KR101315202B1 - Composition for inducing a bone differentiation and method for enhancing a bone differentiation using the same - Google Patents

Abstract

The present invention relates to a synthetic peptide or a homologue thereof for promoting bone differentiation and osteogenesis comprising an amino acid sequence derived from an osteogenic protein, which increases the differentiation inducing effect upon induction of bone cell differentiation of stem cells, The present invention relates to a synthetic peptide for inducing bone differentiation of stem cells, comprising the amino acid sequence of SEQ ID NOS: 1 to 13, And at least one peptide selected from the group consisting of OP5, OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17 and OP18 having homologous sequences or homologues thereof.
The composition for inducing osteogenic differentiation of the present invention constituted as described above provides a synthetic peptide for promoting bone formation having a specific sequence and the synthetic peptide has a remarkable effect in promoting the differentiation of stem cells (MSCs) into osteoblasts The pharmaceutical composition of the present invention comprising the synthetic peptide as an active ingredient is excellent in the treatment of osteoporosis, bone defect disease and / or osteoarthritis, and thus can replace the expensive BMP in the past, The increase method makes it easier and cheaper to induce bone differentiation.

Description

TECHNICAL FIELD The present invention relates to a composition for inducing osteogenic differentiation and a method for enhancing a bone differentiation using the same.

The present invention relates to a composition for inducing osteogenic differentiation of stem cells and a method for increasing osteogenic differentiation using the same. More particularly, the present invention relates to a composition for inducing osteogenic differentiation of stem cells, A synthetic peptide or a homologue thereof for promoting bone differentiation and osteogenesis, a composition or a pharmaceutical composition for inducing bone differentiation comprising the synthetic peptide as an active ingredient, a medium composition containing the same, and a bone formation differentiation method using the same.

The cells that make up the bones are osteocytes that maintain the homeostasis of the bones, osteoblasts that form the bones, osteoclasts that absorb the bones, and cartilage cells that play the joint functions of the ends of the bones (chondrocyte), and in the bone that has been grown, the cancellous bone is absorbed by the osteoclasts, and on the other surface, the new bone is formed by the osteoblasts and is reformed. When the balance between osteogenesis and bone resorption is broken, the disease progresses to osteopathy. Osteoporosis, non-union fracture, osteonecrosis, osteomalacia, osteoporosis, There are deficiencies. In addition, avascular necrosis of the femoral head is a refractory osteoarthritis in which the bone is destroyed by stopping the blood supply to the femoral head that constitutes the hip joint due to trauma, alcohol and steroid use. 20,000 per year, and 4,000-5,000 per year in Korea. The incidence is generally 20-50 years old, with an average rate in the late 30s. However, no special treatment has been developed other than the surgical method of arthroplasty.

Osteoporosis is also referred to as osteoporosis or osteoporosis. It refers to a metabolic bone disease in which the bone mass is significantly reduced compared with that of a normal person, and a major decrease in the bone constituent is the main lesion. In general, the condition of osteoporosis itself is often asymptomatic or mild. However, once a fracture occurs, it is generally difficult to treat and it is difficult to recover enough even after the operation of osteosynthesis.

As a preventive and / or therapeutic agent for bone diseases including osteoporosis, fracture, etc., there has been proposed a pharmaceutical composition containing a calcium preparation, an estrogen preparation, a selective estrogen receptor modulating agent (SERM), an active vitamin D3 preparation, a vitamin K preparation, an ipriflavone preparation, a calcitonin preparation, a bisphosphonate preparation, (BMP), fibroblast growth factor (FGF), etc. have been clinically used or clinical applications have been tried. However, even though a large number of medicines are used in this way The number of patients with bone disease is increasing year by year. Therefore, it is urgently required to develop a new concept of therapeutic agent for efficiently treating such refractory bone diseases.

Traditional therapies for bone disease include autografting, allografting, and artificial bone grafting. However, complications such as infections at the site of bone harvesting, hematomas (autogenous bone graft) (Allograft), and a problem that does not result in fundamental bone formation (artificial bone graft). Therefore, studies on tissue engineering bone formation to treat refractory bone diseases have been actively researched. For the formation of bone tissue by tissue engineering, osteoprogenitor cells that induce bone formation, supporters capable of multiplying and differentiating these cells at appropriate positions, and bone-inducing growth factors promoting induction of bone formation are required.

Bone morphogenetic protein (BMP), a member of the transforming growth factor (TGF) β-phase and superfamily, promotes differentiation from osteoblast progenitor cells to osteoblasts, in vivo) and in vitro (in In vitro , it has been reported to have an effect of increasing bone formation (see Mol Biol Cell. 1999; 10: 3801-13, Bone. 2001; 28: 491-8), in particular BMP-2 protein induced bone formation. Although it has an excellent effect on promoting bone formation with protein, BMP having such an excellent effect requires a large amount due to its short half-life in order to have a satisfactory effect, and thus it is not used for many patients due to the high cost of administration. There is this.

Therefore, various studies have been attempted to solve such shortcomings. Especially, it has been studied whether or not the effect of BMP or the like can be obtained by using a specific partial peptide sequence of known BMP, and it has been studied continuously It is true. For example, Korean Patent Application No. 2007-0055211, entitled " Injection Type Aggregate Biomaterial Containing Bone Formation Promoting Peptide ", discloses an implantable aggregate containing biodegradable peptide- Wherein the present invention relates to a gel forming agent selected from the group consisting of chitosan, alginic acid, silk fibroin, propylene glycol, propylene glycol alginic acid, poloxamer, chondroitin sulfate and combinations thereof. Wherein the bone formation promoting peptide essentially containing the above amino acid sequence is bound or mixed. The peptide used herein is an extract of the amino acid sequence of the active site in the protein constituting the extracellular matrix, and the peptide is the amino acid sequence YRLKRSKS (SEQ ID NO: 1) at the position of the bone sialoprotein 1 (BSP1) 85-105 of the rabbit, KMFHVSNAQYPGA (SEQ ID NO: 2), YRLKRSKSKMFHVSNAQYPGA (SEQ ID NO: 3), amino acid sequence of human bone sialoprotein I149-169, YGLRSKS (SEQ ID NO: 4), KKFRRPDIQYPDAT (SEQ ID NO: 5), YGLRSKSKKFRRPDIQYPDAT And contains the amino acid sequence GLRSKSKKFRRPDIQYPDATDEDITSHM (SEQ ID NO: 7) of human bone sialoprotein I (Osteopontin) 150-177 position.

As another invention, Korean Patent Application No. 2010-0018148 entitled "Oligopeptide for Enhancing Osteoblast Differentiation Potency ", which has developed oligo peptide having a very high reaction specificity for BMP-specific receptor, PEP7 (SEQ ID NO: 1), PEP71 (SEQ ID NO: 2), PEP72 (SEQ ID NO: 3) and PEP73 (SEQ ID NO: 3) as oligopeptides for enhancing the osteoblast differentiation ability in order to increase the degree of calcification, SEQ ID NO: 4), PEP74 (SEQ ID NO: 5), PEP75 (SEQ ID NO: 6) and PEP76 (SEQ ID NO: 7).

In addition to the above-mentioned patent applications, Biomaterials 26 (2005) 5648-5657 discloses that P-15 peptides can regulate cell number and tissue structure by enhancing embryonic cell adhesion and controlling apoptosis (ABM) / P-15 complex can be used as an injectable bioequivalent substrate to promote bone regeneration ( Hanks T, Atkinson & lt ; RTI ID = 0.0 & gt ; BL . Comparison of cell viability on anorganic bone matrix with or without P-15 cell binding peptide . Biomaterials 2004; 25 (19): 4831-6.) ( Nguyen H, Qian JJ , Bhatnagar RS , Li S. Enhanced cell attachment and osteoblastic activity by P-15 peptide - coated matrix in hydrogels . Biochem Biophys Res Commun 2003; 311 (1): 179-86) , and in "Regulatory Peptides 142 (2007) 16-23", OGP and OGP (10-14) are osteogenic when administered to normal and osteogenic test animals. And increase spongy bone density and encourage fracture treatment, and in culturing osteoblasts and other bone marrow basal cells, OGP regulates proliferation, alkaline phosphatase activity and matrix mineralization ( Bab I, Gazit D, Chorev) M, Muhlrad A, Shteyer A, Greenberg Z, Namdar M, Kahn A. Histone H4 - related 오세제성 growth peptide (OGP): a novelcirculating stimulator of osteoblastic activity . EMBO J 1992; 11: 1867-73) .

In addition, in "Biomaterials 30 (2009) 3532-3541 ", OPD peptides are identified from BMP-2 and have affinity for BMP receptors, thereby inducing the differentiation of human bone marrow stem cells into osteoblasts Mundy et al. In "J Korean Med Sci 2005; 20: 438-44" first reported evidence that statins can have an effect of assimilation on bone, where statins there is disclosed that enables significantly stimulated new bone formation in a rodent, and statins are reported to act on the lipid synthesis pathway in osteoblastic cells, and promote the expression of bone morphogenetic protein -2 (BMP-2) ( Mundy , GR, Regulation of bone formation by bone morphogenetic proteins and other growth factors . Clin Orthop Relat Res (324), 24 (1996)) . BMP-2 upregulation is thought to modulate the osteogenic effects of statins. Additionally, the statins inhibit the binding of bisphosphonates from osteoblasts through inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA), essential for normal osteoblastic function, leading to reduced protein prenylation. Have been reported to act on the same upstream pathway ( Chen , PY et al ., Simvastatin promotes osteoblast viability and differentiation via Ras / Smad / Erk / BMP- 2 signaling pathway. Nutr Res 30 (3), 191.).

As described above, the conventional bone formation mechanisms and materials that act on these mechanisms and contribute effectively to bone formation have been continuously studied and the results of these studies have been obtained to some extent. However, The present inventors have conducted intensive studies to fulfill this need, and as a result, they have completed the present invention as a result of intensive study to find out a substance that can induce bone differentiation and provide it at a low cost more effectively. .

Patent Document 1: Korean Patent Application No. 2007-0055211. Patent Document 2: Korean Patent Application No. 2010-0018148.

Non-Patent Document 1: Biomaterials 26 (2005) 5648-5657. Non-Patent Document 2: Regulatory Peptides 142 (2007) 16-23. Non-Patent Document 3: Biomaterials 30 (2009) 3532-3541. Non-Patent Document 4: J Korean Med Sci 2005; 20: 438-44.

DISCLOSURE Technical Problem Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and it is a primary object of the present invention to provide a novel peptide for increasing the differentiation inducing effect upon induction of bone cell differentiation of stem cells, will be.

Another object of the present invention is to provide a synthetic peptide for promoting bone formation, which is composed of an amino acid sequence derived from BMP, and which is effective for promoting bone formation and is economical.

It is still another object of the present invention to provide a composition comprising the novel synthetic peptide as an active ingredient, and in particular, a composition effective for inducing bone differentiation of stem cells.

It is still another object of the present invention to provide a pharmaceutical composition and a medium composition for inducing bone differentiation of stem cells and to provide a method for increasing bone differentiation using the same.

The present invention may also be directed to accomplish these and other objects, which can be easily derived by those skilled in the art from the overall description of the present specification, in addition to the above-mentioned and obvious objects.

The above objects of the present invention are also achieved by the present invention, in a state in which not only the critical receptor-binding sequence of BMP-2 but also their osteogenic region is still completely unknown, although the bone formation characteristics of BMP- Has a remarkable effect in promoting the differentiation of osteoblasts of stem cells (MSC) having a specific amino acid sequence according to the present invention, and that the pharmaceutical composition of the present invention comprising the synthetic peptide as an active ingredient is effective for osteoporosis, A bone defect disease and / or an osteoarthritis of the present invention.

In order to accomplish the above object, the present invention provides a synthetic peptide for inducing bone differentiation of stem cells, comprising:

It is characterized by consisting of one or more peptides or homologues thereof selected from OP5, OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the following sequence:

OP5: DWIVAGSGDWIVA-NH ₂

OP7: DWIVGSGDWIV-NH ₂

OP8: DWIGSGDWIV-NH ₂

OP9: DWIGSGDWI-NH ₂

OP10: DWYGFGDW-NH ₂

OP11: DWGSGDW-NH ₂

OP12: DWYGFDW-NH ₂

OP13: DWYFGDW-NH ₂

OP14: DWYFDW-NH ₂

OP15: DWGSGD-NH ₂

OP16: DWGSGW-NH ₂

OP17: DWYGFD-NH ₂

OP18: DWYGFW-NH ₂

Where G is Gly, V is Val, I is Ile, F is Phe, Y is Tyr, W is Trp, S is Ser, and D is Asp.

According to another configuration of the present invention, the osteogenic differentiation peptide is characterized in that the peptide of OP5, OP10, OP12 or OP14 having the above sequence.

According to another configuration of the present invention, the osteogenic differentiation peptide is characterized in that the peptide of OP5 or OP10 having the above sequence.

According to another aspect of the invention, homologues of the OP5 are the N-, C- terminal, such as _{DWIVAGSGDWIVA, DWIVAGSGDWIVA-NH 2, N} -Ac-DWIVAGSGDWIVA-NH 2, N-Ac-DWIVAGSGDWIVA, N-formyl-DWIVAGSGDWIVA Substituted < / RTI >

According to another embodiment of the present invention, the homologues of OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, As well as substituted ones.

To achieve these and other advantages and in accordance with the purpose of the present invention, as embodied and broadly described herein,

As an active ingredient, one or more peptides or homologues thereof selected from OP5, OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the above sequence can be pharmaceutically acceptable. And excipients.

According to another configuration of the present invention, the active ingredient is characterized in that the peptide of OP5 or OP10 having the above sequence.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating osteoporosis comprising:

As an active ingredient, one or more peptides or homologues thereof selected from OP5, OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the above sequence can be pharmaceutically acceptable. And excipients.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating a bone defect disease comprising:

As an active ingredient, one or more peptides or homologues thereof selected from OP5, OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the above sequence can be pharmaceutically acceptable. And excipients.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating hypophosphoremia, comprising:

As an active ingredient, one or more peptides or homologues thereof selected from OP5, OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the above sequence can be pharmaceutically acceptable. And excipients.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating veterinary medicine, comprising:

As an active ingredient, one or more peptides or homologues thereof selected from OP5, OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the above sequence can be pharmaceutically acceptable. And excipients.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating osteonecrosis of the femoral head comprising:

As an active ingredient, one or more peptides or homologues thereof selected from OP5, OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the above sequence can be pharmaceutically acceptable. And excipients.

Pharmaceutical composition for the prevention or treatment of osteoarthritis of the present invention for achieving the above another object is:

As an active ingredient, one or more peptides or homologues thereof selected from OP5, OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the above sequence can be pharmaceutically acceptable. And excipients.

The composition for inducing osteogenic differentiation of the present invention constituted as described above provides a synthetic peptide for promoting bone formation having a specific sequence and the synthetic peptide has a remarkable effect in promoting the differentiation of stem cells (MSCs) into osteoblasts The pharmaceutical composition of the present invention comprising the synthetic peptide as an active ingredient is excellent in the treatment of osteoporosis, bone defect disease and / or osteoarthritis, and thus can replace the expensive BMP in the past, The increase method is a useful invention which can induce bone differentiation easily and inexpensively.

FIG. 1 is a photograph of a staining test of the stem cell bone differentiation effect of the synthetic peptide OP5 obtained according to the present invention using ALP (alkaline phosphatase) as an early differentiation marker of osteogenic cells and Vonkossa staining method as a calcification marker, wherein 10% FBS is a stem cell culture medium (negative control), ODM is a bone differentiation medium (positive control), and ODM + OP5 is a bone differentiation medium (test group) supplemented with OP5 peptide.
Figure 2 is a graph showing the comparison of the ALP activity, an early differentiation marker of osteoblasts in order to compare the stem cell bone differentiation effect of the synthetic peptide according to the present invention, where "control" is a negative control, BOPD, OGP and BMP -2 is a substance for which bone regeneration is known.
FIG. 3 is a graph showing the results of ARS (Alizarin Red S) staining for evaluating the degree of calcification of extracellular matrix in order to confirm the stem cell bone differentiation effect of synthetic peptides according to the present invention, wherein "control" BOPD, OGP, and BMP-2 are known to be bone regeneration.
FIGS. 4 and 5 are graphs comparing and comparing the osteogenic stem cell bone differentiation effect of the OP synthetic peptide according to the present invention.
FIGS. 6 and 7 illustrate the results of a synthetic peptide material according to the present invention. In order to evaluate the effectiveness of the synthetic peptide material, a paraffin block was cut to a thickness of 5 μm using an animal skull to obtain a tissue specimen, and then H & E (hematoxylin and eosin) And MT (masson trichrome), where the arrow indicates the incision in the direction of the sagittal suture, and the image on the right is the enlarged image of the square on the left.
FIG. 8 is a large bar graph showing the results of calculating the percentages of the newly formed new bone area based on the negative control (10% DMEM) after each measurement.

Hereinafter, the present invention will be described in more detail by way of preferred embodiments with reference to the accompanying drawings.

According to a preferred embodiment of the present invention, in order to provide a composition for inducing osteogenic differentiation according to the present invention, the present invention firstly comprises: 1) establishing culture conditions used for induction of osteogenic differentiation of stem cells; 2) OP5 and the like were added under the above-mentioned culture conditions to obtain an effective bone formation inducing effect.

According to another preferred embodiment of the present invention, the oligopeptides represented by OP5 to OP18 described above can be easily synthesized according to methods known to those skilled in the art. The oligopeptides used in the following examples of the present invention are solid phase And synthesized by using a synthesizer.

In the following Examples, experiments on the bone differentiation effect of OP5 of the present invention were performed on the control group and the results thereof were disclosed.

Significant results were obtained in diesel treated with OP5 according to the present invention when inducing bone differentiation of stem cells (MSC).

The food, food additive or medicinal composition containing the composition for preventing or treating osteoporosis according to the present invention can be prepared, and the preparation can further comprise suitable carriers, excipients and diluents commonly used. In addition, the composition according to the present invention may be administered alone or in combination with other pharmacologically active ingredients for enhancing the efficacy as a therapeutic agent for osteoporosis.

The composition for preventing or treating osteoporosis according to the present invention may be a carrier, an excipient and a diluent which may be contained in a food, a food additive or a pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch , Hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethylcellulose, As shown in FIG.

According to another embodiment, the composition for preventing or treating osteoporosis according to the present invention may be used as oral formulations such as powders, granules, tablets, suspensions, emulsions and syrups according to a conventional method. In addition, the pharmaceutical composition containing the composition of the present invention as an active ingredient can be administered orally or parenterally, and the dosage form thereof may be varied depending on the method of use, and thus is not limited thereto.

The oral formulations are meant to include both solid and liquid formulations for oral administration and solid formulations for oral administration may include tablets, pills, powders, granules, capsules, and the like, At least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, It is possible.

Hereinafter, although an Example demonstrates this invention more concretely, of course, the scope of the present invention is not limited to these Examples.

Example  1. Stem Cells Bone vertebrae  Evaluation: Dyeing test evaluation

In this study, the effect of the synthetic oligopeptides obtained according to the present invention on the differentiation of osteogenic cells was examined using ALP (alkaline phosphatase) and ARS (Alizarin Red S) staining, which are early markers of differentiation of osteogenic cells.

Osteogenic differentiation medium containing ascorbic acid, β-glycerophosphate and Dexamethasone was used as a positive control to compare the differentiation ability of osteogenic cells ( Develop . Grewth Differ . 50,553-564, 2008. ) and 10% FBS (culture medium for stem cell culture containing 10% FBS) was used as a negative control group. The experimental group was prepared by adding the synthetic peptide according to the present invention to each medium. MSCs were plated at 4 × 10 ³ cells per well on a 12-well culture plate and cultured at 37 ° C. in a CO ₂ incubator. The culture medium was replaced with fresh medium every 3 days, and the osteoblast performance was compared by ALP, ARS and Vonkossa staining at 14 days of culture. The results are shown in Fig.

As can be seen from FIG. 1, in the present study, the synthetic peptide OP5 according to the present invention was significantly stained with ALP, ARS and Vonkossa staining in the OP5-treated group compared with the positive control group ODM. Thus, it can be confirmed that the synthetic peptide according to the present invention remarkably improves the ability to differentiate stem cells into osteoblasts.

Example  2. Synthesis Peptides  Assessment of substance effectiveness: Initial Cell division ability  Evaluation of Bone Marrow Stem Cells Bone vertebrae  evaluation)

The purpose of this study is to observe the effect of synthetic oligopeptide according to the present invention on osteoblast differentiation by comparing ALP (alkaline phosphatase) activity, an early differentiation marker of osteoblasts.

In order to compare the osteoblast differentiation ability of the synthetic peptides according to the present invention, the MSCs were inoculated into 4 wells of 3 × 10 ³ / well cells in 96 well plates and cultured for 1 day, followed by the synthesis peptides OP5 to OP18 of the present invention in differentiation medium ODM, Positive controls BMP-2, simvastatin and OP3 were respectively treated and incubated in 37 ℃, CO ₂ incubator. The medium was changed every 3 days and ALP activity was measured on day 4 of culture and the results are shown in FIG. 2.

In this study, the synthetic peptides according to the present invention showed about 35% increased ALP activity compared to the conventional BMP-2, and thus, the synthetic peptides according to the present invention can be found to significantly improve the differentiation ability of osteoblasts.

Example  3. Synthesis Peptides  Evaluation of the efficacy of the substance: Calcification ability  evaluation

The degree of calcification of the extracellular matrix was observed in this study to confirm whether osteoblast osteoblastization progressed well by synthetic peptides according to the present invention.

The culture conditions for evaluating the calcification ability were the same as in Example 2, and the degree of calcification was evaluated by ARS staining at 14 days of culture, and the results are shown in FIG. From the 14th day of cultivation, the calcification ability of the synthetic peptide OP5 according to the present invention was about 4 times higher than that of BMP-2, simvastatin and OP3, and OP6 to OP18 were also significantly higher. From these results, it can be confirmed that the bone morphogenesis by the synthetic oligopeptide according to the present invention can be improved.

Example  4. Synthetic peptide  Evaluation of the efficacy of the substance: Bone vertebrae  evaluation

In this study, in order to confirm and compare the excellent MSC bone-dividing ability of OP-based synthetic peptides according to the present invention, bone-dividing activity was measured using adipose derived stem cells under the same experimental conditions, And 5, respectively. Here, since the differentiation rate of ADSC is slow and the value of ALP or ARS is lower than that of MSC, the value is higher than that of the control group as a result of progressing with synthetic peptide of OP series, And the synthetic peptides according to the present invention have excellent bone differentiation effect.

Example  5. Synthesis Peptides  Evaluation of the efficacy of substances: Evaluation of animal skull utilization efficacy

In this embodiment, in vitro using a four-day-old age of the skull of the mouse in order to evaluate the efficacy of the new bone formed in the synthetic peptide according to the invention (in vitro ) were evaluated.

For evaluation of in vitro bone formation efficacy, the skull of a 4-day-old mouse is removed, and the half is separated based on the sagittal suture. The two-piece skulls were stabilized for about 15 hours in a culture medium for stem cell culture containing 10% FBS. After stabilization, add OP5 (1uM) and OP10 (0.01uM, 1uM) to the osteogenic differentiation medium to the skull. As a negative control group, skulls treated with medium for stem cell culture containing 10% FBS were used, and skulls treated with differentiation medium (ODM) were used as a positive control group. The culture medium was replaced with fresh medium every 3 days.

After 14 days of incubation, the cranial tissue is fixed in 4% paraformaldehyde solution for 24 hours. After the fixation, it is demineralized for 24 hours using 10% EDTA (pH 7.2-7.4) solution. After the demineralization process, the tap water is washed with water, dehydrated using ethanol, embedded in a paraffin solution, and a block is formed. The dehydration and embedding process was performed using a tissue stain processor machine.

The paraffin blocks were cut to a thickness of 5 μm to obtain a histological specimen. H & E (hematoxylin and eosin) staining and MT (masson trichrome) staining were performed to determine the degree of new bone formation. The results are shown in FIGS. 6 and 7, respectively. In the H & E staining shown in FIG. 6, the new bone is a red-stained portion. In the MT staining method shown in FIG. As a result of the experiment, it can be confirmed that bone formation is significantly increased in the skull tissue treated with the synthetic peptide according to the present invention, compared to the skull tissue cultured in the positive control group (ODM). The area of the newly formed new bone was measured, and the result was calculated as a percentage based on the negative control (10% DMEM) as a large bar graph, and the percentage of new bone increased as compared with the positive control (ODM) As shown in Fig. As can be seen from FIG. 8, OP5 and OP10 according to the present invention significantly increase the formation of new bone than the positive control (ODM).

As can be seen from the above examples, the synthetic oligopeptides OP5 to OP18 according to the present invention showed an increase in the activity of the phosphorylated enzyme compared with ODM, which is the most commonly used solution to induce bone regeneration, in the case of OP5, 30% OP10 increased by 25%. In addition, it showed an increase of 25% compared to BOPD and OGP, which are known bone regeneration substances.

In the evaluation of calcification ability, which is an important experiment for bone regeneration, the increase of activity was 30% for OP5 and 25% for OP10. In addition, it showed an increase of 25% compared to BOPD and OGP, which are known bone regeneration substances.

In addition, in the evaluation test of in vitro bone formation using an animal skull, 7.2% of OP5 (1 uM) and 17% of OP10 (1 uM) were used in the osteoclast differentiation medium (ODM) And the area was increased.

Therefore, it can be seen from the above results that the synthetic peptides OP5 to OP18 according to the present invention having the above sequence, particularly OP5 and OP10, can form a useful composition for inducing bone differentiation, which effectively functions to induce bone cell differentiation of stem cells .

Attach an electronic file to a sequence list

Claims (16)

Induction of bone differentiation of stem cells characterized by consisting of one or more peptides selected from OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the amino acid sequence of SEQ ID NOS: 2 to 13 Synthetic peptides. According to claim 1, wherein the osteogenic differentiation peptides for inducing osteogenic differentiation peptides characterized in that the peptide of OP10 having the amino acid sequence of SEQ ID NO: 5. Pharmaceutically acceptable excipients with one or more peptides selected from OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the amino acid sequence of SEQ ID NOs: 2 to 13 as an active ingredient Osteodifferentiation induction composition comprising a. The method of claim 3, wherein the peptide is a composition for inducing bone differentiation, characterized in that the peptide of OP10 having the amino acid sequence of SEQ ID NO: 5. Pharmaceutically acceptable excipients with one or more peptides selected from OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the amino acid sequence of SEQ ID NOs: 2 to 13 as an active ingredient Pharmaceutical composition for the prevention or treatment of osteoporosis, characterized in that it comprises a. According to claim 5, wherein the peptide is a pharmaceutical composition for the prevention or treatment of osteoporosis, characterized in that the peptide of OP10 having the amino acid sequence of SEQ ID NO: 5. Pharmaceutically acceptable excipients with one or more peptides selected from OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the amino acid sequence of SEQ ID NOs: 2 to 13 as an active ingredient Pharmaceutical composition for the prevention or treatment of bone defect disease, characterized in that it comprises a. According to claim 7, wherein the peptide is a pharmaceutical composition for the prevention or treatment of bone defect disease, characterized in that the peptide of OP10 having the amino acid sequence of SEQ ID NO: 5. Pharmaceutically acceptable excipients with one or more peptides selected from OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the amino acid sequence of SEQ ID NOs: 2 to 13 as an active ingredient Pharmaceutical composition for the prevention or treatment of hypophosphatase, characterized in that it comprises a. The pharmaceutical composition for preventing or treating hypophosphatase according to claim 9, wherein the peptide is a peptide of OP10 having an amino acid sequence of SEQ ID NO: 5. Pharmaceutically acceptable excipients with one or more peptides selected from OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the amino acid sequence of SEQ ID NOs: 2 to 13 as an active ingredient Pharmaceutical composition for the prevention or treatment of Begett disease, characterized in that it comprises a. The pharmaceutical composition of claim 11, wherein the peptide is a peptide of OP10 having an amino acid sequence of SEQ ID NO: 5. 12. Pharmaceutically acceptable excipients with one or more peptides selected from OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the amino acid sequence of SEQ ID NOs: 2 to 13 as an active ingredient Pharmaceutical composition for the prevention or treatment of femoral head avascular necrosis, characterized in that it comprises a. The pharmaceutical composition of claim 13, wherein the peptide is a peptide of OP10 having an amino acid sequence of SEQ ID NO: 5. Pharmaceutically acceptable excipients with one or more peptides selected from OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 having the amino acid sequence of SEQ ID NOs: 2 to 13 as an active ingredient Pharmaceutical composition for the prevention or treatment of osteoarthritis, characterized in that it comprises a. The pharmaceutical composition for preventing or treating osteoarthritis of claim 15, wherein the peptide is a peptide of OP10 having an amino acid sequence of SEQ ID NO: 5.

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