Bone formation composition and its uses

Abstract

Provided is a composition having a cell-derived physiological activity. The osteogenic composition according to the present invention contains a processed product of megakaryocytes or a culture thereof.

Description

[Technical Field]

[0001] This invention relates to a bone-forming composition and its uses. [Background technology]

[0002] As therapeutic agents for regenerative medicine, attempts are being made to develop cells that promote tissue regeneration, such as mesenchymal stem cells, as cell therapies. Furthermore, it is believed that these tissue regeneration-promoting cells accelerate tissue regeneration by releasing physiologically active proteins such as growth factors.

[0003] Furthermore, in regenerative medicine in dentistry, platelet-rich plasma (PRP) is used in bone augmentation (regeneration) therapy, etc. However, because PRP is prepared by separation and centrifugal concentration from isolated human blood, the platelet count is not standardized, and its efficacy is unclear. [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] Therefore, the present invention aims to provide a composition having cell-derived osteogenic activity. [Means for solving the problem]

[0005] To achieve the above objective, the bone-forming composition of the present invention (hereinafter also referred to as "the composition") includes a processed product of megakaryocytes or their cultures.

[0006] The bone formation kit of the present invention (hereinafter also referred to as the "kit") comprises a bone formation composition and a bone formation scaffold material, wherein the bone formation composition is the bone formation composition of the present invention. [Effects of the Invention]

[0007] According to the present invention, a composition having cell-derived osteogenic activity can be provided. [Brief explanation of the drawing]

[0008]

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[0009] <Osteogenic composition> The bone-forming composition of the present invention comprises, as described above, a processed product of megakaryocytes or their culture. The composition of the present invention is characterized by comprising a processed product of megakaryocytes or their culture, and other components and conditions are not particularly limited. According to the composition of the present invention, for example, a composition having cell-derived physiological activity can be provided. According to the composition of the present invention, for example, bone formation can be induced by promoting differentiation into osteoblasts.

[0010] In the present invention, "bone formation" may mean any of the following: an increase in existing bone, i.e., "bone augmentation" (bone regeneration); the formation of new bone, i.e., "bone regeneration"; or the recovery of lost (defective) bone or its portion, i.e., "bone regeneration". For this reason, the bone formation composition of the present invention may also be called a bone augmentation (bone regeneration) composition, a bone regeneration composition, and / or a bone regeneration composition.

[0011] The compositions of the present invention are presumed to induce bone formation by promoting, accelerating (promoting induction), enhancing, strengthening, and / or reinforcing the differentiation induction of osteoblasts, as described later. For this reason, the compositions of the present invention can also be described as compositions that promote, accelerate (promoting induction), enhance, strengthen, and / or reinforcing the differentiation induction of osteoblasts, or as compositions that induce, promote, accelerate (promoting induction), enhance, strengthen, and / or reinforcing bone formation.

[0012] In this invention, "megakaryocyte" refers to the largest cell present in the bone marrow in living organisms, and means a cell that releases platelets or has equivalent functions. The aforementioned cell having equivalent functions means a cell that has the ability to produce platelets. In this invention, megakaryocytes may be megakaryocytes before multinucleation (polyploidization), i.e., immature megakaryocytes or megakaryocytes in the proliferative phase, or megakaryocytes after multinucleation (multinucleated megakaryocytes). Specifically, the megakaryocyte may be, for example, a promegakaryblast, megakaryblast, promegakaryocyte, or mature megakaryocyte. The number of chromosome sets in the post-multinucleated megakaryocyte should be greater than 2 sets, and specifically, 16 to 32 sets.

[0013] The origin of the megakaryocytes is not particularly limited, but examples include humans and non-human animals. Examples of non-human animals include primates such as monkeys, gorillas, chimpanzees, and marmosets, as well as mice, rats, dogs, cats, rabbits, sheep, horses, and guinea pigs. The origin of the megakaryocytes may be the same as or different from the target population of the composition of the present invention.

[0014] In the present invention, megakaryocytes can be identified by cell surface markers. When the megakaryocytes are of human origin, the cell surface markers include CD41a, CD42a, and CD42b. That is, the megakaryocytes are cells that are positive for CD41a, CD42a, and CD42b. When the megakaryocytes are of human origin, the cell surface markers may be at least one selected from the group consisting of, for example, CD9, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD123, CD131, and CD203c.

[0015] The megakaryocytes may be megakaryocytes isolated from living organisms, or megakaryocytes induced from cells less differentiated than megakaryocytes, such as pluripotent cells (hereinafter also referred to as "progenitor cells"). The term "cells less differentiated than megakaryocytes" means cells that have the ability to differentiate into megakaryocytes.

[0016] If the megakaryocytes are megakaryocytes isolated from a living organism, they can be isolated from bone marrow, for example, since they are present in bone marrow. In this case, the megakaryocytes may also include other cells of living origin.

[0017] If the megakaryocyte is a megakaryocyte induced from a progenitor cell, then the megakaryocyte is as described below. in vitro They can be induced by [method]. In this case, the megakaryocytes may include the progenitor cells. Examples of the progenitor cells include hematopoietic stem cells, hematopoietic progenitor cells, CD34-positive cells, megakaryocyte-erythroid progenitor cells (MEPs), megakaryocyte progenitor cells, etc. The progenitor cells may be isolated from bone marrow, umbilical cord blood, peripheral blood, etc., or induced from pluripotent cells such as ES cells (embryonic stem cells), induced pluripotent stem cells (iPS cells), nuclear transplantation ES cells (ntES cells), germline stem cells, somatic stem cells, embryonic tumor cells, etc.

[0018] When the megakaryocytes are megakaryocytes induced from progenitor cells, immortalized megakaryocytes are preferred. Compared to megakaryocytes induced by other megakaryocyte induction methods, for example, immortalized megakaryocytes exhibit higher homogeneity in the differentiation stage of the cells, thus suppressing variations in the component composition of the resulting processed product. The immortalized megakaryocytes are, for example, megakaryocytes induced by introducing oncogenes and Polycomb genes, or oncogenes, Polycomb genes, and apoptosis suppressor genes, into the progenitor cells, as described later.

[0019] The aforementioned "oncogenes" refer to genes capable of inducing cell carcinogenesis in living organisms, and examples include MYC family genes such as c-MYC, N-MYC, and L-MYC, SRC family genes, RAS family genes, RAF family genes, protein kinase family genes such as c-kit (CD117), PDGFR (platelet growth factor receptor), and Abl (Abelson murine leukemia viral oncogene homolog).

[0020] The aforementioned "Polycomb genes" refer to genes known to negatively regulate CDKN2a (cyclin-dependent kinase inhibitor 2A, INK4a / ARF) and function to avoid cellular senescence (see references 1-3 below). Specific examples of Polycomb genes include, for instance, BMI1 (Polycomb complex protein BMI-1, polycomb group RING finger protein 4 (PCGF4), RING finger protein 51 (RNF51)), Mel18 (Polycomb group RING finger protein 2), Ring (Ring Finger Protein) 1a / b, Phc (Polyhomeotic Homolog) 1 / 2 / 3, Cbx (Chromobox) 2 / 4 / 6 / 7 / 8, Ezh2 (Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit), Eed (Embryonic Ectoderm Development), Suz12 (SUZ12 Polycomb Repressive Complex 2 Subunit), HADC (Histone deacetylases), Dnmt (DNA (cytosine-5)-methyltransferase) 1 / 3a / 3b, etc. Reference 1: Hideyuki Oguro et al., "Control of stem cell aging by Polycomb group protein complex," Regenerative Medicine, 2007, Vol. 6, No. 4, pp. 26-32. Reference 2: Jesus Gil et.al, “Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all”, Nature Reviews Molecular Cell Biology, 2007, vol.7, pages 667-677 Reference 3: Soo-Hyun Kim et.al., “Absence of p16 INK4a and truncation of ARF tumor suppressors in chickens”, PNAS, 2003, vol.100, No.1, pages 211-216

[0021] The aforementioned "apoptosis suppressor genes" refer to genes that have the function of suppressing apoptosis in cells, and examples include BCL2 (B-cell lymphoma 2), Bcl-xL (B-cell lymphoma-extra large), Survivin (Baculoviral IAP Repeat Containing 5), and MCL1 (BCL2 Family Apoptosis Regulator).

[0022] The immortalized megakaryocytes are preferably megakaryocytes containing exogenous BMI1, MYC, and Bcl-xL genes. "Exogenous" means introduced into the cell from outside the cell. The exogenous genes may be located on the cell's chromosomes, or in the nucleus or cytoplasm. The exogenous genes can be detected, for example, by measuring their number. If the genes are on autosomes, there are two copies of the gene on each autosome, resulting in two copies in a single cell. Therefore, if the exogenous genes are absent, two copies of the gene will be detected in a single cell. On the other hand, if the exogenous genes are present, three or more copies will be detected in a single cell. In this case, the exogenous genes can be detected, for example, using PCR with primers, probes, or a combination thereof. If the exogenous genes have a tag sequence or a selection marker, detection of the exogenous genes may be performed by detecting the tag sequence or the selection marker. Furthermore, the exogenous genes can be detected, for example, against the protein translated from the gene using an antibody.

[0023] The megakaryocyte culture is, for example, a culture produced by culturing the megakaryocytes. The megakaryocyte culture can be carried out, for example, by culturing the megakaryocytes in the presence of a culture medium, as described later.

[0024] Since the megakaryocyte culture is obtained by culturing the megakaryocytes, it is, for example, a mixture containing the megakaryocytes and platelets produced from the megakaryocytes as cellular components. The cellular components mean cells and platelets. As mentioned above, the megakaryocytes can be induced from cells that are less differentiated than the megakaryocytes. Therefore, if the megakaryocytes used to produce the megakaryocyte culture include megakaryocytes induced from cells that are less differentiated than the megakaryocytes, the megakaryocyte culture may also contain cells that are less differentiated than the megakaryocytes.

[0025] In the present invention, the megakaryocyte culture may be the culture obtained by culturing the megakaryocytes itself, or it may be a processed version of the culture. Examples of processing the culture include removing the liquid fraction, extracting the cellular component fraction, and changing the composition of cellular components including platelets. Examples of changing the composition of cellular components include removing cells and / or platelets from the mixture, extracting cells and / or platelets from the mixture, and adding cells and / or platelets to the mixture.

[0026] The term "platelet" refers to a cellular component in the blood that is positive for CD41a and CD42b. For example, platelets lack a nucleus and are smaller in size compared to megakaryocytes. Therefore, platelets and megakaryocytes can be distinguished, for example, by the presence or absence of a nucleus and / or size. Platelets are known to play an important role in thrombus formation and hemostasis, and are also involved in tissue regeneration after injury and the pathophysiology of inflammation. Furthermore, when platelets are activated by bleeding or other factors, receptors for cell adhesion factors such as Integrin αIIBβ3 (glycoprotein IIb / IIIa; a complex of CD41a and CD61) are expressed on their membranes. When platelets are activated, they aggregate, and fibrin coagulates due to various blood coagulation factors released from the platelets, forming a thrombus and promoting hemostasis. In this invention, the origin of the platelets is the same as that of the megakaryocytes.

[0027] In the present invention, the processed product may be prepared from megakaryocytes or from a culture of megakaryocytes. Furthermore, when prepared from a culture of megakaryocytes, the culture of megakaryocytes may be processed. Specifically, the processed product may be, for example, a processed product of the cell fraction of the megakaryocytes or their culture, or a processed product of a processed product obtained by processing the megakaryocytes or their culture. The processing in the preparation of the processed product is not particularly limited and may include, for example, processing to change the density of cellular components such as concentration processing; extraction processing to extract cellular components such as drying processing, freezing processing, freeze-drying processing, solvent processing, surfactant processing, enzyme processing, and protein fraction extraction processing; crushing processing such as grinding processing and pulverization processing; and so on. Specific examples of the processed products include, for example, extracts from concentrates, dried products, frozen products, freeze-dried products, solvent-treated products, surfactant-treated products, enzyme-treated products, protein fractions, sonicated products, etc. of megakaryocytes or their cultures; crushed products such as ground products and pulverized products; extracts from concentrates, dried products, frozen products, freeze-dried products, solvent-treated products, surfactant-treated products, enzyme-treated products, protein fractions, sonicated products of the cell fractions of megakaryocytes or their cultures; crushed products such as ground products and pulverized products; and so on. The processed product may consist of one type of processed product or a mixture of two or more types of processed products. The mixture is not particularly limited and can be a mixture of any combination and ratio of processed products.

[0028] The processed product includes, for example, at least one of one or more growth factors and growth factor receptors. Furthermore, the processed product has physiological activities such as cell proliferation-promoting activity and osteoblast differentiation-promoting activity. Moreover, the processed product can be produced, for example, by processing the megakaryocytes or their cultures, as described above. Therefore, in the present invention, the processed product can be defined, for example, using the following conditions (1) to (3). The processed product may be defined by any of the following conditions (1) to (3), by a combination of conditions, or by all of the conditions. As a specific example, the processed product can be defined, for example, by a combination of conditions. (conditions) (1) Content of growth factors and / or growth factor receptors; (2) The physiological activity of the treated product; (3) Method for manufacturing processed products (Combination of conditions) Condition (1), (2), or (3); Conditions (1) and (2), condition (1) and (3), or condition (2) and (3); Conditions (1), (2), and (3)

[0029] (1) Condition (1) Condition (1), as described above, is a condition relating to the content of growth factors and / or growth factor receptors. Condition (1) may specify the content of the growth factors or the content of the growth factor receptors, or it may specify the content of the growth factors and the content of the growth factor receptors. Furthermore, the growth factors used in the provision of condition (1) may be one type or two or more types. The growth factor receptors used in the provision of condition (1) may be one type or two or more types.

[0030] In the above condition (1), the growth factors include basic fibroblast growth factor (bFGF), insulin-like growth factor-binding protein-1 (IGFBP-1), insulin-like growth factor-binding protein-2 (IGFBP-2), insulin-like growth factor-binding protein-3 (IGFBP-3), insulin-like growth factor-binding protein-6 (IGFBP-6), placental growth factor (PIGF), vascular endothelial growth factor (VEGF), endocrine gland-derived vascular endothelial growth factor (EG-VEGF), differentiation growth factor-15 (GDF-15), amphiregulin (AR), bone morphogenetic protein-5 (BMP-5), bone morphogenetic protein-7 (BMP-7), hepatic growth factor (HGF), TGFβ1 (transforming growth factor β1), etc.

[0031] In the above condition (1), the growth factor receptors include stem cell factor receptors (SCFR), epidermal growth factor receptors (EGFR), vascular endothelial growth factor receptor 2 (VEGFR2), and the like.

[0032] The aforementioned content may be, for example, the weight of each growth factor and each growth factor receptor in the processed product, or the weight of each growth factor and each growth factor receptor relative to the total protein weight of the processed product (content per total protein), but the latter is preferred.

[0033] The total protein weight can be determined, for example, by the BCA protein quantification method. The BCA protein quantification method is a protein quantification method that utilizes the coordination bond between a monovalent copper ion and two molecules of bicinchoninic acid. The sample used for the BCA protein quantification method preferably does not contain, for example, reducing agents and / or copper ion chelating agents. The BCA protein quantification method can be carried out in accordance with Reference 4 below, and commercially available kits may be used, for example. As a kit for the BCA protein quantification method, the Pierce® BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific, Inc.) can be used. Reference 4: Shunji Hase et al., "Experimental Methods in Protein Science: Starting from Simple Principles 1 - Making Proteins: Extraction, Purification, and Synthesis," Kagaku Dojin, December 13, 2008.

[0034] The total protein concentration in the treated product can be appropriately set, for example, by the number of cells used in the treatment and the volume of solvent in the treated product. The total protein concentration in the treated product can be relatively increased, for example, by increasing the number of cells used in the treatment, decreasing the volume of solvent in the treated product, or by extracting from megakaryocytes. Conversely, the total protein concentration in the treated product can be relatively decreased, for example, by reducing the number of cells used in the treatment, increasing the volume of solvent in the treated product, or by extracting from a megakaryocyte culture. As a specific example, the number of cells used in the treatment is 1 × 10⁻⁶. 8 The sample is a cell, and when the volume of solvent in the treated product is 100 μl, the total protein concentration in the treated product is, for example, 0.1 to 200 mg / ml. The solvent is, for example, an aqueous solvent as described later.

[0035] The weights of the growth factors and growth factor receptors can be determined, for example, by the sandwich ELISA method. The sandwich ELISA method can be carried out in accordance with Reference 5 below, and commercially available kits may be used, for example. Examples of sandwich ELISA kits include Quantibody® Human Growth Factor Array 1 (manufactured by RayBiotech). Reference 5: Biochemical Measurement Research Group (ed.), "Immunoassays: From Basics to Advanced Techniques," Kodansha, December 20, 2014.

[0036] Examples of the content of the growth factor and the growth factor receptor in the treated product include the following:

[0037] When the growth factor is bFGF, the processed product contains, for example, 2,000 to 20,000 pg, 5,000 to 20,000 pg, or 10,000 to 20,000 pg of bFGF per 1 mg of total protein. By containing bFGF, the processed product exhibits cell proliferation-promoting activity and osteoblast differentiation-promoting activity, as described below.

[0038] If the growth factor is IGFBP-1, the processed product contains, for example, 0-200 pg, 0.01-200 pg, 0.01-100 pg, or 0.01-50 pg of IGFBP-1 per 1 mg of total protein.

[0039] If the growth factor is IGFBP-2, the processed product contains, for example, 8,000 to 80,000 pg, 10,000 to 80,000 pg, or 20,000 to 80,000 pg of IGFBP-2 per 1 mg of total protein.

[0040] If the growth factor is PIGF, the processed product contains, for example, 1 to 60 pg, 1 to 30 pg, or 1 to 20 pg of PIGF per 1 mg of total protein.

[0041] If the growth factor is VEGF, the processed product contains, for example, 20-800 pg, 20-600 pg, or 20-400 pg of VEGF per 1 mg of total protein.

[0042] If the growth factor is GDF-15, the processed product contains, for example, 1,000 to 10,000 pg, 1,000 to 5,000 pg, or 2,000 to 5,000 pg of GDF-15 per 1 mg of total protein.

[0043] If the growth factor is AR, the processed product contains, for example, 0-16 pg, 0.01-16 pg, 0.1-16 pg, or 1-16 pg of AR per 1 mg of total protein.

[0044] If the growth factor is HGF, the processed product contains, for example, 0-100 pg, 0.01-100 pg, 0.01-50 pg, or 0.01-30 pg of HGF per 1 mg of total protein.

[0045] If the growth factor is BMP-7, the processed product contains, for example, 0 to 1000 pg or 0.01 to 1000 pg of BMP-7 per 1 mg of total protein.

[0046] If the growth factor receptor is SCFR, the processed product contains, for example, 200-2000 pg, 300-1500 pg, or 400-1000 pg of SCFR per 1 mg of total protein.

[0047] If the growth factor receptor is EGFR, the processed product contains, for example, 0-60 pg, 0.01-60 pg, 1-50 pg, 1-45 pg, or 10-40 pg of EGFR per 1 mg of total protein.

[0048] When the growth factor receptor is VEGFR2, the processed product contains, for example, 20-400 pg, 50-350 pg, or 100-300 pg of VEGFR2 per 1 mg of total protein.

[0049] As described above, condition (1) may be defined by the content of one or more types of growth factors, or by the content of one or more types of growth factor receptors, or by any combination of these contents. In this case, condition (1) is defined by at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve conditions selected from the group consisting of the following conditions (A1) to (A9) and (B1) to (B3). As specific examples, the following combinations of growth factor content and / or growth factor receptor content can be exemplified. (Combination of growth factor content and / or growth factor receptor content) One of the following conditions applies: (A1)~(A9) and (B1)~(B3): (A1) bFGF content, (A2) IGFBP-1 content, (A3) IGFBP-2 content, (A4) PIGF content, (A6) VEGF content, (A6) GDF-15 content, (A7) AR content, (A8) HGF content, (A9) BMP-7 content, (B1) SCFR content, (B2) EGFR content, or (B3) VEGFR2 content; Any two of the following conditions: (A1)~(A9) and (B1)~(B3): (A1) and (A2), (A1) and (A3), (A1) and (A4), (A1) and (A5), (A1) and (A6), (A1) and (A7), (A1) and (A8), (A1) and (A9), (A1) and (B1), (A1) and (B2), (A1) and (B3), (A2) and (A3), (A2) and (A4), (A2) and (A5), (A2) and (A6), (A2) and (A7), (A2) and (A8), (A2) and (A9), (A2) and (B1), (A2) and (B2), (A2) and (B3), (A3) and (A4), (A3) and (A5), (A3) and (A6), (A3) and (A7), (A3) and (A8), (A3) and (A9), (A3) and (B1), (A3) and (B2), (A3) and (B3), (A4) and (A5), (A4) and (A6), (A4) and (A7), (A4) and (A8), (A4) and (A9), (A4) and (B1), (A4) and (B2), (A4) and (B3), (A5) and (A6), (A5) and (A7), (A5) and (A8), (A5) and (A9), (A5) and (B1), (A5) and (B2), (A5) and (B3), (A6) and (A7), (A6) and (A8), (A6) and (A9), (A6) and (B1), (A6) and (B2), (A6) and (B3), (A7) and (A8), (A7) and (A9), (A7) and (B1), (A7) and (B2), (A7) and (B3), (A8) and (A9), (A8) and (B1), (A8) and (B2), (A8) and (B3), (A9) and (B1), (A9) and (B2), (A9) and (B3), (B1) and (B2), (B1) and (B3), or (B2) and (B3).

[0050] The content per total protein may be adjusted, for example, by adding or removing other proteins other than the growth factor and growth factor receptor, depending on the intended use of the composition of the present invention. Examples of these other proteins include proteins that do not affect the activity of the growth factor and growth factor receptor, such as serum albumin such as human serum albumin and gamma globulin such as human gamma globulin. Examples of protein removal include removal using a column or removal using an antibody.

[0051] (2) Condition (2) Condition (2), as described above, is a condition relating to the physiological activity of the treated product. In condition (2), the physiological activity of the treated product means, for example, activity that regulates the function of cells, tissues, or organs. Examples of cell functions include proliferation and differentiation.

[0052] The physiological activity of the treated product may include, for example, cell proliferation-promoting activity, differentiation-promoting activity into osteoblasts, and differentiation-promoting activity from bone progenitor cells into osteoblasts. In the cell proliferation-promoting activity, the cells are not particularly limited and may include, for example, mesenchymal stem cells. In the differentiation-promoting activity into osteoblasts, the bone progenitor cells may include, for example, mesenchymal stem cells, osteoblasts, etc. The treated product may have, for example, one activity or multiple activities.

[0053] The mesenchymal stem cells are cells that have the ability to self-renew and differentiate into bone, cartilage, and adipocytes. The mesenchymal stem cells can be identified by cell surface markers. For example, the mesenchymal stem cells are positive for CD73, CD90, and CD105, and negative for CD14, CD34, and CD45.

[0054] The osteoblasts are cells capable of synthesizing and secreting bone matrix. These osteoblasts can be identified, for example, as alkaline phosphatase-positive and osteocalcin-positive cells.

[0055] The proliferation-promoting activity of the cells may be such that, for example, the proliferative capacity of the cells is improved compared to a control group that is similar except that the composition of the present invention is not added, and for example, the proliferative capacity of the cells may be decreased from the start. In this case, the "proliferation-promoting activity" can also be said to be, for example, the suppression of the decrease in proliferative activity. As a specific example, the proliferative activity of the mesenchymal stem cells decreases with each passage. Since the decrease in the proliferative activity of the mesenchymal stem cells can be suppressed according to the composition of the present invention, for example, it can be said that the composition of the present invention exhibits proliferation-promoting activity. The proliferation-promoting activity of the cells can be measured, for example, under culture conditions in which the target cells proliferate. The culture conditions can be appropriately set, for example, according to the type of cell.

[0056] The differentiation-promoting activity to osteoblasts described above should, for example, be sufficient if the differentiation ability to osteoblasts is improved compared to a control group that is similar except that the composition of the present invention is not added. The improvement in differentiation ability may mean, for example, that the differentiation rate to osteoblasts is increased, or that the proportion of cells that differentiate into osteoblasts is increased. For example, refer to Japanese Patent Application Publication No. 2012-120529 for details on the differentiation to osteoblasts. As a specific example, a method for inducing osteoblasts from mesenchymal stem cells can be carried out, for example, based on Example 2 described below.

[0057] (3) Conditions (3) Condition (3), as described above, is a condition relating to the method of manufacturing the treated product. For the method of manufacturing the treated product in the composition of the present invention, refer to the description of the method of manufacturing the composition of the present invention described later.

[0058] The composition of the present invention, in vitro You can use it as well, in vivo It may be used in this way.

[0059] The composition of the present invention in vitro When used in this manner, the target of administration may be, for example, cells, tissues, organs, etc., and the cells may be, for example, cells collected from living organisms, cultured cells, etc.

[0060] The composition of the present invention in vivo When used in this manner, the target of administration may be, for example, humans or non-human animals other than humans. Examples of non-human animals include mice, rats, rabbits, dogs, sheep, horses, cats, goats, monkeys, guinea pigs, etc.

[0061] The conditions for use (administration conditions) of the composition of the present invention are not particularly limited, and the administration form, timing of administration, dosage, etc. can be appropriately set depending on the type of target to be administered, etc.

[0062] The composition of the present invention in vitroWhen used, the composition of the present invention can be used, for example, by adding it to the culture medium of the target progenitor cells. The composition of the present invention may be added, for example, to the maintenance medium used to maintain the progenitor cells, or to the culture medium used to differentiate the progenitor cells into osteoblasts, or to both the maintenance medium and the differentiation medium. According to the composition of the present invention, for example, by maintaining the progenitor cells in a maintenance medium containing the composition, differentiation can be promoted when the progenitor cells are subsequently differentiated into osteoblasts. The final concentration of total protein derived from the composition of the present invention in the maintenance medium is, for example, 10-1000 μg / ml, 10-500 μg / ml, or 10-300 μg / ml. The final concentration of total protein derived from the composition of the present invention in the differentiation medium is, for example, 10-1000 μg / ml, 10-500 μg / ml, or 10-300 μg / ml.

[0063] The composition of the present invention in vivo When used in a clinical setting, the dosage can be appropriately determined depending on the type of target, symptoms, age, and method of administration. For example, when administered to mice, the daily dose of the composition, and the total amount of protein derived from the composition, are not particularly limited and can be appropriately set depending on the application, for example, but a specific example is 10 ng to 100 mg. When administered to humans, the daily dose of the composition, and the total amount of protein derived from the composition, are not particularly limited and can be appropriately set depending on the application, for example, but a specific example is 10 ng to 100 g. The number of daily doses can be, for example, 1 to 5 times, 1 to 3 times, 1 time or 2 times.

[0064] The dosage form of the composition of the present invention is not particularly limited. in vivo When administering the drug, it may be administered orally or parenterally. Parenteral administration may include, for example, intravenous injection, intramuscular injection, transdermal administration, subcutaneous administration, intradermal administration, enteral administration, rectal administration, vaginal administration, nasal administration, pulmonary administration, intraperitoneal administration, local administration, etc.

[0065] The dosage form of the composition of the present invention is not particularly limited and can be appropriately determined, for example, depending on the administration method. The dosage form may be, for example, liquid or solid.

[0066] The compositions of the present invention may, for example, optionally contain additives. The additives are preferably pharmaceutically acceptable additives or pharmaceutically acceptable carriers.

[0067] The method for producing the composition of the present invention (hereinafter also referred to as the "production method") includes a processing step for processing megakaryocytes or their cultures. In the production method of the present invention, the object of processing in the processing step is megakaryocytes or their cultures. For this reason, the production method of the present invention may include, prior to the processing step, a megakaryocyte induction step for inducing megakaryocytes from cells less differentiated than megakaryocytes and / or a production step for producing megakaryocyte cultures.

[0068] In the megakaryocyte induction step, the method for inducing megakaryocytes is not particularly limited and can be carried out by known induction methods. For example, the method for inducing megakaryocytes can be, for example, the method for inducing immortalized megakaryocytes described in International Publication No. 2011 / 034073 (US Patent Application Publication No. 2012 / 0238023), International Publication No. 2012 / 157586 (US Patent Application Publication No. 2014 / 0127815), International Publication No. 2014 / 123242 (US Patent Application Publication No. 2016 / 0002599), etc.; the method for inducing megakaryocytes described in Reference 6 below; etc., which are incorporated herein by reference as constituting a part of this specification. For example, in the megakaryocyte induction step, for example, the oncogene and the Polycomb gene may be forced to be expressed in cells that are less differentiated than the megakaryocytes. As a result, in the megakaryocyte induction step, for example, immortalized megakaryocytes that proliferate indefinitely can be obtained. Furthermore, for example, by releasing the forced expression of the immortalized megakaryocytes, the immortalized megakaryocytes can be induced into multinucleated megakaryocytes and platelet production can be made. Alternatively, in the megakaryocyte induction step, for example, the apoptosis suppressor gene may be forced into the megakaryocyte precursor cells. As a result, in the megakaryocyte induction step, immortalized megakaryocytes can be obtained. Furthermore, for example, by releasing the forced expression of the immortalized megakaryocytes, multinucleated megakaryocytes can be induced from the immortalized megakaryocytes and platelet production can be made. Reference 6: Ann-Kathrin Borger et.al., “Generation of HLA-Universal iPSC-Derived Megakaryocytes and Platelets for Survival Under Refractoriness Conditions”, Mol. Med., 2016, vol. 22, pages 274-288

[0069] In the megakaryocyte induction step, for example, the oncogene, the Polycomb gene, and the apoptosis suppressor gene may be forcibly expressed. In this case, the forcible expression of the oncogene, the Polycomb gene, and the apoptosis suppressor gene may be performed simultaneously or separately. Specifically, in the megakaryocyte induction step, after forcibly expressing the oncogene and the Polycomb gene, the forcible expression may be released, and then the apoptosis suppressor gene may be forcibly expressed; or the oncogene, the Polycomb gene, and the apoptosis suppressor gene may be forcibly expressed; or the oncogene and the Polycomb gene may be forcibly expressed, and then the apoptosis suppressor gene may be expressed. As a result, the megakaryocyte induction step can yield the immortalized megakaryocytes. Furthermore, for example, by releasing the forcible expression of the immortalized megakaryocytes, multinucleated megakaryocytes can be induced from the immortalized megakaryocytes, and platelets can be produced.

[0070] The megakaryocyte induction step preferably includes, for example, a first expression step in which oncogenes and Polycomb genes are forcibly expressed in cells less differentiated than megakaryocytes, a second expression step in which apoptosis-suppressing genes such as the Bcl-xL gene are forcibly expressed in the undifferentiated cells, and a deactivation step in which all of the forcible expression is deactivated, since this can improve the efficiency of introducing each gene.

[0071] The forced expression and de-expression of each gene can be carried out by known methods, such as those described in International Publication No. 2011 / 034073, International Publication No. 2012 / 157586, International Publication No. 2014 / 123242, or Reference 7 below, or by similar methods, which are incorporated herein by reference as part of this specification. Specifically, the forced expression and de-expression of each gene can be carried out, for example, using a drug-responsive gene expression induction system. Examples of such gene expression induction systems include the Tet-on® system and the Tet-off® system. When using the Tet-on system, for example, in the forced expression step, culture is carried out in the presence of a gene expression-inducing drug such as tetracycline or doxycycline, and in the de-expression step, culture is carried out in the absence of the drug. Reference 7: Nakamura S et al, “Expandable megakaryocyte cell lines enable clinically applicable generation of platelets from human induced pluripotent stem cells.”, Cell Stem Cell, 2014, vol.14, No.4, pages 535-548

[0072] Next, the production process involves producing a culture of megakaryocytes. This production process can be carried out, for example, by culturing the megakaryocytes in the presence of a culture medium. The culture of megakaryocytes may be carried out, for example, on feeder cells or without feeder cells. The megakaryocytes can be cultured without feeder cells, for example, because they can be cultured in suspension. The megakaryocyte culture includes, for example, the platelets.

[0073] The culture conditions for the megakaryocytes are not particularly limited, and the usual culture conditions for megakaryocytes can be used. For example, the culture temperature is, for instance, about 35 to 42°C, about 36 to 40°C, or about 37 to 39°C. The CO2 concentration is, for example, about 5 to 15%. The O2 concentration is, for example, about 15 to 25% or about 20%. The culture period is not particularly limited, for example, about 1 day to 2 weeks or about 4 to 8 days.

[0074] The culture medium is not particularly limited, and examples include known media suitable for platelet production from megakaryocytes and similar media. Specifically, the culture medium can be prepared using, for example, a culture medium used for animal cell culture as a base medium. Examples of the base medium include single media or mixtures thereof such as IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI1640 medium, Fischer's medium, Neurobasal® Medium (manufactured by Thermo Fisher Scientific). The culture medium may contain, for example, serum or plasma, or it may be a serum-free medium that does not contain these. Preferably, the serum and plasma are of the same origin as the megakaryocytes. Specifically, if the megakaryocytes are of human origin, it is preferable that the serum and plasma are also of human origin.

[0075] The culture medium may contain, for example, other components. These other components are not particularly limited and include, for example, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, monothioglycerol (MTG), lipids, amino acids (e.g., L-glutamine), ascorbic acid, heparin, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines, etc. These other components may be, for example, one type or two or more types. The cytokines are, for example, substances that promote the differentiation of hematopoietic cells, and specific examples include vascular endothelial growth factor (VEGF), thrombopoietin (TPO), various TPO-like substances, stem cell factor (SCF), ITS (insulin-transferrin-selenite) supplements, ADAM inhibitors, FLT inhibitors, WNT inhibitors, ROCK inhibitors, aromatic hydrocarbon receptor (AhR) inhibitors, etc. The culture medium is preferably an IMDM medium containing, for example, serum, insulin, transferrin, serine, thiolglycerol, ascorbic acid, and TPO. The culture medium may further contain, for example, SCF, and may further contain heparin. The concentrations of the other components are not particularly limited. The concentration of TPO is, for example, about 10 ng / ml to about 200 ng / ml, and about 50 ng / ml to about 100 ng / ml. The concentration of SCF is, for example, about 10 ng / ml to about 200 ng / ml, and about 50 ng / ml. The concentration of heparin is, for example, about 10 U / ml to about 100 U / ml, and about 25 U / ml. The culture medium may further contain, for example, phorbol ester (e.g., phorbol-12-myristo-13-acetate; PMA).

[0076] Next, the processing step involves processing the megakaryocytes or their culture. In this processing step, biomacromolecules such as proteins are extracted, for example, by disrupting the cell membranes of the cells contained in the megakaryocytes or their culture. Specific examples of the processing in this step are not particularly limited and include, for example, processing to change the density of cellular components, such as concentration; extraction processing to extract cellular components, such as drying, freezing, freeze-drying, solvent processing, surfactant processing, enzyme processing, protein fractionation extraction, and sonication; and crushing processing, such as grinding and pulverization. The processing performed in this step may be one type or two or more types. Furthermore, the processing may be performed once or two or more times.

[0077] The concentration treatment can be carried out, for example, by centrifugation of the megakaryocytes or their culture. The centrifugation conditions can be, for example, conditions that precipitate cells or platelets. The drying treatment can be carried out, for example, by drying the megakaryocytes or their culture with a dry spray, drum dryer, etc. The freeze-drying treatment can be carried out, for example, using a freeze-dryer. In the solvent treatment, the solvent is, for example, an organic solvent such as phenol or chloroform; or an aqueous solvent such as water, physiological saline, or buffer solution. When an aqueous solvent is used as the solvent, the solvent treatment is preferably carried out in combination with, for example, a surfactant treatment, an enzyme treatment, and / or sonication treatment described later. The solvent treatment can be carried out, for example, by mixing the megakaryocytes or their culture with the solvent. In the surfactant treatment, the surfactant may be, for example, an ionic surfactant such as sodium lauryl sulfate; a nonionic surfactant such as NP-40, Triton X-100, Tween 20, or n-Dodecyl-β-D-maltopyranoside; or an amphoteric surfactant such as CHAPS. The concentration of the surfactant may be, for example, a concentration capable of disrupting the cell membranes of the cellular components in the megakaryocyte or its culture. The surfactant treatment can be carried out, for example, by contacting the megakaryocyte or its culture with the surfactant in the presence of an aqueous solvent. The contact with the surfactant may be carried out, for example, at a temperature of about 0 to about 10°C. The aqueous solvent may be, for example, water, physiological saline, or a buffer solution. The enzyme in the enzyme treatment may be, for example, a peptidase or a protease. The enzyme treatment can be carried out, for example, by contacting the megakaryocyte or its culture with the enzyme in the presence of the aqueous solvent. The conditions for the enzyme treatment may be, for example, conditions under which the enzyme exhibits activity. The protein fraction extraction process can be carried out, for example, by applying osmotic shock, freeze-thaw cycles, etc., to the megakaryocytes or their culture. The sonication can be carried out, for example, using an ultrasonic generator. The conditions for the sonication can be, for example, conditions that disrupt cells.

[0078] The processing conditions and processing time in each processing step can be appropriately determined, for example, depending on the type of processing. Furthermore, in the processing step, the total protein concentration in the processed product can be adjusted, for example, by adjusting the amount of aqueous solvent.

[0079] Prior to the above treatment, the megakaryocytes or their culture may be processed (pre-treated). In this case, the megakaryocyte culture may be the culture obtained by culturing the megakaryocytes itself, or a processed mixture may be used. Examples of processing the culture include removing the liquid fraction, extracting the cellular component fraction, and changing the composition of cellular components including platelets. Examples of changing the composition of cellular components include removing cells and / or platelets from the mixture, extracting cells and / or platelets from the mixture, and adding cells and / or platelets to the mixture.

[0080] If the manufacturing method of the present invention includes the above-mentioned pretreatment, the manufacturing method of the present invention may also include a removal step of removing platelets from the megakaryocytes or their culture. In this case, the processing step is carried out using megakaryocytes or their culture from which the platelets have been removed, or the removed platelets, as the megakaryocytes or their culture. Megakaryocytes after platelet release have, for example, a high bFGF content. Therefore, the manufacturing method of the present invention can produce a composition with a high bFGF content by, for example, removing the platelets. By removing the platelets, the platelets can be separated from other cell fractions. Therefore, the removal step can also be called, for example, a platelet separation step or a platelet-to-other-cell component separation step. In the removal step, the method for separating platelets from the megakaryocyte culture can be carried out by known methods such as the method described in, for example, International Publication No. 2017 / 065280 (US Patent Application Publication No. 2018 / 282697), or by similar methods, which are incorporated herein by reference as forming part of this specification.

[0081] The platelet removal rate (separation rate) in the removal step is, for example, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. The platelet removal rate is, for example, 60-90%.

[0082] The manufacturing method of the present invention may include a storage step for storing the megakaryocytes or their culture, the megakaryocytes or their culture from which the platelets have been removed, or the removed platelets. Examples of storage in the storage step include refrigeration (about 1 to about 10°C) and freezing (about -200 to about -4°C). The storage period in the storage step is not particularly limited. Freezing is preferred because the storage step can, for example, also serve as the freezing treatment in the processing step.

[0083] The manufacturing method of the present invention can be carried out as follows, for example. However, the present invention is not limited in any way to the following examples. First, the culture medium containing the megakaryocytes or their culture is concentrated by centrifugation to concentrate the cellular components. The centrifugation treatment is, for example, under conditions in which the cellular components precipitate. Specifically, the centrifugation treatment can be carried out by centrifugation at 1000 to 3000 × g for 5 to 20 minutes. Next, the precipitate is collected after centrifugation and the obtained precipitate is subjected to a freezing treatment by rapid freezing. The freezing treatment can be carried out, for example, by contacting the precipitate with a liquefied gas such as liquid nitrogen. Furthermore, the precipitate after freezing is subjected to a surfactant treatment by dissolving it in an aqueous solvent containing the surfactant. The obtained solution is centrifuged to precipitate impurities. The centrifugation treatment is, for example, under conditions in which impurities such as cell membranes precipitate. As a specific example, the centrifugation process can be carried out by centrifugation at 10,000 to 20,000 × g for 3 to 10 minutes. After centrifugation, the protein is present in the supernatant, and the processed product can be obtained by recovering the supernatant as a protein fraction.

[0084] The compositions of the present invention can be used, for example, as osteoblast differentiation-promoting compositions, as described later. For methods of using the present invention, refer to the descriptions of cell proliferation-promoting compositions and osteoblast differentiation-promoting compositions described later. Furthermore, the compositions of the present invention are presumed to be suitably used for, for example, jawbone defects due to jawbone tumors, cysts, osteonecrosis, trauma, and skeletal disorders (ectodermal dysplasia, cleft lip and palate, etc.); alveolar bone defects such as periodontal disease or associated alveolar bone defects, dental caries or trauma; osteoporosis such as primary osteoporosis (postmenopausal osteoporosis, senile osteoporosis, etc.) and secondary osteoporosis (osteogenesis imperfecta, etc.); intractable fractures due to hyperthyroidism, Cushing's syndrome, hypogonadism, trauma, etc.; rib jointing by thoracotomy; bone defects by craniotomy; and systemic bone defects due to tumor resection or cystectomy.

[0085] <Osteoblast differentiation promoting composition> The osteoblast differentiation-promoting composition of the present invention comprises the composition of the present invention as described above. The differentiation-promoting composition of the present invention is characterized by comprising the composition of the present invention, and other components and conditions are not particularly limited. According to the differentiation-promoting composition of the present invention, differentiation into osteoblasts, in particular, differentiation from mesenchymal stem cells to osteoblasts can be promoted. The differentiation-promoting composition of the present invention can be described by reference to the description of the composition of the present invention described above.

[0086] The differentiation-promoting composition of the present invention, in vitro You can use it as well, in vivo It may be used in this way.

[0087] The differentiation-promoting composition of the present invention in vitro When used in this manner, the target of administration may be, for example, cells, tissues, organs, etc., and the cells may be, for example, cells collected from living organisms, cultured cells, etc.

[0088] The differentiation-promoting composition of the present invention in vivoWhen used in this manner, the target of administration may be, for example, humans or non-human animals other than humans. Examples of non-human animals include mice, rats, rabbits, dogs, sheep, horses, cats, goats, monkeys, guinea pigs, etc.

[0089] The conditions for use (administration conditions) of the differentiation-promoting composition of the present invention are not particularly limited, and the administration form, timing of administration, dosage, etc. can be appropriately set depending on the type of target to be administered, etc. The administration conditions of the differentiation-promoting composition of the present invention can be described by referring to the description of the composition of the present invention above.

[0090] The differentiation-promoting composition of the present invention in vitro When used, the differentiation-promoting composition of the present invention can be used, for example, by adding it to the culture medium of the target progenitor cells. The differentiation-promoting composition of the present invention may be added, for example, to the maintenance medium used to maintain the progenitor cells, or to the culture medium used to differentiate the progenitor cells into osteoblasts, or to both the maintenance medium and the differentiation medium. According to the differentiation-promoting composition of the present invention, for example, by maintaining the progenitor cells in a maintenance medium containing the differentiation-promoting composition, differentiation can be promoted when the progenitor cells are subsequently differentiated into osteoblasts. The final concentration of total protein derived from the differentiation-promoting composition of the present invention in the maintenance medium is, for example, 10-1000 μg / ml, 10-500 μg / ml, or 10-300 μg / ml. The final concentration of total protein derived from the differentiation-promoting composition of the present invention in the differentiation medium is, for example, 10-1000 μg / ml, 10-500 μg / ml, or 10-300 μg / ml.

[0091] The differentiation-promoting effect of progenitor cells on osteoblasts can be evaluated, for example, by determining whether the differentiation of mesenchymal stem cells into osteoblasts is promoted when mesenchymal stem cells and the test substance are present together, compared to the absence of the test substance (control group). Specifically, the differentiation-promoting effect on osteoblasts can be evaluated using the concentration of calcium ions in the culture medium of the cell group after the mesenchymal stem cells have been induced to differentiate into osteoblasts as an indicator. The method for inducing the differentiation of mesenchymal stem cells into osteoblasts and the method for measuring the calcium ion concentration can be carried out in accordance with Example 1(6) described below. Furthermore, in the above evaluation, for example, if the calcium concentration in the group coexisting with the test substance is 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less compared to the calcium concentration in the group without the test substance, then the test substance can be evaluated as having a differentiation-promoting effect on osteoblasts.

[0092] The differentiation-promoting composition of the present invention has, for example, the activity of promoting the differentiation of mesenchymal stem cells into osteoblasts, which suppresses the calcium concentration to 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less in the differentiation induction assay of mesenchymal stem cells into osteoblasts. The differentiation induction assay of mesenchymal stem cells into osteoblasts can be carried out in accordance with Example 1(6) described below.

[0093] The differentiation-promoting composition of the present invention in vivo When used in this context, the dosage can be appropriately determined based on factors such as the type of target, symptoms, age, and method of administration. For example, when administered to humans, the daily dose of the differentiation-promoting composition, and the total amount of protein derived from the differentiation-promoting composition, are not particularly limited and can be appropriately set according to their intended use. The number of daily doses can be, for example, 1 to 5 times, 1 to 3 times, 1 time, or 2 times.

[0094] The dosage form of the differentiation-promoting composition of the present invention is not particularly limited. in vivo When administering the drug, it may be administered orally or parenterally. Parenteral administration may include, for example, intravenous injection, intramuscular injection, transdermal administration, subcutaneous administration, intradermal administration, enteral administration, rectal administration, vaginal administration, nasal administration, pulmonary administration, intraperitoneal administration, local administration, etc.

[0095] The dosage form of the differentiation-promoting composition of the present invention is not particularly limited and can be appropriately determined, for example, depending on the administration method. The dosage form may be, for example, a liquid or a solid.

[0096] The differentiation-promoting composition of the present invention may, for example, contain additives as needed. The additives are preferably pharmaceutically acceptable additives or pharmaceutically acceptable carriers.

[0097] <Bone Formation Kit> The bone formation kit of the present invention comprises a bone formation composition and a bone formation scaffold material, wherein the bone formation composition is the bone formation composition of the present invention. The kit of the present invention is characterized by comprising the bone formation composition of the present invention, and other steps and conditions are not particularly limited. The kit of the present invention can easily induce bone formation by combining the composition of the present invention and the scaffold material. The kit of the present invention can be described by reference to the description of the composition and differentiation-promoting composition of the present invention.

[0098] In the kit of the present invention, the scaffold material can be, for example, a scaffold material used for bone or cartilage regeneration. The scaffold material can also be, for example, a material to which osteocytes or osteoblasts, or their precursor cells, can attach, proliferate, and / or differentiate. The scaffold material is, for example, a structure (structure) having a three-dimensional hollow structure or porous structure, and specific examples include porous bodies, porous supports, porous three-dimensional structures (structures), gels (hydrogels), sponge structures, etc.

[0099] Since the scaffolding material is implanted in a living body together with, for example, the composition of the present invention, it is preferable that it exhibits biocompatibility and / or biodegradability. "Biocompatibility" means, for example, that it has affinity with tissues and / or organs in a living body and does not cause foreign body reactions or rejection reactions. "Biodegradability" means, for example, that its constituent components can be broken down in a living body.

[0100] Examples of the components of the scaffolding material include proteins such as collagen, gelatin, albumin, keratin, fibrin, and fibroin; polysaccharides such as agarose, dextran, carboxymethylcellulose, xanthan gum, chitosan, chondroitin sulfate, heparin, hyaluronic acid, and alginic acid; and polyglycolic acid (PGA), polylactic acid (PLA), copolymers of polyglycolic acid and polylactic acid, polyhydroxybutyric acid, polydioxanone, polyethylene glycol (PEG), polycaprolactone, polybutylene succinate, calcium phosphate (e.g., β-tricalcium phosphate), calcium carbonate, hydroxyapatite, polyether ketone, and polyether ether ketone. The components may be used individually or in combination of multiple types.

[0101] The scaffolding material can be prepared, for example, by crosslinking or compounding the constituent components of the scaffolding material.

[0102] Examples of the aforementioned scaffolding materials include NeoBone (made of hydroxyapatite, manufactured by Aimdic MMT), APACERAM (registered trademark, made of hydroxyapatite, manufactured by HOYA Corporation), Bonark (made of collagen / calcium octaphosphate, manufactured by Toyobo Co., Ltd.), Osferion (made of βTCP, manufactured by Olympus Terumo Biomaterials Corporation), Cytotrans Granule (made of carbonate apatite, manufactured by GC Corporation), and Refit (made of collagen / apatite, manufactured by HOYA Corporation).

[0103] In the kit of the present invention, the composition and the scaffolding material may exist independently or as a single unit. In the latter case, the kit of the present invention may also be called, for example, a bone-forming scaffolding material. When the composition and the scaffolding material are a single unit, the composition may, for example, be adsorbed onto the surface of the scaffolding material or be incorporated within the scaffolding material.

[0104] The kit of the present invention may, for example, include a bioactive substance in addition to the above composition. The bioactive substance is, for example, a substance that promotes bone formation or the differentiation of osteoblasts or osteocytes. Examples of the bioactive substance include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), brain-derived neurotrophic factor (BDNF), growth and differentiation factor-5 (GDF5), erythropoiesis-promoting factor (EPO), transforming growth factor (TGF), and bone morphogenetic protein (BMP). The bioactive substance may be used individually or in combination of multiple types.

[0105] The kit of the present invention can be used, for example, by implanting (burial, embedding) the composition and scaffolding material at a desired location in the target of administration where bone formation is to be induced. Specifically, if the composition and the scaffolding material are separate in the kit of the present invention, it can be used by impregnating the scaffolding material with the composition and then implanting the scaffolding material. Alternatively, if the composition and the scaffolding material are separate in the kit of the present invention, it may be used by implanting the scaffolding material and then introducing the composition of the present invention to the implantation site. Furthermore, if the composition and the scaffolding material are integrated in the kit of the present invention, it can be used by implanting the scaffolding material.

[0106] In the kit of the present invention, the content of the composition can be set, for example, according to the dosage of the composition of the present invention.

[0107] <Composition to suppress the activation of immune cells> In another aspect, the present invention provides a composition having activation inhibitory activity for cell-derived immune cells. In this case, the immune cell activation inhibitory composition of the present invention (hereinafter also referred to as the "activation inhibitory composition") comprises a processed product of megakaryocytes or their cultures. The activation inhibitory composition of the present invention is characterized by comprising a processed product of megakaryocytes or their cultures, and other components and conditions are not particularly limited. According to the activation inhibitory composition of the present invention, for example, a composition having activation inhibitory activity for cell-derived immune cells can be provided. The activation inhibitory composition of the present invention can be described by reference to the descriptions of the compositions, differentiation-promoting compositions, and bone formation kits of the present invention.

[0108] The activation-inhibiting composition of the present invention can, for example, suppress the production of inflammatory cytokines in immune cells. For this reason, the activation-inhibiting composition of the present invention can also be called, for example, a composition for suppressing the production of inflammatory cytokines in immune cells.

[0109] In the present invention, "activation of immune cells" may be used to mean, for example, the induction of the expression of an activation marker in immune cells, or the induction of the expression of cytokines such as inflammatory cytokines. Examples of such inflammatory cytokines include TNF-α, IL-1β, IL-6, and IFN-γ.

[0110] The inhibitory effect on immune cell activation can be evaluated, for example, by determining whether the concentration of inflammatory cytokines in the culture medium after culturing the immune cells can be suppressed when the immune cells and the test substance are present together under conditions that activate the immune cells, compared to the absence of the test substance (control group). Specifically, the inhibitory effect on immune cell activation can be evaluated based on the rate of suppression of inflammatory cytokine production. The type of immune cell, the activation conditions, and the amount of inflammatory cytokine production can be measured in accordance with Example 5 described below. In the evaluation, for example, if the suppression rate of production of one or more cytokines is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more, the test substance can be evaluated as having an inhibitory effect on immune cell activation.

[0111] The activation-inhibiting composition of the present invention has an immune cell activation-inhibiting effect, for example, in the immune cell activation assay, which suppresses the concentration of at least one inflammatory cytokine to 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less compared to the control group. The immune cell activation assay can be carried out in accordance with Example 5 described below.

[0112] In the present invention, the immune cells include, for example, lymphocytes such as T cells; macrophages such as inflammatory macrophages; and so on.

[0113] The activation inhibitory composition of the present invention is in vitro You can use it as well, in vivo It may be used in this way.

[0114] The activation-inhibiting composition of the present invention in vitroWhen used in this manner, the target of administration may be, for example, cells, tissues, organs, etc., and the cells may be, for example, cells collected from living organisms, cultured cells, etc.

[0115] The activation-inhibiting composition of the present invention in vivo When used in this manner, the target of administration may be, for example, humans or non-human animals other than humans. Examples of non-human animals include mice, rats, rabbits, dogs, sheep, horses, cats, goats, monkeys, guinea pigs, etc.

[0116] The conditions for use (administration conditions) of the activation inhibitory composition of the present invention are not particularly limited, and the administration form, timing of administration, dosage, etc. can be appropriately set depending on the type of target to be administered, etc.

[0117] The activation-inhibiting composition of the present invention in vitro When used, the activation inhibitory composition of the present invention can be used, for example, by adding it to the culture medium of the target cells. in vitro When used in the culture medium, the final concentration of total protein derived from the activation inhibitory composition of the present invention in the culture medium is, for example, 10-1000 μg / ml, 10-500 μg / ml, or 10-300 μg / ml.

[0118] The activation-inhibiting composition of the present invention in vivo When used in this context, the dosage can be appropriately determined based on factors such as the type of target, symptoms, age, and method of administration. For example, when administered to humans, the daily dose of the activation inhibitory composition, and the total amount of protein derived from the activation inhibitory composition, are not particularly limited and can be appropriately set according to their intended use. The number of daily doses can be, for example, 1 to 5 times, 1 to 3 times, 1 time, or 2 times.

[0119] The administration conditions and dosage forms of the activation-inhibiting composition of the present invention can be described by reference to the description of the composition of the present invention above.

[0120] <Anti-inflammatory composition> In another aspect, the present invention provides a composition having cell-derived anti-inflammatory activity. In this case, the anti-inflammatory composition of the present invention comprises a processed product of megakaryocytes or their cultures. The anti-inflammatory composition of the present invention is characterized by comprising a processed product of megakaryocytes or their cultures, and other components and conditions are not particularly limited. According to the anti-inflammatory composition of the present invention, for example, a composition capable of suppressing inflammation can be provided. The anti-inflammatory composition of the present invention can be described by reference to the descriptions of the compositions of the present invention, differentiation-promoting compositions, bone formation kits, and activation-inhibiting compositions.

[0121] In the present invention, "anti-inflammatory" means suppressing inflammation, and can be investigated using indicators such as suppression of the induction of inflammatory cytokine expression or suppression of the production of inflammatory cytokines.

[0122] The anti-inflammatory composition of the present invention, in vitro You can use it as well, in vivo It may be used in this way.

[0123] The target population, administration conditions, and dosage form of the anti-inflammatory composition of the present invention can be described by referring to the above-described descriptions of the composition of the present invention and the composition for inhibiting the activation of immune cells.

[0124] <Bone formation method> The bone formation method of the present invention uses the bone formation composition of the present invention. The bone formation method of the present invention is characterized by the use of the composition of the present invention, and other steps and conditions are not particularly limited. According to the bone formation method of the present invention, bone formation can be induced, for example, by promoting differentiation into osteoblasts, in particular by promoting differentiation from mesenchymal stem cells to osteoblasts. The bone formation method of the present invention can be described by reference to the description of the composition, differentiation-promoting composition, and bone formation kit of the present invention.

[0125] The bone formation method of the present invention includes, for example, an administration step of administering the bone formation composition of the present invention to a target subject. The bone formation method of the present invention allows, in vivoWhen bone formation is performed, the administration site of the composition of the present invention can be any desired site for bone formation. In the bone formation method of the present invention, the target and administration conditions can be described by referring to the description of the target and administration conditions of the composition of the present invention.

[0126] In the bone formation method of the present invention, the bone formation kit of the present invention may be used as the bone formation composition of the present invention.

[0127] <Methods for promoting osteoblast differentiation> The method for promoting the differentiation of osteoblasts of the present invention (hereinafter also referred to as the "differentiation promotion method") uses the cell differentiation promotion composition of the present invention. The differentiation promotion method of the present invention is characterized by the use of the differentiation promotion composition of the present invention, and other steps and conditions are not particularly limited. According to the differentiation promotion method of the present invention, differentiation into osteoblasts, in particular, differentiation from mesenchymal stem cells to osteoblasts can be promoted. The differentiation promotion method of the present invention can be described by reference to the description of the composition, differentiation promotion composition, bone formation kit, and bone formation method of the present invention.

[0128] The differentiation promotion method of the present invention is, for example, in vitro You can do it this way, in vivo This may be carried out in the present invention. The differentiation-promoting method of the present invention includes, for example, an administration step of administering the differentiation-promoting composition of the present invention to a target subject.

[0129] The differentiation promotion method of the present invention in vitro When implemented, the differentiation-promoting method of the present invention includes, for example, a differentiation step of differentiating progenitor cells into osteoblasts in the presence of the differentiation-promoting composition. The differentiation-promoting method of the present invention may also include, prior to the differentiation step, a maintenance step of maintaining and culturing the progenitor cells in the presence of the differentiation-promoting composition. The target population and administration conditions of the differentiation-promoting composition of the present invention can be described, for example, by referring to the description of the target population and administration conditions for the differentiation-promoting composition of the present invention.

[0130] <Methods for suppressing the activation of immune cells> In another aspect, the present invention provides a method for suppressing the activation of immune cells. The method for suppressing the activation of immune cells of the present invention (hereinafter also referred to as the "activation suppression method") uses the immune cell activation suppression composition of the present invention. The activation suppression method of the present invention is characterized by the use of the activation suppression composition of the present invention, and other steps and conditions are not particularly limited. According to the activation suppression method of the present invention, the activation of immune cells can be suppressed. The activation suppression method of the present invention can be described by reference to the descriptions of the composition, differentiation promoting composition, bone formation kit, activation suppression composition, anti-inflammatory composition, bone formation method, and differentiation promoting method of the present invention.

[0131] The activation suppression method of the present invention is, for example, in vitro You can do it this way, in vivo This may be carried out by the present invention. The activation inhibition method of the present invention includes, for example, an administration step of administering the activation inhibition composition of the present invention to a target subject.

[0132] The present invention's method for suppressing activation in vitro When implemented, the activation inhibition method of the present invention includes, for example, a culture step of culturing immune cells in the presence of the activation inhibition composition. The target recipient, administration conditions, and dosage form of the activation inhibition composition of the present invention can be described, for example, by referring to the description of the target recipient, administration conditions, and dosage form of the activation inhibition composition of the present invention.

[0133] <Methods to suppress inflammation> In another embodiment, the present invention provides a method for suppressing inflammation. The method for suppressing inflammation of the present invention (hereinafter also referred to as the "suppression method") uses the anti-inflammatory composition of the present invention. The method for suppressing inflammation of the present invention is characterized by the use of the anti-inflammatory composition of the present invention, and other steps and conditions are not particularly limited. Inflammation can be suppressed according to the method for suppressing inflammation of the present invention. The method for suppressing inflammation of the present invention can be described by reference to the descriptions of the composition, differentiation-promoting composition, bone formation kit, activation-inhibiting composition, anti-inflammatory composition, bone formation method, differentiation-promoting method, and activation-inhibiting method of the present invention.

[0134] The present invention provides, for example, a method for suppressing inflammation, in vitro You can do it this way, in vivo This may be carried out in the present invention. The method for suppressing inflammation of the present invention includes, for example, an administration step of administering the anti-inflammatory composition of the present invention to a target subject.

[0135] The present invention's method for suppressing inflammation in vitro When implemented, the method for suppressing inflammation of the present invention includes, for example, a culture step of culturing the target subject in the presence of the anti-inflammatory product. The target subject, administration conditions, and dosage form of the anti-inflammatory composition of the present invention can be described, for example, by referring to the description of the target subject, administration conditions, and dosage form in the anti-inflammatory composition of the present invention.

[0136] <Use of composition> The present invention relates to a composition or use thereof, comprising a processed megakaryocyte or culture thereof as an active ingredient, for use in bone formation. The present invention relates to a composition or use thereof, comprising a processed megakaryocyte or culture thereof as an active ingredient, for use in promoting the differentiation of osteoblasts. The present invention relates to a composition or use thereof, comprising a processed megakaryocyte or culture thereof as an active ingredient, for use in suppressing the activation of immune cells. The present invention relates to a composition or use thereof, comprising a processed megakaryocyte or culture thereof as an active ingredient, for use in suppressing inflammation. The present invention relates to the use of a composition, comprising a processed megakaryocyte or culture thereof as an active ingredient, for producing a bone formation composition. Furthermore, the present invention relates to the use of a composition, comprising a processed megakaryocyte or culture thereof as an active ingredient, for producing a composition that promotes the differentiation of osteoblasts. The present invention relates to the use of a composition, comprising a processed megakaryocyte or culture thereof as an active ingredient, for producing a composition that suppresses the activation of immune cells. The present invention relates to the use of a composition, comprising a processed megakaryocyte or culture thereof as an active ingredient, for producing an anti-inflammatory composition. The present invention can be further described by reference to the descriptions of the composition, differentiation-promoting composition, bone formation kit, activation-inhibiting composition, anti-inflammatory composition, bone formation method, differentiation-promoting method, activation-inhibiting method, and inflammation-inhibiting method of the present invention. [Examples]

[0137] The present invention will be described in detail below using examples, but the present invention is not limited to the embodiments described in the examples.

[0138] [Example 1] The composition of the present invention was prepared and confirmed to contain growth factors and growth factor receptors, and to have cell proliferation-promoting activity and differentiation-promoting activity into osteoblasts.

[0139] (1) Production of immortalized megakaryocyte cells Immortalized megakaryocytes were prepared using the following procedure.

[0140] (1-1) Preparation of hematopoietic progenitor cells from iPS cells Human iPS cells (TKDN SeV2 and NIH5: iPS cells derived from human fetal dermal fibroblasts established using Sendai virus) were differentiated into hematopoietic cells according to the method described in Reference 8 below. Specifically, human ES / iPS cell colonies were co-cultured with C3H10T1 / 2 feeder cells for 14 days in the presence of 20 ng / ml VEGF (R&D SYSTEMS) to produce hematopoietic progenitor cells (HPCs). The culture conditions were 37°C, 20% O2, and 5% CO2 (unless otherwise specified, the same conditions were used hereafter). Reference 8: Takayama N. et al., “Transient activation of c-MYC expression is critical for efficient platelet generation from human induced pluripotent stem cells”, J. Exp. Med., 2010, vo.13, pages 2817-2830

[0141] (1-2) Gene transfer systems The gene transfer system utilized a lentiviral vector system. The lentiviral vector is a tetracycline-regulated Tet-on® gene expression induction system vector. It was prepared by rearranging the mOKS cassette of LV-TRE-mOKS-Ubc-tTA-I2G (see reference 9 below) with c-MYC, BMI1, or BCL-xL. The vectors into which c-MYC, BMI1, or BCL-xL were introduced were named LV-TRE-c-Myc-Ubc-tTA-I2G, LVTRE-BMI1-Ubc-tTA-I2G, and LV-TRE-BCL-xL-Ubc-tTA-I2G, respectively. The c-MYC, BMI1, and BCL-xL viruses were prepared by gene transfer into 293T cells using the aforementioned lentiviral vector. By infecting target cells with the obtained virus, the c-MYC, BMI1, and BCL-xL genes are introduced into the target cells' genome sequences. These genes, stably introduced into the genome sequence, can be forced to be expressed by adding doxycycline (clontech#631311) to the culture medium. Reference 9: Kobayashi, T. et al., “Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells.”, Cell, 2010, vol.142, No.5, pages 787-799

[0142] (1-3) Infection of hematopoietic progenitor cells with cMYC and BMI1 virus On a 6-well plate pre-seed with C3H10T1 / 2 feeder cells, 5 × 10⁻¹⁵ HPC obtained by the method described in (1-1) above are placed. 4Cells were seeded to a cell / well ratio, and c-MYC and BMI1 were forced to express using lentiviral assays with BMI1 and c-MYC viruses. Six wells were used for each cell line. Specifically, virus particles were added to the culture medium to achieve an MOI (multiplicity of infection) of 20, and infection was carried out by spin infection (centrifugation at 32°C, 900 rpm, 60 minutes). This spin infection was performed twice, 12 hours apart. The culture medium was prepared by adding 50 ng / ml Human thrombopoietin (TPO) (R&D SYSTEMS), 50 ng / ml Human Stem Cell Factor (SCF) (R&D SYSTEMS), and 2 μg / ml Doxycycline (Dox, clontech #631311) to a base medium (IMDM (Iscove's Modified Dulbecco's Medium) (Sigma-Aldrich) containing 15% Fetal Bovine Serum (GIBCO), 1% Penicillin-Streptomycin-Glutamine (GIBCO), 1% Insulin, Transferrin, Selenium Solution (ITS-G) (GIBCO), 0.45 mmol / l 1-Thioglycerol (Sigma-Aldrich), and 50 μg / ml L-Ascorbic Acid (Sigma-Aldrich)). This resulted in a culture medium (hereinafter referred to as differentiation medium), to which Protamine was further added to reach a final concentration of 10 A culture medium containing the additive at a concentration of μg / ml was used.

[0143] (1-4) Preparation and maintenance culture of megakaryocyte autoproliferating strains Using the methods described in (1-3) above, the day on which infection with c-MYC and BMI1 viruses was carried out was designated as infection day 0. Megakaryocyte autoproliferating strains were then created by culturing HPCs into which the c-MYC gene and the BMI1 gene had been introduced, as follows. Forced expression of the c-MYC gene and the BMI1 gene was performed by adding DOX to the culture medium to a concentration of 1 μg / ml DOX.

[0144] • Day 2 to Day 11 of infection On day 2 of infection, virus-infected hematopoietic cells obtained by the above method were collected by pipetting, centrifuged at 1200 rpm for 5 minutes to remove the supernatant, and then resuspended in fresh differentiation medium and seeded on fresh C3H10T1 / 2 feeder cells (6-well plate). Passaging was performed on day 9 of infection by the same procedure. At the time of reseeding, the cell count was measured and then 1 × 10⁶ cells were seeded. 5 C3H10T1 / 2 feeder cells were seeded at a rate of cells / 2ml / well (6-well plate).

[0145] • Day 12 to Day 13 of infection The same procedure as on the second day of infection was performed. After counting the number of cells, 3 × 10⁻⁶ cells were collected. 5 C3H10T1 / 2 feeder cells were seeded on a 100mm dish so that the cell density was 10ml / 100mm dish.

[0146] • Day 14 of infection Virus-infected blood cells were collected, and the cells were 1.0 × 10⁶ 5 For each cell, 2 μl, 1 μl, and 1 μl of anti-human CD41a-APC antibody (BioLegend), anti-human CD42b-PE antibody (eBioscience), and anti-human CD235ab-pacific blue (BioLegend) antibody were used to react the hematopoietic cells with the antibodies. After the reaction, the cells were analyzed using FACS Verse® (BD Biosciences). Cells with a CD41a positivity rate of 50% or higher on day 14 of infection were designated as megakaryocyte autoproliferating cells.

[0147] (1-5) BCL-xL virus infection of megakaryocyte autoproliferating strains On the 14th day after the infection, BCL-xL was introduced into the megakaryocyte self-proliferating strain by the lentivirus method using the BCL-xL virus. Virus particles were added to the medium to reach an MOI of 10, and infection was carried out by spin infection (centrifugation at 32 °C, 900 rpm for 60 minutes). The forced expression of the BCL-xL gene was carried out by adding DOX to the medium to a concentration of 1 μg / ml DOX.

[0148] (1-6) Generation and maintenance culture of immortalized megakaryocyte strains · From the 14th day to the 18th day after infection The megakaryocyte self-proliferating strain into which the BCL-xL gene was introduced obtained by the method of (1-5) above was collected and centrifuged at 1200 rpm for 5 minutes. After the centrifugation, the precipitated cells were suspended in a new differentiation medium and then seeded on new C3H10T1 / 2 feeder cells at 2×10 5 cells / 2ml / well (6-well plate).

[0149] · 18th day after infection: Subculture The megakaryocyte self-proliferating strain after the introduction of the BCL-xL gene was collected. After measuring the cell number, it was seeded at 3×10 5 cells / 10ml / 100mm dish.

[0150] · 24th day after infection: Subculture The megakaryocyte self-proliferating strain after the introduction of the BCL-xL gene was collected. After measuring the cell number, it was seeded at 1×10 5 cells / 10ml / 100mm dish. Thereafter, subculture was carried out in the same manner every 4-7 days for maintenance culture. At the time of subculture, after suspending in a new differentiation medium, it was seeded.

[0151] On the 24th day after infection, the megakaryocyte self-proliferating strain into which BCL-xL was introduced was collected, and 1.0×10 5Immunostaining was performed on each cell using 2 μl, 1 μl, and 1 μl of anti-human CD41a-APC antibody (BioLegend), anti-human CD42b-PE antibody (eBioscience), and anti-human CD235ab-Pacific Blue (Anti-CD235ab-PB; BioLegend) antibody, respectively, followed by analysis using FACS Verse®. Cells with a CD41a positivity rate of 50% or higher on day 24 after infection were designated as immortalized megakaryocyte cell lines. These cells that were able to proliferate for more than 24 days after infection were designated as immortalized megakaryocyte cell lines SeV2-MKCL and NIH5-MKCL.

[0152] The obtained SeV2-MKCL and NIH5-MKCL were cultured statically in 10cm dishes (10ml / dish). The culture medium consisted of IMDM as the base medium, with the following components added (concentrations are final concentrations). The culture conditions were 37°C and 5% CO2. FBS (Sigma #172012 lot.12E261) 15% L-Glutamin (Gibco #25030-081) 2mmol / l ITS (Gibco #41400-045) 100x dilution MTG (monothioglycerol, sigma #M6145-25ML) 450μmol / l Ascorbic acid (sigma #A4544) 50 μg / ml Puromycin (sigma #P8833-100MG) 2μg / ml SCF (Wako Pure Chemical #193-15513) 50ng / ml TPO-like active substance 200 ng / ml

[0153] (2) Production of megakaryocyte cultures Forced expression was removed by culturing in a DOX-free medium. Specifically, the immortalized megakaryocyte cell lines (SeV2-MKCL and NIH5-MKCL) obtained by the method described in (1) above were washed twice with PBS(-) and suspended in the platelet production medium described below. The cell seeding density was 1.0 × 10⁻⁶. 5 The value was set to cells / ml.

[0154] The aforementioned platelet production medium was prepared by adding the following components to IMDM as the base medium (concentrations are final concentrations). human plasma A6% L-Glutamin (Gibco #25030-081) 4mmol / l ITS (Gibco #41400-045) 100x dilution MTG (monothioglycerol, sigma #M6145-25ML) 450μmol / l Ascorbic acid (sigma #A4544) 50 μg / ml SCF (Wako Pure Chemical #193-15513) 50ng / ml TPO-like active substance 200 ng / ml ADAM inhibitor 15 μmol / l GNF351(Calbiochem #182707)500nmol / LY39983(Chemscene LLC #CS-0096)500nmol / l Urokinase 5U / ml Low molecular weight heparin (SANOFI, Clexane) 1U / ml

[0155] Then, by culturing the cells in the aforementioned platelet production medium for 6 days to induce platelet production, megakaryocyte cultures were produced.

[0156] (3) Production of purified platelets Platelets were produced (purified) from the megakaryocyte culture obtained in (2) above using the following procedure. The same purification process was performed twice.

[0157] (3-1) Concentration of megakaryocyte cultures The megakaryocyte culture obtained in (2) above was introduced into a culture bag. The culture bag was then connected to a concentration system as shown in Figure 1. In Figure 1, washing and preservation solution bags 1 and 2 contain the washing and preservation solution. The washing and preservation solution used was bicarbonate infusion (bicarbon infusion, manufactured by Otsuka Pharmaceutical Co., Ltd.) to which 20% ACD and 2.5% human serum albumin were added, and the pH was adjusted to 7.2 with NaOH. Then, according to Table 1 below, the megakaryocyte culture was concentrated using a hollow fiber membrane (Plasma Flow OP, manufactured by Asahi Kasei Medical Co., Ltd.), and the resulting concentrated megakaryocyte culture was collected in a storage bag.

[0158] [Table 1]

[0159] (3-2) Centrifugation of platelets First, using a sterile joining device, the waste liquid bag of the ACP215 disposable set was replaced with a collection bag. The collection bag used was a Highcalic IVH bag (Terumo HC-B3006A). Next, 10% of ACD-A solution (Terumo Corporation) was added to the concentrated megakaryocyte culture. After the addition, the concentrated solution with ACD-A solution added was injected into a cell bag. The cell bag used was a Highcalic IVH bag (Terumo HC-B3006A).

[0160] Next, using a sterile joining device, the cell bag containing the culture with ACD-A solution added was joined to the ACP215 disposable set. Then, the ACP215 was started in service mode and the rotation speed was set to 2500 rpm (350 × g). The ACP215 was started and the culture from the cell bag was introduced into the separation bowl at approximately 100 ml / min. The liquid components flowing out of the separation bowl were collected in a recovery bag. After introducing the entire amount of culture from the cell bag into the separation bowl, another 500 ml of washing and preservation solution was introduced into the separation bowl. After introducing the washing and preservation solution into the separation bowl, centrifugation was stopped and the recovery bag containing the recovered solution (the recovered liquid components including platelets) was detached using a tube sealer.

[0161] A new ACP215 disposable set was attached to a collection bag containing the recovery solution (including platelets) using the sterile joining device described above. The ACP215 was started in normal mode. The program setting was set to WPC, and the ACP215 disposable set with the attached collection bag was set up according to the instrument's instructions. The collection bag containing the recovery solution was placed on the stand.

[0162] Next, the centrifugal speed of the ACP215 was changed to 5000 rpm (1398.8 × g), and centrifugation was started. When the recovered liquid began to be introduced into the separation bowl, the injection method was changed from automatic to manual. Specifically, the recovered liquid was introduced into the separation bowl at an introduction rate of approximately 100 ml / min. After the entire amount of recovered liquid was added to the separation bowl, an additional 500 ml of washing and storage solution was added.

[0163] (3-3) Washing of platelets The washing was performed using 2000 ml of the aforementioned washing and storage solution, according to the ACP215 program.

[0164] (3-4) Platelet recovery Following the ACP215 program, 200 ml of washed platelets were collected into a platelet preparation bag.

[0165] (3-5) Separation of platelets Platelets were separated from the platelet preparation bag using the hollow fiber membrane by a conventional method and collected in a recovery bag.

[0166] (4) Production of extracts As the megakaryocytes or their cultures, the immortalized megakaryocyte cell line obtained by the method in (1) above, platelets collected in the platelet preparation bag obtained in (3-5) above, and megakaryocyte cultures from which platelets have been removed collected in the drainage bag (hereinafter collectively referred to as "raw materials") were used. For the megakaryocyte cultures from which platelets have been removed, four separately prepared samples were used (platelet-removed megakaryocyte cultures 1-4).

[0167] Each raw material was washed twice with a washing solution. The washing solution used was prepared by adding ACD-A solution to bicarnate infusion to a concentration of approximately 20 (v / v)%, then adding NaOH to adjust the pH to a range of 7.0-7.4. After washing, each raw material was centrifuged at 2000 × g for 10 minutes at room temperature (approximately 25°C). After centrifugation, the precipitate was collected and frozen using liquid nitrogen. Next, cell lysis buffer was added to the frozen precipitate and dissolved by shaking at 50 rpm at 4°C for 30 minutes. The cell lysis buffer used was a commercially available cell lysis buffer (2× Cell Lysis Buffer, RayBiotech, Cat. No.: AA-LYS) with a protease inhibitor cocktail (Protease Inhibitor Cocktail, RayBiotech, Cat. No.: AA-PI) added.

[0168] The resulting solution was centrifuged at 14000 × g for 5 minutes at 4°C. After centrifugation, the supernatant was collected as the processed material for the example. The total protein concentration of the processed materials obtained from each raw material was measured using the Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific). As a result, when using an immortalized megakaryocyte cell line, the concentration was 2.2 × 10⁻⁶. 7 2.604 mg of total protein was extracted from the cells. In addition, when using platelets, 2.5 × 10⁻⁶ mg was extracted. 8 1.187 mg of total protein was extracted from the cells. Furthermore, when using platelet-free megakaryocyte cultures 1-4, 1.97 × 10⁶ was extracted. 8 cells, 5.25×10 8 cells, 3.64×10 8 cells, 6.22×10 8Total protein concentrations of 2.8, 5.944, 3.6, and 4.496 mg were extracted from the cells, respectively. After adjusting the total protein concentration to 5 mg / ml, the concentrations of growth factors (bFGF, IGFBP-1, IGFBP-2, PIGF, VEGF, GDF-15, AR, BMP-7, HGF) and growth factor receptors (SCFR, EGFR, VEGFR2) (pg / 5 mg of total protein) were measured for each treated sample using the Quantibody® Human Growth Factor Array 1 (RayBiotech). These results are shown in Table 2 below. Note that Table 2 also represents concentrations per 5 mg of total protein.

[0169] [Table 2]

[0170] (5) Confirmation of cell proliferation-promoting activity Human adipose tissue-derived mesenchymal stem cells are placed in a 10cm dish, measuring 2.4-4.8 x 10 cm. 5 Cells were seeded in a 10ml culture medium per dish. The mesenchymal stem cells used were commercially available human adipose tissue-derived mesenchymal stem cells (Takara Bio, Cat. No.: C-12977) that had been subcultured twice and then harvested. The culture medium was prepared by adding a composition prepared from platelet-free megakaryocyte culture 2 (Mesenchymal Stem Cell Growth Medium 2, Takara Bio, Cat. No.: C-28009) after freezing and thawing. The composition was added to the culture medium so that the total protein concentration derived from the composition in the medium reached a predetermined concentration (0, 125, 250, or 500 μg / ml). This medium served as a maintenance medium.

[0171] After seeding, the mesenchymal stem cells were cultured for 3 days at 37°C under humid conditions of 5% CO2. After this culture, the mesenchymal stem cells were harvested, seeded under the same conditions, and cultured again for 3 days at 37°C under humid conditions of 5% CO2. Next, the mesenchymal cells were harvested after the culture and the number of cells was counted. The relative value of the number of cells at harvest was calculated using the number of cells at seeding as the baseline (1), and this was used as the relative value of the proliferation activity. In addition, the time required for the cells to proliferate once (doubling time) was calculated based on the number of cells at seeding and the number of cells at harvest. These results are shown in Figure 2.

[0172] Figure 2 is a graph showing the proliferation activity of cells. In Figure 2, (A) shows the proliferation activity, and (B) shows the doubling time. In Figure 2(A), the horizontal axis shows the total protein concentration derived from the composition, and the vertical axis shows the relative value of the proliferation activity. In Figure 2(B), the horizontal axis shows the total protein concentration derived from the composition, and the vertical axis shows the doubling time, with the numerical values ​​in the figure indicating the doubling time. As shown in Figure 2(A), when the composition of the present invention was added, the proliferation activity of mesenchymal stem cells increased in a manner dependent on the total protein concentration derived from the composition. Although not shown, the doubling time after the third passage of mesenchymal stem cells without the addition of the composition was 21 hours. As shown in Figure 2(B), when the composition of the present invention was not added, the doubling time of mesenchymal stem cells was extended by approximately 1.5 times. In contrast, as shown in Figure 2(B), when the composition of the present invention was added, the time required for mesenchymal stem cells to proliferate once was shortened in a manner dependent on the total protein concentration derived from the composition. Furthermore, when mesenchymal stem cells were cultured in a medium without the composition of the present invention, the doubling time increased with the number of passages. In contrast, when the composition of the present invention was added, the increase in the doubling time was almost suppressed. Therefore, it was found that the composition of the present invention exhibits cell proliferation-promoting activity and can suppress the decrease in proliferation activity.

[0173] (6) Confirmation of differentiation-promoting activity The mesenchymal stem cells were maintained in the same manner as in (5) above. Next, the culture medium in each well was replaced with an equal volume of differentiation medium (Day 0 of differentiation initiation). The differentiation medium was prepared by adding a composition prepared from platelet-free megakaryocyte culture 2, which had been frozen and then thawed, to Mesenchymal Stem Cell Osteogenic Differentiation Medium (Takara Bio, Cat. No.: C-28013). The composition was added in such a way that the total protein concentration derived from the composition in the differentiation medium was a predetermined concentration (0, 125, or 250 μg / ml), and the combination with the total protein concentration derived from the composition in the maintenance medium was one of the combinations shown in Table 3 below.

[0174] [Table 3]

[0175] After seeding, the mesenchymal stem cells were cultured for 14 days at 37°C under moist conditions of 5% CO2. During this culture, the culture medium was changed every 3 or 4 days. On day 7 after differentiation initiation, alkaline phosphatase expression was confirmed for each sample by staining with a staining kit (TRACP & ALP double-stain Kit, Takara Bio, Cat. No.: MK300). The negative control was performed in the same manner except that only the maintenance medium was added instead of the differentiation medium. The results are shown in Figure 3.

[0176] Figure 3 shows the staining pattern for ALP. As shown in Figure 3, all samples were ALP-positive, confirming that differentiation into osteoblasts was induced. Furthermore, when the composition of the present invention was added to the maintenance medium or differentiation medium, compared to when the composition of the present invention was not added to the maintenance medium or differentiation medium, and to the negative control, mesenchymal stem cells after culture showed strong ALP staining, suggesting that differentiation into osteoblasts was promoted.

[0177] Furthermore, on day 14 after the start of differentiation, the formation of bone matrix was confirmed in each sample by staining with alizarin red S for calcium. The negative control was performed in the same manner except that only the maintenance medium was added instead of the differentiation medium. The results are shown in Figure 4.

[0178] Figure 4 shows the staining pattern of calcium. As shown in Figure 4, in samples where the composition of the present invention was not added to either the maintenance medium or the differentiation medium, and in the negative control, calcium was hardly stained. In contrast, in samples where the composition of the present invention was added to at least one of the maintenance medium or the differentiation medium, calcium was stained, confirming that bone matrix formation was occurring. In other words, it was found that the composition of the present invention promotes differentiation into osteoblasts.

[0179] Furthermore, the calcium ion concentration in the culture medium was measured on days 10, 14, and 18 after the start of differentiation. The calcium ion concentration in the culture medium was measured using an ion-selective electrode (BIOPROFILE® FLEX, manufactured by NOVA BIOMEDICAL). The calcium ion concentration in the culture medium at the start of culture was 0.88 mmol / l. When the mesenchymal stem cells differentiate into osteoblasts and osteocytes, they utilize the calcium in the culture medium to form the bone matrix. Therefore, differentiation into osteoblasts and osteocytes can be evaluated using a decrease in calcium ion concentration as an indicator. These results are shown in Figure 5.

[0180] Figure 5 is a graph showing the calcium ion concentration in the culture medium. In Figure 5, the horizontal axis shows the measured values ​​of the total protein concentration derived from the composition in the maintenance medium and differentiation medium, and the calcium ion concentration on each measurement day, while the vertical axis shows the calcium ion concentration. As shown in Figure 5, on day 10 of differentiation, the sample to which the composition of the present invention was added had a calcium ion concentration equal to or lower than that of the sample to which the composition of the present invention was not added. Furthermore, on day 14 of differentiation, the sample to which the composition of the present invention was added showed a significantly lower calcium ion concentration compared to the sample to which the composition of the present invention was not added. And on day 18 of differentiation, the sample to which the composition of the present invention was added had a calcium ion concentration equal to or lower than that of the sample to which the composition of the present invention was not added. From these results, it was found that when the composition of the present invention was added, differentiation into osteoblasts and osteocytes progressed earlier, and calcium uptake from the culture medium started earlier. From these results, it was found that the composition of the present invention has differentiation-promoting activity towards osteoblasts.

[0181] [Example 2] We have confirmed that the composition of the present invention has cell proliferation-promoting activity and differentiation-promoting activity into osteoblasts.

[0182] (1) Confirmation of cell proliferation-promoting activity The mesenchymal stem cells were cultured twice, then harvested and cryopreserved. After thawing the cryopreserved cells, the cells were cultured once, and the proliferation activity and doubling time were calculated in the same manner as in Example 1(5), except that the total protein concentration derived from the composition in the maintenance medium was added to a predetermined concentration (0, 0.2, 1.3, 31.3, 62.5, or 125 μg / ml). These results are shown in Figure 6.

[0183] Figure 6 is a graph showing the proliferation activity of cells. In Figure 6, (A) shows the proliferation activity, and (B) shows the doubling time. In Figure 6(A), the horizontal axis shows the total protein concentration derived from the composition, and the vertical axis shows the relative value of the proliferation activity. In Figure 6(B), the horizontal axis shows the total protein concentration derived from the composition, and the vertical axis shows the doubling time. As shown in Figure 6(A), when the composition of the present invention was added, the proliferation activity of mesenchymal stem cells increased in a manner dependent on the total protein concentration derived from the composition, and in particular, when the total protein concentration was 31.3 μg / ml or higher, the proliferation activity of mesenchymal stem cells increased significantly. Although not shown, the doubling time after the third passage of mesenchymal stem cells without the addition of the composition was 17 hours. As shown in Figure 6(B), when the composition of the present invention was not added, the doubling time of mesenchymal stem cells was extended by approximately 1.5 times. In contrast, as shown in Figure 6(B), when the composition of the present invention was added, the time required for a single proliferation of mesenchymal stem cells was shortened in a manner dependent on the total protein concentration derived from the composition. In particular, when the total protein concentration was 31.3 μg / ml or higher, the time required for a single proliferation of mesenchymal stem cells was significantly shortened. Furthermore, when cultured in a medium without the composition of the present invention, the doubling time of mesenchymal stem cells increased as the number of passages increased. In contrast, when the composition of the present invention was added, the extension of the doubling time was almost suppressed, and this effect was particularly pronounced when the total protein concentration derived from the composition was 31.3 μg / ml or higher. Therefore, it was found that the composition of the present invention exhibits cell proliferation-promoting activity and can suppress the decrease in proliferation activity.

[0184] (2) Confirmation of differentiation-promoting activity The mesenchymal stem cells were maintained in the same manner as in Example 2(1) above. Next, the culture medium in each well was replaced with an equal volume of differentiation medium (Day 0 of differentiation initiation). The differentiation medium was prepared by adding a composition prepared from platelet-depleted megakaryocyte culture 2, which had been frozen and then thawed, to mesenchymal stem cell osteoblast differentiation medium. The composition was added such that the total protein concentration derived from the composition in the differentiation medium was a predetermined concentration (0, 0.2, 1.3, 31.3, 62.5, or 125 μg / ml), and the combination with the total protein concentration derived from the composition in the maintenance medium was one of the combinations shown in Table 4 below.

[0185] [Table 4]

[0186] After seeding, the cells were cultured in the same manner as in Example 1(6). Next, on days 14 and 18 after the start of differentiation, the formation of bone matrix was confirmed for each sample by staining with calcium in the same manner as in Example 1(6). The results are shown in Figures 7 and 8.

[0187] Figures 7 and 8 show the staining images of calcium. As shown in Figures 7 and 8, in samples where the composition of the present invention was not added to either the maintenance medium or the differentiation medium, and in the negative control, calcium was hardly stained. In contrast, in samples where the composition of the present invention was added to at least one of the maintenance medium or the differentiation medium, calcium was stained, confirming that bone matrix formation was occurring. In other words, it was found that the composition of the present invention promotes differentiation into osteoblasts.

[0188] Furthermore, the calcium ion concentration in the culture medium was measured on days 6, 10, 14, and 18 after the start of differentiation, in the same manner as in Example 1(6). These results are shown in Figure 9.

[0189] Figure 9 is a graph showing the calcium ion concentration in the culture medium. In Figure 9, the horizontal axis shows the measured values ​​of the total protein concentration derived from the composition in the maintenance medium and differentiation medium, and the calcium ion concentration on each measurement day, while the vertical axis shows the calcium ion concentration. As shown in Figure 9, on days 6 and 10 of differentiation, the samples to which the composition of the present invention was added had calcium ion concentrations equal to or lower than those of the samples to which the composition of the present invention was not added. Furthermore, on day 14 of differentiation, the samples to which the composition of the present invention was added showed a significantly lower calcium ion concentration compared to the samples to which the composition of the present invention was not added, and this was particularly pronounced in the samples where the total protein concentration derived from the composition in the differentiation medium was 1.3 μg / ml. Finally, on day 18 of differentiation, the samples to which the composition of the present invention was added had calcium ion concentrations equal to or lower than those of the samples to which the composition of the present invention was not added. From these results, it was found that when the composition of the present invention is added, differentiation into osteoblasts and osteocytes progresses earlier, and calcium uptake from the culture medium starts earlier. Furthermore, it was found that the differentiation-promoting activity of the composition of the present invention can be obtained even under conditions where the total protein concentration derived from the composition is as low as 0.2 μg / ml. These results indicate that the composition of the present invention has differentiation-promoting activity for osteoblasts.

[0190] [Example 3] We have confirmed that the composition of the present invention has cell proliferation-promoting activity and differentiation-promoting activity into osteoblasts.

[0191] (1) Confirmation of cell proliferation-promoting activity Human bone marrow-derived mesenchymal stem cells (BMMSC, PromoCell, Cat No. C-12974) were divided into 5 × 10⁻¹⁴ cells. 4Cells were seeded in 24-well plates at a density of cells / well and cultured for 7 days. The culture medium was changed every 3 days by replacing 2 / 3 of the medium with fresh medium. When culture is started at the above seeding density, the cell density reaches 80% confluence in about 7 days. The basic medium for BMMSC was DMEM medium (low-glucose) containing 10% FBS and 1% antibiotic (Ab).

[0192] Twenty-four hours after the start of culture, the composition derived from the platelet-removed megakaryocyte culture prepared in Example 1(4) was added to the basic medium so that the total protein concentration derived from the composition in the basic medium reached a predetermined concentration (0, 125, 250, or 500 μg / ml). Then, on days 3, 5, or 7 after the addition of the composition, the cell count was calculated using a cell counting kit (WST8, Cell Counting Kit-8, Dojindo, Cat No. 347-07621) according to the attached protocol. In addition, ALP activity was examined by measuring the absorbance at 405 nm using p-nitrophenyl phosphate (SIGMAFAST® p-nitrophenyl phosphate tablets, SIGMA, Cat No. N1891). The ALP activity value per cell was then calculated. These results are shown in Figures 10 and 11.

[0193] Figure 10 is a graph showing the number of cells. In Figure 10, the horizontal axis represents the number of days after the addition of the composition, and the vertical axis represents the number of cells. The concentration in the figure represents the total protein concentration (the same applies below). As shown in Figure 10, when cells were cultured in a medium containing the composition of the present invention, cell proliferation was promoted in a concentration-dependent manner. In the group to which 500 μg / ml was added, the number of cells decreased on day 7, but this is because the cells reached confluence on day 5.

[0194] Next, Figure 11 is a graph showing ALP activity. In Figure 11(A), the horizontal axis shows the number of days after the addition of the composition, and the vertical axis shows ALP activity (absorbance OD405). In Figure 11(B), the horizontal axis shows the number of days after the addition of the composition, and the vertical axis shows ALP activity (ALP activity value per cell). As shown in Figure 11(B), in the case of well-level culture, ALP activity reached a maximum on day 5 and decreased on day 7. On the other hand, as shown in Figure 11(A), in the case of cell-level culture, ALP activity decreased over time under all culture conditions. Generally, in MSCs, differentiation progresses along with proliferation, so ALP activity increases over time. However, in the presence of the composition of the present invention, strong proliferation is induced while ALP activity is suppressed, so it was presumed that differentiation induction was suppressed. From these findings, it was found that the composition of the present invention exhibits cell proliferation-promoting activity.

[0195] (2) Confirmation of differentiation-promoting activity Human bone marrow-derived mesenchymal stem cells were cultured for 7 days in the same manner as in Example 3(1) above.

[0196] After the aforementioned culture, the culture medium in each well was replaced with the basic medium, or with a basic medium (differentiation medium) containing 100 μg / ml ascorbic acid and the composition as an osteoblast differentiation factor, and cultured for 2 weeks to induce osteoblasts. The medium was changed every 3 days by replacing 2 / 3 of the medium with fresh medium. The composition was added so that the total protein concentration derived from the composition in the basic medium reached a predetermined concentration (0, 125, 250, or 500 μg / ml). The composition used was derived from the platelet-free megakaryocyte culture prepared in Example 1(4).

[0197] Based on the induction start date, cell count and ALP activity were measured on days 7, 10, or 14. The cell count was calculated using the cell count kit according to the attached protocol. ALP activity was investigated by measuring the absorbance at 405 nm using p-nitrophenyl phosphate (SIGMAFAST® p-nitrophenyl phosphate tablets, manufactured by SIGMA, Cat No. N1891). The ALP activity value per cell was then calculated. These results are shown in Figures 12 and 13.

[0198] Figure 12 is a graph showing the number of cells. In Figure 12, the horizontal axis represents the number of days after the start of osteoblast differentiation induction, and the vertical axis represents the number of cells. As shown in Figure 12(A), when osteoblast differentiation factors were not included, an increase in the number of cells over time was observed when cultured in a medium containing the composition of the present invention. On the other hand, as shown in Figure 12(B), when osteoblast differentiation factors were included, the decrease in the number of cells over time was suppressed in a concentration-dependent manner when cultured in a medium containing the composition of the present invention.

[0199] Next, Figure 13 is a graph showing ALP activity. In Figures 13(A) and (C), the horizontal axis shows the number of days after the start of osteoblast differentiation induction, and the vertical axis shows ALP activity (absorbance OD405). In Figures 13(B) and (D), the horizontal axis shows the number of days after the addition of the composition, and the vertical axis shows ALP activity (ALP activity value per cell). As shown in Figures 13(A) and (B), when osteoblast differentiation factors were not included, culturing in a medium containing the composition of the present invention increased ALP activity over time, but no difference in ALP activity was observed between the medium of the comparative example (0 μg / ml) that did not contain the composition of the present invention. On the other hand, as shown in Figures 13(C) and (D), when osteoblast differentiation factors were included, culturing in a medium containing the composition of the present invention resulted in a significant increase in ALP activity at 14 days after the start of differentiation induction compared to culturing in the medium of the comparative example (0 μg / ml) that did not contain the composition of the present invention. Furthermore, regarding the comparative example's culture medium (0 μg / ml), the increase in absorbance observed 10 days after the start of differentiation induction is presumed to be due to an increase in the number of cells due to cell proliferation. These results indicate that the composition of the present invention can promote the differentiation induction of osteoblasts even in an ascorbic acid-mediated osteoblast differentiation induction system. In addition, it was found that the composition of the present invention promotes the differentiation induction to osteoblasts induced by osteoblast differentiation factors. Therefore, it can be said that the composition of the present invention has differentiation-promoting activity to osteoblasts.

[0200] [Example 4] We have confirmed that the composition of the present invention promotes bone formation and bone regeneration in vivo.

[0201] As a bone formation model, a model in which bone formation is performed on the skull of a nude mouse was used. Specifically, the composition of the present invention, along with artificial bone material, was used as a transplant sample and transplanted onto the skull (subperiosteal) of a nude mouse (8 weeks old BALB / cAJCL-nu / nu) under general anesthesia (3-component mixture). Specifically, the following four groups were established. • Negative control group (n=5) Transplant samples prepared solely from 25 mg of β-TCP (β-tricalcium phosphate) granules (β-TCP granules, OSferion G1, Oympus Terumo) were transplanted. • Positive control group (n=9) 25 mg of β-TCP granules and platelet-rich plasma (PRP) were transplanted. For the platelet-rich plasma, platelets were concentrated from mouse peripheral blood to a concentration six times that of peripheral blood, and the transplant sample was prepared and transplanted. Example 4A (n=5) 25 mg of β-TCP granules and the same number of cells (1.65 × 10⁶) as the positive control. 8 A composition (approximately 2 mg of total protein) prepared from a platelet-removed megakaryocyte culture containing (1) was used to prepare and transplant a transplant sample. Example 4B (n=16) 25 mg of β-TCP granules and 5 times the number of cells (8.25 × 10⁻¹) of the positive control. 8 A transplant sample was prepared and transplanted using a composition prepared from a platelet-removed megakaryocyte culture containing (1) cells.

[0202] Each artificial bone material was prepared as follows: • Negative control group On a dappen glass, 25 mg of sterile TCP was mixed with 50 μl of bovine fibrinogen (10 mg / ml) (Sigma, F8630), and 5 μl of bovine thrombin (100 U / ml) (Sigma, T9549) was added to induce gelation. These steps were performed in the animal center to allow for rapid transplantation after gelation. • Positive control group In a low-protein-adsorbent tube, 100 μl of the platelet-rich plasma (PRP) was impregnated into 25 mg of β-TCP granules. After impregnation, it was mixed with 10 μl of autologous serum (10 mg / ml), and 5 μl of thrombin (100 U / ml) was added to induce gelation. These steps were performed in the animal center in order to allow for rapid transplantation after gelation. • Example 4A Using a low protein adsorption tube, 8 μl of the composition of the present invention was mixed with 32 μl of sterilized water (DW), and then impregnated into 25 mg of TCP. After the impregnation, it was mixed with 10 μl of fibrinogen (10 mg / ml), and 5 μl of thrombin (100 U / ml) was added to cause gelation. In order to transplant immediately after gelation, these steps were carried out in an animal center. · Example 4B Using a low protein adsorption tube, 40 μl of the composition of the present invention was impregnated into 25 mg of TCP. After the impregnation, it was mixed with 10 μl of fibrinogen (10 mg / ml), and 5 μl of thrombin (100 U / ml) was added to cause gelation. In order to transplant immediately after gelation, these steps were carried out in an animal center.

[0203] After the transplantation, at the 4th or 8th week, the transplantation samples including the surrounding skull were removed, and histological observation by HE staining was performed. In addition, for the regions where bone formation or bone proliferation occurred, the bone formation amount and bone remodeling amount were examined by counting in pixel units using software (ImageJ). The bone formation amount is the newly formed neonatal bone part in the transplantation sample, and the bone remodeling amount is the target region including neonatal bone, bone marrow, and the artificial bone around them. The bone formation amount and bone proliferation amount were corrected by the bone formation amount and bone proliferation amount in the negative control group. These results are shown in FIGS. 14 and 15.

[0204] FIG. 14 is a photograph showing bone formation. In FIG. 14, each photograph shows the results of Example 4A, Example 4B, the positive control group, and the negative control group from left to right. Also, in the photographs of each group, the upper row is a photograph taken with an objective lens of magnification 20 times, the lower row is a photograph taken with an objective lens of magnification 100 times, and is an enlarged photograph of the region indicated by the broken line in the upper row. As shown in FIGS. 14(A) to (C), in Example 4(A) and (B) and the positive control, bone was newly formed in the region indicated by dark gray around the remaining part of the light gray artificial bone.

[0205] Next, FIG. 15 is a graph showing the amount of bone formation and the amount of bone growth. In FIG. 15, (A) shows the amount of bone formation, and (B) shows the amount of bone growth. In FIG. 15(A), the horizontal axis represents the period after transplantation of the graft material, and the vertical axis represents the amount of bone formation. In FIG. 15(B), the horizontal axis represents the period after transplantation of the graft material, and the vertical axis represents the amount of bone growth. As shown in FIGS. 14 and 15, Example 4A showed the same amount of new bone formation and bone growth including bone marrow and artificial bone around the new bone as the positive control group from 4 to 8 weeks after transplantation. Furthermore, in Example 4B, at 4 weeks after transplantation, an amount of bone formation and bone growth exceeding that of the positive control group was observed, but at 8 weeks after transplantation, the amount of bone formation and bone growth was the same as that of the positive control group. This was presumably because in Example 4B, at 4 weeks after transplantation, the plateau of the amount of bone formation and bone growth already reached the same level as the positive control group and Example 4A.

[0206] [Example 5] It was confirmed that the composition of the present invention has anti-inflammatory activity.

[0207] (1) Activation of immune cells Activation of immune cells was carried out as shown in FIGS. 16(A) and 17(A). Specifically, first, a phosphate buffer containing 15 ng / ml of anti-human CD3 antibody (manufactured by eBioscience, Cat No.: 2045756) and 5 ng / ml of anti-human CD28 antibody (manufactured by eBioscience, Cat No.: 1928813) was seeded in a 24-well plate and incubated overnight (about 12 hours) to immobilize these antibodies on the plate. Then, peripheral blood mononuclear cells were isolated from peripheral blood collected from volunteers using Ficoll (Histopaque (registered trademark) 1077, Sigma Aldrich). The peripheral blood mononuclear cells were seeded in the 24-well plate at a density of 2.5×10 6 cells / wel and the culture was started. The medium for the peripheral blood mononuclear cells was RPMI medium (manufactured by gibco, Cat No.: 20621736) containing 10% FBS and 1% Ab.

[0208] One hour after the start of the culture, the composition (iMDF) was added to each well so that the total protein concentration derived from the composition in the culture medium reached a predetermined concentration (0, 125, 250, or 500 μg / ml). The composition used was the platelet-removed megakaryocyte culture prepared in Example 1(4). After adding the composition, the cells were cultured for a further 1 or 3 hours. After the culture, the peripheral blood mononuclear cells were collected, and then total RNA was extracted from the peripheral blood mononuclear cells using an mRNA extraction reagent (Trizol®, Thermo Fisher Scientific). cDNA was synthesized using the obtained RNA and a reverse transcriptase (SuperScript® III First-Strand Synthesis System, Thermo Fisher Scientific).

[0209] (2) Measurement of cytokine expression levels The obtained cDNA, the primer set described below, and DNA polymerase (Takara-Taq, Takara Bio Inc.) were used to measure the mRNA expression levels of each gene using a qPCR instrument (Mx3000P QPCR System, Agilent Technologies, Inc.). After the measurement, the relative expression levels of each gene were calculated based on the expression level of the internal standard gene (GAPDH gene). The negative control was performed in the same manner, except that anti-CD3 and anti-CD28 antibodies were not immobilized (unstimulated). These results are shown in Figures 16 and 17.

[0210] TNF-α primer set Forward primer (SEQ ID NO: 1) 5'-AATGGCGTGGAGCTGAGA-3' Reverse primer (SEQ ID NO: 2) 5'-TAGACCTGCCCAGACTCGG-3' IFN-γ primer set Forward primer (SEQ ID NO: 3) 5'-CTGTTACTGCCAGGACCCAT-3' Reverse primer (SEQ ID NO: 4) 5'-ACACTCTTTTGGATGCTCTGGT-3' • Primer set for IL-1β Forward primer (SEQ ID NO: 5) 5'-CACAGACCTTCCAGGAGAAT-3' Reverse primer (SEQ ID NO: 6) 5'-TTCAACACGCAGGACAGGTA-3' • Primer set for IL-6 Forward primer (SEQ ID NO: 7) 5'-GTACATCCTCGACGGCATC-3' Reverse primer (SEQ ID NO: 8) 5'-AGCCACTGGTTCTGTGCCT-3' GAPDH Primer Set Forward primer (SEQ ID NO: 9) 5'-GGAGTCCACTGGCGTCTTCAC-3' Reverse primer (SEQ ID NO: 10) 5'-GCTGATGATCTTGAGGCTGTTGTC-3'

[0211] Figures 16 and 17 are schematic diagrams and graphs illustrating the experimental overview and cytokine gene expression levels. In Figures 16(B)-(E) and 17(B)-(E), the horizontal axis represents the sample type, and the vertical axis represents the relative expression level of each gene. As shown in Figures 17(B)-(E), the composition of the present invention suppressed the induction of TNF-α, IFN-γ, IL-1β, and IL-6 expression by peripheral blood mononuclear cells in a concentration-dependent manner at 3 hours post-stimulation. On the other hand, as shown in Figures 16(B)-(E), the composition of the present invention suppressed the induction of TNF-α, IL-1β, and IL-6 expression by peripheral blood mononuclear cells in a concentration-dependent manner at 1 hour post-stimulation. Although the induction of IFN-γ expression was not suppressed at 1 hour post-stimulation, it was suppressed at 3 hours post-stimulation. This indicates that the suppression of IFN-γ expression induction by the composition of the present invention appears at a later timing compared to other inflammatory cytokines. Since the anti-CD3 antibody and anti-CD28 antibody used in this embodiment activate T cells, it was presumed that the composition of the present invention suppresses the activation of immune cells such as T cells by suppressing the induction of cytokine gene expression.

[0212] [Example 6] We have confirmed that the composition of the present invention promotes bone formation and bone regeneration in the site of a skull defect in vivo.

[0213] As a bone formation model, we used a model in which bone formation was performed in a skull defect of a nude rat. Specifically, a 4 mm defect was created in the skull of a nude rat (8 weeks old, F344 / NJcl-rnu / nu) under general anesthesia (3-component mixture), and the composition of the present invention was used as a transplant sample together with artificial bone material and transplanted. Specifically, the following two groups were established. • Negative control group (n=17) A 4 mm diameter octacalcium phosphate (OCP) / collagen (Col) support was impregnated with PBS, and the prepared transplant sample was transplanted. Example 6 (n=8) 8.25 × 108 A composition (approximately 25 mg of total protein) prepared from platelet-depleted megakaryocyte cultures equivalent to one platelet-rich plasma cell was impregnated and adsorbed onto an OCP / Col carrier to prepare a transplant sample, which was then transplanted.

[0214] Each artificial bone material was prepared as follows: • Negative control group OCP granules (diameter 300-500 μm) and 3% porcine-derived telocolazen were mixed, and the mixture was freeze-dried. After freeze-drying, thermal crosslinking was performed to produce an OCP / Col carrier (OCP carrier, containing 77% OCP by weight; diameter 5 mm, thickness 1 mm). • Example 6 20 μl of the composition of the present invention (8.25 × 10 8 An OCP carrier was impregnated with platelet-rich plasma (equivalent to 10

[0215] For the negative control group and Example 6, the graft sample, including the surrounding skull, was excised at 4 or 8 weeks, relative to the time of transplantation at the defect site. μCT imaging was then performed on the excised area. After imaging, specimens were prepared from the excised area and histological observation using HE staining or Masson trichrome staining, as well as immunohistochemistry. Furthermore, the bone mass and bone mineral content of the newly formed bone (neoplastic bone) from the graft material were compared with the bone mass (B) of the bone excised at the time of creating the defect site. S Bone mass (B) of newly formed bone from graft material R ) ratio (B R / B S ) and bone density were evaluated. In the above evaluation, 3D bone analysis software (TRI / 3D-BON; manufactured by Ratoc System Engineering) was used to evaluate bone morphology (bone mass: BV (cm)). 3 ), Bone mineral content: BMC (mg), Bone density: BMD (mg / cm 3 )) was measured. The bone volume (B) of the bone excised when creating the defect site was measured. S Bone mass (B) of newly formed bone from graft material R ) ratio (B R / B S) was calculated as a ratio when the bone loss was set to 100%. These results are shown in FIGS. 18 and 19.

[0216] FIG. 18 is a photograph showing bone formation at 4 weeks after transplantation. In FIG. 18, in each photograph, the upper row shows the negative control group, and the lower row shows the results of the group of Example 6. Also, in the photographs of each group, from the left, there are photographs of the entire excised part, the left side of the defect site, the right side of the defect site, and a cross-section of the excised part including the defect site. As shown in FIG. 18, in the group of Example 6, it was found that bone was newly formed at the defect site of the skull.

[0217] Next, FIG. 19 is a graph showing bone mass, bone mineral content, the ratio of bone loss to newly formed bone, and bone density at 4 weeks after transplantation. In FIG. 19, (A) shows bone mass, (B) shows bone mineral content, (C) shows the ratio of bone loss to newly formed bone (B R / B S ), and (D) shows bone density. In FIG. 19(A), the horizontal axis shows the type of experimental group, and the vertical axis shows bone volume (BV) (cm 3 ). In FIG. 19(B), the horizontal axis shows the type of experimental group, and the vertical axis shows bone mineral content (BMC) (mg). In FIG. 19(C), the horizontal axis shows the type of experimental group, and the vertical axis shows the ratio of bone loss to newly formed bone (%). In FIG. 19(D), the horizontal axis shows the type of experimental group, and the vertical axis shows bone mineral density BMD (mg / cm 3 ). As shown in FIG. 19, compared with the negative control group, in Example 6, the bone mass, bone mineral content, the ratio of bone loss to newly formed bone, and bone density increased on both the left and right sides of the defect. From the above, it was found that the composition of the present invention can newly form bone at the defect site of the skull.

[0218] From the above, it was found that the composition of the present invention can induce bone formation and bone growth, and can promote bone formation and bone growth by increasing the content of its components.

[0219] Although the present invention has been described above with reference to embodiments and examples, the present invention is not limited to the above embodiments and examples. Various modifications to the configuration and details of the present invention can be understood by those skilled in the art within the scope of the present invention.

[0220] This application claims priority based on Japanese Patent Application No. 2020-180042, filed on 27 October 2020, and incorporates all of its disclosures herein.

[0221] <Note> Some or all of the above embodiments and examples may be described as follows, but are not limited to the following. (Note 1) A bone-forming composition comprising a processed product of megakaryocytes or their cultures. (Note 2) The bone-forming composition according to Appendix 1, wherein the processed product is an extract of the cell fraction of megakaryocytes or their cultures. (Note 3) The processed product is a bone-forming composition according to Appendix 1 or 2, containing 2,000 to 20,000 pg of basic fibroblast growth factor (bFGF) per 1 mg of total protein. (Note 4) The processed product is a bone-forming composition according to any one of the appendices 1 to 3, containing 8,000 to 80,000 pg of insulin-like growth factor-binding protein-2 (IGFBP-2) per 1 mg of total protein. (Note 5) The processed product is a bone-forming composition according to any one of the appendices 1 to 4, containing 1 to 60 pg of placental growth factor (PIGF) per 1 mg of total protein. (Note 6) The processed product is a bone-forming composition according to any one of the appendices 1 to 5, containing 200 to 2000 pg of stem cell factor receptors (SCFRs) per 1 mg of total protein. (Note 7) The processed product is a bone-forming composition according to any one of the appendices 1 to 6, containing 20 to 800 pg of vascular endothelial growth factor (VEGF) per 1 mg of total protein. (Note 8) The processed product is a bone-forming composition according to any one of the appendices 1 to 7, containing 20 to 400 pg of vascular endothelial growth factor receptor 2 (VEGFR2) per 1 mg of total protein. (Note 9) The processed product is a bone-forming composition according to any one of the appendices 1 to 8, containing 1,000 to 10,000 pg of differentiation and growth factor-15 (GDF-15) per 1 mg of total protein. (Note 10) The processed product is a bone-forming composition according to any one of the appendices 1 to 9, comprising 0 to 1000 pg of bone morphogenetic protein-7 (BMP-7) per 1 mg of total protein. (Note 11) The processed product is a bone-forming composition according to any one of the appendices 1 to 10, comprising 0 to 16 pg of amphiregulin (AR) per 1 mg of total protein. (Note 12) The processed product is a bone-forming composition according to any one of the appendices 1 to 11, comprising 0 to 60 pg of epidermal growth factor receptor (EGFR) per 1 mg of total protein. (Note 13) The processed product is a bone-forming composition according to any one of the appendices 1 to 12, containing 0 to 100 pg of liver growth factor (HGF) per 1 mg of total protein. (Note 14) The processed product is a bone-forming composition according to any one of the appendices 1 to 13, comprising 0 to 200 pg of insulin-like growth factor-binding protein-1 (IGFBP-1) per 1 mg of total protein. (Note 15) A bone-forming composition according to any one of the appendices 1 to 14, having cell proliferation-promoting activity. (Note 16) The bone-forming composition described in Appendix 15, wherein the cells are mesenchymal stem cells. (Note 17) A bone-forming composition according to any one of the appendices 1 to 16, which has the ability to promote the differentiation of osteoblast precursor cells into osteoblasts. (Note 18) The osteoblast precursor cells are mesenchymal stem cells, as described in Appendix 17, for the bone-forming composition. (Note 19) The bone-forming composition according to any one of the appendices 1 to 18, wherein the megakaryocyte culture is a culture from which platelets have been removed. (Note 20) The megakaryocytes are immortalized megakaryocytes, as described in any of Appendix 1 to 19 of the bone-forming composition. (Note 21) The bone formation composition according to Appendix 20, wherein the immortalized megakaryocytes are megakaryocytes containing exogenous BMI1 gene, MYC gene, and Bcl-xL gene. (Note 22) The megakaryocytes mentioned above are in vitro A bone-forming composition according to any one of the appendices 1 to 21, wherein megakaryocytes are induced by [the specified method]. (Note 23) The megakaryocytes are derived from pluripotent cells, as described in any of Appendix 1 to 22. (Note 24) The pluripotent cells are induced pluripotent stem (iPS) cells, as described in Appendix 23 of the bone formation composition. (Note 25) The aforementioned processed material is Process megakaryocytes or their cultures, The bone-forming composition according to any one of the appendices 1 to 24, wherein the treatment is a concentration treatment, a drying treatment, a freeze treatment, a freeze-drying treatment, a solvent treatment, a surfactant treatment, an enzyme treatment, a protein fractionation and extraction treatment, an ultrasonic treatment, and / or a crushing treatment. (Note 26) The aforementioned processed material is Platelets are removed from the megakaryocytes or their cultures. The bone-forming composition according to Appendix 25, comprising processing megakaryocytes or cultures from which the platelets have been removed. (Note 27) The aforementioned processed material is The megakaryocytes or cultures from which the platelets have been removed are stored. The bone-forming composition according to Appendix 26, for processing the preserved megakaryocytes or their cultures. (Note 28) The aforementioned processed material is The megakaryocyte or its culture is stored, The bone-forming composition according to Appendix 27, wherein the stored megakaryocytes or their cultures are subjected to a crushing treatment. (Note 29) The composition comprises a bone formation composition and a bone formation scaffold material. The bone formation kit is a bone formation composition described in any of the appendices 1 to 28. (Note 30) The scaffolding material is the kit described in Appendix 29, which exhibits biocompatibility and / or biodegradability. (Note 31) The kit according to Appendix 29 or 30, wherein the scaffolding material comprises at least one selected from the group consisting of collagen, gelatin, albumin, keratin, dextran, carboxymethylcellulose, xanthan gum, chitosan, chondroitin sulfate, heparin, hyaluronic acid, polyglycolic acid, polylactic acid, copolymer of polyglycolic acid and polylactic acid, polyhydroxybutyric acid, polydioxanone, polycaprolactone, polybutylene succinate, calcium phosphate, hydroxyapatite, polyether ketone, and polyether ether ketone. (Note 32) The scaffolding material is a kit according to any one of the appendices 29 to 31, having a porous structure. (Note 33) A kit containing a bioactive substance, as described in any of the appendices 29 to 32. (Note 34) A kit as described in any of Appendix 29 to 33, comprising at least one selected from the group consisting of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), brain-derived neurotrophic factor (BDNF), growth and differentiation factor-5 (GDF5), erythropoiesis-promoting factor (EPO), transforming growth factor (TGF), and bone morphogenetic protein. (Note 35) A composition that promotes the differentiation of osteoblasts, comprising the bone formation composition described in any of Appendix 1 to 28. (Note 36) A composition for inhibiting the activation of immune cells, comprising a processed product of megakaryocytes or their cultures. (Note 37) The aforementioned processed product is an extract of the cell fraction of megakaryocytes or their cultures, which is the immune cell activation inhibitory composition described in Appendix 36. (Note 38) The processed product is an immune cell activation inhibitory composition as described in Appendix 36 or 37, comprising 2,000 to 20,000 pg of basic fibroblast growth factor (bFGF) per 1 mg of total protein. (Note 39) The aforementioned processed product is an immune cell activation inhibitory composition according to any one of the appendices 36 to 38, comprising 8,000 to 80,000 pg of insulin-like growth factor-binding protein-2 (IGFBP-2) per 1 mg of total protein. (Note 40) The processed product is an immune cell activation inhibitory composition according to any one of the appendices 36 to 39, comprising 1 to 60 pg of placental growth factor (PIGF) per 1 mg of total protein. (Note 41) The processed product is an immune cell activation inhibitory composition according to any one of the appendices 36 to 40, comprising 200 to 2000 pg of stem cell factor receptors (SCFRs) per 1 mg of total protein. (Note 42) The processed product is an immune cell activation inhibitory composition according to any one of the appendices 36 to 41, comprising 20 to 800 pg of vascular endothelial growth factor (VEGF) per 1 mg of total protein. (Note 43) The aforementioned processed product is an immune cell activation inhibitory composition according to any one of the appendices 36 to 42, comprising 20 to 400 pg of vascular endothelial growth factor receptor 2 (VEGFR2) per 1 mg of total protein. (Note 44) The processed product is an immune cell activation inhibitory composition according to any one of the appendices 36 to 43, containing 1,000 to 10,000 pg of differentiation and growth factor-15 (GDF-15) per 1 mg of total protein. (Note 45) The processed product is an immune cell activation inhibitory composition according to any one of the appendices 36 to 44, comprising 0 to 16 pg of amphiregulin (AR) per 1 mg of total protein. (Note 46) The processed product is an immune cell activation inhibitory composition according to any one of the appendices 36 to 45, comprising 0 to 60 pg of epidermal growth factor receptor (EGFR) per 1 mg of total protein. (Note 47) The processed product is an immune cell activation inhibitory composition according to any one of the appendices 36 to 46, comprising 0 to 100 pg of liver growth factor (HGF) per 1 mg of total protein. (Note 48) The processed product is an immune cell activation inhibitory composition according to any one of the appendices 36 to 47, comprising 0 to 200 pg of insulin-like growth factor-binding protein-1 (IGFBP-1) per 1 mg of total protein. (Note 49) The immune cell is a T cell, according to the immune cell activation inhibitory composition according to any one of the appendices 36 to 48. (Note 50) The aforementioned megakaryocytes are immortalized megakaryocytes, wherein the immune cell activation inhibitory composition is as described in any one of the appendices 36 to 49. (Note 51) The megakaryocytes mentioned above are in vitro A composition for inhibiting the activation of immune cells, which are megakaryocytes induced by any one of the appendices 36 to 50. (Note 52) An anti-inflammatory composition comprising an immune cell activation inhibitory composition described in any one of the appendices 36 to 51. (Note 53) A method for promoting the differentiation of osteoblasts using a bone-forming composition described in any of Appendix 1 to 28. (Note 54) The bone-forming composition is in vitro or in vivo A method for promoting the differentiation of osteoblasts, as described in Appendix 53, for use in this context. (Note 55) A method for promoting the differentiation of osteoblasts, using the osteoblast differentiation-promoting composition described in Appendix 35. (Note 56) The osteoblast differentiation-promoting composition is in [[ID=9�]]vitro or in vivo A method for promoting the differentiation of osteoblasts, as described in Appendix 55, for use in this context. (Note 57) A method for suppressing the activation of immune cells, using an immune cell activation suppression composition described in any of Appendix 36 to 51. (Note 58) The immune cell activation inhibitory composition, in vitro or in [[ID=1Q2]]vivo A method for promoting the differentiation of osteoblasts, as described in Appendix 57, for use in this context. (Note 59) A method for suppressing inflammation using the anti-inflammatory composition described in Appendix 52. (Note 60) The aforementioned anti-inflammatory composition, in vitro or in vivo The method for suppressing inflammation, as described in Appendix 59, to be used in [the case]. (Note 61) A composition containing, as an active ingredient, a processed product of megakaryocytes or their cultures for use in bone formation. (Note 62) A composition containing megakaryocytes or a treated culture thereof as an active ingredient for use in promoting the differentiation of osteoblasts. (Note 63) A composition containing a processed product of megakaryocytes or their cultures as an active ingredient for use in suppressing the activation of immune cells. (Note 64) A composition containing a processed product of megakaryocytes or their cultures as an active ingredient for use in suppressing inflammation. [Industrial applicability]

[0222] As described above, the present invention provides a composition having bone-forming activity derived from porosinocytes. Furthermore, the composition of the present invention has, for example, cell proliferation-promoting activity and differentiation-promoting activity into osteoblasts. For this reason, the present invention is extremely useful in the pharmaceutical field, the field of regenerative medicine, and the like.

Claims

1. Includes processed products of thrombocytopenic megakaryocytes or thrombocytopenic megakaryocyte cultures, The processed product is an extract of the cell fraction of the megakaryocyte or the megakaryocyte culture. The megakaryocytes are immortalized megakaryocytes in this bone-forming composition.

2. Includes processed products of thrombocytopenic megakaryocytes or thrombocytopenic megakaryocyte cultures, The processed product is an extract of the cell fraction of the megakaryocyte or the megakaryocyte culture. The megakaryocytes are megakaryocytes induced in vitro, wherein the composition is an osteogenic composition.

3. The bone-forming composition according to claim 1 or 2, wherein the processed product contains 2,000 to 20,000 pg of basic fibroblast growth factor (bFGF) per 1 mg of total protein.

4. The bone-forming composition according to any one of claims 1 to 3, wherein the processed product contains 8,000 to 80,000 pg of insulin-like growth factor-binding protein-2 (IGFB-2) per 1 mg of total protein.

5. The bone-forming composition according to any one of claims 1 to 4, wherein the processed product contains 1 to 60 pg of placental growth factor (PIGF) per 1 mg of total protein.

6. The bone-forming composition according to any one of claims 1 to 5, wherein the processed product contains 200 to 2000 pg of stem cell factor receptor (SCFR) per 1 mg of total protein.

7. The bone-forming composition according to any one of claims 1 to 6, wherein the processed product contains 20 to 800 pg of vascular endothelial growth factor (VEGF) per 1 mg of total protein.

8. The bone-forming composition according to any one of claims 1 to 7, wherein the processed product contains 20 to 400 pg of vascular endothelial growth factor receptor 2 (VEGFR2) per 1 mg of total protein.

9. The bone-forming composition according to any one of claims 1 to 8, wherein the processed product contains 1,000 to 10,000 pg of differentiation and growth factor-15 (GDF-15) per 1 mg of total protein.

10. The bone-forming composition according to any one of claims 1 to 9, wherein the processed product contains 0 to 16 pg of amphiregulin (AR) per 1 mg of total protein.

11. The bone-forming composition according to any one of claims 1 to 10, wherein the processed product contains 0 to 60 pg of epidermal growth factor receptor (EGFR) per 1 mg of total protein.

12. The bone-forming composition according to any one of claims 1 to 11, wherein the processed product contains 0 to 100 pg of liver growth factor (HGF) per 1 mg of total protein.

13. The bone-forming composition according to any one of claims 1 to 12, wherein the processed product contains 0 to 200 pg of insulin-like growth factor-binding protein-1 (IGFB-1) per 1 mg of total protein.

14. A bone-forming composition according to any one of claims 1 to 13, having cell proliferation-promoting activity.

15. The bone-forming composition according to any one of claims 1 to 14, which has the ability to promote the differentiation of osteoblast precursor cells into osteoblasts.

16. The bone-forming composition according to any one of claims 2 to 15, wherein the megakaryocytes are immortalized megakaryocytes.

17. The osteogenic composition according to any one of claims 1 or 3 to 16, wherein the megakaryocytes are megakaryocytes induced in vitro.

18. The megakaryocytes or megakaryocyte cultures after platelet removal are obtained by removing platelets from megakaryocytes or megakaryocyte cultures, according to any one of claims 1 to 17.

19. The bone formation composition according to claim 18, wherein the megakaryocyte or megakaryocyte culture is obtained by culturing megakaryocytes for 1 day to 2 weeks to produce platelets.

20. The composition comprises a bone formation composition and a bone formation scaffold material. The bone-forming composition is the bone-forming composition according to any one of claims 1 to 19, in the bone-forming kit.

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