JP5885206B2 - Bone formation promoter containing glycogen - Google Patents

Description

  The present invention relates to an osteogenic agent, an osteogenesis promoting agent, a tooth germ growth promoting agent, a root formation promoting agent, a periodontal tissue regeneration promoting agent and the like containing glycogen as an active ingredient. The present invention relates to an osteogenesis agent, an osteogenesis promoter, tooth germ development promotion, tooth root formation promotion, periodontal tissue regeneration promotion, and the like, and relates to a novel use of glycogen.

  Bone tissue regeneration therapy currently performed in regenerative medicine includes a method of transplanting autologous bone or a bone substitute material, a method of promoting bone formation by locally administering a factor such as cytokine. The former is a general method as a bone augmentation method. However, there is a problem that the invasion to the body accompanying bone collection is large and the amount of bone that can be collected is limited. Therefore, in recent years, the method of locally administering the latter exogenous factor has also been widely used. In particular, as a cytokine therapy, platelet-derived growth factor (PDGF), bone morphogenetic protein (BMP) And basic fibroblast growth factor (bFGF) have attracted attention. BMP is a proteinous factor that differentiates undifferentiated mesenchymal cells into osteoblasts. In the United States, recombinant proteins for clinical application have been produced and commercialized. In addition, bFGF has a potent angiogenic action and proliferation-inducing ability of mesenchymal cells, promotes the production of extracellular matrix and has a bone-increasing action. Therefore, it is expected to be clinically applied as a fracture healing promoter and periodontal tissue regenerative drug. Has been. However, each factor requires a large amount for actual bone defect treatment, but the safety problem of the gene transfer method that is effective for mass production has not been completely cleared at present. Also, in terms of cost, there are many problems when widely used in general medical and dental clinics. Accordingly, there has been a demand for the development of an osteogenesis promoter that can be mass-produced and can be provided to clinical sites at low cost and has high safety.

Glycogen is the main storage polysaccharide in animals, fungi, yeasts and bacteria. Glycogen is soluble in water and becomes a milky white solution. The molecular structure of animal glycogen is well studied. Natural glycogen is branched by α-1,6-glucoside bonds from sugar chains that are linearly linked via α-1,4-glucoside bonds of glucose (glucose), and is also branched into a network. It is a homoglucan that forms a structure. Natural glycogen is composed of α-1,4-glucoside-linked chains having an average degree of polymerization of about 10 to about 14 linked by α-1,6-glucoside bonds. There are various theories about the molecular weight of natural glycogen, but it is about 10 ⁵ to about 10 ⁸ . Natural glycogen exists as particles having a molecular weight of about 10 ⁷ (β particles) or larger particles (α particles) formed by aggregation of β particles. The structure of bacterial glycogen is thought to be similar to that of animal glycogen. A certain kind of plant (for example, sweet corn) also has a glucan having a structure similar to glycogen, and is called plant glycogen (phytoglycogen).

  In Non-Patent Document 1, it is reported that glycogen is present in osteoblast progenitor cells, and glycogen disappears when differentiated into osteoblasts. However, it is not known that administration of glycogen can contribute to bone formation.

  Non-Patent Document 1 and Non-Patent Document 2 describe that a glycogen positive site and an alkaline phosphatase (differentiation marker for osteoblast) positive site almost coincide. In vivo, it has been suggested that "the appearance of cells containing glycogen may be involved in bone formation". However, there is no description of the event when glycogen is applied externally.

  Non-Patent Document 3 describes that glycogen of 5000 to 6500 kDa activates immune cells. However, it does not describe promoting proliferation. However, there is no description regarding osteoblasts.

  Patent Document 1 discloses a fibroblast proliferating agent and an external preparation for skin containing the same, and describes that glycogen has a fibroblast proliferating effect. Only what is used as a coating agent is described. However, there is no description regarding osteoblasts.

  Patent Document 2 discloses an epidermal cell activator and an ATP production promoter, and discloses that glycogen promotes ATP production of epidermal cells. However, there is no description regarding osteoblasts.

  As described above, glycogen is a polysaccharide consisting of glucose, stored in the liver and muscles of animals, and plays an important role as an energy source for cells. Recently, we have succeeded in developing a production method using an enzyme capable of obtaining a large amount of glycogen having high purity and high structural stability (Non-patent Document 4 and Patent Document 3). Analysis of bioactive effects is ongoing. As a result, enzyme-synthesized glycogen has almost the same chemical structure and physical properties as natural glycogen, but is less susceptible to degradation by α-amylase (Non-Patent Document 5), and further has an immunostimulatory function (Non-Patent Document 3) and moisturizing. It is becoming clear that there exists an effect | action (patent document 4). However, there are no reports on bone formation.

Japanese Unexamined Patent Publication No. 11-255657 Japanese Unexamined Patent Publication No. 2003-321373 Japanese Patent No. 4086312 Japanese Unexamined Patent Publication No. 2009-227632

Hayato Oshima “Functional significance of glycogen in tooth development” Journal of Niigata Dental Society 29 (2) 185-186: 1999 Yoda et al. “Localization of glycogen and glucose transporters during mouse tooth germ development” The 51st Annual Meeting of the Dental Society of Japan 2009 (Niigata) R. Kakutani, et al. (2007) Carbohydr Res 342, 2371-2379. H. Kajiura, et al. (2008) Biocatal. Biotransform. 26, 133-140. H. Takata, et al. (2009) Carbohydr. Res. 344, 654-659.

  The present invention finds that a specific type of glycogen promotes bone formation, and provides a novel bone formation agent, bone formation promoter, tooth germ growth promoter, tooth root formation promoter, and periodontal tissue regeneration promoter. Completed.

  The present invention solves the problem of side effects of active ingredients relating to therapeutic agents or preventive agents for diseases that require bone formation or bone formation proliferation such as osteoporosis currently on the market, and has an effective osteogenesis promoting effect, To provide a safe compound or composition with reduced side effects and to provide a biocompatible material to promote bone formation.

Accordingly, the present invention provides the following.
(1) A composition for at least one selected from the group consisting of osteogenesis containing glycogen having a weight average molecular weight of less than about 8700 kDa, promotion of osteogenesis, promotion of tooth germ development, promotion of root formation, and promotion of periodontal tissue regeneration object.
(2) The composition according to item 1, wherein the weight average molecular weight is about 8000 kDa or less.
(2A) The composition according to item 1 or 2, wherein the weight average molecular weight is about 5000 kDa or less.
(3) The composition according to item 1, 2 or 2A, wherein the weight average molecular weight is about 3000 kDa or more and about 8000 kDa or less.
(3A) The composition according to item 1, 2, 2A or 3, wherein the weight average molecular weight is about 3000 kDa or more and about 5000 kDa or less.
(4) The composition according to any one of items 1, 2, 2A, 3 or 3A, wherein the weight average molecular weight is about 4900 to about 7000 kDa.
(4A) The composition according to any one of Items 1, 2, 2A, 3, 3A or 4, wherein the weight average molecular weight is about 4900 to about 5000 kDa.
(5) The composition according to any one of Items 1, 2, 2A, 3, 3A, 4 or 4A, wherein the glycogen includes enzyme-synthesized glycogen (ESG).
(6) The glycogen is ESG-5M (enzymatic synthetic glycogen having a weight average molecular weight of about 5200 kDa), ESG-3M (enzymatic synthetic glycogen having a weight average molecular weight of about 3200 kDa), ESGB (enzymatic synthetic glycogen having a weight average molecular weight of about 5000 kDa), 6. The composition according to any one of items 1, 2, 2A, 3, 3A, 4, 4A or 5, comprising at least one selected from the group consisting of enzymatically synthesized glycogen and mussel glycogen having a weight average degree of polymerization of about 7000 kDa.
(7) The composition according to any one of items 1, 2, 2A, 3, 3A, 4, 4A, 5 or 6, wherein the bone formation or bone formation promotion is achieved by promoting osteoblast proliferation. object.
(8) The composition according to any one of items 1, 2, 2A, 3, 3A, 4, 4A, 5, 6 or 7, wherein the composition is a food, an additive, a medical device, or a medicine.
(9) for at least one selected from the group consisting of bone formation, bone formation promotion, tooth germ development promotion, tooth root formation promotion, and periodontal tissue regeneration promotion, comprising glycogen having a weight average molecular weight of less than about 8700 kDa Medicine.
(10) The medicament according to item 9, further comprising a pharmaceutically acceptable excipient.
(11) The medicament according to item 9 or 10, further comprising a drug for bone formation.
(12) The medicament according to any one of items 9 to 11, wherein the medicament is administered for 1 to 10 days.
(13) for at least one selected from the group consisting of bone formation, osteogenesis promotion, tooth germ development promotion, tooth root formation promotion, and periodontal tissue regeneration promotion, comprising glycogen having a weight average molecular weight of less than about 8700 kDa Medical device.
(14) The medical device according to item 13, which is a bone cement, a stent, a catheter, a bone filling material or an implant.
(15) A method for producing a medicament for at least one selected from the group consisting of bone formation, bone formation promotion, tooth germ development promotion, tooth root formation promotion, and periodontal tissue regeneration promotion, wherein the weight average molecular weight is Mixing a glycogen that is less than about 8700 kDa with a pharmaceutically acceptable excipient.
(16) A method for producing a medical device for at least one selected from the group consisting of bone formation, bone formation promotion, tooth germ development promotion, tooth root formation promotion, and periodontal tissue regeneration promotion, the weight average molecular weight Including the step of including a glycogen having a molecular weight of less than about 8700 kDa in the medical device or the medical device material.
(17) At least one selected from the group consisting of forming bone, promoting bone formation, promoting tooth germ development, promoting tooth root formation, and promoting periodontal tissue regeneration. A method wherein a glycogen having a weight average molecular weight less than about 8700 kDa is delivered to a site in need of bone formation or promotion of bone formation to a patient in need of bone formation or promotion of bone formation. Administering the step of administering.
(18) To produce a food, additive, medical device or medicament for at least one selected from the group consisting of bone formation, bone formation promotion, tooth germ development promotion, tooth root formation promotion, and periodontal tissue regeneration promotion Of glycogen having a weight average molecular weight of less than about 8700 kDa.
(19) A glycogen having a weight average molecular weight of less than about 8700 kDa for at least one selected from the group consisting of bone formation, bone formation promotion, tooth germ development promotion, tooth root formation promotion, and periodontal tissue regeneration promotion.
(20) The glycogen according to item 19, wherein the glycogen is provided in the form of a food, an additive, a medical device or a medicine.

  In all these aspects, it is understood that each embodiment described herein (eg, molecular weight, etc.) can be applied in other aspects, as long as applicable.

  While multiple embodiments are disclosed, still other embodiments of the invention will be apparent to those skilled in the art from the following detailed description. As will be apparent to those skilled in the art, the present invention can be modified in various obvious aspects, all without departing from the spirit and scope of the present invention. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not as restrictive.

  The present invention has demonstrated that certain glycogens are useful for bone formation. The finding that such a material compatible with a living body is useful for bone formation is expected to be applied in the pharmaceutical industry and can achieve an effect not found in the prior art.

FIG. 1 is a graph showing a growth curve of osteoblasts (MC3T3-E1) after glycogen addition. Black squares indicate glycogen (ESM-5M), and diamonds indicate controls. * Indicates statistical significance (p <0.05). It turns out that the glycogen addition group has increased significantly compared with control by the 5th day. The concentration of glycogen used is 300 μg / ml. The result of an osteoblast (MC3T3-E1) proliferation promotion effect test of various glycogen is shown. Cell growth was observed over time using various types of glycogen (300 μg / ml) or control, and the cell number was recorded. The left group shows the first day and the right group shows the third day. In each group, control (no pattern), ESG-B (gray), ESG-3M (dashed pattern), ESG-20M (check pattern), and natural glycogen (NSG (Corn), black) are shown from the left. FIG. 3 shows the MC3T3-E1 cell proliferation test. Cell growth was observed over time using various glycogens (500 μg / ml) or controls, and the number of cells was recorded. For NSG (mussel), statistical significance (p <0.05) was also observed. Each group shows the results on the 3rd, 5th, 7th and 10th days from the left. In each group, control (black), ESG-B (dashed line pattern), ESG-20M (check), natural glycogen (mussel; diagonal line), and natural glycogen (rabbit; white) are shown from the left. It is the result of having analyzed the ALP and OC gene expression change of MC3T3-E1 by enzyme synthesis glycogen addition by RT-PCR method. 2% agarose gel electrophoresis, ethidium bromide stained image. Increased expression of ALP mRNA and OC mRNA is observed in the glycogen added group. The concentration of glycogen used is 300 μg / ml. Each lane has no glycogen stimulation on the first day (1 day) from the left (-), glycogen stimulation on the first day (+; 300 μg / ml), no glycogen stimulation on the third day (-), 3 Glycogen stimulation on day (+; 300 μg / ml), no glycogen stimulation on day 5 (−), glycogen stimulation on day 5 (+; 300 μg / ml), glycogen stimulation on day 7 None (−) and glycogen stimulation on day 7 (+; 300 μg / ml). It is a figure which shows the mineralization formation of MC3T3-E1 by enzyme synthesis glycogen addition. The left shows a non-addition group, and the right shows an addition group. In the Kossa-stained image, the dark brown part indicates calcium deposition (arrow). The bar represents 250 μm. Calcium deposition is confirmed only in the glycogen addition group on the 7th day of culture. The concentration of glycogen used is 300 μg / ml. FIG. 6 is a photograph showing the effect of enzyme-synthesized glycogen on bone regeneration: an in vivo experiment. A hole was made in the skull of a mouse, and it was examined whether or not bone regeneration promotion occurred by injecting ESG and applying CT-Scan several days later. The left shows control, and the right shows addition of glycogen (ESG 070223-5M). The arrow indicates the location where glycogen was added. Significant promotion of bone regeneration is observed when glycogen is added. The concentration of glycogen used is 300 μg / ml. It is a photograph which shows the influence on tooth-germ development by the enzyme synthesis glycogen addition in an organ culture system. Mandibular first and second molar tooth germs were collected from mouse fetuses at 16.5 days of gestation and cultured in vitro. Enzyme-synthesized glycogen (5000 kDa) was added to 500 μg / ml and cultured for 4 days, and then the state of tooth germ development was observed. The addition of glycogen clearly promoted growth, and large and well-differentiated tooth germs were formed.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Thus, it should be understood that singular articles (eg, “a”, “an”, “the”, etc. in the case of English) also include the plural concept unless otherwise stated. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the above field unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition)
In the present specification, “glycogen” is a sugar having D-glucose as a structural unit, which is linked only by α-1,4-glucoside bond and α-1,6-glucoside bond, and has a molecular weight. The thing which is 1 million Da or more. In particular, the product used in the present invention has a Mw of 500,000 Da when a product obtained by allowing pullulanase of 50 U / g substrate to act under the following conditions (see Japanese Patent No. 4086312) by the MALLS method. Mw is 500,000 Da or more when the product obtained when the α-amylase of 300 U / g substrate is allowed to act under the following conditions (see Japanese Patent No. 4086312) is analyzed by the MALLS method. Say sugar.

  Test conditions of the test method with pullulanase: The activity of pullulanase is expressed as 1 U in an amount necessary to generate a reducing power corresponding to 1 μmol of glucose per minute using pullulan as a substrate. As the pullulanase, for example, the enzyme agent derived from Bacillusbrevis manufactured by Daiwa Kasei Co., Ltd., or the crystallase PL45, the pullulanase derived from Bacillus acidopullulyticus manufactured by SigmaAldrich, or the pullulanase derived from Klebilla pneumoniae manufactured by the same company is used. it can. As the reaction temperature and reaction pH, a temperature and pH most suitable for the reaction of each enzyme are selected.

  Test conditions of the test method using α-amylase: α-amylase includes α-amylase derived from human salivary gland (eg, Type XIII-A manufactured by Sigma Aldrich) and α-amylase derived from porcine pancreas (eg, Type manufactured by Sigma Aldrich). IA can be used, 1U of α-amylase activity is defined as the amount of enzyme that liberates 1.0 mg maltose in 3 minutes using starch as a substrate at 20 ° C. and pH 6.9.

  In the present specification, “weight average molecular weight (Mw)” and number average molecular weight (Mn) are explained as follows. That is, a polymer such as glycogen is generally not a uniform molecule but a mixture of molecules of various sizes, and its molecular weight is evaluated by a number average molecular weight (Mn) or a weight average molecular weight (Mw). Mn is the total mass of the system divided by the number of molecules contained in the system. In other words, it is an average by a fraction. On the other hand, Mw is an average by weight fraction. If it is a completely uniform substance, Mw = Mn. However, since a polymer generally has a molecular weight distribution, Mw> Mn. Therefore, the greater the Mw / Mn is, the greater the molecular weight non-uniformity (the molecular weight distribution is wider). In the present invention, the weight average molecular weight is mainly used for glycogen.

  The glycogen used in the present invention is known to be stored in the liver or skeletal muscle of animals, and is not particularly limited. The origin is exemplified by animals, but it is also known that plants or microorganisms produce glycogen or a substance having a structure similar to glycogen, and these can all be used as glycogen of the present invention. Furthermore, glycogen used in the present invention can be synthesized using an enzyme. For example, sucrose phosphorylase (EC 2.4.1.7), α-glucan phosphorylase (EC 2.4.1.1), and branching enzyme (EC 2.4.1.18) are added to sugar and primer molecules (malto-oligo and the like and dextrin). ) And a method of allowing a branching enzyme (EC 2.4.1.18) to act on short-chain amylose. Glycogen synthesized by these methods is also known to have the same chemical structure and physical structure as natural glycogen, and these enzymatically synthesized glycogens can also be used in the stabilization method of the present invention. In addition to using natural or synthetic glycogen, derivatives thereof or equivalents thereof may be used. Such derivatives or equivalents can be produced, for example, by the following method.

  The glycogen of the present invention can be separated into fractions having an appropriate molecular weight distribution by separation methods well known to those skilled in the art, for example, chromatographic separation (for example, gel filtration chromatography, HPLC), membrane separation, and the like. In addition, a method such as precipitation using a solvent (for example, methanol or ethanol) may be used alone or in combination to separate into fractions having an appropriate molecular weight distribution. Natural or synthetic glycogen obtained by etherification, esterification, cross-linking and grafting by methods well known to those skilled in the art can be used. As a derivatization method, a method usually used for modification of starch can be used (Biochemical Experimental Method 19, “Starch / Related Sugar Experimental Method”: Nakamura et al., Academic Publishing Center, pp. 273-303, 1986. ). As an example of phosphorylation, phosphorylated glycogen can be obtained by reacting the glycogen of the present invention with phosphorus oxychloride in dimethylformamide.

  As used herein, “bone formation” refers to the formation of new bone by osteoblasts. Bone formation can be actually observed with the naked eye, confirmed by X-ray photography or the like, and can be evaluated based on an index. As an index of bone formation, increase in expression of alkaline phosphatase, promotion of osteoblast proliferation, mineralization, and the like can be mentioned. It is understood that “bone formation” in the present specification includes not only bone formation but also tooth formation and the like, as normally understood by those skilled in the art.

  As used herein, “promoting osteogenesis” includes both making a state without bone formation a certain state and accelerating the existing bone formation state.

In addition to those exemplified in the examples described later, the activity for promoting bone formation is 0.1 to 10.0 μM in the medium concentration in the cultured 2.0 × 10 ⁴ cells / well osteoblast cell line MC3T3-E1. Thus, the compound was added and cultured for 4 days, and alkaline phosphatase (ALP) activity (Japanese Patent Laid-Open No. 2003-155236), which is a differentiation index of osteoblasts, and the amount of DNA (C. Labarca and K.) as an index of cell number. Paigen, Analytical Biochemistry 102: 344-352, 1980) and measuring the ALP activity relative to the amount of DNA. A significantly higher ALP activity compared to the control indicates a higher osteogenesis promoting activity of the compound.

  Alternatively, the osteogenesis promoting activity can be determined for 4 weeks by administering a gastric sonde to a laboratory animal such as a rat (including a disease model animal) or feeding a test feed in addition to the calcification experiment described in the Examples. The animals are reared as described above and can be determined by measuring changes in bone components of the diaphysis and metaphyseal tissue of the animal's femur using the calcium content, alkaline phosphatase activity, and DNA content as indicators. The amount of calcium in the bone tissue was determined by washing the bone tissue with a 0.25M sucrose solution and drying at 100 ° C. for 6 hours, measuring the dry weight, adding concentrated nitric acid and decomposing at 120 ° C. for 24 hours. The amount is quantified with an atomic absorption photometer. Alkaline phosphatase activity was determined by washing bone tissue with a cold 0.25M sucrose solution, crushing in 6.5 mM barbital buffer (pH 7.4), sonicating for 60 seconds, centrifuging at 3000 rpm, and supernatant fraction. Can be measured by a known method (Methods of Enzymatic Analysis, Vol. 1-2, Academic Press, New York, pp 856-860, 1965). The amount of DNA was determined by washing bone tissue with cold 0.25M sucrose solution, removing water, crushing in 0.1N sodium hydroxide solution, extracting, centrifuging at 3000 rpm, and measuring the supernatant fraction with DNA The sample can be measured by a known method (J. Biol. Chem. 214: 39-77, 1955).

  As used herein, “tooth germ” refers to a cell population that is the basis of teeth and periodontal tissues, and includes enamel organs, tooth papilla, and dental follicles. Therefore, in this specification, “promoting tooth germ development” means that tooth germ development is promoted. In addition to those exemplified in the examples described later, (1) tooth germ development is fast (the whole tooth germ The developmental stage is progressing, the individual differentiation of the tooth germ epithelium and mesenchymal cells is progressing), and (2) whether the size of the tooth germ is large can be evaluated as an index. It is judged whether or not the development of the tooth germ is earlier than normal, and it can be said that the development stage of the whole tooth germ is advanced as early as possible, and the individual differentiation of the tooth germ epithelium and mesenchymal cells is advanced. It can be judged by judging whether the size of the tooth germ corresponds to the promotion of tooth germ development. Whether or not the size of the tooth germ is large can be used as a judgment index based on whether or not it is larger than the average value of the sizes of the respective tooth types. For the average tooth size, refer to, for example, Kotaro Fujita, 22nd Edition, Anatomy of teeth (ISBN4-307-45007-8). In the tooth germ organ culture system, a case where the tooth size is large compared to the size of the tooth germ of the group not added with glycogen simultaneously cultured can be referred to as “promoting tooth germ development”. For tooth germ development, for example, Antonio Nanci edited by 7th edition, TenCate's Oral Histology: Development, Structure, and Function (ISBN4-263-45444-8) can be referred to. Strictly speaking, tooth germ growth promotion includes bone formation or actions that are not included in the bone formation action. However, when considering the action in vivo, the tooth germ is synchronized with the surrounding alveolar bone. Since it grows up, it can be said to be substantially included in the promotion of bone formation.

  In this specification, “periodontal tissue” is a general term for surrounding tissues that support teeth. It includes four tissues: soft tissue gingiva and periodontal ligament, and hard tissues, cementum and alveolar bone. Therefore, in this specification, “periodontal tissue regeneration” means regeneration of periodontal tissue. Evaluation of “periodontal tissue regeneration” is not limited to those exemplified in the examples described later, and the regeneration level of each component can be evaluated by an animal experimental model for regeneration of gingiva, periodontal ligament, cementum and alveolar bone it can. An example of such an evaluation method can be performed based on, for example, S. Murakami, et al. J Periodont Res 2003; 38; 97-103 (see, for example, FIG. 4 on page 101). Regarding bFGF and periodontal tissue regeneration, S. Murakami, Periodontology 2000, Vol. 56, 2011, 188-208 and the like can be referred to.

  In the present specification, the “tooth root” refers to a portion in the alveolar bone at the lower part of the tooth. Therefore, in this specification, "protrusion of tooth root formation" means that the formation of tooth root is promoted in an arbitrary form. In addition to those exemplified in the examples described later, the evaluation of the promotion of tooth root formation can be performed by using, for example, (1) a fast root elongation rate and (2) a long tooth root as an index. Regarding the root formation, Ohshima H., J. Oral. Biosci. 50 (3), 147-153 (2008) and the like can be referred to. Strictly speaking, tooth root formation promotion includes bone formation or actions that are not included in bone formation. However, when considering the action in vivo, the root grows in synchronization with the surrounding alveolar bone. Therefore, it can be said that it is substantially included in the promotion of bone formation.

  As used herein, “enzyme-synthesizing glycogen (ESG)” refers to glycogen synthesized by an enzyme. A method of synthesizing glycogen using an enzyme is described in, for example, WO2006 / 035848 and Patent 4086312. The obtained enzyme-synthesized glycogen is highly pure and has a controlled molecular weight and degree of branching. Furthermore, enzyme-synthesized glycogen has an excellent feature that it has higher resistance to various digestive enzymes than natural glycogen. This enzyme-synthesized glycogen also has a moisturizing effect, skin function improving effect, and prevention of skin aging that are equal to or higher than those of natural glycogen.

  The enzyme-synthesized glycogen referred to in this specification includes the enzyme-synthesized glycogen described in WO2006 / 035848. That is, this is produced by a step of producing glycogen by allowing a branching enzyme having the ability to synthesize glycogen to act on a substrate in a solution, and the substrate is mainly α-1,4-glucoside bond Α-glucan having a degree of polymerization of 4 or more linked in the above, the substrate is starch debris, dextrin debranches or enzyme-synthesized amylose, and the weight average molecular weight of the glycogen is 1,000,000 Da or more. When a pullulanase of 50 U / g substrate is allowed to act at 60 ° C. for 30 minutes, the product obtained by the MALLS method has a weight average molecular weight of 500,000 Da or more, and the glycogen contains 300 U / g substrate. The product obtained when α-amylase was allowed to act at 37 ° C. for 30 minutes was analyzed when the product was analyzed by the MALLS method. Average molecular weight of glycogen is 500,000 Da or more.

  As a method of discriminating between ESG and natural glycogen, there is a method of measuring a resistant starch (Resistant Starch). ESG is quantified to be about 20% "Resistant Starch content" when quantified with Megazymes' Resistant Starch assay kit. This is because there is a relatively large portion that is resistant to α-amylase in the molecular core portion of ESG. In the case of NSG, since the ratio of molecules having such a resistant core portion is small, the quantitative value of this kit can be identified by being very low.

  The MALLS method is an HPLC gel filtration analysis method using a multi-angle light scattering detector and a differential refractometer as a detector. Appl. Glycosci. 50, 15-20, 2003. The weight average molecular weight of the glycogen is 1 million to 100 million Da, more preferably 1 million to 50 million Da, more preferably 2 million to 30 million Da, and even more preferably 3 million to 2000 Can be tens of thousands. Used for glycogen synthesis, branching enzyme having the ability to synthesize glycogen Aquifex aeolicus, Aquifex pyrophilus, Rhodothermus obamensis, Rhodothermus marinus, Bacillus stearothermophilus, Bacillus caldovelox, Bacillus thermocatenulatus, Bacillus caldolyticus, Bacillus flavothermus, Bacillus acidocaldarius, Bacillus caldotenax, Bacillus Smithii, Thermosynechococcus elongatus and Esc It can be derived from a bacterium selected from the group consisting of erichia coli.

  Enzyme-synthesizing glycogen that can be used in the present invention is a polymer having substantially the same size and shape as natural glycogen. Furthermore, the enzyme-synthesized glycogen used in the present invention has a lower content of impurities such as electrolytes and higher safety to the skin than natural glycogen. In addition, since the arrangement of branched bonds in the molecule is slightly different, the solution has high stability over time, high resistance to biological decomposition of enzymes, microorganisms, and the like, and high resistance to chemical and physical decomposition. From such characteristics, the combined use with various auxiliary components can synergistically enhance the original effect. The amount of the enzyme-synthesized glycogen can be any amount.

  In the present specification, “osteoblast” is an osteogenic cell made of mesenchymal tissue, and is divided from an undifferentiated mesenchymal cell into a precursor osteoblast and a young osteoblast to a mature osteoblast. Formed. The bone matrix is formed from osteoblasts, which are encapsulated in the bone matrix as bone cells. Type I collagen, alkaline phosphatase (ALP), and osteonectin are produced between the precursor cell stage and the mature cell stage, and osteopontin when differentiated to young osteoblasts, bone sialoprotein (BSP) when differentiated to mature cells, Osteocalcin (BGP) begins to be produced respectively (H. Komori, The Bone 12: 49-59, 1998). The size is 20-30 μm, and the shape is a cubic or columnar cell. Osteoblasts exist on the surface of bone tissue and actively secrete bone matrix proteins such as collagen. Crystals of calcium phosphate and hydroxyapatite are deposited on the matrix protein, resulting in a hard bone tissue. The deposition of calcium phosphate crystals is called “calcification”. Osteoblasts on the bone surface and the bone matrix immediately after the start of calcification are called osteoids, and osteoids increase when there are calcification disorders such as osteomalacia. Osteoblasts are embedded in this matrix and eventually become bone cells. Normally, the cell nucleus is located on the opposite side of the bone, the Golgi and the rough endoplasmic reticulum are developed in the cytoplasm, and the matrix is actively synthesized and secreted on the side of the bone. Osteoblasts show high alkaline phosphatase activity (ALP) and are used as histochemical biochemical markers for osteoblasts. Alternatively, any of the cell markers described above can be used. In vitro, osteoblast proliferation can be observed using osteoblast cell lines such as MC3T3-E1.

  The composition of the present invention is used as an additive for the purpose of culture, medical devices, foods, health foods, functional foods, etc. in addition to pharmaceuticals.

  As used herein, the term “medicament” is interpreted in the broadest sense in the art, and includes any drug, including any drug under the Pharmaceutical Affairs Law, quasi-drug, etc. It is understood to encompass drugs, compositions, etc. of use. Examples thereof include applications in the medical field, dental field, etc., for example, treatment adjuvants (for example, those administered to affected areas), bone fillers (for example, directly filling bone defects), bones, etc. Use as a main component or subcomponent in a filling material or the like, or application to a membrane, a tooth extraction wound protective agent, and the like can also be mentioned. Usually, the medicine contains solid or liquid excipients, and may contain additives such as disintegrants, flavoring agents, delayed release agents, lubricants, binders, coloring agents and the like as necessary. Examples of pharmaceutical forms include, but are not limited to, tablets, injections, capsules, granules, powders, fine granules, sustained release formulations and the like.

  As used herein, the term “medical device” is interpreted in the broadest sense in the art, and includes any device or device. In addition to medical devices under the Pharmaceutical Affairs Law, devices, devices for any application intended for bone formation, It is understood to encompass instruments. Examples of the medical device include bone cement, a stent, a catheter, a bone filling material, and an implant.

  In the present specification, the “food” has a meaning that is routinely used in the art, refers to all foods that can be eaten by humans, and an embodiment includes processed products. For example, components such as glycogen of the present invention can be mixed in processed foods such as confectionery, dairy products, and processed cereal products. In addition, “health food” and “functional food” have the meanings commonly used in the industry, and are specially used to prevent bone loss and bone fragility that show symptoms or signs of osteoporosis. Refers to a type of food that is prescribed in the above and distinct from pharmaceuticals or general foods. Examples of such foods include, but are not limited to, foods that are taken by a patient for a certain period of time before treatment of teeth and bones.

  As used herein, “additive” refers to any drug added for any purpose to the main component. For example, an additive for promoting growth can be exemplified. Examples of the additive of the present invention include, but are not limited to, an additive for promoting growth (culture additive and the like) in dental embryo organ culture for research use.

  As used herein, “treatment” or “prevention” means to significantly improve, alleviate or prevent bone loss and bone vulnerability before and after the onset of osteoporosis by promoting bone formation. The present invention promotes osteoblast differentiation by promoting the formation of mature osteoblasts, which in turn promotes bone formation and improves or alleviates the imbalance between bone formation and bone resorption by osteoclasts. Is done.

(Preferred embodiment)
Preferred embodiments of the present invention are listed below. The embodiments provided below are provided for a better understanding of the present invention, and the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention with reference to the description in the present specification.

  In one aspect, the present invention provides a composition for promoting bone formation or osteogenesis comprising glycogen having a weight average molecular weight of less than about 8700 kDa (wherein the weight average molecular weight is a significant figure) Is displayed in two digits.) Thus, the present invention provides for bone formation, bone formation promotion such as treatment of bone diseases, fractures, bone damage, and other bone abnormalities (these may be promoted calcification, osteoblast proliferation, indicators), bone formation Methods, compositions, foods, additives, medical devices, pharmaceuticals, and the like for promoting, promoting tooth germ development, promoting root formation, and / or promoting periodontal tissue regeneration are provided. This is accomplished by administering glycogen to a subject, such as a mammal, resulting in stimulation or enhancement of calcification. Specific examples of such methods include, for example, food to be ingested by a patient for a certain period of time before treatment of teeth and bones, therapeutic adjuvants to be administered to affected areas, and growth promotion in tooth germ organ culture for research purposes. However, it is not limited to these.

  As the glycogen used in the present invention, any glycogen having a weight average molecular weight of less than about 8700 kDa can be used. It has not been conventionally known that glycogen having a specific molecular weight has osteogenic activity, and it can be said that this is an unexpected effect.

  In various embodiments, the upper limit of the weight average molecular weight of glycogen used in the present invention is about 8700 kDa, about 8600 kDa, about 8500 kDa, about 8400 kDa, about 8300 kDa, about 8200 kDa, about 8100 kDa, about 8000 kDa, about 7900 kDa, about 7800 kDa. , About 7700 kDa, about 7600 kDa, about 7500 kDa, about 7400 kDa, about 7300 kDa, about 7200 kDa, about 7100 kDa, about 7000 kDa, about 6900 kDa, about 6800 kDa, about 6700 kDa, about 6600 kDa, about 6200 kDa, about 6200 kDa, about 6200 kDa 6100 kDa, about 6000 kDa, about 5900 kDa, about 5800 kDa, about 5700 kDa, about 5600 kDa, about 5 00kDa, about 5400kDa, may be about 5300kDa or the like. According to the present invention, since an effect is observed even at a molecular weight of about 7000 kDa, the upper limit of the weight average molecular weight of glycogen used in the present invention can be preferably about 8000 kDa or more.

  The lower limit of the weight average molecular weight of glycogen used in the present invention is about 1000 kDa, about 1100 kDa, about 1200 kDa, about 1300 kDa, about 1400 kDa, about 1500 kDa, about 1600 kDa, about 1700 kDa, about 1800 kDa, about 1900 kDa, which is the lower limit of glycogen itself. , About 2000 kDa, about 2100 kDa, about 2200 kDa, about 2300 kDa, about 2400 kDa, about 2500 kDa, about 2600 kDa, about 2700 kDa, about 2800 kDa, about 2900 kDa, about 3000 kDa, about 3100 kDa, about 3300 kDa, about 3300 kDa, about 3300 kDa 3600 kDa, about 3700 kDa, about 3800 kDa, about 3900 kDa, about 4000 kDa, about 4100 kDa, 4200KDa, about 4300KDa, about 4400KDa, about 4500KDa, about 4600KDa, about 4700KDa, about 4800KDa, may be about 4900kDa like. These lower limits can be arbitrarily combined with any numerical value of the upper limits described herein. For example, such a range can be exemplified by about 3000 kDa to about 8000 kDa, but not limited thereto, in consideration of manufacturing.

  In certain embodiments, the weight average molecular weight used in the present invention is about 3000 kDa or more and about 8000 kDa or less. Each of these weight average molecular weight glycogens has been demonstrated to promote osteoblast proliferation and ALP expression and actual animal experiments, or promote dental healing, and such effects are It has been found that other glycogens (for example, glycogen of Patent Document 1 (Kuppie phytoglycogen)) are not recognized, and it can be said that this is an unexpected effect.

  In a preferred embodiment, the weight average molecular weight used in the present invention is from about 4900 to about 7000 kDa. Although not wishing to be bound by theory, this range is preferable because calcification in animal experiments, promotion of formation of tooth germ or promotion of tooth healing in organ culture, etc. have been confirmed. is there.

  In one embodiment, the glycogen used in the present invention comprises enzymatically synthesized glycogen (ESG).

  In another embodiment, glycogen used in the present invention can be synthesized based on the method of Patent No. 4086312, for example, ESG-5M (enzymatic synthetic glycogen having a weight average molecular weight of about 5200 kDa) used in the examples, ESG-3M (enzymatic synthetic glycogen with a weight average molecular weight of about 3200 kDa), ESGB (enzymatic synthetic glycogen with a weight average molecular weight of about 5000 kDa), ESG-7M (enzymatic synthetic glycogen with a weight average polymerization degree of about 7000 kDa), mussel glycogen (Laboratoires, France) Manufactured by Serobiologiques, imported by Yamakawa Trading Co., Ltd. (also available from Sigma Aldrich), oyster glycogen (Wako Pure Chemical Industries, Ltd., or Sigma Aldrich, Nacalai Tesque Co., Ltd.) Hand possible), Funagai (Slipper Limpet) glycogen (Sigma Aldrich), is such as bovine liver glycogen (Nacalai Tesque, Inc. or Sigma Aldrich).

  Without wishing to be bound by theory, it is believed that the effects of glycogen of the present invention on osteogenesis or osteogenesis are achieved, at least in part, by promoting osteoblast proliferation.

(Medicine, etc.)
In one aspect, the invention includes osteogenesis, osteogenesis promotion (which can be promoted calcification, osteoblast proliferation, indicator), glycogen comprising a glycogen having a weight average molecular weight of less than about 8700 kDa. The present invention provides a medicament for promoting tooth germ development, promoting root formation, and / or promoting periodontal tissue regeneration. Here, as various embodiments of glycogen contained in the medicament of the present invention, those used for a composition for promoting osteogenesis or osteogenesis, such as those described in the section of these compositions in the present specification, are described. Any of those described can be used. The medicament of the present invention can be used in the form of a treatment adjuvant or the like administered to the affected area, but is not limited thereto.

  In one embodiment, the medicament of the present invention further comprises a pharmaceutically acceptable excipient. When the composition of the present invention is used as a medicine or a medicine, examples of its dosage form include tablets, powders, fine granules, granules, coated tablets, sustained-release preparations, capsules, injections and the like. The medicinal product may contain additives such as excipients, if necessary, binders, disintegrants, lubricants, flavoring agents, coloring agents, delayed release agents and the like. In the case of oral preparations, excipients such as lactose, corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose, etc., binders such as polyvinyl alcohol, polyvinyl ether, methylcellulose, hydroxypropylcellulose, gum arabic, tragacanth, gelatin , Shellac, polyvinylpyrrolidone, block copolymer, etc., as a disintegrating agent, such as starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectin, etc., as a lubricant, eg, magnesium stearate For example, cocoa powder, mint oil, cinnamon powder and the like can be used as flavoring agents such as talc, polyethylene glycol, silica and hydrogenated vegetable oil, but are not limited thereto. If necessary, a coating for obtaining a sustained release or enteric preparation can be applied. In the case of injectable preparations, pH adjusters, solubilizers, tonicity agents, buffering agents and the like are used, but are not limited thereto.

  In the medicament of the present invention, further medicaments which may be contained are variously considered depending on the purpose. For example, those which are currently used as a therapeutic agent for osteoporosis, such as a calcium agent (trade name aspara CA, carticol) , Calcium lactate, etc.), vitamin D preparations (trade names such as alpha roll, one alpha, locartrol, and high titrol), female hormone preparations (trade name such as estreol), ipriflavones (trade names such as osten and asost), vitamin K2 preparations (For example, Graque etc.), bisphosphonates (trade name Fasax, risedronate, tiludronate, ibandronate, etc.) and the like can be mentioned, but are not limited thereto.

  In addition to the above therapeutic agents or preventive agents, various therapeutic agents for diseases such as osteoporosis including osteogenesis promoters have been reported. For example, such drugs include pterocarpan [(5αR, 11αR) -3-hydroxy-9,10-dimethoxypterocarpan] (Japanese Patent Laid-Open No. 2003-155236) (pterocarpan is an astragalus plant of the leguminous family) Or estrogen agonists / antagonists such as droloxifene, or drugs that promote bone formation and increase bone mass (such as sodium fluoride, parathyroid hormone, growth hormone or growth hormone secretory), or the like Of polyphosphonates (alendronade, cimadronade, etc.) and / or statins (simvastatin, bravastatin, cerivastatin, etc.) (JP 2001-253827); phosphodiesterase I Xanthines having V inhibitory activity (JP-A-9-169665); lutein hormones and anti-estrogenic substances (estradiol derivatives, triphenylethylene derivatives, benzothiophene derivatives, etc.) (JP-A-6-31930); monocytes-macrophages A colony formation stimulating factor (Japanese Patent Laid-Open No. 5-17367) and the like are not limited thereto.

  Alternatively, examples of the osteogenic component include bone morphogenetic proteins (BMP and the like). The BMP in the present invention is not particularly limited as long as it has an activity of promoting osteogenesis by inducing differentiation into undifferentiated mesenchymal cells into osteoblasts. For example, BMP-1 , BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-12 (above, homodimers), or heterodimers of these BMPs Or a variant (ie, a protein having an amino acid sequence in which one or more amino acids are deleted, substituted and / or added in the amino acid sequence of a naturally occurring BMP and having the same activity as that of a naturally occurring BMP) Etc. Or in the case of such a protein, the nucleic acid which codes it may be sufficient. For example, it may be the same as the base sequence encoding naturally occurring BMP, or one having one or more bases deleted, substituted and / or added in the base sequence encoding naturally occurring BMP. Moreover, you may make it use these things combining 1 type (s) or 2 or more types.

  In one embodiment, the medicament of the present invention is administered over 1 day to 10 days, or over 3 to 10 days. Although not wishing to be bound by theory, it is because it is a period in which the proliferation effect of osteoblasts is well promoted. It is understood that this period depends on the number of cells present at the site where glycogen is administered, and those skilled in the art can appropriately determine depending on the state of the site to be administered (for example, the state of cell confluence). This can be changed.

  In the present specification, an “effective amount” of a drug or the like means an amount capable of exerting the intended drug effect of the drug. In the present specification, among such effective amounts, the minimum concentration may be referred to as the minimum effective amount. Such minimum effective amounts are well known in the art, and usually the minimum effective amount of a drug is determined by those skilled in the art or can be determined as appropriate by those skilled in the art. In order to determine such an effective amount, an animal model or the like can be used in addition to actual administration. The present invention is also useful in determining such effective amounts.

  As used herein, “pharmaceutically acceptable carrier” refers to a substance that is used when producing agricultural chemicals such as pharmaceuticals or animal drugs, and does not adversely affect active ingredients. Such pharmaceutically acceptable carriers include, for example, antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffering agents, delivery agents. Vehicles, excipients and / or agricultural or pharmaceutical adjuvants include, but are not limited to:

(Medical devices, etc.)
In another aspect, the invention includes osteogenesis, osteogenesis promotion (these can be promoted calcification, osteoblast proliferation, an indicator), osteogenesis comprising glycogen having a weight average molecular weight of less than about 8700 kDa The present invention provides a medical device for promoting tooth germ development, promoting root formation, and / or promoting periodontal tissue regeneration. Here, as various embodiments of glycogen contained in the medical device of the present invention, the composition used for osteogenesis or osteogenesis, a pharmaceutical, etc., for example, these compositions or Any of those described in the pharmaceutical section can be used.

  In one embodiment, the medical device of the present invention may be, but is not limited to, bone cement, stent, catheter, bone prosthesis or implant.

  In one embodiment, the medical device of the present invention is intended to be applied to a bone defect site, eg, a site resulting from an injury, a defect caused during surgery, an infection, a malignant tumor or a developmental malformation. Has been. Implants that are appropriately sized and shaped as needed can be used to repair simple and complex fractures and poor fusion, external and internal fixation, joint remodeling like arthrodesis, general arthroplasty, Hip cup arthroplasty, femoral head replacement and total joint replacement, spinal repair including spinal fusion and internal fixation, eg defect filling, isolated resection, laminectomy, spinal tumor resection, anterior neck Tumor surgery such as head and chest surgery, spinal cord injury repair, scoliosis, lordosis and kyphosis treatment, fracture intermaxillary fixation, orthognathic surgery, temporomandibular joint replacement, alveolar ridge enlargement and re-surgery It can be used for a wide range of orthopedic, neurosurgical, oral and maxillofacial surgical procedures such as shaping, implantable bone grafting, implant replacement and modification, sinus lift and the like. Specific bones that can be repaired or replaced with bone-derived implants in this specification include ethmoid bone, frontal bone, nasal bone, occipital bone, parietal bone, temporal bone, mandible, maxilla, cheekbone, cervical vertebra, Thoracic vertebrae, lumbar vertebrae, sacrum, ribs, sternum, clavicle, scapula, humerus, calcaneus, ulna, carpal, palmar, phalange, iliac, sciatic, pubic, femur, tibia, rib, patella, rib, Includes tarsal and metatarsal bones.

  In one embodiment, the device of the present invention may have a biocompatible substrate (matrix). This substrate can be used as a bone formation field (scaffold) for osteogenesis differentiated from undifferentiated mesenchymal cells, or as a field for healing or tooth germ formation (scaffold). Can be. The form of the substrate is preferably a block body (lump). The block body (for example, a sintered body) has shape stability, and when an osteogenesis treatment device is implanted in a living body, the osteogenesis treatment device can be prevented from dissipating from the implantation site at an early stage. In addition, since bone formation can proceed along the shape of the block body, it is particularly effective when the transplant site is a relatively large bone defect or the like.

  In one embodiment, the form of the substrate may be appropriately selected according to the application site (transplantation site) of the osteogenesis treatment device. For example, powder, granule, pellet (small block) Or the like. In the case of using the substrate in such a form, a composition mixed with an active ingredient such as glycogen can be used as an osteogenic treatment device, and the osteogenic treatment device is filled (stuffed) into the bone defect portion. Can be used.

  Moreover, in one embodiment, it is preferable that a base | substrate is a porous thing (porous body). By using a porous body as a substrate, an active ingredient such as glycogen can be more easily and reliably supported on the substrate, and cells involved in bone formation can easily enter the substrate, which is advantageous for bone formation. It is.

  In this case, the porosity is not particularly limited, but is preferably about 30 to 95%, more preferably about 55 to 90%. By setting the porosity within the above range, cells that are involved in bone formation can be more easily penetrated into the substrate while maintaining the mechanical strength of the substrate suitably. can do.

  Further, the constituent material of the substrate is not particularly limited as long as it has biocompatibility. For example, hydroxyapatite, fluorapatite, carbonate apatite, dicalcium phosphate, tricalcium phosphate, tetracalcium phosphate , Calcium phosphate compounds such as octacalcium phosphate, ceramic materials such as alumina, titania, zirconia, yttria, various metal materials such as titanium or titanium alloys, stainless steel, Co-Cr alloys, Ni-Ti alloys, etc. 1 type or 2 types or more of these can be used in combination. Among these, the constituent material of the substrate is preferably a ceramic material (so-called bioceramics) such as a calcium phosphate compound, alumina, zirconia, and particularly, a material mainly composed of hydroxyapatite or tricalcium phosphate is frequently used. Is done.

  Since hydroxyapatite and tricalcium phosphate have the same structure as the mineral main component of bone, they have particularly excellent biocompatibility. Moreover, since it has both positive and negative charges, particularly when liposomes are used as carriers, the liposomes can be stably supported on a substrate for a long time. As a result, an active ingredient such as glycogen adsorbed or encapsulated in the liposome is also stably held on the substrate for a long time, contributing to more rapid bone formation. Moreover, since it has high affinity with osteoblasts, it is preferable for maintaining new bone.

  Such a substrate can be produced (manufactured) by various methods. The case where a porous block body made of a ceramic material is produced as an example will be described as an example. Such a porous block body is made of, for example, a slurry containing a ceramic material powder and transplanted into a bone defect or the like. It can be produced by obtaining a molded body formed into a shape corresponding to a part by, for example, compression molding or the like, and sintering (firing) the molded body. The osteogenic treatment device as described above can be produced (manufactured) by mixing glycogen with a substrate.

  When using a powder, granule, pellet or the like as the substrate, for example, a bone-forming device is produced by molding a kneaded material obtained by kneading the substrate, a binder and the liquid as described above. You can also

(Food, etc.)
In one aspect, the invention includes osteogenesis, osteogenesis promotion (which can be promoted calcification, osteoblast proliferation, indicator), glycogen comprising a glycogen having a weight average molecular weight of less than about 8700 kDa. The present invention provides an additive, a food, a health food, or a functional food for promoting tooth germ development, promoting root formation, and / or promoting periodontal tissue regeneration. Here, as various embodiments of the glycogen contained in the additive, food, health food or functional food of the present invention, it is used for a composition for forming bone or promoting bone formation, a medicine, a medical device, etc. For example, any of those described in the sections of these compositions, medicaments, medical devices and the like of the present specification can be used. Such additives, foods, health foods or functional foods include, for example, foods to be ingested by patients for a certain period of time before treatment of teeth and bones, or for growth promotion in tooth germ organ culture for research purposes. However, it is not limited to these.

  When the present invention is used as an additive, food, health food or functional food, they are used for promoting osteogenesis for the prevention of diseases or conditions that require osteogenic growth activity such as osteoporosis. For example, ingredients such as glycogen of the present invention are added to processed foods such as confectionery, dairy products and processed cereals together with appropriate excipients, and additives such as flavoring agents and coloring agents are added as necessary. Can be mixed. In the case of “health food” and “functional food”, for example, a compound of the present invention or a mixture thereof is enclosed in a gelatin capsule, or a drink containing the compound of the present invention or a mixture thereof is prepared, and osteoporosis symptoms or It can be used as a health maintenance food for preventing bone loss and fragility of bone showing signs, that is, for promoting bone formation. The amount of the active ingredient added is usually an amount corresponding to about 0.1 mg to about 5,000 mg per day for an adult, but is not limited to this range. For foods to be ingested by a patient for a period of time prior to tooth or bone treatment, capsules containing the compound of the present invention or a mixture thereof, or a beverage containing the compound of the present invention or a mixture thereof can be prepared and used, and In the case of research use, in the case of tooth germ organ culture, the growth promoting additive is prepared by, for example, dissolving the compound of the present invention directly in the culture solution or adding a separately dissolved product to the culture solution. Can be used. In the case of such a capsule, it can be enclosed in a capsule together with an excipient or a lubricant. In the case of beverages, sweeteners, acidulants, pH adjusters, fragrances, colorants and the like can be mixed.

(Production methods for pharmaceuticals, etc.)
In one embodiment, the method of the present invention comprises bone formation, bone formation promotion (these may be promoted calcification, osteoblast proliferation, index), bone formation promotion, tooth germ development promotion, tooth root formation promotion, And a method of producing a medicament for promoting periodontal tissue regeneration, comprising the step of mixing glycogen having a weight average molecular weight of less than about 8700 kDa with a pharmaceutically acceptable excipient. Additives, foods, health foods, functional foods and the like can be produced in the same manner. In that case, it can replace with a pharmaceutically acceptable excipient | filler and can use the secondary component according to the objective. Here, various embodiments of glycogen included in the present invention include bone formation, bone formation promotion (these can be used as calcification promotion, osteoblast proliferation promotion, and index), bone formation promotion, tooth germ development promotion. Used in compositions, medicaments, medical devices, etc. for promoting root formation and / or promoting periodontal tissue regeneration, for example, as described in the section of these compositions, medicaments or medical devices herein Any that are present can be used.

  The present invention relates to bone formation, bone formation promotion (which can be used as calcification promotion, osteoblast proliferation promotion, index), bone formation promotion, tooth germ development promotion, tooth root formation promotion, and / or periodontal tissue regeneration promotion. There is provided a method for producing a medical device for use in which a glycogen having a weight average molecular weight of less than about 8700 kDa is included in the medical device or the material of the medical device. As the medical device or the material of the medical device, any material known in the art can be used. Also in the case of a medical device, a secondary component according to the purpose can be used instead of a pharmaceutically acceptable excipient as in the case of a medicine. Here, various embodiments of glycogen included in the present invention include bone formation, bone formation promotion (these can be used as calcification promotion, osteoblast proliferation promotion, and index), bone formation promotion, tooth germ development promotion. Used in compositions, medicaments, medical devices, etc. for promoting root formation and / or promoting periodontal tissue regeneration, for example, as described in the section of these compositions, medicaments or medical devices herein Any that are present can be used.

(Bone formation, treatment and prevention)
In one aspect, the present invention is from the group consisting of forming bone, promoting bone formation, promoting tooth germ development, promoting tooth root formation, and promoting periodontal tissue regeneration. A method for at least one selected, which comprises bone formation, bone formation promotion (these may be calcification promotion, osteoblast proliferation promotion, indicator), bone formation promotion, tooth germ development promotion Glycogen having a weight average molecular weight of less than about 8700 kDa is used to promote bone formation, bone formation (promoting mineralization, promoting osteoblast proliferation, A method comprising administering to be delivered to a site in need of bone formation promotion, tooth germ development promotion, tooth root formation promotion, and / or periodontal tissue regeneration promotion. Here, various embodiments of glycogen included in the present invention include bone formation, bone formation promotion (these can be used as calcification promotion, osteoblast proliferation promotion, and index), bone formation promotion, tooth germ development promotion. Used in compositions, medicaments, medical devices, etc. for promoting root formation and / or promoting periodontal tissue regeneration, for example, as described in the section of these compositions, medicaments or medical devices herein Any that are present can be used. The present invention provides methods and compositions for the treatment of bone diseases, fractures, bone damage, and other bone abnormalities. At least in part, glycogen is administered to a subject such as a mammal, and bone formation, bone formation promotion (these can be used as calcification promotion, osteoblast proliferation promotion, index), bone formation promotion, tooth germ It is achieved by a process that leads to the promotion of events associated with promotion of growth, promotion of root formation, and / or promotion of periodontal tissue regeneration, for example stimulation or enhancement of calcification.

  In the bone formation or bone formation promoting method of the present invention, a patient who needs treatment, treatment or prevention such as a disease or treatment requiring bone formation such as fracture, osteoporosis, and the like can be targeted. The administration method such as oral administration, intravenous administration, intraarterial administration, intramuscular administration, intraperitoneal administration, and rectal administration is appropriately selected according to the patient's condition, age, sex and the like. The dose of the active ingredient is usually about 0.1 mg to about 5,000 mg per day for an adult, and is not limited to this range, but is appropriately selected according to the patient's condition, age, sex and the like. Glycogen used in the present invention is considered to be non-toxic or low-toxic. In the method of promoting tooth germ growth, tooth root formation and / or periodontal tissue regeneration of the present invention, such as diseases or conditions that require tooth germ growth promotion, tooth root formation promotion and / or periodontal tissue regeneration promotion, etc. Patients requiring treatment, treatment or prevention can be targeted, and for this patient, an administration method such as oral administration and intravenous administration is appropriately selected according to the patient's condition, age, sex and the like. The dose of the active ingredient is usually about 0.1 mg to about 5,000 mg per day for an adult, and is not limited to this range, but is appropriately selected according to the patient's condition, age, sex and the like. Glycogen used in the present invention is considered to be non-toxic or low-toxic. To promote tooth germ development, root formation, and / or periodontal tissue regeneration, when implants or teeth are replanted or transplanted, glycogen is applied during the treatment to heal the pulp and periodontal tissues. The usage method of promoting is considered. Therefore, promotion of tooth germ development, promotion of tooth root formation, and / or promotion of periodontal tissue regeneration is expected to be positioned as a combined therapy in implant or tooth replantation or transplantation.

  In another aspect, the present invention relates to bone formation, bone formation promotion (these may be promoted calcification, osteoblast proliferation, indication), bone formation, tooth germ development, tooth root formation promotion, and / or Provided is the use of glycogen having a weight average molecular weight of less than about 8700 kDa for the manufacture of a food, additive, medical device or medicament for promoting periodontal tissue regeneration. Alternatively, the present invention provides glycogen having a weight average molecular weight of less than about 8700 kDa for bone formation or promoting bone formation.

  The amount of the active ingredient used in the treatment method or prevention method of the present invention depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, cell form or type, etc. In view of this, it can be easily determined by those skilled in the art. The frequency with which the treatment method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration every day to once every several months (for example, once a week to once a month). It is preferable to administer once a week to once a month while observing the course.

  The type and amount of the drug used in the treatment or prevention method of the present invention is determined based on the purpose of use, target disease (type, severity) based on information obtained by the method of the present invention (for example, information on the disease). Etc.) can be easily determined by those skilled in the art in consideration of the patient's age, weight, sex, medical history, the form or type of the site of the subject to be administered, and the like. The frequency with which the monitoring method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. The frequency of monitoring the disease state includes, for example, daily-once every several months (for example, once a week-once a month). It is preferable to perform monitoring once a week to once a month while observing the progress.

  The present invention may be used as a kit or the like, in which case instructions may be accompanied. In the present specification, the “instruction” describes the treatment method of the present invention and the like for a person who performs administration such as a doctor or a patient. This instruction describes a word that instructs to administer the medicine of the present invention to an appropriate site (for example, a bone defect) at an appropriate amount and at an appropriate time. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc. in the United States) where the present invention is implemented, and is approved by the supervisory authority. It is clearly stated that it has been received. The instruction is a so-called package insert and is usually provided as a paper medium, but is not limited thereto. For example, a form such as an electronic medium (for example, a homepage or e-mail provided on the Internet) is used. But it can be provided.

  If desired, more than one drug may be used in the treatment of the present invention. When two or more drugs are used, substances with similar properties or origins may be used, or drugs with different properties or origins may be used. Information regarding disease levels for methods of administering two or more such drugs can also be obtained by the methods of the present invention.

(General techniques used in this specification)
Unless otherwise specifically indicated, the techniques used herein are within the technical scope of the field, such as glycoscience, medical device manufacturing technology, formulation technology, microfabrication, organic chemistry, biochemistry, Use well-known and conventional techniques in genetic engineering, molecular biology, microbiology, genetics and related fields. Such techniques are well described, for example, in the documents listed below and in references cited elsewhere herein. The culture method used in the present invention is described and supported in, for example, an animal culture cell manual, edited by Seno et al., Kyoritsu Shuppan, 1993, and all of this description is incorporated herein.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

  The present invention will be described below based on examples, but the following examples are provided for illustrative purposes only. Accordingly, the scope of the present invention is not limited to the above-described embodiment and the following examples, but is limited only by the appended claims. Reagents, resins and the like used in the examples shown below can be obtained from Wako Pure Chemicals, Sigma-Aldrich, Kewpie, Yamakawa Trading, Nacalai Tesque, and Invitrogen unless otherwise specified.

(Preparation Examples)
The enzymatically synthesized glycogen used in the examples was produced as follows.

(Manufacture of ESG-5M)
Dextrin (MAX1000, manufactured by Matsutani Chemical Industry Co., Ltd.) was dissolved in 100 mL of 0.01% by weight calcium chloride aqueous solution to 8% by weight, and Pseudomonas-derived isoamylase (Sigma Aldrich) was added at 5000 units per gram of substrate. . After reacting at 50 ° C. for 4 hours and decomposing into short-chain amylose, the temperature was raised to 65 ° C., 5 mL of 1 M citrate buffer (pH 6.7), Aquefex aeolicus-derived branching enzyme (5000 units / g substrate) And Thermus aquaticus-derived amylomaltase (2 units / g substrate) were added and allowed to react for 48 hours. The pH of the reaction solution was adjusted to about 3.5 with hydrochloric acid, and the reaction was stopped by heating at 100 ° C. for 15 minutes. 100 mL of ethanol was added to this reaction solution, and the precipitate was collected by centrifuging at about 10,000 × g for 5 minutes, and further washed with 100 mL of 50% ethanol three times. The precipitate was dissolved in 50 mL of distilled water and freeze-dried to obtain powdered enzyme-synthesized glycogen (ESG-5M).

(Manufacture of ESG-3M)
It was produced in the same manner as ESG-5M except that the reaction time with isoamylase was 3.5 hours.

(Manufacture of ESG-7M)
It was produced in the same manner as ESG-5M except that the reaction time of isoamylase was 4.5 hours.

(Manufacture of ESG-8M)
It was produced in the same manner as ESG-5M except that the reaction time of isoamylase was changed to 5 hours.

(Manufacture of ESG-20M)
It was produced in the same manner as ESG-5M except that the reaction time of isoamylase was 9 hours.

(Manufacture of ESG-B)
It was manufactured by the method shown in Non-Patent Document 3.

Example 1
In this example, osteoblast proliferation promoting effects were examined for various glycogens shown below.

(Effect of promoting osteoblast proliferation)
Alkaline phosphatase was used as an osteoblast differentiation marker.

  Bone marrow cell proliferation promoting effect was measured using MC3T3-E1.

(Method and material)
ESG-5M (Lot 070223-5M)
ESG-3M (Lot 070312-3M)
ESGB (Lot 050225) (also known as GlyB)
ESG-20M (Lot 070312-20M)
ESG-8M (Lot 070315-8M)
NSG (Corn) (Lot ZG7002) (also known as Phytoglycogen)
NSG (Mussel) (Lot 127K3775) (aka Sigma Type VII)
NSG (Rabbit river) (Lot 039K3775) (also known as Sigma Type III)
(Cell proliferation experiment)
The following experiment was performed using an osteoblast-like cell line (MC3T3-E1) established from a mouse skull. The medium was calcified medium by adding β-sodium glycerophosphate, L-ascorbic acid, and dexamethasone in α-minimum essential medium (α-MEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin. Cell culture was performed under 5% CO ₂ /95% air conditions. In the same calcification medium, 300 μg / ml of enzyme-synthesized glycogen (average molecular weight of about 5000 kDa) was added or cultured under the non-addition condition and used for the experiment. The culture solution was changed every two days.

  In the cell proliferation experiment, each of 5000 MC3T3-E1 cells was seeded in a 6-well plate, and cells were collected with a trypsin-EDTA solution after 1 day, 3 days, and 5 days, and the total number of cells was measured with a cell count plate. did. Three measurements were performed under each condition, and statistical analysis was performed. As a result, in the glycogen-added medium, a significant increase in the number of cells was confirmed on the fifth day of culture compared with the non-added group (FIG. 1). Therefore, it was shown that the synthetic glycogen in the medium promotes the proliferation of osteoblast-like cells.

(result)
The results are shown in Table 1 below (in the table, ESG: enzyme-synthesized glycogen; NSG: natural glycogen) and FIGS. As shown in Table 1, an osteoblast proliferation promoting effect was observed at least at a weight average molecular weight of about 3000 kDa to about 6400 kDa.

  From the above, it was found that only ESG and NSG of less than about 8700 kDa have an osteoblast proliferation promoting effect. This was an effect that was not expected at all from the findings of Non-Patent Documents 1 and 2. Although not wishing to be bound by theory, it is thought that the mechanism is related to the occurrence of increased expression of GLUT-1 (glucose transporter). Such a mechanism has not been anticipated from the prior art, and is an example showing one of the remarkable effects of the present invention.

(Example 2: Gene expression search)
Total RNA was extracted from MC3T3-E1 cultured for 1 day, 3 days, 5 days or 7 days in a medium with or without synthetic glycogen (ESG-5M), and cDNA was synthesized and subjected to PCR reaction to produce osteoblasts. Changes in gene expression were analyzed for factors involved in differentiation and calcification (alkaline phosphatase (ALP), osteocalcin (OC)).

MC3T3-E1 cultured in a 6-well plate was extracted with 1 ml of Trisol (manufactured by Invitrogen) according to the product protocol. CDNA was synthesized from 2 μg of total RNA using a cDNA synthesis kit (Roche Applied Science). As specific primers for ALP gene and OC gene, ALP-Forward (5′-CCA GCA GGT TTC TCT CTT GG-3 ′ (SEQ ID NO: 1)) and ALP-Reverse (5′-CTG GGA GTC TCA TCC TGA TGC -3 '(SEQ ID NO: 2)), OC-Forward (5'-CTT GGT GCA CAC CTA GCA GA-3' (SEQ ID NO: 3)) and OC-Reverse (5'-ACC TTA TTG CCC TCC TGC TT-3 '(SEQ ID NO: 4)) was used for PCR amplification with PTC-100 ^™ Programmable Thermal Controller (manufactured by MJ Research). First, after heat denaturation at 94 ° C. for 2 minutes, 30 cycles of 94 ° C., 1 minute heat denaturation, 62 ° C., 1 minute annealing, 72 ° C., 1 minute extension reaction were performed. Internal standards include β-actin (Forward: 5′-TGG AAT CCT GTG GCA TCC ATG AAA C-3 ′ (SEQ ID NO: 5), Reverse: 5′-TAA AAC GCA GCT CAG TAA CAG TCC G-3 ′ (sequence No. 6)) was used. After electrophoresis on a 2% agarose gel, the PCR product was stained with ethidium bromide and photographed.

(result)
The results are shown in Table 2 below (in the table, ESG: enzyme-synthesized glycogen; NSG: natural glycogen) and FIG. As shown in Table 2, at least a weight average molecular weight of about 3000 kDa to about 6400 kDa was found to be effective in promoting the expression of alkaline phosphatase, which is an osteoblast differentiation marker.

  As a result, ALP gene expression was started in the glycogen added group from the third day of culture, and an increase in the same gene expression was confirmed in the added group on the fifth and seventh days. In addition, osteocalcin, an osteoblast marker, was found to have increased gene expression in the synthetic glycogen addition group on the seventh day of culture (FIG. 4). Therefore, it was clarified that the synthetic glycogen in the medium enhances differentiation into osteoblasts and calcification ability.

(Example 3)
In this example, various glycogens were examined for the presence of a calcification promoting effect.

  MC3T3-E1 cultured in a medium supplemented with or without synthetic glycogen (ESG-5M) for 7 days (confluent state) was fixed with a 4% paraformaldehyde solution and subjected to Kossa staining as an indicator of calcification.

  The cultured cells after fixation with 4% paraformaldehyde were washed with distilled water and then reacted in 5% silver nitrate solution for 1 hour under indirect light. After washing with distilled water, a fixing reaction was performed with 5% sodium thiosulfate solution for 3 minutes. After washing with distilled water again, nuclear staining was performed with a Cologne Echloth funnel, sealed with a cover glass using a water-soluble mounting agent, and observed with an optical microscope.

(result)
As a result, deposition of microcalcifications was confirmed only in the glycogen added group (FIG. 5), and it was shown that synthetic glycogen in the medium promotes osteoblast differentiation and calcium deposition of MC3T3-E1.

  Enzymatic synthetic glycogen has been shown to promote the growth, differentiation and calcification ability of osteoblast-like cell line MC3T3-E1 in an in vitro experimental system, but the specific molecular mechanism is still unclear. . Regarding the presence or absence of enzymatically synthesized glycogen uptake into cells, MC3T3-E1 was incubated for 1 hour with biotinylated enzyme synthetic glycogen added to the medium, reacted with FITC-streptavidin, and examined with a fluorescence microscope. The cytoplasmic fluorescence reaction was observed. As a result, clear uptake of synthetic glycogen was confirmed. On the other hand, after culturing for 1 day, 3 days, 5 days, and 7 days under the same conditions, the reaction with FITC-streptavidin was observed, and when the fluorescence reaction in the cytoplasm was observed with a fluorescence microscope, the apparent uptake of synthetic glycogen was It was not confirmed. From these results, it has been inferred that glycogen molecule uptake into cells is a stimulus, which may cause some activation of intracellular signal transduction, promote cell proliferation and differentiation, and lead to bone formation.

(Example 4: Effect of enzyme-synthesized glycogen on bone regeneration: in vivo experiment In this example, it was observed whether the bone regeneration (formation) effect was achieved by glycogen of the present invention, two on the left and right sides of the mouse parietal bone. , Filled with collagen gel with and without glycogen added, and evaluated bone formation, drilled into the skull of mice, injected ESG, and applied CT-Scan several days later, It was investigated whether bone regeneration promotion occurred.

  A percutaneous flap was formed on the parietal portion of the ICR mouse subjected to deep anesthesia to expose the parietal bone, and a total of two left and right fossa sinuses were formed with a dental turbine with a 4 mm diameter Lindemanber. After implanting collagen gel (Cellmatrix; manufactured by Nitta Gelatin Co., Ltd.) only in the control side bone cavity and collagen gel mixed with 500 μg / ml ESG-5M in the bone cavity in the experiment side, the skin periosteal flap was inserted. It was returned and sutured to finish the operation. Two weeks later, the mouse was perfused and fixed with 4% paraformaldehyde, the head was cut off, and a micro CT (Elescan; manufactured by Nippon Steel ELEX Co., Ltd.) was photographed with a slice width of 15 μm. The photographed images were three-dimensionally constructed with NDTView (manufactured by Sony Corporation) and 3D bone (manufactured by Ratok System Engineering Co., Ltd.).

(result)
The results are shown in FIG. As shown in FIG. 6, the effectiveness was confirmed in ESG 070223-5M. Originally, glycogen is a natural molecule that is abundant in shellfish such as oysters and scallops, and food such as corn. The enzyme-synthesized glycogen used in the present invention has almost the same chemical structure as natural glycogen and has no cytotoxicity. It is a compound that can be safely used in the human body. Therefore, according to the present invention, a new bone formation promoter containing enzyme-synthesized glycogen as an active ingredient can be provided, and can be widely applied to the treatment of diseases associated with bone defects such as fractures and periodontal diseases. That is, application to bone formation promotion, calcification promotion, osteoblast proliferation promotion, tooth germ development promotion, tooth root formation promotion, and periodontal tissue regeneration promotion can be expected. Furthermore, since enzyme-synthesized glycogen is expected to be able to be taken orally because it is not easily degraded by α-amylase, it can be administered not only locally to the bone defect site but also systemically as an osteogenesis promoter. It is expected to be.

(Example 5: Experimental example showing effectiveness as a beverage for promoting dental healing)
(A. Effects in pups)
Enzyme-synthesized glycogen aqueous solution (weight average polymerization degree about 7000 kDa (ESG-7M (ESG-7M (lot 070808-7M)) prepared as described in the preparation examples in ICR mice <CLEA Japan> from day 0 of pregnancy. )) 2 mg / mL) (ESG group) <This aqueous solution was prepared by dissolving 200 mg of enzymatically synthesized glycogen in 100 ml of sterile water. > Was orally administered (free drinking) and born mice were bred until 3 weeks of age. As a model for tooth replantation, the teeth of pup mice were removed and re-implanted at the same site to examine the healing of dental pulp and the differentiation of odontoblasts.

  As a result, in the ESG group, healing of dental pulp and differentiation of odontoblasts were promoted. Specifically, as can be seen from the comparison of the percentage of odontoblast differentiation in the rightmost column of Table 3 that reached the entire pulp, ESG significantly increased pulp healing, odontoblasts in all experimental examples. Differentiation was promoted.

  In all the mice used in this experiment, 19 cases (86.4%) of 22 cases in the water group and 11 cases (42.3%) of the ESG group caused root fractures at the time of tooth extraction. %), And significantly less in the ESG group. This suggests that strong teeth were formed in the ESG group.

  (Tooth replanting experiment)

^{* 1 Indicates} that healing is progressing toward the right (no [curing effect is low]) <only root apex <whole root <crown + root [high healing effect]].

  Thus, the above results revealed that the composition of the present invention is effective in promoting tooth germ development. In addition, in this example, in addition to the bone formation or the bone formation promotion shown in the above example, it became clear that it also promoted the healing of the dental pulp, and the bone formation or bone around the tooth (alveolar bone) In addition to promoting formation, it has been shown to comprehensively promote tooth germ development.

  In addition, even when the weight average polymerization degree used in this example is about 7000 kDa, an effect related to bone formation that promotes the healing of teeth was found, so that it is less than about 8700 kDa used for the purpose of the present invention. It will be appreciated that glycogen may be of the order of about 7000 kDa.

(B. Effects in mother mice)
ICR mice <CLEA Japan> from 3 weeks of age, water (water group) or an enzyme-synthesized glycogen aqueous solution prepared as described in the preparation examples (weight average degree of polymerization of about 7000 kDa, 2 mg / mL) (ESG group) < This aqueous solution is prepared by dissolving 200 mg of enzyme-synthesized glycogen in 100 ml of sterilized water (orally drinking), and after 1 week, the mouse's teeth are removed and re-implanted at the same site to recover the dental pulp healing, ivory By examining the status of blast differentiation, it is possible to confirm whether there is a healing promoting effect when enzyme-synthesized glycogen is used.

(Example 6: Promotion of formation of tooth germ in organ culture)
An ICR mouse (CLEA Japan) was made pregnant, and a mouse fetus extracted at 16.5 gestation was used as the fetus of this example. Specifically, the mouse fetus on the 15th day of gestation was obtained from CLEA Japan and used in this example. Mandibular first and second molar tooth germs were collected from the fetus and cultured in vitro. More specifically, tooth germ organ culture was performed using the Trowell method in a DMEM medium containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 100 μg / ml ascorbic acid. ESGB (5000 kDa) prepared as described in the preparation examples was directly added to the medium so as to be 500 μg / mL, cultured for 4 days, and then the state of tooth germ formation was observed. ESG clearly promoted growth and formed large tooth germs (see FIG. 7). The average width of the first molar after the culture was 0.63 mm in the control group and 0.72 mm in the ESG group (both n = 7).

  From the above results, it was confirmed that the present invention has an effect on the promotion of tooth germ development. In addition, in this example, in addition to the bone formation or osteogenesis promotion shown in the above example, it is also clear that it has an action of promoting the development of only the tooth germ part, and the bone formation or osteogenesis of the surrounding bone In addition to promotion etc., it has been shown to comprehensively promote tooth germ development.

(Example 7: Measurement of molecular weight range, cell level experiment)
Cell proliferation experiments and gene expression studies using an osteoblast-like cell line (MC3T3-E1) in the same manner as in Examples 1, 2, and 3, using enzyme-synthesized glycogen having an average molecular weight of about 2000 kDa, about 7000 kDa, and about 12000 kDa. Investigate calcification promoting effect. In this way, it can be confirmed whether or not there is a significant effect when enzyme-synthesized glycogen of about 2000 kDa, about 7000 kDa and about 12000 kDa is used.

(Example 8: Measurement of molecular weight range, animal experiment)
Experiments similar to Example 4 are performed using enzyme-synthesized glycogen having an average molecular weight of about 2000 kDa, about 7000 kDa, and about 12000 kDa, and the bone regeneration effect is examined. As a result, it can be confirmed whether or not there is a significant bone regeneration effect when enzyme-synthesized glycogen of about 2000 kDa, about 7000 kDa and about 12000 kDa is used.

(Example 9: Tablet formulation example)
For the pharmaceutical ingredient identified by the present invention, a tablet having the following composition is produced by a conventional method.
100 mg of the compound of the present invention
Lactose 60mg
Potato starch 30mg
Polyvinyl alcohol 2mg
Magnesium stearate 1mg
Tar pigment Trace amount.

  When glycogen is used using such a tablet, it can be confirmed whether there is a significant bone regeneration effect.

(Example 10: Formulation example of injection)
Sodium chloride 0.9g
Enzyme-synthesized glycogen 5g
100 ml of purified water
The above solid components are dissolved in purified water to give an injection.

  It can be confirmed whether or not there is a significant bone regeneration effect when glycogen is used using such an injection.

(Example 11: Dental application example)
In this embodiment, as a dental application example, a blood vessel or a cell is drawn from a surrounding tissue by being inserted into a wound, and a dosage form used for regeneration of a new tissue is carried out. The following preparation examples are shown.
Enzyme-synthesized glycogen 5mg
Saline 0.1ml
Collagen gel sponge appropriate amount
(Example 12: Application example of medical device)
In this embodiment, as an application example of a medical device, a dosage form of a bone prosthetic material is implemented. The following preparation examples are shown.
Enzyme-synthesized glycogen 100mg
β-type tricalcium phosphate (β-TCP) 190mg
Dextran 100mg
Saline 0.4ml
(Example 13: Example of food to be ingested by a patient for a certain period before treatment of teeth and bones)
In the present embodiment, as an application example of food, a food form that is ingested by a patient for a certain period before the treatment of teeth and bones is implemented. The following preparation examples are shown.

Enzyme-synthesized glycogen 5g
Citric acid 0.03g
Sucralose 0.005g
Perfume appropriate amount Purified water 100ml.

(Example 14: Example of treatment adjuvant administered to affected area)
In this example, as an application example of the treatment auxiliary agent, a treatment auxiliary agent administered to the affected area is carried out. The following preparation examples are shown.

Sodium chloride 0.9g
Enzyme-synthesized glycogen 5g
100 ml of purified water.

(Example 15: Example of growth promoting additive in tooth germ organ culture for research use)
In this example, as an application example of the additive, an additive for promoting growth in tooth germ organ culture is carried out. The following preparation examples are shown.

Enzyme-synthesized glycogen 5mg
DMEM medium 8.9ml
Fetal calf serum 1ml
Penicillin Streptomycin 0.1ml
L ascorbic acid 1 mg.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

  This application claims priority to Japanese Patent Application No. 2010-207293 filed on September 15, 2010, the entire contents of which are specifically described in this specification. It should be understood that this should be incorporated as part of the specification as if it were.

  The present invention has demonstrated that certain glycogens are useful for bone formation. The discovery that such a biocompatible material is useful for bone formation is expected to be applied in the pharmaceutical industry, and in the field of medicine and the like that can achieve effects not found in the prior art. There is usability.

SEQ ID NO: 1: PCR primer used in Example 2 (ALP-Forward)
SEQ ID NO: 2: PCR primer used in Example 2 (ALP-Reverse)
SEQ ID NO: 3: PCR primer used in Example 2 (OC-Forward)
SEQ ID NO: 4: PCR primer used in Example 2 (OC-Reverse)
SEQ ID NO: 5: PCR primer used in Example 2 (β-actin-Forward)
SEQ ID NO: 6: PCR primer used in Example 2 (β-actin-Reverse)

Claims (16)

Bone formation comprising glycogen weight average molecular weight of less than 8 700 kDa, osteogenic, tooth germ growth promotion, root formation promoting, and periodontal tissue composition for at least one selected from the group consisting of promoting regeneration met The composition is an additive, medical device or medicament . The composition of claim 1 wherein the weight average molecular weight is less than 8 000kDa. The composition of claim 1 wherein the weight average molecular weight is less than 3 000KDa or 7 000kDa. The composition of claim 1 wherein the weight average molecular weight of 4 900~ 7 000kDa. The composition according to any one of claims 1 to 4 , wherein the glycogen comprises enzymatically synthesized glycogen (ESG). The composition according to claim 1, wherein the glycogen includes enzyme-synthesized glycogen having a weight average molecular weight of 3 200 kDa to 7000 kDa . The composition according to any one of claims 1 to 6, wherein the bone formation or bone formation promotion is achieved by promoting osteoblast proliferation. A medicament for at least one selected from the group consisting of bone formation, bone formation promotion, tooth germ development promotion, tooth root formation promotion, and periodontal tissue regeneration promotion, comprising glycogen having a weight average molecular weight of less than 8 700 kDa. The medicament according to claim 8 , further comprising a pharmaceutically acceptable excipient. The medicament according to claim 8 or 9 , further comprising an agent for bone formation. The said pharmaceutical is administered over 1 day-10 days, The pharmaceutical as described in any one of Claims 8-10 characterized by the above-mentioned. A medical device for at least one selected from the group consisting of bone formation, bone formation promotion, tooth germ development promotion, tooth root formation promotion, and periodontal tissue regeneration promotion, comprising glycogen having a weight average molecular weight of less than 8 700 kDa. Bone cement, stents, catheters, a bone prosthetic material or implant, a medical device of claim 1 2. A method for producing a medicament for at least one selected from the group consisting of bone formation, bone formation promotion, tooth germ development promotion, tooth root formation promotion and periodontal tissue regeneration promotion, the weight average molecular weight being less than 8 700 kDa Mixing a glycogen which is a pharmaceutically acceptable excipient. A method for producing a medical device for at least one selected from the group consisting of bone formation, bone formation promotion, tooth germ development promotion, tooth root formation promotion, and periodontal tissue regeneration promotion, having a weight average molecular weight of 8 700 kDa Including the step of including a glycogen that is less than or equal to the medical device or the material of the medical device. Bone formation, bone formation promoting, tooth germ growth promotion, root formation promoting, and at least for one of the added vulcanizing agent is selected from the group consisting of periodontal tissue regeneration-promoting, for the production of medical devices or pharmaceutical, weight average Use of glycogen having a molecular weight of less than 8 700 kDa.