JP3646162B2 - 軟骨組織の再生用移植体 - Google Patents
軟骨組織の再生用移植体 Download PDFInfo
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- JP3646162B2 JP3646162B2 JP2001204013A JP2001204013A JP3646162B2 JP 3646162 B2 JP3646162 B2 JP 3646162B2 JP 2001204013 A JP2001204013 A JP 2001204013A JP 2001204013 A JP2001204013 A JP 2001204013A JP 3646162 B2 JP3646162 B2 JP 3646162B2
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- composite material
- cells
- porous
- chondrocytes
- sheet
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- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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Description
【産業上の利用分野】
本発明は、変形性関節症などの疾患や事故などの原因による軟骨創傷を修復するために用いられる軟骨組織の再生に関する。
【0002】
【従来の技術】
変形性関節症は整形外科分野において高頻度見られる疾患で、しばしば高度の機能障害をきたす。人工関節手術が外科的治療の主体を成すが、現在の金属や高分子ポリマーを材料とする人工関節部分は感染・磨耗・緩み・破損といった問題を有する。組織移植の場合では、ドナーの不足という問題に加え、ドナーが他人の場合、免疫応答に基づく拒絶反応という問題もある。このような種々の問題点の存在により、現在では、再生医工学的な手法による治療法は理想的であると考えれ、軟骨組織の再生に関する研究が盛んに行われている。再生医工学的な手法により軟骨組織を再生するためには、軟骨細胞あるいは軟骨細胞に分化する幹細胞が増殖するための足場として、また、形成している生体組織の支持体としての3次元的な多孔質性の担体材料が必要である。このような担体材料は多孔質性や生体親和性や生体吸収性などの条件が要求され、従来、ポリ乳酸(PLA)やポリグリコール酸(PGA)や乳酸とグリコール酸との共重合体(PLGA)のような生体吸収性合成高分子、あるいはコラーゲンなどの天然高分子で調製した3次元的な多孔質性担体材料がよく用いられている。
【0003】
しかしながら、上記の生体吸収性合成高分子からなるものは、機械強度に優れているものの、疎水性であり、またその大きい隙間、割れ目のため、大部分の細胞は隙間を通過してしまい、その上に載らず、軟骨細胞あるいは軟骨細胞に分化する幹細胞を播種することが極めて困難であり、このため、有効な細胞の播種率が得られず、これら細胞を大量に支持担体に集積することができないため、軟骨組織の再生効率が低く、実用上大きな障害となっていた。
一方、生体由来の天然高分子の多孔質性材料である例えばコラーゲンスポンジは、親水性で、細胞との相互作用が非常に優れており、細胞の播種の容易なものであるが、機械強度が低く、また柔らかくて捩れやすいので、臨床では取り扱いにくいという問題点を包含していた。
【0004】
【発明が解決しようとする課題】
本発明は、従来技術のこのような問題点を解決することを課題とするものである。 具体的には、良好な生体親和性を有し、軟骨細胞あるいは軟骨細胞に分化する幹細胞の播種が容易で、播種効率が良好であり、そのためこれら細胞を大量に支持担体に集積することが可能であって、軟骨組織の再生効率が良好であるとともに、機械的強度も高く、臨床においても取扱い易い、軟骨細胞あるいは軟骨細胞に分化する幹細胞の支持担体を提供することにあり、さらに、このような材料に軟骨細胞あるいは軟骨細胞に分化する幹細胞を含有せしめた、軟骨組織を再生するための生体内移植体を提供しようとするものである。
【0005】
【課題を解決するための手段】
本発明は、上記の課題を解決するためになされたものであり、以下(1)〜(4)からなるものである。
(1)生体吸収性合成高分子からなるメッシュ体あるいは多孔質体の内部構造マトリックス内に、さらに天然高分子からなる、連続する細孔を有する多孔質構造体が形成されている複合体材料からなるものであって、該天然高分子の多孔質構造体が、上記メッシュ体あるいは多孔質体に浸漬又は塗布された天然高分子水溶液を凍結乾燥することにより形成されたものであることを特徴とする、軟骨細胞あるいは軟骨細胞に分化する幹細胞の担持用担体。
(2)複合材体がシート状物である、(1)に記載の軟骨細胞あるいは軟骨細胞に分化する幹細胞の担持用担体。
(3)複合体材料が請求項2に記載のシート状物を積層またはロール状に巻いたものである、(1)に軟骨細胞あるいは軟骨組織に分化する幹細胞の担持用担体。
(4)請求項1〜3のいずれか1項記載の軟骨細胞あるいは軟骨細胞に分化する幹細胞の担持用担体に、軟骨細胞あるいは軟骨細胞に分化する幹細胞を担持した、軟骨組織再生に用いるための移植体。」
【0006】
以下、本発明をさらに詳述する。
本発明における軟骨細胞あるいは軟骨細胞に分化する幹細胞を担持する担体は、生体吸収性合成高分子のメッシュ体あるいは多孔質体における表面およびその内部構造マトリックス内にコラーゲン等の天然高分子からなる多孔質構造体をさらに形成した複合体材料により構成される。
【0007】
本発明に用いられる生体吸収性合成高分子のメッシュ体あるいは多孔質体は、主として本発明の複合体材料の機械的強度を増大させるために用いられ、メッシュ体は、織物、織布又は不織布等からなるものでよい。また、多孔質体は、発泡剤を利用する発泡成形法、あるいは多孔質化剤除去法等周知の方法により得ることができる。この多孔質体の発泡成型法においては、高分子化合物に発泡剤を添加し発泡剤を発泡させた後、上記高分子を硬化させる。高分子溶液中に、水溶性の糖類あるいは塩類を添加し、硬化後、該水溶性物質を水で洗浄除去すればよい。
メッシュのアミ目の大きさあるいは多孔質体の孔の大きさは大きくなればなるほど、機械的強度は低下するものの、メッシュ単位当たりの天然高分子多孔質構造体の細孔密度が高くなり、播種細胞はこの細孔に保持されるので、複合体における播種細胞数を増大でき、軟骨組織の再生が効率的になる。
したがって、そのメッシュのアミ目の大きさあるいは多孔質体の孔の大きさは、移植される生体内の場所等に応じて、求められる機械強度あるいは弾力性、あるいは軟骨組織の再生速度等を勘案して適宜定められる。
【0008】
メッシュ体あるいは多孔質体を形成する生体吸収性合成高分子としては、ポリ乳酸、ポリグリコール酸、乳酸とグリコール酸の共重合体、ポリリンゴ酸、ポリ−ε−カプロラクトンなどのポリエステル或いはセルロース、ポリアルギン酸などの多糖類等を挙げることができる。本発明において好ましく使用される生体吸収性合成高分子はポリ乳酸、ポリグリコール酸、乳酸とグリコール酸の共重合体である
【0009】
本発明の天然高分子多孔質構造体は、生体に由来するもので、生体親和性を示すものであれば、何れも使用できるが、コラーゲン、ゼラチン、フィブロネクチン、及びラミニンから選ばれた1種以上のもの、特にコラーゲンが好ましく使用される。コラーゲンにはI、II、III、IV型のものがあるが、本発明においてはこれらの何れも使用できる。
天然高分子多孔質構造体の細孔は、播種細胞の増殖及び組織再生の足場とするものであり、細孔は連続していることが好ましい。その大きさは1〜300μm、好ましくは20〜100μm程度とするのがよい。
また、本発明においては、厚みは、生体複合材料の使用態様によって適宜定めればよいが、通常0.1〜5mm、好ましくは0.1〜1mmである。その空隙率は、通常80%以上である。
【0010】
本発明の複合体材料は、生体吸収性合成高分子のメッシュ体あるいは多孔質体の内部構造マトリックス内、すなわちメッシュ体のアミ目あるいは多孔質体の孔内に、コラーゲン等の天然高分子からなる多孔質体をさらに形成したものであり、本発明の複合体材料は種々の方法により製造することができるが、例えば、前記生体吸収性合成高分子のメッシュ体あるいは多孔質体と天然高分子多孔質構造体とを架橋結合させることにより得ることができる。
この方法は、(1)生体吸収性合成高分子のメッシュ体あるいは多孔質体にコラーゲン等の天然高分子材料の溶液を付着、含浸せしめた後、(2)凍結乾燥し、次いで、(3)生成する生体複合材料をガス状の化学架橋剤で処理するものである。
上記工程(1)においては、前記生体吸収性合成高分子メッシュ体を前記生体由来の天然高分子水溶液で処理する。処理方法としては種々のものがあるが、浸漬法や塗布法が好ましく採用される。
浸漬法は、生体由来の天然高分子水溶液の濃度や粘度が低い場合に有効であり、具体的には、生体由来の天然高分子の低濃度水溶液に生体吸収性合成高分子メッシュ体を浸漬することにより行われる。
塗布法は、生体由来の天然高分子水溶液の濃度や粘度が高く、浸漬法が適用できないときに有効であり、具体的には、生体由来の天然高分子の高濃度水溶液を生体吸収性合成高分子メッシュ体に塗布することにより行われる。
【0011】
生体吸収性合成高分子メッシュ体あるいは多孔質体に天然高分子溶液が含浸、付着した複合物は、次いで(2)凍結乾燥に付される。
凍結乾燥は、上記複合物を凍結し、これを真空減圧下で凍結乾燥するものであるが、かかる工程により、生体由来の天然高分子が多孔質化され、生体吸収性合成高分子のメッシュ体あるいは多孔質体と天然高分子多孔質構造体との複合体材料が形成される。
【0012】
凍結乾燥の方法は、従来公知の方法がそのまま適用できる。凍結温度は、通常−20℃以下である。凍結乾燥圧力は、凍結された水が気体となる減圧条件を設定すればよく、通常、0.2Torr程度の減圧下に調製される。
凍結乾燥された複合体材料は、ついで(3)の架橋工程に付される。この工程は、複合生体材料を構成する生体由来の天然高分子多孔体をガス状の架橋剤により、架橋化し、天然高分子の多孔質構造体を固めると共に合成高分子メッシュ体との結合力を高め、所望とする架橋複合体材料の多孔質構造を安定するための十分な弾力と強度を与えるために必要なものである。
【0013】
一般に、架橋化方法としては、紫外線照射処理による光架橋や熱架橋などの物理的架橋法、溶液状の架橋化剤やガス状の架橋化剤を用いる化学架橋法などが知られているが、本発明においては、ガス状の架橋化剤を用いる方法が最も望ましい。
【0014】
すなわち、紫外線照射処理による光架橋や熱架橋などの物理的架橋法では、その架橋化工程において、架橋度は限られて、更には複合生体材料を構成する生体吸収性合成高分子の変質や分解を惹起する恐れがあり、また化学架橋法でも、溶液状の架橋化剤を用いる方法では、その架橋過程で天然高分子が溶解する恐れがあるからである。なお、天然高分子の架橋化剤溶液の溶解を防止するめに、溶液状の架橋化剤による架橋に先だって光架橋や熱架橋を施す方法を採用したとしても、前記したように光や熱により天然高分子の分解や変質が生じてしまうので望ましくない。
【0015】
これに対して、ガス状の架橋剤を用いる方法は、上記のような問題点の全てが克服され、分解や変質を生じることなく、天然高分子が所望の形態で架橋され、3次元化されると共に合成高分子メッシュ体との結合力を高められ、目的とするに足る十分な強度と弾力を有する架橋化された複合体材料を得ることができる。
本発明で用いられる架橋剤としては、従来公知のものが何れも使用できる。好ましく使用される架橋剤は、グルタルアルデヒド、ホルムアルデヒド、パラホルムアルデヒドのようなアルデヒド類、特にグルタルアルデヒドである。
【0016】
本発明の架橋化は、前記したように、上記の架橋剤をガス状にして用いる。
具体的には、上記天然高分子多孔質体を架橋するに際し、一定温度で一定濃度の架橋剤水溶液で飽和した架橋剤の蒸気の雰囲気下で一定時間架橋を行う。
架橋温度は、生体吸収性合成高分子メッシュ体が溶解せず、且つ架橋剤の蒸気が形成できる範囲内で選定すればよく、通常、20℃〜50℃に設定される。
架橋時間は、架橋剤の種類や架橋温度にもよるが、上記天然高分子多孔質体の親水性や生体吸収性を阻害せず、かつ生体移植時にこのものが溶解しないような架橋固定化が行われる範囲に設定するのが望ましい。
架橋時間が短くなると、架橋固定化が不十分となり、移植後生体内で天然高分子多孔質体が短時間で溶解する恐れがあり、また架橋時間が長いほど架橋化が進むが、架橋時間があまり長過ぎると、親水性が低くなり、軟骨細胞あるいは軟骨細胞に分化する幹細胞の複合体材料に対する播種密度が低くなり、細胞の贈殖及び組織再生が効率よく行われないほか、生体吸収性も低下する等の問題点を生じるので好ましくない。
【0017】
本発明の複合体材料においては、例えば生体吸収性合成高分子の多孔質体を用いる場合、移植部位に対応する立体形状にあらかじめ該多孔質体を成形し、この多孔質体の孔に天然高分子の多孔質構造体を形成せしめてもよいが、このような方法は操作が簡便であり、機械的強度も優れている反面、軟骨細胞あるいは軟骨細胞に分化する幹細胞が、播種の際、上記天然高分子の多孔質構造体の内奧部の細孔に到達しにくく、これら細胞の播種密度が低くなるということがある。
本発明の複合材料の望ましい形状は、シート状の形状であり、このような形状の生体吸収性合成高分子のメッシュ体あるいは多孔質体の内部構造マトリックス内、すなわちメッシュ体のアミ目あるいは多孔質体の孔内に、天然高分子の多孔質構造体を形成させる。このシート状物の全体の厚さは、通常0.1〜5mm、好ましくは0.1〜1mmが好ましく、また、上記天然高分子多孔質構造体の厚さは適宜調製できるが、生体吸収性合成高分子のメッシュ体あるいは多孔質体とほぼ同じ厚さに形成するのが好ましい。その多孔質構造体の空隙率は、通常80%以上である。なお、本明細書においてシート状物というときは、フィルム状のものないし膜状のものも包含する。
【0018】
例えば、図1(a)に示されるような、本発明のシート状の複合体材料を製造するには、シート状の生体吸収性合成高分子のメッシュ体あるいは多孔質体を生体由来の天然高分子の水溶液の中央に位置させて凍結し、凍結乾燥する。これにより、生体吸収性合成高分子のメッシュ体あるいは多孔質体が天然高分子多孔質構造体中にサンドイッチされているシート状の複合体材料が形成される。 また、図1(b)に示されるような、シート状の生体吸収性合成高分子のメッシュ体あるいは多孔質体を生体由来の天然高分子水溶液の上面或いは下面で凍結し、凍結乾燥すると、片面が生体吸収性合成高分子メッシュ体あるいは多孔質体で、他面が天然高分子多孔質構造体であるシート状複合体材料が形成される。
なお、図1(a)および(b)は、模式図であり、これらによれば、天然高分子多孔質構造体が生体吸収性合成高分子のメッシュ体あるいは多孔質体の表面に形成されているように記載しているが、図4の電子顕微鏡写真からも明らかなように、実際には、天然高分子多孔質構造体は、生体吸収性合成高分子のメッシュ体のアミ目あるいは多孔質体の孔内にも形成される。
【0019】
本発明において用いる軟骨細胞および軟骨細胞に分化する幹細胞は常法により生体組織から調製する。
軟骨細胞は、生体軟骨組織をコラーゲナーゼ、トリプシン、リバラーゼ、プロティナーゼ等の酵素処理により、細胞外マトリックスを分解処理し、次いで血清培地を添加し、遠心して、軟骨細胞を単離する。単離した軟骨細胞を培養フラスコに播き、10%ウシ胎児血清,4500mg/Lグルコース、584mg/Lグルタミン、0.4mMプロリンおよび50mg/Lアスコルビン酸を含有するDMEM培地(DMEM血清培地)で培養する。十分な細胞数になるまで、2〜3回継代培養し、この継代培養した細胞をトリプシン処理により回収し、播種用細胞液とする。
また、軟骨細胞に分化する幹細胞は、骨髄抽出液をパーコール(percoll)からなる密度勾配媒体を用いた密度勾配遠心法により遠心して単離する。これらの細胞を培養フラスコに播き、DMEM血清培地で十分な細胞数となるまで、2〜3回継代培養する。継代培養した細胞をトリプシン処理により回収し、播種用細胞液とする。
【0020】
本発明の複合体材料に、軟骨細胞あるいは軟骨細胞に分化する幹細胞を播種するには、 上記複合体材料を培養液で濡らしておき、この複合体材料に上記播種用細胞液を含浸するか、あるいは上記複合体材料に直接播種用細胞液を含浸することにより行う。
上記播種用細胞液の細胞濃度は、1×106cells/ml〜5×107cells/mlが望ましく、複合体材料の体積以上の容量の細胞液を播種することが望ましい。
【0021】
本発明の軟骨組織を再生するための移植体は、軟骨細胞の場合、上記複合体材料に上記播種用細胞液を含浸した後、さらに、培養液を添加し、該複合体中の軟骨細胞をDMEM血清培地で、37℃、5%CO2雰囲気下のインキュベータにおいて培養増殖させることにより、当該移植体を得る。
幹細胞の場合は、さらに軟骨細胞への分化工程が必要であり、上記複合体材料に上記軟骨細胞に分化する幹細胞の播種用細胞液を含浸した後、DMEM血清培地で1〜2週間培養増殖させた後、4500mg/Lグルコース、584mg/Lグルタミン、0.4mMプロリンおよび50mg/Lアスコルビン酸に加え、トランスフォーミング増殖因子−β3(TGF−β3)を含有するDMEM培地(分化培地)で1〜2週間培養し、分化させて当該移植体を得る。
以下、本発明の複合体材料がシート状物である場合において、軟骨細胞あるいは軟骨細胞に分化する幹細胞を該シート状物に播種し、軟骨組織を再生するための移植体を得る手法の一例について具体的に述べる。
【0022】
例えば、本発明のシート状複合体材料を清潔で無菌なシャーレ等の容器中に入れ、該シート状複合体材料を培養液で濡らしてから、播種用細胞液を上から滴下する。
細胞を播種する回数は特に限らないが、1、2回が望ましい。2回播種する時、1回目が上から行って、2回目はシート状複合体材料を裏返してから行う。1回目と2回目の間が24時間置いたほうが望ましい。
なお、この際、播種される細胞が、シート状複合体材料から漏れ出さないようにするため、シート状複合体材料の縁をゴム等のリングで囲っておくことが望ましい。
次いでシート状複合体材料中に播種用細胞液が含浸された状態で、インキュベータ中において37℃、5%CO2雰囲気下で4時間静置して培養する。その後、ゴムのリングを取り出し、多量の培養液を入れて、培養した後、軟骨組織の再生用移植体を得る。
【0023】
本発明においてシート状の複合体材料を使用する利点は、複合体材料中に形成されるコラーゲンスポンジ等の天然高分子の多孔質構造体が薄いことに起因する。薄い多孔質構造体においては、上記播種用細胞液が多孔質体の細孔にもれなく含浸でき、結果として複合体材料において保持される細胞の密度が高まり、軟骨組織の再生が速やかにかつ効率的に行われることになる。
軟骨細胞あるいは軟骨細胞に分化する幹細胞を播種した複合体材料をシート状のままで用いると、薄い軟骨組織を再生することが可能であるが、図2に示すように、これら細胞を播種した複合体材料を積層して用いることもできる。この場合において再生される軟骨の厚みはシート状複合体材料の積層する枚数により調整できる。この図2のものは、積層されたシート状複合体材料の各々において上記細胞の播種が行われているので、播種された細胞の密度は、1枚のシート状複合体材料と変わらずに高く、これを移植体として生体内に移植すれば、軟骨組織の再生が良好に行われる。
【0024】
また、図3に示すように、細胞を播種したシート状複合体材料をロール状に巻いて、円筒状の形状にすることもできる。この場合においては、再生される軟骨の長さは、ロールの高さで、直径はロールを巻く回数で調整できる。さらに、本発明においては、軟骨組織の欠損部の形状に合わせて、上記シート状物を適宜変形あるいは集合させることも可能である。
前記シート状複合材料の積層物あるいはロール状物等の種々の形状の移植体を得るには、これらの成形前の5日間から2週間まで培養したほうが望ましい。
【実施例】
以下、本発明を実施例により更に詳細に説明する。
【0025】
実施例1
機械強度が高い生体吸収性高分子である乳酸とグリコール酸との共重合体(PLGA)メッシュ体を0.5wt%のウシI型アテロコラーゲン酸性水溶液(pH=3.0)に浸漬し、-80℃で12時間凍結した。次にこの凍結物を、真空減圧下(0.2 Torr)で24時間凍結乾燥し、PLGAメッシュ体とコラーゲンスポンジとのシート状の未架橋複合体材料を製造した。
得られた未架橋複合生体材料を37℃で、25wt%のグルタルアルデヒド水溶液で飽和したグルタルアルデヒド蒸気で4時間架橋処理した後、リン酸緩衝液で10回洗浄した。さらに、0.1Mグリシン水溶液に4時間浸漬し、リン酸緩衝液で10回洗浄した後、蒸留水で3回洗浄し、-80℃で12時間凍結した。これを真空減圧下(0.2 Torr)で24時間凍結乾燥し、本発明のPLGAメッシュ体とコラーゲンスポンジとのシート状の架橋複合体を得た。
この材料を金でコーティングし、それらの構造を走査型電子顕微鏡(SEM)で観察した。その結果を図4に示す。
【0026】
実施例2
実施例1で得た、PLGAメッシュ体とコラーゲンスポンジとのシート状の架橋複合体材料を幅が5.0mm、長さが20.0mmの大きさに切り、サンプルを作製した。このサンプルを2−[4−(2−ヒドロキシエチル)−1−ピペラジル]エタンスルホン(HEPES)緩衝液(pH7.4)に浸漬し濡れた状態で、静的引張試験を行った。その結果を表1に示す。
【0027】
【表1】
*比較サンプル:実施例1で用いたPLGAメッシュ体単独
**比較サンプル:0.5wt%のウシI型アテロコラーゲン酸性水溶液(pH=3.0)単独を実施例1と同様の多孔架橋処理することにより得られたコラーゲン単独スポンジ
表1から、本発明のPLGAメッシュ体とコラーゲンスポンジとの架橋複合体材料は、HEPES緩衝液で濡れた状態で、コラーゲンスポンジのみからなる生体材料に比べより高い引張強度を示し、また、PLGAメッシュとほぼ同じな引張強度を示すことが判る。
【0028】
【実施例3】
実施例1で作成したPLGAメッシュ体とコラーゲンスポンジとのシート状架橋複合体を 酸化エチレンガスで滅菌した。
一方、ウシ肘関節の軟骨から薄い軟骨片をメスで剃りおろし、細かく刻んだ後、0.2(w/v)%のコラーゲナーゼを含有するDMEM培地中で37℃で12時間インキュベートした。そして、ポアサイズが70μmのナイロンフィルターで濾過した上澄みを2000rpmで5分間遠心し、抗生物質と10%ウシ胎児血清を含有するDMEM血清培地で2回洗浄した後、ウシ肘の軟骨細胞を得た。得られた軟骨細胞をDMEM血清培地で37℃、5%CO2雰囲気下で培養した。2回継代培養した軟骨細胞を0.025%トリプシン/0.01%EDTA/PBS(-)で剥離・採集し、1×107cells/ml細胞液を調製した。
次に、酸化エチレンガスで滅菌した上記PLGAメッシュ体とコラーゲンスポンジとのシート状架橋複合体をDMEM血清培地で濡らし、複合体(膜)の縁をゴムのリングで囲って、1.3ml/cm2細胞液を滴加した。インキュベータ中で、37℃、5%CO2雰囲気下で、4時間静置して培養した。その後、ゴムのリングを取り外し、多量の培養液を入れて、培養を続けた。培地を三日間ごとに交換した。
1週間培養した後、複合体をヌードマウスの背中の皮下に移植した。移植後6週の時点で検体を採収し、HE(ヘマトオキシリンとエオシン)染色、safranin-O染色を行った。また、検体よりm−RNAを回収しRT-PCRにより関節軟骨組織に特有にみられるタイプ II コラーゲン、アグリカンの発現解析を行った。
【0029】
その結果、ヌードマウスの背中の皮下に移植した検体は、図5に示すように6週間後表面光沢があり、色が乳白色であることが観察された。
また、検体をHE染色とsafranin-O染色を行った結果、図6に示すように、小窩内小円形細胞とSafranin-O染色性細胞外マトリックスが認められた。さらに検体から抽出したm−RNA試料中にタイプ II コラーゲンやアグリカンを発現するm−RNAが検出同定され、これらのことにより、再生した組織が関節軟骨組織であることを確認した。
【0030】
【発明の効果】
本発明において、軟骨細胞あるいは軟骨細胞に分化する幹細胞の担持担体として使用する複合体材料は、生体吸収性合成高分子メッシュ体と生体由来の天然高分子多孔質体が強固に架橋複合化された構造を有するものである。
生体吸収性合成高分子メッシュ体は、メカニカル骨格として機能し、有利な形状制御性、優れた機械的強度及び取扱いの容易性を賦与し、また、天然高分子多孔質体は、軟骨細胞の増殖あるいは軟骨に分化する幹細胞の分化および増殖に極めて優れており、本発明の上記複合体材料は、これらの優れた特性を併せ持つものである。また、特にシート状に成形された複合体材料は、播種効率が良好で、複合体材料中に担持せしめられる上記細胞の密度が極めて高く、これにより、この複合体材料に軟骨細胞の増殖あるいは軟骨細胞に分化する幹細胞を担持せしめて、生体に移植した場合、軟骨組織の再生は速やかで、極めて効率的である。したがって、本発明の複合体材料およびこれに軟骨細胞あるいは軟骨細胞に分化する幹細胞を担持せしめた移植体は、軟骨組織の再生手段として極めて有用なものである。
【図面の簡単な説明】
【図1】(a)および(b)は、本発明に係る代表的なシート状複合体材料の模式断面図。
【図2】は、積層されたシート状複合体材料の縦方向模式断面図。
【図3】は、ロール状に巻かれたシート状複合体材料の横方向模式断面図。
【図4】は、本発明のるシート状複合体材料の電子顕微鏡写真。
【図5】は、再生したウシ関節軟骨組織の外観写真。
【図6】は、再生したウシ関節軟骨組織の組織学的な染色写真。
Claims (4)
- 生体吸収性合成高分子からなるメッシュ体あるいは多孔質体の内部構造マトリックス内に、さらに天然高分子からなる、連続する細孔を有する多孔質構造体が形成されている複合体材料からなるものであって、該天然高分子の多孔質構造体が、上記メッシュ体あるいは多孔質体に浸漬又は塗布された天然高分子水溶液を凍結乾燥することにより形成されたものであることを特徴とする、軟骨細胞あるいは軟骨細胞に分化する幹細胞の担持用担体。
- 複合体材料がシート状物である、請求項1に記載の軟骨細胞あるいは軟骨組織に分化する幹細胞の担持用担体。
- 複合体材料が請求項2に記載のシート状物を積層またはロール状に巻いたものである、請求項1の軟骨細胞あるいは軟骨組織に分化する幹細胞の担持用担体。
- 請求項1〜3のいずれか1項記載の軟骨細胞あるいは軟骨細胞に分化する幹細胞の担持用担体に、軟骨細胞あるいは軟骨細胞に分化する幹細胞を担持せしめた、軟骨組織再生に用いるための移植体。
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Publication number | Priority date | Publication date | Assignee | Title |
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US5263984A (en) * | 1987-07-20 | 1993-11-23 | Regen Biologics, Inc. | Prosthetic ligaments |
GB2215209B (en) * | 1988-03-14 | 1992-08-26 | Osmed Inc | Method and apparatus for biodegradable, osteogenic, bone graft substitute device |
US6080194A (en) * | 1995-02-10 | 2000-06-27 | The Hospital For Joint Disease Orthopaedic Institute | Multi-stage collagen-based template or implant for use in the repair of cartilage lesions |
US5916585A (en) * | 1996-06-03 | 1999-06-29 | Gore Enterprise Holdings, Inc. | Materials and method for the immobilization of bioactive species onto biodegradable polymers |
GB9704749D0 (en) * | 1997-03-07 | 1997-04-23 | Univ London | Tissue Implant |
AU3097999A (en) * | 1998-03-18 | 1999-10-11 | University Of Pittsburgh | Chitosan-based composite materials containing glycosaminoglycan for cartilage repair |
AU2001234623A1 (en) * | 2000-01-28 | 2001-08-07 | Orthogene, Inc. | Gel-infused sponges for tissue repair and augmentation |
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2001
- 2001-07-04 JP JP2001204013A patent/JP3646162B2/ja not_active Expired - Lifetime
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2002
- 2002-07-03 EP EP02254659A patent/EP1273312B1/en not_active Expired - Fee Related
- 2002-07-03 DE DE60214477T patent/DE60214477T2/de not_active Expired - Lifetime
- 2002-07-03 US US10/188,890 patent/US20030012805A1/en not_active Abandoned
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EP1273312B1 (en) | 2006-09-06 |
EP1273312A2 (en) | 2003-01-08 |
US20030012805A1 (en) | 2003-01-16 |
DE60214477T2 (de) | 2007-04-19 |
JP2003010309A (ja) | 2003-01-14 |
DE60214477D1 (de) | 2006-10-19 |
EP1273312A3 (en) | 2003-02-05 |
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