 and FIG. 7, first, the normal extracellular vesicles and the silicate-induced extracellular vesicles loaded with the hydrogel are solidified in a culture dish, and then the diabetic cell model is cultured on the hydrogel loaded with the normal extracellular vesicles and / or the silicate-induced extracellular vesicles after hardening at a quantity of 10,000 pieces / hole, and the serum-free DMEM is added and cultured, and the cell proliferation rate is analyzed on the 1st, 3rd, and 7th days, respectively. Here, the hydrogel group is the first control group (Ctl), the hydrogel loaded with the normal extracellular vesicles is the second control group (EV), and the hydrogel loaded with the silicate-induced cells is the experimental group (CSEV).

[0054] From the results of FIG. 7, the cell number of the diabetic model cells cultured using the hydrogel carrying the silicate-induced extracellular vesicles (CSEV) is greater than that of the first control group (Ctl) and the second control group from the third day. Here, a significant difference occurs from the seventh day of culture onwards. This result can be mutually corroborated with the result of FIG. 5A of Experiment 1, and the effect of the silicate-induced extracellular vesicles (CSEV) in inducing cell proliferation of the diabetic cell model is demonstrated.

[0055] In experiment 4, the effects of the hydrogel loaded with the normal extracellular vesicles and the silicate-derived extracellular vesicles (CSEVs) on the expression levels of each indicator factor in the diabetic cell model are compared, where each indicator factor includes inflammation-related factors TNG-alpha, IL-1, and IL-6; tissue regeneration-related factors ColI, Angiogenin, EGF, FGF-2, and PDGF-BB; anti-inflammatory-related factors IL-8 and IL-10; and collagen factor (ColI) as an indicator of wound healing.

[0056] The diabetic model cells were cultured in the hydrogel as the first control group (Ctl), the hydrogel carrying normal extracellular vesicles as the second control group (EV), and the hydrogel carrying silicic acid-induced extracellular vesicles as the experimental group (CSEV). The cells were collected, proteins were extracted and quantified, and 20 μg of protein was taken from each group. The expression levels of each of the indicator factors in the first control group (Ctl) were used as the reference value to analyze the second control group (EV) and the experimental group (CSEV) using cytokine array technology.

[0057] The results in FIG. 8 show that the expression levels of inflammation-related factors TNG-α, IL-1, and IL-6 in the diabetic cell model cultured by the experimental group (CSEV) are significantly reduced compared with the second control group (EV). The expression of tissue regeneration-supporting related factors ColI, Angiogenin, EGF, FGF-2, and PDGF-BB is also improved, the expression of anti-inflammatory related factors IL-8 and IL-10 is also improved, and the expression of collagen factor (ColI) that supports wound healing is also improved. From these results, it can be concluded that the silicate-induced extracellular vesicles (CSEV) can induce the expression of indicator factors such as anti-inflammation, tissue regeneration, and cell repair in the diabetic cell model cells.

[0058] Furthermore, the expression of miRNAs in the diabetic model cells cultured in the first control group (Ctl) and the experimental group (CSEV) was compared by RNA sequencing, and miRNAs were isolated using RNAiso (TaKaRa, Japan), and the subsequent sequence analysis was completed by Shanghai OEBiotech Co., Ltd. Through the miR-Basev.21 database (http: / / www.mirbase.org / ), miRNAs contributing to angiogenesis, anti-inflammation, and wound healing were compared and identified. The differential expression analysis (DEG) algorithm in R language was used to calculate miRNAs with significant differences (p<0.05), and three independent replicate experiments were performed for each group. Then, miRanda software was used to predict the targets of significantly expressed miRNAs in animals using parameters S≧150, ΔG≦-30 kcal / mol, and 5' factor pairs. Subsequently, the gene locations of different miRNA targets were analyzed using Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) methods.

[0059] By analyzing the log2 fold change of the data, the ratio value between the normalized signal values ​​is obtained, and in Figures 9A to 9c, the expression of each of the miRNAs is compared between the diabetic model cells cultured by the experimental group (CSEV) and the first control group (Ctl). The miRNA-31, which is an indicator of angiogenesis, anti-inflammation, and wound healing, in the diabetic model cells cultured by the experimental group (CSEV), all have significant expression, demonstrating that the silicate-induced extracellular vesicles (CSEV) can induce the expression of indicator genes (miRNAs) such as anti-inflammation, tissue regeneration, and cell repair in the diabetic model cells.

[0060] Here, the establishment of a diabetic animal model refers to the experimental step method in the literature, and induces the occurrence of diabetes in experimental animals by the chemical preparation alloxan monohydrate. The principle of alloxan-induced diabetes is to selectively destroy the bata cells that secrete insulin in the pancreas and reduce the insulin content. The alloxan monohydrate used (alloxan monohydrate, ≥ 98%, Sigma Aldrich Chemical, Saint Louis, MO, USA) is in powder form. Since it is prone to deterioration when exposed to air, it is sealed and stored in a refrigerator, maintaining the temperature at 2-8 °C. The preparation method of the drug is to mix 5.26 g of the drug with 100 ml of sterile saline to obtain a 5% reagent (pH = 7), which is then filtered through a 0.22 μm filter (the above process is operated on ice), and immediately collected using intravenous injection (dose: 100 mg / kg). Blood glucose levels are measured every two days, with the goal of keeping blood glucose levels above 300 mg / dl throughout the entire experiment; if this is not achieved within one week, alloxan is administered.

[0061] In experiment 5, the effect of the silicate-derived extracellular vesicles (CSEV) on wound repair in a diabetic animal model is compared. After successfully establishing a diabetic animal model, a 1.5 cm 2 The wound was formed to a depth reaching the muscle layer to provide a malignant wound model.

[0062] The first control group (Ctl) was a group that did not receive any treatment, the second control group (Col) was a group that used only hydrogel as a wound dressing, the third control group (Ev@Col) was a wound dressing formed by the hydrogel carrying the normal extracellular transport, and the experimental group (CSEV@Col) was a wound dressing formed by the hydrogel carrying the silicate-derived extracellular vesicles. At the second and third week after the start of treatment, the wound tissue was removed along the wound edge and fixed with 10% formalin. It was then embedded in paraffin and formed into 6 μm tissue sections, which were stained with hematoxylin-eosin (H&E) and Sirius red staining (PRS). The thickness of the wound epithelium tissue, the amount of granulation tissue, and whether structures that mature skin should have (hair follicles, sebaceous glands, etc.) were observed and quantitatively analyzed by H&E staining. Sirius red staining allows the amount and arrangement pattern of collagen fibers at the wound site to be compared and the repair state of wound tissue to be evaluated.

[0063] As shown in Figure 10, compared with the other groups, the wound of the experimental group (CSEV@Col) became completely flat from the second week, and PRS staining showed that the dermis layer showed a uniform distribution (indicated by arrow A) at the wound site due to the proliferation of dermal fibroblasts and the secretion of a large amount of collagen fibers that filled the wound defect.

[0064] At week 3, PSR staining showed complete expression of muscle texture in the muscle layer (arrow B). H&E staining clearly showed that hair follicles were generated and arranged in an orderly fashion after wound tissue repair (arrow C), demonstrating that the silicate-induced extracellular vesicles (CSEVs) contribute to cell proliferation and wound repair in wound tissue of diabetic animal models.

[0065] Based on the above experimental results, it is proven that the silicate-induced extracellular vesicle material provided by the present invention has extremely high biological safety, contributes to wound repair, and can achieve the effect of promoting wound healing and repair. [Explanation of symbols]

[0066] A Arrow B Arrow C Arrow

Claims

1. Extracellular vesicles formed by silicate-induced secretion in stem cells that aid in cell repair.

2. The extracellular vesicles that assist in cell repair according to claim 1, wherein the stem cells are cultured in a silicic acid medium supplemented with a silicate component.

3. The extracellular vesicles that assist in cell repair according to claim 2 , wherein the stem cells include human adipose stem cells, bone marrow stem cells, dental pulp stem cells, or Wortonglia mesenchymal stem cells.

4. The extracellular vesicle that assists in cell repair described in claim 1, wherein the silicate is calcium silicate.

5. The extracellular vesicles that support cell repair according to claim 1, wherein the silicate medium comprises a medium that is mesenchymal stem cell medium (MSCM) or Dulbecco's Modified Eagle Medium (DMEM).

6. The extracellular vesicle for assisting cell repair according to claim 1, comprising a membrane surface having a potential lower than -15 mV.

7. The extracellular vesicle that assists cell repair according to claim 6, wherein the potential of the membrane surface is -20 mV to -30 mV.

8. The extracellular vesicles that support cell repair described in claim 1, having a total protein content of more than 4 μg / 109.

9. The extracellular vesicles that aid in cell repair described in claim 1, wherein at least 40% of the extracellular vesicles express at least one of the transmembrane protein CD9, the transmembrane protein CD63, and the transmembrane protein CD81.

10. A method for producing extracellular vesicles that support cell repair, the extracellular vesicles being formed by inducing secretion in stem cells with silicate, Taking 1g of calcium silicate, adding it to 10-100ml of culture medium, soaking for at least 24 hours, and collecting the clear liquid to prepare a silicate culture medium; Culturing the stem cells in the silicate medium; isolating the extracellular vesicles secreted by the stem cells; A method for producing extracellular vesicles that assist in cell repair, comprising:

11. The method for producing extracellular vesicles that assist in cell repair according to claim 10, wherein the medium comprises a medium that is mesenchymal stem cell medium (MSCM) or Dulbecco's Modified Eagle Medium (DMEM).

12. A preparation that aids in cell repair, comprising extracellular vesicles formed by inducing secretion in stem cells with silicate.

13. A formulation for assisting in cell repair, comprising a hydrogel that retains extracellular vesicles formed by inducing secretion in stem cells with silicate.

14. The ratio of the extracellular vesicles (CSEV) in the hydrogel is 10 ９ ~10 １４ The preparation for supporting cell repair according to claim 13, wherein the concentration is 1 / mL.

15. Use of a preparation for wound repair, comprising extracellular vesicles formed by inducing secretion from stem cells with silicate.

16. 16. Use of a preparation for wound repair according to claim 15, wherein the wound repaired by said preparation is a difficult-to-treat wound caused by diabetes.

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