WO2020226043A1 - Bone-like tissue and method for producing same - Google Patents

Bone-like tissue and method for producing same Download PDF

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WO2020226043A1
WO2020226043A1 PCT/JP2020/017005 JP2020017005W WO2020226043A1 WO 2020226043 A1 WO2020226043 A1 WO 2020226043A1 JP 2020017005 W JP2020017005 W JP 2020017005W WO 2020226043 A1 WO2020226043 A1 WO 2020226043A1
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bone
tissue
dimensional
producing
gel
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PCT/JP2020/017005
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French (fr)
Japanese (ja)
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英見 栗原
幹人 加治屋
奈央 小松
総太 本池
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国立大学法人広島大学
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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
    • 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
    • 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
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • 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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids

Definitions

  • the present invention relates to a bone-like tissue and a method for producing the same, and particularly to a bone-like tissue containing no artificial material and a method for producing the same.
  • MSCs mesenchymal stem cells having pluripotency and self-proliferation ability are considered as cells suitable for tissue regeneration therapy, and in recent years, MSCs and artificial scaffolding materials have been mixed. Attempts have been actively made to produce and supply three-dimensional bone-like tissues corresponding to autologous bones.
  • MSCs are mixed with hydroxyapatite, polylactic acid, chitosan, atelocollagen gel, hyaluronic acid gel, etc., and this is used as a bone differentiation-inducing medium (dexamethasone, ascorbic acid, and ⁇ -glycerophosphate) established in an experimental system of two-dimensional culture. It is known that a three-dimensional implant having high bone regeneration ability can be obtained by culturing in (additional medium) (see Non-Patent Document 1).
  • MSCs are three-dimensionally cultured without using artificial materials, and a bone matrix is used. A new culture method is needed to produce and induce the bone cells contained therein.
  • MSCs spheroids mainly for cell-cell adhesion and extracellular matrix (ECM) produced by MSCs.
  • ECM extracellular matrix
  • the present invention has been made in view of the above problems, and an object of the present invention is to enable bone tissue formation while culturing MSCs three-dimensionally without using an artificial scaffold material, which is effective for bone regeneration therapy.
  • the purpose is to obtain a three-dimensional bone-like tissue.
  • the present inventors by culturing a three-dimensional mesenchymal stem cell agglomerate in a gel using a bone differentiation-inducing medium.
  • the present invention has been completed by finding that a three-dimensional bone-like tissue composed of a bone matrix on which minerals are deposited and bone cells contained in the bone matrix can be produced without using an artificial scaffold material.
  • the method for producing a bone-like tissue comprises a step of culturing a three-dimensional mesenchymal stem cell agglomerate in a gel using a bone differentiation-inducing medium, and comprises an artificial scaffold material. Is not used.
  • bone-like tissue it is suitable for transplantation into a bone defect portion containing a mineral-deposited bone matrix and bone cells contained in the bone matrix and not containing a foreign substance such as an artificial scaffold material. Bone-like tissue can be easily obtained.
  • the method for producing a bone-like tissue includes a step of adhering and culturing mesenchymal stem cells in a culture vessel to obtain a cell sheet, a step of peeling the cell sheet from the culture vessel, and the peeled cell sheet. It is preferable to further include a step of suspend-culturing the cells to obtain a three-dimensional mesenchymal stem cell mass.
  • the gel is preferably a gel containing a polysaccharide as a main component.
  • the gel does not contain an extracellular matrix (extracellular matrix) component other than the extracellular matrix (extracellular matrix) component generated from the embedded cells. Is preferable.
  • the bone differentiation-inducing medium preferably contains ascorbic acid and ⁇ -glycerophosphate.
  • the bone differentiation-inducing medium preferably contains dexamethasone.
  • the bone-like tissue according to the present invention contains a bone matrix in which minerals are deposited and bone cells contained in the bone matrix, does not contain an artificial scaffold material, and the bone cells form a network. It is a feature. Moreover, it is preferable that the bone-like tissue according to the present invention does not contain blood vessels.
  • the bone-like tissue since it contains a bone matrix and bone cells contained in the bone matrix but does not contain a foreign body artificial scaffold material, it is suitable for transplantation to a bone defect and bone regeneration therapy. Can be effectively used for.
  • the pharmaceutical composition according to the present invention is characterized by being a pharmaceutical composition for bone regeneration containing the bone-like tissue according to the present invention.
  • the pharmaceutical composition according to the present invention since it contains the above-mentioned bone-like tissue, it is suitable for transplantation to a bone defect and can be effectively used for bone regeneration therapy.
  • the bone-like tissue according to the present invention and the method for producing the same, it is possible to obtain a three-dimensional bone-like tissue effective for bone regeneration therapy, which contains a bone matrix on which minerals are deposited and bone cells contained in the bone matrix.
  • FIG. 6 is a photograph showing the results of alizarin red staining or HE staining of paraffin-embedded sections of three-dimensional bone-like tissue obtained by embedding C-MSCs in a gel and culturing in a concentration-optimized bone differentiation-inducing medium.
  • 3 is a photograph showing the results of alizarin red staining or HE staining of paraffin-embedded sections of cell agglomerates obtained by suspension-culturing C-MSCs using a conventional bone differentiation-inducing medium.
  • 6 is a photograph showing the results of alizarin red staining or HE staining of paraffin-embedded sections of cell agglomerates obtained by suspension culture of C-MSCs using concentration-optimized bone differentiation induction culture.
  • 6 is a photograph showing the results of alizarin red staining or HE staining of paraffin-embedded sections of three-dimensional bone-like tissue obtained by embedding C-MSCs in a gel and culturing in a conventional bone differentiation-inducing medium. It is a photograph obtained by micro CT imaging on a three-dimensional bone-like tissue obtained by embedding C-MSCs in a gel and culturing in a concentration-optimized bone differentiation-inducing medium.
  • the bone matrix protein was added to the paraffin-embedded section of the three-dimensional bone-like tissue obtained by embedding C-MSCs in a gel and culturing in a concentration-optimized bone differentiation-inducing medium.
  • It is a photograph showing the result of immunostaining (a) shows the result of staining COL1, (b) shows the result of staining OPN, and (c) shows the result of staining OCN.
  • FIGS. 1 to (c) are photographs showing the state of the transplanted portion 4 weeks after subcutaneously transplanting the three-dimensional bone-like tissue into the SCID mouse, and (a) shows the results of micro CT imaging in the subcutaneous transplanted portion of the mouse. It is a photograph which shows, (b) is a photograph which shows the result of having taken out the subcutaneous transplant part of a mouse together with three-dimensional bone-like tissue, and HE-stained the section prepared after decalcification, and (c) is the photograph which AZAN-stained the section. It is a photograph showing the result.
  • FIGS. 1 to (c) are photographs showing the state of the transplanted portion 8 weeks after subcutaneously transplanting the three-dimensional bone-like tissue into the SCID mouse, and (a) shows the results of micro CT imaging in the subcutaneous transplanted portion of the mouse. It is a photograph which shows, (b) is a photograph which shows the result of having taken out the subcutaneous transplant part of a mouse together with three-dimensional bone-like tissue, and HE-stained the section prepared after decalcification, and (c) is the photograph which AZAN-stained the section. It is a photograph showing the result. It is a photograph which shows the state which the three-dimensional bone-like tissue was transplanted to the bone defect part of the calvaria of a nude rat.
  • 6 is an HE-stained photograph showing the bone defect showing the result 4 weeks after the defect was prepared when the three-dimensional bone-like tissue was not transplanted into the calvaria defect of the SCID mouse.
  • 6 is an HE-stained photograph showing the bone defect showing the result 4 weeks after transplanting the three-dimensional bone-like tissue into the calvaria defect of the SCID mouse.
  • (A) is an HE-stained photograph showing the bone defect 8 weeks after transplanting the three-dimensional bone-like tissue into the calvaria defect of a nude rat, and (b) and (c) are respectively. It is an enlarged view of the area surrounded by the square shown in (a).
  • (A) is an AZAN-stained photograph showing the bone defect 8 weeks after transplanting the three-dimensional bone-like tissue into the calvaria defect of a nude rat, and (b) and (c) are respectively. It is an enlarged view of the area surrounded by the square shown in (a).
  • the bone-like tissue according to the present invention is characterized by containing a mineral-deposited bone matrix and bone cells contained in the bone matrix, not containing an artificial scaffold material, and the bone cells forming a network. It is a thing. Further, preferably, the bone-like tissue according to the present invention does not contain blood vessels.
  • the bone matrix is an accumulation of substances produced by cells having bone-forming ability and secreted extracellularly.
  • cells having bone-forming ability and secreted extracellularly There are, but not limited to, calcium phosphate and the like, which include proteins such as osteocalcin, osteopontin, osteonectin and collagen.
  • bone cells are cells that are most abundant in bone, and are cells that are contained in bone matrix and form a network with each other to form bone together with bone matrix.
  • the formation of a network means that bone cells are three-dimensionally connected to each other. In particular, it refers to a state in which proteins related to the cytoskeleton such as F-actin are expressed in bone cells, and the cells extend protrusions and connect to each other to form a three-dimensional network.
  • the mineral specifically includes calcium, magnesium and phosphorus.
  • Mineral deposition in the present embodiment means a state in which at least calcium is deposited on the bone matrix, and in this case, magnesium or phosphorus may be additionally deposited.
  • a method of staining tissue sections with alizarin red which is a pigment that binds to calcium, can be used, but of course, the method is not limited to this, and the presence of minerals in the bone matrix is not limited to this. Other methods can be used as long as the method can be confirmed.
  • the artificial scaffold material is an artificial scaffold material conventionally used for three-dimensional cell culture, for example, hydroxyapatite, tricalcium phosphate apatite, polylactic acid, chitosan, atelocollagen.
  • artificial scaffold material conventionally used for three-dimensional cell culture, for example, hydroxyapatite, tricalcium phosphate apatite, polylactic acid, chitosan, atelocollagen.
  • examples include, but are not limited to, gels, hyaluronic acid gels, and the like.
  • the bone-like tissue according to the present embodiment does not contain the artificial scaffold material, that is, does not contain foreign substances for the bone tissue, the bone cells are well networked in the bone matrix, and for bone regeneration therapy. Extremely excellent as a transplant material. Further, in the bone-like tissue according to the present embodiment, it is preferable that the bone cells are derived from artificially cultured mesenchymal stem cells. Further, the bone-like tissue according to the present embodiment is preferably produced by the method for producing a bone-like tissue according to the present invention. Further, the bone-like tissue according to the present embodiment is preferably prepared by an in vitro culture system according to the method for producing a bone-like tissue according to the present invention, and therefore does not contain blood vessels in the tissue.
  • the method for producing a bone-like tissue according to the present invention is artificial by culturing a three-dimensional mesenchymal stem cell agglomerate in a gel using a bone differentiation-inducing medium without using an artificial scaffold material. This is a method for producing a three-dimensional bone-like tissue that does not contain a scaffold material.
  • the artificial scaffold material is, as described above, an artificial scaffold material conventionally used for three-dimensional cell culture, for example, hydroxyapatite or phosphoric acid.
  • examples include, but are not limited to, tricalcium apatite, polylactic acid, chitosan, atelocollagen gel, hyaluronic acid gel and the like.
  • mesenchymal stem cells are collected from tissues such as, but not limited to, the spinal cord, adipose tissue, placenta tissue, umbilical band tissue, and dental pulp of animals such as humans. Also included are mesenchymal stem cells derived from induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).
  • iPS cells induced pluripotent stem cells
  • ES cells embryonic stem cells
  • the three-dimensional mesenchymal stem cell aggregate is a cell aggregate composed of a plurality of mesenchymal stem cells, and is in the form of a two-dimensional flat sheet. Rather, it is a cell clump having a three-dimensional granular shape.
  • mesenchymal stem cells are adherently cultured, they proliferate two-dimensionally, that is, they proliferate in a sheet shape to form a cell sheet.
  • the mesenchymal stem cells are embedded in a gel and cultured. Before the process, the cells are adherently cultured in a culture vessel and propagated to form a sheet (cell sheet).
  • the cell sheet After the cell sheet is peeled from the culture vessel, the cell sheet is suspended and cultured to form a three-dimensional mesenchymal stem cell mass. After that, it is preferable to culture the cell agglomerates in a gel-embedded state using a bone differentiation-inducing medium.
  • the growth medium until this cell agglomeration is obtained is not particularly limited as long as it is a medium containing a factor capable of proliferating mesenchymal stem cells and inducing the production of extracellular matrix such as ascorbic acid.
  • High glucose DMEM manufactured by Sigma
  • Prime-XV MSC expansion XSFM (manufactured by Irvine) containing no heterologous animal protein can be used. Can be used.
  • the constituent components of the gel are not particularly limited as long as the gel can be cultured by embedding a three-dimensional mesenchymal stem cell aggregate inside the gel, but the polysaccharide is polysaccharide.
  • the gel contains the above as a main component, and for example, Vitrogel (manufactured by The Well Bioscience) can be used.
  • gels containing polysaccharides as the main component gels containing extracellular matrix (extracellular matrix) such as collagen and laminin as the main components can also be used, but since they contain a large amount of cell adhesion factors, they can be used from cell clumps.
  • the gel used is preferably a gel that does not contain extracellular matrix components, because cell migration occurs and the resulting three-dimensional bone-like tissue becomes smaller. Therefore, even in the culturing process, it is preferable that the gel does not contain extracellular matrix components other than the extracellular matrix components generated from the embedded cells.
  • the bone differentiation-inducing medium may contain ascorbic acid and ⁇ -glycerophosphate. It is preferable and preferably contains dexamethasone. A recombinant protein of BMP2 or Wnt3a may be contained instead of dexamethasone.
  • MSCgo Osteogenic differentiation medium manufactured by Biological Industries, which does not contain heterologous animal protein, can also be used.
  • the pharmaceutical composition according to the present invention is a pharmaceutical composition used for bone regeneration including the bone-like tissue according to the present invention.
  • the pharmaceutical composition according to one embodiment of the present invention is used for the purpose of bone regeneration, and applicable bone is not limited and can be applied to any bone. Although not particularly limited to the following, it can be suitably applied to bone regeneration therapy for patients with intractable fractures, periodontitis, or extensive bone defects after osteotomy.
  • the pharmaceutical composition according to the present embodiment may contain various pharmaceutically acceptable additives in addition to the bone-like tissue according to the present invention.
  • the pharmaceutical composition according to the present embodiment is not particularly limited in its form, but is preferably powdery or granular so that it can be directly transplanted to a bone defect portion having various shapes.
  • Example 1 Culture of mesenchymal stem cells and preparation of three-dimensional bone-like tissue] (Preparation of C-MSCs) Human bone marrow-derived mesenchymal stem cells (MSCs, obtained from the Institute of Physical and Chemical Research) were seeded on 96-well plates (manufactured by Corning) at a cell density of 5.0 ⁇ 104 cells / well, 10% FBS, 100 U / ml penicillin and 100 ⁇ g.
  • MSCs Human bone marrow-derived mesenchymal stem cells
  • ECM is produced by culturing for 4 days in a culture medium containing 50 ⁇ g / ml of ascorbic acid (manufactured by Sigma) in a growth medium (GM medium) composed of High glucose DMEM (manufactured by Sigma) containing / ml streptomycin. I let you. Then, it was adhered to a 96-well plate, and the sheet-shaped cell population (cell sheet) was bluntly exfoliated. Specifically, the cell sheet can be isolated from the plate by moving a thin rod around the inner wall of the well while applying it to the edge of the cell sheet adhered to the plate. By doing so, a cell sheet composed of ECM and MSCs was suspended.
  • the obtained MSCs / ECM complex was transferred to an ultra-low binding plate (manufactured by Iwaki), and 100 nM dexamethasone, 10 mM ⁇ -glycerophosphate and 50 ⁇ g / ml ascorbic acid were added to the above GM medium to induce bone differentiation.
  • the cells were cultured in the medium for 3 days.
  • C-MSCs which are spherical cell clumps having a diameter of about 600 ⁇ m to 800 ⁇ m, were obtained.
  • the C-MSCs obtained as described above are embedded in a hydrogel containing a polysaccharide as a main component (Vitrogel 3D-RGD (500 ⁇ l / 48well): manufactured by The Well Bioscience), and placed in the GM medium on a 48-well plate.
  • 250 ⁇ l of bone differentiation-inducing medium concentration-optimized bone differentiation-inducing medium containing 10 nM dexamethasone, 5 mM ⁇ -glycerophosphate and 50 ⁇ g / ml ascorbic acid was added and cultured for 14 days (Example).
  • the medium was added on the gel.
  • a three-dimensional bone-like tissue which is a substantially spherical grain having a diameter of about 1 mm.
  • the bone differentiation-inducing medium (Comparative Example 1) or the concentration-optimized bone differentiation-inducing medium (Comparative Example 2) in a suspended state without embedding C-MSCs in the gel as described above. was cultured for 14 days.
  • spherical calcified product was obtained in Comparative Example 1, and no significant change was observed from the spherical cell clump in Comparative Example 2.
  • FIGS. 6 (a) to 6 (c) and 7 The results observed with a laser microscope are shown in FIGS. 6 (a) to 6 (c) and 7 respectively.
  • the three-dimensional bone-like tissue according to this example showed X-ray impermeableness as a whole like human bone tissue. Furthermore, in the three-dimensional bone-like tissue according to this example, as shown in FIGS. 6 (a) to 6 (c), the expression of COL1, OPN, and OCN coincided with the portion deeply stained with HE shown in FIG. It was observed (the white part inside the three-dimensional bone-like tissue in FIGS. 6 (a) to 6 (c)). Further, as shown in FIG. 7, in the three-dimensional bone-like tissue according to the example, F-actin is totally expressed in cells, and each cell forms a network while protruding protrusions, and bone cells. It showed the characteristics as. From the above results, it is considered that a three-dimensional bone-like tissue having the same characteristics as human bone tissue was obtained in this example.
  • Example 2 Evaluation of autonomous bone formation ability of three-dimensional bone-like tissue
  • the transplanted three-dimensional bone-like tissue was taken out together with the skin and decalcified, and then HE-stained and AZAN-stained.
  • AZAN staining young bones are dyed blue and mature bones are dyed red. Bone-like tissue was observed by HE staining 4 weeks after transplantation as shown in FIG. 8 (b), but as shown in FIG. 8 (c), it is a weak bone substrate that stains blue with AZAN staining. It has been shown.
  • 8 weeks after transplantation a tissue dyed red by AZAN staining was observed along with deep staining by HE staining (Fig. 9 (b)), and thus it became a mature bone-like tissue. I was able to confirm that it was there.
  • the three-dimensional bone-like tissue according to this example has the ability to form bone autonomously.
  • Example 3 Transplantation of three-dimensional bone-like tissue into immunodeficient mouse / rat calvaria defect
  • the effect was examined using an immunodeficient SCID mouse and a nude rat calvaria defect model. evaluated. The method and results will be described below.
  • a bone defect having a diameter of 1.6 mm was prepared using a round bar on the calvaria of SCID mice.
  • a bone defect having a diameter of 8 mm was prepared on the calvaria of a nude rat using a trephin bar.
  • one solid bone-like tissue as a granule was directly transplanted to the bone defect of the SCID mouse and 64 to the bone defect of the nude rat without using an artificial scaffold material or the like (see FIG. 10).
  • the case where the three-dimensional bone-like tissue was not transplanted to the bone defect portion of the SCID mouse was used as a comparative example.
  • a three-dimensional bone-like tissue composed of a bone matrix and bone cells encapsulated while living in the bone matrix and forming a network with each other can be obtained.
  • the bone-like tissue according to the present invention obtained by such a method exhibits an excellent bone regeneration effect, and does not contain an artificial scaffold material, that is, does not contain a foreign substance material, and thus is a transplant material for bone regeneration therapy. Can be suitably used as.

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Abstract

The method that produces bone-like tissue according to the present disclosure cultures a three-dimensional mesenchymal stem cell aggregate embedded in a gel using bone differentiation induction medium. This method does not use an artificial scaffold material.

Description

骨様組織及びその製造方法Bone-like tissue and its manufacturing method
　本発明は、骨様組織及びその製造方法に関し、特に人工材料を含まない骨様組織及びその製造方法に関する。 The present invention relates to a bone-like tissue and a method for producing the same, and particularly to a bone-like tissue containing no artificial material and a method for producing the same.
　従来から、重篤な骨折や、歯周炎又は腫瘍を原因とした骨摘出術後における広範囲に及ぶ骨欠損を示す患者に対する骨再生療法として、オトガイや腸骨から骨を分離して移植する自家骨移植が行われている。これは、骨欠損部に骨細胞及び骨基質を供給するため、理想的な骨再生療法といえるが、その採取量には限りがあり、実際には骨組織が不足して治療困難となるケースが多い。 Traditionally, as a bone regeneration therapy for patients with severe bone fractures or extensive bone defects after osteotomy due to periodontitis or tumor, autologous bone grafts are separated from the mussel and ilium. Bone grafting is being done. This is an ideal bone regeneration therapy because it supplies bone cells and bone matrix to the bone defect, but the amount collected is limited, and in reality there is a shortage of bone tissue, making treatment difficult. There are many.
　一方、多分化能及び自己増殖能を有する間葉系幹細胞（ＭＳＣｓ：Mesenchymal stem cell）は、組織再生療法に適した細胞として考えられており、近年、ＭＳＣｓと人工足場材料とを混和することで、自家骨に相当する立体的骨様組織を作製及び供給する試みが盛んになされている。例えば、ハイドロキシアパタイト、ポリ乳酸、キトサン、アテロコラーゲンゲル、ヒアルロン酸ゲルなどとＭＳＣｓを混和し、これを二次元培養の実験系で確立されている骨分化誘導培地（デキサメタゾン、アスコルビン酸及びβグリセロフォスフェート添加培地）で培養することで、骨再生能の高い立体的移植体が得られることが知られている（非特許文献１を参照。）。 On the other hand, mesenchymal stem cells (MSCs) having pluripotency and self-proliferation ability are considered as cells suitable for tissue regeneration therapy, and in recent years, MSCs and artificial scaffolding materials have been mixed. Attempts have been actively made to produce and supply three-dimensional bone-like tissues corresponding to autologous bones. For example, MSCs are mixed with hydroxyapatite, polylactic acid, chitosan, atelocollagen gel, hyaluronic acid gel, etc., and this is used as a bone differentiation-inducing medium (dexamethasone, ascorbic acid, and β-glycerophosphate) established in an experimental system of two-dimensional culture. It is known that a three-dimensional implant having high bone regeneration ability can be obtained by culturing in (additional medium) (see Non-Patent Document 1).
　しかしながら、上記のいずれの方法においても、骨に類似する立体的骨様組織の創生には至らず、自家骨移植に相当するほどの骨再生効果は現在のところ得られていない。その原因として、人工足場材料は細胞が産生する骨基質となることはなく、生体にとっては異物でしかないことが考えられる。また、人工材料を足場とする限り、ＭＳＣｓをどれほど骨分化培地で培養しても、骨基質内に内包され、ネットワーク形成をする骨細胞にまで誘導することは不可能であるためといえる。すなわち、より効果的な骨再生療法として応用可能な、自家骨に相当する立体的骨様組織を作製するためには、人工材料を用いることなく、ＭＳＣｓを３次元的に培養し、骨基質を産生させ、それに内包される骨細胞にまで誘導する新規の培養方法が必要となる。 However, none of the above methods has led to the creation of three-dimensional bone-like tissue similar to bone, and the bone regeneration effect equivalent to autologous bone grafting has not been obtained at present. It is considered that the cause is that the artificial scaffold material does not become a bone matrix produced by cells, but is only a foreign substance for the living body. Further, as long as the artificial material is used as a scaffold, it can be said that no matter how much MSCs are cultured in the bone differentiation medium, it is impossible to induce them into bone cells that are encapsulated in the bone matrix and form a network. That is, in order to prepare a three-dimensional bone-like tissue corresponding to autologous bone, which can be applied as a more effective bone regeneration therapy, MSCs are three-dimensionally cultured without using artificial materials, and a bone matrix is used. A new culture method is needed to produce and induce the bone cells contained therein.
　これまでに実際に、人工材料を用いないＭＳＣｓの代表的な３次元培養法として、細胞間接着を主とするＭＳＣｓスフェロイドや、ＭＳＣｓが産生した細胞外基質（ＥＣＭ：extracellular matrix）から構築される立体的細胞集塊（Ｃ－ＭＳＣｓ）を骨分化培地（デキサメタゾン（１００ｎＭ）＋βグリセロフォスフェート（１０ｍＭ）＋アスコルビン酸（５０μｇ／ｍｌ））で培養することにより、立体的骨様組織を作製する試みがなされてきた。しかし、ＭＳＣｓスフェロイド、Ｃ－ＭＳＣｓのいずれにも、ミネラルの沈着は誘導できるが、適切な骨基質形成と骨細胞の誘導は達成できなかった（非特許文献２及び３を参照。）。 So far, as a typical three-dimensional culture method of MSCs that does not actually use artificial materials, it is constructed from MSCs spheroids mainly for cell-cell adhesion and extracellular matrix (ECM) produced by MSCs. Attempt to prepare steric bone-like tissue by culturing steric extracellular matrix (C-MSCs) in bone differentiation medium (dexametazone (100 nM) + β-glycerophosphate (10 mM) + ascorbic acid (50 μg / ml)) Has been done. However, although mineral deposition could be induced in both MSCs spheroids and C-MSCs, appropriate bone matrix formation and bone cell induction could not be achieved (see Non-Patent Documents 2 and 3).
　上述したように、人工足場材料とＭＳＣｓとの併用は、骨再生療法の分野において多くの期待を集めているが、骨に相当する立体骨様組織の作製技術は未だ開発されていない。効果的な骨再生療法に適用可能な立体的骨様組織を作製及び供給するためには、人工足場材料を用いずに、ＭＳＣｓを３次元的に培養しながら、骨組織形成まで達成させる必要がある。 As mentioned above, the combined use of artificial scaffolding materials and MSCs has attracted many expectations in the field of bone regeneration therapy, but a technique for producing a three-dimensional bone-like tissue corresponding to bone has not yet been developed. In order to produce and supply three-dimensional bone-like tissue applicable to effective bone regeneration therapy, it is necessary to achieve bone tissue formation while culturing MSCs three-dimensionally without using artificial scaffolding material. is there.
　本発明は、前記問題に鑑みてなされたものであり、その目的は、人工足場材料を用いずに、ＭＳＣｓを３次元的に培養しながら、骨組織形成までできるようにし、骨再生療法に有効な立体的骨様組織を得ることにある。 The present invention has been made in view of the above problems, and an object of the present invention is to enable bone tissue formation while culturing MSCs three-dimensionally without using an artificial scaffold material, which is effective for bone regeneration therapy. The purpose is to obtain a three-dimensional bone-like tissue.
　前記の目的を達成するために、本発明者らは、鋭意研究の結果、立体的な間葉系幹細胞集塊をゲルに包埋した状態で、骨分化誘導培地を用いて培養することにより、人工足場材料を用いること無く、ミネラルが沈着した骨基質と該骨基質に内包された骨細胞からなる立体的骨様組織を作製できることを見出して本発明を完成した。 In order to achieve the above object, as a result of diligent research, the present inventors by culturing a three-dimensional mesenchymal stem cell agglomerate in a gel using a bone differentiation-inducing medium. The present invention has been completed by finding that a three-dimensional bone-like tissue composed of a bone matrix on which minerals are deposited and bone cells contained in the bone matrix can be produced without using an artificial scaffold material.
　具体的に、本発明に係る骨様組織の製造方法は、立体的な間葉系幹細胞集塊をゲルに包埋した状態で、骨分化誘導培地を用いて培養するステップを備え、人工足場材料を用いないことを特徴とする。 Specifically, the method for producing a bone-like tissue according to the present invention comprises a step of culturing a three-dimensional mesenchymal stem cell agglomerate in a gel using a bone differentiation-inducing medium, and comprises an artificial scaffold material. Is not used.
　本発明に係る骨様組織の製造方法によると、ミネラルが沈着した骨基質と該骨基質に内包された骨細胞とを含み、人工足場材料といった異物を含まない骨欠損部への移植に適した骨様組織を簡便に得ることができる。 According to the method for producing bone-like tissue according to the present invention, it is suitable for transplantation into a bone defect portion containing a mineral-deposited bone matrix and bone cells contained in the bone matrix and not containing a foreign substance such as an artificial scaffold material. Bone-like tissue can be easily obtained.
　本発明に係る骨様組織の製造方法は、間葉系幹細胞を培養容器で接着培養して細胞シートを得るステップと、前記細胞シートを前記培養容器から剥離するステップと、前記剥離された細胞シートを浮遊培養して立体的な間葉系幹細胞集塊を得るステップとをさらに備えていることが好ましい。 The method for producing a bone-like tissue according to the present invention includes a step of adhering and culturing mesenchymal stem cells in a culture vessel to obtain a cell sheet, a step of peeling the cell sheet from the culture vessel, and the peeled cell sheet. It is preferable to further include a step of suspend-culturing the cells to obtain a three-dimensional mesenchymal stem cell mass.
　本発明に係る骨様組織の製造方法において、前記ゲルは、多糖を主成分とするゲルであることが好ましい。 In the method for producing bone-like tissue according to the present invention, the gel is preferably a gel containing a polysaccharide as a main component.
　また、本発明に係る骨様組織の製造方法において、前記ゲルは、前記包埋された細胞から生じた細胞外基質（細胞外マトリクス）成分以外の細胞外基質（細胞外マトリクス）成分を含まないことが好ましい。 Further, in the method for producing bone-like tissue according to the present invention, the gel does not contain an extracellular matrix (extracellular matrix) component other than the extracellular matrix (extracellular matrix) component generated from the embedded cells. Is preferable.
　また、本発明に係る骨様組織の製造方法において、前記骨分化誘導培地は、アスコルビン酸及びβグリセロフォスフェートを含むことが好ましい。 Further, in the method for producing bone-like tissue according to the present invention, the bone differentiation-inducing medium preferably contains ascorbic acid and β-glycerophosphate.
　また、本発明に係る骨様組織の製造方法において、前記骨分化誘導培地は、デキサメタゾンを含むことが好ましい。 Further, in the method for producing bone-like tissue according to the present invention, the bone differentiation-inducing medium preferably contains dexamethasone.
　一方、本発明に係る骨様組織は、ミネラルが沈着した骨基質と該骨基質に内包された骨細胞とを含み、人工足場材料を含まず、前記骨細胞はネットワーク形成をしていることを特徴とする。また、本発明に係る骨様組織は、血管を含まないことが好ましい。 On the other hand, the bone-like tissue according to the present invention contains a bone matrix in which minerals are deposited and bone cells contained in the bone matrix, does not contain an artificial scaffold material, and the bone cells form a network. It is a feature. Moreover, it is preferable that the bone-like tissue according to the present invention does not contain blood vessels.
　本発明に係る骨様組織によると、骨基質と該骨基質に内包された骨細胞とを含みながら、異物である人工足場材料を含まないため、骨欠損部への移植に適し、骨再生療法に有効に用いられ得る。 According to the bone-like tissue according to the present invention, since it contains a bone matrix and bone cells contained in the bone matrix but does not contain a foreign body artificial scaffold material, it is suitable for transplantation to a bone defect and bone regeneration therapy. Can be effectively used for.
　また、本発明に係る医薬組成物は、上記本発明に係る骨様組織を含む骨再生のための医薬組成物であることを特徴とする。 Further, the pharmaceutical composition according to the present invention is characterized by being a pharmaceutical composition for bone regeneration containing the bone-like tissue according to the present invention.
　本発明に係る医薬組成物によると、上記のような骨様組織を含むため、骨欠損部への移植に適し、骨再生療法に有効に用いられ得る。 According to the pharmaceutical composition according to the present invention, since it contains the above-mentioned bone-like tissue, it is suitable for transplantation to a bone defect and can be effectively used for bone regeneration therapy.
　本発明に係る骨様組織及びその製造方法によると、ミネラルが沈着した骨基質と該骨基質に内包された骨細胞とを含む骨再生療法に有効な立体的骨様組織を得ることができる。 According to the bone-like tissue according to the present invention and the method for producing the same, it is possible to obtain a three-dimensional bone-like tissue effective for bone regeneration therapy, which contains a bone matrix on which minerals are deposited and bone cells contained in the bone matrix.
Ｃ－ＭＳＣｓをゲルに包埋して濃度至適化骨分化誘導培地で培養して得られた立体骨様組織のパラフィン包埋切片をアリザリンレッド染色又はＨＥ染色した結果を示す写真である。6 is a photograph showing the results of alizarin red staining or HE staining of paraffin-embedded sections of three-dimensional bone-like tissue obtained by embedding C-MSCs in a gel and culturing in a concentration-optimized bone differentiation-inducing medium. Ｃ－ＭＳＣｓを従来の骨分化誘導培地を用いて浮遊培養して得られた細胞集塊のパラフィン包埋切片をアリザリンレッド染色又はＨＥ染色した結果を示す写真である。3 is a photograph showing the results of alizarin red staining or HE staining of paraffin-embedded sections of cell agglomerates obtained by suspension-culturing C-MSCs using a conventional bone differentiation-inducing medium. Ｃ－ＭＳＣｓを濃度至適化骨分化誘導培を用いて浮遊培養して得られた細胞集塊のパラフィン包埋切片をアリザリンレッド染色又はＨＥ染色した結果を示す写真である。6 is a photograph showing the results of alizarin red staining or HE staining of paraffin-embedded sections of cell agglomerates obtained by suspension culture of C-MSCs using concentration-optimized bone differentiation induction culture. Ｃ－ＭＳＣｓをゲルに包埋して従来の骨分化誘導培地で培養して得られた立体骨様組織のパラフィン包埋切片をアリザリンレッド染色又はＨＥ染色した結果を示す写真である。6 is a photograph showing the results of alizarin red staining or HE staining of paraffin-embedded sections of three-dimensional bone-like tissue obtained by embedding C-MSCs in a gel and culturing in a conventional bone differentiation-inducing medium. Ｃ－ＭＳＣｓをゲルに包埋して濃度至適化骨分化誘導培地で培養して得られた立体骨様組織に対するマイクロＣＴ撮影により得られた写真である。It is a photograph obtained by micro CT imaging on a three-dimensional bone-like tissue obtained by embedding C-MSCs in a gel and culturing in a concentration-optimized bone differentiation-inducing medium. （ａ）～（ｃ）はＣ－ＭＳＣｓをゲルに包埋して濃度至適化骨分化誘導培地で培養して得られた立体骨様組織のパラフィン包埋切片に対して、骨基質タンパク質の免疫染色を行った結果を示す写真であり、（ａ）はＣＯＬ１を染色した結果を示し、（ｂ）はＯＰＮを染色した結果を示し、（ｃ）はＯＣＮを染色した結果を示す。In (a) to (c), the bone matrix protein was added to the paraffin-embedded section of the three-dimensional bone-like tissue obtained by embedding C-MSCs in a gel and culturing in a concentration-optimized bone differentiation-inducing medium. It is a photograph showing the result of immunostaining, (a) shows the result of staining COL1, (b) shows the result of staining OPN, and (c) shows the result of staining OCN. Ｃ－ＭＳＣｓをゲルに包埋して濃度至適化骨分化誘導培地で培養して得られた立体骨様組織のパラフィン包埋切片に対してＦ－ａｃｔｉｎの免疫染色を行った結果を示す写真である。Photograph showing the results of immunostaining of F-actin on paraffin-embedded sections of three-dimensional bone-like tissue obtained by embedding C-MSCs in a gel and culturing in a concentration-optimized bone differentiation-inducing medium. Is. （ａ）～（ｃ）はＳＣＩＤマウスに立体骨様組織を皮下移植した４週間後の移植部の状態を示す写真であり、（ａ）はマウスの皮下移植部においてマイクロＣＴ撮影をした結果を示す写真であり、（ｂ）はマウスの皮下移植部を立体骨様組織ごと取り出し、脱灰後に作製された切片をＨＥ染色した結果を示す写真であり、（ｃ）は当該切片をＡＺＡＮ染色した結果を示す写真である。(A) to (c) are photographs showing the state of the transplanted portion 4 weeks after subcutaneously transplanting the three-dimensional bone-like tissue into the SCID mouse, and (a) shows the results of micro CT imaging in the subcutaneous transplanted portion of the mouse. It is a photograph which shows, (b) is a photograph which shows the result of having taken out the subcutaneous transplant part of a mouse together with three-dimensional bone-like tissue, and HE-stained the section prepared after decalcification, and (c) is the photograph which AZAN-stained the section. It is a photograph showing the result. （ａ）～（ｃ）はＳＣＩＤマウスに立体骨様組織を皮下移植した８週間後の移植部の状態を示す写真であり、（ａ）はマウスの皮下移植部においてマイクロＣＴ撮影をした結果を示す写真であり、（ｂ）はマウスの皮下移植部を立体骨様組織ごと取り出し、脱灰後に作製された切片をＨＥ染色した結果を示す写真であり、（ｃ）は当該切片をＡＺＡＮ染色した結果を示す写真である。(A) to (c) are photographs showing the state of the transplanted portion 8 weeks after subcutaneously transplanting the three-dimensional bone-like tissue into the SCID mouse, and (a) shows the results of micro CT imaging in the subcutaneous transplanted portion of the mouse. It is a photograph which shows, (b) is a photograph which shows the result of having taken out the subcutaneous transplant part of a mouse together with three-dimensional bone-like tissue, and HE-stained the section prepared after decalcification, and (c) is the photograph which AZAN-stained the section. It is a photograph showing the result. ヌードラットの頭蓋冠の骨欠損部に立体骨様組織を移植した状態を示す写真である。It is a photograph which shows the state which the three-dimensional bone-like tissue was transplanted to the bone defect part of the calvaria of a nude rat. ＳＣＩＤマウスの頭蓋冠骨欠損部に立体骨様組織を移植しなかった場合における欠損作製から４週間後の結果を示す当該骨欠損部を示すＨＥ染色写真である。6 is an HE-stained photograph showing the bone defect showing the result 4 weeks after the defect was prepared when the three-dimensional bone-like tissue was not transplanted into the calvaria defect of the SCID mouse. ＳＣＩＤマウスの頭蓋冠骨欠損部に立体骨様組織を移植して４週間後の結果を示す当該骨欠損部を示すＨＥ染色写真である。6 is an HE-stained photograph showing the bone defect showing the result 4 weeks after transplanting the three-dimensional bone-like tissue into the calvaria defect of the SCID mouse. （ａ）はヌードラットの頭蓋冠骨欠損部に立体骨様組織を移植して８週間後の結果を示す当該骨欠損部を示すＨＥ染色写真であり、（ｂ）及び（ｃ）は、それぞれ（ａ）に示す四角で囲む領域の拡大図である。(A) is an HE-stained photograph showing the bone defect 8 weeks after transplanting the three-dimensional bone-like tissue into the calvaria defect of a nude rat, and (b) and (c) are respectively. It is an enlarged view of the area surrounded by the square shown in (a). （ａ）はヌードラットの頭蓋冠骨欠損部に立体骨様組織を移植して８週間後の結果を示す当該骨欠損部を示すＡＺＡＮ染色写真であり、（ｂ）及び（ｃ）は、それぞれ（ａ）に示す四角で囲む領域の拡大図である。(A) is an AZAN-stained photograph showing the bone defect 8 weeks after transplanting the three-dimensional bone-like tissue into the calvaria defect of a nude rat, and (b) and (c) are respectively. It is an enlarged view of the area surrounded by the square shown in (a).
　以下、本発明を実施するための形態を図面に基づいて説明する。以下の好ましい実施形態の説明は、本質的に例示に過ぎず、本発明、その適用方法或いはその用途を制限することを意図するものではない。 Hereinafter, a mode for carrying out the present invention will be described with reference to the drawings. The following description of preferred embodiments is merely exemplary and is not intended to limit the invention, its application methods or its uses.
　本発明に係る骨様組織は、ミネラルが沈着した骨基質と該骨基質に内包された骨細胞とを含み、人工足場材料を含まず、骨細胞はネットワーク形成をしていることを特徴とするものである。また、好ましくは本発明に係る骨様組織では、血管を含まない。 The bone-like tissue according to the present invention is characterized by containing a mineral-deposited bone matrix and bone cells contained in the bone matrix, not containing an artificial scaffold material, and the bone cells forming a network. It is a thing. Further, preferably, the bone-like tissue according to the present invention does not contain blood vessels.
　本発明の一実施形態に係る骨様組織において、本実施形態に係る骨様組織において、骨基質とは、骨形成能を有する細胞によって産生されて細胞外に分泌された物質が蓄積したものであり、以下のものに限定されないが、リン酸カルシウム等であって、オステオカルシンやオステオポンチン、オステオネクチン、コラーゲン等のタンパク質を含むものである。また、骨細胞とは、骨に最も多く存在する細胞であって、骨基質に内包されて細胞同士が互いにネットワークを形成して骨基質と共に骨を構成する細胞である。ここで、ネットワークの形成とは、骨細胞同士が立体的に互いに繋がった状態を形成していることをいう。特に、骨細胞にＦ－ａｃｔｉｎ等の細胞骨格に関わるタンパク質が発現しており、細胞同士が突起を延ばして互いに接続して立体的なネットワークを形成した状態をいう。 In the bone-like tissue according to one embodiment of the present invention, in the bone-like tissue according to the present embodiment, the bone matrix is an accumulation of substances produced by cells having bone-forming ability and secreted extracellularly. There are, but not limited to, calcium phosphate and the like, which include proteins such as osteocalcin, osteopontin, osteonectin and collagen. In addition, bone cells are cells that are most abundant in bone, and are cells that are contained in bone matrix and form a network with each other to form bone together with bone matrix. Here, the formation of a network means that bone cells are three-dimensionally connected to each other. In particular, it refers to a state in which proteins related to the cytoskeleton such as F-actin are expressed in bone cells, and the cells extend protrusions and connect to each other to form a three-dimensional network.
　本実施形態において、ミネラルとは、具体的にカルシウム、マグネシウム及びリンを含む。本実施形態におけるミネラルの沈着とは、骨基質に少なくともカルシウムが沈着している状態を意味し、この場合、付加的にマグネシウム又はリンが沈着していてもよい。ミネラルの沈着の有無の確認には、例えばカルシウムに対して結合する色素であるアリザリンレッドを用いて、組織切片を染色する方法が利用できるが、当然にこれに限られず、骨基質におけるミネラルの存在を確認できる方法であれば他の方法を利用することもできる。 In this embodiment, the mineral specifically includes calcium, magnesium and phosphorus. Mineral deposition in the present embodiment means a state in which at least calcium is deposited on the bone matrix, and in this case, magnesium or phosphorus may be additionally deposited. To confirm the presence or absence of mineral deposition, for example, a method of staining tissue sections with alizarin red, which is a pigment that binds to calcium, can be used, but of course, the method is not limited to this, and the presence of minerals in the bone matrix is not limited to this. Other methods can be used as long as the method can be confirmed.
　本実施形態に係る骨様組織において、人工足場材料とは、従来から３次元的細胞培養に用いられる人工的な足場材料であり、例えばハイドロキシアパタイト、リン酸三カルシウムアパタイト、ポリ乳酸、キトサン、アテロコラーゲンゲル、ヒアルロン酸ゲル等が挙げられるがこれらに限られない。 In the bone-like tissue according to the present embodiment, the artificial scaffold material is an artificial scaffold material conventionally used for three-dimensional cell culture, for example, hydroxyapatite, tricalcium phosphate apatite, polylactic acid, chitosan, atelocollagen. Examples include, but are not limited to, gels, hyaluronic acid gels, and the like.
　本実施形態に係る骨様組織は、上記人工足場材料を含まず、すなわち骨組織にとっての異物を含まないため、骨基質内で骨細胞が良好にネットワーク形成されており、骨再生療法のための移植材料として極めて優れている。また、本実施形態に係る骨様組織において、骨細胞は人工的に培養された間葉系幹細胞由来であることが好ましい。また、本実施形態に係る骨様組織は、本発明に係る骨様組織の製造方法によって、製造されたものであることが好ましい。また、本実施形態に係る骨様組織は、本発明に係る骨様組織の製造方法等による生体外の培養系で作製されることが好ましく、従ってその組織中に血管を含まない。 Since the bone-like tissue according to the present embodiment does not contain the artificial scaffold material, that is, does not contain foreign substances for the bone tissue, the bone cells are well networked in the bone matrix, and for bone regeneration therapy. Extremely excellent as a transplant material. Further, in the bone-like tissue according to the present embodiment, it is preferable that the bone cells are derived from artificially cultured mesenchymal stem cells. Further, the bone-like tissue according to the present embodiment is preferably produced by the method for producing a bone-like tissue according to the present invention. Further, the bone-like tissue according to the present embodiment is preferably prepared by an in vitro culture system according to the method for producing a bone-like tissue according to the present invention, and therefore does not contain blood vessels in the tissue.
　本発明に係る骨様組織の製造方法は、人工足場材料を用いずに、立体的な間葉系幹細胞集塊をゲルに包埋した状態で骨分化誘導培地を用いて培養することによって、人工足場材料を含まない立体的骨様組織を作製する方法である。 The method for producing a bone-like tissue according to the present invention is artificial by culturing a three-dimensional mesenchymal stem cell agglomerate in a gel using a bone differentiation-inducing medium without using an artificial scaffold material. This is a method for producing a three-dimensional bone-like tissue that does not contain a scaffold material.
　本発明の一実施形態に係る骨様組織の製造方法において、人工足場材料とは、上記の通り、従来から３次元的細胞培養に用いられる人工的な足場材料であり、例えばハイドロキシアパタイト、リン酸三カルシウムアパタイト、ポリ乳酸、キトサン、アテロコラーゲンゲル、ヒアルロン酸ゲル等が挙げられるがこれらに限られない。また、本実施形態に係る骨様組織の製造方法において、間葉系幹細胞は、以下に限定されないが、例えばヒト等の動物の髄、脂肪組織、胎盤組織、臍帯組織、歯髄等の組織から採取されたもの、並びに人工多能性幹細胞（ｉＰＳ細胞）由来及び胚性幹細胞（ＥＳ細胞）由来の間葉系幹細胞も含む。 In the method for producing a bone-like tissue according to an embodiment of the present invention, the artificial scaffold material is, as described above, an artificial scaffold material conventionally used for three-dimensional cell culture, for example, hydroxyapatite or phosphoric acid. Examples include, but are not limited to, tricalcium apatite, polylactic acid, chitosan, atelocollagen gel, hyaluronic acid gel and the like. Further, in the method for producing bone-like tissue according to the present embodiment, mesenchymal stem cells are collected from tissues such as, but not limited to, the spinal cord, adipose tissue, placenta tissue, umbilical band tissue, and dental pulp of animals such as humans. Also included are mesenchymal stem cells derived from induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).
　また、本実施形態に係る骨様組織の製造方法において、立体的な間葉系幹細胞集塊とは、複数の間葉系幹細胞で構成された細胞集塊であり、２次元的な平たいシート状ではなく、３次元的な粒状等の形状の細胞集塊である。通常、間葉系幹細胞を接着培養すると、二次元的に増殖し、すなわちシート状に増殖して細胞シートが形成されるが、本実施形態では、間葉系幹細胞をゲルに包埋して培養する前に、培養容器で接着培養して増殖させてシート状（細胞シート）にし、当該細胞シートを培養容器から剥離した後に、細胞シートを浮遊培養して立体的な間葉系幹細胞集塊を得て、当該細胞集塊をゲルに包埋した状態で骨分化誘導培地を用いて培養することが好ましい。この細胞集塊を得るまでの増殖培地としては、間葉系幹細胞を増殖できて、アスコルビン酸等の細胞外基質の産生を誘導する因子を含む培地であれば特に限定はされないが、例えば、上記因子の他にＦＢＳや所定の抗生物質を含むＨｉｇｈ　ｇｌｕｃｏｓｅ　ＤＭＥＭ（Sigma社製）を用いることができ、この他にも、例えば異種動物タンパク不含のPrime-XV MSC expansion XSFM(Irvine社製)を用いることができる。 Further, in the method for producing bone-like tissue according to the present embodiment, the three-dimensional mesenchymal stem cell aggregate is a cell aggregate composed of a plurality of mesenchymal stem cells, and is in the form of a two-dimensional flat sheet. Rather, it is a cell clump having a three-dimensional granular shape. Normally, when mesenchymal stem cells are adherently cultured, they proliferate two-dimensionally, that is, they proliferate in a sheet shape to form a cell sheet. In this embodiment, the mesenchymal stem cells are embedded in a gel and cultured. Before the process, the cells are adherently cultured in a culture vessel and propagated to form a sheet (cell sheet). After the cell sheet is peeled from the culture vessel, the cell sheet is suspended and cultured to form a three-dimensional mesenchymal stem cell mass. After that, it is preferable to culture the cell agglomerates in a gel-embedded state using a bone differentiation-inducing medium. The growth medium until this cell agglomeration is obtained is not particularly limited as long as it is a medium containing a factor capable of proliferating mesenchymal stem cells and inducing the production of extracellular matrix such as ascorbic acid. In addition to the factors, High glucose DMEM (manufactured by Sigma) containing FBS and a predetermined antibiotic can be used. In addition, for example, Prime-XV MSC expansion XSFM (manufactured by Irvine) containing no heterologous animal protein can be used. Can be used.
　本実施形態に係る骨様組織の製造方法において、ゲルは、その内側に立体的な間葉系幹細胞集塊を包埋して培養ができるものであればその構成成分は特に限定されないが、多糖を主成分とするゲルであることが好ましく、例えばＶｉｔｒｏｇｅｌ（The Well Bioscience社製）を用いることができる。また、ゲルの硬さは適宜調整可能である。Ｖｉｔｒｏｇｅｌの場合、ゲル固形化させる際にその濃度を薄めることで硬さを調整できる。例えばＶｉｔｒｏｇｅｌ：ＰＢＳ＝１：１で作製したものは硬く、１：５で作製すると柔らかく、半固形の状態になる。立体骨様組織の誘導にはＶｉｔｒｏｇｅｌ：ＰＢＳ＝１：３とすることが好ましい。また、多糖を主成分とするゲル以外に、コラーゲンやラミニンなどの細胞外基質（細胞外マトリクス）を主成分とするゲルも使用可能ではあるが、細胞接着因子を多く含むため、細胞集塊から細胞の遊出が生じ、得られる立体骨様組織が小さくなるため、用いるゲルは細胞外マトリクス成分を含まないゲルであることが好ましい。従って、培養過程においても、当該ゲルには包埋された細胞から生じた細胞外マトリクス成分以外の細胞外マトリクス成分は含まれないことが好ましい。 In the method for producing bone-like tissue according to the present embodiment, the constituent components of the gel are not particularly limited as long as the gel can be cultured by embedding a three-dimensional mesenchymal stem cell aggregate inside the gel, but the polysaccharide is polysaccharide. It is preferable that the gel contains the above as a main component, and for example, Vitrogel (manufactured by The Well Bioscience) can be used. Moreover, the hardness of the gel can be adjusted as appropriate. In the case of Vitrogel, the hardness can be adjusted by diluting the concentration when solidifying the gel. For example, a product prepared with Vitrogel: PBS = 1: 1 is hard, and a product prepared with 1: 5 is soft and semi-solid. Vitrogel: PBS = 1: 3 is preferable for the induction of three-dimensional bone-like tissue. In addition to gels containing polysaccharides as the main component, gels containing extracellular matrix (extracellular matrix) such as collagen and laminin as the main components can also be used, but since they contain a large amount of cell adhesion factors, they can be used from cell clumps. The gel used is preferably a gel that does not contain extracellular matrix components, because cell migration occurs and the resulting three-dimensional bone-like tissue becomes smaller. Therefore, even in the culturing process, it is preferable that the gel does not contain extracellular matrix components other than the extracellular matrix components generated from the embedded cells.
　本実施形態に係る骨様組織の製造方法において、骨分化誘導培地は、周知の種々の骨分化誘導培地を用いることができるが、骨分化誘導培地はアスコルビン酸及びβグリセロフォスフェートを含むことが好ましく、また、デキサメタゾンを含むことが好ましい。デキサメタゾンの代わりにＢＭＰ２やＷｎｔ３ａのリコンビナントタンパク質を含んでいてもよい。また、異種動物タンパク不含のMSCgo Osteogenic differentiation medium (Biological Industries社製)も使用できる。 In the method for producing bone-like tissue according to the present embodiment, various well-known bone differentiation-inducing media can be used as the bone differentiation-inducing medium, but the bone differentiation-inducing medium may contain ascorbic acid and β-glycerophosphate. It is preferable and preferably contains dexamethasone. A recombinant protein of BMP2 or Wnt3a may be contained instead of dexamethasone. In addition, MSCgo Osteogenic differentiation medium (manufactured by Biological Industries), which does not contain heterologous animal protein, can also be used.
　本発明に係る医薬組成物は、上記本発明に係る骨様組織を含む骨再生の用途で用いられる医薬組成物である。 The pharmaceutical composition according to the present invention is a pharmaceutical composition used for bone regeneration including the bone-like tissue according to the present invention.
　本発明の一実施形態に係る医薬組成物は、骨再生を目的として用いられ、適用可能な骨は限定されず如何なる骨にも適用可能である。以下に特に限定されないが、難治骨折や歯周炎、又は骨摘出術後の広範囲骨欠損症例に対する骨再生療法等に好適に適用可能である。 The pharmaceutical composition according to one embodiment of the present invention is used for the purpose of bone regeneration, and applicable bone is not limited and can be applied to any bone. Although not particularly limited to the following, it can be suitably applied to bone regeneration therapy for patients with intractable fractures, periodontitis, or extensive bone defects after osteotomy.
　本実施形態に係る医薬組成物は、上記本発明に係る骨様組織の他に医薬的に許容可能な種々の添加剤を含んでいても構わない。本実施形態に係る医薬組成物は、その形態については特に限定されないが、種々の形状の骨欠損部に直接に移植可能となるように粉状又は粒状であることが好ましい。 The pharmaceutical composition according to the present embodiment may contain various pharmaceutically acceptable additives in addition to the bone-like tissue according to the present invention. The pharmaceutical composition according to the present embodiment is not particularly limited in its form, but is preferably powdery or granular so that it can be directly transplanted to a bone defect portion having various shapes.
　以下に、本発明に係る骨様組織及びその製造方法について詳細に説明するための実施例を示す。 An example for explaining in detail the bone-like tissue and the method for producing the same according to the present invention is shown below.
　［実施例１：間葉系幹細胞の培養及び立体骨様組織の作製］
　（Ｃ－ＭＳＣｓの作製）
　ヒト骨髄由来間葉系幹細胞（ＭＳＣｓ、理化学研究所より入手）を９６ｗｅｌｌプレート（Corning社製）に５．０×１０４ｃｅｌｌｓ／ｗｅｌｌの細胞密度で播種し、１０％ＦＢＳ、１００Ｕ／ｍｌのペニシリン及び１００μｇ／ｍｌのストレプトマイシンを含むＨｉｇｈ　ｇｌｕｃｏｓｅ　ＤＭＥＭ（Sigma社製）からなる増殖培地（ＧＭ培地）に５０μｇ／ｍｌのアスコルビン酸（Sigma社製）を加えた培養液で４日間培養し、十分なＥＣＭを産生させた。その後、９６ｗｅｌｌプレートに接着し、シート状となった細胞集団（細胞シート）を鈍的に剥離した。具体的には、細い棒体をプレートに接着した細胞シートの縁に当てながらウェルの内壁に沿って一周移動させることによって、細胞シートをプレートから隔離することができる。そうすることで、ＥＣＭとＭＳＣｓとから構成された細胞シートを浮遊させた。この得られたＭＳＣｓ／ＥＣＭ複合体をultra-low bindingプレート（Iwaki社製）に移し、上記ＧＭ培地に１００ｎＭのデキサメタゾン、１０ｍＭのβグリセロフォスフェート及び５０μｇ／ｍｌのアスコルビン酸を加えた骨分化誘導培地で３日間培養した。その結果、直径６００μｍ～８００μｍ程度の球形の細胞集塊であるＣ－ＭＳＣｓが得られた。 [Example 1: Culture of mesenchymal stem cells and preparation of three-dimensional bone-like tissue]
(Preparation of C-MSCs)
Human bone marrow-derived mesenchymal stem cells (MSCs, obtained from the Institute of Physical and Chemical Research) were seeded on 96-well plates (manufactured by Corning) at a cell density of 5.0 × 104 cells / well, 10% FBS, 100 U / ml penicillin and 100 μg. Sufficient ECM is produced by culturing for 4 days in a culture medium containing 50 μg / ml of ascorbic acid (manufactured by Sigma) in a growth medium (GM medium) composed of High glucose DMEM (manufactured by Sigma) containing / ml streptomycin. I let you. Then, it was adhered to a 96-well plate, and the sheet-shaped cell population (cell sheet) was bluntly exfoliated. Specifically, the cell sheet can be isolated from the plate by moving a thin rod around the inner wall of the well while applying it to the edge of the cell sheet adhered to the plate. By doing so, a cell sheet composed of ECM and MSCs was suspended. The obtained MSCs / ECM complex was transferred to an ultra-low binding plate (manufactured by Iwaki), and 100 nM dexamethasone, 10 mM β-glycerophosphate and 50 μg / ml ascorbic acid were added to the above GM medium to induce bone differentiation. The cells were cultured in the medium for 3 days. As a result, C-MSCs, which are spherical cell clumps having a diameter of about 600 μm to 800 μm, were obtained.
　（Ｃ－ＭＳＣｓからの立体骨様組織誘導）
　上記のようにして得られたＣ－ＭＳＣｓを、多糖を主成分とするハイドロゲル（Vitrogel 3D-RGD（500μl/48well）:The Well Bioscience社製）に包埋し、４８ｗｅｌｌプレートにおいて上記ＧＭ培地に１０ｎＭのデキサメタゾン、５ｍＭのβグリセロフォスフェート及び５０μｇ／ｍｌのアスコルビン酸を加えた骨分化誘導培地（濃度至適化骨分化誘導培地）を２５０μｌ加えて１４日間培養した（実施例）。具体的には、ウェル内にＣ－ＭＳＣｓを内包する上記ゲルを入れた後に、該ゲルの上に上記培地を添加した。その結果、直径が約１ｍｍの略球形の粒体である立体骨様組織が得られた。一方、比較例として、Ｃ－ＭＳＣｓを上記のようなゲルで包埋すること無く、浮遊状態のままで骨分化誘導培地（比較例１）又は濃度至適化骨分化誘導培地（比較例２）を用いて１４日間培養した。その結果、比較例１では球形の石灰化物が得られ、比較例２では球形の細胞集塊から大きな変化が見られなかった。また、比較例３として、Ｃ－ＭＳＣｓを上記の通りゲル包埋し、従来の骨分化誘導培地にて培養した。各実施例及び比較例で得られた立体骨様組織又は細胞集塊の組織学的評価について以下に説明する。 (Three-dimensional bone-like tissue induction from C-MSCs)
The C-MSCs obtained as described above are embedded in a hydrogel containing a polysaccharide as a main component (Vitrogel 3D-RGD (500 μl / 48well): manufactured by The Well Bioscience), and placed in the GM medium on a 48-well plate. 250 μl of bone differentiation-inducing medium (concentration-optimized bone differentiation-inducing medium) containing 10 nM dexamethasone, 5 mM β-glycerophosphate and 50 μg / ml ascorbic acid was added and cultured for 14 days (Example). Specifically, after putting the gel containing C-MSCs in the well, the medium was added on the gel. As a result, a three-dimensional bone-like tissue, which is a substantially spherical grain having a diameter of about 1 mm, was obtained. On the other hand, as a comparative example, the bone differentiation-inducing medium (Comparative Example 1) or the concentration-optimized bone differentiation-inducing medium (Comparative Example 2) in a suspended state without embedding C-MSCs in the gel as described above. Was cultured for 14 days. As a result, spherical calcified product was obtained in Comparative Example 1, and no significant change was observed from the spherical cell clump in Comparative Example 2. In addition, as Comparative Example 3, C-MSCs were gel-embedded as described above and cultured in a conventional bone differentiation-inducing medium. The histological evaluation of the three-dimensional bone-like tissue or cell clumps obtained in each Example and Comparative Example will be described below.
　（立体骨様組織の組織学的評価）
　上記各培養によって得られた実施例及び比較例の立体骨様組織又は細胞集塊の石灰化物沈着程度を観察するために、それらを１％ホルムアルデヒドで固定し、パラフィン包埋後、５μｍの切片を作製しアリザリンレッド染色を行った（比較例３を除く）。また、それらの組織学的構造を観察するために、得られたサンプルを１０％エチレンジアミン四酢酸によって脱灰処理後に、５μｍの切片を作製しＨＥ染色を行った。その結果を図１～４に示す。また、上記実施例に係る立体骨様組織に対してＣＴ撮影を行った結果を図５に示す。さらに、上記実施例に係る立体骨様組織の切片に対して、常法を用いて、骨基質タンパク質のＣＯＬ１、ＯＰＮ及びＯＣＮの免疫染色並びに細胞骨格のＦ－ａｃｔｉｎの免疫染色を行い、共焦点レーザー顕微鏡で観察した結果をそれぞれ図６（ａ）～（ｃ）及び図７に示す。 (Histological evaluation of three-dimensional bone-like tissue)
In order to observe the degree of calcification of the three-dimensional bone-like tissue or cell clumps of the examples and comparative examples obtained by each of the above cultures, they were fixed with 1% formaldehyde, embedded in paraffin, and 5 μm sections were formed. It was prepared and stained with alizarin red (excluding Comparative Example 3). In addition, in order to observe their histological structure, the obtained sample was decalcified with 10% ethylenediaminetetraacetic acid, and then a 5 μm section was prepared and subjected to HE staining. The results are shown in FIGS. 1 to 4. In addition, FIG. 5 shows the results of CT imaging of the three-dimensional bone-like tissue according to the above embodiment. Furthermore, immunostaining of bone matrix proteins COL1, OPN and OCN and immunostaining of cytoskeleton F-actin were performed on the sections of the three-dimensional bone-like tissue according to the above examples using a conventional method, and confocal. The results observed with a laser microscope are shown in FIGS. 6 (a) to 6 (c) and 7 respectively.
　図１に示すように、Ｃ－ＭＳＣｓをゲルに包埋して濃度至適化骨分化誘導培地で培養した実施例では、豊富な骨基質と、アリザリンレッドに染色された骨基質に沈着したミネラルが観察され、ＨＥ染色像ではＨＥに濃染する骨様基質とこれを取り囲む骨芽細胞用細胞、さらにその内部には骨細胞様細胞が認められた。すなわち、実施例では、骨細胞様細胞を含む骨基質様組織からなる細胞集塊、すなわち立体骨様組織が得られたことが確認された。これに対して、比較例１では図２に示すように、アリザリンレッドに染色されるミネラルの沈着が観察されたが、脱灰切片にして組織学的に観察すると、骨基質構造は認められず、細胞の多くは核の収縮によって示される細胞死の状態であった。細胞死の原因は、骨分化誘導に用いられる添加因子による過度な石灰化効果であると考えられる。一方、デキサメタゾン及びβグリセロフォスフェートの濃度が低い濃度至適化骨分化誘導培地を用いた比較例２では図３に示すように、細胞死は減少したものの、ＥＣＭへのミネラルの沈着は得られなかった。 As shown in FIG. 1, in the example in which C-MSCs were embedded in a gel and cultured in a concentration-optimized bone differentiation-inducing medium, abundant bone matrix and minerals deposited on the bone matrix stained with Arizarin red Was observed, and in the HE-stained image, a bone-like matrix that was deeply stained in HE, osteoblast-like cells surrounding the bone-like substrate, and bone cell-like cells inside were observed. That is, in the examples, it was confirmed that a cell mass composed of bone matrix-like tissue containing bone cell-like cells, that is, a three-dimensional bone-like tissue was obtained. On the other hand, in Comparative Example 1, as shown in FIG. 2, deposition of minerals stained with alizarin red was observed, but when decalcified sections were histologically observed, no bone matrix structure was observed. Many of the cells were in a state of cell death indicated by contraction of the nucleus. The cause of cell death is considered to be the excessive calcification effect of the additive factor used to induce bone differentiation. On the other hand, in Comparative Example 2 using the optimized bone differentiation-inducing medium in which the concentrations of dexamethasone and β-glycerophosphate were low, as shown in FIG. 3, cell death was reduced, but mineral deposition in ECM was obtained. There wasn't.
　以上から、Ｃ－ＭＳＣｓをゲルに包埋して骨分化誘導に用いられる添加因子の濃度を低減した濃度至適化骨分化誘導培地で培養した実施例では、良好な立体骨様組織が得られたが、ゲル包埋培養を行わずに浮遊培養を行った比較例１及び２では、立体骨様組織が得られないことが明らかとなった。すなわち、良好な立体骨様組織を得るためには、Ｃ－ＭＳＣｓをゲルに包埋して培養することが必要であることが示唆される。 From the above, in the example in which C-MSCs were embedded in a gel and cultured in a concentration-optimized bone differentiation-inducing medium in which the concentration of an additive factor used for inducing bone differentiation was reduced, a good three-dimensional bone-like tissue was obtained. However, it was clarified that the three-dimensional bone-like tissue could not be obtained in Comparative Examples 1 and 2 in which suspension culture was performed without gel embedding culture. That is, it is suggested that it is necessary to embed C-MSCs in a gel and culture them in order to obtain good three-dimensional bone-like tissue.
　また、ゲル包埋培養を行う一方、従来の骨分化誘導培地を用いた比較例３を脱灰切片にして組織学的に観察すると、図４に示すように、実施例と比較して多くの細胞が核の収縮によって示される細胞死の状態であり、骨基質量も少ない状態であった。従って、良好な立体骨様組織を得るには、Ｃ－ＭＳＣｓをゲル包埋培養するのに加えて、骨分化誘導に用いられる添加因子濃度を低減させて至適化することが好ましいと考えられる。 In addition, while performing gel-embedded culture, when Comparative Example 3 using a conventional bone differentiation-inducing medium was used as a decalcified section and observed histologically, as shown in FIG. 4, many were compared with Examples. The cells were in a state of cell death indicated by contraction of the nucleus, and the bone mass was also low. Therefore, in order to obtain a good three-dimensional bone-like tissue, it is considered preferable to optimize the concentration of additive factors used for inducing bone differentiation in addition to gel-embedded culture of C-MSCs. ..
　また、本実施例に係る立体骨様組織は、図５に示すように、全体的にヒトの骨組織と同様にＸ線不透過性を示した。さらに、本実施例に係る立体骨様組織において、図６（ａ）～（ｃ）に示すように、図１に示すＨＥに濃染する部分に一致して、ＣＯＬ１、ＯＰＮ、ＯＣＮの発現が認められた（図６（ａ）～（ｃ）における立体骨様組織の内側の白色の部分）。さらに、図７に示すように、実施例に係る立体骨様組織において、Ｆ－ａｃｔｉｎが細胞に全体的に発現しており、各々の細胞が突起を出しながらネットワーク形成をしており、骨細胞としての特徴を示していた。以上の結果から、本実施例において、ヒトの骨組織と同等の特徴を有する立体骨様組織が得られたと考えられる。 In addition, as shown in FIG. 5, the three-dimensional bone-like tissue according to this example showed X-ray impermeableness as a whole like human bone tissue. Furthermore, in the three-dimensional bone-like tissue according to this example, as shown in FIGS. 6 (a) to 6 (c), the expression of COL1, OPN, and OCN coincided with the portion deeply stained with HE shown in FIG. It was observed (the white part inside the three-dimensional bone-like tissue in FIGS. 6 (a) to 6 (c)). Further, as shown in FIG. 7, in the three-dimensional bone-like tissue according to the example, F-actin is totally expressed in cells, and each cell forms a network while protruding protrusions, and bone cells. It showed the characteristics as. From the above results, it is considered that a three-dimensional bone-like tissue having the same characteristics as human bone tissue was obtained in this example.
　［実施例２：立体骨様組織の自律的な骨形成能の評価］
　次に、上記のようにして得られた立体骨様組織の自律的な骨形成能の評価を行った。その方法及び結果について以下に示す。 [Example 2: Evaluation of autonomous bone formation ability of three-dimensional bone-like tissue]
Next, the autonomous bone-forming ability of the three-dimensional bone-like tissue obtained as described above was evaluated. The method and results are shown below.
　まず、免疫不全動物であるＳＣＩＤマウスの皮膚を切開した。その後、人工足場材料を用いることなく、１６個の上記本実施例に係る立体骨様組織を上記ＳＣＩＤマウスに対して皮下移植した。マイクロＣＴにて皮下での硬組織の存在有無を確認したところ、図８（ａ）に示すように、移植４週後にＸ線不透過性を示す立体骨様組織が認められ（図中の白丸部分）、図９（ａ）に示すように、移植８週後にはその不透過度が向上していた（図中の白丸部分）。 First, the skin of SCID mice, which are immunodeficient animals, was incised. Then, 16 three-dimensional bone-like tissues according to the present example were subcutaneously transplanted into the SCID mice without using the artificial scaffolding material. When the presence or absence of hard tissue under the skin was confirmed by micro CT, as shown in FIG. 8 (a), three-dimensional bone-like tissue showing X-ray impermeable was observed 4 weeks after transplantation (white circles in the figure). Part), as shown in FIG. 9 (a), the opacity was improved 8 weeks after transplantation (white circle part in the figure).
　次に、移植された立体骨様組織を皮膚ごと取り出して脱灰した後、ＨＥ染色及びＡＺＡＮ染色を施した。なお、ＡＺＡＮ染色では幼弱骨は青く染まり、成熟骨は赤く染まる。図８（ｂ）に示すように移植４週後のＨＥ染色では骨様組織が観察されたが、図８（ｃ）に示すように、それはＡＺＡＮ染色に青く染まる幼弱な骨基質であることが示された。一方、移植８週後には、ＨＥ染色による濃染（図９（ｂ））と共に、ＡＺＡＮ染色に赤く染まる組織が認められた（図９（ｃ））ことから、成熟した骨様組織となっていることが確認できた。 Next, the transplanted three-dimensional bone-like tissue was taken out together with the skin and decalcified, and then HE-stained and AZAN-stained. In AZAN staining, young bones are dyed blue and mature bones are dyed red. Bone-like tissue was observed by HE staining 4 weeks after transplantation as shown in FIG. 8 (b), but as shown in FIG. 8 (c), it is a weak bone substrate that stains blue with AZAN staining. It has been shown. On the other hand, 8 weeks after transplantation, a tissue dyed red by AZAN staining was observed along with deep staining by HE staining (Fig. 9 (b)), and thus it became a mature bone-like tissue. I was able to confirm that it was there.
　以上の結果から、本実施例に係る立体骨様組織は、自律的に骨形成する能力を有することが示された。 From the above results, it was shown that the three-dimensional bone-like tissue according to this example has the ability to form bone autonomously.
　［実施例３：立体骨様組織の免疫不全マウス・ラット頭蓋冠骨欠損への移植］
　次に、上記のようにして得られた立体骨様組織を骨欠損部に移植することによる骨再生効果を検討するために、免疫不全ＳＣＩＤマウス及びヌードラット頭蓋冠欠損モデルを用いてその効果を評価した。その方法及び結果を以下に説明する。 [Example 3: Transplantation of three-dimensional bone-like tissue into immunodeficient mouse / rat calvaria defect]
Next, in order to examine the bone regeneration effect by transplanting the three-dimensional bone-like tissue obtained as described above into the bone defect portion, the effect was examined using an immunodeficient SCID mouse and a nude rat calvaria defect model. evaluated. The method and results will be described below.
　（方法）
　まず、ＳＣＩＤマウスの頭蓋冠にラウンドバーを用いて直径１．６ｍｍの骨欠損を作製した。一方、ヌードラットの頭蓋冠にトレフィンバーを用いて直径８ｍｍの骨欠損を作製した。その後、ＳＣＩＤマウスの骨欠損部には１個、ヌードラットに骨欠損部には６４個の粒体としての立体骨様組織を、人工足場材料などを用いることなく直接移植した（図１０を参照）。なお、ＳＣＩＤマウスの骨欠損部に立体骨様組織を移植しない場合を比較例とした。ＳＣＩＤマウスでは移植の４週間後、ヌードラットでは移植の８週間後に、動物を屠殺し、頭蓋冠を取り出して、上記と同様に脱灰後に連続切片を作製してＨＥ染色を行い骨組織の観察を行った。また、移植の８週間後のヌードラットのサンプルについては、ＡＺＡＮ染色も行って観察した。 (Method)
First, a bone defect having a diameter of 1.6 mm was prepared using a round bar on the calvaria of SCID mice. On the other hand, a bone defect having a diameter of 8 mm was prepared on the calvaria of a nude rat using a trephin bar. Then, one solid bone-like tissue as a granule was directly transplanted to the bone defect of the SCID mouse and 64 to the bone defect of the nude rat without using an artificial scaffold material or the like (see FIG. 10). ). In addition, the case where the three-dimensional bone-like tissue was not transplanted to the bone defect portion of the SCID mouse was used as a comparative example. After 4 weeks of transplantation in SCID mice and 8 weeks after transplantation in nude rats, the animals were sacrificed, the calvaria was taken out, and after decalcification as described above, continuous sections were prepared and HE-stained to observe the bone tissue. Was done. Nude rat samples 8 weeks after transplantation were also observed by AZAN staining.
　（結果）
　ＳＣＩＤマウスモデルにおいて、図１１に示すように立体骨様組織を移植しなかった比較例では骨の再生は起こらないのに対し、図１２に示すように立体骨様組織を移植した実施例では骨再生が観察された。また、ヌードラットモデルにおいても図１３に示すように、立体骨様組織の移植により、ＨＥ染色では欠損両端をつなぐ骨様組織で満たされていることが観察された。また図１４に示すように、ＡＺＡＮ染色においてもほとんどの部分が赤く染まっており、すなわちこれらの骨様組織が成熟骨からなることが確認された。従って、骨欠損部における立体骨様組織の移植により骨再生が起こることが確認された。 (result)
In the SCID mouse model, bone regeneration did not occur in the comparative example in which the three-dimensional bone-like tissue was not transplanted as shown in FIG. 11, whereas bone was transplanted in the example in which the three-dimensional bone-like tissue was transplanted as shown in FIG. Regeneration was observed. In addition, as shown in FIG. 13, in the nude rat model, it was observed that the bone-like tissue connecting the two ends of the defect was filled with HE staining by transplantation of the three-dimensional bone-like tissue. Further, as shown in FIG. 14, it was confirmed that most of the AZAN staining was also dyed red, that is, these bone-like tissues consisted of mature bone. Therefore, it was confirmed that bone regeneration occurs by transplantation of three-dimensional bone-like tissue in the bone defect.
　上記実施例１～３の結果から、間葉系幹細胞をゲルに包埋して所定の骨誘導培地で培養することにより、人工足場材料が無くても、内部には骨細胞様細胞を含む骨基質様組織からなる細胞集塊、すなわち立体骨様組織が得られることが明らかとなった。また、得られた立体骨様組織を骨欠損部に移植することにより有効に骨再生を誘導することが明らかとなった。 From the results of Examples 1 to 3 above, by embedding mesenchymal stem cells in a gel and culturing in a predetermined bone induction medium, bone containing bone cell-like cells inside even without an artificial scaffold material. It was clarified that a cell clump composed of a matrix-like tissue, that is, a three-dimensional bone-like tissue was obtained. In addition, it was clarified that the obtained three-dimensional bone-like tissue was transplanted to the bone defect to effectively induce bone regeneration.
　以上の通り、本発明に係る骨様組織の製造方法によると、骨基質と該骨基質に生存しながら内包されて互いにネットワーク形成をした骨細胞からなる立体骨様組織が得られる。また、そのような方法で得られた本発明に係る骨様組織は、優れた骨再生効果を示し、また、人工足場材料を含まず、すなわち異物材料を含まないため、骨再生療法の移植材料として好適に用いることができる。 As described above, according to the method for producing bone-like tissue according to the present invention, a three-dimensional bone-like tissue composed of a bone matrix and bone cells encapsulated while living in the bone matrix and forming a network with each other can be obtained. In addition, the bone-like tissue according to the present invention obtained by such a method exhibits an excellent bone regeneration effect, and does not contain an artificial scaffold material, that is, does not contain a foreign substance material, and thus is a transplant material for bone regeneration therapy. Can be suitably used as.

Claims (9)

  1. 　ミネラルが沈着した骨基質と該骨基質に内包された骨細胞とを含み、人工足場材料を含まず、
    　前記骨細胞はネットワーク形成をしていることを特徴とする人工の骨様組織。     Contains mineral-deposited bone matrix and bone cells encapsulated in the bone matrix, without artificial scaffolding material
    An artificial bone-like tissue characterized in that the bone cells form a network.
  2. 　血管を含まないことを特徴とする請求項１に記載の骨様組織。 The bone-like tissue according to claim 1, which is characterized by not containing blood vessels.
  3. 　請求項１又は２に記載の骨様組織を含む骨再生のための医薬組成物。 A pharmaceutical composition for bone regeneration containing the bone-like tissue according to claim 1 or 2.
  4. 　請求項１又は２に記載の骨様組織を製造する方法であって、
    　立体的な間葉系幹細胞集塊をゲルに包埋した状態で、骨分化誘導培地を用いて培養するステップを備え、
    　人工足場材料を用いないことを特徴とする骨様組織の製造方法。     The method for producing a bone-like tissue according to claim 1 or 2.
    A step of culturing a three-dimensional mesenchymal stem cell mass in a gel using a bone differentiation-inducing medium is provided.
    A method for producing bone-like tissue, which is characterized by not using an artificial scaffold material.
  5. 　間葉系幹細胞を培養容器で接着培養して細胞シートを得るステップと、
    　前記細胞シートを前記培養容器から剥離するステップと、
    　前記剥離された細胞シートを浮遊培養して立体的な間葉系幹細胞集塊を得るステップとをさらに備えていることを特徴とする請求項４に記載の骨様組織の製造方法。     Steps to obtain a cell sheet by adhesively culturing mesenchymal stem cells in a culture vessel,
    The step of peeling the cell sheet from the culture vessel and
    The method for producing a bone-like tissue according to claim 4, further comprising a step of suspending and culturing the detached cell sheet to obtain a three-dimensional mesenchymal stem cell mass.
  6. 　前記ゲルは、多糖を主成分とするゲルであることを特徴とする請求項４又は５に記載の骨様組織の製造方法。 The method for producing a bone-like tissue according to claim 4 or 5, wherein the gel is a gel containing a polysaccharide as a main component.
  7. 　前記ゲルは、前記包埋された細胞から生じた細胞外マトリクス成分以外の細胞外マトリクス成分を含まないことを特徴とする請求項４～６のいずれか１項に記載の骨様組織の製造方法。 The method for producing bone-like tissue according to any one of claims 4 to 6, wherein the gel does not contain extracellular matrix components other than extracellular matrix components generated from the embedded cells. ..
  8. 　前記骨分化誘導培地は、アスコルビン酸及びβグリセロフォスフェートを含むことを特徴とする請求項４～７のいずれか１項に記載の骨様組織の製造方法。 The method for producing a bone-like tissue according to any one of claims 4 to 7, wherein the bone differentiation-inducing medium contains ascorbic acid and β-glycerophosphate.
  9. 　前記骨分化誘導培地は、デキサメタゾンを含むことを特徴とする請求項４～８のいずれか１項に記載の骨様組織の製造方法。 The method for producing bone-like tissue according to any one of claims 4 to 8, wherein the bone differentiation-inducing medium contains dexamethasone.
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CN116942915A (en) * 2023-08-09 2023-10-27 山东大学齐鲁医院 Bone cell gel culture method and application thereof in bone echinococcosis bone defect
CN116942915B (en) * 2023-08-09 2024-02-13 山东大学齐鲁医院 Bone cell gel culture method and application thereof in bone echinococcosis bone defect

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