JP4756816B2 - Method for manufacturing bone regenerative prosthesis - Google Patents
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Description
【０００１】
【産業上の利用分野】
本発明は、骨再生補綴物の製造方法に関する。
【０００２】
【従来の技術】
1. 背景
大きな骨欠損を修復する方法として，以前は金属やセラミックスなどの材料で補強，または補填する方法が用いられていた。
【特許文献１】
例えば特開昭５６−１８８６４号公報の様な、表面にリン酸カルシウム被覆層を有するチタン、金などの金属インプラントが示される。
しかしこの方法には，自家骨が傷つく，炎症が起こる，若年者には用いられない，などの問題があった。そこで近年，骨髄又は骨膜由来の間葉系幹細胞を生体親和性の高い材料に播種し，欠損部に移植することで，骨の新生を促す治療法が用いられている。
【特許文献２】
特開昭58-58041号公報には、連続気孔を有するリン酸カルシウム化合物からなる海綿状多孔体にコラーゲンを含浸させた骨充填材が開示されているが、全てが異物であり十分な補綴能力を発揮するものには至っていない。
この治療法において，細胞を播種する足場である担体に求められる性質は以下の3点である。１．細胞を保持できる，２．新生骨に置換される，３．内部の細胞が壊死しない。ところが、新生骨への置換が進みにくい、内部の細胞が壊死を起こすなどの問題がある。血管が導入されにくいのもこの方法の問題点である。
【特許文献３】
特開平10-108575号公報
この文献には、球形の多孔質セラミックス粒子の製造方法が記載されており、当該粒子を用いた骨充填材が示されている。ビーズは生体親和性・細胞適合性に優れ，表面積が大きいため，より速やかに新生骨に置換されると考えられる。
しかし、顆粒状のビーズでは、補綴物としての、形態を形成するには、更に、型枠を用いる必要があり、取り扱いには、煩雑さが残るものである。移植部位からの脱けだすことも多い。
【０００３】
【本発明が解決しようとする課題】
目的
本発明は，内部まで細胞が存在し，壊死を起こさない細胞担体の開発を目的とし、細胞部位に効率良く栄養素が行き渡るような構造を検討した上で、その構造が応用性を持つための方法を検討した。
【０００４】
【課題を解決するための手段】
本発明者らは、鋭意研究の結果、多孔質セラミックス粒子（ビーズの一形態）を一層敷き詰めた状態で骨髄由来細胞を播種すると，細胞外マトリックスの接着力でシート状の組織である担体（ビーズシート）（図1）が形成可能であることを知見し、本発明に到達したものである。図１（a）は,実際のビーズシートをピンセットＰで持ち上げた図であり、図１（b）は,ビーズシートＢを側面から模式的にとらえた図である。図１（c）は、ビーズシートＢをピンセットで持ち上げたところを写真で示した図１（a）を線図により模式的に示した図である。図１で示すビーズシートＢの厚みＡは、おおよそ500μmであるが、一例であり、それ以上の厚さ又はそれ以下の厚さであっても良い場合もある。例えば、ビーズを細胞が完全に覆ってしまった状態で、厚みがあるシートを形成しても良い場合もある。
【０００５】
更に本発明者らは、担体内部の細胞の壊死を防ぐには，栄養の分散が必要であり，それを実現するうえでビーズシートのシート形状は有用である。これを用いて，ある程度の大きさを持ちながら，内部まで細胞が存在し壊死しない担体として「骨再生ユニット」を作製する。
即ち、細胞とビーズ状、粉末状、ブロック状、多孔質状、表面に凹凸を有する形態の連結用部材とを結合し、シート状の補綴物用、細胞培養用担体を得ると共に、新たな細胞培養方法を提案するに至った。表面に凹凸を有する形態とは、例えば、多孔質までとはいわないが、１乃至複数の窪みをもつ形態が例示される。多孔質状とは、少なくとも一つの孔を持つものを含む場合もあり、少なくとも細胞が、連結用部材の表面に付着的な状態を有する連結部材であれば、いかなるものでも良く、その形状も限られるものではない。
当該連結用部材は、多孔質粒子、少なくとも表面に凹凸のある粒子、ガラス粒子、生理活性物質、有機化合物質、無機化合物質から選ばれた１乃至複数のもので形成されればよく、単独の他、例えば、無機化合物質と有機化合物質の複合部材により形成されても良い。
【０００６】
本発明は、連結用部材を細胞によって連結する構成を一例として取り得るものであって、本発明における連結用部材としては、例えば無機化学物質の一つであるセラミックスが適当である。又当該連結用部材の比重は、おおよそ３程度が好ましい。又当該連結用部材としてビーズ状のものを選択した場合は、その粒径は１００〜５００μmが保形性等の点で好ましい場合もある。
その他有機化学物質であるポリ乳酸、ポリグリコール酸などの高分子樹脂、PRPのような生理活性物質、絹のような動物由来繊維等でも可能な場合もあり、特にこれら無機乃至有機物質に限定されない。
【０００７】
本発明におけるセラミックスは、生体親和性を有するものであれば良く、例えば、アルミナ、ジルコニア、リン酸カルシウム系化合物が示され、その中でも、リン酸カルシウム系セラミックスが生体親和性において、優れている点で、好ましく、より具体的には、ハイドロキシアパタイト、α−リン酸三カルシウム、β−リン酸三カルシウムが例示され、その中でも、難溶解性を有するβ−リン酸三カルシウムが細胞を培養する基材として長時間使用するためには優れている。有機化合物素材としては水酸基、カルボキシル基、アミノ基などの官能基がある樹脂素材が適当であるが、プラズマ処理などにより、カルボキシル基などの前記官能基を導入することができるため特に限定されない。生理活性物質としてはPRPなどの成長因子を含んだもの、コラーゲンのような生体素材、ガラスのような無機物でも細胞接着性のすぐれているものが適当である。
当該プラズマ処理によれば、非生分解性ポリマーによるシート部材を生分解性ポリマーによる部材で挟み込み、更に連結部材と、細胞よりなるビーズシートで挟み込み結合させるような、多層担体を形成する際有効である。
【０００８】
細胞を結合するための素材の大きさは、１00 〜500μｍが好ましく、空隙率は、10 〜40 ％が細胞との結合速度、等の点から好ましい。その製法は、特に限定されないが、例えばセラミックスの場合は、特開昭６２−３６０８３号公報に記載された手法や、特開平10-108575号公報に開示された手法が用いられる。
本発明で示される細胞は、骨髄由来細胞を含む幹細胞、ES細胞、さらに成熟期細胞としては骨芽細胞、破骨細胞、骨細胞（成長因子を含む場合もある）肝細胞、繊維芽細胞等の動物細胞、植物細胞等が例示されるが、これらに限るものではない。
これらの細胞は、生体に補綴する場合は、自家細胞が好ましいが、これに限らず、少なくとも、拒絶反応の程度が影響のないものであれば、良く、これら細胞は、１種類ではなく多数の種類を複合的に用いたものであっても良い場合もある。
本発明は、厚さをビーズ状、粒子状またはブロック状、表面に凹凸を有する形態、多孔質状の直径または高さとしたシート材、もしくは繊維状のものの繊維質の幅のシートを形成できるほか、当該シートの製法によれば、そのまま３次元細胞培養すらも可能とするのであり、細胞培養方法としても有用である。尚、連結用部材中に、血管誘導因子等を含浸させたものであっても良い。
【０００９】
培養方法例としては、次の行程が示される。
１．ビーズ（多孔質粒子）を敷き詰める。
その場合、粒子が隣接する程度、好ましくは〜200μｍの間隔に敷き詰められればよい。
２．次に目的とする細胞を播種する。
この播種は、例えば、敷き詰められたビーズに一様に接触する程度に行われる。
３．その状態で、その他培養するための成分培地、成長因子、血清などを添加し、温度36〜37℃で、時間24〜72時間培養する。
【００１０】
本発明では、細胞とセラミックス粒子を結合させたシート状にしたものにより、容積の大きな補綴物、を形成可能とするが、その積層する枚数としては、例えば、3〜6枚が好ましいがこれに限るものではない。
本発明では、複数の積層構成を形成する際は、例えばポリ乳酸等の高分子樹脂よりなるシート（厚み0.001〜10mm）を介在させて積層させる。
高分子シートを介して、当該ビーズシートを積層させる場合、分散させたビーズ上に細胞を播種した状態に高分子シートを乗せその上に上述した３つの工程に基づいてビーズと細胞の配列を形成する。所望の積層数までこれを繰り返した後、培養成分の添加により、温度37℃、時間0.001〜1000時間培養してもよい。好ましくは1日〜1週間が適当である。
本発明は、更に、当該担体内部に血管を導入する構成をとり得る。例えば、 b-FGF:basic fibroblast growth factor、a-FGF:acidic fibroblast growth factor、EGF、VEGF、IGF、TGF-β、Angiopoietin-1,2等の血管誘導因子を添加することや、血管内皮細胞など血管細胞を播種することで、担体内部に、セラミックス粒子の空隙を利用して導入することを可能とし、より実用化された生体移植用担体の製造を可能とする。
【００１１】
本発明は、複数のシートを積層する際、生理活性物質、生分解性素材、有機化合物、繊維状素材、細胞、細胞成長因子を含む機能層を設けることで、血管を誘導する誘導性により補綴後の生体結合性が高い補綴物或いは、細胞培養担体が得られるが、その際、機能層を設けた補綴物の製造方法の一例を示す。
最初、連結用部材と細胞の組み合わせよりなるシートを上述の製法に従って複数枚製造する。これら複数枚のシートを重ねた状態で、コラーゲン、 ポリ乳酸等の機能用材料を含む溶液に浸す。
すると、溶液中で個々のシートは、その縁部が湾曲しているため、シート間に隙間が生じているので、このシート間の間隙にコラーゲンが入り込み、機能性部材を介した担体、試験片が形成されるのである。
この製法によれば、特に機能層を重ねるための種々の工程（例えば、積層と乾燥を繰り返す工程）の必要が無く、簡単に積層部材を形成することができる。機能層は、その他の手法で介在させても良い場合もある。
又、当該溶液に、血管誘導因子等を含ませることで、容易に複合的な機能層を形成させることができる。
機能層には、細胞の為の栄養成分を含ませるほか、後述する成長因子等を含ませたものであってもよい。
更に本発明では、上述した血管内皮細胞等の血管誘導能を有する因子等を機能層に含ませることで、担体内部に血管が入り込み、細胞が壊死せず、新生骨への置換を促進させるなど生体組織として事実上同化した状態を形成することができる。
【００１２】
本発明の製造方法の一例を以下に示す
ビーズの作製
ビーズは、特開平10-108575号公報に記載された手法に基づき、β―リン酸三カルシウムを原料としたスラリーを液体窒素に滴下後、これを乾燥、焼成して多孔質状の粒子を得た。
（1）ビーズシートの作製方法
ビーズシートを利用するために，シートが形成される条件を明らかにし，安定した作製を可能にする作製方法を確立する必要があった。そこで，2cm×2cmの面積を持つウェルディッシュにビーズを敷き詰め，そこにそれぞれ１０ 6 ,１０ 7 ,１０ 8 の細胞を播種し，シート化するかを調べた。また直径1cmの円形のウェルでも同様の実験を行った。
【００１３】
（2）骨細胞への分化
ビーズシートは骨髄由来細胞を用いて作製するが，これらの細胞の中にはまだどの組織の系統にも分化していない未分化の間葉系幹細胞が含まれていると考えられる。適当な成長因子を付加することで，この未分化の細胞に骨系の分化を促すような環境を与え，ビーズシートの変化を調べた。加えた成長因子を以下に示す。
100nM dexamethasone
0．05mM ascorbate 2-hosphate
10mM β-glycerophosphate
【００１４】
（3）「骨再生ユニット」の作製・移植
ビーズシート（厚さビーズの粒径前後）とPLLA不織布（厚さ１cm）を交互に積層・接着し，培養した。PLLA不織布は生分解性材料であり，これをビーズシート間に挟むことで，栄養が運搬される隙間を持たせることができる。さらに生体内では，この隙間に血管の新生が期待できる。この点を検証するために，作製した骨再生ユニットをヌードマウス皮下に移植・回収し，染色によってその結果を調べた。
【００１５】
結果と考察
（1）ビーズシートの作製方法
形成条件を調べた結果，2cm×2cmのウェルに対して１０ 6 の細胞を播種するのが適当であると分かった。また使用するビーズの径は150〜460μm，容器はチャンバースライドＣＤ（図2），培養日数は３日が適しているとわかり，この条件でビーズシートを作製した。チャンバースライドＣＤを用いて，シートＢを空気中に出さずにシャーレＳＲに移動する作製方法を確立し，ビーズシートＢの安定した作製が実現された。
【００１６】
（2）骨細胞への分化
成長因子を加えたことでビーズシートの強度は明らかに向上した。これをアリザリンレッド染色し，カルシウムＣを検出した（図3）。図３（a）は、写真図であり、図３（b）は、（a）の模式図である。これらの結果から，シート上の細胞が骨系に分化している可能性は高い。三週間の培養の結果では、骨分化の証拠であるアルカリフォスファターゼ染色で染色される事実を確認し、骨細胞への分化が行われていることを確認した。
【００１７】
（3）骨再生ユニット
染色の結果，骨再生ユニット中では移植から3週間の後でも内部まで細胞が生存していることが確認された（図４）。 図４（a）は、写真図であり、図4（b）は、（a）の模式図である。マウス皮下より回収したユニットの表面の様子から，材料の工夫などによって血管侵入する担体の作製は十分実現可能である。
【００１８】
【発明の効果】
結論
以上のように，連結部材と細胞からなるシートの作製方法を確立し，内部まで細胞が壊死しない担体の開発には成功した。これは血管侵入を前提とした担体という，新たな概念を導くものであり、長期間の使用に耐え得る補綴物、細胞培養基材及び簡易で、しかも効率的な３次元の細胞培養を実現する。
【図面の簡単な説明】
【図１】本発明の一実施例を示す図である
【図２】図１で示した実施例を説明するための図である
【図３】図１で示した実施例を説明するための図である
【図４】図１で示した実施例を説明するための図である[0001]
[Industrial application fields]
The present invention relates to a method for manufacturing a bone regeneration prosthesis .
[0002]
[Prior art]
1. Background As a method for repairing large bone defects, a method of reinforcing or supplementing with a material such as metal or ceramics has been used.
[Patent Document 1]
For example, metal implants such as titanium and gold having a calcium phosphate coating layer on the surface as disclosed in JP-A-56-18864 are shown.
However, this method has problems such as injury to the autologous bone, inflammation, and inability to be used by young people. Therefore, in recent years, a treatment method that promotes bone renewal by seeding bone marrow or periosteum-derived mesenchymal stem cells on a material having high biocompatibility and transplanting the material into a defect part has been used.
[Patent Document 2]
Japanese Laid-Open Patent Publication No. 58-58041 discloses a bone filling material in which a sponge-like porous body made of a calcium phosphate compound having continuous pores is impregnated with collagen. It hasn't come to what it does.
In this treatment method, the following three points are required for a carrier that is a scaffold for seeding cells. 1. Cells can be maintained, 2. It is replaced with new bone, 3. The cells inside do not necrotize. However, there are problems such as replacement of new bone is difficult to proceed, and internal cells cause necrosis. Another problem with this method is that it is difficult to introduce blood vessels.
[Patent Document 3]
In this document, a method for producing spherical porous ceramic particles is described, and a bone filler using the particles is shown. Because the beads are excellent in biocompatibility and cytocompatibility and have a large surface area, they are considered to be replaced with new bone more rapidly.
However, in the case of granular beads, in order to form a form as a prosthesis, it is necessary to further use a formwork, and handling remains complicated. There are many cases of taking off from the transplant site.
[0003]
[Problems to be solved by the present invention]
the purpose
The present invention aims at development of a cell carrier in which cells exist to the inside and does not cause necrosis, and after examining a structure in which nutrients are efficiently distributed to a cell site, a method for making the structure applicable It was investigated.
[0004]
[Means for Solving the Problems]
As a result of intensive studies, the present inventors have found that when bone marrow-derived cells are seeded in a state in which porous ceramic particles (one form of beads) are further spread, a carrier (beads) that is a sheet-like tissue by the adhesion of the extracellular matrix Sheets (FIG. 1) were found to be formed, and the present invention was achieved. FIG. 1A is a diagram in which an actual bead sheet is lifted by tweezers P, and FIG. 1B is a diagram in which the bead sheet B is schematically captured from the side. FIG. 1 (c) is a diagram schematically showing FIG. 1 (a), which is a photograph showing the bead sheet B lifted with tweezers. The thickness A of the bead sheet B shown in FIG. 1 is approximately 500 μm, but is only an example, and may have a thickness greater than or equal to that. For example, a thick sheet may be formed in a state where the cells are completely covered with the beads.
[0005]
Furthermore, the present inventors need nutrient dispersion to prevent cell necrosis inside the carrier, and the sheet shape of the bead sheet is useful for realizing it. Using this, a “bone regeneration unit” is produced as a carrier that has a certain size but does not have necrosis due to cells inside.
In other words, cells and beads, powders, blocks, porouss, and connecting members in the form of irregularities on the surface are combined to obtain a sheet-like prosthesis and cell culture carrier, and new cells It came to propose the culture method. The form having irregularities on the surface is, for example, not porous, but a form having one or more depressions is exemplified. The porous shape may include those having at least one hole, and any shape may be used as long as at least cells are attached to the surface of the connecting member. It is not something that can be done.
The connecting member may be formed of one or more selected from porous particles, at least uneven particles on the surface, glass particles, physiologically active substances, organic compound substances, and inorganic compound substances. In addition, for example, it may be formed of a composite member of inorganic compound and organic compound.
[0006]
In the present invention, a configuration in which the connecting members are connected by cells can be taken as an example, and as the connecting member in the present invention, for example, ceramic which is one of inorganic chemical substances is suitable. The specific gravity of the connecting member is preferably about 3. Further, when a bead-shaped member is selected as the connecting member, the particle size may be preferably 100 to 500 μm from the viewpoint of shape retention.
Other organic chemical substances such as polylactic acid and polyglycolic acid, bioactive substances such as PRP, animal-derived fibers such as silk may be possible, and are not particularly limited to these inorganic or organic substances. .
[0007]
The ceramic according to the present invention may be any material as long as it has biocompatibility. For example, alumina, zirconia, and calcium phosphate compounds are shown, and among them, calcium phosphate ceramics are preferable in terms of biocompatibility. More specifically, hydroxyapatite, α-tricalcium phosphate, and β-tricalcium phosphate are exemplified. Among them, β-tricalcium phosphate having poor solubility is used as a base material for culturing cells for a long time. Excellent for use. A resin material having a functional group such as a hydroxyl group, a carboxyl group, or an amino group is suitable as the organic compound material, but is not particularly limited because the functional group such as a carboxyl group can be introduced by plasma treatment or the like. Suitable physiologically active substances include growth factors such as PRP, biological materials such as collagen, and inorganic substances such as glass that have excellent cell adhesion.
The plasma treatment is effective for forming a multilayer carrier in which a sheet member made of a non-biodegradable polymer is sandwiched between members made of a biodegradable polymer, and is further sandwiched and joined by a connecting member and a bead sheet made of cells. is there.
[0008]
The size of the material for binding the cells is preferably 100 to 500 μm, and the porosity is preferably 10 to 40% from the viewpoint of the binding speed with the cells. The production method is not particularly limited. For example, in the case of ceramics, the method described in JP-A-62-36083 and the method disclosed in JP-A-10-108575 are used.
The cells shown in the present invention include stem cells and bone marrow-derived cells, ES cells, and mature cells such as osteoblasts, osteoclasts, bone cells (which may contain growth factors) hepatocytes, fibroblasts, etc. However, the present invention is not limited to these.
These cells are preferably autologous cells when prosthetic in a living body. However, the cells are not limited to this, as long as at least the degree of rejection is not affected. In some cases, a combination of types may be used.
The present invention can form a sheet material having a thickness of beads, particles or blocks, a surface with irregularities on the surface, a porous diameter or height, or a fibrous sheet having a fiber width. According to the method for producing the sheet, even three-dimensional cell culture can be performed as it is, which is also useful as a cell culture method. The connecting member may be impregnated with a blood vessel inducing factor or the like.
[0009]
As an example of the culture method, the following steps are shown.
1. Spread beads (porous particles).
In that case, the particles may be spread so as to be adjacent to each other, preferably at intervals of ˜200 μm.
2. Next, the target cells are seeded.
This sowing is performed, for example, to such an extent that the seeds are uniformly contacted.
3. In that state, other mediums for culturing, growth factors, serum and the like are added and cultured at a temperature of 36 to 37 ° C. for 24 to 72 hours.
[0010]
In the present invention, it is possible to form a prosthesis having a large volume by using a sheet in which cells and ceramic particles are combined. For example, 3 to 6 sheets are preferably stacked. It is not limited.
In the present invention, when a plurality of laminated structures are formed, they are laminated with a sheet (thickness 0.001 to 10 mm) made of a polymer resin such as polylactic acid interposed therebetween.
When laminating the bead sheets via a polymer sheet, the polymer sheet is placed in a state where cells are seeded on the dispersed beads, and an array of beads and cells is formed on the polymer sheet based on the above-described three steps. To do. After repeating this to the desired number of layers, the cells may be cultured at a temperature of 37 ° C. for 0.001-1000 hours by adding culture components. One day to one week is preferable.
The present invention can further be configured to introduce blood vessels inside the carrier. For example, b-FGF: basic fibroblast growth factor, a-FGF: acidic fibroblast growth factor, EGF, VEGF, IGF, TGF-β, Angiopoietin-1, 2, etc. By seeding vascular cells, it is possible to introduce the inside of the carrier using the voids of the ceramic particles, and it is possible to manufacture a carrier for living transplantation that has been put to practical use.
[0011]
The present invention provides a prosthesis that induces blood vessels by providing a functional layer containing a physiologically active substance, a biodegradable material, an organic compound, a fibrous material, a cell, and a cell growth factor when laminating a plurality of sheets. An example of a method for producing a prosthesis provided with a functional layer is shown below, although a later prosthesis or a cell culture carrier having high biobinding properties can be obtained.
First, a plurality of sheets made of a combination of a connecting member and cells are manufactured according to the above-described manufacturing method. In a state where these multiple sheets are stacked, they are immersed in a solution containing functional materials such as collagen and polylactic acid.
Then, since the edges of the individual sheets in the solution are curved, gaps are formed between the sheets, so that collagen enters the gaps between the sheets, and the carrier and test piece via the functional member. Is formed.
According to this manufacturing method, there is no need for various steps (for example, a step of repeating lamination and drying) for stacking the functional layers, and a laminated member can be easily formed. The functional layer may be interposed by other methods.
Moreover, a complex functional layer can be easily formed by including a blood vessel inducing factor or the like in the solution.
In addition to containing nutrients for cells, the functional layer may contain growth factors, which will be described later.
Furthermore, in the present invention, by including in the functional layer a factor having a blood vessel inducing ability such as the above-mentioned vascular endothelial cell, the blood vessel enters the carrier, the cell is not necrotized, and replacement with new bone is promoted. It is possible to form a virtually assimilated state as a living tissue.
[0012]
An example of the production method of the present invention is as follows. Preparation of beadsBeads, based on the technique described in JP-A-10-108575, after dropping a slurry of β-tricalcium phosphate as a raw material into liquid nitrogen, This was dried and fired to obtain porous particles.
(1) Production method of bead sheet In order to use a bead sheet, it was necessary to clarify the conditions under which the sheet is formed and to establish a production method that enables stable production. Therefore, beads were spread on a well dish having an area of 2 cm × 2 cm, and 10 6 , 10 7 , and 10 8 cells were seeded there to examine whether to form a sheet. The same experiment was performed on a circular well having a diameter of 1 cm.
[0013]
(2) Differentiation into bone cells Although bead sheets are prepared using bone marrow-derived cells, these cells contain undifferentiated mesenchymal stem cells that have not yet differentiated into any tissue lineage. It is thought that there is. By adding an appropriate growth factor, this undifferentiated cell was given an environment that promotes the differentiation of the bone system, and changes in the bead sheet were examined. The added growth factors are shown below.
100nM dexamethasone
0. 05mM ascorbate 2-hosphate
10 mM β-glycerophosphate
[0014]
(3) Production and transplantation of “bone regeneration unit” Bead sheets (thickness before and after the particle size of beads) and PLLA nonwoven fabric (thickness 1 cm) were alternately laminated and adhered, and cultured. PLLA nonwoven fabric is a biodegradable material that can be sandwiched between bead sheets to provide a gap for nutrients to be transported. Furthermore, in the living body, the formation of blood vessels can be expected in this gap. In order to verify this point, the prepared bone regeneration unit was transplanted and collected subcutaneously in nude mice, and the results were examined by staining.
[0015]
Results and Discussion (1) Preparation method of bead sheet As a result of examining the formation conditions, it was found that it is appropriate to seed 10 6 cells in a 2 cm × 2 cm well. Moreover, it was found that the diameter of beads to be used was 150 to 460 μm, the container was a chamber slide CD (FIG. 2), and the number of culture days was 3 days. Using the chamber slide CD, a production method for moving the sheet B to the petri dish SR without taking it into the air was established, and stable production of the bead sheet B was realized.
[0016]
(2) Differentiation into bone cells The strength of the bead sheet was clearly improved by adding growth factors. This was stained with alizarin red to detect calcium C (FIG. 3). FIG. 3 (a) is a photograph, and FIG. 3 (b) is a schematic diagram of (a). From these results, it is highly possible that the cells on the sheet have differentiated into the bone system. The results of three weeks of culture confirmed the fact that the cells were stained with alkaline phosphatase staining, which is evidence of bone differentiation, and confirmed that differentiation into bone cells was performed.
[0017]
(3) Bone regeneration unit As a result of staining, it was confirmed that cells survived to the inside even after 3 weeks from transplantation in the bone regeneration unit (Fig. 4). FIG. 4A is a photograph, and FIG. 4B is a schematic diagram of (a). From state of the surface of the unit recovered from mouse subcutaneous, preparation of the carrier which vascular invasion, such as by devising the material Ru sufficiently realizable der.
[0018]
【The invention's effect】
Conclusion As described above, we have established a method for producing a sheet consisting of a connecting member and cells, and have succeeded in developing a carrier that does not necrotize cells. This leads to a new concept of a carrier premised on blood vessel invasion, realizing a prosthesis that can withstand long-term use, a cell culture substrate, and a simple yet efficient three-dimensional cell culture. .
[Brief description of the drawings]
FIG. 1 is a diagram showing an embodiment of the present invention. FIG. 2 is a diagram for explaining the embodiment shown in FIG. 1. FIG. 3 is a diagram for explaining the embodiment shown in FIG. FIG. 4 is a diagram for explaining the embodiment shown in FIG.
Claims (7)
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| JP2003064063A JP4756816B2 (en) | 2003-03-10 | 2003-03-10 | Method for manufacturing bone regenerative prosthesis |
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| JP2003064063A JP4756816B2 (en) | 2003-03-10 | 2003-03-10 | Method for manufacturing bone regenerative prosthesis |
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| JP4756816B2 true JP4756816B2 (en) | 2011-08-24 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| GB2438565B (en) | 2005-03-30 | 2011-02-16 | Univ Nagoya Nat Univ Corp | Cell culture in spherical droplets |
| KR101744040B1 (en) | 2010-03-01 | 2017-06-07 | 후지필름 가부시키가이샤 | Cell structure comprising cells and macromolecular blocks having biocompatibility |
| JP5990298B2 (en) * | 2011-08-31 | 2016-09-14 | 富士フイルム株式会社 | Cell structure for cell transplantation and cell assembly for cell transplantation |
| JP5876787B2 (en) * | 2011-08-31 | 2016-03-02 | 富士フイルム株式会社 | Cell structure for cell transplantation and cell assembly for cell transplantation |
| JP2016136848A (en) * | 2015-01-26 | 2016-08-04 | 富士フイルム株式会社 | Chemical evaluating method and chemical screening method |
| JP6854904B2 (en) | 2017-08-30 | 2021-04-07 | 富士フイルム株式会社 | Angiogenic agents and their manufacturing methods |
| JP6941175B2 (en) * | 2017-08-30 | 2021-09-29 | 富士フイルム株式会社 | Device for cell transplantation and its manufacturing method |
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| JPH03266980A (en) * | 1989-03-16 | 1991-11-27 | W R Grace & Co | Base material for cell culture and production of cell aggregate using same |
| CA2018228C (en) * | 1989-06-05 | 1996-02-27 | Nancy L. Parenteau | Cell culture systems and media |
| JP3635316B2 (en) * | 1996-11-25 | 2005-04-06 | 株式会社アドバンス | Method for producing spherical ceramic porous body |
| JP3360810B2 (en) * | 1998-04-14 | 2003-01-07 | ペンタックス株式会社 | Method for producing bone replacement material |
| US7405078B2 (en) * | 2000-03-16 | 2008-07-29 | Cellseed Inc. | Bed material for cell culture, method for co-culture of cell and co-cultured cell sheet obtainable therefrom |
| DE60143892D1 (en) * | 2000-07-21 | 2011-03-03 | Cellseed Ind | HEART MUSCLES-SIMILAR CELL LAYER, THREE-DIMENSIONAL CONSTRUCT, HEART MUSCLE-SIMILAR TISSUE, AND METHOD OF MANUFACTURE |
| JP2003047461A (en) * | 2001-07-31 | 2003-02-18 | Astec:Kk | Method for carrying out high-density cell culture of cell by using apatite sheet, culturing device and cell culture module |
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