KR102179155B1 - Osteogenesis method by Jade extract in ADSC - Google Patents

Abstract

The present invention relates to a method for differentiating bone cells of stem cells by a jade extract. More specifically, the present invention relates to a method for differentiating bone cells of stem cells by a jade extract and a composition for restoring skin cells or treating arthritis and a cosmetic composition containing bone cells manufactured by the method, wherein the method comprises the following steps: 1) treating the jade extract to skin cells to discharge activated functional ingredients; 2) separating a supernatant of a medium containing the functional ingredients; 3) treating the separated medium supernatant to stem cells and culturing the same to differentiate into bone cells; and 4) separating the differentiated bone cells. When using the method for differentiating bone cells, adipose-derived stem cells can be differentiated into bone cells. Therefore, the promotion of bone cell differentiation according to the present invention can be effectively used for bone development of infants in a growth period and prevention of osteoporosis in the middle-aged. In addition, bone cells obtained according to the present invention can be usefully used in the treatment of skin disease such as general scars and burn scars, and bone metabolic diseases such as bone defect part procedures.

Description

Bone cell differentiation method using jade extract {Osteogenesis method by Jade extract in ADSC}

The present invention relates to a method for differentiating bone cells using a jade extract, and more particularly, to a method for differentiating bone cells of stem cells using a secretion product of skin cells activated by the jade extract.

Jade is derived from piedra de ijada, meaning a jewel that prevents or cures kidney disease when Spain conquered Latin America.

Jade is the essence of the heavens and the earth from ancient times, and it is believed to have medicinal properties and to defeat mischief if it is worn in the body. Also, jade is used as a basic material for body decoration and clothing decoration. Jade is commonly used by grinding and trimming gemstones into small circles and then punching holes. It is used in various forms such as necklaces, rings and bracelets. The types of jade used since ancient times include agate, glass, amber, seokwoonghwang, jade, cheonhaseokok, and topaz.

Jade is divided into jadeite (硬玉) and purgatory (軟玉). Jadeite is a lead pyroxene mineral belonging to the pyroxene family, and is a monoclinic material composed of silicic acid and soda, and its hardness is a dense mass like crystal and light. Is black, cyan, green, and has translucent properties. Nephrite Jade is a monoclinic fissile mineral of Inosilicates, and is divided into serpentine superbasic purgatory among the paleo-soil marbles. The quality of purgatory is determined by the finer structure, that is, the fine structure of openwork-yanggiseok sperm, which is made up of sockjo (束組) and fibers, and it is known that the finer the fiber is, the better the quality.

On the other hand, 90% of modern adult diseases are caused when lipid peroxide, which occurs when unsaturated fatty acids are oxidized, causes disorders in the blood circulation system.In our human body, an enzyme called SOD (Glutathione Peroxidase, GSH-PX) that removes lipid peroxidation It exists and stays healthy. However, as adults become older, the induction capacity of these enzymes decreases, and as lipid peroxide in the body is deposited on the walls of blood vessels, it has been found to gradually become adult diseases.

Jade has the property of emitting a large amount of far-infrared rays of 5-20㎛, which is a growing ray from the ore itself. The far-infrared rays of the absorbed light act strongly on the polymerized rings of unsaturated fatty acids, lipid peroxides, and contaminated water in the body. Since the double and triple bonds of these contaminants are separated by far-infrared rays emitted from the jade, the physiological activity of the human body is increased and promoted.

According to a report published in the prestigious German medical literature (KRAFF der KRISTALLE und der EDELSTEINE by Mauda Palmer Die Verborgene), jadeite and purgatory are two different ores that contain silicon and oxygen like most gemstones. . However, while jadeite is formed of granular crystals, purgatory differs in that it is composed of countless crystals such as fibers and hair and fine grain aggregates. In particular, jadeite has been reported to have a great effect on the healing of diseases caused by high blood pressure, diabetic circulatory disorders, heart disease, and kidney disorders, since jadeite is mainly composed of three components beneficial to the human body, namely calcium, iron, and magnesium.

However, although jade is known to have excellent medical effects as described above due to its rarity, it is mostly used only as a jewelry accessory for personal decoration such as necklaces, rings, and bracelets. Also, since jade is a high-priced product, the development of jade as a general household product is relatively weak. Therefore, it is necessary to research and develop jade so that jade can be used in various ways in everyday life.

Meanwhile, it was confirmed that bone marrow-derived adult stem cells differentiate into osteogenic cells (Friedenstein AJ et al., Transplantation., 6:230-247, 1968), and stem cells isolated from bone marrow are cultured to differentiate into osteoblasts and proliferate. There is progress in the treatment method, and the possibility of clinical application through it is increasing (Ohgushi H. et al., J. Biomed Mater Res., 48:913-927, 1999). Therefore, a method of differentiating bone cells from stem cells is being actively studied.

Among tissue engineering, a series of techniques that allow the formation of new parenchymal tissues by implanting a polymer carrier that is completely absorbed by the living body and has affinity with cells is called bio-tissue engineering. Biotissue engineering is a multidisciplinary combination of cell culture, material engineering, and transplant surgery. Such biological tissue engineering has been studied in various fields today, and is being used to treat patients who have been damaged in a specific part of the human body.

Recently, a histo-engineered treatment method in which stem cells are collected from autologous cells of a patient and regenerated into damaged tissues is in the spotlight. In the case of culturing stem cells collected from the patient's autologous cells and inducing differentiation into specific tissues and transplanting them into the defective tissue of the patient, it is useful because there is no transplant rejection reaction. The first successful organ transplant between humans was a kidney transplant between identical twin siblings, attempted by Joseph Murray in 1954. After many developments since then, human organ transplantation is expanding to all areas of human organs such as the heart, liver, pancreas, and lungs from the late 1970s with the development of new immunosuppressants and anesthesia and surgical techniques.

The development of transplantation surgery played a very important role in promoting human health, but the number of donated organs gradually became significantly less than the number of organs needed, reaching the limit. To solve this problem, organs from animals were transplanted to humans. I have studied and looked at so-called xenotransplantation, but when used for a long period of time, problems such as rejection and infection occur and must be exchanged frequently, and even if there are no problems such as rejection or infection, there is a problem that is significantly inferior in functioning to maintain life. there was.

In order to solve this problem, biomaterials with excellent biocompatibility without rejection in the human body have been developed, or bio-tissue engineering approaches have been studied to replace damaged organs from the patient's own cells.

First, artificial biomaterials with excellent biocompatibility include natural materials that exist in the human body or can be obtained from nature, and artificial materials represented by various polymers. Natural materials include chitin and chitosan, and artificial materials include synthetic polymers such as polyurethane and silicone rubber. These materials have the advantage of being able to change physicochemical surface properties or mechanical properties in various ways. It has the disadvantage that it cannot be used permanently in the human body due to coagulation of Therefore, in order to compensate for this, many studies have been conducted on bio-tissue engineering methods to replace damaged organs from the patient's own cells.

The ideal healing modality of bone grafts is complete host ossification of the graft material. However, it is known that bone healing patterns differ depending on the origin and treatment process of the graft material. For this reason, interest in the use of adult stem cells that have the ability to differentiate specifically to specific tissues and organs after in vivo transplantation in the process of tissue regeneration, and that can metastasize to cells of other tissues differently from the original cell characteristics, has attracted attention. Is losing.

Stem cells are a type of parent cell in which different cells or organs that make up the human body grow, and are also called stem cells. Stem cells include embryonic stem cells, which can be made using human embryos, and adult stem cells, such as bone marrow cells that constantly make blood cells. Embryonic stem cells can differentiate into all cells and tissues that make up the human body, but their use is limited for ethical reasons, whereas adult stem cells are derived from umbilical cord blood or bone marrow and blood of mature adults, and are mesenchymal stem cells and hematopoietic stem cells. Exists as. Adult stem cells are cells with differentiation flexibility that can differentiate specifically to specific tissues and organs after transplantation in vivo and can metastasize into cells of other tissues different from the original cell characteristics. Is widely used in

Already in human and animal studies, it has been confirmed that bone marrow-derived stem cells among adult stem cells are differentiated into osteogenic cells (Friedenstein AJ et al., Transplantation., 6:230-247, 1968). There is progress in the method of culturing stem cells isolated from bone marrow to differentiate and proliferate into osteoblasts, and the possibility of clinical application through this is increasing (Ohgushi H. et al., J Biomed Mater Res., 48:913-927, 1999 ). In recent years, a method of differentiating bone cells from mesenchymal stem cells is being actively studied.

However, autologous bone graft requires bone harvesting from patients with reduced basic physical strength and immunity.There are problems that may result in complications such as infection, pain, and hematoma at the bone collection site. Even if it is considered to collect bones from a donor who does not have a bone marrow, there is a problem that the number of bone marrow donors is limited. Therefore, there has been an urgent need to develop a method for differentiating stem cells taken from other body parts of patients into bone cells.

Accordingly, the present inventors are striving to develop a method for inducing the differentiation of bone cells from stem cells, and in particular, while paying attention to the possibility of the bone differentiation promoting function of the jade extract and devising various methods to verify its actual effect, jade extract Was first treated on skin cells, and the supernatant of the medium containing the functional ingredients of the skin cells secreted by stimulation of the extract was obtained and treated with adipose-derived stem cells (ADSC). As a result, bone differentiation of ADSC was analyzed by analyzing gene expression. The present invention was completed by confirming that the composition can induce bone cell differentiation.

An object of the present invention is to provide a method for differentiating stem cells into bone cells from components derived from the skin of a mammal.

The object of the present invention is to obtain a medium supernatant containing functional secretions of activated skin cells by treating the jade extract on skin cells, and then to treat this on adipose-derived stem cells and analyze the expression of bone genes to confirm the function of promoting bone differentiation. And, to provide a composition for treatment and cosmetics using the obtained bone cells.

In order to achieve the above object, the present invention comprises the steps of: 1) treating the jade extract to skin cells (Fibroblast) to discharge the activated functional ingredients; 2) separating the supernatant of the medium containing the functional ingredient; 3) treating and culturing the separated medium supernatant on stem cells to differentiate into bone cells; And 3) separating the differentiated bone cells. It provides a method for differentiating bone cells of stem cells using the jade extract.

In addition, the present invention provides a composition for restoring skin cells or treating arthritis containing bone cells prepared by the method and a functional cosmetic composition containing bone cells prepared by the method.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of: 1) treating the jade extract to skin cells (Fibroblast) to discharge the activated functional ingredients; 2) separating the supernatant of the medium containing the functional ingredient; 3) treating the separated medium supernatant to stem cells and culturing them to differentiate into bone cells; And 3) separating the differentiated bone cells. It provides a method for differentiating bone cells of stem cells using a jade extract.

In the method for differentiation of stem cells from stem cells by the jade extract of the present invention, the jade extract is preferably extracted with hot water at 70° C. for 2 hours and concentrated under reduced pressure at a concentration of 50 to 500 ug/mL, and the stem cells Is preferably an adipose-derived stem cell (ADSC), and the culture in step 3) is preferably performed for 1 to 10 days.

The present invention relates to a function of promoting bone differentiation of a jade extract based on the fact that the secretion of skin cells activated by the jade extract has an activity of differentiating adipose-derived stem cells into bone cells. Specifically, in order to promote bone differentiation of the jade extract, after treating the skin cells with the jade extract, a medium supernatant containing the functional ingredients of the skin cells secreted due to the extract stimulation was obtained and treated with adipose-derived stem cells (ADSC). After that, it was confirmed by gene amplification and expression analysis that it induces bone differentiation of ADSC.

In addition, the present invention provides a composition for restoring skin cells or treating arthritis containing bone cells prepared by the method and a functional cosmetic composition containing bone cells prepared by the method.

In the cosmetic composition of the present invention, the composition is skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, It is preferable to be used in cosmetics selected from the group consisting of packs, soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, press powders, loose powders, and eye shadows, and the composition is cosmetology or skin It is preferable to further include an auxiliary agent that is commonly used in the scientific field and includes an amount generally used, wherein the auxiliary agent is a fatty substance, an organic solvent, a solubilizing agent, a thickening agent, a gelling agent, an emollient, an antioxidant, a suspending agent. , Stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments It is more preferable to include at least one selected from the group consisting of hydrophilic or lipophilic active agents and lipid vesicles.

In addition, the content of the active ingredient in the functional cosmetic composition varies depending on the type of use, purpose of use, method of use, formulation, etc., but, for example, 0.01 to 8% by weight, preferably 0.5 to 5% by weight of a cosmetic agent is contained, and an ointment or cream The agent is 0.01 to 12% by weight, preferably 5 to 12% by weight, and the remainder is filled with additives and solvents required for each formulation.

In addition to the functional cosmetic composition containing the bone cells of the present invention, in addition to glycolic acid and vitamin C, it may contain various additives such as a soft meat and a moisturizer, an emollient, a humectant, a lubricant, and a preservative.

The additives include spermaceti, stearyl alcohol, polyethylene glycol 4000, glycerin, cetanol, sodium lauryl sulfate, ethyl paraoxybenzoate, butyl paraoxybenzoate, and propylene glycol, and these additives are added respectively or purchased commercially. It can be added in the form of ingredients contained in the possible base.

Sperm wax is obtained by refining the waxy solid component in the tofu of whales. It is added to other bases to give the ointment an appropriate consistency and can be added in an appropriate amount. Stearyl alcohol (stearyl alcohol) acts as a lubricant and can be added in an appropriate amount. Polyethylene glycol 4000 is a representative water-soluble base, a polymer of ethylene oxide and water. It is well soluble in water, has good mixing properties with most pharmaceuticals, and has excellent absorption of the main drug. It can be used in an appropriate amount.

Glycerin can be used in an appropriate amount as an emollient and a desiccant. Cetanol may be used in an appropriate amount as an emulsifier and thickener. Sodium lauryl sulfate (sodium lauryl sulfate) may be used in an appropriate amount as a wetting emollient to aid in mixing the composition. Ethyl paraoxybenzoate and butyl paraoxybenzoate may be included in an effective amount as a preservative for sustaining the effectiveness of the formulation. The preservatives exhibit activity in a wide pH range, and their effectiveness is increased by using the two types together. Propylene glycol may be used in an amount of 5 to 10% by weight as a surfactant, and it helps the skin absorption of glycolic acid and vitamin C.

When the bone cell differentiation method of the present invention is used, adipose-derived stem cells can be differentiated into bone cells. Therefore, the promotion of bone cell differentiation according to the present invention can be effectively used for the prevention of osteoporosis in the middle and old age and bone development of the growing infant of the jade extract, and the bone cells obtained according to the present invention can be used for skin diseases such as general scars and burn scars and bone defects. It can be usefully used in the treatment of bone metabolic diseases such as minor procedures.

1 is a table schematically showing the overall experimental process of the present invention.
2 and 3 are graphs showing flow cytometry graphs (FIG. 2) and cell activity (FIG. 3) of skin cells under the jade extract as a cytotoxicity experiment for jade extract of skin cells.
4 to 6 show the expression of bone cell differentiation genes by the jade extract.
7 shows the differentiation of bone cells by activated skin cells.
8 is a test report showing the number of viable cells present in the jade powder used in the present invention.
9 is a component analysis test report of the extract of the present invention.
10 is a graph showing changes in cell morphology.
11 and 12 are photographs of low magnification (FIG. 11) and high magnification (FIG. 12) of cell morphology change.
13 is a graph showing the cytotoxicity experiment of jade powder.
14 is a graph showing changes in the concentration of calcium ions in cells.

Hereinafter, the present invention will be described.

First, jade powder was extracted with hot water and concentrated under reduced pressure to prepare jade extracts of various concentrations. In addition, skin cells (Fibroblast) were cultured, and the jade extract concentration to be optimally treated was determined by treating the cultured skin cells with the jade extract at various concentrations.

The supernatant of the culture medium of the skin cells treated with the jade extract was separated, treated with pre-cultured adipose-derived stem cells (ADSC), and analyzed for bone cell differentiation for each cell for 1 to 10 days.

As a result, it was confirmed that various bone differentiation markers and bone differentiation genes were expressed in cells cultured for 7 days. In other words, skin cells activated by the jade extract secrete a substance that promotes bone differentiation, which promotes bone differentiation of ADSC.

Hereinafter, preferred embodiments according to the present invention will be described in more detail by presenting them in more detail. However, the following embodiments are provided so that the present invention may be sufficiently understood by those of ordinary skill in the art, and may be modified in various other forms, and the present invention is limited by the above embodiments. It is not.

<Example 1> Determination of skin cell treatment concentration of jade extract

First, skin cells (Fibroblast) (Korea Cell Line Bank) were treated with jade extract in culture medium (DMEM, Hyclone), and cultured to 80% cell density at 37°C and 5% CO2.

The jade extract was used by extracting jade powder with hot water (70° C., 2 hours) and a vacuum concentrator (WEV-1001V, Korea Science), and the concentration of each jade extract (50, 100, 300, 500 ug/mL) was applied to the skin. After the cells were treated for 24 hours, cell viability was measured (Annexin V FITC and PI, Invitrogen) (FIG. 3). Cell viability analysis was performed with a flow cytometer (Calbuir, BD science) and an analysis program (FlowJo 10.6, BD science) (FIG. 2).

As a result of measuring the cell viability for the jade extract of the skin cells, the optimal concentration to be treated on the skin cells was determined to be 150 ug/mL.

<Example 2> Separation of the supernatant of skin cell treatment medium of jade extract

As a result of Example 1, the skin cells were treated with 150 ug/mL of the jade extract, and the supernatant of the fibroblasts treated with the jade extract was separated with a 1 ml micropipette.

<Example 3> Culture of adipose-derived stem cells (ADSC) and treatment of skin cell supernatant

ADSC (StemPro™ Human Adipose-Derived Stem Cells, Thermofisher) was cultured in a culture medium (StemPro™ MSC SFM XenoFree, Gibco) at 37°C and 5% CO2 to 80% cell density. 100uL of the supernatant of the skin cell culture medium treated with the jade extract (150ug/mL) obtained in Example 2 was taken, treated with ADSC, and cultured for 1-10 days, and then each cell was taken and analyzed for bone differentiation as follows. .

<Example 4> Analysis of bone differentiation of adipose-derived stem cells (ADSC)

The bone differentiation analysis of ADSC was performed using antibodies of osteoponin (osteoponin-PE, biotechne), osteocalcin (osteocalcin-FITC, biotechne), and sclerotin (SOST-Alexa647, Novusbio), which are bone differentiation markers. And analysis program (FlowJo 10.6, BD science). In addition, the expression of the bone differentiation genes Runx2 and DLx5 genes was analyzed by PCR. In PCR analysis, all RNAs were extracted using Ribospin reagent (GeneAll, Seoul, Korea). For cDNA synthesis, Maxime™ RT-Premix (iNtRON, Seong-nam, Korea) was used, and gene amplification (AccuPower PCR premix, Bioneer) was performed using the prepared PCR Primer (Bioneer, Deajeon, Korea). Gene expression analysis was performed using Chemidak (ibright FL1000, thermofisher). After staining with antibodies corresponding to the bone differentiation markers, the type and number of bone cells were counted using a fluorescence microscope (Ts2-Nikon, Japan) (FIGS. 4 to 6 ).

As a result of analyzing the expression of the genes Runx2 and Dlx5, which are expressed in the early stages of bone differentiation, the amount of Runx2 expression in cells cultured for 7 days increased by 4.19 times compared to the control, and the expression level of Dlx5 increased by 1.24 times. This can be confirmed that the skin cells activated by the jade extract secreted a substance that promotes bone differentiation, thereby promoting the bone differentiation of ADSC.

As a result of flow cytometric analysis of osteocalcin and osteoponin expressed in osteogenic cells, the expression levels of these proteins increased by 1.7 times compared to the control in cells cultured for 3 days and 7 days, and more than doubled in cells cultured for 10 days. As a result of checking at the protein level, it was confirmed that bone differentiation was promoted by the jade extract, similar to the PCR result.

As a result of confirming bone differentiation cells with a fluorescence microscope, osteoblast (osteocyte) increased up to 2 times compared to the control on days 3, 7, and 10, and in the case of osteocyte (osteocyte), 4,4 times on day 3, 10 times on day 7, On the 10th day, it increased 13 times (Fig. 7). 7 shows the differentiation of bone cells by activated skin cells.

<Example 5> Control of immune response of jade powder using macrophage cell line

First, 0.1 g of jade powder was diluted in 10 ml of PBS, centrifuged, and the supernatant was collected, passed through a 0.45 uM filter, and diluted in DMEM medium to be used. Using the Raw246.7 cell line (mouse macrophage), the immune activity of jade powder was investigated by repeating at least 3 times per group. The treated LPS concentration was treated with 20 ng/ml for 6 hours.

Macrophages were treated with LPS alone or with jade powder as described above. At this time, LPS-pre Jade is jade powder before legislation and post jade is jade powder after legislation.

Calcium ion concentration was measured by fluorescence after diluting 1uM Calcium Green-1 ^TM in the culture medium and incubating for 3 hours, and the cytotoxicity test was performed by Tryphan Blue exclusion test. For statistical processing, student't test and one-way ANOVA were used, and statistically, a value of P <0.05 was considered a statistically significant difference.

The change in cell morphology after LPS treatment is shown in FIG. 10. As a result, there was no statistically significant morphological difference between each group. At this time, FSC-H (Forward sidescatter-Height) is a measurement of cell size, SSC-H (Sidescatter-Height) is a measurement of intracellular structure change, LPS-pre Jade is jade powder before legislation, and post Jade is jade powder after legislation. . Changes in cell morphology were confirmed at low magnification (FIG. 11) and high magnification (FIG. 12).

Cytotoxicity experiments of jade powder were performed (Fig. 13), and the change in the concentration of calcium ions in the cells (Fig. 14) was measured. As a result, it was observed that the intracellular calcium ion homeostasis was broken during LPS treatment. When treated with jade powder, the result of protection from the above phenomena was observed. Cellular activity at the time of treatment with LPS and at the time of simultaneous treatment with LPS-okgaru is shown in Table 1 below.

Cell viability (%) Control 90.18±3.08 LPS 87.70±2.89 Jade powder after LPS-legal 84.65±2.64

In conclusion, it was observed that the homeostasis of calcium ions in Raw246.7 cell line (mouse macrophage) was broken when LPS treatment, and at this time, it was observed that treatment with jade powder after the legislation alleviated the destruction of calcium ion homeostasis. In addition, it was confirmed that jade powder and LPS after legislation did not cause abnormal morphological changes and cytotoxicity when treated to Raw 247.7 cells.

<Production Example>

A lotion, emulsion, and essence containing the osteogenic bone cell composition of Examples 4 and 5 were prepared.

<Preparation Example 1> Preparation of lotion containing bone cell composition

Mix and dissolve 0.05 g of polypyrrolidone, 0.1 g of oleyl alcohol, 0.2 g of polyoxyethylene monooleate, 0.2 g of fragrance, 0.1 g of paraoxybenzoate methyl ester, a small amount of antioxidant, and a small amount of pigment in 8 g of 95% ethanol. . 10 g of the bone cell composition and 5 g of glycerin were dissolved in 85.33 g of purified water, and the mixture was added and stirred to obtain a skin-improving lotion.

<Production Example 2> Preparation of emulsion containing bone cell composition

Cetyl alcohol 1.2g, squalane 10g, vaseline 2g, paraoxybenzoic acid ethyl ester 0.2g, glycerin monoesterade 1g, polyoxyethylene (20 mole addition) monooleate 1g and fragrance 0.1g were heated and mixed and dissolved at 70℃. , 0.5 g of the bone cell composition obtained in Example 3, 5 g of dipropylene glycol, 2 g of polyethylene glycol-1500, 0.2 g of triethanolamine, and 76.2 g of purified water were dissolved by heating at 75°C. Both were mixed, emulsified, and then cooled to obtain an oil-in-water (O/W) type emulsion with skin improvement effect.

<Preparation Example 3> Preparation of essence containing bone cell composition

95% ethyl alcohol 5g, polyoxyethylene sorbitan monooleate 1.2g, kitulose 0.3g, sodium hyaluronate 0.2g, vitamin E-acetate 0.2g, sodium licorice 0.2g, paraoxybenzoate ethyl ester 0.1g, practice 1 g of the bone cell composition obtained in Example 3 and an appropriate amount of pigment were mixed to obtain a cosmetic liquid having an effect of improving skin.

<Preparation Example 4> Preparation of a composition in which glycolic acid and vitamin C are added to the bone cell composition

45 g of glycolic acid (99% glycolic acid, manufactured by Aldrich) and 30 g of vitamin C (L-Ascorbic acid, Sigma) were well mixed with 450 g of the bone cell composition.

Above, the present invention has been described in detail with reference to a preferred embodiment, but the present invention is not limited to the above embodiment, and various modifications by those of ordinary skill in the art within the scope of the technical idea of the present invention This is possible.

Claims (6)

1) treating the skin cells (Fibroblast) with a jade extract having a concentration of 50 to 500 ug/mL to discharge the activated functional ingredients;
2) separating the supernatant of the medium containing the functional ingredient;
3) treating and culturing the separated medium supernatant on adipose-derived stem cells (ADSC) to differentiate into bone cells; And
4) Separating the differentiated bone cells; Bone cell differentiation method of stem cells by a jade extract comprising.
The method of claim 1, wherein the jade extract extracts jade with hot water at 70° C. for 2 hours and concentrates under reduced pressure.
delete The method of claim 1, wherein the culture in step 3) is performed for 1 to 10 days.
delete delete

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