KR100759388B1 - Method of osteoblastic differentiation of alveolar bone marrow-derived adult stem cell - Google Patents

Abstract

      The present invention is an osteoblast differentiation method of adult stem cells derived from alveolar bone marrow. More specifically, adult stem cells isolated from alveolar bone marrow are treated with osteoinduction agents, that is, fast dichloromethane fraction or micro-macro structure of abnormal calcium phosphate It relates to osteoblast differentiation method. Clinical application for periodontal and bone tissue regeneration by increasing the stability and promoting bone differentiation as a result of treating fast dichloromethane fraction and micro-macro-structured ideal calcium phosphate salts on alveolar bone marrow-derived adult stem cells Useful for

Alveolar bone marrow-derived adult stem cells, fast fraction, MBCP

Description

Method of osteoblastic differentiation of alveolar bone marrow-derived adult stem cell}

Figure 1a is a picture of the cell surface antigen analysis with a FACS caliber flow cytometer, Figure 1b is a picture of morphology observation with an optical microscope to determine the morphological characteristics of the cells,

Figure 2 is a value and graph measuring the basic phosphatase activity when treated with dexamethasone (dexextasone: DEX) and fast dichloromethane fractions in alveolar bone marrow-derived adult stem cells,

Figure 3a is a plate diagram showing the calcium accumulation when the alveolar bone marrow-derived adult stem cells treated with DEX and fast dichloromethane fractions,

Figure 3b is a value and graph measuring the calcium accumulation when the DEX and fast dichloromethane fractions in alveolar bone marrow-derived adult stem cells,

4 is a diagram confirming the expression of BSP and OC by treating the alveolar bone marrow-derived adult stem cells with DEX and fast dichloromethane fractions by Western blot,

5 is a numerical value and graph measuring cell proliferation when MBCP is treated to alveolar bone marrow-derived adult stem cells,

6 is a numerical value and a graph measuring collagen synthesis when MBCP was treated to alveolar bone marrow-derived adult stem cells,

7 is a value and a graph of measuring the basic phosphatase activity when the MBCP treatment of alveolar bone marrow-derived adult stem cells,

8 shows Western blot expression of BSP and OC when alveolar bone marrow-derived adult stem cells were treated with MBCP.

The present invention is an osteoblast differentiation method of adult stem cells derived from alveolar bone marrow, and more specifically, dichloromethane fraction or micro-macro biphasic calcium phosphate of fast dichloromethane fractions in adult stem cells isolated from alveolar bone marrow (micro-macro biphasic calcium phosphate). It relates to an osteoblast differentiation method of treating: MBCP).

Hard tissue in the human body (hard tissue) is largely classified into bones and teeth, a disease that occurs due to the problem of the periodontal disease. Periodontal disease is an infectious disease of chronic inflammation accompanied by the destruction of the periodontal tissue. Loss of the alveolar bone caused by this disease leads to a decrease in the supportive tissue, which is the largest cause of extraction in adults. Therefore, it can be said that the purpose of periodontal treatment is to remove various bacteria of plaque or tartar that cause the disease, and to regenerate lost tooth support tissue (Page RC, J Periodontol ., 64: 744,753, 1993). As the treatment of the disease, alveolar bone graft using various bone materials in addition to general periodontal treatment is preferred.

When materials used for bone grafts are classified according to the source, they can be divided into autologous bone, allogeneic bone, xenograft bone, and synthetic bone.

Firstly, autologous bone graft is the implantation of bone from the patient's own body, which is the most ideal implant or limit of bone graft, additional surgery for bone graft, damage to the donor site during bone extraction and the possibility of complications after the procedure. There are disadvantages.

Second, allogeneic bone is donated by organs (bones) from brain lions and made by tissue banks, which compensates for the disadvantages of autologous bones, but the possibility of disease transmission from donors and immune rejection may be a problem.

Third, xenografts are collected and used from other individuals, and very little absorption occurs in tissues, and may remain for a long time, but bone induction capacity is lower than that of allogenes, and controversy continues.

Fourth, synthetic bone was developed as a biocompatible material that can replace copper bone and xenograft. Synthetic bone graft has been used in the periodontal area, such as natural coral, tricalcium phosphate, and hydroxyapatite (Barney VC et al ., J Periodontol , 57 (12): 764-770, 1986). Among these, hydroxyapatite, which is a non-absorbable synthetic bone, is classified into dense hydroxyapatite and porous hydroxyapatite. Foroum GM , et al , J Periodontol . , 53 (12): 719-725, 1982; Moskow BS, et al , J. Peridontol , 54 (8): 455-462, 1983), porous hydroxyapatite has a space similar to that of bone tissue, allowing the inflow and growth of cells into the space (Roy DN, et al, Nature , 247). (438): 220-222, 1974). Biphasic calcium phosphate (BCP) was developed as a graft with hydroxyapatite and β-tricalcium phosphate (Gauthier O., et al , Bionaterials , 19: 133-). 139,1998). In clinical practice, micro-macro biphasic calcium phosphate (MBCP) having a micro-macro structure has been recently developed and widely used to give a implant a space for forming a contact surface for cell adhesion. MBCP is a porous implant with a chemical structure of 60:40 mixed with stable hydroxyapatite and highly soluble β-3 calcium phosphate. Due to the chemical properties of the graft, hydroxyapatite provides a support until the new bone tissue remains structurally stable, and β-3 calcium phosphate is rapidly dissolved so that the new osteoblasts are transferred between the grafts. To spread well into the space. In addition, the countless pores in the implants enable ion exchange and form new contact surfaces for cell adhesion through the precipitation of bone crystals, and assist in blood vessel formation and remodeling and growth of new bone. When the implant is implanted in the bone defect, the interaction between the implant and the biomaterial occurs (Iwasaki Y. et. al . , Biomaterials , 23: 3421-4327, 2002), but there are no reports of promoting the process of bone differentiation.

The most ideal healing aspect of bone graft is complete host ossification of the implant. However, the healing pattern of alveolar bone is known to be different according to the origin and processing of the implant. For this reason, there is an interest in the use of adult stem cells that have the ability to differentiate into specific tissues and organs after transplantation in vivo during periodontal tissue regeneration, and also to metastasize to cells of other tissues, unlike the original cellular characteristics. Are gathering.

Stem cells (Stem cells) are a kind of blast cells that grow different cells or organs that make up the human body, also called hepatocytes. Stem cells include adult stem cells, such as embryonic stem cells that can be made from human embryos, and bone marrow cells that constantly make blood cells. Embryonic stem cells can differentiate into all the cells and tissues that make up the human body, but their use is limited for ethical reasons, while adult stem cells are derived from umbilical cord blood or bone marrow and blood of mature adults. Exists as. Adult stem cells are cells that have differentiation flexibility that can differentiate into specific tissues and organs after transplantation in vivo, and can metastasize to cells of other tissues different from the characteristics of the original cells. It is widely used in.

Already in human and animal studies, bone marrow-derived stem cells from adult stem cells have been differentiated into osteogenic cells (Friedenstein AJ et al ., Transplantation ., 6: 230-247, 1968). Advances in the differentiation and proliferation of stem cells isolated from bone marrow into osteoblasts have increased the possibility of clinical application (Ohgushi H. et. al ., J Biomed Mater Res ., 48: 913-927, 1999).

Adult stem cells have been collected and studied from the iliac bone and tibia, but they are associated with secondary wound shapes and postoperative pain. There is a need for an alternative method to obtain adult stem cells more easily. In recent years, alveolar bone-derived adult stem cells have higher osteogenic capacity than iliac stem-derived adult stem cells (Matsubara T., et. al ., J bone Miner Res . , 20 (3): 399-409, 2005).

Meanwhile, in order to differentiate adult stem cells into osteoblasts, dexamethasone (hereinafter referred to as DEX) was mainly used (Shigeyuki K. et. al ., Artif Organ . , 28: 72-82, 2004). However, DEX has been reported to have side effects such as impaired immune response during prolonged body administration (Carlsten H. et. al ., Inflamm Res . 45: 26-30, 1996; Yaube M. et al ., Inflamm Res ., 49: 548-552,2000), which have long-term effects on the cells and are of concern for unexpected side effects when used clinically. In order to reduce these side effects, when the adult stem cells are differentiated into osteoblasts, an alternative method is required.

Recently, herbal medicines widely used in oriental medicine have been found to be stable and effective in humans by scientific research methods (Hisha H., et. al ., Blood , 90 (3): 1022-1030, 1997). Representative herbal preparations include safflower seed, angelica, and sokdan.

Safflower seed has been shown to be effective in strengthening fractures, bone fractures, clavicles, bone diseases and fragile bones. Safflower seed extracts increase osteoblast cell proliferation, basic phosphatase activity, new bone formation and maturation. It has been reported to have an excellent effect on bone formation, regeneration and healing process (Yoon Dong-hwan et al., Journal of Korean Periodontology, 28 (4): 769-786, 1998), and angelica sinensis ) has been shown to increase osteoblast proliferation and differentiation (Yang Q., et. al ., Clinica Chimica Acta ., 71 (1): 88-94, 2002).

Phlomis umbrosa ) is one of the many herbal medicines used in oriental medicine. It is known to strengthen the liver and kidneys and to smooth the flow of blood to connect muscles and bones. In addition, the speed fraction was reported that bone regeneration activity and the availability of zero-destructive periodontal disease in bone can influence the differentiation of cells (such as yiyoungjun, Journal of Periodontal 33: 0.259 to 269, 2003), speed Has been reported to increase the number of cells, increase the activity of basic phosphatase, increase the expression of bone protein, and skull regeneration effect. Influence, Ph.D., Wonkwang University, 2003; Park, Sang, Park, Ph.D.

Therefore, the present inventors have isolated the adult stem cells from the bone marrow of human alveolar bone and treated the fast fraction or MBCP, and confirmed that the alveolar bone marrow-derived adult stem cells promoted the differentiation into osteoblasts without toxicity of the cells. Was completed.

It is a main object of the present invention to provide an osteoblast differentiation method through the treatment of osteoblast differentiation in adult stem cells derived from alveolar bone marrow.

       In order to achieve the above object, the present invention comprises the steps of: i) separating adult stem cells from the bone marrow of the alveolar bone; Ii) culturing the stem cells; And iii) treating the stem cells with osteoblast differentiation inducing agents to differentiate the alveolar bone-derived adult stem cells into osteoblasts.

       The present invention also provides an agent for inducing differentiation using MBCP as an active ingredient.

The present invention also provides an agent for inducing differentiation of fast dichloromethane fraction as an active ingredient.

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

       The present invention provides a method for isolating adult stem cells from bone marrow bones of alveolar bone and treating the stem cells with rapid dichloromethane fraction and MBCP to differentiate into osteoblasts.

       Hereinafter, the present invention will be described in detail.

       In the present invention, adult stem cells are preferably used adult stem cells collected from the alveolar bone marrow, alveolar bone marrow can be collected from all mammals including humans.

       A sufficient amount of adult stem cells for bone regeneration can be incubated with an amount of 0.1 to 3 ml extracted from the patient's alveolar bone marrow, without the need for additional surgery for conventional methods of harvesting bone marrow from the iliac bone. Bone marrow is taken from the site during tooth extraction or during periodontal surgery to prepare adult stem cells for future use.

 In the preferred embodiment of the present invention, the adult stem cells are cells that have been passaged 3,6,9 after separation of cells.

In the present invention, the adult stem cells are adult stem cells derived from iliac bone (Kotobuki N., al ., Artif The same adult stem cells as Organ ., 28 (1): 33-39, 2004) were provided using a cell surface antigen and cell morphology measurement method. As a result of the embodiment of the present invention it was confirmed that the adult stem cells derived from the alveolar bone marrow and the adult stem cells derived from the long bone.

Fast-acting fractions used in the present invention, the methanol extracts of the fast-distributed fractions were sequentially obtained using n-hexane, dichloromethane and n-butanol to obtain respective fractions, and a double dichloromethane fraction was used as the fast-acting fraction of the present invention. The dichloromethane fraction has been shown to have osteogenic induction effect of human fetal osteoblasts in Korean Patent Application No. 2004-57670, filed by the present inventors, but induces the differentiation of adult stem cells into osteoblasts It has not been revealed yet.

        Fast dichloromethane fractions were treated in each of 3, 6, and 9 passages of the adult stem cells and examined for bone differentiation. DEX used as a control is a substance used in the differentiation method of the conventional osteoblasts, it is known that there is a problem causing side effects such as impaired immune response when used for a long time. Treatment of fast dichloromethane fractions into adult stem cells means the fast dichloromethane fraction is added at a concentration of 0.5-100 ng / ml, with a concentration of 10 ng / ml being most preferred.

First, the activity of the basic phosphatase was measured, and thus whether the adult stem cell differentiation into osteoblasts was started. Basic phosphatase is a 160 kDa osteoblast marker and is expressed in osteoblasts, prosthetic cells, osteoblasts, osteosarcoma cells and the like. The enzyme is involved in the early stages of bone cell differentiation, and locally increases phosphate ion concentrations to convert proteins into phosphoproteins, which are known to play a key role in bone mineralization, as they have a calcium-binding propensity. The activity of this enzyme is high in cells with high capacity, but low in cells without bone formation ability, and this enzyme has been reported to be useful as a marker for cells with bone formation ability (Wlodarski KH,et al ., Calcif Tissue int, 39: 382-385, 1986). As a result of the embodiment of the present invention, when treated with the fast dichloromethane fraction, the significance was slightly lower than DEX, it was measured that the basic phosphatase activity is significantly increased significantly compared to the control (see Fig. 2). Therefore, even when administering the fast dichloromethane fraction, which is a herbal medicine, it was found that the process of differentiation of adult stem cells into osteoblasts begins.

Fast dichloromethane fractions were also administered to passaged adult stem cells to measure calcium accumulation, which is closely related to bone formation. In the present embodiment, an alizarin red sulfate (AR-S) staining method, which is a staining method for determining calcium accumulation concentration, was used. AR-S staining shows a specific reaction to calcium salts to form a red complex of calcium liver. Effective for measurement. Example Results It was measured that calcium accumulation increased sufficiently significantly when the fast dichloromethane fraction was treated (see FIG. 3).

In the present invention, the expression of Bone Sialoprotein (hereinafter referred to as BSP) and osteocalcin (hereinafter referred to as OC) was measured by Western blot. BSP accounts for 15% of bone's total non-relative protein and is associated with osteocalcification and remodeling. In addition, the protein is associated with the attachment of osteoclasts to the bone matrix, and is known to play an important role in the process of osteocalcification, bone resorption, and dentin and chalkiness. Using this property, it can be used as a measure of bone mineralization and differentiation that can control mineral crystal formation (Shimizu-sasaki E., et. al , J Biol Chem ., 276 (5459-5466, 2001). BSP is involved in the late stages of osteoblast differentiation. OC, on the other hand, is a vitamin K-dependent protein that is produced in osteoblasts and accounts for about 10-20% of the comparative proteins in the bone matrix. The protein is highly binding to calcium and is produced in osteoblasts and plays an important role in bone resorption and bone formation. In the bone structure, the protein is tightly bound to hydroxyapatite and calcium. Increasing osteoblast activity increases the expression of OC in osteoblasts. This property can be used as an indicator to measure osteoblast activity during osteoblastogenesis by measuring OC expression (Marazuela. M., et al ., Calcif Tissue Int ., 52 (6): 419-421, 1993), OC is also involved in late stages of osteoblast differentiation. Since the expression of BSP and OC was increased in the results of the present invention, it was found that the fast dichloromethane fraction also affected the late stages of osteoblast differentiation, so that the adult stem cells functioned completely into osteoblasts. (See Figure 4). From the above results, it can be seen that the fast dichloromethane fraction administered as an osteoclast of the adult stem cells shows a significant significant increase.Also, the fast dichloromethane fraction is administered as the alveolar bone marrow-derived stem like other bone differentiation promoters. The cells can be seen that differentiation proceeds to osteoblasts.

     The present invention is to isolate the adult stem cells derived from the alveolar bone marrow and provide a differentiation inducing agent by treating the stem cells with MBCP. At this time, the treatment refers to culturing the adult stem cells on the MBCP, or to the adult stem cells by crushing or segmenting the MBCP. As a result of measuring the cell proliferation by treating MBCP with adult stem cells derived from alveolar bone marrow, it was determined that it did not affect cell proliferation (see FIG. 5). Thus, the osteoblast differentiation agent using MBCP as an active ingredient has no cytotoxicity. Confirmed.

     In the present invention, collagen synthesis was measured when the adult stem cells were treated with MBCP. Bone is an optical special connective tissue, 33% of which is organic, 28% of which is collagen type 1, and 5% of non-conjugated bone matrix proteins such as soteonectin, osteocalcin, etc., of which collagen is an important function in bone formation Do this. Collagen type 1 is formed in osteoblasts, and minerals are formed in and around the collagen fibers to form bone tissue, and thus, collagen synthesis can be measured to determine the effectiveness of bone formation. In the results of the embodiment of the present invention, when the adult CPCP treated with the adult stem cells, the increase was about 7 times compared to the control (see Figure 6). Collagen is mainly formed in osteoblasts and plays an important role in bone tissue formation. Thus, it was proved that the osteoblast differentiation agent using MBCP as an active ingredient has a positive effect on bone formation.

     In addition, when the adult stem cells were treated with MBCP, basic phosphatase activity was increased by about 5 times compared to the control group (see FIG. 7). In addition, it was measured that the expression of BSP and OC significantly increased (see FIG. 8). It can be seen that MBCP promotes osteogenic action on the adult stem cells.

In another aspect, the present invention provides a bone differentiation inducing agent containing MBCP or fast dichloromethane fraction as an active ingredient. The composition of the present invention, in addition to the above-described active ingredient can be prepared by including at least one pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include polymeric compounds such as polylactic acid and polyglycolic acid, collagen, haaluronic acid, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and these components One or more components can be mixed and used, and other conventional additives, such as an antioxidant, a buffer solution, and bacteriostatic agents, can be added as needed. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by an appropriate method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA. Osteoblasts differentiated from alveolar bone marrow-derived adult stem cells of the present invention can be used as a cell therapy for bone tissue regeneration lost due to various diseases. Bone loss in the dental field has been mainly caused by periodontal disease, and has been treated through bone graft, but the bone graft can be replaced or supplemented by differentiating adult stem cells into osteoblasts by the method of the present invention. have. In addition, cell therapy can be appropriately used for trauma, accidental fractures, bone defects, and disease-related bone defects.It is also very effective in bone regeneration and transplantation for implantation after extraction with periodontal disease. Can be used.

When the osteoblasts differentiated using the osteodermal differentiation agent of the present invention are applied to the treatment, the number of cells to be administered is about 2 × 10 ⁶ to 2 × 10 ⁷ and is administered once during surgery for bone regeneration. Cell therapy can also be used alone, can be used in combination with one or more other bone therapy, and can be used in conjunction with bone graft during bone regeneration using bone graft material. When used in conjunction with a bone graft, the cells are cultured in an appropriate skeleton to make a bio construct. Skeleton can be used as a synthetic bone product (MBCP, etc.), allogeneic bone (ICB, etc.) or PLGA (innopol) currently available clinically, and gel type atelocollagen, hyaluronic acid, alginate, etc. may be considered. The culture medium is not added at the time of human administration, and the bioconstitution may be administered after being cleanly washed with PBS.

The bone differentiation inducing agent of the present invention can be used alone in the treatment of bone regeneration in periodontal disease and bone defects alone or in combination with methods using surgery, hormone therapy, drug treatment and biological response modifiers.

     As described above, it can be seen that treatment of fast dichloromethane fraction or MBCP in adult stem cells derived from alveolar bone marrow promotes differentiation into osteoblasts. have.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

< Example  1> Alveolar bone marrow-derived adult stem cells ( Alveolar bone marrow - derived adult  stem cell :Below AMASC Culture

0.1 to 3 ml of blood was collected from the patient's alveolar bone marrow, and the blood was mixed with 9 ml of α-MEM containing 20% FBS and antibiotics and treated at 37 ° C., 5% CO ₂ , 95% air and 100% humidity. After incubation for 5 days in a mixed incubator, foreign substances and blood were removed with α-MEM without FBS. The medium was changed at 2 days intervals until sufficient proliferation of the cells was apparent with α-MEM containing 20% FBS. In this example, cells cultured in 3,6,9 passages were used.

Therefore, unlike the conventional method of harvesting adult stem cells from the long bone, the amount of harvest is small when the adult stem cells are separated from the alveolar bone marrow, and a sufficient amount of adult stem cells for bone regeneration can be treated even with a small amount. It was confirmed that autologous adult stem cells could be used.

< Example  2> cell surface antigen ( Cell surface antigen ) And cell morphology measurements

To analyze Cell surface antigens, AMASCs were incubated with 1 × 10 ⁵ cells, then separated with 0.05% trypsin and resuspended in PBS. 50 μl of the supernatant in the cell suspension was reacted with a fluorescent-conjugated anti-CD antibody in the dark for 30 minutes, washed three times with PBS, and washed with a FACS calibur flow cytometer (Beckton Dickinson). , CA, USA). Antibodies used in the present invention are CD-29-FITC (Serotec, Oxford, UK), CD 44-FITC (Serotec, Oxford, UK), and CD 34-PE (Dinona, Seoul, Korea) and the like, and isotype control (control) ) Is an anti-human mouse IgG antibody (Dinona, Seoul, Korea). It was then used to observe the morphology to determine the morphological characteristics of AMASC.

As shown in FIG. 1A, most of the cells responded negatively to CD34, a positive antigen against hematopoietic stem cells, and positive for CD 29 and CD 44, which are positive markers of mesenchymal stem cells. The reaction was shown. In FIG. 1B the cell morphology depicts the fibroblast-like morphology of the spindle shape.

As a result, it was confirmed that the adult stem cells collected and cultured from the alveolar bone were the same cells as the iliac stem adult stem cells.

< Example  3> from fast Fraction  extraction

Extraction of fractions from the fast run was used the method described in Korean Patent Application No. 2004-57670. Sokdan, purchased from Iksan, Korea, was washed, dried, and cleaned. 1.2 kg of fine fasteners were repeatedly extracted with 5 L of methanol for 2 hours, filtered, and the filtrate was concentrated under reduced pressure to obtain 196 g of methanol extract. The resulting methanol extract was dissolved in 60% aqueous methanol solution and partitioned sequentially using n-hexane and dichloromethane. The 60% methanol aqueous solution fraction was concentrated under reduced pressure again, and the obtained residue was dissolved in distilled water and then partitioned using n-butanol. For each solvent fraction, the solvent was removed using a vacuum concentrator, and n-hexane, dichloromethane and n-butanol fractions were obtained in 4.5g, 6.7g and 56.0g, respectively. The dichloromethane fraction of the fraction was used in the treatment of adult stem cells in the present invention because it showed a significant effect in the bone formation test results of human fetal osteoblasts.

< Example  4> Fast Dichloromethane Fraction  According to treatment AMASC Basic phosphatase of Alkalinee Phosphateas , ALP Activity measurement

AMASCs were aliquoted into 1 × 10 ⁵ cells in 6-well plates to differentiate into osteoblasts, and then incubated for 5 days in a mixed incubator at 37 ° C., 5% CO ₂ , 95% air and 100% humidity. The experimental group was treated with 10 µg / ml insulin, 10 mM β-glycerophosphate, 50 µg / ml vitamin C (ascorbic acid) and 10 ng / ml fraction of fast dichloromethane, and 10 µg / ml for the positive control group. Insulin, 10 mM β-glycerophosphate, 50 μg / ml vitamin C, and 100 nM DEX were added to culture 3,6,9 passages. After incubation, the medium was removed, cells were separated by trypsin-EDTA, and then centrifuged at 1,500 rpm for 6 minutes. The supernatant was removed and suspended in an ultrasonic grinder with the addition of 0.2 ml of distilled water. 0.2 ml of 0.1 M glycine NAOH buffer (pH 10.4), 0.1 ml of 15 mM p-nitrophenly phosphate (pNPP, Sigma), 0.1% tryptone X-100 / salin triton X-100 / saline) and 0.1 ml of distilled water were mixed and incubated at 37 ° C. for 30 minutes. The reaction was stopped by adding 0.6 mL of 0.1N NaOH. The cultured cells were transferred to a 96-well plate, and the degree of hydrolysis of p-NPP was confirmed in an ELISA reader of 410 nm wavelength, and p-nitrophenol (p-NP, Sigma) was used as a reference value. Protein concentration was measured using a BCA protein assay reagent (Pierce, USA), and ALP activity was expressed as nM / 30min / mg of protein. This example was repeated three times.

In the analysis of the examples, the mean and standard error were calculated using SPSS version 10.0, and their statistical significance was pretested using one-way ANOVA, and the post test was performed using the Turkey method (P <0.05). .

As shown in FIG. 2 and Table 1, the basic phosphatase activity of the alveolar bone marrow-derived adult stem cell 3 subgroup was 0.037 ± 0.008, DEX 0.353 ± 0.033, and fast dichloromethane fraction 0.317 ± 0.015. In addition, the fast dichloromethane fraction was lower than the DEX administration, but there was no significant difference. The basic phosphatase activity of the sixth passage group was 0.015 ± 0.008, DEX 0.473 ± 0.025, and 0.396 ± 0.015 fast dichloromethane fractions, which was the same as the three passages. 0.05). There was also a significant difference between DEX and fast dichloromethane fractions. Basic phosphatase activity of the 9th passage group was 0.033 ± 0.005, DEX 0.163 ± 0.021, and fast dichloromethane fraction 0.212 ± 0.020. Both groups showed significant increase compared to the control group. Unlike the other passages, the fast dichloromethane fraction showed a tendency to increase basic kinase activity, but there was no statistical significance. In addition, in the 9th passage group, the basic kinase activity was decreased as compared with the 3rd and 6th passage group, and in particular, it was confirmed that the osteogenic differentiation ability of DEX was greatly decreased.

Therefore, the result of the measurement of basic phosphatase activity showed that even if the fast dichloromethane fraction, which is a herbal preparation, did not reach DEX, the adult stem cells differentiated into osteoblasts.

Control DEX PR 3rd generation 0.037 ± 0.008 0.353 ± 0.033 * 0.317 ± 0.022 * 6th Fleet 0.051 ± 0.008 0.473 ± 0.025 * 0.396 ± 0.015 * # 9th Fleet 0.033 ± 0.005 0.163 ± 0.021 * 0.212 ± 0.020 *

< Example  5> fast Dichloromethane Fraction  According to treatment AMASC Accumulation of Calcium

Six-well plates were aliquoted into 1 × 10 ⁵ cells and cultured. Experimental group and positive control group were added and cultured under the same conditions as in Example 3. 4mM / L NaHPO ₄ was added on day 23 to induce mineralization of the extracellular matrix, and on day 25 the medium was removed and washed with PBS. Fix with ice-cold 70% ethanol at 4 ° C. for 1 hour, remove ethanol and stain with 40 mM / L alizarin red-sulfate (AR-S, Sigma) for 10 minutes at room temperature. To visually compare and observe the stained area, photographed with a digital camera (Nikin E995, Micon Corporation, Japan), and then quantitatively compare it with 10% (10 mM / L sodium phosphate, pH 7.0). w / v) The staining site was dissolved using a solution containing cetylpyridinium chloride, and the concentration of AR-S was read at an absorbance of 562 nm, and the same AR-S standard curve was used for the same solution. It was. This example was repeated three times.

In the analysis of the Examples, SPSS 10.0 was used to calculate the mean and standard error, and their statistical significance was pretested using one-way ANOVA, and the post test was Turkey (P <0.05).

As shown in FIG. 3A, both DEX and fast dichloromethane fractions showed a markedly increased calcification nodule compared to the control. Fast dichloromethane fractions did not reach DEX during the entire incubation period, but it was found to cause a lot of calcium accumulation. In the 3,6,9 subgroups, nodule formation was apparent in both DEX and fast dichloromethane fractions. When the fast dichloromethane fractions were treated, nodules were no larger than DEX, but nodules were formed by the same mechanism as DEX. In the sixth subgroup, no large nodules were seen as in the third subgroup, but both groups showed very dark staining and numerous sintered sections. In other words, the increased passage did not show very active calcium accumulation as in the third passage, but if the incubation period was longer in the sixth passage, it would be possible to form a large nodule as in the third passage. . In the 9th passage group, the calcium formation ability was slightly decreased as compared with the 6th passage group. The DEX treatment resulted in more calcium accumulation formation than the fast dichloromethane fraction. As shown in Figure 3b and Table 2, the calcium accumulation of the third generation of alveolar bone marrow-derived adult stem cell group was 114.225 ± 6.275, DEX 833.630 ± 21.615, fast dichloromethane fraction 718.315 ± 17.915, respectively, about 7-fold and about the control group. 6-fold increase. Calcium accumulation in the sixth subgroup was increased by 7 times compared with the control group, 84.310 ± 14.990, DEX 671.175 ± 28.175, and fast dichloromethane fraction 631.095 ± 17.915. Calcium accumulation in 9 passages was increased by 5 and 3 times, respectively, to the control group 112.560 ± 12.560, DEX 589.360 ± 11.940, and fast dichloromethane fraction 449.100 ± 27.100. In this experiment, both DEX and fast dichloromethane fractions significantly increased significantly.

Therefore, the addition of fast dichloromethane fraction to alveolar bone-derived adult stem cells was found to have the same mechanism as DEX, a traditional bone differentiation inducer, and used as a bone tissue regeneration agent that had an effect on bone nodule formation in bone regeneration. I think there is a possibility.

Control DEX PR 3rd generation 114.225 ± 6.275 833.630 ± 21.615 ＊ 718.315 ± 17.915 ＊ 6th Fleet 84.310 ± 14.990 671.175 ± 28.175 ＊ 631.095 ± 30.665 ＊ 9th Fleet 112.560 ± 12.560 589.360 ± 11.940 449.100 ± 27.100 ＊ #

< Example  6> Fast Dichloromethane Fraction  According to treatment AMASC of Weston Blot

The experimental group and the positive control group were cultured under the same conditions as in Example 3. AMASCs differentiated for 25 days were separated by 0.25% trypsin-EDTA. The separated AMASC was washed with PBS, followed by lysis buffer [10 mM Na ₂ HPO ₄ (pH 7.2), 0.9% NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 0.2% sodium azide, 0.004% sodium fluoride] was extracted, and the protein concentration was measured by mixing 50: 1 copper (II) sulfate (Sigma) with BCA solution (Sigma). 15% SDS-polyacrylamide gel electrophoresis was performed using 50 μg of protein extracted from the control and experimental groups, followed by transfer to PVDF membrane (Millipore Corp., Bedford, MA, USA). PVDF membranes were treated with each blocking solution (Zymed, USA) for 1 hour at room temperature to prevent binding of nonspecific antibodies. Subsequently, the cells were reacted with primary antibodies of Osteocalcin (Biogenesis, Kingston, NH, USA) and Bone Sialoprotein (BSP) (Chemicon, Temecula, CA, USA) for 90 minutes. After reacting with the primary antibody, the cells were washed once with PBS and treated with anti-mouse or anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60 minutes. It was then washed twice with PBS for 7 minutes, and reacted with ECL kit (Amersham Biosciences, Buckinghamshir, UK) for 1 minute and exposed to Hyperfilm-MP (Amersham Biosciences, Bukinghamshir, UK).

As shown in FIG. 4, BSP and OC, which are representative expression proteins of bone differentiation, were increased when DEX and fast dichloromethane fractions were administered over the entire passage group. In the third passage, the expression levels of DEX and fast dichloromethane fractions were similar and increased. In the sixth passage, the expression of BSP and OC was slightly increased in the DEX treatment than in the fast dichloromethane fraction. In the 9th passage, unlike the 3 and 6 passages, the expression of the fast dichloromethane fraction decreased significantly compared with the DEX treatment. The reduction of OC and BSP expressions compared to DEX by the fast dichloromethane fraction in the 9th passage was similar to the case where calcium accumulation was significantly decreased in the 9th passage compared to the 3,6 passage.

Therefore, it was confirmed that the fast dichloromethane fraction influences the late stage of the differentiation of alveolar bone-derived adult stem cells into osteoblasts.

< Example  7> MBCP  According to treatment AMASC Cell proliferation

The cultured AMASC was separated by 0.25% trypsin-EDTA and suspended in the culture solution. The experimental group was divided into 1 × 10 ⁴ cells on the MBCP surface of 10 mm × 5 mm, and the control group secured the same space as the experimental group after MBCP. Dissociated to 1 × 10 ⁴ cells in the absence. After incubation in a mixed incubator at 37 ° C., 5% CO ₂ , 95% air and 100% humidity for 24 hours to allow cells to attach, unattached cells were removed with medium. After incubation for 1, 3 and 5 days, the number of cells was calculated using a hemocytometer and repeated three times.

In the analysis of the Examples, SPSS 10.0 was used to calculate the mean and standard error, and their statistical significance was pretested using one-way ANOVA, and the post test was Turkey (P <0.05).

As shown in FIG. 5 and Table 3, no significant difference was observed in cell proliferation between the experimental group and the control group in all 1, 3, and 5 day groups. Cell proliferation was observed in the culture for 5 days, but it was difficult to be attributed to MBCP.

Therefore, MBCP did not have any cytotoxicity.

1 day 3 days 5 days Control 1.20 ± 0.06 2.58 ± 0.12 4.30 ± 0.13 MBCP 1.11 ± 0.04 2.68 ± 0.17 4.65 ± 0.08

< Example  8> MBCP  According to treatment AMASC Of collagen synthesis

Group is then dispensed, and the free space, such as, the test group the control group to get the 1 × 10 ⁶ cells in the MBCP of 10mm × 5mm and then dispensed to a 1 × 10 ⁶ cells in the absence MBCP respectively 20% FBS 50 μg / ml vitamin C and 10 mM sodium β-glycerophosphate-containing α-MEM were incubated at two-day intervals. After culturing for 5 to 7 days, only the cells were collected from the medium and centrifuged at 1,500 rpm for 5 minutes to remove dead cells, and then 3 ml of 10N HCl was added, and each cell was separated by trypsin-EDTA and centrifuged. After separation, the supernatant was removed and 3 ml 6N HCl was added. Each sample was filtered after hydrolysis at 110 ° C. for 10-24 hours. Samples were divided into two and 100 μl was added. After drying completely, 50 µl of methanol was added to react at 60 ° C until the remaining hydrochloric acid was removed. 1.2ml 50% isopropanol was added to dissolve the remaining precipitate, followed by reaction for 10 minutes by mixing with 200µl chloramine-T solution. 1.2 ml Ehrlich reaction reagent was added to the mixture, and then incubated at 50 ° C. for 90 minutes, and then cooled at room temperature. Collagen synthesis measurements were measured by spectrophotometer (Spectrophotometer, Beckman, DU-550, USA) at 558 nm using a standard curve as a standard indicator. This example was repeated three times.

 In the analysis of the Examples, SPSS 10.0 was used to calculate the mean and standard error, and their statistical significance was pretested using one-way ANOVA, and the post test was Turkey (P <0.05).

As shown in Figure 6 and Table 4, the control group 0.300 ± 0.018, experimental group 2.300 ± 0.247 in the 5 days group treated with MBCP significantly increased collagen synthesis in the experimental group about 7 times, 0.446 ± 0.019, In experimental group 3.330 ± 0.257, collagen synthesis was also increased by about 7 times.

Therefore, it can be seen that MBCP promotes collagen synthesis of adult stem cells and promotes differentiation into osteoblasts.

Control MBCP 5 days 0.300 ± 0.018 2.300 ± 0.247 ＊ 7 days 0.446 ± 0.019 3.330 ± 0.257 ＊

< Example  9> MBCP  According to treatment AMASC Of basic phosphatase activity

The experimental group divided AMASC into 1 × 10 ⁶ in 10 mm × 5 mm MBCP, and the control group divided the cells into 1 × 10 ⁶ cells without MBCP after 20% FBS, 50 μg / ml vitamin C, α-MEM containing 10 mM sodium β-glycerophosphate was mixed and incubated in a mixed incubator at 37 ° C., 5% CO ₂ , 95% air and 100% humidity. After incubation for 5 to 7 days, the medium was removed, cells were separated with trypsin-EDTA, and then centrifuged at 1,500 rpm for 6 minutes. The supernatant was removed, and 0.2 ml of distilled water was added and suspended by an ultrasonic mill. 0.2 ml of 0.1 M glycine NAOH buffer (pH 10.4), 0.1 ml of 15 mM p-nitrophenly phosphate (pNPP, Sigma) in 0.1 ml of each cell suspension, 0.1% tryptone X-100 / salin 100 / saline) and 0.1ml of distilled water were mixed and incubated at 37 ° C for 30 minutes. The reaction was stopped by adding 0.6 ml of 0.1N NaOH. The cultured cells were transferred to a 96-well plate, and the degree of hydrolysis of p-NPP was confirmed in an ELISA reader having a wavelength of 410 nm, and p-nitrophenol (p-NP, Sigma) was used as a reference value. Protein concentration was measured using a BCA protein assay reagent (Pierce, USA), and basic kinase activity was expressed as nM / 30min / mg of protein. This example was repeated three times.

In the analysis of the Examples, SPSS 10.0 was used to calculate the mean and standard error, and their statistical significance was pretested using one-way ANOVA, and the post test was Turkey (P <0.05).

As shown in FIG. 7 and Table 5, the control group 0.040 ± 0.009, the experimental group 0.198 ± 0.028 of the MBCP treated 5 days increased by about 5 times, even in the 7 days group 0.034 ± 0.012, experimental group 0.189 ± 0.019 about 5 times To some extent, basic phosphatase activity was measured.

Therefore, it was found that adult stem cells derived from alveolar bone marrow were differentiated into osteoblasts when MBCP was treated.

Control MBCP 5 days 0.040 ± 0.009 0.198 ± 0.028 ＊ 7 days 0.034 ± 0.012 0.189 ± 0.019 ＊

< Example  10> MBCP  According to treatment AMASC of Weston Blot

The experimental group was used to separate AMASC cultured on the surface of MBCP for 25 days with 0.25% trypsin-EDTA, and the control group was used to separate AMASC cultured in the environment without MBCP. The separated AMASC was washed with PBS, followed by lysis buffer [10 mM Na ₂ HPO ₄ (pH 7.2), 0.9% NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 0.2% sodium azide , 0.004% sodium fluoride] was extracted, and the protein concentration was measured by mixing 50: 1 copper (II) sulfate (Sigma) with BCA solution (Sigma). 15% SDS-polyacrylamide gel electrophoresis was performed using 50 μg of protein extracted from the control and experimental groups, followed by transfer to PVDF membrane (Millipore Corp., Bedford, MA, USA). PVDF membranes were treated with each blocking solution (Zymed, USA) for 1 hour at room temperature to prevent binding of nonspecific antibodies. Subsequently, the primary antibody of osteocalcin (Biogenesis, Kingston, NH, USA) and Bone Sialoprotein (BSP) (Chemicon, Temecula, CA, USA) was reacted for 90 minutes. After reacting with the primary antibody, the cells were washed once with PBS and treated with anti-mouse or anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 60 minutes. It was then washed twice with PBS for 7 minutes, and reacted with ECL kit (Amersham Biosciences, Buckinghamshir, UK) for 1 minute and exposed to Hyperfilm-MP (Amersham Biosciences, Bukinghamshir, UK).

As shown in FIG. 8, OC and BSP expression tended to increase in the experimental group to which MBCP was added compared to the control group.

Therefore, it was confirmed that MBCP influences the late stage of osteoblast differentiation.

As described above, the method of extracting adult stem cells derived from alveolar bone of the present invention and treating them with differentiation dichloromethane fractions or MBCP as osteoinduction agents to differentiate into osteoblasts is to collect a small amount of adult stem from the alveolar bone marrow. It is possible to cultivate a large amount of osteoblasts with the cells, and has the effect of significantly promoting the differentiation process by overcoming the cytotoxicity and side effects of the immune system, and the osteoblasts differentiated by the above method are used to treat periodontal disease and trauma and Cell therapy for damaged bone tissue regeneration in case of accidental fractures and bone defects due to disease can be used safely and usefully in the clinic.

Claims (8)

       Iii) separating adult stem cells from the bone marrow of the alveolar bone;        Ii) culturing the stem cells; And        Iii) treating said stem cells with at least one osteoinduction agent selected from the group consisting of fast dichloromethane fractions and micro-macro biphasic calcium phosphate (MBCP);        Method for differentiating adult stem cells derived from alveolar bone into osteoblasts. delete           The method of claim 1 wherein the fast dichloromethane fraction is treated at a concentration of 0.5-100 ng / ml.         The method of claim 1, wherein the adult stem cells are cultured in MBCP, an osteoinduction agent, differentiation of alveolar bone-derived adult stem cells into osteoblasts. Osteoblast differentiation agent containing MBCP as an active ingredient. Osteodifferentiation inducer comprising a fast dichloromethane fraction as an active ingredient. Osteoblast differentiation inducing agent comprising MBCP as an active ingredient. Osteoblast differentiation inducer comprising a fast dichloromethane fraction as an active ingredient.

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