KR102088714B1 - Composition for promoting osteogenic differentiation comprising salicylate as effective component - Google Patents

Abstract

The present invention relates to a composition for promoting osteogenic differentiation of stem cells derived from periosteum comprising salicylic acid as an active ingredient. A composition for promoting osteogenic differentiation of adult stem cells and a method for promoting differentiation from the adult stem cells to osteocyte using the same of the present invention may be useful in the field of cell therapy for bone-related diseases.

Description

Composition for promoting osteogenic differentiation comprising salicylate as effective component

The present invention relates to a composition for promoting osteogenic differentiation of periosteum-derived stem cells containing salicylic acid as an active ingredient.

Recently, a rapid aging society is in progress, and as a result, the number of patients with degenerative bone diseases such as osteoarthritis and osteoporosis is also increasing. In addition, regenerative medicine is actively being studied to develop new control and treatment methods for bone-related diseases caused by aging. Regenerative medicine is a treatment method that replaces damaged or defective tissues and organs by creating new tissues or organs by using biocompatible materials or stem cells alone or in combination. Mesenchymal stem cells (MSCs) derived from mesenchymal cells are self-proliferative and can be differentiated into bone cells, chondrocytes, and adipocytes, so they are actively researched as a material for regenerative medicine targeting degenerative bone diseases. Is becoming. Bone marrow-derived mesenchymal stem cells (BMMSCs) have been traditionally used in bone regeneration studies using differentiation induction from MSCs to osteoblasts or bone cells. However, BMMSCs have the disadvantage that there is a risk of pain and infection accompanying stem cell extraction by bone marrow inhalation. Recently, while supplementing these shortcomings, periosteum-derived mesenchymal stem cells (POMSCs) containing periosteum origin including osteoprogenitor cells, which are important for bone regeneration, are also used as research materials for regenerative medicine. .

Interestingly, in recent studies on stem cell differentiation and metabolism, glycolysis decreases gradually while the differentiation of stem cells progresses, whereas oxidative phosphorylation occurs as the differentiation occurs. It is reported to increase. Oxidative oxidation is a biochemical reaction that occurs in the mitochondria and is an important process for the production of ATP, a biological energy. This oxidative oxidation is achieved by five oxidative phosphorylation complexes (OXPHOS complexes) present in the mitochondrial inner membrane. The 13 transmembrane protein subunits, which are essential for the function of OXPHOS complexes in mammals, are encoded in the mitochondrial DNA, transcribed and translated within the mitochondria, and inserted into the mitochondrial lining. Therefore, it can be said that mitochondrial DNA is essential for ATP production by oxidative oxidation. However, studies on the effect of mitochondrial biosynthesis and oxidative oxidation or the effect of mitochondria on differentiation in the bone cell differentiation process of POMSCs are still incomplete.

Salicylate is a major component of aspirin, one of the oldest medicines, and has been widely used as an anti-inflammatory, analgesic and antipyretic agent. In a recent study, salicylic acid treatment was reported to induce mitochondrial biosynthesis by regulating the expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) in animal cells (Biochem Biophys Res Commun. 2017 Sep 16; 491 (2): 436-441). Although many studies have been conducted to identify the stem cell differentiation process of stem cells using POMSCs, attempts to analyze the bone cell differentiation process from the perspective of mitochondrial biosynthesis and energy metabolism are still insufficient. Thus, in the present invention, the effect of salicylic acid, which has an effect of enhancing mitochondrial biosynthesis, on the bone cell differentiation process of POMSCs was studied.

On the other hand, Korean Patent Publication No. 2017-0044333 discloses a composition for promoting bone cell formation comprising '3,4'-dihydroxyflavone as an active ingredient, and Korean Registered Patent No.1594341 discloses' benzoyl aconin. Disclosed is a composition for inducing bone cell differentiation of mesenchymal stem cells and a method for inducing bone cell differentiation using the same, but there is no description of a composition for promoting bone cell differentiation comprising salicylic acid of the present invention as an active ingredient.

The present invention was derived by the above-described demands, and the present inventors cultivated adult stem cells derived from periosteum by adding salicylic acid to a bone cell differentiation-inducing culture at a concentration of 200 μM and 1 mM, and in a culture solution not containing salicylic acid. It was observed that the activity of ALP, a marker for osteoblast differentiation, was significantly increased in adult stem cells cultured in a culture medium containing salicylic acid at a concentration of 1 mM compared to the cultured adult stem cells, and the biosynthesis of intracellular mitochondria was also significantly increased. , Through the above results, the present invention was completed by confirming that the treatment of salicylic acid can promote the initial process of osteoblast differentiation of adult stem cells derived from periosteum.

In order to solve the above problems, the present invention provides a composition for promoting osteogenic differentiation of adult stem cells comprising salicylic acid or a salt thereof as an active ingredient.

In addition, the present invention comprises the step of culturing the periosteum-derived adult stem cells in a bone cell differentiation-inducing culture medium containing salicylic acid or a salt thereof as an active ingredient, to promote differentiation from periosteum-derived adult stem cells to bone cells. How to order.

In the present invention, it was suggested that salicylic acid can be a substance capable of controlling differentiation from adult stem cells to bone cells. In addition, by confirming that salicylic acid can also increase the biosynthesis of mitochondria along with an increase in the activity of ALP, a marker for bone cell differentiation, substances that regulate mitochondrial biosynthesis or function may also affect the process of osteoblast differentiation of adult stem cells It has been suggested that these substances can be applied as new modulators for bone cell differentiation or regenerative medicine. Therefore, the composition for promoting bone stem differentiation of adult stem cells of the present invention and a method using the same may be useful in the field of cell therapy for bone-related diseases in the future.

1 is an analysis of the effect of salicylic acid (salicylate) on proliferation (A) and survival (B) of adult stem cells (POMSCs) of periosteal origin.
Figure 2 is an analysis of the effect of salicylic acid on bone cell differentiation of POMSCs, confirming the activity of ALP (alkaline phosphatase) in cell lysates on days 5 and 10 of culture. *; P <0.05.
3 is that the analysis of the effect of salicylic acid on mitochondrial biosynthetic POMSCs for differentiation of bone cells, the flow cytometric analysis (A) and its quantification (B) a graph of POMSCs with Mitotracker Green FM ^® of mitochondrial staining reagent. *; P <0.05, **; P <0.01.
FIG. 4 is a graph (A) and quantification (B) of the image visualizing the biosynthesis of mitochondria during bone cell differentiation of POMSCs using the mitochondrial staining reagent Mitotracker ^® Green FM. **; P <0.01.

In order to achieve the object of the present invention, the present invention provides a composition for promoting osteoblast differentiation (osteogenic differentiation) of adult stem cells comprising salicylic acid or a salt thereof as an active ingredient.

In the composition according to an embodiment of the present invention, the salicylic acid may be preferably included in the composition at a concentration of 0.5 to 1.5 mM, and more preferably, may be included in the composition at a concentration of 1 mM, but is not limited thereto. When the salicylic acid is contained in less than 0.5 mM, the effect of promoting the differentiation of adult stem cells into bone cells is not apparent within 5 days of culture, and when salicylic acid is contained in 2 mM or more, it promotes bone cell differentiation compared to the salicylic acid-free group. It was confirmed that the effect was insufficient.

In the composition of the present invention, the salt of salicylic acid refers to an organic acid salt of a colorless salicylic acid (2-hydroxybenzoic acid), which is one of aromatic oxycarboxylic acids, but is not limited thereto, sodium salicylate, salicylic acid Choline salicylate, methyl salicylate, or magnesium salicylate.

In the composition for promoting osteoblast differentiation of adult stem cells according to an embodiment of the present invention, the salicylic acid may be preferably sodium salicylate, but is not limited thereto.

In the composition of the present invention, the adult stem cells may be adult stem cells derived from periosteum, but are not limited thereto.

The term 'adult stem cell' is a cell extracted from umbilical cord blood (umbilical cord blood) or bone marrow and blood of an adult who has grown up, and is a primitive cell immediately before being differentiated into cells of specific organs such as bone, liver, and blood. to be. There are hematopoietic stem cells, mesenchymal stem cells and neural stem cells, which are in the spotlight as materials for regenerative medicine. Adult stem cells are difficult to proliferate and have a strong tendency to differentiate easily. Instead, various types of adult stem cells can be used to regenerate the organs required by actual medicine, as well as to differentiate according to the characteristics of each organ after transplantation. It has the characteristic. However, it is known that it is impossible to differentiate into cells of all tissues like embryonic stem cells. In addition, since adult stem cells are extracted from already grown body tissues such as bone marrow and brain cells, unlike embryonic stem cells extracted from human embryos, there is an advantage to avoid ethical disputes.

The periosteum is a fibrous connective tissue that covers the bone except the surface of the joint, and consists of two layers, the inner and outer layers. It protects the bones and serves to control the growth of the bones, and bone fractures are formed during the fracture, which also causes the bones to adhere.

The composition for promoting bone cell differentiation of adult stem cells of the present invention has an effect of increasing the activity of the alkaline phosphatase (ALP), a bone cell differentiation marker, by treatment with salicylic acid.

The present invention also includes the step of culturing the periosteum-derived adult stem cells in a bone cell differentiation-inducing culture medium containing salicylic acid or a salt thereof as an active ingredient, to promote differentiation from periosteum-derived adult stem cells to bone cells. How to order.

In the method according to an embodiment of the present invention, the salicylic acid may be included in the bone cell differentiation-inducing culture solution preferably at a concentration of 0.5 to 1.5 mM, and more preferably included in the bone cell differentiation-inducing culture solution at a concentration of 1 mM. However, it is not limited thereto.

In addition, in the method of the present invention, the salicylic acid may be preferably sodium salicylate (sodium salicylate), but is not limited thereto.

In a method according to an embodiment of the present invention, the bone cell differentiation-inducing culture medium contains salicylic acid at a concentration of 0.5 to 1.5 mM and L-ascorbic acid 2-phosphate at 45 to 55 μg / ml. ), 8 to 12 nM of dexamethasone (dexamethasone) and 8 to 12 mM of β-glycerol phosphate (β-glycerophosphate), preferably 1 mM of salicylic acid, 50 μg / ml of L-ascorbic acid 2 -Phosphate, 10 nM of dexamethasone and 10 mM of β-glycerol phosphate may be composed of, but is not limited to.

In addition, the method for promoting differentiation from periosteum-derived adult stem cells to bone cells according to the present invention may further include a step of determining whether the differentiated cells have differentiated into desired cells, wherein the determination is differentiation. It may be performed by confirming whether the cell expresses the mRNA or protein of the bone cell specific gene. Whether the mRNA or protein is expressed can be confirmed through various methods known in the art.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Materials and methods

Cell culture and osteoclast differentiation of adult stem cells (POMSCs) of periosteal origin

With the consent of the patient in accordance with the provisions of the Ethics Committee of Gyeongsang National University Hospital (GNUH 2014-05-012), a periosteum tissue sample was obtained from the jaw bone during extraction of the wisdom tooth and POMSCs were isolated (Arch Oral Biol. 2007 Oct; 52 (10): 983-9.). Cell culture was performed in a culture condition of 37 ° C and 5% CO ₂ using 10% fetal bovine serum and 1% penicillin / streptomycin added DMEM culture medium (Dulbecco's modified Eagle's medium). In order to induce bone cell differentiation, DMEM culture medium was added with 50 μg / ml L-ascorbic acid 2-phosphate, 10 nM dexamethasone, and 10 mM β-glycerophosphate in an osteogenic differentiation induction medium (hereinafter referred to as OM culture medium). Cells were cultured. In the cell culture for bone cell differentiation, the OM culture was replaced every 3 days.

Analysis of proliferation and viability of POMSCs

To analyze the effect of salicylic acid on cell proliferation and viability, sodium salicylate (catalog no.A17056, Thermo Fisher Scientific, Waltham, USA) after inoculation of POMSCs into 24-well plates at a concentration of 2 × 10 ⁴ cells / well The cells were cultured in OM culture treated with 200 μM and 1 mM concentration, respectively. On the 5th and 10th day of cell culture, cell proliferation ability was analyzed by counting cells using a hemocytometer, and cell viability was analyzed by MTT assay.

Alkaline phosphatase (ALP) activity assay

To investigate the effect of salicylic acid on bone cell differentiation of POMSCs, the activity of ALP, an early marker of bone cell differentiation, was analyzed. After inoculating POMSCs into 24-well plates at a concentration of 2 × 10 ⁴ cells / well, salicylic acid was cultured in OM culture treated with 200 μM and 1 mM, respectively. Bone cell differentiation induced ALP activity was analyzed on days 5 and 10 of cell culture. Briefly, cell lysis was first performed using NP40 Cell Lysis Buffer (Life Technologies, Carlsbad, USA). After adding the buffer and the ALP substrate, p-nitrophenyl phosphate (Thermo Fisher Scientific, Waltham, USA) was added to some cell lysates obtained therein, followed by enzymatic reaction for 10 minutes. Thereafter, ALP activity was analyzed by measuring absorbance at a wavelength of 405 nm using a microplate reader (Molecular Devices, San Jose, USA). For some other cell lysates, protein concentrations were analyzed by Bradford assay. To correct the absorbance according to the number of cells, the final ALP activity was determined by calculating the absorbance per protein of the same amount in the cell lysate.

Mitochondrial biosynthesis analysis

OM containing salicylic acid (200 μM or 1 mM) after inoculation of POMSCs in a 24-well plate at a concentration of 2 × 10 ⁴ cells / well to investigate the effect of salicylic acid on mitochondrial biosynthesis during the bone cell differentiation process of POMSCs Cells were cultured in culture. After 2 weeks of bone cell differentiation induced cell culture, Mitotracker ^® Green FM (Life Technologies, Carlsbad, USA), a fluorescent dye (dye) that specifically stains intracellular mitochondria, was treated with POMSCs differentiated for 20 minutes at a concentration of 200 nM. . Next, after the cells were obtained through trypsin treatment, they were suspended in PBS and analyzed by flow cytometry. In addition, after POMSCs were cultured on a chamber slide under the same conditions as above, the intracellular mitochondria were stained with Mitotracker ^® Green FM fluorescent dye, and observed with an Observer.Z1 (ZEISS, Oberkochen, Germany) microscopy microscope. Quantitative analysis was performed using the Image J program (NIH, Bethesda, USA).

Statistical analysis

All experiments were performed at least 3 times independent repetition, and the results obtained from the repetition experiments were analyzed by variance using Graphpad Prism 7 software (GraphPad, La Jolla, USA), and expressed as mean ± standard deviation. Statistical analysis of differences in mean values of each experimental group determined that it was statistically significant when p <0.05.

Example 1. Analysis of the effect of salicylic acid on cell proliferation and viability of POMSCs

To investigate the effect of salicylic acid on cell proliferation and viability of POMSCs, salicylic acid was treated with POMSCs at a concentration of 200 μM and 1 mM, respectively, and cultured for 5 days and 10 days in bone cell differentiation OM culture. Thereafter, cell counting and MTT assay were performed. Compared to the control group not treated with salicylic acid, the number of cells proliferated in the cell culture period between 5 and 10 days in the group treated with 200 μM and 1 mM salicylic acid was found to be similar (FIG. 1A). The results of MTT assay also showed that salicylic acid (200 μM or 1 mM) treatment had no significant effect on the viability of POMSCs (FIG. 1B). The results indicated that salicylic acid did not affect the proliferation and viability of POMSCs at a concentration of at least 200 μM and 1 mM.

Example 2. Analysis of the effect of salicylic acid on the induction of osteoblast differentiation of POMSCs

In order to investigate the effect of salicylic acid on the induction of bone cell differentiation of POMSCs, POMSCs were cultured with a bone cell differentiation OM culture supplemented with 200 μM and 1 mM salicylic acid. After 5 and 10 days of induction of osteoblast differentiation, the activity of ALP, well-known as an early osteoblast differentiation marker, was measured. As shown in Figure 2, compared to the control group using a conventional DMEM culture medium that does not have the effect of inducing bone cell differentiation, the OM culture group that induces bone cell differentiation has ALP activity of about 2 times and 10 days in culture for 5 days. In culture, it increased about 4 times. These results clearly show that the POMSCs used in the present invention have the ability to differentiate into osteoblasts, an early stage of bone cell differentiation, in response to OM culture. In addition, treatment with 1 mM salicylic acid in the OM culture increased ALP activity by about 2.5 times and 5 times compared to the control group using DMEM culture in both cell culture for 5 days and 10 days. The relatively low 200 μM salicylic acid treatment was not different in the 5-day culture, but it was observed that the ALP activity was slightly increased in the 10-day culture. Although not shown in Figure 2, the higher concentration of 2 mM salicylic acid treatment did not show an increase in bone cell differentiation induction compared to the OM culture group. Taken together, these results suggest that salicylic acid treatment can facilitate the initial process of osteoblast differentiation of POMSCs.

Example 3. Analysis of the effect of salicylic acid on the mitochondrial biosynthesis in the process of osteoblast differentiation of POMSCs

In order to investigate the effect of salicylic acid on mitochondrial biosynthesis in the process of bone cell differentiation of POMSCs, POMSCs were cultured for 2 weeks with bone cell differentiation OM culture containing salicylic acid. The cultured cells were treated with Mitotracker ^® Green FM fluorescent dye to stain intracellular mitochondria. The stained POMSCs were quantitatively analyzed for intracellular mitochondria by flow cytometry. In FIG. 3A, compared to the control group not treated with salicylic acid, the fluorescence value increased at both concentrations of 200 μM and 1 mM salicylic acid, and these results indicated that the amount of intracellular mitochondria increased. In Figure 3B, the flow cytometry experiment results are presented quantitatively. On the other hand, by staining the cells in the differentiation-induced bone POMSCs as above under the same conditions in chamber slides and after the acid treatment accompanied, Mitotracker Green ^® the POMSCs incubated with FM fluorescent dye and observed with a fluorescence microscope (FIG. 4A). Two groups treated with salicylic acid (200 μM or 1 mM) showed a markedly increased green fluorescence compared to the control group, indicating an increase in intracellular mitochondria. In FIG. 4B, the green fluorescence of the fluorescence microscopy was quantified by an Image J program and shown in a graph. Taken together, these experimental results, which analyzed the amount of intracellular mitochondria after induction of osteoblast differentiation, indicated that salicylic acid treatment increased mitochondrial biosynthesis during the osteoblast differentiation process of POMSCs.

Claims (7)

A composition for promoting osteogenic differentiation of adult stem cells derived from periosteum containing sodium salicylate at a concentration of 0.5 to 1.5 mM as an active ingredient. delete delete Comprising the step of culturing periosteum-derived adult stem cells in a bone cell differentiation-inducing culture medium containing sodium salicylate at a concentration of 0.5-1.5 mM as an active ingredient, to promote differentiation from periosteum-derived adult stem cells to bone cells. As a method,
The bone cell differentiation-inducing culture medium was 0.5-1.5 mM sodium salicylate, 45-55 μg / ml L-ascorbic acid 2-phosphate, 8-12 nM dexamethasone and 8 Method consisting of ~ 12 mM β-glycerol phosphate (β-glycerophosphate). delete delete 5. The method of claim 4, wherein the method further comprises determining whether the differentiated cells have differentiated into desired cells.

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