WO2023075571A1 - Use of polysaccharides derived from strains of enterococcus genus - Google Patents

Abstract

The present invention relates to use of polysaccharides derived from strains of an Enterococcus genus, wherein polysaccharides derived from strains of Enterococcus genus can promote bone differentiation of stem cells and thus can be used to promote osteogenesis, and can be used for the prevention or treatment of bone diseases that are treatable by bone regeneration.

Description

Uses of polysaccharides derived from strains of the genus Enterococcus

The present invention relates to the use of a polysaccharide derived from a strain of the genus Enterococcus.

Bone is the body's hard connective tissue and has several functions, including protecting various organs, producing red and white blood cells, storing minerals, and supporting the body. Bone has an intrinsic ability to repair itself. However, self-healing of bone is often difficult after severe bone loss has occurred. Bone regeneration is important in reconstructive surgery, and bone differentiation of stem cells may provide a tool for repairing bone defects.

Stem cells are used for tissue regeneration after injury or disease because they have the ability to self-renew and differentiate into a variety of cell types. Dental pulp stem cells (DPSCs) are neural crest-derived mesenchymal stem cells that can differentiate into adipogenic, osteogenic, chondrogenic and myogenic cells. DPSCs can be isolated from dental pulp tissues in a non-surgical way. DPSCs have the potential to be used to treat some diseases such as neurological diseases, immunodeficiency diseases and cartilage diseases.

On the other hand, exopolysaccharides are microbial polysaccharides that form capsular membranes around cell walls as part of microbial cell walls or accumulate during fermentation in the form of mucilage on the outside of cell walls, and are primary or secondary metabolites. EPS is composed mostly of polysaccharides (exopolysaccharides) and proteins, but also contains other macromolecules such as DNA, lipids and humic substances. EPS is a component of bacterial colonization and remains attached to the outer surface of cells or secreted into the growth medium.

An object of the present invention is to provide a composition for promoting osteogenesis comprising a polysaccharide derived from a strain of the genus Enterococcus.

An object of the present invention is to provide a pharmaceutical composition for preventing or treating bone diseases.

1. A composition for promoting osteogenesis comprising a polysaccharide derived from Enterococcus genus strain.

2. The composition for promoting bone formation according to 1 above, wherein the Enterococcus genus strain is Enterococcus faecium or Enterococcus lactis .

3. The step of centrifuging the growth medium containing the polysaccharide according to 1 above; Separating the supernatant of the growth medium and culturing by adding trichloroacetic acid to the separated supernatant; A composition for promoting bone formation obtained by a method for producing a polysaccharide comprising the steps of mixing and culturing the cultured supernatant with absolute ethanol to obtain a precipitate and dialysis of the precipitate.

4. The composition for promoting bone formation according to 1 above, wherein the concentration of the polysaccharide is 0.5 to 2 μg/ml.

5. The composition for promoting bone formation according to 1 above, which promotes the induction of osteogenic differentiation of stem cells.

6. The composition for promoting bone formation according to 5 above, wherein the stem cells are dental pulp stem cells.

7. The composition for promoting bone formation according to 1 above, which is used for at least one of artificial bone, artificial joint, bone fixation material, bone substitute, bone restoration material, bone filling material, or bone graft material.

8. A pharmaceutical composition for preventing or treating bone diseases treatable by bone formation or bone regeneration, comprising a polysaccharide derived from Enterococcus genus strain.

9. The pharmaceutical composition for preventing or treating bone disease according to 8 above, wherein the Enterococcus genus strain contains a polysaccharide of Enterococcus faecium or Enterococcus lactis .

10. The step of centrifuging the growth medium containing the polysaccharide according to 8 above; Separating the supernatant of the growth medium and culturing by adding trichloroacetic acid to the separated supernatant; A pharmaceutical composition for preventing or treating bone disease, obtained by a method for preparing a polysaccharide comprising the steps of mixing the cultured supernatant with absolute ethanol to obtain a precipitate and dialysis the precipitate.

11. The pharmaceutical composition for preventing or treating bone diseases according to 8 above, wherein the concentration of the polysaccharide is 0.5 to 2 μg/ml.

12. The bone disease according to 8 above, periodontal disease, periodontitis, alveolar bone disease, osteoporosis, osteomalacia, osteopenia, bone atrophy atrophy, osteoarthritis, rheumatoid arthritis, osteolysis, fibrous dysplasia, Paget's disease, osteoogenesis imperfect and hypercalcemia. At least one selected from the group consisting of, a pharmaceutical composition for preventing or treating bone disease.

The polysaccharide derived from the Enterococcus genus strain of the present invention can promote bone formation through the induction of osteogenic differentiation of stem cells.

The composition containing polysaccharides derived from Enterococcus spp. of the present invention can be applied to promote bone formation, and can be applied to bone regeneration and treatment or prevention of bone diseases requiring bone formation.

1 to 4 show the results of characterization of hDPSCs (human dental pulp stem cells) at passages 3 and 8 by fluorescence-activated cell sorting (FACS) (Figs. 1 and 2 are passages 3 (Passage 3), Fig. 3 and 4 are passage 8).

5 to 10 show that Enterococcus faecium L15 extract (L 15 extract) promotes osteogenic differentiation of hDPSCs. Figure 5 shows the cell viability (%) according to the L15 extract treatment at various concentrations. 6 is a bright field micrograph (EVOS™ XL Core Imaging System, scale bar = 200 μm) after ALP staining of hDPSCs treated with or without L15 extract. 7 shows calcium deposition through ARS staining of hDPSCs treated with or without the L15 extract. 8 shows the relative expression levels of osteogenic differentiation marker genes (RUNX2, ALP and COL1A1) analyzed by real-time PCR. GAPDH was used for normalization. 9 shows the relative expression levels of osteogenic differentiation marker proteins (BMP4, RUNX2 and ALP). FIG. 10 is a result of quantitatively analyzing the expression intensity of each protein compared to GAPDH shown in FIG. 9 . Error bars represent standard deviation of the mean (*p < 0.05, **p < 0.01, ***p < 0.001. N = 3). Extract: Enterococcus pesium L15 extract treatment group, Control: control group not treated with anything.

11 shows a size exclusion chromatogram of Enterococcus faecium L15-derived extracellular polysaccharide (L15 EPS).

Figure 12 shows the survival rate of hDPSCs when exposed to various concentrations of L15 EPS using osteogenic differentiation medium for (a) 3 days and (b) 7 days (***p < 0.001. Scale bar = 200 μm. N = 3).

13 to 17 show that Enterococcus faecium L15-derived extracellular polysaccharide (L15 EPS) promotes osteogenic differentiation of hDPSCs. 13 is a bright field micrograph (EVOS™ XL Core Imaging System, scale bar = 200 μm) after ALP staining of hDPSCs treated with or without L15 EPS. 14 shows calcium deposition through ARS staining of hDPSCs treated with or without L15 EPS. 15 shows the relative expression levels of osteogenic differentiation marker genes (RUNX2, ALP and COL1A1) analyzed by real-time PCR. GAPDH was used for normalization. 16 shows the relative expression levels of osteogenic differentiation marker proteins (BMP4, RUNX2 and ALP). 17 is a result of quantitatively analyzing the expression intensity of each protein compared to GAPDH shown in FIG. 16. Error bars represent standard deviation of the mean (*p < 0.05. N = 3). EPS: Enterococcus faecium L15-derived extracellular polysaccharide treated group, Control: a control group not treated with anything.

18 to 24 show that L15 EPS activates the p38 MAPK pathway. 18 is a Western blot analysis result of MAPK (ERK, JNK, and p38) and Wnt signaling pathways in hDPSCs treated with L15 EPS. 19 to 20 are quantitative analysis results of phosphorylation levels, cytoplasmic and nuclear β-catenin expression levels in FIG. 18 . 21 shows the results of western blot analysis on the expression levels of p38 and p-p38 after inhibition of the p38 MAPK pathway. 22 is a quantitative analysis result of each protein expression level of FIG. 21 . 23 shows the results of Western blot analysis on the expression levels of osteogenic differentiation marker proteins (BMP4, RUNX2, and ALP) after inhibition of the p38 MAPK pathway. 24 is a quantitative analysis result of each protein expression level in FIG. 23 (*p < 0.05, **p < 0.01, ***p < 0.001. N = 3). EPS: Enterococcus faecium L15-derived extracellular polysaccharide treatment group, SB: p38 inhibitor SB203580 (SB) treatment group, SB+EPS: SB and EPS combined treatment group, Control: control group without any treatment.

25 to 27 show the effect of L15 EPS on primary osteoblast precursors and bone formation. Figure 25 is an ALP staining micrograph of primary osteoblast precursors (obtained from calvaria of ICR neonates) treated with or without L15 EPS for 7 days (scale bar = 200 μm). 26 shows the results of real-time PCR analysis of the expression of osteogenic differentiation marker genes (RUNX2, COL1A1, ALP) in primary osteoblast precursors. 27 shows the thickness of calvaria cultured in hDPSC culture medium treated with or without 10 μg/mL L15 EPS. Evaluated by H&E staining (scale bar = 25 μm, *p < 0.05. N = 3).

28 shows whether various types of lactic acid bacteria extracts promote osteogenic differentiation of hDPSCs. Digital images after treatment with L7.5, L15, L28, L30, L31 or M34 extracts and after ALP salting and bright field micrographs of L15 and L28 are shown (scale bar = 200 μm).

29 shows the presence or absence of calcium deposition in hDPSC cultures after treatment with Enterococcus lactis L28 strain-derived extracellular polysaccharide (EPS). This is a digital image after ARS staining.

The present invention relates to the use of a polysaccharide derived from a strain of the genus Enterococcus.

The present invention provides a composition for promoting osteogenesis comprising a polysaccharide derived from Enterococcus genus strain.

Polysaccharide refers to a compound substance in which several sugars are attached, and is a component of carbohydrates, and is not limited to the type of polymeric polysaccharide produced and released by microorganisms, including lactic acid bacteria, during the growth process. Specifically, extracellular polysaccharide ( exopolysaccharides, EPS), but is not limited thereto.

The polysaccharide of the present invention is sufficient as long as it is derived from Enterococcus genus strain, and the strain type is not limited.

The Enterococcus genus strain may be, for example , Enterococcus faecium or Enterococcus lactis.

According to one embodiment, the polysaccharide derived from Enterococcus genus strain is a polysaccharide derived from Enterococcus pesium L-15. Enterococcus pesium L-15 is a strain deposited at the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on March 15, 2018, and the accession number is KCTC13498BP.

Name of depositor: Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC)

Accession number: KCTC13498BP

Entrusted date: 20180315

The polysaccharide derived from the Enterococcus genus strain of the present invention is obtained by centrifuging the growth medium containing the Enterococcus genus strain as follows; Separating the supernatant of the growth medium; culturing by adding trichloroacetic acid to the separated supernatant; Obtaining a precipitate by mixing and culturing the cultured supernatant with absolute ethanol; And it may be obtained by a method for producing a polysaccharide derived from a strain of the genus Enterococcus comprising dialysis of the precipitate.

Centrifugation of the growth medium containing the strain of Enterococcus may be performed at 8,000 to 12,000 xg for 15 to 25 minutes.

When cultured by adding trichloroacetic acid to the separated supernatant, culture conditions may be incubation at 34 to 38° C. for 30 minutes to 2 hours.

Proteins and nucleic acids may be denatured by adding trichloroacetic acid to the separated supernatant and culturing the supernatant.

The mixing volume ratio of the cultured supernatant and absolute ethanol may be 1: 1.5 to 3. The mixed culture conditions may be incubation at 3 to 5°C for 1 to 2 days. A precipitate is formed through the mixed culture.

Dialysis may be carried out at 3 to 5 °C for 18 to 54 hours after dissolving the mixed culture precipitate in ddH ₂ O. Dialysis can remove trace amounts of protein in the precipitate.

A step of freeze-drying the precipitate after dialysis may be further included.

The polysaccharide derived from the Enterococcus genus strain of the present invention can promote bone formation by inducing bone differentiation of stem cells.

Stem cells capable of inducing osteogenic differentiation by the polysaccharide derived from the strain of the genus Enterococcus of the present invention may be mesenchymal stem cells (MSC).

Mesenchymal stem cells of the present invention may be derived from neural crest, bone marrow, adipose tissue, or peripheral blood, but are not limited thereto.

The stem cells may be dental pulp stem cells (DPSC), for example, dental pulp stem cells derived from neural crest.

Dental pulp stem cells refer to stem cells present in dental pulp tissues, and are multipotent stem cells necessary for dental pulp and dentin regeneration, and can differentiate into various tissues such as bone, adipose tissue, neurons, and dentin. Specifically, the dental pulp stem cells may be human dental pulp stem cells (hDPSC) isolated from human teeth, and may be autologous, allogenic, or xenogenic cells.

According to one embodiment, the polysaccharide derived from the Enterococcus genus strain can increase the expression level of bone formation-related genes in stem cells. For example, it can increase RUNX2, ALP, and COL1A1 gene expression levels.

According to one embodiment, the polysaccharide derived from Enterococcus genus strain can increase the expression level of bone formation-related proteins in stem cells. For example, the expression levels of BMP4, RUNX2, and ALP proteins can be increased.

According to one embodiment, polysaccharides derived from strains of the genus Enterococcus can activate the p38 mitogen-activated protein kinases (MAPK) pathway of stem cells.

Specifically, the polysaccharide derived from Enterococcus genus strain can increase the phosphorylation of p38 in stem cells.

The concentration of the polysaccharide in the composition for promoting bone formation is 0.5 to 25 μg/ml, 0.5 to 20 μg/ml, 0.5 to 15 μg/ml, 0.5 to 10 μg/ml, 0.5 to 5 μg/ml, 0.5 to 2 μg/ml. ml, 0.5 to 1 μg/ml, 1 to 10 μg/ml, 1 to 5 μg/ml, 1 to 3 μg/ml, 1 to 2 μg/ml, or 1 to 1.5 μg/ml, but is limited thereto It is not.

The composition for promoting bone formation of the present invention may be used, for example, for at least one of artificial bone, artificial joint, bone fixation material, bone substitute, bone restoration material, bone filling material, and bone graft material.

For example, since a bone graft material or a bone substitute can be used to fill a space in a bone tissue in a narrow sense or to replace a part of a bone or tooth in a broad sense, the composition for promoting bone formation can be compressed, compressed, or pressed. It can be used in the form of putty, paste, moldable strip, block, chip, etc. using methods such as contact, packing, pressing, and hardening, and using chemical additives, it can be used in the form of gel, granule, paste, tablet, pellet, etc. It can be formulated and used, and can also be used in powder form.

The present invention provides a pharmaceutical composition for the prevention or treatment of bone disease, which contains a polysaccharide derived from Enterococcus genus strain and is treatable by bone formation or bone regeneration.

Enterococcus genus ( Enterococcus genus ) The strain-derived polysaccharide is the same as that described in the bone formation promoter described above.

The polysaccharide derived from Enterococcus genus strain may be prepared by the method described above.

According to one embodiment, the polysaccharide derived from Enterococcus genus strain is a polysaccharide derived from Enterococcus pesium L-15 (accession number KCTC13498BP).

The pharmaceutical composition of the present invention can be used for the prevention or treatment of bone diseases requiring an increase in bone density due to bone loss, and bone diseases treatable by bone formation or bone regeneration.

For example, the diseases include periodontal disease, periodontitis, alveolar bone disease, osteoporosis, osteomalacia, osteopenia, bone atrophy, and osteoarthritis. , At least one selected from the group consisting of rheumatoid arthritis, osteolysis, fibrous dysplasia, Paget's disease, bone formation imperfect and hypercalcemia It may be, but is not limited thereto.

According to one embodiment, the pharmaceutical composition may increase bone thickness or promote alveolar bone regeneration by inducing bone regeneration or bone formation by being administered to a subject in need of promoting bone formation.

The pharmaceutical composition according to the present invention may be administered in a pharmaceutically effective amount.

In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit or risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and activity of the drug of the patient's disease , sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitantly used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, sex, and weight, and is generally 0.1 mg to 100 mg per 1 kg of body weight, preferably 0.2 mg to 17 mg per day or every other day. It may be administered or divided into 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, severity of obesity, gender, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

When formulating the composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches, etc., and these solid preparations are suitable for the dead lactic acid bacteria Propionibacterium freudenreiki MJ2 (Propionibacterium freudenreichii KCCM12272P) of the present invention It is prepared by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, or syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspension solutions, emulsions, freeze-dried formulations, suppositories, and the like.

Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending solvents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerol, gelatin, and the like may be used.

The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

Hereinafter, the present invention will be described in more detail through examples.

method

1. Preparation of Enterococcus faecium L15 (E. faecium L15) extract

E. faecium strain L15 (KCTC13498BP, “L15”) was obtained from NeoRegen Biotech (Suwon, Gyeonggi-do). L15 extract preparation process is described in “Kim H, Park S, Kim K, Ku S, Seo J, Roh S. Enterococcus faecium L-15 cell-free extract improves the chondrogenic differentiation of human dental pulp stem cells. Int J Mol Sci. 2019 ;20(3):624.” This strain was originally isolated from a traditional Korean rice fermented food containing flounder. L15 was cultured in TSB (tryptic soy broth) medium (Hardy Diagnostics, Santa Maria, CA, USA). ) for 18 hours at 35° C. The cultured L15 was harvested, washed three times with phosphate-buffered saline (PBS) and resuspended in double deionized water (ddH2O), and the washed L15 was sonicated on ice for 30 minutes. (Sonics, Stratford, CT, USA), centrifuged at 12,000xg for 10 min to remove cell debris, supernatant was passed through a 0.45 μm filter and lyophilized overnight at -80 °C, then It was reconstituted in PBS for use in experiments.

2. Purification of EPS from E. faecium and E. lactis

E. faecium and E. lactis-derived exopolysaccharides (EPS) were purified using ethanol precipitation.

E. faecium L15 and E. lactis L28 were cultured in TSB (tryptic soy broth) medium (Hardy Diagnostics, Santa Maria, CA, USA) for 18 hours at 35°C, respectively. Cultured L15 and L28dmf were separated from the growth medium by centrifugation at 10,000xg for 20 minutes. After centrifugation, the supernatant was collected, denatured proteins and nucleic acids by adding 14% trichloroacetic acid to the final concentration, and then incubated at 37°C for 1 hour. The solution at a ratio of 2:1 was incubated at 4° C. for 1 day to allow aggregation. The precipitate was dissolved in ddH ₂ O and dialyzed at 4° C. for 24-48 hours to remove trace amounts of protein. The precipitate was lyophilized at -80 °C and then reconstituted with PBS for use in experiments.

3. EPS fractionation

Size exclusion chromatography was performed on the crude EPS obtained by purification as above. The purified crude EPS was applied to a HiLoad™ 16/600 Superdex 200 (GE Healthcare, Chicago, IL, USA) column equilibrated with PBS. Elution was performed at a flow rate of 1 ml min ^-1 , and a fraction of 2 ml was obtained using a fraction collector.

4. Isolation and culture of human dental pulp stem cells (hDPSCs)

Human maxillary central supernumerary teeth (n = 3) were extracted at Seoul National University Dental Hospital according to IRB-approved guidelines. The extracted tooth was cut short around the cemento-enamel junction (CEJ) using a cutting disc. The pulp tissue was exposed and gently separated from the crown. The pulp tissue was minced with a scalpel blade into 1 mm ² pieces and transferred to a 12-well culture dish. Cells were then grown in Minimum essential medium eagle—alpha modification (α-MEM; Hyclone Laboratories Inc., Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories Inc.) and 5% CO ₂ . and cultured in an incubator at 37°C. The culture medium was changed every 3 days.

5. Characterization of hDPSCs using Fluorescence Activated Cell Sorting (FACS)

FACS was performed to identify hDPSCs. At passages 3 and 8, hDPSCs were isolated and resuspended in cold PBS containing 5% FBS. Cells were stained with CD10-fluorescein isothiocyanate (FITC), CD29-Alexa 488, CD44-FITC, CD73-FITC, CD90-FITC, D105-FITC, CD14-allophycocyanin (APC), CD34- Incubated for 30 min on ice with monoclonal antibodies to Alexa 647, CD45-APC and CD31-APC.

Analysis was performed using the FACSVerse (Becton Dickinson, Franklin Lakes, NJ, USA).

6. Osteogenic Differentiation of hDPSCs

hDPSCs were grown in α-MEM supplemented with 10% FBS (Hyclone Laboratories Inc.), 100 nM dexamethasone (Sigma-Aldrich), 10 mM β-glycerophosphate (Sigma-Aldrich) and 0.05 mM ascorbic acid 2-phosphate (Sigma-Aldrich). (Hyclone Laboratories Inc.) was seeded at 15,000 cells/cm ² in a 12-well culture dish in an osteogenic differentiation medium. The medium was replaced every 2 to 3 days, and osteogenic differentiation proceeded for 28 days.

7. Cell viability assay

Cell viability was measured using the EZ-Cytox kit (Daeil Lab Service, Seoul, Korea) based on the water-soluble tetrazolium salt (WST) method. hDPSCs were seeded in 96-well plates at a density of 1×10 ⁴ cells per well. Cells were cultured with various concentrations of L15 extract for 3 days and cultured in osteogenic differentiation medium containing L15 EPS for 3 and 7 days. WST solution was added to each well. The mixture was incubated at 37° C. for 30 minutes. The absorbance of each well was measured at 450 nm using an Emax Plus Microplate reader (Molecular Devices, Sunnyvale, CA, USA).

8. Reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR

RT-PCR and real-time PCR were used to quantify gene expression. Total RNA was extracted from the pellet using the PureLink™ RNA Mini kit (Life Technologies, Camarillo, CA, USA). cDNA synthesis was performed using M-MLV reverse transcriptase (Promega Corporation, Fitchburg, WI, USA) according to the manufacturer's instructions. Real-time PCR was performed using SYBR Pre-mix Ex Taq™ II (Takara, Tokyo, Japan) and 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). Primers used are listed in Table 1. The PCR reaction was performed at 95°C for 30 seconds followed by 40 amplification cycles of 95°C for 5 seconds and 60°C for 34 seconds. A comparative CT method was used to measure the expression level. Glyceralde-hyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene for normalization.

Table 1 relates to primer sequences used for real-time PCR.

Distinction	gene	forward primer	reverse primer
human genes	Runx2	CAGACCAGCAGCACTCCATA (SEQ ID NO: 1)	TTCAATATGGTCGCCAAACA (SEQ ID NO: 7)
	COL1A1	GCGAGAGCATGACCGATGGA (SEQ ID NO: 2)	GCGGATCTCGATCTCGTTGGA (SEQ ID NO: 8)
	ALP	GGGATAAAGCAGGTCTTGGGGTGC (SEQ ID NO: 3)	CGCTTGGTCTCGCCAGTACTTGG (SEQ ID NO: 9)
	GAPDH	ACATGTTCCAATATGATCCC (SEQ ID NO: 4)	TGGACTCCACGACGTACTCA (SEQ ID NO: 10)
mouse gene	Runx2	Same as human primer (SEQ ID NO: 1)	Same as human primer (SEQ ID NO: 7)
	COL1A1	Same as human primer (SEQ ID NO: 2)	Same as human primer (SEQ ID NO: 8)
	ALP	CCAACTCTTTTTGTGCCAGAGA (SEQ ID NO: 5)	GGCTACATTGGTGTTGAGCTTTT (SEQ ID NO: 11)
	GAPDH	AGGTCGGTGTGAACGGATTTG (SEQ ID NO: 6)	TGTAGACCATGTAGTTGAGGTCA (SEQ ID NO: 12)

9. Alizarin Red S (ARS) Staining

Osteogenic differentiation evaluation used ARS staining to visualize calcium deposits. The differentiated cells were fixed with 4% paraformaldehyde (PFA), stained with alizarin red solution (Sigma-Aldrich), and then photographed with a digital camera (Canon, Tokyo, Japan) using an inverted microscope (EVOS™ XL Core Imaging system, Thermo Scientific™, Waltham, MA, USA).

10. Alkaline phosphatase (ALP) staining

ALP staining was performed using the StemAb Alkaline Phosphatase Staining Kit II (Reprocell, Beltsville, MD, USA) according to the manufacturer's instructions. hDPSCs and mouse calvaria-derived osteoblasts were treated for 7 days with or without the L15 extract or extracellular polysaccharide (EPS). Cells were fixed for 2 minutes using fixative solution, washed twice with PBS, and incubated for 30 minutes with ALP staining solution. The ALP staining solution was removed and staining was observed with an inverted microscope (EVOS™ XL Core Imaging System; Thermo Scientific™, Waltham, MA, USA).

11. Western blot analysis

Cytoplasmic and nuclear proteins were extracted with NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific™) containing proteinase inhibitor (MedChemExpress, Monmouth Junction, NJ, USA) and phosphatase inhibitor (MedChemExpress). Cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted with the following indicated antibodies. bone morphogenetic protein-4 (BMP4; Cusabio Life Science, Wuhan, China), runx family transcription factor 2 (Runx2; Novus Biologicals, Centennial, Colorado, USA), ALP (R&D Systems, Minneapolis, MN, USA), extracellular signal Regulated kinase (ERK; Cell Signaling Technology), phospho-ERK (p-ERK; Cell Signaling Technology), c-Jun N-terminal kinase (JNK; Cell Signaling Technology), p-JNK (Cell Signaling Technology), p38 ( Cell Signaling Technology), p-p38 (Cell Signaling Technology), β-catenin (Cell Signaling Technology), Lamin B1 (Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (BioLegend, SanDiego, CA, USA) . GAPDH was used as a housekeeping gene for normalization. Lamin B1 was used to normalize nuclear β-catenin.

12. Isolation of Calvaria-derived osteoblast precursors

Primary osteoblast precursors were isolated from mouse calvaria. Calvaria were isolated from 4-day-old mice and digested with 0.25% trypsin and 0.2% collagenase at 37°C for 30 min. The released cells were plated in 100 mm dishes, grown in α-MEM (Hyclone Laboratories Inc.) supplemented with 10% FBS (Hyclone Laboratories Inc.), and cultured in an incubator under 5% CO ₂ and 37°C conditions. After 3 days, adherent cells were used as osteoblast precursors. Osteoblast precursors were used for osteogenic differentiation using L15 extracellular polysaccharide (EPS).

13. Mouse calvarial organ culture and hematoxylin and eosin staining

At postnatal day 4, calvaria from ICR mice were cultured on grids in 12-well culture plates. Calvaria were cultured in hDPSC culture medium with or without 10 μg/mL L15 EPS. The medium was changed every 2 days and calvarias were harvested on the 7th day. Calvaria were fixed in 4% PFA for 24 hours and decalcified in 14% EDTA for 2 days. After decalcification, calvaria were embedded in paraffin wax. Blocks were trimmed to a depth of 800 μm and cut sagittal to a thickness of 10 μm (Leica Microsystems, Wetzlar, Germany) at the midline. The cut tissue was stained with hematoxylin and eosin (H&E) and bright field micrographs were captured with an Olympus BX50 microscope (Olympus, Tokyo, Japan). Bone thickness was measured on sagittal slices obtained at a specific location (1 mm away from the midline suture of calvaria) using Image Pro software (Media Cybernetics Inc., Silver Spring, MD, USA).

14. Statistical Analysis

Results were expressed as mean ± SD. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey post hoc test and Student's t-test using GraphPad Prism V5.0 software (GraphPad Software, La Jolla, CA, USA). *p<0.05, **p<0.01, ***p<0.001 were defined as statistical significance.

result

1. Characteristics of human dental pulp stem cells (hDPSCs)

To investigate the characteristics of hDPSCs, cell surface antigen cluster (cluster of differentiation, CD) markers of mesenchymal, hematopoietic and endothelial stem cells were analyzed by fluorescence-activated cell sorting (FACS), respectively (Figs. 1 to 4, Table 2 and 3). At passage 3 of hDPSCs, the cells expressed high levels of mesenchymal stem cell markers but low levels of hematopoietic and endothelial stem cell markers (Tables 2 and 3). The ratio of surface marker expression in hDPSCs at passage 8 was similar to that at passage 3 (Table 3). Therefore, passage 3–8 cells were used for osteogenic differentiation.

Tables 2 and 3 quantify the percentage of cells expressing each of the stem cell CD markers measured in FIGS. 1 and 2 . (n=3).

division	CD10	CD29	CD44	CD73	CD90	CD105
passage number 3	68.67±7.56	99.02 ±0.55	99.15 ±0.59	99.10 ±0.55	99.07 ±0.48	80.45 ±17.99
passage number 8	76.66±8.76	99.08 ±0.90	99.21 ±0.78	99.16 ±0.82	95.18 ±5.14	74.29 ±33.73

division	CD14	CD31	CD34	CD45
passage number 3	31.16±14.55	1.24±0.17	4.5±2.7	1.24±0.32
passage number 8	33.10±9.91	1.89±0.61	5.75±2.29	1.84±0.65

2. Effect of L15 extract on hDPSC survival

As shown in Fig. 5, the survival rate of hDPSCs was significantly decreased when the L15 extract was treated with 5 μg/mL or more (p < 0.001). This suggested that an L15 extract concentration of 1 μg/mL was non-toxic to the cells, and this concentration was used for subsequent osteogenic differentiation.

3. Effect of L15 extract on promoting osteogenic differentiation of hDPSCs

After culturing hDPSCs with the L15 extract for 7 days, ALP staining was performed. More intense ALP staining was observed in the L15 extract treatment group compared to the control group (FIG. 6). Calcium deposition was examined by ARS staining after osteogenic differentiation for 28 days. Enhanced calcium deposition was observed in the L15 extract treatment group compared to the control group (FIG. 7). The expression levels of RUNX2, alkaline phosphatase (ALP), and type I collagen (COL1A1), which are bone formation-related genes, were investigated by real-time PCR. The expression levels of RUNX2, ALP and COL1A1 increased significantly after treatment with the L15 extract (FIG. 8; p < 0.05, p < 0.01). The effect of the L15 extract on the levels of bone formation-related proteins (Bone morphogenetic protein 4 (BMP4), RUNX2, ALP) was confirmed by Western blotting (FIG. 9). BMP4, RUNX2 and ALP expressions were significantly increased in the L15 extract treatment group compared to the control group (FIG. 10).

4. Selection of the optimal EPS concentration that does not show cytotoxicity

The purified extracellular polysaccharide (EPS) of E. faecium L15 was fractionated using size exclusion chromatography. As shown in FIG. 11, L15 EPS appeared as a single peak corresponding to the low molecular weight fraction. A water-soluble tetrazolium salt (WST) assay was performed to determine the effect of L15 EPS on the viability of hDPSCs. Six concentrations of L15 EPS (0, 1, 2.5, 5, 10 and 25 μg/ml) were treated for 3 days. There was no significant difference between groups (Fig. 12 (a)). The same concentration of L15 EPS was treated with osteogenic differentiation medium for 7 days. The viability of hDPSCs was significantly decreased by treatment with 2.5 μg/mL or more L15 EPS (FIG. 12 (b)). This result suggested that continuous treatment with 2.5 μg/ml L15 EPS was toxic to the osteogenic differentiation of hDPSCs, and 1 μg/ml L15 EPS was used in subsequent experiments.

5. E. faecium L15-derived EPS (L15 EPS) promotes osteogenic differentiation of hDPSCs

After treatment with L15 EPS for 7 days, ALP staining was performed. ALP staining was observed to be more intense in the L15 EPS treated group than in the control group (FIG. 13). Calcium accumulation was examined using ARS staining after 28 days of osteogenic differentiation. Calcium deposition was significantly increased in the L15 EPS treatment group compared to the control group (FIG. 14). The expression levels of RUNX2, ALP, and COL1A1, which are bone formation-related genes, were significantly up-regulated in the L15 EPS-treated group compared to the control group (FIG. 15). The protein expression levels of BMP4, RUNX2 and ALP were significantly increased in the L15 EPS treatment group (FIGS. 16 and 17; p < 0.05).

6. hDPSC osteogenic differentiation promotion pathway of L15 EPS (p38 MAPK pathway)

The effect of L15 EPS on pathway protein (ie, ERK, p-ERK, JNK, p-JNK, p38, p-p38, cytoplasmic β-catenin and nuclear β-catenin) levels was confirmed by Western blot analysis. (FIG. 18). Since nuclear translocation of β-catenin is a key feature of Wnt pathway activation, nuclear and cytoplasmic fractionation of β-catenin was performed. Expression levels of ERK, p-ERK, JNK, p-JNK, cytoplasmic β-catenin and nuclear β-catenin did not change (FIGS. 18 to 20). The expression of p-p38 was upregulated in the L15 EPS treatment group (FIG. 19). The p-p38/p38 ratio was significantly increased in the L15 EPS treated group compared to the control group (FIG. 19). These results showed that the p38 MAPK pathway was activated after L15 EPS treatment.

7. Inhibition of L15 EPS-induced osteogenic differentiation of hDPSCs through inhibition of the p38 MAPK pathway

The role of p38 in the osteogenic differentiation of hDPSCs induced with L15 EPS was further analyzed using the p38 inhibitor SB203580 (SB). The efficiency of SB was confirmed by Western blot (FIG. 21). SB significantly decreased the p-p38/p38 ratio (FIG. 22). The ratio of p-p38/p38 was significantly up-regulated in the EPS-treated group compared to the control group (FIG. 22). Quantitative analysis showed that the ratio of p-p38/p38 was suppressed in the SB + EPS treatment group (FIG. 22). After osteogenic differentiation for 14 days, RUNX2 and ALP protein expression levels were significantly downregulated in the SB + EPS treated group compared to the EPS treated group (FIG. 23, 24).

8. Effect of L15 EPS on osteoblast progenitors in calvaria

To examine whether L15 EPS improves the osteogenic differentiation of osteoblast precursors, isolated osteoblast precursors were cultured in an osteogenic medium treated with or without L15 EPS for 7 days. ALP intensity was stronger in the L15 EPS treated group compared to the control group (FIG. 25). After 14 days of osteogenic differentiation into L15 EPS, mRNA levels were analyzed by qPCR. The mRNA levels of RUNX2, COL1A1 and ALP were significantly up-regulated with L15 EPS (FIG. 26).

9. Confirmation of promotion of bone formation by L15 EPS in a calvarial organ culture model

The effect of L15 EPS on bone formation was confirmed using calvarial ex vivo organ culture. Through H&E staining, it was confirmed that L15 EPS increased bone thickness (FIG. 27). These results indicate that L15 EPS can induce osteogenesis.

10. Whether EPS derived from various strains promotes osteogenic differentiation

(1) Whether extracts derived from various strains promote osteogenic differentiation

Enterococcus faecium L15 (L15) and Enterococcus lactis L28 (L28) extracts, as well as Lactococcus lactis L7.5 (L7.5), Lactobacillus plantarum L30 (L30, KCTC 3107), and Bifidobacterium longum L31 (which belong to other genera). L31) and Lactobacillus pentosus MS4 (MS4) strain extracts were tested to confirm the bone formation promoting effect.

The L15 and L28 strains were cultured in TSB (tryptic soy broth) medium, and the L7.5, L30, L31 and MS4 strains were cultured in MRS broth medium at 35° C. for 18 hours, respectively. The cultured strains were harvested, washed three times with phosphate-buffered saline (PBS), and resuspended in double deionized water (ddH2O). Washed strains were sonicated (Sonics, Strat-ford, CT, USA) for 30 minutes on ice. It was centrifuged at 12,000xg for 10 minutes to remove cell debris. The supernatant was passed through a 0.45 μm filter and lyophilized overnight at -80 ° C to obtain extracts derived from each strain. Then, it was reconstituted with PBS for use in experiments. Each extract was treated with hDPSCs in the same manner as described above (each extract treated for 7 days, extract concentration: 1 ug/ml) and cultured for 28 days, and then osteogenic differentiation was observed by ALP staining.

As a result, as shown in FIG. 28, osteogenic differentiation was observed only in the group treated with the extracts of L15 and L28 strains belonging to the genus Enterococcus.

Through this, it was confirmed that extracts of strains other than Enterococcus (or the extracellular polysaccharide contained therein) had no osteogenic differentiation promoting effect.

(2) Confirmation of the bone formation-promoting effect of L28-derived extracellular polysaccharide by observing calcium deposition

L28-derived extracellular polysaccharide (L28 EPS) was treated with hDPSCs in the same manner as described above (L28 EPS treatment for 7 days, concentration of L28 EPS: 1 ug/ml) and cultured for 28 days, followed by calcium deposition through ARS staining. It was confirmed whether

As a result, as shown in FIG. 29 , calcium deposition was confirmed in the L28 EPS-treated group, unlike the control group that was not treated with anything. Through this, it was found that not only Enterococcus pesium but also the exopolysaccharide derived from the Enterococcus lactis strain corresponding to the genus Enterococcus had an osteogenic differentiation promoting effect.

Claims (12)

1. Enterococcus genus ( Enterococcus genus ) A composition for promoting osteogenesis comprising a strain-derived polysaccharide.
2. The method according to claim 1, Enterococcus genus strain Enterococcus faecium ( Enterococcus faecium ) or Enterococcus lactis ( Enterococcus lactis ) of, the composition for promoting bone formation.
3. The method according to claim 1, wherein the polysaccharide comprises the steps of centrifuging a growth medium containing Enterococcus strain;

   Separating the supernatant of the growth medium and culturing by adding trichloroacetic acid to the separated supernatant;

   Obtaining a precipitate by mixing and culturing the cultured supernatant with absolute ethanol and

   A composition for promoting bone formation obtained by a method for producing a polysaccharide comprising the step of dialysis of the precipitate.
4. The method according to claim 1, wherein the concentration of the polysaccharide is 0.5 to 2 μg / ml, the composition for promoting bone formation.
5. The composition for promoting bone formation according to claim 1, which induces bone differentiation of stem cells.
6. The composition for promoting bone formation according to claim 5, wherein the stem cells are dental pulp stem cells.
7. The composition for promoting bone formation according to claim 1, which is used for at least one of artificial bone, artificial joint, bone fixation material, bone substitute, bone restoration material, bone filling material, or bone graft material.
8. Enterococcus genus ( Enterococcus genus ) A pharmaceutical composition for the prevention or treatment of bone diseases that includes a strain-derived polysaccharide and is treatable by bone formation or bone regeneration.
9. The pharmaceutical composition for preventing or treating bone diseases according to claim 8, wherein the Enterococcus genus strain is Enterococcus faecium or Enterococcus lactis .
10. The method according to claim 8, wherein the polysaccharide comprises centrifuging a growth medium containing Enterococcus strain;

    Separating the supernatant of the growth medium and culturing by adding trichloroacetic acid to the separated supernatant;

    Obtaining a precipitate by mixing and culturing the cultured supernatant with absolute ethanol and

    A pharmaceutical composition for preventing or treating bone diseases obtained by a method for producing a polysaccharide comprising the step of dialysis of the precipitate.
11. The pharmaceutical composition for preventing or treating bone diseases according to claim 8, wherein the concentration of the polysaccharide is 0.5 to 2 μg/ml.
12. The method according to claim 8, wherein the bone disease is periodontal disease, periodontitis, alveolar bone disease, osteoporosis, osteomalacia, osteopenia, bone atrophy , osteoarthritis, rheumatoid arthritis, osteolysis, fibrous dysplasia, Paget's disease, osteogenesis imperfect and hypercalcemia At least one selected from, a pharmaceutical composition for preventing or treating bone disease.

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