JP7385664B2 - 生体移植材料 - Google Patents
生体移植材料 Download PDFInfo
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- JP7385664B2 JP7385664B2 JP2021536993A JP2021536993A JP7385664B2 JP 7385664 B2 JP7385664 B2 JP 7385664B2 JP 2021536993 A JP2021536993 A JP 2021536993A JP 2021536993 A JP2021536993 A JP 2021536993A JP 7385664 B2 JP7385664 B2 JP 7385664B2
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- gelatin
- stem cells
- bone
- cells
- granules
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Classifications
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Description
<1> ゼラチン含有顆粒と、幹細胞とを含む、骨再生剤。
<2> ゼラチンが、コラーゲンに特徴的なGly-X-Yで示される配列の繰り返しを有し、ここで、複数個のGly-X-Yはそれぞれ同一でも異なっていてもよく、式中、X及びYはそれぞれ独立にアミノ酸の何れかを示し、ゼラチンの分子量が2KDa以上100KDa以下である、<1>に記載の骨再生剤。
<3> ゼラチン含有顆粒の1つの大きさが100μm以上2000μm以下である、<1>又は<2>に記載の骨再生剤。
<4> 幹細胞が、間葉系幹細胞、iPS細胞又はES細胞である、<1>から<3>の何れか一に記載の骨再生剤。
<5> 幹細胞が、脂肪由来幹細胞、骨髄由来幹細胞又は歯髄由来幹細胞である、<1>から<4>の何れか一に記載の骨再生剤。
<6> ゼラチン含有顆粒1mgあたりの幹細胞の数が1.0×103個以上である、<1>から<5>の何れか一に記載の骨再生剤。
<7> 幹細胞の70%以上が、ゼラチン含有顆粒の表面又は表面の孔に付着している、<1>から<6>の何れか一に記載の骨再生剤。
<8> 顎裂部に用いる、<1>から<7>の何れか一に記載の骨再生剤。
<B> ゼラチン含有顆粒と、幹細胞とを含む、骨再生の治療において使用するための治療剤。
<C> 骨再生剤の製造のための、ゼラチン含有顆粒と幹細胞との組み合わせの使用。
本発明の骨再生剤は、ゼラチン含有顆粒と、幹細胞とを含む。
本発明の骨再生剤は、後記の実施例におけるラット頭頂骨欠損モデルにおいて実証される通り、骨再生速度を向上することができる。また、本発明の骨再生剤によれば、後記の実施例における顎裂部再建モデルにおいて実証される通り、元々骨がない箇所でも良好な骨再生が可能である。
ゼラチンとしては、天然由来ゼラチン又はリコンビナントゼラチンの何れでもよいが、リコンビナントゼラチンが好ましい。
リコンビナントゼラチンとは、遺伝子組み換え技術により作られたゼラチン類似のアミノ酸配列を有するポリペプチドもしくは蛋白様物質を意味する。
この最小アミノ酸配列の含有量は、細胞接着・増殖性の観点から、タンパク質1分子中3~50個が好ましく、さらに好ましくは4~30個、特に好ましくは5~20個である。最も好ましくは12個である。
好ましくは、ゼラチンはテロペプタイドを有さない。
好ましくは、ゼラチンは、アミノ酸配列をコードする核酸により調製された実質的に純粋なポリペプチドである。
(1)配列番号1に記載のアミノ酸配列からなるペプチド;
(2)配列番号1に記載のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ生体親和性を有するペプチド;または
(3)配列番号1に記載のアミノ酸配列と80%以上(さらに好ましくは90%以上、特に好ましくは95%以上、最も好ましくは98%以上)の配列同一性を有するアミノ酸配列からなり、かつ生体親和性を有するペプチド;
の何れかである
本発明で用いるゼラチンのGRAVY値を上記範囲とすることにより、親水性が高く、かつ、吸水性が高くなる。
本発明におけるゼラチン含有顆粒とは、その成分の全部又は一部がゼラチンである顆粒を意味する。ゼラチン含有顆粒としては、例えば、ゼラチンのみからなる顆粒、無機成分にゼラチンをコーティングした顆粒などが挙げられる。二種以上のゼラチン含有顆粒を混合して用いてもよい。ゼラチン含有顆粒としては、ゼラチンのみからなる顆粒が好ましい。また、無機成分とは、1族のアルカリ金属、2族のアルカリ土類金属、3~12族の遷移金属、13~15族の典型金属、および、ホウ素、ケイ素、ゲルマニウム等の半金属の元素を含む成分をいう。無機成分は、セラミックス、チタン、ステンレス、ヒドロキシアパタイト、β-リン酸三カルシウムなど、生体適合性が高いものが好ましい。
無機成分にゼラチンをコーティングした顆粒は、例えば、無機成分の顆粒をゼラチン水溶液に浸漬させ、風乾または凍結乾燥によって無機成分の顆粒にゼラチンをコーティングすることにより、製造することができる。
(式2)は、ゼラチン含有顆粒1g当たりのリジン量(モル等量)を示す。
(式中、Asはサンプル吸光度、Abはブランク吸光度、Vは反応液量(g)、wはゼラチン含有顆粒質量(mg)を示す。)
(式3)は、1分子あたりの架橋数を示す。
本発明では幹細胞を使用する。
幹細胞とは、自ら増殖する能力(自己複製能)と、特定の細胞に分化する能力(多分化能)とを有する細胞である。
幹細胞の数は、ゼラチン含有顆粒1個当たり、2.0×102個以上であることが好ましく、5.0×102個以上であることがより好ましく、2.0×103個以上であることがさらに好ましく、5.0×103個以上であることがよりさらに好ましく、上限は特に限定されないが、一般的には2.0×105個以下であり、2.0×104個以下でもよい。
本発明の骨再生剤は、ゼラチン含有顆粒と幹細胞とを一緒に混合することによって製造することができる。例えば、96ウエルプレート等のプレート上においてゼラチン含有顆粒に、幹細胞を含む細胞懸濁液を浸潤させて、所定の時間培養する。その後、適当な培地を含む培養容器中に上記の細胞を浸潤させたゼラチン含有顆粒を入れ、振動を与えることによって、本発明の骨再生剤を製造することができる。
図1から分かるように、楕円形で示す細胞は、ゼラチン含有顆粒を使用する場合の方が少ない。
具体的には、ゼラチン含有顆粒+幹細胞の場合、上記の通り、ゼラチン含有顆粒1mg当たりの細胞数は、1.0×103個以上であることが好ましく、一般的には2.0×105個以下である。一方、花弁状ブロック+幹細胞の場合、例えば特許文献2では、花弁状ブロック1mg当たり、1.0×106個(=細胞1個当たり0.001μgのブロック)であり、顆粒またはブロック当たりの細胞数に、数十~数千倍の差がある。
<1>リコンビナントペプチド(リコンビナントゼラチン)
リコンビナントペプチド(リコンビナントゼラチン)として以下のCBE3を用意した(国際公開WO2008/103041号公報に記載)。
CBE3:
分子量:51.6kD
構造:GAP[(GXY)63]3G
アミノ酸数:571個
RGD配列:12個
イミノ酸含量:33%
ほぼ100%のアミノ酸がGXYの繰り返し構造である。CBE3のアミノ酸配列には、セリン、スレオニン、アスパラギン、チロシンおよびシステインは含まれていない。CBE3はERGD配列を有している。
等電点:9.34
GRAVY値:-0.682
1/IOB値:0.323
アミノ酸配列(配列表の配列番号1)(国際公開WO2008/103041号公報の配列番号3と同じ。但し末尾のXは「P」に修正)
GAP(GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPP)3G
厚さ5mm、直径98mmのアルミ製円筒カップ状容器を用意した。円筒カップは曲面を側面としたとき、側面は1mmのアルミで閉鎖されており、底面(平板の円形状)も5mmのアルミで閉鎖されている。一方、上面は開放された形をしている。また、側面の内部にのみ、肉厚3mmのテフロン(登録商標)を均一に敷き詰め、結果として円筒カップの内径は90mmになっている。以後、この容器のことを円筒形容器と呼称する。
愛知学院大学歯学部附属病院・口腔外科の外来にて、患者とその保証人において、抜去乳歯及び歯髄の提供・研究への使用についての同意書に署名頂いた後、抜去歯から歯髄摘出した。
3継代目まで培養した乳歯歯髄由来幹細胞(以下、DDPSCs)を含む細胞を回収し、1.0×105cells/18μl、5.0×104cells/18μl、1.0×104cells/18μlの細胞懸濁液を作製した。窒素雰囲気下で135℃、4.75時間処理した熱架橋体Fuji Bone Graft(ゼラチン顆粒)を3mg用意し、96well plate上で、先程準備した細胞懸濁液を浸潤させ、37℃の環境下の炭酸ガスインキュベータ(SANYO MCO-18AIC(UV))(37℃環境下 CO2:5%)内にて10分間培養した。その後、Cell reactor15ml(Greiner bio-one)に、PRIME-XV(登録商標)MCS XSFM(P/S1%)10mlと、先ほど浸潤させたゼラチン顆粒3mgを入れ、Bio shaker(37℃環境下)にて24時間振動(振動数118rpm/min)を与えて移植体(ゼラチン顆粒+細胞)の調製を行った。
移植用の動物として、17匹のnudeラット(F344/NJcL-rnu/rnu nude rat、雄、10週齢、0.3~0.5kg)を用いた。ラットの麻酔には、マウス・ラット等小動物実験用簡易吸入麻酔装置NARCOBIT-E(2型)を用いて、麻酔薬として、イソフルラン吸入麻酔液(ファイザー)を用い、吸入麻酔により麻酔した(導入が4.5%、 維持が3.5%の濃度)。No15メスにてラットの頭蓋皮膚及び骨膜を切開し、剥離して頭蓋骨を露出させた。一定の骨欠損を作製するために、トレフィンバーφ4.0mm/5.0mm(Hanger & Meisinger)にて、頭蓋骨の中央の縫合部を避けるようにして、頭頂骨の左側の中央に位置するように、直径5mmの円形の骨欠損を作成した。
表1及び図2に示す結果から、各移植群において骨形成量の増加が認められた。また、DDPCs1×105cells移植ラットにおいては、DDPCs1×104cells移植ラットに比べ移植後1週における骨形成量に有意に高い値が認められた(t値=0.038)。
3種混合麻酔0.5ml/100g(0.3mg/kgのDOMITOR(登録商標)(Nippon Zenyaku Kogyo Co.,Ltd,Japan),4.0mg/kgのmidazolam(SANDOZ,Japan)、5.0mg/kgのVetorphale(登録商標)(Meiji Seika Pharma Co.,Ltd,Japan)及び2.9ml/kgのOtsuka Normal Saline(Otsuka Pharmaceutical Co.,Ltd,Japan)を用いて腹腔内麻酔を行う。次に、移植体を移植する空洞を作成する(図3)。まずは、移植部位を作成する側の頬粘膜に絹糸をかけ、モスキートにて伸展し、口蓋部となる術野の視野を確保する。口蓋部の口蓋粘膜の切開にはNo.15のメスをもちいる。切開は、切歯から第一大臼歯方向にむけて、切歯骨体上に骨膜下の骨までの深さで長さ10mmの切開を加える(図3A)。切歯骨から粘膜骨膜弁を剥離すると切歯骨体の皮質骨の表面が観察できる。骨膜まで剥離ができていないと骨が観察できない。切開部の骨膜に沿って、粘膜骨膜を切歯骨側から口蓋骨に沿って、鼻腔方向に向けて剥離を続けると、陥入している口蓋裂が観察できる。この陥入している口蓋裂部に、移植体を移植することになる。次に、鼻中隔側の骨膜を鼻腔側に向けて剥離を続ける。次に、口蓋裂を覆っていた口蓋部の粘膜を粘膜と骨膜に分離する(図3B)。剥離した切歯骨・口蓋骨側の骨膜と鼻中隔側の骨膜を、鼻腔側に滅菌綿球を用いて押し込む(図3C)。メジャーにて口蓋裂部の深さを計測し、口腔粘膜から5mm程度の深さが得られたところで、骨膜を鼻腔側に陥入させることを停める。ここに、インプラント体を移植する部位ができるので、ここに移植体を移植する。
Claims (4)
- ゼラチン含有顆粒と、幹細胞とを含む、骨再生剤であり、
前記ゼラチン含有顆粒の表面に前記幹細胞が付着しており、
幹細胞が、脂肪由来幹細胞、骨髄由来幹細胞又は歯髄由来幹細胞であり、
ゼラチン含有顆粒1mgあたりの幹細胞の数が1.0×103個以上であり、
ゼラチン含有顆粒の1つの大きさが100μm以上2000μm以下である、
骨再生剤、ただし、リコンビナントゼラチンの花弁状ブロックと間葉系幹細胞とを用いたモザイク細胞塊を含む軟骨再生材料は除く。 - ゼラチンが、コラーゲンに特徴的なGly-X-Yで示される配列の繰り返しを有し、ここで、複数個のGly-X-Yはそれぞれ同一でも異なっていてもよく、式中、X及びYはそれぞれ独立にアミノ酸の何れかを示し、ゼラチンの分子量が2KDa以上100KDa以下である、請求項1に記載の骨再生剤。
- 幹細胞の70%以上が、ゼラチン含有顆粒の表面又は表面の孔に付着している、請求項1又は2に記載の骨再生剤。
- 顎裂部に用いる、請求項1から3の何れか一項に記載の骨再生剤。
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