WO2002017983A1

WO2002017983A1 - Medicinal compositions for forming tissue around bone or tooth, process for preparing the same, injections for forming tissue around bone or tooth and process for preparing the same

Info

Publication number: WO2002017983A1
Application number: PCT/JP2001/007289
Authority: WO
Inventors: Minoru Ueda; Ken-Ichiro Hata; Yoshitaka Hibino; Kunihiko Okada
Original assignee: Osteogenesis Co., Ltd.
Priority date: 2000-08-28
Filing date: 2001-08-24
Publication date: 2002-03-07
Also published as: AU2001280164A1

Abstract

Medicinal compositions for forming a tissue around a bone or a tooth which are highly effective in regenerating a bone tissue, can be handled conveniently and have a high safety. A medicinal composition for forming a tissue around a bone or a tooth, which is in the form of a paste at using, is prepared by mixing mesenchymal stem cells capable of differentiating into bone cells, extracellular matrix of these cells and ß-TCP having an average particle size of from 1 to 5 νm.

Description

Description Pharmaceutical composition for forming bone or periodontal tissue and method for preparing the same, and injection for forming bone or periodontal tissue and method for preparing the same

The present invention relates to a pharmaceutical composition for forming bone or periodontal tissue and a method for preparing the same, which can be used for repairing and regenerating bone or periodontal tissue, and an injection for forming bone or periodontal tissue and a method for preparing the same. Background art

Attempts have been made to repair and regenerate bone tissue and cartilage tissue damage and defects by tissue engineering. The regeneration of bone tissue (cartilage tissue) by tissue engineering combines the cells that form bone tissue, the substrate that replaces new bone tissue, and growth factors that control the proliferation and differentiation of cells involved in bone tissue regeneration. It is intended to regenerate bone tissue (cartilage tissue) by using it, and to apply functions related to the repair mechanism of normal bone tissue in the living body. In particular, focusing on growth factors, repairing growth factors together with substrates Many attempts have been made to regenerate bone tissue by directly transplanting to the desired bone defect. Among growth factors, bone morphogenetic protein (BMP) has been widely studied. BMP is a growth factor that has been discovered as a substance exhibiting ectopic osteoinductive activity in the demineralized bone matrix, and acts on undifferentiated mesenchymal stem cells to cause osteoblasts or cartilage. Differentiate into blasts. As a carrier for using BMP, glass fiber, hydroxyapatite, ceramics, collagen, synthetic polymer, and the like are being studied. For bone formation implants combining BMP and carrier, see For example, it has been proposed in Japanese Patent Application Laid-Open Nos. 7-24632-35 and 10-151188.

On the other hand, the use of cell-hybrid artificial bone, which combines cells cultured in a test tube with biomaterials, is being studied. In this method, for example, an osteogenic cell collected from bone marrow is cultured in a three-dimensional scaffold having a defect-like form to be regenerated, thereby producing a cell-incorporated artificial bone. Is implanted into the defect. Caplan et al. Found that mesenchymal stem cells were present in the bone marrow and showed that ectopic osteoinduction was observed when the bone marrow was mixed with porous calcium phosphate ceramics and transplanted (Ohguchi, H., Goldgerg, VM: Heterotopic osteogenesis m porous ceramics muced by marrow cells. J. orthop. Res. 7: 568-578 (1989)). Further studies show that bone formation can be induced in vitro by seeding bone marrow cells on porous ceramics in a culture dish and culturing in the presence of dexamethasone. (Hanada, K., Dennis, JE: Stimulatery effect of basic fibroblast growth factor and bone morphogenetic protem-2 on osteogenetic differentation of rat bone marrow-derived mesenchymal stem cells. J. Bone Mineral Res. 12: 1606-1614 (1997)).

Also, regarding the use of a transplantation material containing bone marrow cells or osteoblasts and a calcium phosphate-based compound, Japanese Patent Application Laid-Open Nos. Hei 3-45267 / 07, 197-14733 / 07, and It has been proposed in Kaihei 10—2 439 996 and Japanese Patent No. 2752387.

The only method that has been widely applied clinically to bone tissue regeneration is the periodontal tissue regeneration method called GTR (guides tissue regeneration). This method involves inserting a shielding membrane (GTR membrane) between the root surface and the gingival flap to block the invasion of cells from the connective tissue and epithelium of the gingiva to the tissue defect, and to form periodontal ligament cells and osteoblasts Cells are periodontal It provides a place to regenerate the organization. Polytetrafluoroethylene film and the like are generally used for the GTR film, but since these materials are non-bioabsorbable, secondary surgery is required to remove the GTR film. Absorbent membranes made of synthetic polymers and membranes made of collagen with excellent biocompatibility have been developed, but as of now, the former has low affinity for tissues, and the latter does not have sufficient strength. There is a problem. It has also been pointed out that the GTR method has a limited regeneration ability for large alveolar bone defects.

Even in the method of regenerating cartilage, attempts have been made to transplant a combination of chondrocytes and a carrier.

As described above, tissue engineering has attempted to regenerate bone tissue (cartilage tissue) or periodontal tissue in many cases. However, knowledge on the regeneration mechanism of bone tissue (cartilage tissue) or periodontal tissue may not be sufficient. There are few effective regeneration methods that can be applied clinically, and the development of a new bone or periodontal tissue regeneration method is desired. Such a regeneration method is required not only to have a high effect of regenerating bone or periodontal tissue, but also to have good operability and to be safe for a living body. Disclosure of the invention

The present invention has been made as a result of intensive studies in view of the above problems, and in order to provide a novel bone or periodontal tissue regeneration method applicable to clinical use, the bone or periodontal tissue used in the method is provided. It is intended to provide a pharmaceutical composition for tissue formation. The configuration of the first aspect of the present invention is as follows.

A pharmaceutical composition for forming bone or periodontal tissue which has fluidity at the time of use, comprising mesenchymal stem cells having acquired the ability to differentiate into bone cells, and an inorganic bioabsorbable material. Also preferably, a pharmaceutical composition for forming a bone or periodontal tissue, which further contains a gelling agent and is configured to gel after application. According to such a configuration, since it includes cells that have acquired the ability to differentiate into bone cells, direct regeneration of bone tissue by itself can be expected. In addition, inorganic bioabsorbable materials used as cell carriers or scaffolds will be replaced with bone tissue in the future, and their safety is high. Furthermore, since it has fluidity at the time of use, it does not need to be molded in advance in accordance with the shape of the bone or periodontal tissue defect, and is versatile, and its handling is easy. As described above, according to the configuration of the first aspect of the present invention, a bone or periodontal tissue defect can be effectively repaired and regenerated, and bone or bone having high operability and safety can be obtained. A pharmaceutical composition for periodontal tissue formation is provided. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a view showing a phase-contrast micrograph of the bone marrow cells on the third day of the initial culture in Example 1.

FIG. 2 is a view showing a phase-contrast micrograph of the bone marrow cells on day 10 of the subculture in Example 1.

FIG. 3 is a view showing a phase-contrast micrograph showing a state of bone marrow cells on day 14 of subculture in Example 1. Bone marrow cells are observed in a polygonal shape, and extracellular matrix-like structures can be observed around the cells.

FIG. 4 is a view showing a stained image obtained by staining the cells on day 14 of the subculture in Example 1 with alkaline phosphatase. It can be seen that the cells are partially aggregated and form calcified nodules.

FIG. 5 is a photograph showing the results of transplantation of the cell-containing (3-TCP paste) in Example 3. The tissue image of the demineralized HE (hematoxylen-geosin) stained portion of the transplanted portion 8 weeks after transplantation is shown. (100 times). The part missing in white is decalcified) 3-TCP. A regular lamellar structure is observed in the formed bone tissue, the bone cavities are narrowed, and mature bone tissue is generally observed. FIG. 6 is a diagram showing a table summarizing the osteogenic ability of each sample in Example 4. C and E represent the control group and the stimulation group, respectively.

FIG. 7 is a graph summarizing the measurement results in Example 5. The alkaline phosphatase (ALP) activity in each group when cultured under the conditions of an extension rate of 15% (mechal5%) and 5 cycles / min (5 cycles / min, about 0.083 Hz) is shown. SC1 and SC2 were the groups that did not use an induction medium and did not receive a stretching stimulus. The groups without a group, indl + Sl to indl + S4, represent the groups to which a stretching stimulus is applied using an induction medium, respectively.

FIG. 8 is a graph showing a summary of measurement results in Example 5. It shows the activity of ALPHA in each group when cultured under the conditions of an extension rate of 15% (mechal5%) and 10 cycles / min (10 cycles / min, about 0.167 Hz). SC1 and SC2 were a group that did not use an induction medium and did not receive a stretching stimulus, SC + S1 and SC + S2 were a group that added only a stretching stimulus without using an induction medium, and indl and ind2 were a group that used an induction medium and applied a stretching stimulation The groups without a group, indl + Sl to indl + S4, represent the groups to which a stretching stimulus is applied using an induction medium, respectively.

FIG. 9 is a diagram showing a graph summarizing the measurement results in Example 5. The alkaline phosphatase (ALP) activity in each group when cultured under the conditions of an extension rate of 20% (mecha 20%) and 5 cycles / min (5 cycles / min, about 0.083 Hz) is shown. SC1 and SC2 do not use induction medium and do not stimulate, SC + S1 and SC + S2 do not use induction medium and apply only extension stimulation, indl and ind2 use induction medium and do not stimulate extension The groups, indl + Sl to indl + S4, represent the groups to which the stretching stimulus is applied using the induction medium. BEST MODE FOR CARRYING OUT THE INVENTION In the first aspect of the present invention, a mesenchymal stem cell that has acquired a differentiation ability to a bone cell is used, but “acquired a differentiation ability to a bone cell” is an undifferentiated state. Is the state in which the cells are oriented to differentiate into bone cells. Here, bone cells include osteoblasts, osteoclasts, and chondroblasts.

In addition, as the mesenchymal stem cells having acquired the differentiation ability to bone cells, not only autologous cells but also allogeneic allogeneic cells can be used. In particular, human mesenchymal stem cells can be used.

Mesenchymal stem cells that have acquired the ability to differentiate into bone cells (hereinafter referred to as “osteoblast-acquiring cells”) are transformed under the conditions that induce undifferentiated mesenchymal stem cells to differentiate into bone cells. It can be prepared by culturing. For example,) 3-glycerophosphate> dexamethasone, L-ascorbic acid

By culturing undifferentiated mesenchymal stem cells in a medium containing (L-ascorbic acid), differentiation into bone cells can be induced. Of course, the culture conditions are not limited to these, and any known conditions for inducing differentiation into bone cells can be used.

Investigation of culture conditions for inducing differentiation into bone cells shows that culturing the cells in the presence of physical stimulation (mechanical stress) promotes differentiation into bone cells. I understood. From this, it can be said that giving physical stimulation to cells at the time of induction of differentiation is one preferable condition. For example, in the presence of physical stimuli, undifferentiated mesenchymals in a medium containing 3-glycerophosphate (β-glycerophosphate), dexamethasone (Dexamethason), and L-ascorbic acid (L-ascorbic acid) By culturing the lineage stem cells, efficient differentiation can be induced. Here, the physical stimulation refers to the pressure from outside the cell, pulling force or the like is applied, for example, a gas in the culture vessel (air, C_〇 _2, etc.) periodically ', continuous, or Intermittent The cells can be supplied with a desired external pressure, etc., through the culture medium. Wear. In addition, the cells are seeded on a stretchable membrane or the like (for example, a silicon membrane), and the membrane to which the cells adhere is expanded or contracted periodically, continuously, or intermittently, thereby stretching and stimulating the cells to stretch. Stimulation can be given. Also, a physical stimulus can be given to the cells by giving a desired flow to the medium. In addition, it is possible to provide physical stimulation using ultrasound. Incidentally, a desired physical stimulus can be given by arbitrarily combining these methods.

Sources of undifferentiated mesenchymal stem cells include bone marrow, dental pulp, and cord blood. After collecting them according to a conventional method, undifferentiated mesenchymal cells are selected based on the presence or absence of adhesiveness. That is, undifferentiated mesenchymal stem cells can be obtained by selecting cells having adhesive properties from cells contained in bone marrow and the like.

It is preferable that the pharmaceutical composition for forming bone or periodontal tissue of the present invention (hereinafter, referred to as “the pharmaceutical composition of the present application”) is obtained by adding the extracellular matrix of the cells together with the cells having the ability to differentiate into bone. This is because the extracellular matrix is expected to serve as a scaffold for the bone differentiation-acquiring cells at the site to which the pharmaceutical composition of the present invention is applied, and it is considered that the bone differentiation-acquiring cells are likely to be established at the application site. In addition, it also serves as a scaffold for bone cells existing around the application site, so that high bone inducing ability can be expected. In addition, factors such as BMP contained in the extracellular matrix include the bone cell differentiation-acquiring cells themselves contained in the pharmaceutical composition of the present invention, and bone cells around the application site or stem cells having the ability to differentiate into bone cells. This is because it can be expected to promote the growth, proliferation, and differentiation of E. coli. The extracellular matrix here is a matrix (matrix) that surrounds the cells that have acquired the osteoblastic capacity.

Not only the extracellular matrix of autologous cells but also the extracellular matrix of allogeneic cells of the same type can be used. This is because BMPs and the like contained in such extracellular matrix are of the same species, so that the same effect can be expected even when allogeneic cells are used. As described above, not only the extracellular matrix of autologous cells but also the extracellular matrix of allogeneic cells can be used, which facilitates the preparation of the extracellular matrix of cells having the ability to acquire bone differentiation potential. When the extracellular matrix is added, the cells with the ability to acquire bone differentiation potential added simultaneously may not be living cells. This means that it is not necessary to treat the cells with bone differentiation potential in the state of living cells, which is desirable from the viewpoint of handling. For example, after obtaining cells having bone differentiation potential, those obtained by freeze-drying or the like can be prepared and used as the cells having bone differentiation potential and its extracellular matrix.

The content of the bone differentiation-acquiring cells in the pharmaceutical composition of the present invention is preferably such that 1 × 10 ⁵ or more cells are present in 1 ml of the composition, more preferably 1 × 10 ⁶ to 1 × 10 10. Preferably, there are ^seven cells. This is because by setting such a cell content, bone formation can be effectively induced.

Although the type of the inorganic bioabsorbable material is not particularly limited,) 3_tricalcium phosphate (hereinafter, referred to as "3-TCP"), α-tricalcium phosphate (hereinafter, referred to as "0; -TCPJ"), phosphorus A material selected from the group consisting of tetracalcium acid, octacalcium phosphate, and amorphous calcium phosphate can be used, and these materials can be used alone, as well as optionally. Two or more selected types may be used in combination. Preferably,) any one of 3-TC） or α-TCΡ, or a combination of these at an arbitrary ratio is used. Used as an inorganic bioabsorbable material.

The inorganic bioabsorbable material can be obtained by a known method. Also, commercially available inorganic biomaterials can be used. 3) As TCP, for example, one manufactured by Olympus Optical Co., Ltd. can be used.

The inorganic bioabsorbable material is preferably in the form of a powder having a particle size such that the pharmaceutical composition of the present invention becomes fluid when used.

The powdery inorganic bioabsorbable material can be prepared by crushing and processing the inorganic bioabsorbable material processed to an appropriate size to a desired particle size. The average particle diameter of the inorganic bioabsorbable material should be 0.5 im to 50 m. Is preferred. More preferably, the average particle size is 0.5! 1 to 10 inorganic bioabsorbable materials. Still more preferably, the average particle size is 1! 5 to 5 organic bioabsorbable materials are used. It is also possible to use a combination of a plurality of types of inorganic bioabsorbable materials having different particle diameters.

It is preferable that the inorganic bioabsorbable material is contained in an amount of 50% by weight to 75% by weight based on the whole pharmaceutical composition of the present invention.

The fluidity of the pharmaceutical composition of the present invention is mainly determined by the particle size and the content of the inorganic bioabsorbable material, and the desired fluidity can be obtained by appropriately adjusting both. Can be. In addition, when a thickener described below is added, the fluidity can be adjusted also by the amount of the thickener added.

Here, the degree of fluidity of the pharmaceutical composition of the present invention at the time of use is not particularly limited, and may be slurry, paste, clay, high-viscosity fluid, or the like. It is preferably in the form of a paste. By making it into a paste, it becomes a pharmaceutical composition for forming bone or periodontal tissue having excellent plasticity. Therefore, it can be applied without being previously formed into the shape of the application portion. That is, application to the application section can be easily performed. In addition, it becomes a pharmaceutical composition for forming bone or periodontal tissue, which has good fixation properties at the application part.

In use, it is preferable that the fluidity is such that it can be injected using an injection container, and such fluidity facilitates application to the application section. In addition, desired fluidity can be obtained depending on the application section. For example, when injecting under the periosteum, it is preferable to make the liquid more fluid (a state having a lower viscosity).

The pharmaceutical composition of the present invention only needs to have fluidity at least at the time of use, and may be in the form of powder or solid before use. Therefore, the pharmaceutical composition of the present invention can be used in a frozen state. The lyophilized state can be used as the pharmaceutical composition of the present invention. Frozen or lyophilized before use By doing so, long-term storage becomes possible, and handling before use becomes easy. Furthermore, since the antigenicity can be reduced by freezing or freeze-drying, it is safe to use allogeneic cells instead of autologous cells as mesenchymal stem cells that have acquired the ability to differentiate into bone cells. The performance is improved.

The flowability of the pharmaceutical composition of the present invention can be adjusted by adding a thickener. As thickeners, thickener polysaccharides such as sodium alginate, glycerin, and serine can be used, but from the viewpoint of safety and bone formation ability, they are highly biocompatible and bioabsorbable. It is preferable to use a substance having biodegradability or biodegradability. By adding glycerin, etc., the effect of preventing frost damage can also be obtained.

It is preferable that the pharmaceutical composition of the present invention contains a gelling agent so as to be gelled after application. According to such a configuration, gelation occurs immediately after application, so that fixability at the application site is improved, and bone or periodontal tissue is repaired or regenerated effectively. In addition, since it has fluidity during use and gels at the application site after use (after application), it is not necessary to mold the application site in advance, and versatility is improved.

As the gelling agent, it is preferable to use one having high biocompatibility, for example, collagen or fibrin glue can be used. Various collagens can be selected and used, but it is preferable to use collagen suitable for the application purpose (applied tissue) of the pharmaceutical composition for forming bone or periodontal tissue of the present invention. When the purpose is to regenerate bone tissue, for example, type I collagen can be used. The collagen used is preferably soluble (acid-soluble collagen, alkali-soluble collagen, enzyme-soluble collagen, etc.).

The pharmaceutical composition of the present application may contain an aqueous solvent. That is, at least an osteogenic differentiation-capable cell and an inorganic bioabsorbable material may be mixed in an aqueous solvent. Aqueous solvents include sterile water, saline, A phosphate buffer or the like can be used. In addition, a part of the culture solution used for obtaining the cells having bone differentiation potential can be used as a solvent.

The pharmaceutical composition of the present application may contain, in addition to the above components, a stabilizer, a preservative, a pH adjuster, and the like. It can also include growth factors, especially osteoinductive factors (BMP).

The pharmaceutical composition of the present application is prepared by mixing the above components. It can also be prepared by the preparation method in the second aspect of the present invention described later. Furthermore, it can be prepared, for example, by the following method. First, a block of an inorganic bioabsorbable material is prepared in a culture solution, and skeletal cells are cultured so as to be adsorbed on the block to form an inorganic bioabsorbable material-bone cell complex. Then, this is crushed, pulverized, or the like to obtain a pharmaceutical composition of the present application comprising a mixture of bone cells and an inorganic bioabsorbable material. The pharmaceutical composition of the present invention thus prepared contains, in addition to the bone cells, the extracellular matrix of the cells. In the case of the above-mentioned preparation method, a thickener, a gelling agent (collagen, fubulin glue, etc.), a stabilizing agent, a preservative, a pH regulator, a growth factor and the like can be separately added. Further, the finally obtained mixture of each component may be frozen or lyophilized. Freezing or freeze-drying can be performed according to a conventional method.

When the pharmaceutical composition of the present invention is prepared in a frozen state or a lyophilized state, it is made into a state having desired fluidity at the time of use. If frozen, thaw to return to the state before freezing. At this time, a desired fluidity can be adjusted by adding a physiological saline or the like. On the other hand, in the case of the freeze-dried state, a solvent such as physiological saline is added to obtain a fluidity before the freeze-drying treatment or a state having a desired fluidity.

By enclosing the pharmaceutical composition of the present invention in an injection container, an injection for bone or periodontal tissue formation (hereinafter referred to as “the injection of the present application”) can be obtained. For example, having liquidity The pharmaceutical composition of the present invention prepared in this state is sealed in an injection container, and then frozen or freeze-dried to obtain an injection of the present invention. The use of such an injection makes handling easier. That is, a desired effect can be expected by injecting the present injection transdermally or transmucosally without adding lime to the skin or mucous membrane as in the past when applying. Such an injection of the present invention is used after the bone or periodontal tissue forming pharmaceutical composition encapsulated therein is brought into a state having desired fluidity in the same manner as described above. The type of the injection container is not particularly limited, and for example, a commercially available syringe can be used. A second aspect of the present invention provides a method for preparing a pharmaceutical composition for forming bone or periodontal tissue having fluidity during use, which comprises the following steps a) to c).

a) selecting mesenchymal stem cells having adhesiveness from a mesenchymal stem cell source, b) culturing the mesenchymal stem cells selected in step a) under conditions that induce differentiation into bone cells,

c) a step of mixing a mesenchymal stem cell that has acquired the differentiation ability into a bone cell in step b) with an inorganic bioabsorbable material in an aqueous solvent to form a flowable composition. In step a), cells having adhesive properties are selected from a mesenchymal stem cell source. Sources of mesenchymal stem cells include bone marrow, dental pulp, and cord blood, as described above. After collecting them according to a conventional method, cells having adhesive properties are selected. Selection of cells having adhesive properties is performed, for example, by seeding a suspension of bone marrow cells or the like in a flask or culture dish, culturing for a desired period of time, and exchanging the medium. This is performed by removing cells and the like. Preferably, this selection operation is performed several times to reduce the incorporation of non-adhesive components.

In step b), the mesenchymal stem cells selected in step a) are cultured under conditions that induce differentiation into bone cells. That is, in step b), undifferentiated mesenchymal stem cells are oriented to differentiate into bone cells. For example, 0-glyceric phosphate (| 3 By culturing in a medium containing dexamethasone (L-ascorbic acid) and L-ascorbic acid (L-ascorbic acid), differentiation into bone cells can be induced. Of course, the culture conditions are not limited to these, and any known conditions for inducing differentiation into bone cells can be employed.

Alkaline phosphatase activity and osteocalcin content are used as indicators of differentiation from undifferentiated mesenchymal stem cells to bone cells. Alkaline phosphatase activity is expressed from the early stage of differentiation, and osteocalcin is known to be expressed in mature osteoblasts. Therefore, by measuring the expression levels of these, it is possible to determine whether or not the cells have the ability to differentiate into bone cells, and to know the degree of differentiation. .

Between step a) and step b), it is preferable that the mesenchymal stem cells obtained in step a) be cultured and expanded while maintaining the ability to differentiate into bone cells. By performing this operation, the number of cells can be increased, and it is easy to mix a desired number of cells with the inorganic bioabsorbable material in step c) described later. Regarding the culture of undifferentiated mesenchymal cells, it has been pointed out that by repeating the passage in accordance with a known culture method, the number of cells differentiated into bone cells decreases thereafter. In other words, it is said that it is difficult to culture undifferentiated mesenchymal cells for a long period of time while maintaining the ability to differentiate into bone cells in the future. Therefore, as a result of repeated studies on the conditions for culturing undifferentiated mesenchymal stem cells while maintaining the ability to differentiate into bone cells, it was found that culturing in the presence of physical stimuli (mechanical stress) resulted It has been found that a decrease in the ability to differentiate into bone cells due to repeated generations can be suppressed. Therefore, in order to culture and proliferate the mesenchymal stem cells obtained in step a) in an undifferentiated state, a step of culturing the mesenchymal stem cells selected in step a) in the presence of physical stimulation (Step a-1)) is preferably performed.

Physical stimulation refers to the application of external pressure, tension, etc. to a cell. Eg to periodic gas (air, co _2, etc.) into the culture vessel, by continuous or Komu Ri intermittently feeding, can provide the desired external pressure such as the cell via the culture medium. In addition, the cells are seeded on a stretchable membrane or the like (for example, a silicon membrane), and the membrane to which the cells adhere is expanded or contracted periodically, continuously, or intermittently to stimulate the cells to expand or contract. , Can give extension stimulation. Also, by applying a desired flow to the medium, physical stimulation can be applied to the cells. In addition, physical stimulation can be provided using ultrasound. Incidentally, a desired physical stimulus can be applied by arbitrarily combining these methods.

In step c), the mesenchymal stem cells, which have acquired the differentiation potential into bone cells in step b), and an inorganic bioabsorbable material are mixed in an aqueous solvent to form a fluid composition, that is, fluidity A bone or periodontal tissue forming pharmaceutical composition having the following formula is formed. Further, the mesenchymal stem cells having acquired the differentiation ability to the bone cells according to b), the extracellular matrix of the cells, and a bioabsorbable material are mixed in an aqueous solvent to form a fluid composition. You can also. When the composition for forming bone or periodontal tissue is prepared by adding the extracellular matrix to form a flowable composition in this manner, the bone or periodontal tissue forming pharmaceutical composition can be used at the site where the composition has been applied. The extracellular matrix is expected to serve as a scaffold for bone differentiation-acquiring cells, and it is thought that the bone differentiation-acquiring cells are likely to be established at the application site. In addition, it also serves as a scaffold for bone cells existing around the application site, and can be expected to have high bone inducing ability. In addition, factors such as BMP contained in the extracellular matrix may be used to obtain the bone differentiation-acquiring cells themselves contained in the pharmaceutical composition for forming bone or periodontal tissue, and the bone cells or bone cells around the application site. It is expected to promote the growth, proliferation, and differentiation of stem cells that have the ability to differentiate into cells. Here, the extracellular matrix refers to a matrix (matrix) that surrounds the cells having bone differentiation potential.

In step c), a gelling agent is further mixed to form a flowable composition such that the composition for forming bone or periodontal tissue prepared by the method of the present invention gels after application. Preferably. According to such a configuration, a composition for forming bone or periodontal tissue which gels immediately after application is prepared. Therefore, it is possible to prepare a pharmaceutical composition for forming a bone or a periodontal tissue, which can improve the fixability at an application site and can effectively repair or regenerate the bone or the periodontal tissue.

It is preferable to use a gelling agent having a high biocompatibility, for example, collagen or fipurin glue. Various collagens can be selected and used, but it is preferable to use collagen suitable for the application purpose (applied tissue) of the pharmaceutical composition for forming bone or periodontal tissue of the present invention. When the purpose is to regenerate bone tissue, for example, type I collagen can be used. The collagen used is preferably soluble (acid-soluble collagen, alkali-soluble collagen, enzyme-soluble collagen, etc.).

Resulting flowable composition in step c) in 1 m 1, the it is preferred that 1 X 1 0 ⁵ or more cells are mixed bone based differentiation capacitation cells so that the presence, and et al Preferably, the cells are mixed so that 1 × 10 ⁶ to 1 × 10 ⁷ cells are present.

Although the type of the inorganic bioabsorbable material is not particularly limited, a material selected from the group consisting of / 3-TCP, a-TCP, tetracalcium phosphate, octacalcium phosphate, and amorphous calcium phosphate is used. Can be used. These materials can be used alone, or two or more arbitrarily selected materials may be used in combination. Preferably, 0-TCP is used as the inorganic bioabsorbable material.

The inorganic bioabsorbable material can be obtained by the method described in the first aspect of the present invention. As for the particle diameter and properties of the inorganic bioabsorbable material, those described in the first aspect of the present invention are also applied in the second aspect. The mixing amount of the inorganic bioabsorbable material depends on the total flowable composition obtained as a result of step c). Preferably, it is 50% by weight to 75% by weight.

As the aqueous solvent, sterilized water, physiological saline, phosphate buffer and the like can be used. Also, a part of the culture solution used in step b) can be used.

The degree of fluidity of the composition obtained as a result of step c) is not particularly limited, and may be paste, clay, high-viscosity fluid, or the like. Preferably, it is in the form of a paste. By forming the paste, a composition having excellent plasticity is obtained. Therefore, it can be applied without being previously formed into the shape of the application portion. That is, a pharmaceutical composition for forming bone or periodontal tissue which can be easily applied is prepared. In addition, a pharmaceutical composition for forming bone or periodontal tissue having good fixation properties at the application site is prepared.

In addition, when used, it is preferably fluid enough to be injected using an injection container. With such fluidity, application to the application site is further facilitated.

In addition, desired fluidity can be obtained depending on the application site. For example, when injecting under the periosteum, it is preferable to make the liquid more fluid (low viscosity).

A thickener can also be added in step c). As the thickening agent, thickening polysaccharides such as sodium alginate, glycerin, petrolatum, etc. can be used.However, from the viewpoints of safety and no or bone formation ability, it has high biocompatibility and bioabsorbability. Alternatively, a biodegradable material is preferably used. Addition of glycerin and the like also has the effect of preventing frost damage. Further, a stabilizer, a preservative, a pH adjuster, and the like can be added. In addition, growth factors, in particular osteoinductive factors (BMP), can be added.

After step c), a step (step) of freezing the flowable composition obtained in step c) can be performed. The frozen pharmaceutical composition for bone or periodontal tissue formation obtained by such a step is suitable for long-term storage. It can be stored with stable quality until use. Further, once frozen, the antigenicity of the fluid composition can be reduced, and immune rejection when applied (transplanted) to a living body can be reduced. The freezing treatment can be performed according to a conventional method. The frozen pharmaceutical composition for forming bone or periodontal tissue is thawed and used at the time of use. At this time, the desired fluidity can be adjusted by adding an aqueous solvent such as physiological saline.

After step c), a step of freeze-drying the flowable composition obtained in step c) (step e)) can be performed. The freeze-dried pharmaceutical composition for bone or periodontal tissue formation obtained by such a step is suitable for long-term storage similarly to the above-mentioned frozen one, and has a stable quality until use. Can be saved. Also, a decrease in antigenicity can be expected. The freeze-drying treatment can be performed according to a conventional method. The lyophilized pharmaceutical composition for bone or periodontal tissue formation is used after it has been made to have a fluidity before lyophilization or a desired fluidity by adding an aqueous solvent such as physiological saline. Is done.

From the pharmaceutical composition for forming bone or periodontal tissue obtained by the above-mentioned preparation method, an injection for forming bone or periodontal tissue can be prepared by performing the following steps. A) enclosing the osteogenic pharmaceutical composition obtained by the above-mentioned preparation method in an injection container; and

B) a step of freezing or freeze-drying the pharmaceutical composition for forming bone or periodontal tissue enclosed in the injection container obtained in step A).

The type of injection container is not particularly limited, and for example, a commercially available syringe can be used. Freezing or freeze-drying can be performed according to a conventional method.

A third aspect of the present invention provides a method for preparing cells having the ability to differentiate into bone cells, and a method for preparing bone cells. As described in the second aspect of the present invention, the present inventors dissociate into bone cells by culturing the cells under a physical stimulus. It has been found that more subcultures are possible without losing chemopotency. The third aspect of the present invention has been made based on such findings, and is a method for preparing a cell having the ability to differentiate into bone cells, comprising the following steps.

i) a step of selecting mesenchymal stem cells having adhesion from a mesenchymal stem cell source; and ii) culturing the mesenchymal stem cells selected in step i) in the presence of a physical stimulus.

According to the above-described preparation method, more subcultures can be performed while maintaining the differentiation ability to bone cells, and as a result, a large amount of cells can be obtained. The elements in steps i) and ii) here are the same as those in steps a) and a-1) in the second aspect of the present invention, and therefore, description thereof will be omitted. In addition, well-known or well-known culture conditions can be adopted as culture conditions other than the physical stimulation.

A third aspect of the present invention is a method for preparing a bone cell, comprising the following steps.

i) selecting a mesenchymal stem cell having adhesiveness from a mesenchymal stem cell source, ii) culturing the mesenchymal stem cell selected in step i) in the presence of a physical stimulus, and

iii) culturing under conditions that induce differentiation into bone cells.

According to such a preparation method, a large amount of bone cells can be obtained. At the same time, a large amount of extracellular matrix of bone cells can be obtained.

Each element of step iii) is the same as step b) in the second aspect of the present invention, and a description thereof will be omitted.

The bone cell obtained by the preparation method of the third aspect of the present invention can be used, for example, as a mesenchymal stem cell that has acquired the ability to differentiate into a bone cell in the first aspect of the present invention. A fourth aspect of the present invention is a method for preparing bone cells, which comprises culturing mesenchymal stem cells in the presence of a physical stimulus when the cells are induced to differentiate into bone cells. The bone cells obtained by such a preparation method can be used, for example, as mesenchymal stem cells that have acquired the ability to differentiate into bone cells in the first aspect of the present invention.

As described above, as the mesenchymal stem cells, those prepared from bone marrow, dental pulp, cord blood, etc. according to a conventional method can be used as described above. After collection, cells cultured in the presence of a physical stimulus can also be used.

[Example 1] Preparation of bone marrow cells

(1-1) Removal of rat femur

The lower body of a male Fischer (7-week-old) rat anesthetized by a ether was shaved. After shaving, the remaining hair in the shaved area was completely removed with a depilatory cream. Subsequently, the hair removal cream and the hair remaining on the shaved part were sufficiently washed away. Thereafter, excess water was sufficiently removed. Next, the rat was fixed on the stomach, and the back of the lower body was thoroughly wiped with hibiden for disinfection. After disinfection, the back of the lower body was incised into a cross shape, and the skin at the incision was sufficiently peeled off and fixed to expose the thigh. The femur was exposed by exfoliating muscles and the like from the head of the femur. At this time, the soft tissue attached to the bone was removed as much as possible. Subsequently, the ligaments and the like at the joints were cut, and the femur was extracted. The extracted large fe bone was eagle minimal essential me mm (GIBCO Laboratories Life Technologies, NY USA) (15% Fetal Bovine Serum (GIBCO Laboratories Life Technologies, NY USA), lOOU / ml penicillin G (Meiji Seika Co., Ltd.), 100 g / ml streptomycin (Sigma Chemical Co., St Louis, USA) containing 0.25 β g / ml amphotericins B (GIBCO Laboratories Life Technologies, NY USA).

(1-2) Collection of bone marrow cells The basic medium was aspirated into a syringe for lOcc (manufactured by Terumo Corporation). The femur obtained in (1-1) was transferred to a petri dish, and excess soft tissue attached to the femur was removed. After that, the femur was transferred to another Petri dish, and both ends (bone head) were cut off. The femur whose both ends were cut was held on a centrifuge tube, and in this state, a syringe needle prepared in the bone marrow cavity was once passed through. Then, the needle was lifted upward, and about 5 cc of the basal medium in the syringe was slowly poured. Next, the femur was turned upside down, and the remaining basal medium was poured from the opposite stump. By such an operation, a suspension of bone marrow cells was collected in a centrifuge tube.

(1-3) Seeding bone marrow cells

Add an appropriate amount of basal medium to the suspension of bone marrow cells collected in the centrifuge tube in (1-2). (If bone marrow cells collected from two femurs per rat are used, the total amount of (Approximately 30 cc to 4 Occ), and the mixture was sufficiently stirred. Then, a suspension of bone marrow cells was seeded in a flask (80 cm ² , Greiner labortechnik Germany).

(1-4) Selection and culture of adherent cells

The flask in which the bone marrow cells were seeded was transferred into Incube overnight, and cultured under the conditions of 5% CO ₂ and 37. The medium was replaced 24 hours after seeding of the bone marrow cells in order to remove the blood cells contained in the culture solution and to select and culture the adherent cells. Thereafter, culturing was continued for about 7 days in the culture solution until the cells became subconfluent. During that time, medium exchange was performed once every two days. The state of the cells on the third day of the initial culture is shown in FIG.

Subsequently, the cells were detached using 0.05% trypsin, inoculated so that the area ratio became 2 to 4 times, and subcultured.

(1-5) Induction of bone marrow cell differentiation

First, after culturing for about 14 days from the passage, it was confirmed that subconfluence was reached, and the culture broth was added to 3-glycerophosphate (Sigma Chemical Co., St Louis, US), Dexamethasone (Sigma Chemical Co., St Louis , USA), and VitaminC phosphate (L-ascorbic acid phosphate magnesium salt n- hydrate, Sigma Chemical Co., St Louis, USA) and, ^{respectively, 10 mM, 10 · 8 M} , And the culture was continued at 80 μg / ml. The medium was exchanged every two days, and each time, 3-glycerophosphate, Dexamethasone, and Vitamin C phosphate (jj-ascorbic acid phosphate magnesium salt n-hydrate) were added to the medium as described above. In this way, the cells were cultured for about 2 weeks until calcification was confirmed in the flask.

The state of the cells on day 10 of the subculture is shown in FIG. FIG. 3 shows the state of the cells after further culturing for 4 days (subculture day 14). Bone marrow cells are observed in a polygonal shape, and extracellular matrix-like structures can be observed around the cells. In addition, the photograph in FIG. 4 shows cells on the 14th day of subculture stained with alkaline phosphatase. It can be seen that the cells are partially aggregated and form calcified nodules. Immediately, positive reaction of lipophosphatase can be observed mainly in the nodules.

(1-6) Recovery of cell components

The medium in the flask was removed except for a small amount of the medium in the flask. The cells were then detached from the flask using Cell'Scraper (Nunc Inter Med). The detached cells were transferred to a centrifuge tube (FALCON ECTON DICKINSON USA) together with the medium, and subjected to centrifugation (1500 rpm, 5 min). The supernatant was aspirated and the cell components were collected.

[Example 2] Preparation of osteogenic pharmaceutical composition (cell-containing -TCP paste)

Preparation of (2-1) / 3-TCP powder

/ 3 -TCP (manufactured by Olympus Optical Co., Ltd., porosity: 90%) was mechanically pulverized to adjust the particle diameter to an average of about 2 xm. The 3-TCP powder thus prepared —Treclave sterilized.

(2-2) Preparation of β-TCP paste containing cells

An amount of sterile water smaller than the amount required finally) and 3) -TCP powder were mixed by stirring to form a paste. If more viscosity is required, sodium alginate solution can be used instead of sterile water.

On the other hand, the cell components recovered in (1-6) were suspended using a small amount of sterile water to obtain a cell suspension.

The 3-TCP paste and the cell suspension were stirred and mixed to obtain a cell-containing) 3-TCP paste. At this time, sterile water was used to adjust the content of | 3-TCP to about 67% by weight based on the whole] -TCP paste. This gives a paste-like) 3-TCP paste. The cell-containing j3-TCP paste thus prepared can be frozen or lyophilized for storage.

[Example 3] Bone formation using 13-TCP paste containing cells

The cell-containing) 3-TCP paste obtained in Example 2 was transferred to a 5 cc syringe. On the other hand, the back of a 7-year-old male male rat (7 weeks old) anesthetized with ether was shaved. After shaving, the remaining hair in the shaved area was completely removed with a depilatory cream. Subsequently, the hair removal cream and the hair remaining on the shaved part were sufficiently washed away. Thereafter, excess water was sufficiently removed. Next, the rats were fixed on their stomachs, and the surgical field was thoroughly wiped with hibiden to disinfect them. Thereafter, approximately 3 cc of the cell-containing (3-TCP paste) obtained in Example 2 was injected subcutaneously into the back using a syringe.

Then, the bone formation at the cell-containing) -TCP paste injection site was observed with an optical microscope. Figure 5 shows the results. Fig. 5 is a demineralized HE (hematoxylene eosin) -stained tissue image (100-fold) of the transplanted portion 8 weeks after transplantation of the cell-containing i8-TCP paste. The part missing in white is decalcified / 3-TCP. On the formed bone tissue Has a regular lamellar structure, narrowed bone lacunae, and an overall mature bone structure is observed.

[Example 4] Comparison of bone formation ability by different culture methods

The bone marrow cells obtained in Example 1 were subcultured in the presence and absence of physical stimulation, and the bone formation ability of each of the cultured cells was compared.

(4-1) Culture in the presence of physical stimulus

A culture device capable of intermittently applying a physical stimulus by changing the air pressure was prepared. That is, a pump that repeatedly repeats decompression and pressurization through a tube was attached to a closed space (box-shaped container), and this was used as a culture device. In such a device, the enclosed space is repeatedly depressurized and pressurized at a speed of about 10 Hz to about 1 to 1.2 atm, and physical stimulation can be applied to cultured cells in a petri dish installed inside. it can.

Using the above culture apparatus, the bone marrow cells prepared in Example 1 above were cultured (stimulation group). First, the cells prepared in Example 1 were seeded on a culture dish (bone marrow cells obtained from one femur were seeded on a culture dish of 80 cm ² ). Subsequently, the medium was replaced 24 hours after the seeding of the bone marrow cells in order to remove the hemocyte cells contained in the culture medium and to select and culture the adherent cells. After that, the culture was continued for about 10 days until it became subconfluent (primary culture). Subsequently, the cells were detached using 0.05% trypsin, and the cells cultured in one Petri dish were divided into two Petri dishes and inoculated (two times in area ratio) for subculture. Physical stimulation was performed for 8 hours on Z days during the primary culture and every day from the day following each passage. In addition, the medium was changed every day, and the culture dish was taken out of the closed space except at the time of stimulation and ventilated.

For the control group, primary culture and subculture were performed using a general-purpose incubator instead of the culture device. Other conditions are the same as those of the stimulus group.

Under the above conditions, each of the stimulation group and the control group was cultured up to 6 passages. (4-2) Preparation for transplantation

The cells after each subculture obtained by the above method were seeded in a 3-TCP block, respectively, and further cultured. In this case, the medium includes -glycerophosphate (Sigma Chemical Co., St Louis, US), Dexamethasone (Sigma Chemical Co., St Louis, USA), and VitaminC osphate (L-ascorbic acid phosphate magnesium salt n-hydrate, Sigma Chemical Co., St Louis, USA) were added to 10 mM, 10-8 M, and 50 Wg / ml, respectively. Addition of these induces differentiation into osteoblasts. The stimulus group was also given a physical stimulus by the above-mentioned method even during culture using the | 9 "TCP block.

(4-1-3) Transplant

By the following method:-Bone marrow cells cultured in TCP were transplanted together with i3-TCP blocks into syngeneic rats (7 weeks old, male). First, under general anesthesia with pentobarbi, the back skin was incised to expose the fascia. Then, the cell-containing / 3-TCP block was left on the fascia, and then sutured.

(4-4) Evaluation of bone formation ability

Four weeks after transplantation, macroscopic and decalcified specimens of the cell-containing β-TCP block were prepared, and the amount of bone formation was evaluated histologically. A plurality of tissue specimens were prepared for each sample, and the amount of new bone tissue in these was used as the amount of bone formation of the sample. The amount of bone formation of the sample using the primary cultured cells was defined as 100, and the amount of bone formation (area ratio) of each sample was defined as the bone formation ability of each sample. Fig. 6 shows a table summarizing the osteogenic ability of each sample. In FIG. 6, C and E represent a control group and a stimulation group, respectively. As shown in the figure, it can be seen that in the stimulus group, the decrease in bone formation ability due to the passage is suppressed significantly as compared to the control group. In other words, it was shown that by subculturing in the presence of physical stimulus, subculture can be repeated while maintaining the osteogenic ability. [Example 5] Examination of promotion effect of bone differentiation induction by difference in culture method at the time of differentiation induction Using human mesenchymal stem cells, the effect of physical stimulation on differentiation induction was examined. First, iliac bone marrow fluid was collected from a human iliac bone using a bone marrow puncture needle into a lOcc syringe (manufactured by Terumo Corporation), and the basic medium (eagle minimal essential medium (GIBCO Laboratories Life Technologies, NY USA, 15% Fetal Bovine

Serum (GIBCO Laboratories Life Technologies, NY USA), lOOU / ml penicillin

G (manufactured by Meiji Seika Co., Ltd.), lOO ig / ml streptomycin (Sigma Chemical Co., St.

Louis, USA), 0.25 g / ml amphotericine B, containing GIBCO Laboratories Lite Technologies, NY USA).

Subsequently, an appropriate amount (concentration: 30%) of a basal medium was added to the suspension of bone marrow fluid collected in the centrifuge tube, and then seeded in a flask (80 cm ² , Greiner labortec nik Germany). Then, the flask seeded with bone marrow fluid was transferred to an incubator, and cultured under the conditions at 5% C0 _2, 37. The culture medium was replaced 24 hours after seeding the bone marrow cells in order to remove the blood cells contained in the culture medium and to select and culture the adherent cells. Thereafter, culturing was continued for about 7 days in the culture medium until the cells became subconfluent. During that time, the medium was changed once every three days. In addition, MSCGM, 50 U / ml penicillin G, and 50 pg / ml streptomycin (manufactured by Poietics) were added to the culture solution. Subsequently, the cells were detached using 0.05% trypsin, inoculated at an area ratio of 2 to 4 times, and subcultured.

In the culture of bone marrow cells (human mesenchymal stem cells) obtained in this way) 3

GlyceroDhoshate Sigma chemical Uo., St Louis, US), Dexamethasone

(Sigma Chemical Co., St Louis, USA) and VitaminC pliosphate (L-ascorbic acid phosphate magnesium salt n-hydrate, 'Sigma Chemical Co., St Louis, USA) at 10 mM, 100 nM, and 0.05 mM, respectively. (Induction medium And the culture was continued. After changing to the induction medium, cell expansion stimulation was applied using an expansion stimulator (manufactured by Scalatec Corporation). The stimulus was changed at an extension rate of 10 to 20% and a frequency of 0.05 to 0.2 Hz, and the osteoblast differentiation effect was evaluated under each condition. As controls, there were prepared a group to which no extension stimulus was applied without using an induction medium, a group to which no extension stimulus was applied using an induction medium, and a group to which only extension stimulus was used without using an induction medium. The evaluation was performed by measuring the alkaline phosphatase (ALP) activity in the culture medium one week after the change to the induction medium.

Figures 7 to 9 show graphs summarizing the ALP activity of each group. Figure 7 shows the results under the conditions of an extension rate of 15% (mecal 5%) and 5 cycles (5 cycles / min, about 0.083Hz). Similarly, FIGS. 8 and 9 show the results under the conditions of an extension rate of 15% (mechal5%), 10 cycles / min (10 cycles / min, about 0.167 Hz), and an extension rate of 20% (mec a20%), The results are for five cycles (5 cycles / min, about 0.083 Hz). In each graph, SC1 and SC2 show the results of the group without using the induction medium and no extension stimulus, and SC + S1 and SC + S2 show the results of the group without the induction medium and only the extension stimulus.ぴ ind2 is the result of the group using an induction medium and no extension stimulus was applied, and indl + Sl to indl + S4 is the result of the group using an induction medium and extension stimulation was applied.

As shown in each graph, it can be seen that ALP is significantly increased by the application of the stretching stimulus when the induction medium is used. In the group to which the stretching stimulation was added, a slight suppression of cell proliferation was also observed (data not shown overnight). On the other hand, when the induction medium was not used, there was almost no change in ALP activity due to the application of the stretching stimulus. No significant change was observed in cell number (data not shown). From the above results, it was shown that the addition of a stretching stimulus (physical stimulation) at the time of inducing chemical differentiation (using an induction medium) has an effect of promoting differentiation into bone cells. The slight decrease in cell proliferation is considered to reflect the effect of mesenchymal stem cells becoming more differentiated into osteoblasts. On the other hand, when no induction medium was used, bone differentiation was observed. It is predicted from the fact that stimulation of cell extension itself has no effect of promoting differentiation into osteoblasts.

The present invention is not limited to the description of the embodiment and the example of the above invention. Various modifications are included in the present invention without departing from the scope of the claims and within the scope of those skilled in the art. Industrial applicability

The pharmaceutical composition for forming bone or periodontal tissue of the present invention can be applied to various fields that require repair and regeneration of bone tissue or periodontal tissue. For example, the present invention can be applied to regeneration of bone tissue (cartilage tissue) at a bone (cartilage) defect caused by trauma or various bone diseases, and reinforcement or supplementation of bone (cartilage). Further, the present invention can be applied to regeneration of alveolar bone and periodontal tissue in a defective portion of alveolar bone due to periodontal disease or the like. As an application method, a bone-forming pharmaceutical composition in a paste form or the like is injected, applied, or the like to an application site.

Since the pharmaceutical composition for forming bone or periodontal tissue of the present invention contains cells that have acquired the ability to differentiate into bone cells, direct regeneration of bone or periodontal tissue by itself can be expected. In addition, inorganic bioabsorbable materials used as cell carriers or scaffolds will be replaced with bone or periodontal tissue in the future, and their safety is high. Furthermore, since it has fluidity, it is not necessary to mold it in advance according to the shape of the bone defect, and it is versatile and easy to handle. Furthermore, since the pharmaceutical composition for forming bone or periodontal tissue of the present invention can be injected transdermally or transmucosally using a needle, it can be applied without opening the wound. In other words, minimally invasive application is possible when applied. In addition, unlike the existing technology, the pharmaceutical composition for forming a bone or periodontal tissue of the present invention eliminates the need to apply the application site at the time of application. Further, according to a third aspect of the present invention, a method for preparing a cell having the ability to differentiate into bone cells is provided. Thus, more subcultures can be carried out while maintaining the ability to differentiate into bone cells. As a result, it is possible to obtain cells that retain the ability to differentiate into large amounts of bone cells. Further, in the third aspect of the present invention, a method for preparing a bone cell is also provided, and a large amount of the bone cell can be obtained. At the same time, it is possible to obtain a large amount of extracellular matrix of bone cells.

Furthermore, according to the method for preparing bone cells in the fourth aspect of the present invention, differentiation induction from mesenchymal stem cells to bone cells can be promoted, and as a result, cells having osteogenic ability can be efficiently used. It can be prepared in a suitable manner.

Claims

The scope of the claims

1. A pharmaceutical composition for forming bone or periodontal tissue which has fluidity at the time of use, comprising mesenchymal stem cells having acquired the ability to differentiate into bone cells, and an inorganic bioabsorbable material.

2. Bone comprising a mesenchymal stem cell that has acquired the ability to differentiate into a bone cell, an extracellular matrix of the mesenchymal stem cell, and an inorganic bioabsorbable material, and having fluidity during use. Or a pharmaceutical composition for periodontal tissue formation.

3. The pharmaceutical composition for forming a bone or periodontal tissue according to claim 1 or 2, wherein the pharmaceutical composition has a fluidity that can be injected using a potting container at the time of use. A pharmaceutical composition for forming bone or periodontal tissue.

4. The inorganic bioabsorbable material is selected from the group consisting of / 3-tricalcium phosphate, α-tricalcium phosphate, tetracalcium phosphate, octacalcium phosphate, and amorphous calcium phosphate. The pharmaceutical composition for forming a bone or periodontal tissue according to any one of claims 1 to 3, wherein the pharmaceutical composition is one or more selected inorganic bioabsorbable materials. .

5. The inorganic bioabsorbable material has an average particle size of 0.5 μπ! The pharmaceutical composition for forming a bone or periodontal tissue according to any one of claims 1 to 4, wherein the pharmaceutical composition has a viscosity of from 50 to πι.

6. The bone or periodontal tissue according to any one of claims 1 to 5, wherein the inorganic bioabsorbable material is contained in an amount of 50% by weight to 75% by weight. Pharmaceutical compositions for formation.

7. The pharmaceutical composition for forming a bone or periodontal tissue according to any one of claims 1 to 6, further comprising a gelling material, wherein the composition gels after application.

8. The pharmaceutical composition for forming a bone or periodontal tissue according to claim 7, wherein the gelling material is collagen and / or fibrin glue.

9. The bone or the bone according to any one of claims 1 to 8, wherein the pharmaceutical composition for forming bone or periodontal tissue is used after being thawed from a frozen state at the time of use. A pharmaceutical composition for periodontal tissue formation.

10. An injection for forming bone or periodontal tissue, wherein the pharmaceutical composition for forming bone or periodontal tissue according to any one of claims 1 to 9 is enclosed in an injection container.

11. A method for preparing a pharmaceutical composition for forming bone or periodontal tissue having fluidity during use, comprising the following steps a) to c):

c) a step of mixing a mesenchymal stem cell that has acquired the differentiation ability into a bone cell in step b) with an inorganic bioabsorbable material in an aqueous solvent to form a flowable composition.

12. The bone or periodontal tissue formation according to claim 11, wherein in step c), collagen and / or fibrin glue are further mixed to form a flowable composition. Method for preparing a pharmaceutical composition for

13. A method for preparing a pharmaceutical composition for forming bone or periodontal tissue having fluidity during use, comprising the following steps a) to c):

c) A fluid composition obtained by mixing a mesenchymal stem cell having acquired the differentiation ability into a bone cell in step b), an extracellular matrix of the cell, and an inorganic bioabsorbable material in an aqueous solvent. Forming a.

14. The method according to claim 13, wherein, in the step (c), a flowable composition is formed by further mixing collagen and fibrin glue. A method for preparing a pharmaceutical composition for forming bone or periodontal tissue.

15. The bone or periodontal tissue according to any one of claims 11 to 14, wherein the following step a-1) is further performed after the step a). A method of preparing a pharmaceutical composition for forming,

a-1) A step of culturing the mesenchymal stem cells selected in step a) in the presence of a physical stimulus.

16. The bone or periodontal tissue forming physician according to any one of claims 11 to 15, wherein the following step d) is further performed after the step c). Preparation method of drug composition,

d) freezing the flowable composition obtained according to step c).

17. The bone or periodontal tissue forming physician according to any one of claims 11 to 15, wherein the following step e) is further performed after the step c). Preparation method of drug composition,

e) freeze-drying the flowable composition obtained in step c).

18. The inorganic bioabsorbable material is selected from the group consisting of) 3-tricalcium phosphate, α-triphosphate phosphate, tetracalcium phosphate, octacalcium phosphate, and amorphous calcium phosphate. The pharmaceutical composition for forming a bone or periodontal tissue according to any one of claims 11 to 17, which is one or more inorganic bioabsorbable materials selected. Preparation method.

19. The inorganic bioabsorbable material has an average particle size of 0.5 μπ! The method for preparing a pharmaceutical composition for forming bone or periodontal tissue according to any one of claims 11 to 18, wherein the pharmaceutical composition is in a range of from 50 to 50πι.

20. The bone or bone according to any one of claims 11 to 19, wherein the inorganic bioabsorbable material is contained in an amount of 50% by weight to 75% by weight. A method for preparing a pharmaceutical composition for periodontal tissue formation.

21. A method for preparing an injection for bone or periodontal tissue formation, comprising the following steps A) and B):

A) A bone or periodontal tissue forming pharmaceutical composition obtained by the preparation method according to any one of claims 11 to 15 and 18 to 20 is enclosed in an injection container. Steps to perform, and

B) Step of freezing or freeze-drying the medical composition for forming bone or periodontal tissue enclosed in the injection container obtained in A).

2 2. A method for preparing cells having the ability to differentiate into bone cells, comprising the following steps: i) selecting mesenchymal stem cells having adhesion from a mesenchymal stem cell source; and ii) step i. A) culturing the mesenchymal stem cells selected by the above) in the presence of a physical stimulus.

23. A method for preparing bone cells, comprising the following steps:

iii) culturing under conditions that induce differentiation into bone cells.

2. A method for preparing bone cells, which comprises culturing mesenchymal stem cells in the presence of a physical stimulus when inducing differentiation into bone cells.