KR20130049793A - Composition for inducing a bone differentiation and method for enhancing a bone differentiation using the same - Google Patents

Abstract

PURPOSE: A composition for inducing bone differentiation of stem cells and a method for enhancing bone differentiation using the same are provided to obtain a synthetic peptide for promoting bone formation. CONSTITUTION: A synthetic peptide for inducing bone differentiation of stem cells contains OP5 peptide with an amino acid sequence in sequence number 1. A composition for including bone differentiation contains the OP5 peptide and pharmaceutically acceptable excipient. A pharmaceutical composition for preventing or treating osteroporosis contains the OP5 peptide and pharmaceutically acceptable excipient. A pharmaceutical composition for preventing or treating bone loss disorders contains the OP5 peptide and pharmaceutically acceptable excipient.

Description

Composition for inducing a bone differentiation and method for enhancing a bone differentiation using the same}

The present invention relates to a composition for inducing bone differentiation of stem cells and a method for increasing bone differentiation using the same, and more specifically, to increase the differentiation inducing effect when inducing osteoblast differentiation of stem cells, consisting of an amino acid sequence derived from a bone-forming protein. It relates to a synthetic peptide or a homologue thereof for promoting bone differentiation and bone formation, a composition or pharmaceutical composition for inducing bone differentiation comprising the synthetic peptide as an active ingredient, a medium composition containing them, and a method for increasing bone differentiation using the same.

The cells that make up bone are osteocytes that maintain bone homeostasis, osteoblasts that form bones, osteoclasts that absorb bones, and cartilage cells that are responsible for joint function at the ends of bones. (chondrocyte), etc., and even in the bone after growth, the cancellous bone is absorbed by the osteoclasts, and the other surface is remodeled as new bones are formed by the osteoblasts. If the balance between bone formation and bone resorption is broken, it progresses to bone disease, and these bone diseases include osteoporosis, non-union fractures, osteonecrosis, osteomalacia, and bone disease. There are deficiencies and the like. In addition, Avascular necrosis of the Femoral Head is a refractory bone disease in which the blood supply to the femoral head of the hip joint is interrupted due to trauma, alcohol, or taking steroids. The incidence rate is 20,000 per year, and 4,000 to 5,000 per year in Korea. Most of them occur in the 20-50 years old, and the average incidence rate is high in the late 30s. However, no special treatment methods have been developed other than surgical methods for artificial joint replacement surgery.

In addition, osteoporosis, also referred to as osteoporosis or osteoporosis, refers to a metabolic bone disease in which the bone mass is significantly reduced compared to a normal person, and the quantity of bone components is reduced as the main lesion. In general, the condition of osteoporosis itself is often asymptomatic or mild, but once a fracture occurs, it is generally difficult to treat, and it is difficult to sufficiently recover even if the osteosynthesis is performed.

As a prophylactic and/or therapeutic agent for bone diseases including osteoporosis and fractures, calcium preparations, estrogen preparations, selective estrogen receptor modulators (SERM), active vitamin D3 preparations, vitamin K preparations, ifriflavone preparations, calcitonin preparations, bisphosphonate preparations, Parathyroid hormone preparations, anabolic hormone preparations, bone morphology inducing factor (BMP), or fibroblast proliferation factor (FGF) have been used clinically or have been attempted clinical applications, but despite the use of a number of such drugs. In addition, the number of patients with bone disease is increasing year by year, and therefore, the development of a new concept of therapeutic agent for efficiently treating such intractable bone disease is urgently required.

Traditional treatments for bone diseases include autografting, allografting, and artificial bone grafting, but induce complications such as infection and hematoma at the bone collection site (autologous bone transplant), from donors. There is a problem with the possibility of disease transmission of the disease (allogeneic bone transplantation), and the inability to generate fundamental bones (artificial bone transplantation). Therefore, tissue engineering research on bone formation to treat intractable bone diseases by solving these shortcomings is actively being conducted. For the formation of bone tissue by tissue engineering, osteoblastic progenitor cells that induce bone formation, a scaffold capable of proliferating and differentiating these cells at appropriate locations, and bone-inducing growth factors that promote the induced differentiation of bone tissue formation are required.

Among these, bone morphogenetic protein (BMP) is a member of the β-superfamily of transforming growth factor (TGF) and promotes differentiation from osteoblast progenitor cells into osteoblasts and promotes the differentiation of osteoblasts into osteoblasts in vivo. in vivo) and is reported Biol (Mol both in vitro (in vitro) showing the effect of increasing bone formation, Cell 1999; 10:. 3801-13, Bone 2001; 28:. 491-8 and reference) are, in particular BMP -2 Protein is a bone formation-inducing protein and has an excellent effect in promoting bone formation, but BMP having such an excellent effect requires a large amount due to a short half-life to exhibit a satisfactory effect. There is a disadvantage that it is not used by many patients.

Therefore, various studies have been attempted to solve these shortcomings. In particular, since a specific partial peptide sequence of a known BMP is used, it has been studied whether or not the same effect as BMP or more can be obtained, and has been continuously studied. Actually. As a study on the promotion of bone formation, for example, Korean Patent Application No. 2007-0055211 is called "injectable bone regeneration material containing bone formation promoting peptide", and is an injection-type bone regeneration material containing bone formation promoting peptide. In the present invention, chitosan, alginic acid, silk fibroin, propylene glycol, propylene glycol alginic acid, poloxamer, chondroitin sulfate, and a gel-forming base selected from the group consisting of a combination thereof, one of SEQ ID NOs: 1 to 7 Disclosed is an injection-type bone regeneration material, characterized in that a bone formation promoting peptide essentially containing the above amino acid sequence is bound or mixed. The bone formation promoting peptide used here is obtained by separating and extracting the amino acid sequence of the active site from the protein constituting the extracellular matrix, and the peptide is the amino acid sequence YRLKRSKS (SEQ ID NO: 1) at positions 85-105 of rabbit bone sialoprotein 1 (BSP1), KMFHVSNAQYPGA (SEQ ID NO: 2), YRLKRSKSKMFHVSNAQYPGA (SEQ ID NO: 3), human bone sialoprotein I149-169 amino acid sequence YGLRSKS (SEQ ID NO: 4), KKFRRPDIQYPDAT (SEQ ID NO: 5), YGLRSKSKKFRRPDIQYPDAT (SEQ ID NO: 6) It essentially contains, and discloses that it contains the amino acid sequence GLRSKSKKFRRPDIQYPDATDEDITSHM (SEQ ID NO: 7) at positions 150-177 of human bone sialoprotein I (Osteopontin).

In another invention, in Korean Patent Application No. 2010-0018148, under the name "oligopeptide that enhances osteoblast differentiation ability", by developing an oligopeptide having very high response specificity for BMP-specific receptors, the degree of osteoblast differentiation and PEP7 (SEQ ID NO: 1), PEP71 (SEQ ID NO: 2), PEP72 (SEQ ID NO: 3), PEP73 ( An oligopeptide applicable to an implant selected from the group consisting of SEQ ID NO: 4), PEP74 (SEQ ID NO: 5), PEP75 (SEQ ID NO: 6) and PEP76 (SEQ ID NO: 7) is disclosed.

In addition, in addition to the above patented inventions, in "Biomaterials 26 (2005) 5648-5657", the P-15 peptide can regulate the number of cells and tissue structure by enhancing the adhesion of embryonic cells and through the regulation of apoptosis. In addition, recent studies suggest that the inorganic bone matrix (ABM)/P-15 complex can be used as an injectable bio-like matrix to promote bone regeneration ( Hanks T, Atkinson 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) discloses, and in "Regulatory Peptides 142 (2007) 16-23", OGP and OGP(10-14) are bone formation when administered to normal and osteopenic experimental animals. And increasing cancellous bone density and encouraging fracture treatment, and in the cultivation of osteoblasts and other bone marrow organ tissue 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 osteogenic growth peptide ( OGP ): a novelcirculating stimulator of osteoblastic activity . EMBO J 1992;11:1867-73.) discloses .

In addition, in "Biomaterials 30 (2009) 3532-3541", the OPD peptide has been identified from BMP-2, and has an affinity for the BMP receptor, thereby inducing the differentiation of organ cells of the human bone marrow into osteoblasts. In "J Korean Med Sci 2005; 20: 438-44", Mundy et al. first reported evidence showing that statins can have an anabolic effect on bone, where statins Discloses that it significantly stimulated new bone formation in rodents, and it has also been reported that statins act on the lipid synthesis pathway in osteoblasts and enhance 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 believed to modulate the osteogenic effects of statins. In addition, statins are of bisphosphonates in osteoblasts through inhibition of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA), which is essential for normal osteoblast function, leading to reduced protein prenylation. It has been reported that it acts 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, conventional bone formation mechanisms and substances that act on these mechanisms and effectively contribute to bone formation have been continuously studied, and the results of these studies have been obtained to some extent, but including the aforementioned patent inventions and research reports. Conventional techniques still require research on materials that can induce bone differentiation more effectively and provide at low cost, and therefore, the present inventors and the like have made intensive research to meet these needs to complete the present invention. Became.

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.

Accordingly, the present invention has been made in view of the above-described conventional circumstances, and the primary object of the present invention is to more effectively and simply provide a new peptide that increases the differentiation-inducing effect when inducing osteoblast differentiation of stem cells. will be.

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

Another object of the present invention is to provide a composition comprising the new synthetic peptide as an active ingredient, particularly effective in inducing osteogenic differentiation of stem cells.

Another object of the present invention is 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 can also aim to achieve these objects and other objects that can be easily derived by a person skilled in the art from the general description of the present specification in addition to the above-described clear objects.

Objects of the present invention described above, although the bone formation characteristics of BMP-2 have been reported, not only the definitive receptor-binding sequence of BMP-2, but also their bone formation regions are still not fully elucidated, according to the present invention. It was found that the synthetic peptide for promoting bone formation having a specific amino acid sequence according to the present invention has a remarkable effect in promoting the differentiation of stem cells (MSC) into osteoblasts, and the pharmaceutical composition of the present invention comprising the synthetic peptide as an active ingredient is osteoporosis, It has been achieved by finding that it has an excellent effect in the treatment of bone defect disease and/or osteoarthritis.

The synthetic peptide for inducing bone differentiation of stem cells of the present invention to achieve the above object is:

It is characterized in that it may consist of one or more peptides selected from OP5, OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 or a homologue thereof 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

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

According to another configuration of the present invention, the homolog of the OP5 is DWIVAGSGDWIVA, DWIVAGSGDWIVA-NH _2, N-Ac-DWIVAGSGDWIVA-NH _{2 ,} N-Ac-DWIVAGSGDWIVA, N-formyl-DWIVAGSGDWIVA, such as N-, C-terminal Includes substituted ones.

According to another configuration of the present invention, homologues of the OP7, OP8, OP9, OP10, OP11, OP12, OP13, OP14, OP15, OP16, OP17, OP18 and the N- and C-terminals of all peptides are OP5 and Includes those substituted together.

The composition for inducing bone differentiation of the present invention for achieving the above other object is:

It is characterized in that it contains a peptide of OP5 having the above sequence as an active ingredient and a pharmaceutically acceptable excipient.

The pharmaceutical composition for preventing or treating osteoporosis of the present invention for achieving the above another object is:

It is characterized in that it contains a peptide of OP5 having the above sequence as an active ingredient and a pharmaceutically acceptable excipient.

The pharmaceutical composition for preventing or treating bone defect disease of the present invention for achieving the above another object is:

It is characterized in that it contains a peptide of OP5 having the above sequence as an active ingredient and a pharmaceutically acceptable excipient.

The pharmaceutical composition for the prevention or treatment of hypophosphatemia of the present invention for achieving the above another object is:

It is characterized in that it contains a peptide of OP5 having the above sequence as an active ingredient and a pharmaceutically acceptable excipient.

The pharmaceutical composition for preventing or treating Besette's disease of the present invention for achieving the above another object is:

It is characterized in that it contains a peptide of OP5 having the above sequence as an active ingredient and a pharmaceutically acceptable excipient.

The pharmaceutical composition for the prevention or treatment of avascular necrosis of the femoral head of the present invention for achieving the above another object is:

It is characterized in that it contains a peptide of OP5 having the above sequence as an active ingredient and a pharmaceutically acceptable excipient.

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

It is characterized in that it contains a peptide of OP5 having the above sequence as an active ingredient and a pharmaceutically acceptable excipient.

The composition for inducing bone differentiation of stem cells of the present invention constituted as described above provides synthetic peptides for promoting bone formation having a specific sequence, and these synthetic peptides have 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 has an excellent effect in the treatment of osteoporosis, bone defect disease and/or osteoarthritis, so that it can not only replace the conventional expensive BMP, but also bone differentiation using the same. Increasing method is a useful invention that can induce bone differentiation more easily and inexpensively.

FIG. 1 is a photograph of a staining test of the stem cell osteodifferentiation effect of the synthetic peptide OP5 obtained according to the present invention using ALP (alkaline phosphatase), which is an initial marker for differentiation of osteogenic cells, and Vonkossa staining, which is a calcification marker, where 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 to which OP5 peptide is added (test group).
2 is a graph showing the comparison of ALP activity, which is an initial differentiation marker of osteoblasts, in order to compare the stem cell osteodifferentiation effect of the synthetic peptide according to the present invention, where "control" is a negative control, BOPD, OGP and BMP -2 is a material with known bone regeneration.
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 effect of stem cell osteodifferentiation of the synthetic peptide according to the present invention, where "control" is negative As a control group, BOPD, OGP and BMP-2 are known substances for bone regeneration.
4 and 5 are graphs confirming and comparing the osteodifferentiation effect of adipose stem cells of the synthetic peptides of the OP series according to the present invention.
6 and 7 are H&E (hematoxylin and eosin) staining to determine the degree of new bone formation after obtaining a tissue sample by cutting a paraffin block into 5 μm thickness using an animal skull to evaluate the effectiveness of the synthetic peptide material according to the present invention. And MT (masson trichrome) staining. Here, the arrow part indicates the incision in the sagittal suture direction, and the enlarged square part of the left photo is the right photo.
FIG. 8 is a large bar graph showing the results of calculating the area of newly formed new bones as a percentage based on the negative control group (10% DMEM) after each measurement.

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

According to a preferred embodiment of the present invention, in order to provide the composition for inducing osteodifferentiation of stem cells according to the present invention, the present invention first includes: 1) establishing a culture condition used for inducing bone differentiation of stem cells, and 2) In the above culture conditions, OP5 or the like was added to obtain an effective effect of inducing bone differentiation.

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

In the following examples, an experiment was conducted on the bone differentiation effect of OP5 of the present invention compared to the control group, and the results were disclosed.

In the induction of bone differentiation of stem cells (MSCs), significant results were obtained compared to the control group in the presence of the OP5 according to the present invention.

A food, food additive or pharmaceutical composition containing the composition for preventing or treating osteoporosis according to the present invention may be prepared, and the preparation may further include an appropriate carrier, excipient, and diluent commonly used. In addition, the composition according to the present invention may be administered alone or in combination with other pharmaceutically active ingredients to enhance efficacy as a therapeutic agent for osteoporosis.

The composition for preventing or treating osteoporosis according to the present invention includes lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch as carriers, excipients, and diluents that can be included in foods, food additives or pharmaceutical compositions. , Gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil It may further include.

According to another embodiment, the composition for preventing or treating osteoporosis according to the present invention may be used in oral formulations such as powders, granules, tablets, suspensions, emulsions, and syrups according to a conventional method. In addition, the pharmaceutical composition comprising the composition of the present invention as an active ingredient may be administered orally or parenterally, and the formulation thereof may vary depending on the method of use, so it is not limited to the above description.

The oral dosage form is meant to include solid preparations and liquid preparations for oral administration, and solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations are included in the composition. It may be prepared by including at least one or more excipients, for example starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, and syrups.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives are included. May be.

Hereinafter, the present invention will be described in more detail by examples, but it is a matter of course that the scope of the present invention is not limited to these examples.

Example 1. Stem Cell Bone differentiation 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 observed by using ALP (alkaline phosphatase) and ARS (Alizarin Red S) staining, which are early differentiation markers of osteogenic cells.

To compare the differentiation ability of osteogenic cells, as a positive control, an osteogenic differentiation medium (ODM) containing ascorbic acid, β-glycerophosphate, and dexamethasone was used ( Develop . Grewth Differ . 50,553-564, 2008. ), 10% FBS (a medium for stem cell culture containing 10% FBS) was used as a negative control. The experimental group was carried out by adding the synthetic peptide according to the present invention to each medium. MSCs were dispensed into a cell culture plate (12 well culture plate) by 4×10 ³ cells per well, and cultured in a _{CO 2} incubator at 37°C. Fresh medium was replaced every 3 days of culture, and bone differentiation ability was compared by ALP, ARS and Vonkossa staining on the 14th day of culture, and the results are shown in FIG. 1.

As can be seen from FIG. 1, it can be seen that in this study, the synthetic peptide OP5 according to the present invention was significantly stained with ALP, ARS, and Vonkossa staining in the experimental group to which OP5 was added when compared to the positive control, ODM. Accordingly, it can be confirmed that the synthetic peptide according to the present invention remarkably improves the differentiation ability of stem cells into osteoblasts.

Example 2. Synthesis Peptide Evaluation of the effectiveness of the substance: initial Cell differentiation Evaluation of bone marrow stem cells Bone differentiation evaluation)

This study is the initial differentiation of osteoblasts. Marker Inn ALP ( alkaline phosphatase ) Synthesis according to the present invention by comparing the activity Oligopeptides This is to observe the effect on the differentiation of osteoblasts.

Synthesis according to the invention Peptide Osteoblast Differentiation To compare, MSC 4 x 10 in a 96 well plate ³ /Well cells are aliquoted and cultured for 1 day ODM In the synthesis of the present invention Peptide OP5 To OP18 , Positive control soldier BMP -2, simvastatin ( simvastatin ) And OP3 By treating each of 37° C., CO ₂ Incubated in an incubator. The medium was changed every 3 days and on the 4th day of culture ALP The activity was measured and the results are shown in FIG. 2.

Synthesis according to the invention in this study Peptide is Conventional BMP About 35% compared to -2 Increased ALP By showing activity, the synthesis according to the invention Peptide is Osteoblastic Differentiation It can be seen that it can be remarkably improved.

Example 3. Synthesis Peptide Evaluation of the effectiveness of the substance: Calcification evaluation

In this study, the degree of calcification of the extracellular matrix was observed in order to confirm whether osteoblast differentiation by the synthetic peptide according to the present invention proceeded well.

The culture conditions for the evaluation of calcification were the same as in Example 2, and the degree of calcification was compared and evaluated by ARS staining on the 14th day of culture, and the results are shown in FIG. 3. As a result of the experiment, on the 14th day of culture, 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 bone differentiation by the synthetic oligopeptides according to the present invention can proceed in an improved manner.

Example 4. Synthetic peptide Evaluation of the effectiveness of the substance: adipose-derived stem cells Bone differentiation evaluation

In this study, in order to confirm and compare the excellent MSC bone differentiation capability of the OP-based synthetic peptide according to the present invention, the bone differentiation capability was measured using Adipose derived stem cells under the same experimental conditions, and the results are shown in FIG. 4. And 5 shown. Here, the differentiation rate of ADSC is slow, so that the value of ALP or ARS is lower than that of MSC, but it shows a higher value compared to the value in the control group as a whole in progression with the synthetic peptide of the OP series, from which the present invention The results of the experiment in MSC that the synthetic peptides according to the above have excellent osteodifferentiation effects are sufficiently supported.

Example 5. Synthesis Peptide Evaluation of the effectiveness of the substance: Evaluation of the efficacy of using animal skulls

In this example, in order to evaluate the efficacy of the new bone formation of the synthetic peptide according to the present invention, in vitro (in In vitro ) bone generation efficacy evaluation experiment was conducted.

For the evaluation of in vitro osteogenic efficacy, the skull of a 4-day-old mouse is excised and separated in half based on a sagittal suture. The skull separated into two pieces was stabilized for about 15 hours in a stem cell culture medium containing 10% FBS. After completion of stabilization, a medium to which OP5 (1uM) and OP10 (0.01uM, 1uM) were added to the osteogenic differentiation medium (ODM) for differentiation, respectively, was added to the skull. As a negative control, a skull treated with a stem cell culture medium containing 10% FBS was used, and as a positive control, a skull treated with a differentiation medium (ODM) was used. Fresh medium was replaced every 3 days during the incubation period.

After incubation for 14 days, the skull tissue is fixed in 4% paraformaldehyde solution for 24 hours. After fixation is complete, demineralization is performed for 24 hours using a 10% EDTA (ethylenediaminetetraacetic acid) pH 7.2~7.4 solution. After the demineralization process is finished, it is washed in tap water, dehydrated using ethanol, and embedded in a paraffin solution to form a block. The dehydration and embedding process was performed using a tissue stain processor.

After the paraffin block was cut to a thickness of 5 μm, a tissue sample was obtained, and H&E (hematoxylin and eosin) staining and MT (masson trichrome) staining were performed to determine the degree of new bone formation, and the results are shown in FIGS. 6 and 7 respectively. In the H&E staining shown in FIG. 6, the new bone is a part dyed in red, and the MT staining method shown in FIG. 7 is a part dyed in blue to determine the degree of formation of the new bone. As a result of the experiment, it can be seen that bone formation is significantly increased in the cranial tissue treated with the synthetic peptide according to the present invention than in the cranial tissue cultured in the positive control medium for differentiation (ODM). After measuring the area of each newly formed new bone, the result of calculating it as a percentage based on the negative control group (10% DMEM) is shown as a large bar graph, and the percentage of the amount of new bone that has increased compared to the positive control group (ODM) is shown as a small blue bar graph. And shown in FIG. 8. As can be seen from FIG. 8, it can be seen from FIG. 8 that OP5 and OP10 according to the present invention significantly increase the generation of new bones than the positive control group (ODM).

As can be seen in each of the above examples, the synthetic oligopeptides OP5 to OP18 according to the present invention have an increase in kinase activity of 30% in the case of OP5 compared to ODM, which is the solution most often used to induce bone regeneration, In the case of OP10, the result was increased by 25%. And, compared to the conventional materials known to bone regeneration BOPD, OGP showed a 25% increase in results.

In the evaluation of calcification ability, which is an important experiment for bone regeneration, the activity increase was increased by 30% in the case of OP5 and 25% in the case of OP10. And, compared to the conventional materials known to bone regeneration BOPD, OGP showed a 25% increase in results.

In addition, in the experiment for evaluating the efficacy of new bone formation in vitro using the skull of an animal, 7.2% for OP5 (1uM) and 17% for OP10 (1uM) than the cranial tissue cultured only in the positive control stem cell differentiation medium (ODM). The result was an increase in the area.

Therefore, from the above results, it can be seen that the synthetic peptides OP5 to OP18 according to the present invention having the above sequence, in particular OP5 and OP10, can form a useful composition for inducing bone differentiation that effectively acts on inducing osteoblast differentiation of stem cells. .

<110> WOORIDUL LIFE SCIENCES LIMITED <120> Composition for inducing a bone differentiation and method for enhancing a bone differentiation using the same <130> P-2011-0501 <160> 13 <170> KopatentIn 2.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 1 Asp Trp Ile Val Ala Gly Ser Gly Asp Trp Ile Val Ala 1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 2 Asp Trp Ile Val Gly Ser Gly Asp Trp Ile Val 1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 3 Asp Trp Ile Gly Ser Gly Asp Trp Ile Val 1 5 10 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 4 Asp Trp Ile Gly Ser Gly Asp Trp Ile 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 5 Asp Trp Tyr Gly Phe Gly Asp Trp 1 5 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 6 Asp Trp Gly Ser Gly Asp Trp 1 5 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 7 Asp Trp Tyr Gly Phe Asp Trp 1 5 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 8 Asp Trp Tyr Phe Gly Asp Trp 1 5 <210> 9 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 9 Asp Trp Tyr Phe Asp Trp 1 5 <210> 10 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 10 Asp Trp Gly Ser Gly Asp 1 5 <210> 11 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 11 Asp Trp Gly Ser Gly Trp 1 5 <210> 12 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 12 Asp Trp Tyr Gly Phe Asp 1 5 <210> 13 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Peptide <400> 13 Asp Trp Tyr Gly Phe Trp 1 5

Claims (8)

A synthetic peptide for inducing bone differentiation of stem cells, characterized by comprising a peptide of OP5 having the amino acid sequence of SEQ ID NO: 1.
1. A composition for inducing bone differentiation comprising a peptide of OP5 having an amino acid sequence of SEQ ID NO: 1 as an active ingredient and a pharmaceutically acceptable excipient.
A pharmaceutical composition for preventing or treating osteoporosis characterized by comprising an OP5 peptide having an amino acid sequence of SEQ ID NO: 1 as an active ingredient and a pharmaceutically acceptable excipient.
A pharmaceutical composition for preventing or treating a bone defect disease, which comprises a peptide of OP5 having an amino acid sequence of SEQ ID NO: 1 as an active ingredient and a pharmaceutically acceptable excipient.
1. A pharmaceutical composition for preventing or treating hypophosphoremia, characterized in that it comprises a peptide of OP5 having an amino acid sequence of SEQ ID NO: 1 as an active ingredient and a pharmaceutically acceptable excipient.
1. A pharmaceutical composition for the prevention or treatment of vesicovirus, which comprises a peptide of OP5 having an amino acid sequence of SEQ ID NO: 1 as an active ingredient and a pharmaceutically acceptable excipient.
A pharmaceutical composition for preventing or treating osteonecrosis of the femoral head characterized by comprising an OP5 peptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient and a pharmaceutically acceptable excipient.
A pharmaceutical composition for preventing or treating osteoarthritis, comprising an OP5 peptide having the amino acid sequence of SEQ ID NO: 1 as an active ingredient and a pharmaceutically acceptable excipient.

Images