JP2984176B2 - Method for culturing bone marrow cells, culture mixture and material for transplantation into hard tissue defect - Google Patents
Method for culturing bone marrow cells, culture mixture and material for transplantation into hard tissue defectInfo
- Publication number
- JP2984176B2 JP2984176B2 JP5351035A JP35103593A JP2984176B2 JP 2984176 B2 JP2984176 B2 JP 2984176B2 JP 5351035 A JP5351035 A JP 5351035A JP 35103593 A JP35103593 A JP 35103593A JP 2984176 B2 JP2984176 B2 JP 2984176B2
- Authority
- JP
- Japan
- Prior art keywords
- cells
- bone marrow
- culture
- collagen
- marrow cells
- Prior art date
- 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.)
- Expired - Fee Related
Links
- 210000002798 bone marrow cell Anatomy 0.000 title claims description 73
- 239000000203 mixture Substances 0.000 title claims description 29
- 238000000034 method Methods 0.000 title claims description 24
- 239000000463 material Substances 0.000 title claims description 22
- 238000012258 culturing Methods 0.000 title claims description 19
- 230000007547 defect Effects 0.000 title claims description 19
- 210000001519 tissue Anatomy 0.000 title claims description 14
- 238000002054 transplantation Methods 0.000 title claims description 13
- 210000004027 cell Anatomy 0.000 claims description 89
- 239000002609 medium Substances 0.000 claims description 65
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 claims description 64
- 239000001506 calcium phosphate Substances 0.000 claims description 51
- 229910000389 calcium phosphate Inorganic materials 0.000 claims description 50
- 235000011010 calcium phosphates Nutrition 0.000 claims description 50
- 239000002245 particle Substances 0.000 claims description 45
- 239000007788 liquid Substances 0.000 claims description 39
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 32
- 102000008186 Collagen Human genes 0.000 claims description 30
- 108010035532 Collagen Proteins 0.000 claims description 30
- 229920001436 collagen Polymers 0.000 claims description 30
- 210000002449 bone cell Anatomy 0.000 claims description 23
- 239000000512 collagen gel Substances 0.000 claims description 21
- 102000012422 Collagen Type I Human genes 0.000 claims description 18
- 108010022452 Collagen Type I Proteins 0.000 claims description 18
- 239000000126 substance Substances 0.000 claims description 18
- 238000012136 culture method Methods 0.000 claims description 17
- 229960005070 ascorbic acid Drugs 0.000 claims description 16
- 230000000694 effects Effects 0.000 claims description 16
- 210000000963 osteoblast Anatomy 0.000 claims description 14
- 210000002997 osteoclast Anatomy 0.000 claims description 12
- 210000004102 animal cell Anatomy 0.000 claims description 11
- 210000001612 chondrocyte Anatomy 0.000 claims description 10
- 235000010323 ascorbic acid Nutrition 0.000 claims description 9
- 239000011668 ascorbic acid Substances 0.000 claims description 9
- 229910000392 octacalcium phosphate Inorganic materials 0.000 claims description 7
- YIGWVOWKHUSYER-UHFFFAOYSA-F tetracalcium;hydrogen phosphate;diphosphate Chemical compound [Ca+2].[Ca+2].[Ca+2].[Ca+2].OP([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O YIGWVOWKHUSYER-UHFFFAOYSA-F 0.000 claims description 7
- GBNXLQPMFAUCOI-UHFFFAOYSA-H tetracalcium;oxygen(2-);diphosphate Chemical compound [O-2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O GBNXLQPMFAUCOI-UHFFFAOYSA-H 0.000 claims description 7
- 238000004113 cell culture Methods 0.000 claims description 4
- 238000009630 liquid culture Methods 0.000 claims description 2
- 239000001963 growth medium Substances 0.000 claims 1
- 239000000843 powder Substances 0.000 description 21
- 210000000988 bone and bone Anatomy 0.000 description 18
- 230000004069 differentiation Effects 0.000 description 15
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 11
- 238000010186 staining Methods 0.000 description 11
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 9
- 102000013563 Acid Phosphatase Human genes 0.000 description 9
- 108010051457 Acid Phosphatase Proteins 0.000 description 9
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 9
- 239000013078 crystal Substances 0.000 description 9
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- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 101000688200 Prevotella intermedia Alkaline phosphatase Proteins 0.000 description 4
- 239000011575 calcium Substances 0.000 description 4
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 4
- 239000002504 physiological saline solution Substances 0.000 description 4
- 239000012679 serum free medium Substances 0.000 description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- 239000007995 HEPES buffer Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
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- 230000012010 growth Effects 0.000 description 3
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- 238000003786 synthesis reaction Methods 0.000 description 3
- 206010003694 Atrophy Diseases 0.000 description 2
- 208000004434 Calcinosis Diseases 0.000 description 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
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- 229910019142 PO4 Inorganic materials 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
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- 210000000845 cartilage Anatomy 0.000 description 2
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- 238000006243 chemical reaction Methods 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
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- VSIIXMUUUJUKCM-UHFFFAOYSA-D pentacalcium;fluoride;triphosphate Chemical compound [F-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O VSIIXMUUUJUKCM-UHFFFAOYSA-D 0.000 description 2
- 210000002379 periodontal ligament Anatomy 0.000 description 2
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- 229950003937 tolonium Drugs 0.000 description 2
- HNONEKILPDHFOL-UHFFFAOYSA-M tolonium chloride Chemical compound [Cl-].C1=C(C)C(N)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 HNONEKILPDHFOL-UHFFFAOYSA-M 0.000 description 2
- 235000019731 tricalcium phosphate Nutrition 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 206010066054 Dysmorphism Diseases 0.000 description 1
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- 241000283073 Equus caballus Species 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- YDHWWBZFRZWVHO-UHFFFAOYSA-H [oxido-[oxido(phosphonatooxy)phosphoryl]oxyphosphoryl] phosphate Chemical compound [O-]P([O-])(=O)OP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O YDHWWBZFRZWVHO-UHFFFAOYSA-H 0.000 description 1
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- 230000003115 biocidal effect Effects 0.000 description 1
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Materials For Medical Uses (AREA)
Description
【０００１】[0001]
【産業上の利用分野】この発明は、所望の骨系細胞を未
分化な骨髄細胞から分化させる、リン酸カルシウム含有
コラーゲンゲルを用いた培養方法とこの培養方法に用い
る培養用混合物に関する。この培養方法は、所望の移植
部位に適応した骨系細胞を体外で（invitro）作ること
ができ、また、骨髄細胞の分化機構や機能を解明するの
にも有用である。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a culture method using calcium phosphate-containing collagen gel for differentiating desired bone cells from undifferentiated bone marrow cells, and a culture mixture used in this culture method. This culture method can produce bone cells adapted to a desired transplant site in vitro, and is also useful for elucidating the differentiation mechanism and function of bone marrow cells.
【０００２】さらに、この発明は、上記培養方法により
得られた移植用材料に関する。この移植用材料は、骨欠
損部などの硬組織欠損部を修復するために移植される。[0002] Furthermore, the present invention relates to a transplant material obtained by the above-mentioned culturing method. The transplant material is transplanted to repair a hard tissue defect such as a bone defect.
【０００３】[0003]
【従来の技術】動物の骨、軟骨を形成する細胞は、それ
ぞれ、骨芽細胞、軟骨芽細胞であり、未分化な骨髄細胞
に由来する。そこで、骨欠損部を修復するために、骨片
を移植する代わりに骨髄細胞を移植することが試みられ
ている。しかし、骨髄細胞は、流動性を有するため移植
部位から流動して目的とする部位で増殖せず、不必要な
部位で増殖することがある。骨髄細胞が流動しないよう
に固定化するために、再構成した繊維状アテロペプチド
コラーゲン、リン酸三カルシウム、骨髄、および、展性
のある調製物を得るのに充分な流体の混合物からなる移
植用材料が提案された（特開昭６２−２６８５６３号公
報）。ここで流体としては生理食塩水が使用されてい
る。2. Description of the Related Art The cells forming the bone and cartilage of animals are osteoblasts and chondroblasts, respectively, and are derived from undifferentiated bone marrow cells. In order to repair a bone defect, transplantation of bone marrow cells instead of bone fragments has been attempted. However, since bone marrow cells have fluidity, they do not flow from a transplant site and proliferate at a target site, but may proliferate at an unnecessary site. For implantation, consisting of a reconstituted fibrous atelopeptide collagen, tricalcium phosphate, bone marrow, and a fluid mixture sufficient to obtain a malleable preparation to immobilize bone marrow cells from flowing A material has been proposed (Japanese Patent Application Laid-Open No. 62-268563). Here, a physiological saline is used as the fluid.
【０００４】骨髄細胞が固定化された移植用材料を生体
に移植すると、体内での（in vivo)骨髄細胞の分化を人
為的に制御することはできず、移植された部位に適した
骨系細胞に分化しないという問題がある。上記移植用材
料は、流体として生理食塩水を用いるので骨髄細胞の体
外での（in vitro）培養を想定したものではない。他
方、骨髄不足の問題を防ぎ、骨欠損部への移植用材料を
安定に供給するために、生体から取り出された骨髄細胞
を体外で培養して骨形成に関与する骨系細胞に分化させ
ることが検討された。[0004] When a transplantation material having bone marrow cells immobilized thereon is transplanted into a living body, it is impossible to artificially control the differentiation of bone marrow cells in the body (in vivo). There is a problem that it does not differentiate into cells. Since the above-mentioned material for transplant uses physiological saline as a fluid, it is not intended for in vitro culture of bone marrow cells. On the other hand, in order to prevent the problem of bone marrow deficiency and to stably supply a material for transplantation to a bone defect, culture of bone marrow cells removed from a living body in vitro and differentiation into bone cells involved in bone formation Was considered.
【０００５】現在報告されている培養方法（培養手技）
では、骨髄組織から骨髄細胞を単離し、種々の添加物を
含む血清含有培地で培養し、軟骨細胞に分化させること
に成功している。体外で骨髄間質細胞の液体培養を行う
と、線維芽細胞様の細胞のコロニーが形成され、コロニ
ー中心部に石灰化沈着物の観察がなされている（Maniat
opoulos 他、Cell Tissue Res(1988)254:317-330）。[0005] Culture methods (cultivation techniques) currently reported
Have successfully isolated bone marrow cells from bone marrow tissue, cultured them in a serum-containing medium containing various additives, and differentiated them into chondrocytes. When liquid culture of bone marrow stromal cells is carried out in vitro, colonies of fibroblast-like cells are formed, and calcified deposits have been observed in the center of the colonies (Maniat
opoulos et al., Cell Tissue Res (1988) 254: 317-330).
【０００６】[0006]
【発明が解決しようとする課題】骨芽細胞や軟骨芽細胞
への分化、形成過程および骨、軟骨の形成機序は未解決
のまま残されているので、従来の細胞培養方法では、体
外で骨髄細胞の分化を制御し、所望の骨系細胞を自在に
作り出すことはできなかった。従って、骨系細胞を安定
して得るために、骨髄細胞の骨系細胞への分化を人為的
に制御できる培養方法が望まれる。The differentiation and formation process of osteoblasts and chondroblasts and the formation mechanism of bone and cartilage remain unresolved. It was not possible to control the differentiation of bone marrow cells and freely produce desired bone cells. Therefore, in order to stably obtain bone cells, a culture method capable of artificially controlling the differentiation of bone marrow cells into bone cells is desired.
【０００７】上記のように細胞培養に用いられる血清は
牛胎仔血清あるいは馬胎仔血清であるため、血清含有培
地で培養された細胞がヒトなどの生体へ移植されたとき
にこれらの血清が強い免疫原生を示し、良好な生体親和
性は期待できない。骨欠損部修復に培養骨髄細胞を使用
するためには、移植部位に適合した細胞を無血清培地で
作り、良好な生体親和性を有する移植担体とともに移植
する必要がある。[0007] As described above, serum used for cell culture is fetal bovine serum or fetal equine serum. Therefore, when cells cultured in a serum-containing medium are transplanted into a living body such as a human, these sera are strongly immunized. It shows virginity and good biocompatibility cannot be expected. In order to use cultured bone marrow cells for repairing a bone defect, it is necessary to prepare cells suitable for the transplantation site in a serum-free medium and transplant them with a transplantation carrier having good biocompatibility.
【０００８】ところが、従来の無血清培地は、骨髄細胞
の培養を行うためには、デキサメサゾンなどのホルモン
を含む必要がある。このようにホルモンを含む無血清培
地を用いて骨髄細胞の培養を行うと細胞が異形化などの
変異を起こすため骨系細胞への分化を制御することがで
きない。この発明は、血清もホルモンも含まない液体培
地を用いて、体外で未分化な骨髄細胞を移植部位に適応
した所望の骨系細胞に分化させることができる骨髄細胞
の培養方法、および、この培養方法に用いる培養用混合
物を提供することを課題とする。However, a conventional serum-free medium needs to contain hormones such as dexamethasone in order to culture bone marrow cells. When culturing bone marrow cells using a serum-free medium containing hormones as described above, the cells undergo mutations such as dysmorphism, so that differentiation into bone cells cannot be controlled. The present invention provides a method for culturing bone marrow cells capable of differentiating undifferentiated bone marrow cells in vitro into desired bone cells adapted to a transplantation site using a liquid medium containing neither serum nor hormone, and this culturing. It is an object to provide a culture mixture for use in the method.
【０００９】この発明は、上記培養方法により得られ、
血清を含まず良好な生体親和性持ち、骨欠損部などの硬
組織欠損部を修復するために移植される移植用材料を提
供することを課題とする。[0009] The present invention is obtained by the above culture method,
An object of the present invention is to provide an implantable material which is free from serum, has good biocompatibility, and is implanted for repairing a hard tissue defect such as a bone defect.
【００１０】[0010]
【課題を解決するための手段】上記課題を解決するため
に、発明者らは、血清もホルモンも含まない液体培地で
骨髄細胞を所望の骨系細胞に分化させる方法を検討し
た。そして、コラーゲンゲル中に化学活性を有するリン
酸カルシウム粉末粒子が分散された複合体を骨髄細胞の
支持体として用いた場合には、リン酸カルシウムから液
体培地にカルシウムイオンとリン酸イオンが溶解するの
で、液体培地が血清もホルモンも含まなくても、骨髄細
胞を所望とする骨系細胞に分化させることができるのを
見い出してこの発明を完成させた。Means for Solving the Problems In order to solve the above problems, the inventors studied a method for differentiating bone marrow cells into desired bone cells in a liquid medium containing neither serum nor hormone. When a complex in which calcium phosphate powder particles having chemical activity are dispersed in a collagen gel is used as a support for bone marrow cells, calcium ions and phosphate ions are dissolved in the liquid medium from calcium phosphate. Found that they could differentiate bone marrow cells into desired bone cells without serum or hormones, and completed this invention.
【００１１】この発明は、コラーゲンゲル中に化学活性
を有するリン酸カルシウム粒子が分散された複合体を骨
髄細胞の支持体として用い、動物細胞培養用液体培地中
で前記骨髄細胞を所望とする骨系細胞に分化させる骨髄
細胞の培養方法を提供する。この発明は、コラーゲンゲ
ル中に化学活性を有するリン酸カルシウム粒子が分散さ
れた複合体からなるゲル状支持体、この支持体に支持さ
れた骨髄細胞、および、前記支持体に含有された動物細
胞培養用液体培地を備えた培養用混合物を提供する。The present invention uses a complex in which calcium phosphate particles having a chemical activity are dispersed in a collagen gel as a support for bone marrow cells, and the bone marrow cells in which the bone marrow cells are desired in a liquid medium for culturing animal cells. The present invention provides a method for culturing a bone marrow cell to be differentiated into the same. The present invention, collagen gate
Calcium phosphate particles is dispersed with a chemical activity in Le
The present invention provides a gel-like support comprising the complex obtained, a bone marrow cell supported on the support, and a culture mixture comprising a liquid medium for culturing animal cells contained in the support.
【００１２】この発明は、コラーゲンゲル中に化学活性
を有するリン酸カルシウム粒子が分散された複合体から
なるゲル状支持体、この支持体に支持され、骨髄細胞か
ら分化した骨系細胞、および、前記支持体に含有された
動物細胞培養用液体培地を有する、硬組織欠損部への移
植用材料を提供する。この発明に用いる液体培地は、動
物細胞の生存や増殖に必要なすべての必須栄養素、エネ
ルギー代謝・触媒作用に必須のビタミンやそのほかの微
量金属など、動物細胞の培養に必須の成分を含むもので
あれば特に限定はない。[0012] The present invention provides a gel support comprising a complex in which calcium phosphate particles having a chemical activity are dispersed in a collagen gel , a bone cell supported on the support and differentiated from bone marrow cells, Provided is a material for transplantation into a hard tissue defect having a liquid medium for culturing animal cells contained in a body. The liquid medium used in the present invention contains all the essential nutrients necessary for the survival and growth of animal cells, vitamins and other trace metals essential for energy metabolism and catalysis, and other components essential for culturing animal cells. There is no particular limitation if it exists.
【００１３】前記液体培地は、従来の骨髄細胞の培養に
必須成分であった血清およびホルモンを含む必要はない
ので、天然培地よりも、全部またはほぼ全部の成分が既
知の合成培地の方が好ましい。合成培地の中でも、動物
細胞の培養に通常使用されるα−ＭＥＭ（ALPAH-MINIMU
M ESSENTIAL MEDIUM）培地が好ましい。α−ＭＥＭ培地
は、従来は血清および／またはホルモンを必ず添加して
使用していたのに対し、この発明では血清もホルモンも
添加しない形で用いる。このようなことが可能になった
のは、骨髄細胞の支持体として後述する特定の複合体を
用いるからである。なお、液体培地は、通常、１０-4〜
１０-7重量％の濃度でペントシリン、ゲンタマイシン、
ファンギーソンなどの抗生物質を含むのが好ましい。前
記範囲を上回ると細胞萎縮を示すおそれがある。Since the liquid medium does not need to contain serum and hormones, which have been essential components in the conventional culture of bone marrow cells, a synthetic medium in which all or almost all components are known is more preferable than a natural medium. . Among the synthetic media, α-MEM (ALPAH-MINIMU) usually used for culturing animal cells is used.
MESSENTIAL MEDIUM) medium is preferred. Conventionally, the α-MEM medium has always been used without the addition of serum and / or hormone. In the present invention, the α-MEM medium is used without adding any serum or hormone. This is possible because a specific complex described later is used as a support for bone marrow cells. In addition, the liquid medium is usually 10 -4 ~
Pentocillin, gentamicin, at a concentration of 10 -7 % by weight
Preferably, it contains an antibiotic such as Funghison. If it exceeds the above range, cell atrophy may be exhibited.
【００１４】この発明に用いる液体培地には、必要に応
じて、アスコルビン酸を添加するのが好ましい。シグマ
・ケミカル・カンパニー（SIGMA CHEMICAL CO.、米国ミ
ズリー州セントルイス）から市販されているα−ＭＥＭ
培地は同社の成分表によればＬ−アスコルビン酸を０．
０５０g/l 含んでいるが、更にアスコルビン酸を添加す
るのである。アスコルビン酸、特に、Ｌ−アスコルビン
酸を含む液体培地で動物細胞の培養を行うと、アスコル
ビン酸がコラーゲンの合成を活性化して細胞増殖を促進
する。アスコルビン酸は、０．００１〜０．００５重量
％、好ましくは０．００４〜０．００７重量％の濃度と
なるように添加される。ただし、ここでの数値範囲に
は、液体培地の成分として元々含まれていたアスコルビ
ン酸は含まれない。前記範囲を下回るとコラーゲンの合
成を活性化する働きが得られず、上回ると液体培地のｐ
Ｈに変動を起こし、細胞萎縮を示すおそれがある。It is preferable to add ascorbic acid to the liquid medium used in the present invention, if necessary. Α-MEM commercially available from SIGMA CHEMICAL CO., St. Louis, Missouri, USA
According to the company's composition table, the medium contains 0.1% L-ascorbic acid.
Although it contains 050 g / l, ascorbic acid is further added. When animal cells are cultured in a liquid medium containing ascorbic acid, particularly L-ascorbic acid, ascorbic acid activates collagen synthesis to promote cell growth. Ascorbic acid is added to a concentration of 0.001 to 0.005% by weight, preferably 0.004 to 0.007% by weight. However, ascorbic acid originally contained as a component of the liquid medium is not included in the numerical range here. When the amount is below the above range, the function of activating collagen synthesis cannot be obtained.
H may fluctuate and show cell atrophy.
【００１５】この発明に用いられる骨髄細胞は、たとえ
ば、動物の骨髄組織から無菌的に取り出した骨髄細胞；
動物の歯髄組織から無菌的に取り出した歯髄細胞；動物
から無菌的に取り出した各種の血球細胞などをコラーゲ
ンゲル上で初代培養してコラーゲンゲルに付着した細胞
を単離したものである。無菌的に取り出された細胞は、
細胞の生育に悪影響を与える夾雑物を除くために、好ま
しくは、上述の液体培地で洗浄され、ＥＧＴＡ処理（Ｃ
ａ2+を取り除き細胞同士の接着を剥がす薬品、たとえ
ば、ＥＧＴＡ（エチレングリコールビス（２−アミノエ
チルエーテル）四酢酸）を溶解した液体培地に３７℃で
短時間細胞を浸す処理）した後、遠心分離などの操作で
細胞成分が回収され、初代培養に供される。ここで初代
培養は、５％ＣＯ2 、３７℃のインキュベーター内でコ
ラーゲンゲル上で行われる。初代培養された細胞は、コ
ラーゲンを消化する酵素（たとえば、コラゲナーゼ）で
コラーゲンゲルを消化してコラーゲンゲルに付着してい
る細胞を単離して、この発明に用いられる。また、低温
保存や凍結保存された骨髄細胞も使用できる。The bone marrow cells used in the present invention include, for example, bone marrow cells aseptically removed from bone marrow tissues of animals;
Dental pulp cells aseptically removed from dental pulp tissue of animals; various blood cells aseptically removed from animals are primarily cultured on collagen gel to isolate cells attached to collagen gel. Aseptically removed cells
In order to remove contaminants that adversely affect cell growth, the cells are preferably washed with the above-mentioned liquid medium and treated with EGTA (C
a2 + is removed from the cell culture medium at 37 ° C. for a short time in a liquid medium containing EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid) dissolved therein to remove the adhesion between cells, eg, EGTA (ethylene glycol bis (2-aminoethyl ether) tetraacetic acid). Cell components are collected by an operation such as separation and used for primary culture. Here, the primary culture is performed on a collagen gel in an incubator at 37 ° C. with 5% CO 2 . The primary cultured cells are used in the present invention after the collagen gel is digested with an enzyme that digests collagen (eg, collagenase) to isolate the cells attached to the collagen gel. In addition, bone marrow cells stored at low temperatures or frozen can be used.
【００１６】この発明に用いる、骨髄細胞の支持体は、
コラーゲンと化学活性を有するリン酸カルシウムの複合
体である。この複合体は、コラーゲンゲル中にリン酸カ
ルシウム粉末粒子が分散してなる含水ゲルであり、良好
な生体親和性を有し、流動性を持たない。前記コラーゲ
ンとしては、酸可溶性コラーゲン、アルカリ可溶性コラ
ーゲン、酵素可溶化コラーゲンなどの可溶性コラーゲン
であって、Ｉ型コラーゲンが用いられる。Ｉ型コラーゲ
ンの中でも、アテロコラーゲンは、生体為害性の原因と
なる分子末端のテロペプタイドが酵素処理により一部ま
たは全部除去されているので生体に対して抗原性をほと
んどまたは全く持たないので好ましい。The support for bone marrow cells used in the present invention is:
It is a complex of collagen and calcium phosphate with chemical activity. This complex is a hydrogel formed by dispersing calcium phosphate powder particles in a collagen gel, has good biocompatibility, and has no fluidity. As the collagen, a soluble collagen such as an acid-soluble collagen, an alkali-soluble collagen, and an enzyme-solubilized collagen, and type I collagen is used. Among the type I collagens, atelocollagen is preferable because it has little or no antigenicity to the living body because telopeptides at molecular terminals that cause harm to the living body are partially or entirely removed by enzymatic treatment.
【００１７】前記リン酸カルシウムとしては、微細構
造、リン酸塩のプロトン化状態、または、水和の程度に
関わらず、Ｃａ2+とリン酸塩イオンから構成され、化学
活性を有する固体物質である。ここで、リン酸カルシウ
ムが化学活性を有するとは、生理条件下、母床の結晶界
面上でカルシウムイオンを解離、加水分解を経てカルシ
ウム欠損水酸アパタイトまたは炭酸アパタイトに結晶転
化する能力を持つということである。この発明に用い
る、化学活性を有するリン酸カルシウムとしては、非結
晶質リン酸カルシウム（以下では「ＡＣＰ」と言うこと
がある）、リン酸八カルシウム（以下では「ＯＣＰ」と
言うことがある）、リン酸四カルシウム（以下では「Ｔ
ｅＣＰ」と言うことがある）およびα−リン酸三カルシ
ウム（以下では「α−ＴＣＰ」と言うことがある）から
なる群から選ばれる少なくとも１つが、目的の骨系細胞
への分化を誘導するので、好ましい。この発明では、リ
ン酸カルシウムは、平均粒径が好ましくは５〜４０μ
ｍ、より好ましくは１０〜３２μｍの粉末または顆粒で
使用される。The calcium phosphate is a solid substance having chemical activity composed of Ca 2+ and phosphate ions regardless of the microstructure, the protonated state of the phosphate, or the degree of hydration. Here, that calcium phosphate has chemical activity means that it has the ability to dissociate calcium ions on the crystal interface of the mother bed under physiological conditions and crystallize to calcium-deficient hydroxyapatite or carbonate apatite through hydrolysis. is there. Examples of the chemically active calcium phosphate used in the present invention include amorphous calcium phosphate (hereinafter sometimes referred to as “ACP”), octacalcium phosphate (hereinafter sometimes referred to as “OCP”), and tetraphosphate. Calcium (hereinafter "T
At least one selected from the group consisting of eCP) and α-tricalcium phosphate (hereinafter sometimes referred to as “α-TCP”) induces differentiation into a target bone cell. Therefore, it is preferable. In the present invention, the calcium phosphate preferably has an average particle size of 5 to 40 μm.
m, more preferably in the form of powders or granules of 10 to 32 μm.
【００１８】前記複合体は、コラーゲン水溶液、濃縮さ
れた液体培地、緩衝液、化学活性を有するリン酸カルシ
ウム粉末を混合して、前記粉末を液中に懸濁させ、得ら
れた懸濁物を３６〜３７℃にしてコラーゲンをゲル化さ
せることにより得られる。得られた複合体では、コラー
ゲンゲル中にリン酸カルシウム粉末の粒子が分散してい
る。複合体を作るために、コラーゲン水溶液、濃縮され
た液体培地、緩衝液、リン酸カルシウム粉末はいちどに
混合されてもよいし、順次混合されてもよいし、２以上
を予め混合した後残りを混合してもよいし、特に混合方
法には限定はない。The complex is prepared by mixing an aqueous collagen solution, a concentrated liquid medium, a buffer, and a calcium phosphate powder having chemical activity, and suspending the powder in the solution. It is obtained by gelling collagen at 37 ° C. In the obtained composite, calcium phosphate powder particles are dispersed in the collagen gel. In order to form a complex, the aqueous collagen solution, the concentrated liquid medium, the buffer, and the calcium phosphate powder may be mixed at once, may be mixed sequentially, or may be pre-mixed with two or more and then mixed with the rest. The mixing method is not particularly limited.
【００１９】液体培地に緩衝液などの液が添加される場
合、液体培地と前記液との混合物が、液体培地の成分を
所定濃度で含むように、濃縮された形で液体培地が使用
される。コラーゲンは、前記懸濁物がゲル化しうるよう
な量、たとえば、全体量に対して５０〜９０％、好まし
くは７０〜８０％の割合（０．３重量％コラーゲン水溶
液に換算した容積割合）で使用される。When a liquid such as a buffer is added to the liquid medium, the liquid medium is used in a concentrated form so that a mixture of the liquid medium and the liquid contains the components of the liquid medium at a predetermined concentration. . Collagen is used in an amount such that the suspension can gel, for example, 50 to 90%, preferably 70 to 80% of the total amount (volume ratio in terms of 0.3% by weight collagen aqueous solution). used.
【００２０】前記緩衝液は、前記複合体に緩衝能を付与
するために添加される。これは、複合体において、リン
酸カルシウムが溶解してｐＨ値を大きく変動するのを防
ぐためである。緩衝液としては、たとえば、ＨＥＰＥＳ
緩衝液などのｐＨ７〜８付近の緩衝液が使用される。緩
衝液の添加量は、混合物がｐＨ７〜８付近において緩衝
能を有するような量で適宜添加され、特に制限はない。The buffer is added to impart a buffering capacity to the complex. This is to prevent calcium phosphate from dissolving and greatly changing the pH value in the complex. As a buffer, for example, HEPES
A buffer such as a buffer having a pH of about 7 to 8 is used. The amount of the buffer solution to be added is appropriately determined so that the mixture has a buffering capacity around pH 7 to 8, and is not particularly limited.
【００２１】化学活性を有するリン酸カルシウム粉末
は、液体培地にリン酸イオンを溶出して、リン酸カルシ
ウムの結晶成長を助け、リン酸八カルシウム、カルシウ
ム欠損ハイドロキシアパタイト、炭酸アパタイトなどの
生成を促進する。リン酸イオンの量が少なすぎるとリン
酸カルシウムの結晶成長が遅延するおそれがあり、リン
酸イオンの量が過剰だとリン酸塩の無機的析出を起こ
し、細胞によるリン酸イオンの石灰化を阻害するおそれ
があるので、化学活性を有するリン酸カルシウム粉末の
量は、通常、コラーゲン水溶液（０．３重量％換算）１
mlあたり１．０〜５．０mg／mlである。Calcium phosphate powder having chemical activity elutes phosphate ions into the liquid medium, helps crystal growth of calcium phosphate, and promotes the formation of octacalcium phosphate, calcium-deficient hydroxyapatite, carbonate apatite and the like. If the amount of phosphate ions is too small, the crystal growth of calcium phosphate may be delayed, and if the amount of phosphate ions is excessive, inorganic precipitation of phosphate will occur, inhibiting the calcification of phosphate ions by cells. Since there is a possibility that the calcium phosphate powder having the chemical activity may be used in an amount of usually 1% aqueous collagen solution (0.3% by weight).
It is 1.0 to 5.0 mg / ml per ml.
【００２２】骨髄細胞は、前記複合体の表面に通常の手
法により播種されたり、あるいは、前記複合体中に通常
の手法（たとえば、複合体の表面に播種された後、その
上に複合体を形成して播種された細胞を覆う方法、ある
いは、複合体を作るための懸濁液に細胞も懸濁させてコ
ラーゲンをゲル化させる方法）により包埋されたりす
る。すなわち、この発明の培養方法は、骨髄細胞を二次
元的に増殖させたり、あるいは、三次元的に増殖させた
りすることができる。The bone marrow cells are seeded on the surface of the complex by a usual method, or the bone marrow cells are seeded on the complex by a normal method (for example, after seeding on the surface of the complex, the complex is placed thereon). (The method of covering the cells formed and seeded, or the method of gelling collagen by suspending the cells in a suspension for forming a complex). That is, the culture method of the present invention allows the bone marrow cells to grow two-dimensionally or three-dimensionally.
【００２３】骨髄細胞を分化させるための培養は、前記
複合体を支持体として用い、前記液体培地、好ましくは
α−ＭＥＭ培地にＬ−アスコルビン酸と上記抗生物質が
添加されたもの、中において、たとえば、温度３６．５
〜３７．０℃、ＣＯ2 濃度２．０〜５．０容積％の培養
用器具（ＣＯ2 インキュベーター）内で行われる。培地
交換は１〜２日ごとに１回の割合で行われる。Culture for differentiation of bone marrow cells is performed by using the above-mentioned complex as a support and in the above-mentioned liquid medium, preferably an α-MEM medium to which L-ascorbic acid and the above antibiotics are added. For example, a temperature of 36.5
３７37.0 ° C., in a culture instrument (CO 2 incubator) having a CO 2 concentration of 2.0 to 5.0% by volume. The medium exchange is performed once every one to two days.
【００２４】この発明の培養方法により骨髄細胞から分
化される骨系細胞は、たとえば、骨芽細胞、破骨細胞、
軟骨芽細胞から選ばれるいずれか１種または２種以上の
共存物である。分化制御は、たとえば、次のようにして
行う。骨髄細胞を骨芽細胞に分化させるためには、ペプ
シン可溶化Ｉ型コラーゲンとＴｅＣＰとの複合体を支持
体として用いる。ＴｅＣＰは、Ｉ型コラーゲン水溶液
（０．３重量％換算）１mlに対して１．０〜５．０mg、
好ましくは２．０〜３．０mgの割合で混合される。培養
日数は、初代培養日数も含めて１０〜１５日である。The bone cells differentiated from bone marrow cells by the culture method of the present invention include, for example, osteoblasts, osteoclasts,
Any one or more selected from chondroblasts. The differentiation is controlled, for example, as follows. To differentiate bone marrow cells into osteoblasts, a complex of pepsin-solubilized type I collagen and TeCP is used as a support. TeCP is used in an amount of 1.0 to 5.0 mg per 1 ml of an aqueous solution of type I collagen (converted to 0.3% by weight),
Preferably, they are mixed at a ratio of 2.0 to 3.0 mg. The culture days are 10 to 15 days including the primary culture days.
【００２５】骨髄細胞を破骨細胞に分化させるために
は、ペプシン可溶化Ｉ型コラーゲンと、ＯＣＰおよび／
またはＡＣＰ（ＯＣＰ：ＡＣＰ＝０：１００〜１００：
０重量比）との複合体を支持体として用いる。ＯＣＰお
よび／またはＡＣＰは、Ｉ型コラーゲン水溶液（０．３
重量％換算）１mlに対して１．０〜５．０mg、好ましく
は１．５〜２．５mgの割合で混合される。培養日数は、
初代培養日数も含めて１５〜２５日である。To differentiate bone marrow cells into osteoclasts, pepsin-solubilized type I collagen, OCP and / or
Or ACP (OCP: ACP = 0: 100 to 100:
0 weight ratio) is used as a support. OCP and / or ACP are prepared from an aqueous type I collagen solution (0.3
It is mixed at a ratio of 1.0 to 5.0 mg, preferably 1.5 to 2.5 mg, per 1 ml of the mixture (% by weight). Culture days
It is 15 to 25 days including the number of days of primary culture.
【００２６】骨髄細胞を骨芽細胞と破骨細胞とに共存分
化させるためには、ペプシン可溶化Ｉ型コラーゲンとα
−ＴＣＰとの複合体を支持体として用いる。α−ＴＣＰ
は、Ｉ型コラーゲン水溶液（０．３重量％換算）１mlに
対して１．０〜５．０mg、好ましくは１．５〜３．０mg
の割合で混合される。培養日数は、初代培養日数も含め
て１２〜２２日である。In order to co-differentate bone marrow cells into osteoblasts and osteoclasts, pepsin-solubilized type I collagen and α
-A complex with TCP is used as a support. α-TCP
Is 1.0 to 5.0 mg, preferably 1.5 to 3.0 mg, per 1 ml of type I collagen aqueous solution (0.3% by weight).
Are mixed at the ratio of The culture days are 12 to 22 days including the primary culture days.
【００２７】骨髄細胞を軟骨芽細胞に分化させるために
は、酸可溶化Ｉ型コラーゲンとα−ＴＣＰとの複合体を
支持体として用いる。α−ＴＣＰは、Ｉ型コラーゲン水
溶液（０．３重量％換算）１mlに対して１．０〜５．０
mg、好ましくは１．５〜３．０mgの割合で混合される。
培養日数は、初代培養日数も含めて１２〜２２日であ
る。In order to differentiate bone marrow cells into chondroblasts, a complex of acid-solubilized type I collagen and α-TCP is used as a support. α-TCP is used in an amount of 1.0 to 5.0 per 1 ml of an aqueous solution of type I collagen (converted to 0.3% by weight).
mg, preferably 1.5 to 3.0 mg.
The culture days are 12 to 22 days including the primary culture days.
【００２８】この発明の培養方法は、経時的に細胞を位
相差顕微鏡で観察し、細胞の分化を形態学的に追跡した
り、あるいは、分化機構を追求したりすることができる
有効な手法である。オステオカルシンの検出などの公知
の手法により骨芽細胞が識別される。酒石酸耐性酸性フ
ォスファターゼ染色などの公知の手法により破骨細胞が
識別される。トルイジンブルー染色などの公知の手法に
より軟骨芽細胞が識別される。この発明は、また、無血
清培地で培養を行っているため、培養上澄みからの骨分
化因子を容易に同定できる。The culturing method of the present invention is an effective technique that allows the cells to be observed with a phase contrast microscope over time to follow the differentiation of the cells morphologically, or to pursue the differentiation mechanism. is there. Osteoblasts are identified by known techniques such as the detection of osteocalcin. Osteoclasts are identified by known techniques such as tartaric acid-resistant acid phosphatase staining. Chondroblasts are identified by a known technique such as toluidine blue staining. In the present invention, since the culture is performed in a serum-free medium, the bone differentiation factor from the culture supernatant can be easily identified.
【００２９】上記の培養方法により、骨髄細胞から分化
した骨系細胞がコラーゲンと化学活性を有するリン酸カ
ルシウムとの複合体に支持された移植用材料が得られ
る。得られた移植用材料は、骨欠損部の修復材、歯根膜
細胞複合化インプラント材などのハイブリッド型の修復
材として、骨折、先天的な骨の欠陥、外科的に生じた骨
の欠損などの骨欠損部、補遺を必要とする骨の構造およ
び歯周の欠陥（たとえば、歯根膜細胞の欠損）などの生
体硬組織の欠損部などに対して、整形外科や歯科などの
医師に良く知られた標準的な外科的手術（たとえば、骨
髄または骨片だけを供給する際に用いられる方法であ
る）を用いて移植される。移植に先立って、組織適合性
をチェックする必要があることは従来の移植用材料と同
様である。According to the above-mentioned culture method, a transplant material in which bone cells differentiated from bone marrow cells are supported by a complex of collagen and calcium phosphate having chemical activity is obtained. The obtained implant material is used as a repair material for bone defects, as a hybrid repair material such as a periodontal ligament cell composite implant material, and is used for fractures, congenital bone defects, and surgically generated bone defects. It is well known to physicians such as orthopedics and dentists for defects in living tissue such as bone defects, bone structures requiring supplements, and periodontal defects (eg, periodontal ligament cell defects). It is implanted using standard surgical procedures (eg, the method used in supplying only bone marrow or bone fragments). Prior to implantation, the need to check for histocompatibility is similar to conventional implant materials.
【００３０】この発明の培養方法により得られた上記移
植用材料は、培養に用いた液体培地を含んだゲルの状態
で生体に移植される。The transplant material obtained by the culturing method of the present invention is transplanted into a living body in the form of a gel containing the liquid medium used for culturing.
【００３１】[0031]
【作用】この発明の骨髄細胞の培養方法は、細胞の支持
体としてコラーゲンゲル中に化学活性を有するリン酸カ
ルシウム粒子が分散された複合体を用いて行うので、無
血清かつ無ホルモンの液体培地で骨髄細胞を培養するこ
とができ、しかも、この培養により骨髄細胞が骨系細胞
へ分化し、あるいは、分化が促進される。コラーゲンと
前記リン酸カルシウムは、リン酸イオンの存在下でゲル
化反応と結晶転化反応により化学的に複合化され、細胞
の分化・増殖、および、骨様組成物の合成が可能とな
る。リン酸イオンは、たとえば、リン酸カルシウムから
溶出したものである。培養条件を適宜変更することによ
り、骨髄細胞が所望の骨系細胞に分化するように制御す
ることができる。In the method for culturing bone marrow cells of the present invention, a complex in which calcium phosphate particles having chemical activity are dispersed in a collagen gel is used as a cell support. The cells can be cultured, and the culture differentiates bone marrow cells into bone cells or promotes differentiation. Collagen and the calcium phosphate are chemically complexed by a gelling reaction and a crystal inversion reaction in the presence of phosphate ions, thereby enabling cell differentiation / proliferation and synthesis of a bone-like composition. The phosphate ions are eluted from, for example, calcium phosphate. By appropriately changing the culture conditions, it is possible to control the bone marrow cells to differentiate into desired bone cells.
【００３２】この発明の培養用混合物は、コラーゲンゲ
ル中に化学活性を有するリン酸カルシウム粒子が分散さ
れた複合体からなる支持体、この支持体に支持された骨
髄細胞、および、リン酸イオンを含む動物細胞培養用液
体培地を備えているので、無血清かつ無ホルモン下で骨
髄細胞（硬組織細胞）を体外で培養するのに適した培養
基材に骨髄細胞が支持されたものとなっている。The culture mixture of the present invention, collagen gate
Calcium phosphate particles is dispersed with a chemical activity in Le
A bone marrow cell (hard tissue) under serum-free and hormonal-free conditions, comprising a support composed of the obtained complex, bone marrow cells supported by this support, and a liquid medium for culturing animal cells containing phosphate ions. Bone marrow cells are supported on a culture substrate suitable for culturing cells outside the body.
【００３３】前記コラーゲンとしてＩ型コラーゲンを用
いることにより、骨、腱、象牙質に認められるコラーゲ
ンと一致するため移植部位への置換性に優れるという利
点がある。前記リン酸カルシウムが、ＡＣＰ、ＯＣＰ、
ＴｅＣＰおよびα−ＴＣＰからなる群から選ばれる少な
くとも１つであると、未分化な細胞を賦活化するエネル
ギーに富むという利点がある。The use of type I collagen as the collagen has the advantage of being superior to the collagen found in bones, tendons and dentin, and thus being excellent in the ability to replace the transplant site. The calcium phosphate is ACP, OCP,
At least one selected from the group consisting of TeCP and α-TCP has an advantage of being rich in energy for activating undifferentiated cells.
【００３４】前記液体培地が合成培地であれば、成分が
分かっているので、無血清かつ無ホルモンとすることが
容易である。前記合成培地がα−ＭＥＭ培地であると、
市販品であるため入手しやすく、この知見を基礎に応用
研究分野へも活用できるという利点がある。前記液体培
地がアスコルビン酸を含んでいると、細胞のタンパク質
合成が促進される。If the liquid medium is a synthetic medium, the components are known, so that it is easy to make serum-free and hormone-free. When the synthetic medium is an α-MEM medium,
Since it is a commercial product, it is easy to obtain and has the advantage that it can be used in applied research fields based on this knowledge. When the liquid medium contains ascorbic acid, protein synthesis of cells is promoted.
【００３５】この発明の移植用材料は、コラーゲンゲル
中に化学活性を有するリン酸カルシウム粒子が分散され
た複合体からなる支持体、および、この支持体に支持さ
れ、骨髄細胞から分化した骨系細胞を有するので、硬組
織に近似した骨様組成物となっており、移植しようとす
る部位に適した骨系細胞が固定されていて流動しないよ
うになっている。このため、この移植用材料を移植する
と、その部位から骨系細胞が流れ去らずに増殖し、良好
な生体硬組織修復能を示すと期待される。The transplant material of the present invention is a collagen gel.
Chemically active calcium phosphate particles are dispersed inside
And a bone-like composition that is differentiated from bone marrow cells and is supported by this support, resulting in a bone-like composition similar to hard tissue, suitable for the site to be transplanted. Bone cells are fixed and do not flow. For this reason, when this transplant material is transplanted, it is expected that the bone cells proliferate without flowing away from the site, and exhibit a good ability to repair living hard tissue.
【００３６】[0036]
【実施例】以下に、この発明の実施例と、この発明の範
囲を外れた比較例とを示すが、この発明は下記実施例に
限定されない。 （実施例１）冷却下で、セルマトリックスタイプＩ−Ｐ
（新田ゼラチン株式会社製のペプシン可溶化Ｉ型コラー
ゲンの０．３重量％水溶液）、５倍濃度のα−ＭＥＭ培
地、および、ＨＥＰＥＳ緩衝液を８：１：１の重量比で
混合し、この混合物を直径３５mmのプラスチックプレー
トに入れた。このプレートを３７℃で１時間放置してコ
ラーゲンゲルシート（厚み２．０〜３．０mm）を作っ
た。EXAMPLES Examples of the present invention and comparative examples outside the scope of the present invention will be shown below, but the present invention is not limited to the following examples. (Example 1) Cell matrix type IP under cooling
(0.3% by weight aqueous solution of pepsin-solubilized type I collagen manufactured by Nitta Gelatin Co., Ltd.) A 5-fold concentration of α-MEM medium and HEPES buffer were mixed at a weight ratio of 8: 1: 1, This mixture was placed in a 35 mm diameter plastic plate. The plate was left at 37 ° C. for 1 hour to produce a collagen gel sheet (2.0 to 3.0 mm thick).
【００３７】５〜６週令のウィスター系雄ラットの骨髄
中から無菌的に摘出された骨髄細胞をコラーゲンゲルシ
ートの上に播種した。播種された細胞を、α−ＭＥＭ培
地（シグマ・ケミカル・カンパニー（SIGMA CHEMICAL C
O.）、米国ミズリー州セントルイス）にＬ−アスコルビ
ン酸（濃度１０ｍＭ、ただし、α−ＭＥＭ培地に元々含
まれているＬ−アスコルビン酸の濃度はこの数値に含ま
れない）、ペントシリン１mg／ml、ゲンタマイシン０．
５mg／ml、ファンギーソン３．０μｇ／mlを添加した液
体培地中で、５％ＣＯ2 、３７℃に調整したＣＯ2 イン
キュベーター内で７日間初代培養を行った。培地交換は
２日に１度の間隔で行った。Bone marrow cells aseptically removed from the bone marrow of 5-6 week old male Wistar rats were seeded on a collagen gel sheet. The seeded cells were placed in an α-MEM medium (SIGMA CHEMICAL C
O.), L-ascorbic acid (concentration 10 mM, but the concentration of L-ascorbic acid originally contained in the α-MEM medium is not included in these figures), pentocillin 1 mg / ml, Gentamicin 0.
Primary culture was performed for 7 days in a CO 2 incubator adjusted to 5% CO 2 and 37 ° C. in a liquid medium supplemented with 5 mg / ml and 3.0 μg / ml of fungison. The medium was changed once every two days.
【００３８】初代培養後、プレート内の浮遊細胞を捨て
た後、コラーゲンゲルに付着した細胞を０．１重量％コ
ラゲナーゼによるコラーゲンの消化（３７℃、１５分
間）で浮遊させ、この消化液中に初代培養細胞を１×１
０4 cells/mlの量で含む細胞懸濁液を得た。冷却下で、
セルマトリックスタイプＩ−Ｐ（新田ゼラチン株式会社
製のペプシン可溶化Ｉ型コラーゲンの０．３重量％水溶
液）、５倍濃度のα−ＭＥＭ培地、および、ＨＥＰＥＳ
緩衝液を８：１：１の体積比で混合し、この混合物にＬ
−アスコルビン酸（濃度１０ｍＭ、ただし、α−ＭＥＭ
培地に元々含まれているＬ−アスコルビン酸の濃度はこ
の数値に含まれない）を混合し、リン酸四カルシウム粉
末粒子（粒径３２μｍ以下）を２．０mg／mlの割合で懸
濁させた懸濁液を直径３５mmのプラスチックプレートに
入れた。このプレートを３７℃で１時間放置して、リン
酸四カルシウム粉末粒子が２．０mg／mlの量で分散され
たコラーゲンゲルシート（厚み２．０〜４．０mm）を作
った。After the primary culture, the floating cells in the plate are discarded, and the cells attached to the collagen gel are suspended by digestion of collagen with 0.1% by weight collagenase (37 ° C., 15 minutes). 1x1 primary cells
0 to obtain a cell suspension in an amount of 4 cells / ml. Under cooling,
Cell matrix type IP (0.3% by weight aqueous solution of pepsin-solubilized type I collagen manufactured by Nitta Gelatin Co., Ltd.), 5-fold concentration α-MEM medium, and HEPES
The buffer was mixed at a volume ratio of 8: 1: 1 and the mixture was added to L
-Ascorbic acid (concentration 10 mM, except for α-MEM
(The concentration of L-ascorbic acid originally contained in the medium is not included in this value), and tetracalcium phosphate powder particles (particle diameter: 32 μm or less) were suspended at a rate of 2.0 mg / ml. The suspension was placed in a 35 mm diameter plastic plate. The plate was left at 37 ° C. for 1 hour to prepare a collagen gel sheet (2.0 to 4.0 mm in thickness) in which tetracalcium phosphate powder particles were dispersed in an amount of 2.0 mg / ml.
【００３９】上記細胞懸濁液をプレートに入れて初代培
養細胞をコラーゲンゲル上に播種した（細胞密度１×１
０4 cells/ml）。初代培養と同じ液体培地を使用し、３
６．５〜３７．０℃、２．０〜３．０容積％のＣＯ2 下
で継代培養した（骨芽細胞に分化させるときは、培養器
内の酸素分圧をやや高く調節する）。培地は２日ごとに
交換した。The above cell suspension was placed on a plate, and primary culture cells were seeded on a collagen gel (cell density: 1 × 1).
0 4 cells / ml). Use the same liquid medium as the primary culture,
The cells were subcultured at 6.5 to 37.0 ° C. under 2.0 to 3.0% by volume of CO 2 (when differentiated into osteoblasts, the oxygen partial pressure in the incubator is adjusted slightly higher). . The medium was changed every two days.
【００４０】継代培養開始後３〜８日目（初代培養開始
後１０〜１５日目）、分化した細胞の確認をするため
に、細胞の一部を取って、ALPase染色およびオステオカ
ルシンの免疫染色を行った。リン酸カルシウム粒子を中
心に線維芽細胞様細胞の重層化したコロニーが認めら
れ、コロニーを形成する細胞表面には、石灰化物の結晶
が見られた。石灰化物を有する細胞は、ALPase陽性を示
し、オステオカルシンの局在を認めた。これらの結果か
ら、実施例１の継代培養細胞は骨芽細胞であることが確
認された。On the 3rd to 8th days after the start of the subculture (10 to 15 days after the start of the primary culture), in order to confirm the differentiated cells, a part of the cells was taken, and ALPase staining and osteocalcin immunostaining were performed. Was done. A stratified colony of fibroblast-like cells centered on calcium phosphate particles was observed, and calcified crystals were observed on the cell surface forming the colony. Cells having calcifications showed ALPase positivity and localization of osteocalcin was recognized. From these results, it was confirmed that the subcultured cells of Example 1 were osteoblasts.
【００４１】（実施例２）実施例１において、細胞懸濁
液の細胞の量を５×１０4 cells/mlに変えたこと、リン
酸四カルシウム粉末粒子の代わりにリン酸八カルシウム
粉末粒子（粒径３２μｍ以下）を用いたこと、継代培養
時のＣＯ2 濃度を５．０容積％に変えたこと以外は実施
例１と同様にして骨髄細胞を培養した。Example 2 In Example 1, the amount of cells in the cell suspension was changed to 5 × 10 4 cells / ml, and instead of tetracalcium phosphate powder particles, octacalcium phosphate powder particles ( Bone marrow cells were cultured in the same manner as in Example 1 except that the particle size was 32 μm or less, and that the CO 2 concentration during subculture was changed to 5.0% by volume.
【００４２】継代培養開始後１３〜１８日目（初代培養
開始後２０〜２５日目）、分化した細胞の確認をするた
めに、細胞の一部を取って、酒石酸耐性酸性フォスファ
ターゼ染色を施した。リン酸カルシウム粒子を中心に形
成された線維芽細胞様細胞のコロニーが多数認められ
た。リン酸カルシウム粒子周囲の細胞は多核巨細胞であ
り、酒石酸耐性酸性フォスファターゼ染色で陽性に染ま
る細胞であった。これらの結果から、リン酸カルシウム
粒子周囲の細胞は、破骨細胞に分化していることが確認
された。On the 13th to 18th days after the start of the subculture (20 to 25 days after the start of the primary culture), in order to confirm the differentiated cells, a part of the cells was removed and stained with tartaric acid-resistant acid phosphatase. did. Many colonies of fibroblast-like cells formed around calcium phosphate particles were observed. The cells around the calcium phosphate particles were multinucleated giant cells, which stained positively by tartaric acid-resistant acid phosphatase staining. From these results, it was confirmed that the cells around the calcium phosphate particles were differentiated into osteoclasts.
【００４３】（実施例３）実施例２において、リン酸八
カルシウム粉末粒子の代わりに非晶質リン酸カルシウム
粉末粒子（粒径３２μｍ以下）を用いたこと以外は実施
例２と同様にして骨髄細胞を培養した。継代培養開始後
１３〜１８日目（初代培養開始後２０〜２５日目）、分
化した細胞の確認をするために、細胞の一部を取って、
酒石酸耐性酸性フォスファターゼ染色を施した。リン酸
カルシウム粒子を中心に形成された線維芽細胞様細胞の
コロニーが多数認められた。リン酸カルシウム粒子周囲
の細胞は多核巨細胞であり、酒石酸耐性酸性フォスファ
ターゼ染色で陽性に染まる細胞であった。これらの結果
から、リン酸カルシウム粒子周囲の細胞は、破骨細胞に
分化していることが確認された。Example 3 Bone marrow cells were prepared in the same manner as in Example 2 except that amorphous calcium phosphate powder particles (having a particle size of 32 μm or less) were used instead of octacalcium phosphate powder particles. Cultured. On the 13th to 18th days after the start of the subculture (20 to 25 days after the start of the primary culture), in order to confirm the differentiated cells, a part of the cells was taken.
Tartaric acid resistant acid phosphatase staining was performed. Many colonies of fibroblast-like cells formed around calcium phosphate particles were observed. The cells around the calcium phosphate particles were multinucleated giant cells, which stained positively by tartaric acid-resistant acid phosphatase staining. From these results, it was confirmed that the cells around the calcium phosphate particles were differentiated into osteoclasts.
【００４４】（実施例４）実施例２において、リン酸八
カルシウム粉末粒子半分の量を非晶質リン酸カルシウム
粉末粒子（粒径３２μｍ以下）に置き換えたこと以外は
実施例２と同様にして骨髄細胞を培養した。継代培養開
始後１３〜１８日目（初代培養開始後２０〜２５日
目）、分化した細胞の確認をするために、細胞の一部を
取って、酒石酸耐性酸性フォスファターゼ染色を施し
た。リン酸カルシウム粒子を中心に形成された線維芽細
胞様細胞のコロニーが多数認められた。リン酸カルシウ
ム粒子周囲の細胞は多核巨細胞であり、酒石酸耐性酸性
フォスファターゼ染色で陽性に染まる細胞であった。こ
れらの結果から、リン酸カルシウム粒子周囲の細胞は、
破骨細胞に分化していることが確認された。Example 4 Bone marrow cells were prepared in the same manner as in Example 2, except that half of the octacalcium phosphate powder particles were replaced with amorphous calcium phosphate powder particles (having a particle size of 32 μm or less). Was cultured. 13 to 18 days after the start of the subculture (20 to 25 days after the start of the primary culture), in order to confirm the differentiated cells, a part of the cells was taken and stained with tartaric acid-resistant acid phosphatase. Many colonies of fibroblast-like cells formed around calcium phosphate particles were observed. The cells around the calcium phosphate particles were multinucleated giant cells, which stained positively by tartaric acid-resistant acid phosphatase staining. From these results, cells around the calcium phosphate particles
It was confirmed that the cells had differentiated into osteoclasts.
【００４５】（実施例５）実施例１において、細胞懸濁
液の細胞の量を３×１０4 cells/mlに変えたこと、リン
酸四カルシウム粉末粒子の代わりにα−リン酸三カルシ
ウム粉末粒子（粒径３２μｍ以下）を用いたこと、継代
培養時のＣＯ2 濃度を５．０容積％に変えたこと以外は
実施例１と同様にして骨髄細胞を培養した。Example 5 In Example 1, the amount of cells in the cell suspension was changed to 3 × 10 4 cells / ml, and α-tricalcium phosphate powder was used instead of tetracalcium phosphate powder particles. Bone marrow cells were cultured in the same manner as in Example 1 except that particles (having a particle size of 32 μm or less) were used and the CO 2 concentration during subculture was changed to 5.0% by volume.
【００４６】継代培養開始後８〜１８日目（初代培養開
始後１５〜２５日目）、分化した細胞の確認をするため
に、細胞の一部を取って、ALPase染色およびオステオカ
ルシンの免疫染色を施し、また、酒石酸耐性酸性フォス
ファターゼ染色を施した。リン酸カルシウム粒子を中心
に形成された線維芽細胞様細胞のコロニーが多数認めら
れた。コロニー辺縁部の細胞には、石灰化物の結晶が見
られた。石灰化物の結晶を含む細胞は、ALPase陽性を示
し、オステオカルシンの局在も認められた。リン酸カル
シウム粒子周囲の細胞は多核巨細胞であり、酒石酸耐性
酸性フォスファターゼ染色で陽性に染まる細胞であっ
た。これらの結果から、コロニー辺縁部の細胞は骨芽細
胞であり、リン酸カルシウム粒子周囲の細胞は、破骨細
胞に分化していることが確認された。すなわち、実施例
４の場合、骨芽細胞および破骨細胞の共存分化を誘導す
ることが示された。8 to 18 days after the start of the subculture (15 to 25 days after the start of the primary culture), in order to confirm the differentiated cells, a part of the cells was taken, and ALPase staining and immunostaining of osteocalcin were performed. And tartaric acid resistant acid phosphatase staining. Many colonies of fibroblast-like cells formed around calcium phosphate particles were observed. Calcified crystals were found in the cells at the periphery of the colony. Cells containing calcified crystals were positive for ALPase, and localization of osteocalcin was also observed. The cells around the calcium phosphate particles were multinucleated giant cells, which stained positively by tartaric acid-resistant acid phosphatase staining. From these results, it was confirmed that the cells at the periphery of the colony were osteoblasts, and the cells around the calcium phosphate particles were differentiated into osteoclasts. That is, in the case of Example 4, it was shown that co-differentiation of osteoblasts and osteoclasts was induced.
【００４７】（実施例６）実施例１において、セルマト
リックスタイプＩ−Ｐの代わりにセルマトリックスタイ
プＩ−Ａ（新田ゼラチン株式会社製の酸可溶化Ｉ型コラ
ーゲンの０．３重量％水溶液）を用いたこと、細胞懸濁
液の細胞の量を４×１０5 cells/mlに変えたこと、リン
酸四カルシウム粉末粒子の代わりにα−リン酸三カルシ
ウム粉末粒子（粒径３２μｍ以下）を用いたこと、継代
培養時のＣＯ2 濃度を５．０容積％に変えたこと以外は
実施例１と同様にして骨髄細胞を培養した。Example 6 In Example 1, cell matrix type IA (0.3% by weight aqueous solution of acid-solubilized type I collagen manufactured by Nitta Gelatin Co., Ltd.) was used instead of cell matrix type IP. Was used, the amount of cells in the cell suspension was changed to 4 × 10 5 cells / ml, and α-tricalcium phosphate powder particles (having a particle size of 32 μm or less) were used instead of tetracalcium phosphate powder particles. Bone marrow cells were cultured in the same manner as in Example 1 except that they were used and that the CO 2 concentration during subculture was changed to 5.0% by volume.
【００４８】継代培養開始後５〜１５日目（初代培養開
始後１２〜２２日目）、分化した細胞の確認をするため
に、細胞の一部を取って、トルイジンブルー染色を施し
て、メタクロマジーの観察を行った。リン酸カルシウム
粒子を中心の細胞のコロニーが認められ、コロニーを形
成する細胞表面には、石灰化物の結晶が見られた。石灰
化物の結晶を有する細胞は、メタクロマジーを示してい
た。これらの結果から、分化した細胞は、軟骨芽細胞で
あることが確認された。On the 5th to 15th day after the start of the subculture (12 to 22 days after the start of the primary culture), in order to confirm the differentiated cells, a part of the cells was taken and subjected to toluidine blue staining. Observation of metachromism was performed. Colonies of cells centered on calcium phosphate particles were observed, and calcified crystals were observed on the cell surface forming the colonies. Cells with calcified crystals showed metachromism. From these results, it was confirmed that the differentiated cells were chondroblasts.
【００４９】（比較例１）アテロペプチドコラーゲンの
生理食塩水溶液（濃度６５mg／ml）４．８ｃｃ、およ
び、β−リン酸三カルシウムとハイドロキシアパタイト
の混合物（７０重量％の多孔性、密度０．６ｇ／ml、
４．２ｃｃ）２．５ｇを良く混合した。この混合物に
１．４ｃｃの骨髄を加えて、７ｃｃの組成物を得た。こ
の組成物は、骨髄１６重量％、コラーゲン３重量％、生
理食塩水５２重量％、β−リン酸三カルシウムとハイド
ロキシアパタイトの混合物２９重量％であった。Comparative Example 1 4.8 cc of a solution of atelopeptide collagen in physiological saline (concentration 65 mg / ml) and a mixture of β-tricalcium phosphate and hydroxyapatite (porosity 70% by weight, density 0.6 g) / Ml,
(4.2 cc) 2.5 g was mixed well. 1.4 cc of bone marrow was added to this mixture to obtain a 7 cc composition. This composition was 16% by weight of bone marrow, 3% by weight of collagen, 52% by weight of physiological saline, and 29% by weight of a mixture of β-tricalcium phosphate and hydroxyapatite.
【００５０】実施例１において、骨髄細胞が播種された
コラーゲンシートの代わりに上記のようにして作られた
組成物を用いたこと以外は実施例１と同様にして骨髄細
胞を培養した。その結果、１週間後、浮遊してきた細胞
だけでなくコラーゲンに付着していた細胞も死んでい
た。The bone marrow cells were cultured in the same manner as in Example 1 except that the composition prepared as described above was used instead of the collagen sheet seeded with bone marrow cells. As a result, one week later, not only the floating cells but also the cells attached to the collagen died.
【００５１】[0051]
【発明の効果】この発明の骨髄細胞の培養方法によれ
ば、体外で（in vitro）、未分化な骨髄細胞を移植部位
に適応した所望の骨系細胞に分化させることができる。
この発明の培養用混合物は、骨髄細胞を上記この発明の
培養方法で培養しやすい形態に整えたものであり、体外
で骨髄細胞の分化を行うのに好適である。According to the method for culturing bone marrow cells of the present invention, undifferentiated bone marrow cells can be differentiated into desired bone cells adapted to the transplantation site in vitro (in vitro).
The culture mixture of the present invention is obtained by preparing bone marrow cells in a form that can be easily cultured by the above-described culture method of the present invention, and is suitable for extracellular differentiation of bone marrow cells.
【００５２】この発明の移植用材料は、骨形成に関与す
る細胞を有する骨様組成物であり、この発明の培養方法
により、体外で安定かつ多量に得られる。骨欠損部など
の硬組織欠損部を修復するために、この発明の移植用材
料を用いれば、移植部位に適した骨系細胞を固定した状
態で移植することができ、取り扱いやすく、修復を効果
的に行うことができる。The material for transplantation of the present invention is a bone-like composition having cells involved in bone formation, and can be obtained in a stable and large amount outside the body by the culture method of the present invention. By using the transplantation material of the present invention to repair a hard tissue defect such as a bone defect, it is possible to transplant the bone tissue in a state where bone cells suitable for the transplantation site are fixed, easy to handle, and effective in repairing Can be done
───────────────────────────────────────────────────── フロントページの続き (72)発明者 萬代 佳宣 大阪府八尾市二俣２丁目22番地 新田ゼ ラチン株式会社大阪工場内 (72)発明者 永冨 功治 大阪府八尾市二俣２丁目22番地 新田ゼ ラチン株式会社大阪工場内 (56)参考文献 特開 平６−343456（ＪＰ，Ａ) 特表 平４−505717（ＪＰ，Ａ) 特公 平２−33388（ＪＰ，Ｂ２) ──────────────────────────────────────────────────続 き Continuing on the front page (72) Yoshinobu Bandai 2--22 Futama, Yao-shi, Osaka Nitta Zelatin Co., Ltd. Osaka Plant (72) Koji Nagatomi 2--22, Futama, Yao-shi, Osaka Nitta (56) References JP-A-6-343456 (JP, A) JP-T4-505717 (JP, A) JP-T2-33388 (JP, B2)
Claims (15)
ン酸カルシウム粒子が分散された複合体を骨髄細胞の支
持体として用い、動物細胞培養用液体培地中で前記骨髄
細胞を所望とする骨系細胞に分化させる骨髄細胞の培養
方法。1. A complex in which calcium phosphate particles having chemical activity are dispersed in a collagen gel is used as a support for bone marrow cells, and the bone marrow cells are differentiated into desired bone cells in a liquid medium for animal cell culture. A method for culturing bone marrow cells to be cultured.
求項１記載の培養方法。2. The culture method according to claim 1, wherein ascorbic acid is added to the liquid medium.
軟骨芽細胞からなる群から選ばれる少なくとも１つの骨
系細胞に分化させる請求項１または２記載の培養方法。3. The culture method according to claim 1, wherein the bone marrow cells are differentiated into at least one bone cell selected from the group consisting of osteoblasts, osteoclasts, and chondroblasts.
とリン酸四カルシウムとの複合体を用い、液体培地とし
てα−ＭＥＭ培地を用いて骨髄細胞を培養し、骨芽細胞
に分化させる請求項３記載の培養方法。4. The method according to claim 1, wherein a bone marrow cell is cultured by using a complex of enzyme-solubilized type I collagen and tetracalcium phosphate as a support, and using α-MEM medium as a liquid medium to differentiate into osteoblasts. 3. The culture method according to 3.
ンとリン酸八カルシウムおよび／または非結晶質リン酸
カルシウムとの複合体を用い、液体培地としてα−ＭＥ
Ｍ培地を用いて骨髄細胞を培養し、破骨細胞に分化させ
る請求項３記載の培養方法。5. A support comprising a complex of enzyme-solubilized type I collagen and octacalcium phosphate and / or amorphous calcium phosphate, and α-ME as a liquid medium.
The culture method according to claim 3, wherein the bone marrow cells are cultured using the M medium to differentiate into osteoclasts.
とα−リン酸三カルシウムとの複合体を用い、液体培地
としてα−ＭＥＭ培地を用いて骨髄細胞を培養し、骨芽
細胞と破骨細胞に分化させる請求項３記載の培養方法。6. A bone marrow cell is cultured by using a complex of enzyme-solubilized type I collagen and α-tricalcium phosphate as a support, and using an α-MEM medium as a liquid medium. The culture method according to claim 3, wherein the cell is differentiated.
α−リン酸三カルシウムとの複合体を用い、液体培地と
してα−ＭＥＭ培地を用いて骨髄細胞を培養し、軟骨芽
細胞に分化させる請求項３記載の培養方法。7. A bone marrow cell is cultured by using a complex of acid-solubilized type I collagen and α-tricalcium phosphate as a support and using α-MEM medium as a liquid medium to differentiate into chondroblasts. The culture method according to claim 3.
ン酸カルシウム粒子が分散された複合体からなるゲル状
支持体、この支持体に支持された骨髄細胞、および、前
記支持体に含有された動物細胞培養用液体培地を備えた
培養用混合物。8. A gel support comprising a complex in which calcium phosphate particles having chemical activity are dispersed in a collagen gel, bone marrow cells supported by the support, and animal cell culture contained in the support A culture mixture with a liquid medium for culture.
項８記載の培養用混合物。9. The culture mixture according to claim 8, wherein the collagen is type I collagen.
カルシウム、リン酸八カルシウム、リン酸四カルシウム
およびα−リン酸三カルシウムからなる群から選ばれる
少なくとも１つである請求項８または９記載の培養用混
合物。10. The culture mixture according to claim 8 or 9, wherein the calcium phosphate is at least one selected from the group consisting of amorphous calcium phosphate, octacalcium phosphate, tetracalcium phosphate, and α-tricalcium phosphate. .
ら１０までのいずれかに記載の培養用混合物。11. The culture mixture according to claim 8, wherein the liquid medium is a synthetic medium.
項１１記載の培養用混合物。12. The culture mixture according to claim 11, wherein the synthetic medium is an α-MEM medium.
求項８から１２までのいずれかに記載の培養用混合物。13. The culture mixture according to claim 8, wherein the liquid medium also contains ascorbic acid.
リン酸カルシウム粒子が分散された複合体からなるゲル
状支持体、この支持体に支持され、骨髄細胞から分化し
た骨系細胞、および、前記支持体に含有された動物細胞
培養用液体培地を有する、硬組織欠損部への移植用材
料。14. A gel support comprising a complex in which calcium phosphate particles having a chemical activity are dispersed in a collagen gel , a bone cell supported by the support and differentiated from bone marrow cells, and A material for transplantation into a hard tissue defect having a contained liquid culture medium for animal cell culture.
び軟骨芽細胞からなる群から選ばれる少なくとも１つで
ある請求項１４記載の移植用材料。15. The transplant material according to claim 14, wherein the osteoblast is at least one selected from the group consisting of osteoblasts, osteoclasts, and chondroblasts.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5351035A JP2984176B2 (en) | 1993-12-30 | 1993-12-30 | Method for culturing bone marrow cells, culture mixture and material for transplantation into hard tissue defect |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP5351035A JP2984176B2 (en) | 1993-12-30 | 1993-12-30 | Method for culturing bone marrow cells, culture mixture and material for transplantation into hard tissue defect |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH07194373A JPH07194373A (en) | 1995-08-01 |
| JP2984176B2 true JP2984176B2 (en) | 1999-11-29 |
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ID=18414605
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|---|---|---|---|
| JP5351035A Expired - Fee Related JP2984176B2 (en) | 1993-12-30 | 1993-12-30 | Method for culturing bone marrow cells, culture mixture and material for transplantation into hard tissue defect |
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| Country | Link |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE112008001609T5 (en) | 2007-06-20 | 2010-09-09 | Hoya Corporation | Repair and treatment of bone defects using drug-induced cells made from chondrocytes capable of hypertrophication and a scaffold |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2198978A1 (en) | 1996-03-01 | 1997-09-01 | Clemens Antoni Van Blitterswijk | Method for the in vitro production of bone |
| WO2002017983A1 (en) * | 2000-08-28 | 2002-03-07 | Osteogenesis Co., Ltd. | 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 |
| JP2002233567A (en) * | 2000-12-06 | 2002-08-20 | Mitsuo Ochi | Tissue equivalent for transplantation and method for producing the same |
| KR100751690B1 (en) * | 2005-06-13 | 2007-08-23 | 세원셀론텍(주) | Bone-producing composition using a mixture of osteoblasts and biological matrix components and a method for producing the same |
| JP2016158827A (en) * | 2015-02-27 | 2016-09-05 | 国立大学法人東北大学 | Bone regeneration material |
| KR102105549B1 (en) * | 2019-07-25 | 2020-04-29 | 주식회사 메디팹 | A manufacturing method for calcium phosphate hydrogel composition containing a constant content of calcium phosphate |
| WO2021015562A1 (en) | 2019-07-25 | 2021-01-28 | 주식회사 메디팹 | Osteoporosis model comprising calcium phosphate hydrogel composition and use thereof |
| JPWO2024189842A1 (en) * | 2023-03-15 | 2024-09-19 |
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1993
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE112008001609T5 (en) | 2007-06-20 | 2010-09-09 | Hoya Corporation | Repair and treatment of bone defects using drug-induced cells made from chondrocytes capable of hypertrophication and a scaffold |
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| Publication number | Publication date |
|---|---|
| JPH07194373A (en) | 1995-08-01 |
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