KR101738087B1 - Composition for Facilitating the Osteogenic Differentiation of Stem Cells Comprising the Extract of Asiasarum heterotropoides as an Effective Ingredient - Google Patents

Abstract

The present invention relates to a composition for promoting bone differentiation of stem cells, and a composition for promoting bone marrow stromal differentiation comprising a sesamin extract as an active ingredient. The sesquin extract has an effect of inducing bone differentiation, And can be usefully used for osteogenesis of stem cells for the purpose of cell therapy.

Description

TECHNICAL FIELD The present invention relates to a composition for promoting bone marrow stromal differentiation,

The present invention relates to a composition for promoting differentiation of stem cells and a method for promoting differentiation using the same.

Recently, the use of stem cells in the field of regenerative medicine using tissue engineering has been proposed as a new field for the treatment of incurable diseases. Therefore, interest in stem cell research has increased, and it has been recognized that stem cells capable of forming a tissue through proliferation and differentiation can solve not only most diseases but also tissue damage.

Stem cells have the ability to self-replicate under undifferentiated conditions and differentiate into specific cells under appropriate conditions. Stem cells can be divided into embryonic stem cells and adult stem cells depending on their origins. Since human embryonic stem cells are derived from embryos that can occur in human life forms, they have excellent cell proliferation and differentiation potential, but have bioethical problems.

On the other hand, adult stem cells have a limited ability to differentiate from embryonic stem cells, but they have been developed into stem cells from bone marrow, blood, brain, and skin, which already exist in various organs of the human body. . As adult stem cells, there are hematopoietic stem cells, mesenchymal stem cells, neuroblastoma, epithelial cells, etc. Hematopoietic stem cells can produce hematopoietic cells and lymphocytes, and are useful for the treatment of immune system diseases including hematologic malignancies. Stem cells are differentiated into bone, cartilage, fat, and fibrous tissue, and are involved in the recovery of each tissue when damaged.

In other words, the term "stem cell" refers to a "undifferentiated" cell that has this differentiation ability but does not yet undergo differentiation. When appropriate conditions are met under such undifferentiated state, stem cells can be differentiated into various tissue cells, and studies for treating diseases using such cells have been actively conducted.

For example, by regulating the culture conditions of mesenchymal stem cells, mesenchymal stem cells are differentiated into osteoblasts, chondroblasts, myoblasts, and nerve cells . Patent Document 1 discloses a technique for differentiating stem cells into osteoblasts using SMOC1 (SPARC-related Modular Calcium-binding protein 1) protein or SMOC1 gene. In addition, studies have been made to differentiate a natural cell extract or compound into a specific cell by adding it to a stem cell culture medium.

On the other hand, Asiasari Radix is a dried whole plant, rhizome or root of Asiasarum heterotropoides or Asarum sieboldii . It is a perennial herbaceous plant. Its rootstock is relatively short and has a fine grain and a diameter of about 1mm. It has an unevenly bent cord shape with a diameter of 3 ~ 5mm and a yellowish brown marginal rhinoceros. It has many roots around 5 ~ 20cm in length. Its color is light brown, long and thin and very shallow vertical wrinkles. Seesin extract has been used for the purpose of local anesthesia, analgesic, antipyretic, shinhae, genomic and diuretic because it exhibits body temperature drop, swelling, antihistaminic action, and cardiac action.

The present inventors have studied whether or not extracts of natural products can be used for the differentiation of stem cells into osteoblasts, and confirmed that stem cells are differentiated into osteoblasts by treating the extract of Seixin with a culture of gingival stem cells. Respectively.

Korean Patent No. 10-1176449

It is an object of the present invention to provide a composition for promoting stem cell bone differentiation using an active ingredient derived from a plant of Asarum genus, and a method for promoting differentiation of stem cells using the composition.

In order to accomplish the above object, one aspect of the present invention provides a composition for promoting bone marrow stromal stem cell differentiation comprising cedma extract.

According to another aspect of the present invention, there is provided a method for promoting stem cell bone differentiation comprising treating a stem cell with a sesquiterpenic extract.

The composition for promoting bone marrow differentiation according to the present invention has an effect of promoting the differentiation of stem cells into osteoblasts, and when the stem cells are introduced into osteoblasts, the composition for promoting bone marrow stromal cells of the present invention is useful Lt; / RTI >

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned can be clearly understood by those skilled in the art from the following description.

FIG. 1 is a photograph showing changes in cell morphology at 1 day after culturing adult stem cells derived from gingiva extract;
A: Control group B: 0.001 占 퐂 / ml, C: 0.01 占 퐂 / ml, D: 0.1 占 퐂 / ml, E: 1 占 퐂 / ml, F: 10 占 퐂 / / Ml.
FIG. 2 is a graph showing cell viability at day 1 after treatment with acinaric acid extract. FIG.
FIG. 3 is a graph showing the degree of ALP activity after acinaric acid extract treatment;
0: positive control (induced in bone differentiation medium); 0.001-10 mu g / ml; Seesin extract treatment group.
FIG. 4 is a graph showing the degree of mineralization after acetic acid extract treatment;
0: positive control (induced in bone differentiation medium); 0.001-10 mu g / ml; Seesin extract treatment group.
FIG. 5 is a graph showing the degree of mineralization after acetic acid extract treatment;
0: positive control (induced in bone differentiation medium); 0.001-10 mu g / ml; Seesin extract treatment group.

First, terms used in the specification of the present invention will be described.

The term " stem cell " referred to in the present invention refers to a cell having pluripotent or totipotent self-renewal capable of differentiating into cells of all tissues of the individual Cells, and includes embryonic stem cells, inducible pluripotent stem cells, and adult stem cells.

The term " embryonic stem cell " refers to a cell obtained by extracting an inner cell mass from a blastocyst embryo immediately before fertilization of the embryo into the uterus of a mother, cultivating the same in vitro, (Pluripotent) or omnipotent (totipotent) self-renewal stem cells (self-renewal) refers to stem cells.

The term ' inducible pluripotent stem cells ' refers to cells that have been induced so that the differentiated cells have pluripotent differentiation potential through an artificial reprogramming process, which is also referred to as iPSC (induced pluripotent stem cells).

The term ' adult stem cells ' is a primitive cell just before differentiation, isolated from the tissues of mammals, including humans, and has the ability to grow indefinitely and various types of cells (eg, adipocytes, cartilage Cells, muscle cells, bone cells, etc.).

In addition, 'differentiation' referred to in the present invention refers to the development of less specific cells into specific cells, and the differentiation causes changes in the size, shape, membrane potential, metabolism activity and signal of the cells. Differentiation occurs mainly during the process of forming a complex tissue in a zygote by a multicellular organism, or when a damaged tissue is restored when it becomes an adult, adult stem cells differentiate into specific cells.

The ' extract ' referred to in the present invention refers to a substance extracted or separated from a raw material by an arbitrary method, and includes an extract obtained from a raw material, a concentrate obtained therefrom, a dried product and a powder of the concentrate, It means.

Also, the term ' medium ' referred to in the present invention means a composition containing nutrients necessary for maintaining growth and survival of cells in vitro .

The term " subculture " referred to in the present invention means a method in which a part of cells is periodically transferred to a new culture container in order to continuously cultivate the cells in a healthy state for a long period of time, and the culture medium is continuously changed while the culture medium is changed . As the number of cells increases in a culture container having a limited space, it is used as a method for increasing the number of healthy cells, since proliferation nutrients are consumed or pollutants are accumulated and cells naturally die after a certain period of time. Usually, Culture vessel) or culturing the cell group separately is referred to as 1 passage.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention provides a composition for stimulating bone marrow differentiation comprising Asiasari radix extract as an active ingredient.

In the present invention, it is preferable to use Asiasarum heterotropoides as a raw material of an effective ingredient of a composition for promoting bone marrow differentiation of stem cells. However, the present invention is not limited to this, but an Asarum genus plant belonging to the above- Plants of other species having a high genetic affinity can also be used as a raw material of the active ingredient of the composition for promoting stem cell bone differentiation of the present invention. The above-mentioned plant of the genus L. or the genus L. can be used without limitation, such as cultivated or commercially available plants.

The mugwort can be any one selected from the group consisting of fine roots, main roots, stems, leaves, hibiscus and fruit, and more preferably at least one of fine roots and main roots, but is not limited thereto.

The composition for promoting bone marrow stromal cell differentiation according to the present invention contains the extract obtained from the above-mentioned sesamin as an active ingredient. The extract can be obtained by any of known extraction methods such as a solvent extraction method, an ultrasonic extraction method, a filtration method and a reflux extraction method, and can be obtained by using a solvent extraction method or a reflux extraction method.

Water, an organic solvent, or a mixture thereof may be used as an extraction solvent for the extraction, and water is preferably used, but is not limited thereto. The organic solvent may be selected from the group consisting of C ₁ -C ₄ lower alcohols, hexane (n-hexane), ether, glycerol, propylene glycol, butylene glycol, ethyl acetate and methyl acetate, As the C ₁ -C ₄ lower alcohol, ethanol or methanol is preferably used, but not limited thereto.

The extraction can be carried out by adding the extraction solvent to the total weight of the sesquic acid by 1 to 20 times, preferably 2 to 10 times, more preferably 3 to 5 times, , But not limited to. When the addition amount of the extraction solvent is out of the above range, the extraction itself is not properly performed or the active ingredient can not be sufficiently secured even if it is extracted.

The extraction can be carried out in a temperature range of 20 to 200 ° C, preferably in a temperature range of 50 to 150 ° C, more preferably in a temperature range of 80 to 120 ° C, but is not limited thereto. Maintaining this temperature range may be effective in preventing decay, discoloration, and kinking.

The extraction can be performed for 0.5 to 20 hours, preferably for 1 to 15 hours, more preferably for 2 to 10 hours, but is not limited thereto. If the extraction time is out of the above range, the extraction itself may not be performed properly or the active ingredient may not be sufficiently secured even if it is extracted.

In addition, the extraction may be repeated several times, preferably one or two to five times, more preferably two to three times, but is not limited thereto.

After such an extraction process, the extract may be further roughened, such as concentrated or lyophilized. Specifically, the concentration may be concentrated under reduced pressure and concentrated using a vacuum rotary concentrator, but is not limited thereto. In addition, the concentration may be performed at 20 to 100 ° C, preferably 50 to 70 ° C, but is not limited thereto. In addition, the drying can be performed by lyophilization, and a vacuum freeze dryer is preferably used, but is not limited thereto. The lyophilization may be carried out in a temperature range of -50 to -100 ° C, preferably -60 to -80 ° C, but is not limited thereto.

The cecin extract obtained through the above process has an effect of promoting the differentiation of stem cells into osteoblasts. In other words, the cecin extract induces stem cells to differentiate into osteoblasts, and the differentiated osteoblasts exhibit high alkaline phosphatase (ALP) activity and form inorganic crystals.

The stem cell may be a stem cell derived from a mammal including a human. In addition, the stem cells may be adult stem cells, more preferably adult stem cells derived from gingivae.

In addition, the composition for promoting bone marrow stromal cell differentiation according to the present invention may contain the sheath extract in an amount of 0.0001 to 50 μg / ml, preferably 0.0005 to 30 μg / ml, But is not limited thereto, in an amount of 0.001 to 10 μg / ml. When the sesquicin extract is out of the above-described content range, the promoting effect of stem cell bone differentiation may be inhibited. In addition, when the extract of Sicin is contained in an amount exceeding 50 μg / ml, the shape of the stem cells may be changed from fusiform to round, and the number of dead cells may increase.

 In a specific example of the present invention, 400 g of cecin is immersed in 2,000 ml of water for 2 hours and heated under reflux for 2.5 hours. The extract of cecinus obtained is then added to the gum-derived adult stem cells at a concentration of 0.001-10 μg / , It was confirmed that the rate of differentiation of stem cells into osteoblasts was improved to show ALP activity in a shorter time than the control group (see FIG. 3), and the mineralization proceeded (see FIGS. 4 and 5).

Another aspect of the present invention provides a culture medium for differentiating stem cells comprising the sesquicline extract.

The sesquicent extract may be contained as one component in a culture medium for stem cell culture.

The culture medium for stem cell culture includes all mediums commonly used for culturing stem cells. For example, DMEM (Dulbeco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F May be a basic medium such as -10, F-12, MEM (Minimal Essential Medium), GMEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), or a medium containing components capable of inducing bone differentiation , But not limited to.

Another aspect of the present invention provides a method for promoting stem cell bone differentiation comprising the step of treating the stem cell with the sesicin extract.

The method of promoting stem cell bone differentiation according to the present invention includes treating the stem cell with the sesicin extract and culturing the stem cell treated with the sesamin extract.

The stem cells to be targeted for promoting bone differentiation may be in an in vitro state, and the sesquicent extract may be processed into stem cells in the form of a culture medium for culturing the stem cells. In addition, stem cells can be cultured in a three-dimensional scaffold to promote bone differentiation. The component constituting the support may be a biocompatible material, and the osteoblast cultured and differentiated in the support may be transplanted into the living body in a state of being attached to the support for bone disease treatment without being separated from the support separately.

The cultivation can be performed in an in-vitro state or an in-vivo state, and can be performed in a CO ₂ incubator, particularly when performed in an in-vitro state.

The culture in the CO ₂ incubator can be carried out in an environment with a CO ₂ concentration of 5 to 15%, preferably 5 to 10%, more preferably 5 to 8% It is not limited. The inside of the CO ₂ incubator may be filled with O ₂ in addition to the CO ₂ .

In addition, the cultivation can be carried out in a temperature range of 25 to 45 DEG C, preferably 30 to 40 DEG C, more preferably 34 to 37 DEG C, but is not limited thereto. When the incubation temperature is outside the above temperature range, differentiation of stem cells is not efficiently promoted, and there is a problem that the number of stem cells to be killed is increased.

In addition, the culturing can be carried out continuously for 1 to 35 days, preferably 1 to 30 days, more preferably 1 to 25 days, but is not limited thereto. When the incubation period exceeds 35 days, as the density of cells in the culture vessel increases, the nutrients are rapidly depleted, the characteristics of the cells are changed, and the differentiation into osteoblasts is not induced. In addition, subculture may be performed during the incubation period to separate the cell groups and cultivate them.

Another aspect of the present invention provides a composition for preventing or treating bone defects comprising osteoblast differentiated into a composition for promoting bone differentiation according to the present invention as an active ingredient.

The differentiated osteoblast of the present invention can be used for various cell treatments. The differentiated osteoblasts can be mixed with various biologic adjuvants such as collagen or hydroxyapatite scaffolds and transplanted into the bone tissue by surgical operation. The therapeutic composition of the present invention may further contain, as an auxiliary component, collagen or an ophthalmic suspension in addition to the above-described differentiated osteoblasts. The osteoclast differentiated in the composition of the present invention can be controlled in number depending on the type of disease, route of administration, age and sex of the patient, and degree of disease, and preferably 1-5 × 10 ⁷ cells / cm < ³ > scaffold. In addition, the therapeutic composition of the present invention can be administered once or several times in divided doses.

In the composition of the present invention, the therapeutic composition is characterized by being used for treating bone diseases. Here, bone disease refers to a bone-related disease that requires bone differentiation and regeneration, and includes traumatic bone loss such as fracture and bone defect after surgery.

Hereinafter, the present invention will be described in detail with reference to Production Examples, Examples and Experimental Examples.

However, the following Production Examples, Examples and Experimental Examples are for illustrating the present invention, and the contents of the present invention are not limited by the following Production Examples, Examples and Experimental Examples.

[ Example  And Experimental Example ]

Preparation of sesquin extract

400 g of dried roots of asiasarum heterotropoides was immersed in 2,000 ml of distilled water for 2 hours and then heated at 100 ° C under reflux for 2.5 hours to obtain a sesquin water extract.

The extract was centrifuged at 5,000 x g for 10 minutes, and the supernatant was concentrated under reduced pressure to 300 ml using a rotary evaporator (Eyela NE-1001, Tokya Rikakikai Co. Ltd., Japan). The concentrate was lyophilized using a lyophilizer (Labconco, USA) to give 65 g of a solid residue (yield: 16% (w / w)).

Isolation and culture of gingival stem cells

Approved by the Ethical Review Committee of the Medical Research Ethics Committee of the Catholic University Medical College Seoul, we obtained normal gingiva tissue from four healthy patients undergoing crown enlargement.

The gingival tissues were immediately placed in sterile phosphate buffered saline (PBS, Welgene) containing 100 U / ml penicillin (Sigma Aldrich, USA) and 100 ug / ml streptomycin (Sigma Aldrich, USA) at 4 ° C. The gingival tissues were de-epithelialized and pulverized, digested with collagenase IV (Sigma Aldrich), and then incubated at 37 ° C in a humidified incubator of 5% CO ₂ and 95% O ₂ . After 24 hours of incubation, unattached cells were washed and replaced with medium based on? -MEM medium. The medium was prepared by mixing 15% fetal bovine serum (Gibco), 100 U / ml penicillin, 100 ug / ml streptomycin, 200 mM L-glutamine (Sigma Aldrich, USA) and 10 mM ascorbic acid 2- phosphate (Sigma Aldrich, ). Thereafter, human adult gingival adult stem cells were established by changing the medium every 2-3 days.

The cells exhibited stem cell characteristics such as colony-forming ability, plastic adherence and various lines of differentiation ability (osteogenesis, lipogenesis, cartilage formation), and the cells were analyzed by flow cytometry for CD44, CD73 , CD90 and CD105, but not CD14, CD45, CD34 and CD19.

Identification of stem cell morphology after treatment with sesqui extract

Example 2 2.0 the stem cells established in 96 well plates × 10 ³ cells / arranged at a density of wells, the stem cells of Example 1 α-MEM media supplemented with a slender extract obtained in (Gibco, USA ) In a humidified incubator of 5% CO ₂ and 95% O ₂ at 37 ° C. The α-MEM medium was prepared by mixing 15% fetal bovine serum (Gibco), 100 U / ml penicillin, 100 μg / ml streptomycin, 200 mM L-glutamine (Sigma Aldrich, USA) and 10 mM ascorbic acid 2- (Control), 0.001, 0.01, 0.1, 1, 10, 100 and 1,000 μg / ml in the α-MEM medium. The morphology of the cells was confirmed on day 1 using an inverted microscope (Leica DM IRM, Germany) and images were obtained.

As a result, in the case of stem cells cultured in a medium containing 0.001 to 10 μg / ml of the sesquin extract, the cell form was formed in the same spindle-shaped form as that of the control without addition of the sesquicin extract on day 1, Whereas the stem cells cultured with the medium containing 1,000 μg / ml of sesquicin extract had rounded morphology of the cells compared to the control without acetic anhydrase extract, It was confirmed that the cells remained (see Fig. 1).

From the above results, it was found that the addition of the extract of the present invention did not affect the morphology of the stem cells for a short period of time. When the extract was used in the stem cell culture, Or may be promoted to be killed, it is necessary to add it at a low content such as 0.001 to 100 占 퐂 / ml.

After treatment with sesqui extract, the survival of stem cells was also confirmed

Stem cells were cultured in the same manner as in Example 3, and live cells were identified by cell counting kit 8 (CCK.8, Japan) at 1 day of culture. Cell counting kit is an assay method for confirming cell viability. It is based on the principle that water-soluble tetrazolium salt is reduced by dehydrogenase in cells in culture medium to become formazan, and the amount of formazan produced is living Since the amount of formazan produced is proportional to the number of cells, the degree of cell viability can be confirmed by confirming the amount of formazan by colorimetric assays. Cells of the first day of culture were separated from the culture vessel to prepare a cell suspension. 100 μl of the cell suspension was added to a 96-well plate, and then 10 μl of CCK-8 solution was added to each well. Gt; 37 C < / RTI > for 2 hours in a humidified incubator of 5% CO ₂ and 95% O ₂ . The viability of the stem cells was confirmed by measuring the spectrophotometric absorbance of the cultured 96-well plate using a microplate reader (BioTek, USA). The analysis was repeated three times.

As a result, when the stem cell density of the control group was set to 100% (100.0 ± 1.8) on the day of culturing, 0.001 μg / ml of the sesquihime extract showed 194.6% ± 60.2% (see FIG. Also, it was confirmed that stem cell survival was high even at a concentration of 0.01 to 10 μg / ml of sesqui extract (see FIG. 2).

From the above results, it can be seen that the survival rate of the stem cells is increased by treatment with the sesquin extract, and the survival rate of the stem cells is particularly high when the sesquin extract is contained in an amount of 0.001 μg / ml.

Determination of Alkaline phosphatase (ALP) activity after treatment with Seixin extract

ALP is known to be a marker of osteoblast activity and acts as a regulator of the transport of inorganic phosphoric acid, cell division and differentiation during the calcification process. Therefore, ALP activity was measured in order to determine whether the sesquin extract induces the differentiation of gingival-derived adult stem cells into osteoblasts.

The sesquin extract-treated group was cultured by treating the sesquin extract in the same manner as in the culture medium of Example 3 above. That is, the stem cells established in Example 2 were cultured in a humidified incubator of 5% CO ₂ and 95% O ₂ in α-MEM medium (Gibco, USA) supplemented with the seschin extract obtained in Example 1 at a temperature of 37 ° C. Lt; / RTI > The α-MEM medium was prepared by mixing 15% fetal bovine serum (Gibco), 100 U / ml penicillin, 100 μg / ml streptomycin, 200 mM L-glutamine (Sigma Aldrich, USA) and 10 mM ascorbic acid 2- Aldrich, USA), and the sesquic phase extract was added to the α-MEM medium to a final concentration of 0 (control), 0.001, 0.01, 0.1, 1 and 10 μg / ml.

As a positive control, the stem cells established in Example 2 were cultured in an osteogenic differentiation inducing medium. Specifically, the second embodiment with a stem cell established at 2.0 × 10 ⁴ in 12 well cell culture plates at a cell density of cells / well, were incubated and then, the next day the medium was replaced with a bone differentiation-inducing medium for one night. The bone formation induction medium was prepared by adding 15% fetal bovine serum (Gibco), 100 μM dexamethasone (Sigma Aldrich, USA), 10 mM ascorbic acid 2-phosphate (Sigma Aldrich, USA) Ml penicillin, 100 占 퐂 / ml streptomycin.

The sesqui extract treatment group and the positive control group were cultured for 7 days and replaced with new bone differentiation induction medium every 3-4 days. Alkaline phosphatase activity assay was performed using a SensoLyte® pNPP Alkaline Phosphatase Assay Kit (AnaSpec, Inc, Canada) on the 7th day of culture according to the manufacturer's manual. Briefly, the cell lysate was mixed with the prepared 10 mM p-nitrophenyl phosphate substrate and incubated at 37 ° C. After incubation, the absorbance of p-nitrophenol was determined spectrophotometrically at 405 nm. The assay was performed five times.

As a result, when the sesquicin extract was added at a concentration of 0.001 to 10 μg / ml, it was confirmed that ALP activity was similar to that of the positive control supplemented with the bone differentiation inducing medium (FIG. 3). Especially, when the extract of Sicin extract was contained at a concentration of 0.001 ㎍ / ㎖, the ALP activity was higher than that of other extracts.

From the above results, it can be seen that bone turnover of stem cells is accelerated by treatment with sesquicin extract. In particular, when the sesquicin extract is contained in an amount of 0.001 μg / ml, it can be understood that the most excellent bone differentiation effect appears.

Check the degree of mineralization after treatment with sesqui extract

Alizarin red binds to inorganic crystals such as calcium. Since osteoblasts form inorganic crystals, the degree of differentiation into osteoblasts can be determined in proportion to the degree of alizarin red staining. The present inventors carried out alizarin red staining after culturing stem cells with a medium containing sesquin extract in order to confirm whether or not the stem cells differentiate into osteoblasts by the sesquin extract.

In the case of achene extract-treated group, the acacia sinensis extract was treated by concentration in the same manner as in the culturing method of Example 5 above. As a positive control, bone differentiation induction medium was treated. Specifically, the stem cells established in Example 2 were placed in a 24-well cell culture plate at a density of 1 x 10 ⁴ cells / well and cultured overnight. The next day, the medium was replaced with the bone differentiation induction medium. The composition of the differentiation-inducing medium was a mixture of 15% fetal bovine serum (Gibco), 100 μM dexamethasone (Sigma Aldrich, USA), 10 mM β-glycerol phosphate (Sigma Aldrich, USA), 10 mM ascorbic acid 2 -Phosphate (Sigma Aldrich, USA), 100 U / ml penicillin and 100 [mu] g / ml streptomycin.

The sesqui extract treatment group and the positive control group were cultured for 21 days and replaced with new bone differentiation induction medium every 3-4 days. Alizarin Red S staining (Sigma Aldrich, USA) was performed to measure calcium formation on days 14 and 21 of culture.

First, Alizarin Red S staining solution was prepared. 2 g of alizarin red S was dissolved in 100 ml of distilled water and the pH was adjusted to 4.1-4.3 to prepare an alizarin red S staining solution, which was then filtered and stored in a dark place.

To immobilize the cultured cells, the medium was removed from the culture vessel and the cells were washed with PBS. PBS was removed and neutralized formalin (10%) was treated to cover the cells. After 30 minutes, the formalin was removed and the cells were washed with distilled water and then distilled water was removed. Alizarin Red S, prepared and stored as above, was added to the fixed cells and stained in a dark room for 45 minutes. After completion of dyeing, alizarin red S was removed, washed 4 times with distilled water, and then phosphate buffer was added. The stained cells were observed with an inverted microscope. In order to quantitatively evaluate the degree of calcium formation, Alizarin Red S bound to calcium was dissolved in 10 mM sodium phosphate containing 10% cetylpyridinium chloride, and the resultant was identified by spectroscopic colorimetry at 562 nm.

As a result, it was confirmed that when the sesquicin extract was added at a concentration of 0.001 μg / ml, minerals were formed similarly to the positive control to which the bone differentiation inducing medium was added, and the sesquicin extract was contained at a concentration of 0.01 to 10 μg / , It was confirmed that the bone formation was not induced as much as the positive control but the mineral was formed (see FIGS. 4 and 5).

From the above results, it can be seen that bone turnover of stem cells is accelerated by treatment with sesquicin extract. In particular, when the sesquicin extract is contained in an amount of 0.001 μg / ml, it can be understood that the most excellent bone differentiation effect appears.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the scope of the invention is not limited to the disclosed exemplary embodiments. It will be possible to change it appropriately.

Claims (14)

A composition for stimulating osteogenic differentiation of stem cells, comprising a sesquicin extract as an active ingredient,
The stem cells are adult stem cells derived from gingiva,
Wherein said bone differentiation differentiates stem cells into osteoblasts. The method according to claim 1,
Wherein the sesquicin extract is extracted with water, an organic solvent or a mixture thereof as an extraction solvent. The method of claim 2,
Wherein the organic solvent is selected from the group consisting of C ₁ -C ₄ alcohols, hexane (n-hexane), ether, glycerol, propylene glycol, butylene glycol, ethyl acetate and methyl acetate. The method of claim 2,
Wherein the extraction solvent is water. delete delete delete The method according to claim 1,
The composition for stimulating osteogenic differentiation of stem cells according to claim 1, wherein the sesquicin extract is contained in an amount of 0.0001 to 50 μg / ml. As a medium for stem cell differentiation comprising a sesquin extract,
The stem cells are adult stem cells derived from gingiva,
Wherein said differentiation is differentiation of stem cells into osteoblasts. A method for promoting bone differentiation of stem cells in vitro comprising treating acini extracts in vitro on stem cells,
The stem cells are adult stem cells derived from gingiva,
Wherein said bone differentiation is differentiation of stem cells into osteoblasts. The method of claim 10,
Wherein the sesquicin extract is extracted with water, an organic solvent or a mixture thereof as an extraction solvent. The method of claim 11,
Wherein the extraction solvent is water. The method of claim 10,
Wherein the treatment time of the sesquicent extract is 1 to 35 days. delete

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