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WO2002017983A1 - Compositions medicinales pour la formation de tissu autour d'un os ou d'une dent, procede de preparation desdites compositions, substances a injecter pour la formation de tissu autour d'un os ou d'une dent et procede de preparation desdites substances - Google Patents

Compositions medicinales pour la formation de tissu autour d'un os ou d'une dent, procede de preparation desdites compositions, substances a injecter pour la formation de tissu autour d'un os ou d'une dent et procede de preparation desdites substances Download PDF

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Publication number
WO2002017983A1
WO2002017983A1 PCT/JP2001/007289 JP0107289W WO0217983A1 WO 2002017983 A1 WO2002017983 A1 WO 2002017983A1 JP 0107289 W JP0107289 W JP 0107289W WO 0217983 A1 WO0217983 A1 WO 0217983A1
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WIPO (PCT)
Prior art keywords
bone
cells
pharmaceutical composition
periodontal tissue
forming
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2001/007289
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English (en)
Japanese (ja)
Inventor
Minoru Ueda
Ken-Ichiro Hata
Yoshitaka Hibino
Kunihiko Okada
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OSTEOGENESIS CO Ltd
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OSTEOGENESIS CO Ltd
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Priority to AU2001280164A priority Critical patent/AU2001280164A1/en
Publication of WO2002017983A1 publication Critical patent/WO2002017983A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3821—Bone-forming cells, e.g. osteoblasts, osteocytes, osteoprogenitor cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/12—Phosphorus-containing materials, e.g. apatite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/3633—Extracellular matrix [ECM]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3645—Connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
    • A61L27/3645—Connective tissue
    • A61L27/365—Bones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3847—Bones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3865—Dental/periodontal tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • 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.
  • 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 bone morphogenetic protein
  • 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.
  • cell-hybrid artificial bone which combines cells cultured in a test tube with biomaterials
  • 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.
  • 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.
  • GTR membrane shielding membrane
  • 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.
  • tissue engineering has attempted to regenerate bone tissue (cartilage tissue) or periodontal tissue in many cases.
  • knowledge on the regeneration mechanism of bone tissue (cartilage tissue) or periodontal tissue may not be sufficient.
  • 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.
  • 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.
  • inorganic bioabsorbable materials used as cell carriers or scaffolds will be replaced with bone tissue in the future, and their safety is high.
  • 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.
  • 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
  • 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.
  • 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.
  • bone cells include osteoblasts, osteoclasts, and chondroblasts.
  • mesenchymal stem cells having acquired the differentiation ability to bone cells not only autologous cells but also allogeneic allogeneic cells can be used.
  • human mesenchymal stem cells can be used.
  • osteoblast-acquiring cells Mesenchymal stem cells that have acquired the ability to differentiate into bone cells.
  • 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
  • differentiation into bone cells By culturing undifferentiated mesenchymal stem cells in a medium containing (L-ascorbic acid), differentiation into bone cells can be induced.
  • the culture conditions are not limited to these, and any known conditions for inducing differentiation into bone cells can be used.
  • 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_ ⁇ 2, etc.) periodically ', continuous, or Intermittent
  • the cells can be supplied with a desired external pressure, etc., through the culture medium. Wear.
  • 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.
  • a physical stimulus can be given to the cells by giving a desired flow to the medium.
  • 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.
  • 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.
  • 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.
  • it also serves as a scaffold for bone cells existing around the application site, so that high bone inducing ability can be expected.
  • 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.
  • the extracellular matrix of autologous cells 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.
  • the extracellular matrix of autologous cells can be used, which facilitates the preparation of the extracellular matrix of cells having the ability to acquire bone differentiation potential.
  • 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.
  • 3_tricalcium phosphate hereinafter, referred to as "3-TCP”
  • ⁇ -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 ⁇ -TC ⁇ , 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.
  • 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.
  • the fluidity can be adjusted also by the amount of the thickener added.
  • 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.
  • the fluidity is such that it can be injected using an injection container, and such fluidity facilitates application to the application section.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical composition of the present invention contains a gelling agent so as to be gelled after application.
  • a gelling agent so as to be gelled after application.
  • gelation occurs immediately after application, so that fixability at the application site is improved, and bone or periodontal tissue is repaired or regenerated effectively.
  • it 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.
  • collagen or fibrin glue can be used as the gelling agent.
  • 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.
  • 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.
  • 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).
  • BMP osteoinductive factors
  • 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.
  • a thickener 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.
  • the pharmaceutical composition of the present invention 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.
  • an injection for bone or periodontal tissue formation (hereinafter referred to as “the injection of the present application”) can be obtained.
  • 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.
  • 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).
  • 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
  • step a) 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.
  • 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.
  • 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 (
  • 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. .
  • the mesenchymal stem cells obtained in step a) be cultured and expanded while maintaining the ability to differentiate into bone cells.
  • 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.
  • 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.
  • a step of culturing the mesenchymal stem cells selected in step a) in the presence of physical stimulation is preferably performed.
  • Physical stimulation refers to the application of external pressure, tension, etc. to a cell.
  • periodic gas air, co 2, etc.
  • Komu Ri intermittently feeding can provide the desired external pressure such as the cell via the culture medium.
  • 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.
  • by applying a desired flow to the medium physical stimulation can be applied to the cells.
  • physical stimulation can be provided using ultrasound. Incidentally, a desired physical stimulus can be applied by arbitrarily combining these methods.
  • 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.
  • 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.
  • 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.
  • the extracellular matrix refers to a matrix (matrix) that surrounds the cells having bone differentiation potential.
  • 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.
  • 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.
  • a gelling agent having a high biocompatibility for example, collagen or fipurin glue.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • growth factors in particular osteoinductive factors (BMP), can be added.
  • 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.
  • step e) 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.
  • 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
  • step 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.
  • 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.
  • step 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.
  • 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.
  • 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.
  • a large amount of bone cells can be obtained.
  • a large amount of extracellular matrix of bone cells can be obtained.
  • 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.
  • 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.
  • 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 ⁇ g / ml amphotericins B (GIBCO Laboratories Life Technologies, NY USA).
  • 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.
  • the cells were detached using 0.05% trypsin, inoculated so that the area ratio became 2 to 4 times, and subcultured.
  • 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.
  • 3-glycerophosphate, Dexamethasone, and Vitamin C phosphate jj-ascorbic acid phosphate magnesium salt n-hydrate
  • 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.
  • 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.
  • 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.
  • 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.
  • sterile water was used to adjust the content of
  • the cell-containing j3-TCP paste thus prepared can be frozen or lyophilized for storage.
  • the cell-containing) 3-TCP paste obtained in Example 2 was transferred to a 5 cc syringe.
  • 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.
  • 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.
  • 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.
  • HE hematoxylene eosin
  • 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.
  • 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.
  • the bone marrow cells prepared in Example 1 above were cultured (stimulation group).
  • 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 ).
  • 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.
  • the culture was continued for about 10 days until it became subconfluent (primary culture).
  • 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.
  • 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.
  • 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.
  • 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
  • Fig. 6 shows a table summarizing the osteogenic ability of each sample.
  • C and E represent a control group and a stimulation group, respectively.
  • 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
  • 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.
  • 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.
  • VitaminC pliosphate L-ascorbic acid phosphate magnesium salt n-hydrate, 'Sigma Chemical Co., St Louis, USA
  • 10 mM 100 nM
  • 0.05 mM 0.05 mM
  • 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).
  • 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).
  • SC1 and SC2 show the results of the group without using the induction medium and no extension stimulus
  • SC + S1 and SC + S2 show the results of the group without the induction medium and only the extension stimulus.
  • ind2 is the result of the group using an induction medium and no extension stimulus was applied
  • indl + Sl to indl + S4 is the result of the group using an induction medium and extension stimulation was applied.
  • 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.
  • 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).
  • 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.
  • a bone-forming pharmaceutical composition in a paste form or the like is injected, applied, or the like to an application site.
  • 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.
  • 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.
  • it 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.
  • 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.
  • 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.
  • a method for preparing a cell having the ability to differentiate into bone cells is provided.
  • more subcultures can be carried out while maintaining the ability to differentiate into bone cells.
  • 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.
  • 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.

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Abstract

L'invention concerne des compositions médicinales permettant la formation d'un tissu autour d'un os ou d'une dent. Ces compositions, à usage pratique et d'une grande innocuité, sont très efficaces en matière de régénération d'un tissu osseux. Cette invention concerne plus particulièrement une composition médicinale pour la formation d'un tissu autour d'un os ou d'une dent, se présentant sous la forme d'une pâte que l'on prépare en mélangeant des cellules souches mésenchymateuses capables de se différencier en cellules osseuses, des matrices extracellulaires desdites cellules et du bêta-tricrésylphosphate (TCP) présentant une taille de particules moyenne comprise entre 1 et 5 ?m.
PCT/JP2001/007289 2000-08-28 2001-08-24 Compositions medicinales pour la formation de tissu autour d'un os ou d'une dent, procede de preparation desdites compositions, substances a injecter pour la formation de tissu autour d'un os ou d'une dent et procede de preparation desdites substances Ceased WO2002017983A1 (fr)

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AU2001280164A AU2001280164A1 (en) 2000-08-28 2001-08-24 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

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006123579A1 (fr) * 2005-05-17 2006-11-23 National University Corporation Nagoya University Procede de preparation cellulaire pour la formation de tissu osseux et application de cellule pour la formation de tissu osseux
US7497686B2 (en) 2004-09-02 2009-03-03 Odontis Ltd. Bone regeneration
WO2010064680A1 (fr) * 2008-12-03 2010-06-10 国立大学法人東京大学 Procédé de fabrication d'os granulaire de culture

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0345267A (ja) * 1989-07-12 1991-02-26 Mitsubishi Materials Corp 骨欠損部及び骨空隙部充てん材
JPH07194373A (ja) * 1993-12-30 1995-08-01 Nitta Gelatin Inc 骨髄細胞の培養方法、培養用混合物および硬組織欠損部への移植用材料
WO1998017791A1 (fr) * 1996-10-23 1998-04-30 Advanced Tissue Sciences, Inc. Production de tissu cartilagineux utilisant des cellules isolees a partir de la gelee de wharton
WO1998022573A1 (fr) * 1996-11-20 1998-05-28 Advanced Tissue Sciences, Inc. Application d'une contrainte par courant de cisaillement a des chondrocytes
JPH10243996A (ja) * 1997-03-07 1998-09-14 Kagaku Gijutsu Shinko Jigyodan 硬組織石灰化促進用生体材料

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0345267A (ja) * 1989-07-12 1991-02-26 Mitsubishi Materials Corp 骨欠損部及び骨空隙部充てん材
JPH07194373A (ja) * 1993-12-30 1995-08-01 Nitta Gelatin Inc 骨髄細胞の培養方法、培養用混合物および硬組織欠損部への移植用材料
WO1998017791A1 (fr) * 1996-10-23 1998-04-30 Advanced Tissue Sciences, Inc. Production de tissu cartilagineux utilisant des cellules isolees a partir de la gelee de wharton
WO1998022573A1 (fr) * 1996-11-20 1998-05-28 Advanced Tissue Sciences, Inc. Application d'une contrainte par courant de cisaillement a des chondrocytes
JPH10243996A (ja) * 1997-03-07 1998-09-14 Kagaku Gijutsu Shinko Jigyodan 硬組織石灰化促進用生体材料

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7497686B2 (en) 2004-09-02 2009-03-03 Odontis Ltd. Bone regeneration
WO2006123579A1 (fr) * 2005-05-17 2006-11-23 National University Corporation Nagoya University Procede de preparation cellulaire pour la formation de tissu osseux et application de cellule pour la formation de tissu osseux
JPWO2006123579A1 (ja) * 2005-05-17 2008-12-25 国立大学法人名古屋大学 骨組織形成用細胞の調製方法、及び骨組織形成用細胞の利用
WO2010064680A1 (fr) * 2008-12-03 2010-06-10 国立大学法人東京大学 Procédé de fabrication d'os granulaire de culture

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