JP4002299B2 - Improved hydrogel for tissue treatment - Google Patents

Description

ここに、ｎは整数である。
架橋結合のために適当なポリホスファゼンは、酸性であり、２または３価カチオンと塩橋を形成し得る多数の側鎖を有する。好ましい酸性側鎖の例は、カルボン酸基、およびスルホン酸基である。加水分解安定性ポリホスファゼンは、Ｃａ2+またはＡｌ3+のような２価または３価カチオンによって架橋されるカルボン酸基を有するモノマーから形成される。イミダゾール、アミノ酸エステルまたはグリコール側鎖基を有するモノマーを導入することによって、加水分解で分解するポリマーを合成できる。例えば、ポリアニオン性[ビス（カルボキシルアトフェノキシ）]ホスファゼン（ＰＣＰＰ）を合成することができ、この者は室温またはそれ以下において、水性媒質中の溶解多価カチオンで架橋結合されて、ヒドロゲルマトリックスを作る。
生分解性ポリホスファゼンは２種の異なるタイプの側鎖、すなわち、多価カチオンと塩橋を形成し得る酸性側鎖、及びｉｎ ｖｉｖｏ条件下加水分解する側基、例えばイミダゾール基、アミノ酸エステル、グリセロール及びグルコシルである。ここで使用される生分解性なる用語は、所望の応用（普通はｉｎ ｖｉｖｏ治療）において、一旦約２５℃−３８℃の温度を有するｐＨ６−８の生理溶液に曝されたときに、許容し得るある期間で溶解または分解するポリマーを意味し、約５年以下、最も好ましくは、約１年以下で、溶解または分解するポリマーを意味する。側鎖の加水分解は、ポリマーの崩壊をもたらす。加水分解性側鎖の例は、非置換または置換のイミジゾール及びアミノ酸エステルであって、基がアミノ結合を介してリン原子に結合しているもの（両Ｒ基がこのように結合しているポリホスファゼンポリマー）である。ポリイミダゾールホスファゼンについては、ポリホスファゼン主鎖上のＲ基のいくつかが、環窒素原子を介して主鎖中のリンに結合されたイミダゾール環である。その他のＲ基は、加水分解に参加しない有機残基、メチルフェノキシ基、またはＡｌｌｃｏｃｋ等のＭａｃｒｏｍｏｌｅｃｕｌｅｓ１０：８２４−８３０（１９７７）の科学文献に記載されているその他の基のようなものである。
種々のタイプのポリホスファゼンの合成及び分析方法は、Ａｌｌｃｏｃｋ Ｈ．Ｒ．等のＩｎｏｒｇ．Ｃｈｅｍ ．１１，２５４８（１９７２）；Ａｌｌｃｏｃｋ等のＭａｃｒｏｍｏｌｅｃｕｌｅ ｓ１６，７１５（１９８３）；Ａｌｌｃｏｃｋ等のＭａｃｒｏｍｏｌｅｃｕｌｅｓ １９，１５０８（１９８６）；ＡｌｌｃｏｃｋらのＢｉｏｍａｔｅｒｉａｌｓ，１９，５００（１９８８）；Ａｌｌｃｏｃｋ等のＭａｃｒｏｍｏｌｅｃｕｌｅｓ，２１，１９８０（１９８８）；Ａｌｌｃｏｃｋ等のＩｎｏｒｃｒ．Ｃｈｅｍ．２１（２），５１５５２１（１９８２）；Ａｌｌｃｏｃｋ等のＭａｃｒｏｍｏｌｅｃｕ ｌｅｓ２２，７５（１９８９）；Ａｌｌｃｏｃｋ等の米国特許４、４４０、９２１、４、４９５１７４および４、８８０、６２２；Ｍａｇｉｌｌ等の米国特許４、９４６、９３８；及びＧｒｏｌｌｅｍａｎ等のＪ．Ｃｏｎｔｒｏｌｌｅｄ Ｒｅ ｌｅａｓｅ ３，１４３（１９８６）に記載されており、これらの教示は、参考として特にここに導入される。
前記のその他のポリマーの合成方法は、当業者に公知である。例えば、Ｃｏｎ ｃｉｓｅ Ｅｎｃｙｃｌｏｐｅｄｉａ ｏｆ Ｐｏｌｙｍｅｒ Ｓｃｉｅｎｃｅ及びＰｏｌｙｍｅｒｉｃ Ａｍｉｎｅｓ ａｎｄ Ａｍｍｏｎｉｕｍ Ｓａｌｔ ｓ，Ｅ．Ｇｏｅｔｈａｌｓ編集（Ｐｅｒｇａｍｅｎ Ｐｒｅｓｓ，Ｅｌｍｓｆｏｒｄ，ＮＹ１９８０）参照。ポリ（アクリル酸）のような多くのポリマーが市販されている。
電荷のある側鎖を有する水溶性ポリマーは、そのポリマーを、反対電荷の多価イオンを含む水性溶液と反応させることにより架橋結合され、ポリマーが酸性側鎖を有するならば、多価カチオンあるいは、ポリマーが塩基性側鎖を有するならば、多価アニオンを用いる。ヒドロゲルを形成するのに酸性側鎖を有するポリマーの架橋結合のために好ましいカチオンは、銅、カルシウム、アルミニウム、マグネシウム、ストロンチウム、バリウム、及び錫のような２価及び３価カチオンであり、アルキルアンモニウム塩、例えば

のようなジ−，トリ−またはテトラ−官能性カチオンも使用できる。これらのアニオンの水性溶液は、ポリマーに添加されて、柔らかい、高度に膨潤したヒドロゲル及び膜を形成する。カチオンの濃度が高いほど、あるいは結合価が大きいほど、ポリマーの架橋結合度が高くなる。０．００５Ｍのような低い濃度で、ポリマーを架橋結合させることが判明した。
ヒドロゲルを形成するのにポリマーを架橋結合させるのに好ましいアニオンは、低分子量ジカルボン酸、例えばテレフタル酸、サルフェートイオン、カーボネートイオンである。これらのアニオンの塩の水性溶液が、ポリマーに添加されて、カチオンの場合のように柔らかい、高度に膨潤したヒドロゲル及び膜を形成する。生相容性ポリマーおよび架橋結合剤は、任意の生理的に相容性の溶媒中に溶解し得る。例えばここでは共通培地Ｍ−１１９を代表溶媒として用いる。
種々のポリカチオンを、ポリマーヒドロゲルを錯化し、安定化して半透性表面膜とするために使用できる。３，０００−１００，０００の好ましい分子量を有するアミンやイミンのような塩基性反応性基を持つポリマーが、使用できる物質の例であり、例えば、ポリエチレンイミン及びポリリシンである。これらは市販されている。一つのポリカチオンは、ポリ（Ｌ−リシン）であり、合成ポリアミンの例は、ポリエチレンイミン、ポリ（ビニールアミン）及びポリ（アリルアミン）である。また多糖類，チトサンのような天然ポリカチオンもある。
ポリマーヒドロゲル上の塩基性表面基と反応して半透膜を形成するのに使用できるポリアニオンとしては、アクリル酸、メタクリル酸及びアクリル酸のその他の誘導体のポリマー及びコポリマー、スルホン化ポリスチレンのようなペンダントＳＯ3Ｈ基を有するポリマー、およびカルボン酸基を有するポリスチレンがある。
本発明における移植剤中で使用が意図されているポリマーには、ここに記載されているヒドロゲルと同様な粘弾性であるヒドロゲルを適当な条件下で形成するその他の天然、合成または変性バイオポリマーを包含する。適当なバイオポリマーとしては、例えば変性アルギン酸塩及びその他のバイオぽりまーがある（例えば、Ｐｕｔｎａｍ ＡＪ；Ｍｏｏｎｅｙ ＤＪ（１９９６）”Ｔｉｓｓｕｅ ｅｎｇｉｎｅｅｒｉｎｇ ｕｓｉｎｇ ｓｙｎｔｈｅｔｉｃ ｅｘｔｒａｃｅｌｌａｒ ｍａｔｒｉｃｅｓ”，Ｎａｔｕｒｅ Ｍｅｄｊｃｉｎｅ，２（７）；８２４−８２６，（１９９６）；Ｍｏｏｎｅｙ，Ｄ．Ｊ．（１９９６），”Ｔｉｓｓｕｅ ｅｎｇｉｎｅｅｒｉｎｇ ｗｉｔｈ ｂｉｏｄｅｇｒａｄａｂｌｅ ｐｏｌｙｍｅｒ ｍａｔｒｉｘｅｓ”，Ｂａｊｐａｉ，Ｐｒａｐｈｕｌｌａ Ｋ（Ｅｄ），Ｐｒｏｃ．Ｓｏｕｔｈ．Ｂｉｏｍｅｄ．Ｅｎｇ．Ｃｏｎｆ．，１５ｔｈ，ＩＥＥＥ，ＮＹ，１９９６；及びＷｏｎｇ，Ｗ．Ｈ．及びＤ．Ｊ．Ｍｏｏｎｅｙ”Ｓｙｎｔｈｅｓｉｓ ａｎｄ ｐｒｏｐｅｒｔｉｅｓ ｏｆ ｂｉｏｄｅｇｒａｄａｂｌｅ ｐｏｌｙｍｅｒｓ ｕｓｅｄ ａｓ ｓｙｎｔｈｅｔｉｃ ｍａｔｒｉｃｅｓ ｆｏｒ ｔｉｓｓｕｅ ｅｎｇｉｎｅｅｒｉｎｇ”Ａｔａｌａ等編集”Ｓｙｎｔｈｅｔｉｃ Ｂｉｏｐｏｌｙｍｅｒ Ｓｃａｆｆｏｌｄｓ（Ｔｉｓｓｕｅ Ｅｎｇｉｎｅｅｒｉｎｇ），Ｂｉｒｋｈａｕｓｅｒ，１９９７，ＩＳＢＮ；０８１７６３９１９５を参照）。
アルギン酸バイオポリマー組成物
好ましい態様において、本発明方法は、酸性バイオポリマーの多価カチオンによる架橋結合によりヒドロゲルを形成することを含み、その際に多価カチオンの有効利用性が弱溶解度塩を溶解させて制御される、可溶性カチオンが架橋結合によって吸収されるにしたがって、可溶性カチオンを補充するためである。多価カチオンの一部は充分溶解度塩によって与えられ、自由に利用できる（速架橋結合イオン）、一方完全架橋結合のために必要な多価カチオンは、弱溶解度塩（遅架橋結合イオン）によって与えられる。この態様に好ましいポリマーは、アルギン酸塩である；しかしながら、本発明の方法は、バイオポリマーであるκ−カラゲナンのような様々な酸性ポリマーにも適用することもできる。以下の説明においてアルギン酸塩は、任意な適切なポリマーの代表である。
好ましいバイオポリマー（アルギン酸塩）と使用するために、好ましい多価イオンは、カルシウムである。ここで、本発明の、架橋カチオンを供給するための多価カチオン源を用いて（ここで、カチオン源は、溶解度が異なるものが選択される）ゲル化の性質と最終的なゲル特性を制御するための方法を、代表的な架橋剤としてカルシウムおよびカルシウム塩を用いて説明する。同様の性質を有するその他の多価イオンは、当然ながら、本発明の予想範囲内である。また、本発明によって提供される組成物は、好ましくは、多価カチオンに応じた可溶性の緩衝系も含み、上記カチオンが二価または三価の金属イオンである場合、通常はリン酸塩である。リン酸塩とアルギン酸塩とは、金属に関して競合するが、それによって、組成物の成分が混合される際に、限定された量の利用可能なカチオンに関してアルギン酸アニオンとリン酸アニオンとが競合し、組成物中でのアルギン酸塩の架橋が先取りされるようになる。
代表例において、生相容性酸性バイオポリマーのアルギン酸塩は、少量の充分可溶性塩化カルシウム及び多量の弱可溶性硫酸カルシウムによって架橋結合される。ヒドロゲルが形成される組成物は、リン酸イオンを比較的高濃度で含み、可溶性カルシウムイオン濃度を緩衝し、アルギン酸塩の完全架橋結合を妨げる。組成物が所望の部位で注入されると、リン酸イオンは周囲の生体液中へ拡散することにより希釈されてしまい、可溶性カルシウムイオンの濃度が局部的に増加する。可溶性カルシウムイオンの濃度が増加するにつれて、アルギン酸塩中で更なる架橋結合をカルシウムが与え、ヒドロゲルが硬化する。類似の金属イオンについても、リン酸アニオンまたはその他の類似の系（例えば、類似の方法で可溶性カチオン濃度を緩衝する生体適合性の金属イオン封鎖剤）で緩衝することによって同様の作用が得られる可能性がある。
アルギン酸塩は、海草から得られるポリマンヌロネート及びポリグルロネートのストレッチの共合体である。適当なアルギン酸塩が市販されている。個々のアルギン酸塩調剤はマンヌロネート：グルロネート（Ｍ／Ｇ）の関数である測定可能な結合用カルシウム容量を有する。Ｍ及びＧ残基の比の変化からもたらされる効果は米国特許４，３５２，８８３号に記載されている、これを参考のためここに導入する。適当な高精製アルギン酸塩は３５／６５のＭ／Ｇ比を有する。
アルギン酸塩は、室温で、水中において２価カチオンによりイオン的に架橋結合され、ヒドロゲルマトリックスを形成することができる。このような穏和な条件のために、アルギン酸塩は、例えばＬｉｍの米国特許４，３５２，８８３号に記載されるようにハイブリドーマ細胞カプセル化のために最も一般的に用いられているポリマーである。Ｌｉｍのプロセスでは、カプセル封入しようとする生物学的材料を含む水溶液を、水溶性ポリマー溶液に懸濁し、この懸濁物を液滴状にし、この液滴を多価カチオンと接触させることによって個々のマイクロカプセルに形成し、次に、このマイクロカプセルの表面をポリアミノ酸で架橋し、カプセル封入された材料の周囲に半透過性の膜を形成する。
ヒドロゲル態の細胞／ポリマー系は、組織処理（エンジニヤリング）のために用いられ、例えば、ヒドロゲル形成を開始させるためにカルシウム塩が添加されたアルギン酸塩溶液中の軟骨細胞のような細胞懸濁物がある。これらの系の典型は、軟骨細胞／アルギン酸カルシウム溶液であり、単離された細胞懸濁物をアルギン酸ナトリウム溶液（例えば、０．１ＭのＫ₂ＨＰＯ₃中に、０．１３５ＭＮａＣｌ，ｐＨ７．４）とかき混ぜ１．０％アルギン酸塩溶液中に２０Ｘ１０６細胞／ｍＬの細胞密度（野生のウシ関節の軟骨の細胞密度の約５０パーセントの細胞密度）を生じさせる。軟骨細胞−アルギン酸ナトリウム懸濁物は使用するまで４℃で氷上に保存しそして注入前に冷軟骨細胞−アルギン酸塩溶液１ｍＬあたり０．２ｇの滅菌ＣａＳＯ₄粉末を添加する。
架橋イオン源として硫酸カルシウムを用いた、配合のゲル形成の特性に関する研究によれば（例えば実施例１を参照）、このような配合物は、ヒトの体温では僅か１６分でゲル化が可能であるが、室温では容易にゲル化しないことが示されている。アルギン酸塩／細胞懸濁物の温度が、妥当な時間内（すなわち、手術室中で、時間ではなく分のレベルで）にゲル形成が開始されるような温度に上昇した場合、組成物のコンシステンシィが注射に適した状態にあるのは、ほんの数分である。このような配合物が室温でゲル化することを防ぐには、注射の前に配合物をインキュベートするための加熱器具の使用が必要である。加えて、混合および注射の問題は、ＣａＳＯ ₄ 粒子周囲に、溶液中のＣａ ⁺² の移動を妨げるゲルが形成され、さらに、アルギン酸塩がカプセル封入されたＣａＳＯ ₄ の塊が形成されて注射器のカニューレを介した注射が妨げられることによって発生する。それゆえに、懸濁物中のさらに迅速なゲル化領域によって形成された塊をふるい分けするために、注射器にフィルターを取り付ける必要がある。これらの特徴は、この配合の臨床での適用における複雑さを増大させ、あらゆるロット間の運搬におけるコンシステンシィとゲル化特性を達成することを難しくしている。
混合物を加熱する必要性及び注射器にフィルターを加える必要性を回避するため、本発明者は組織処理で使用される注入可能ヒドロゲルのための新処方を開発した。この処方は、迅速なゲル形成及びゲル懸濁物の長い注入可能性を与え、より一層一貫した性能を与える。
注射の際に組織平面を分離させるのに十分なコンシステンシィを迅速に形成し、注射器のカニューレが閉塞する問題を回避しつつ溢出を予防するために、本発明は、架橋多価カチオンの２種の源を含むヒドロゲル形成組成物を提供する：ここで、この２種の源とは、一つは、少量のカチオン源（十分に利用可能なカチオンを提供する）、および、もう一方は、多量のカチオン源（ゲルが形成されるにつれて架橋イオンの徐放性を提供する）である。第一のカチオン源は、高い水溶性を有する塩であり、すなわち、少なくとも１ｇ／１００ｍｌ、好ましくは少なくとも３０ｇ／１００ｍｌ、より好ましくは少なくとも６０ｇ／１００ｍｌの溶解度を有する。特に好ましくは、カルシウム塩であり、例えば塩化カルシウムである。第二のカチオン源は、弱溶解性塩であり、すなわち、１ｇ／１００ｍｌ未満、好ましくは０．５ｇ／１００ｍｌ未満、より好ましくは０．３ｇ／１００ｍｌ未満の溶解度を有する。特に好ましくは、カルシウム塩であり、例えば硫酸カルシウムである。冷水及び熱水（１００℃）での硫酸カルシウム２水塩（ＣａＳＯ₄・２Ｈ₂Ｏ）の溶解度は、それぞれ２．４１及び２．２２ｍｇ／ｍＬである。また、塩化カルシウムおよび硫酸カルシウムに類似した水への溶解度を有する多価カチオンのあらゆる２種の塩も、本発明での使用にそれぞれ適している。どちらかの塩単独でも十分な架橋カチオンを提供して十分硬化したアルギン酸塩ヒドロゲルを生産することもあるが、２種の塩の使用により、架橋作用が長期にわたって延長され、それによって部分的に硬化されたヒドロゲル（弱く架橋されたヒドロゲル）が、カニューレを介した注射に適したコンシステンシィを有するようになる期間を拡大させる。
塩化カルシウムが好ましい「迅速な」Ｃａ ⁺² 源の一つであり、その高い溶解度のためにアルギン酸塩のゲル化をスピードアップさせることができる。同等の溶解度を有するその他のカルシウム塩が、同様の様式で作用する。ＣａＣｌ ₂ 単独で作用するかどうかを決定するために、実験を行った。１．０〜６．６ｍｇ／ｍＬの濃度範囲を試験したところ、ＣａＣｌ ₂ ・２Ｈ ₂ Ｏの濃度が３．３ｍｇ／ｍＬに等しいか、またはそれより高い場合、アルギン酸塩とＣａＣｌ ₂ 溶液との境界面でアルギン酸塩が即座にゲル化したが、２．７ｍｇ／ｍＬに等しいか、またはそれより低い場合、アルギン酸塩を１．５％（ｗ／ｖ）の最終濃度で用いた場合、全くゲル化しようとしなかったため、ＣａＣｌ ₂ 単独では、ヒドロゲルを架橋させるには不適切であることを発見した。アルギン酸塩濃度と望ましいゲル強度に応じて、ＣａＣｌ ₂ は、０．５ｍｇ／ｍＬ〜３．０ｍｇ／ｍＬの範囲が考えられる。
典型的なアルギン酸ナトリウムと化学量論的に反応させるのに必要なＣａＳＯ ₄ ・２Ｈ ₂ Ｏの量は、アルギン酸塩１ｍｇあたり０．３０９ｍｇである。典型的な既知の硫酸カルシウムで架橋された配合物は、アルギン酸ナトリウム１ｍｇあたり２０ｍｇのＣａＳＯ ₄ ・２Ｈ ₂ Ｏ粉末、または、必要量の約６５倍を使用している。しかしながら、混合および注射の問題は、ＣａＳＯ ₄ 粒子周囲に、溶液中のＣａ ⁺² の移動を妨げるゲルが形成され、さらに、アルギン酸塩がカプセル封入されたＣａＳＯ ₄ の塊が形成されて注射器のカニューレを介した注射が妨げられることによって発生する。
必要な時間内で許容できるコンシステンシィの均一なゲルを生産する比率が好ましい。ＣａＳＯ ₄ ／ＣａＣｌ ₂ の好ましい割合は、ポリマー（例えば、アルギン酸塩に関するＭ／Ｇ比）、ゲル化に必要な時間、必要な組織強度などに依存する。一例として、アルギン酸塩１ｇあたり０．３０９ｇのＣａＳＯ ₄ ・２Ｈ ₂ Ｏと結合できるアルギン酸ナトリウムにとって、ＣａＣｌ ₂ ・２Ｈ ₂ Ｏ／ＣａＳＯ ₄ ・２Ｈ ₂ Ｏ／アルギン酸塩の割合は、１：１０：１０が可能であり、好ましくは、配合物中に存在する所定のリン酸塩の量と一致している。
適切な組成物としては、室温で約１４分間で、許容できるコンシステンシィのゲルが生じる比率を有するものが可能である。アルギン酸塩３０ｍｇあたり約３ｍｇの塩化カルシウムと３０ｍｇの硫酸カルシウムを示すＣａＳＯ ₄ ／ＣａＣｌ ₂ の割合は、アルギン酸ナトリウム１ｇあたり０．３０９ｇのＣａＳＯ ₄ ・２Ｈ ₂ Ｏと結合するアルギン酸塩との使用に適している。当業者であれば、様々なカルシウム結合能を有するアルギン酸塩の化学量論は容易に決定することができ、さらに、弱溶解度で大量の可溶性の塩の類似のモル比を計算して、これらの代わりの塩の適切な量を決定することができる。アルギン酸塩のＭ／Ｇ比は、Ｃａ結合能を決定する。好ましくは、アルギン酸塩、十分な可溶性の塩および弱溶解度塩、ならびに、場合により可溶性金属イオン封鎖剤の相対的量は、混合物が２０分間以内にゲルを形成し、そのゲルが重力に抗してその形状を保持すると予想されるが、少なくとも４時間（この時間は、室温で混合物をインキュベートするための時間である）は注射器のカニューレを閉塞させることなく注射が可能になるような量と予想される。
上述したように、遅延性の可溶性カチオン源のみを用いた配合物は、アルギン酸塩がカプセル封入された塊に、溶解していない塩と、回収して注射することができるより液状の部分との成分の分配を起こす。このような生成物は、コンシステンシィの点で再現性が低く、十分な特徴付け、十分な制御ができないか、または、変更されて最終配合物に各種の特性を与える可能性がある。迅速な可溶性のカチオン源の使用も同様に、十分に架橋されたゲルが部分的に形成されるにつれてカチオンを混合物全体に分布させることができない場合、仕切りを形成する可能性があり、容易に注射できない。これらの問題は、弱溶解度と大量に利用可能な多価カチオン源とを両方添加することによって回避される場合もある。しかしながら、発明者等は、過剰なカチオン結合能を与えるカチオンを封鎖するアニオンの存在下で、アルギン酸塩を２種のカチオン源と混合することによって、現存するゲルを上回る改善：極めて均一な混合、および、全体にわたり制御された、かつ一様の特性を有する、完全に注射可能な一貫した懸濁物の製造を示す最終配合物が製造されることを発見した。
その結果として、本発明に係るヒドロゲル形成組成物は、場合により、バイオポリマーに利用可能な架橋カチオンの量を緩衝すると予想される成分を含む。緩衝成分は通常、アニオン性金属イオン封鎖剤であり、例えばリン酸アニオンである。アルギン酸塩の迅速で完全な架橋は、Ｃａ ⁺² の結合においてアルギン酸塩と競合する配合のＰＯ ₄ アニオンで妨害され、それにより、ゲル化速度は大きく減少する。リン酸アニオンは、典型的には、約０．１Ｍで存在するが、リン酸濃度は、ゲル化時間を制御できるように０．０３Ｍ〜０．３Ｍの範囲で様々であってよい。また、同様の方法でカルシウムに関してアルギン酸塩と競合すると予想され、そのためにアルギン酸塩がカプセル封入された塩の粒子の形成を予防するその他の生体適合性の金属イオン封鎖剤も本発明で考慮される。
過剰なカチオン結合能力、および、その利用可能なカチオンに関するアニオンとアルギン酸塩との競合は、懸濁物全体にわたり完全な利用可能なカチオンの分配を可能にする。その生成物は、全体にわたり均質であり、一貫して生産され、は、従来の試験プロトコールによって特徴付けが可能であり、生成物を損失させることなく適用に完全に利用可能であり、および、望ましい特性（例えば粘度、ゲル強度、ゲル化時間など）が付与されるように調節することができる。この混合において均一性は、様々な混合様式（例えば、試験管中、磁気式および機械式撹拌器での、混合注射器内での、ボルテックス混合またはハンドミキシングなど）で実証されている。
アルギン酸塩の分解速度は十分に文書で示されておらず、例えば、架橋方法、アルギン酸塩のタイプ、および、投与部位に依存する。所定のアルギン酸塩ゲル配合物は、人体中では、多糖類を認識する酵素が欠けているため極めてゆっくりしか崩壊する。その上、アルギン酸塩は、その分解速度を増加または減少させる様々な物質で修飾および／または架橋することができる。アルギン酸塩は、組織の空間または空隙に注射または適用することができ、さらに望ましい形状に順応するように成形または鋳型加工することができる弱いゲルが製造されるように、２種のカルシウム源を用いて架橋することができる。適切な組成物の製造（ただし、本発明の配合における細胞の添加が省略される場合を除く）が、米国出願番号０８／７６２，７３３で詳細に説明されており、これは、参照によりその全体を開示に含む。
本明細書で説明されている配合物に対するアルギン酸塩の分解と線維形成反応の特徴を合わせると、本発明は、注射された際の寸法を維持し、移動せずに、さらに、現在の組織充填剤より著しく長い期間持続させることが可能な、永続的な組織様の本体を製造する。特に有益な特徴は、この材料は、様々な寸法の針を介して注射可能であることであり、それにより、わずかな外科的処置を介入させるだけで膀胱鏡または内視鏡の適用が可能になる。
細胞源
本発明の組成物は、多数の細胞の効率的な移行のため、及び新組織または組織均等物の創出の目的で移植体接合の促進のために三次元の骨組み内で、遺伝子変更細胞を包含する多重細胞タイプを与えるのに使用できる。それは新組織または組織均等物が生長している間に宿主免疫系を排除することにより、細胞移植体の免疫保護のためにも使用できる。
ここに記載されているように移植できる細胞の例としては、軟骨細胞及び軟骨細胞を形成するその他の細胞、骨芽細胞及び骨芽細胞を形成するその他の細胞、筋肉細胞、繊維芽細胞ならびに器官細胞があり、また単離幹細胞の拡張物も含まれる。本明細書で用いられる場合、器官細胞とは、肝細胞、小島細胞、腸源細胞、腎臓から誘導された細胞、ならびに物質を合成し、分泌しまたは代謝する作用を主とするその他の細胞が包含される。
注入可能ヒドロゲルに含臓されるべき細胞は、ドナーから直接に、ドナーからの細胞の細胞培養から、または確立された細胞培養ラインから得られる。好ましくは態様において、細胞は、ドナーから直接得て、洗浄され、ポリマー物質と組合せて直接に移植される。細胞物質は、解離された単細胞でも、集塊組織（すなわち集合細胞の塊）または解離細胞から試験管内で生じた細胞集合体であってもよい。細胞は、組織培養の当業者に公知の方法で培養できる。
注入後の細胞持続性及び生存性は、走査電子顕微鏡、組織学、及びラジオアイソトープでの定量分析によって測定できる。移植細胞の機能は、上記の技法及び機能測定の組合せによって測定できる。例えば肝細胞の場合に、生体内肝機能研究は、患者の共通胆汁ダクト内にカニューレを配置することによって行うことができる。それにより胆汁は少量づつ捕集できる。胆汁色素は、基本テトラパイロールスを観察する高圧液体クロマトグラフィーにより、あるいは、Ｐ−グルクロニダーゼでの処理または処理なしで、ジアゾ化アゾジパイロールスエチルアンスラニレートとの反応によりアソジパイロールスへ転化した後に薄層クロマトグラフィーにより分析できる。ジコンジュゲート化、またはモノコンジュゲート化ビリルビンも、結合胆汁色素のアルカリンメタノリシスの後に薄層クロマトグラフィーにより測定できる。一般に、機能する移植肝細胞の数が増すにつれて、コンジュゲート化ビリルビンのレベルが増大する。また簡単な肝機能試験は、血液試料において、アルブミン産生のような項目が実施できる。
同様な器官機能試験は、移植後の細胞機能の程度を測定するために、当業者に公知の技法で実施できる。例えば、膵臓の小島細胞は、糖尿病を治すためにインスリンの適切な分泌によってグルコース調節を達成するのに、肝細胞を移植するのに特定的に使用するものと同様な方式で分配できる。その他の内分泌組織も移植できる。標識グルコースを用いての研究、ならびにタンパク分析を用いての研究はポリマー骨組み上の細胞マスを定量するのに使用できる。これらの細胞マスの研究は、どれが適切な細胞マスであるかを決定するために、細胞機能研究と相関させうる。軟骨細胞の場合に、機能は、周囲の結合した細胞のための適当な支持体を与え得る軟骨マトリックス成分（例えば、プロテオグリカン類、コラーゲン類）の合成として定義される。
また、軟骨細胞の前駆細胞、または、多分化能性幹細胞から誘導された軟骨細胞に分化する能力を有する細胞を、軟骨細胞の代わりに用いることもできる。例としては、分化して軟骨細胞を形成する線維芽細胞または間葉幹細胞が挙げられる。本明細書で記載される用語「軟骨細胞」は、このような軟骨細胞の前駆細胞を含む。
混合プロトコール
部分硬化コンシステンシイを有するヒドロゲルを形成するのに反応させる成分についての添加の順序は大きく変化されうる。典型的には、任意に金属イオン封鎖アニオンを含有するアルギン酸塩は、一またはそれ以上の段階でカチオン源と混合される。一般に、生分解性液体ポリマー、例えば次の多価イオン塩との混合時に制御された割合で固化させるように設計されたアルギン酸塩が使用される。個々の成分の濃度及び混合インキュベーションの条件の変化は、（ａ）注入時の材料のコンシステンシイを制御するため、（ｂ）注入可能コンシステンシイを達成するのに必要とされる時間を制御するため、そして（ｃ）適切なキメ及び寸法保持のために受容組織の特定の要件に適合するように生体内最終細胞の性質を制御するために改変しうる。
個々の成分の濃度は、単独または組合せで、適用の前及び後の両方で配合物の種々の性質を変更して、（ａ）注入及び適用のため、（ｂ）移植体の良好な接合ならびに最終ゲル及び交換組織の所要の性質及び機能の創出のため、あるいは（ｃ）配合物の製造、分配及び患者への適用のために、特定の要件に適合するようにできる。例えば、詰った組織（例えば筋肉、皮下）へのヒドロゲル配合物の注入は、材料の流出を防ぐために高粘度を必要とする。粘度の変更は、（１）原料の粘度の選択（例えば、低、中または高粘度アルギン酸塩）、（２）ゲルの濃度（例えば、広範囲のゲル粘度を達成するために０．３％〜３．０％のアルギン酸塩の範囲を使用できる。）、（３）部分架橋結合度を制御するための多価カチ才ン源の量、または（４）カチオン結合のためにアルギン酸塩と競合するために与えられるアニオン濃度、のような多くの機構を、単独または組合せることにより達成できる。種々の細胞タイプによる移植体への良好な侵入、あるいは創出されるべき所望の交換組織の性質は、配合物成分の変更を必要とする。例えば、硬い詰った組織（例えば肝臓、軟骨）の創出は、より大きなポリマー鎖長または濃度（例えば＞１．５％アルギン酸塩及び／または高グルロン含量アルギン酸塩）を必要とする。
注入可能時間及び注入前の配合物の性質の制御は、本発明による治療物の製造、分配及び適用の助けとなる。最終配合物の加工時間、または注入可能コンシステンシイまで配台物を固めるのに必要とされる時間は、多くの方法、例えば与えられる高溶解性カチオンの量及び／またはカチオン結合の競合のために与えられるアニオンの濃度により、さらには濃度調節により、制御できる。例えば、注入可能コンシステンシイの時間は、温度によって、数分間の加工時間（３４〜３８℃）または１月以上（４〜８℃）を許容するように制御できる。温度は、ゲル形成速度において、非常に重要な役割を果たす。温度を上昇させると、例えば３７℃の温度の組織中へ材料を注入するときに起こるように温度を上げると、材料は硬化を進行して、完全架橋結合ヒドロゲルになる。実施例１に示されたように０．１ｘのＣａＳＯ₄・２Ｈ₂Ｏのレベルにおいて（すなわち配合バッチにおいて２０ｍｇ／ｍＬ）、部分硬化ゲルは体温で３２分間で固まり、少なくとも９０分間注入可能である。この３７℃でのＣａＳＯ₄の最良の場合は、注入可能コンシステンシイに達するまでになお過大な時間を必要とし、所望されるものよりも繁雑でありまた所望されるものと矛盾する。
カチオン源は、ゲル化及びゲルの性質を制御するのに選択でき、また分解を制御するのに組み合わせができる。迅速及び遅延架橋結合源を含む本発明による組成物は、硫酸カルシウムのみの配合物と比較して、より迅速な粘度上昇、及び完全架橋されるまでにより長い時間を与える。殊に好ましい態様において、部分硬化ヒドロゲルを形成する混合物は、著量のリン酸アニオンも含むであろう。典型的には、リン酸濃度は約０．１モルであろう。
アルギン酸塩の濃度を増加すると、ゲルがより多くの水を保持する助けとなり、その結果として、より濃厚なペースト及びより強いゲルが得られ、またゲル形成を加速する。好ましいアルギン酸塩レベルは、ヒドロゲルを形成する最終細胞含有懸濁物中で、少なくとも０．７５％、さらに好ましくは１．５％〜２．５％、そして３．０％までである。
本発明の好ましい具体例において、酸性バイオポリマーは、アルギン酸ナトリウムであり、充分可溶性塩は塩化カルシウムであり、そして弱可溶性塩は硫酸カルシウムである。アルギン酸塩は溶液少なくとも０．７５重量％、好ましくは約１．５％の量で存在する。自由溶解性塩は１０〜１５ｍＭのカルシウムイオンを与え、一方弱溶解性塩は、アルギン酸塩溶液中に、もしも完全に溶解されたならば約８倍のカルシウムイオンを供給する量で、分散された粉末の状態で与えられる。換言すれば、二つの塩によって、２価金属架橋結合剤として供給され、すなわち重量で１部：１０部の重量比の塩化カルシウム及び硫酸カルシウムの二つの塩によって与えられる。ヒドロゲル懸濁物を製造する好ましい方法において、２％アルギン酸塩溶液の９部がＭ−１９９のような適当な細胞懸濁媒体の２部と混合され、次いで１．８％の無水塩化カルシウムを含む同じ細胞懸濁媒体に１８％の硫酸カルシウムを懸濁させたもの１部と混合される。最終混合物は良く混合され、組織中への注入のために使用される。粘度の迅速な増加は、全成分を一緒に混合して１５分以内に起こると予想される。この混合物のコンシステンシイは、混合物の形成が重力に抗して保持されるように充分であるべきであるが、少なくとも２４時間は室温において注入可能のままであろう。
注入可能応用のために好ましい粘度は意図された用途によって変るが、（ａ）配合物容器（例えば注射器寸法、ピストン直径、適切な可触のために必要とされる抵抗、または適用速度）、（ｂ）適用装置（カテーテルまたは針の長さ、直径、組成）、（ｃ）受容組織の分離抵抗及び（ｄ）必要とされる分配タイプ（例えば骨または器官表面への局所適用、流出しなでの組織内への注入等）の種々のパラメーターのバランスである。
本発明の意図の範囲には、本発明の組成物の調製及び注入に使用するキットがある。そのようなキットは、組成物の成分を一つまたはそれ以上の容器に収容してあり、処方容器（例えば混合容器または注射器）及び／またはゲル破壊オリフィス（例：カニューレまたは注射針）が含まれる。
治療用途
典型的には、ヒドロゲル溶液は、患者の部位に直接注射され、そこでヒドロゲルが移植材を形成し、周囲組織から線維形成反応を誘導する。線維形成反応によって形成されたゆるい結合組織は、ヒドロゲル粒子を所定位置に保持し、組織形成に血管新生が付随して起こり、血管新生を起こした軟部組織本体が生成し、その容積を占めるコンシステンシィは移植の際に確立される。このようなヒドロゲル移植は、移植材料をその場で鋳型加工することなどの多種多様な再建術に用いることができ、３次元的な組織の欠陥を再建することができる。
あるいは、細胞は、本発明のヒドロゲル組成物に含まれていてもよいし、患者の部位に直接注射してもよく、そこで、ヒドロゲルが、細胞を分散させるマトリックスに硬化する。その他の代替法において、細胞をバイオポリマー溶液に懸濁してもよく、ここで、この溶液を、望ましい解剖学的な形状を有する固定式または膨張式の鋳型に注ぐか、または注射し、続いて硬化させ、細胞を分散させるマトリックスが形成され、これを患者に移植することができる。移植の後に、本ヒドロゲルは、最終的には分解し、生じた組織だけが残留する。このようなヒドロゲル−細胞混合物は、３次元的な組織の欠陥を再建するため、注文に応じて細胞の移植材料を鋳型加工すること、予め挿入された膨張式鋳型または骨組みを充填すること、同様に、一般的には組織移植などの多種多様な再建術に用いることができる。本発明は、このような、本発明に係る組成物を用いた移植を想定している。
軟骨細胞、好ましくは自己由来の軟骨細胞を、ｉｎ ｖｉｖｏで液状の生分解性、生相容性のポリマー物質（例えば凝固可能なアルギン酸塩、またはその他のキャリアー）と混合し、細胞懸濁物を形成する、膀胱尿管逆流および失禁の治療が説明されている。本発明のヒドロゲル懸濁物を、尿路に必要な制御を提供する組織の本体の形成を誘導するのに有効な量で、逆流が起こっている領域、または、充填剤が必要な領域に注射してもよい。このような懸濁物は、カルシウム塩と接触した場合に制御された速度で凝固するように設計された生分解性の液状ポリマー（例えばアルギン酸塩）、グルロン酸とマンヌロン酸とのコポリマーを含む。このようなヒドロゲル組成物は、望ましい部位に注射され、そこで、欠陥を直すための組織様の本体が形成される。また、本発明に係るヒドロゲル組成物は、周囲組織からの細胞の浸潤を補うために、軟骨細胞のような細胞を含んでいてもよい。この細胞の懸濁物には、採取され、高密度になるまで成長させ、必要に応じて時間経過させ、続いてカルシウム塩と接触した場合に制御された速度で凝固するように設計された生分解性の液状ポリマー（例えば、アルギン酸塩、グルロン酸とマンヌロン酸のコポリマー）と混合された軟骨細胞が含まれる。このような細胞は、ヒドロゲル形成組成物と混合され、続いて、望ましい部位に注射され、そこで、それらは増殖し、欠陥を直す。好ましいは、自己由来の軟骨細胞であり、なぜなら、この治療方法は、自己由来の細胞を用いるとＦＤＡの承認を必要としないためである。
アルギン酸塩のような弱く架橋されたポリマー物質をベースとするヒドロゲルを、欠陥を直す膀胱粘膜、例えば尿路に必要な制御を提供する膀胱粘膜の隆起が生じるのに有効な量で、欠陥領域に注射することができる。同様に、生分解性ポリマーは、人体内で組織構造を維持するための注射可能な移植材のための合成基材として役立つ。このようなアルギン酸塩懸濁物は、膀胱鏡を用いて容易に注射することができ、さらに、形成された組織は、閉塞をまったく起こすことなく、膀胱尿管逆流を直すことができる。
逆流の内視鏡的治療に理想的な注射可能な物質は、非抗原性、非移動性であり、かつ容積が安定している天然の充填剤と予想される。この手法は、１５分間未満で、マスク麻酔をする短い時間で、外来患者単位で、入院の必要がまったくなく行うことができる。膀胱のドレーンも膀胱周囲のドレーンも必要ない。全ての手法が内視鏡的に行われ、膀胱は外科的処置を受けないため、術後の不快感がまったくない。患者は、ほとんどすぐに正常な活動レベルに戻ることができる。
本明細書で説明されているゲル材料は、滅菌成分から製造された場所で配合され、分離したユニット（これは、混合して最終的なゲル材料を形成することができる）、または、最終的な架橋生成物を含む予め充填された注射器ののいずれかとして医師に供給することができる。
この材料は、声帯麻痺や軟部組織の損傷の空隙のような用途で、現存の組織充填剤（例えばコラーゲンペースト；Ｔｅｆｌｏｎペースト）の代用として用いてもよい。
実施例
本発明のより完全な理解を促進するために、以下に幾つかの実施例を示す。しかし、これらの実施例に開示された具体的な実施態様に本発明の範囲は制限されず、これらの実施例は単に例証の目的のためだけである。
実施例１
硫酸カルシウム濃度のゲル化時間における影響
この実験は、塩化カルシウムの不存在下で硫酸カルシウム濃度を変化させることの影響を示す。０℃、３７℃および室温（２３℃）で、配合物のその他の内容を変化させないで維持しながら、硫酸カルシウム量を０．０２ｇ／ｍＬ（０．１ｘ）〜０．２ｇ／ｍＬ（１．０ｘ）に変動させた。アルギン酸ナトリウム（Ｍ／Ｇ比が３５／６５のＵＰ ＭＶＧ等級、Ｐｒｏｎｏｖａ製）を、０．７９％の塩化ナトリウムを含有する０．１モルリン酸塩緩衝液（ｐＨ＝７．４）の２％溶液として調製した。アルギン酸ナトリウム溶液をＭ１９９細胞培地で１対１に希釈し、硫酸カルシウム粉末を２０ｍｇ／ｍＬ〜２００ｍｇ／ｍＬの範囲の量で加えた。混合後、試料溶液を、氷浴温度（０℃）、室温（Ｒ．Ｔ．）、または体温（３７℃）のいずれかに保った。マイラーシート上に各試料のアリコットを注入することにより周期的にゲル形成について試験した。
表Ａに結果を示す。ゲル形成時間（Ｘとして示した）および注入阻止時間（記号Ｂ）も示す。表Ａから明らかなように、添加塩化カルシウムの不存在下氷浴上ではゲル形成が観察されなかった。室温では、７０〜１００分の間でゲル形成が起こり、ヒドロゲル注入はその時間の２０分以内で阻止された。３７℃では、硫酸カルシウムの濃度に依存して最初のゲル化が３２分から１６分までに起こり、注入の阻止が最初のゲル化の１５分以内で概ね起こった。
これらの実験は、０℃で、１２０分後までゲル化が起こらなかった（ｎｃ）ことを示す。室温では、０．２ｘについて９０分でゲル化が始まり、１．０ｘについて７０分でゲル化が始まった。体温では、１．０ｘ配合について１６分でゲル化が始まり、ゲル化時間は硫酸カルシウム濃度の減少に伴ってほとんど直線的に減少する。ヒト体温でこの硫酸カルシウム架橋ヒドロゲル配合物は１６分内にゲル化可能性があるが、注入に数分のみしか許されず、それにプラスされて、送達やゲル化特性におけるロット間のばらつきをなくするために加熱装置およびシリンジに装着されるフィルターを加える必要性がある。これらの要求事項の双方とも配合物の臨床用途に複雑性を増しうる。温度はゲル形成速度に非常に重要な役割を果たす。表Ａから、０．１ｘ、すなわち配合されたバッチ中に６０ｍｇのＣａＳＯ₄・２Ｈ₂Ｏの量において、体温で３２分内でゲル化が起こり、少なくとも９０分間は注入できた。３７℃でＣａＳＯ₄のこの最もよい場合のシナリオは、注入までに多くの時間がなお必要であり、目標よりもより複雑でありしかもよりばらつきがあることである。

実施例２
塩化カルシウムと硫酸カルシウムの混合物
好適な配合物では、アルギン酸ナトリウムをｐＨ７．４のＫ2ＨＰＯ4緩衝液に溶解する。Ｍ１９９培地中に細胞を供給し、次いで、アルギン酸塩溶液中に懸濁させた。１．５ｍｌバイアル中に硫酸カルシウム粉末を貯える。Ｍ１９９培地に塩化カルシウムを１８ｍｇ／ｍＬの濃度で溶解させ、別のバイアルに貯える。この応用では、ＣａＣｌ₂溶液をＣａＳＯ₄粉末と混合し、次いで、アルギン酸塩と混合し、シリンジ中に入れ、約１５分間待ち、この配合物は注入される準備が整う。
ボルテックス上での試験管混合に加えて、この配合物のシリンジ混合が同じゲル化特性を呈したことを示す実験も実施した。配合物中に軟骨細胞を加える実験も行い、それが室温で１４分に成功裡にゲル化したが、さらに２時間以上にわたって注入可能性を維持した。後の実験は、この配合物は２週間以上にわたってその注入可能性を維持できることを示す。表２は、この配合物の成分を列挙する。

実施例３
ゲル化時間における温度の影響 塩化カルシウム／硫酸カルシウム塩混合物と混合したときのアルギン酸塩のゲル化に要する時間は温度により変動する。アルギン酸ナトリウム（ＵＰ ＭＶＧ等級、Ｐｒｏｎｏｖａ製）を、０．７９％の塩化ナトリウムを含有する０．１モルリン酸塩緩衝液（ｐＨ＝７．４）の２％溶液として調製した。２．２５ｍｌアルギン酸塩溶液を０．５ｍｌのＭ１９９細胞培養培地と混合した。この混合物に、１８ｍｇの塩化カルシウム／ｍｌおよび４５ｍｇの懸濁した硫酸カルシウム粉末を含有する０．２５ｍｌの細胞培養培地を加えた。混合後、各試料を所定の温度に保持し、ゲル形成について周期的に試験した。二試料におけるゲル形成が検出された時間を添付の表に示す。室温で（２０〜３０℃）で、ゲルは１３〜３０分で形成した。３７℃で、７〜８分ゲル形成にかかった。

実施例４
注入可能なアルギン酸塩ゲルの形成 混合物中の種々の成分の濃度をわずかに変動させてゲル化形成に要する時間の感度を以下に記載するように試験した。アルギン酸ナトリウム、塩化カルシウムおよび硫酸カルシウムの溶液を実施例１および２に記載したとおりにして調製した。但し、一または別の成分を基準値より１６％までプラスまたはマイナスに変動させた。混合後試料を室温に保ち、ゲル形成について周期的に試験をした。
表３にゲル形成時間と注入するのに適したコンシステンシーを維持した期間を示す。表中に、各成分の量について、基準値をＭ、基準値よりも１６％高い値をＨ、そして基準値よりも１６％低い値をＬで示す。これらのパラメーターの変動内で、ゲル化時間が１８分プラスまたはマイナス約１５％は、この範囲内のある特定の成分の変動にわずかしか影響がないことを示す。これらの溶液のすべては約２０分以内でゲル化したが、アルギン酸塩ゲルのコンシステンシーは２４時間後を超えてもなお注入するのに受け入れることができた。同様の方法で調製したこの配合物の試料は配合後１ヶ月を超えても適切な注入可能特性を示した。

実施例５
ＣａＳＯ₄粉末のＣａＣｌ₂／ＣａＳＯ₄混合物に対する比較 この実験は２配合物のゲルおよびゲル化プロセスを定量的に特性化し、最終ゲルの強度を定性的に決定するために行う。上述の実験の間、ゲル化およびゲル化するのに要する時間を目視観察により決定した。粘度計を使用することにより、アルギン酸塩がゲル化を開始するときの粘度変化を直接測定でき、ゲルの相対強度も定性的に決定できる。
ＣａＳＯ₄粉末によりゲル化したアルギン酸塩を実施例１について記述した通りにして調製し、そしてＣａＣｌ₂／ＣａＳＯ₄の混合物によりゲル化したアルギン酸塩を実施例２について記述した通りにして調製した。ＣａＣｌ₂／ＣａＳＯ₄試料をシリンジ混合で調製し、ＣａＳＯ₄粉末試料をボルテックス混合法を用いて調製した。シリンジ混合は、容易に行うことができしかも非常にばらつきのない生成物を得ることのできる非常に簡便な方法である。それは承認された生物医学器具の使用もでき、生成物が環境にさらされることから分離するクローズドシステムを作り上げる。５℃、２５℃および３７℃の温度におけるこれらの二種類の系のゲル化の過程に伴う粘度変化を測定し、結果を図１〜図３にプロットする。
室温では、ＣａＣｌ₂／ＣａＳＯ₄試料がゲル化して１３分で注入できるセットポイント（注入できるセットポイント：崩壊することなく積み重ねができるゲルのコンシステンシー）となった一方、ＣａＳＯ₄単独試料は５６分までゲルに硬化しなかった。ゲル化前のＣａＣｌ₂／ＣａＳＯ₄試料の粘度は約６６５ｃＰであり、ＣａＳＯ₄単独試料は２０２ｃＰであり、前者は後者の約３．３倍程度の濃さである。アルギン酸塩がゲル化を開始するとき、その粘度は急激に増加し、２分以内で１１００ｃＰから３０，０００ｃＰまでとなる非常に高い値（図１）に達する。ゲル化後、ＣａＣｌ₂／ＣａＳＯ₄試料の粘度は、ＣａＳＯ₄単独試料の約２倍である（約４０，０００ｃＰ対２０，０００ｃＰ）。
両配合物のゲル化は体温で促進される。ＣａＣｌ₂／ＣａＳＯ₄試料は４分でゲル化を開始し、ＣａＳＯ₄単独試料は１３分でゲル化を開始する。ゲル化前の粘度は、３７℃でＣａＣｌ₂／ＣａＳＯ₄試料およびＣａＳＯ₄単独試料についてそれぞれ約５２０ｃＰおよび１３５ｃＰである。ゲル化後、ＣａＣｌ₂／ＣａＳＯ₄試料の粘度は１６，６００ｃｐに達することができ、ＣａＳＯ₄単独配合物の粘度よりも約１０倍高く（図２）、ＣａＣｌ₂およびＣａＳＯ₄の混合はより強力なゲルを生成させ、移植用途により良好な組織分離を可能にすることができる。
ゲル化はより低い温度で非常に遅延する。同様に、ＣａＣｌ₂／ＣａＳＯ₄試料は、５℃のすべての時点においてＣａＳＯ₄単独よりも一層粘度が高い（図３）。
ＣａＳＯ₄単独の配合物に比較して、ＣａＣｌ₂およびＣａＳＯ₄の混合物を使用して、１）３〜４倍より高い最初の粘度；２）注入可能なコンシステンシーに到達するのにより短い時間；３）ゲル化後より高い粘度を与え、したがって、より良好な組織分離を与える。実施例６ 組織適応性および注入性特性を有するアルギン酸塩ゲルの組成物を示すために行われた実験 下の表４に列挙した成分を含有する組成物を調製した。この組成物の粘度は混合直後約１０００Ｃｐだった。混合後約１５分でゲル化が起こり、無傷であるが弱い架橋ゲルを生成した（モジュラス＝６．５９ｋｇｆ／ｃｍ²）。２２ゲージ針を通して注入するとき、ゲルは不規則形態片（「クランブル」）に破砕され、ゲル強度の減少をもたらす（モジュラス＝０．２３１ｋｇｆ／ｃｍ²）。
この物質を正常マウスの皮下部に注入し、６ヶ月間移植組織をモニターした。移植組織の組織学的評価をすると、５０ミクロン〜５００ミクロンの範囲の寸法のクランブルを観察した。移植組織の最初の体積は６ヶ月間持続した。経時の組織学的評価はクランブルを包む宿主から緩い結合組織を示し、皮下部に本明細書に記載した組成物のアルギン酸塩ゲルの注入は注入したゲルフラグメント中またはその周りの緩い結合組織の湿潤を促進させることを示す。このゲルは繊維形成応答の特性を示し、この応答により、結合組織は究極的にゲル物質の注入で形成された量のうち相当割合を占める。

理解を明確にするために、本発明を、具体的な実施態様に関連させて例証および実施例により、ある程度詳細に記載したが、その他の態様、利点および修正は本発明に関連する技術分野の当業者に明かであろう。前述の記載および実施例は例証することを目的としており、本発明を制限するものではない。本発明を実施するための上述の態様の修正は、医学、形成外科、組織培養および／または関連分野の熟達者に明かであり、それもまた本発明の範囲内であるものとし、この範囲は請求の範囲にのみ制限される。
本明細書に挙げたすべての刊行物、特許出願は本発明に関連する業界の熟達者の水準を示すものである。すべての刊行物および特許出願は、各個々の刊行物または特許出願を具体的におよび個別に参照として含めると本明細書中に記載した場合と同一の程度を限度として、参照として本明細書に含める。
［本発明の態様］
１．生相容性ポリマーからなる、動物中へ注入するための組成物であって、その生相容性ポリマーが安定な、部分硬化ヒドロゲルを形成することを特徴とする組成物。
２．組成物が、ブロックに成型されたといきに、重力に等しい力の下での流れに抵抗するペーストであり、そのペーストが動物中への注入に適当である、１の組成物。
３．注入可能ペーストが、不規則な形のヒドロゲル粒子の懸濁物から主としてなり、それらの粒子が３０ミクロンから５００ミクロンの直径を有する２の組成物。
４．注入可能ペーストが少なくとも０．１ｋｇｆ／ｃｍ ² のゲル強度を有し、粒子中のヒドロゲルが少なくとも３ｋｇｆ／ｃｍ ² のゲル強度を有する３の組成物。
５．脆いヒドロゲルを作り、そのヒドロゲルをオリフィスに通して、それによりヒドロゲルが破壊されて不規則な形の粒子を形成することにより、組成物が作られ、その組成物が動物中の組織平面を分離するのに充分なコンシステンシィを維持している粒子を含んでいる、１の組成物。
６．生相容性ポリマーがアルギン酸塩である１−５のいずれかの組成物。
７．多価金属カチオンの高度溶解性塩、多価カチオンの弱溶解性塩をさらに含む６の組成物。
８．高度溶解性塩及び弱溶解性塩が共にカルシウム塩である７の組成物。
９．高度溶解性塩が塩化カルシウムであり、弱溶解性塩が銅、カルシウム、アルミニウム、マグネシウム、ストロンチウム、バリウム、錫及びジ−、トリ−またはテトラー官能性有機カチオンからなる群より選択されるカチオンと、低分子量ジカルボン酸イオン、サルフェート及びカーボネートからなる群より選択されるアニオンと、を含む７の組成物。
１０．弱溶解性塩が硫酸カルシウムであり、混合物が塩化カルシウム／硫酸カルシウムを重量基準で０．００７５ないし１．０の比で含む９の組成物。
１１．混合物が重量で少なくとも０．５％のアルギン酸塩、及びアルギン酸塩グラム当たり少なくとも約０．００１ｇの塩化カルシウムを含む９の組成物。
１２．カルシウムイオンの結合のためにアルギン酸塩と競合する金属イオン封鎖剤を更に含む９の組成物。
１３．金属イオン封鎖剤がホスフェートアニオンを含む１２の組成物。
１４．組成物が生細胞をも含む１−１３のいずれかの組成物。
１５．細胞が軟骨細胞及び軟骨組織を形成するその他の細胞、骨芽細胞及び骨を形成するその他の細胞、筋肉細胞、線維芽細胞ならびに器官細胞からなる群より選択される１４の組成物。
１６．細胞が解離細胞及び細胞集合体からなる群より選択される１４−１５のいずれかの組成物。
１７．動物内の構造欠陥を、その治療の必要に応じて治療する方法であって、１−１６のいずれかによる組成物を欠陥の部位で動物中へ移植することからなる該方法。
１８．動物内の構造欠陥を、その治療の必要に応じて治療する方法であって、部分硬化された生分解性、生相容性のヒドロゲルであって、任意に生細胞を含む部分硬化ヒドロゲルを調製し、そのヒドロゲルを圧力下にオリフィス内を通過させて破壊して、ヒドロゲル粒子を含む懸濁物を作り、その懸濁物を欠陥の部位で動物中に移植する、ことからなり、そのヒドロゲルが移植後に、組織を包囲すること及び移植物の脈管化により、繊維形成応答を誘発する、上記治療方法。
１９．動物内の構造欠陥を、その治療の必要に応じて治療する方法であって、バイオポリマーまたは変性バイオポリマーからなり、任意に生細胞を含む生相容性ヒドロゲルを調製し、そのヒドロゲルを圧力下にオリフィス内を通して、組織平面を分離させるのに充分なコンシステンシィを有する流動性組成物を作り、そして、その流動性組成物を欠陥の部位で動物中へ移植する、ことからなる上記方法。
２０．動物中へ注入するのに適当であり、ｉｎ ｖｉｖｏで十分に硬化することになる、部分硬化ヒドロゲルを調製するためのキットであって、２価カチオンにより架橋結合する時にヒドロゲルを形成する、好ましくはアルギン酸塩である、ポリマー、２価の金属カチオンを含む充分な可溶性の塩、好ましくは塩化カルシウム、及び、銅、カルシウム、アルミニウム、マグネシウム、ストロンチウム、バリウム、錫、及びジ−、トリ−またはテトラ−官能性有機カチオンからなる群より選択されるカチオンと、低分子量ジカルボン酸、サルフェートイオン及びカーボネートイオンからなる群より選択されたアニオンとを、有する弱可溶性塩、好ましくは硫酸カルシウム、からなり、それらの成分が、哺乳動物細胞の懸濁物と一緒にされた時に３０分以内に、注入に適し、１．５％の濃度でポリマー、１．５ｍｇ／Ｌの濃度で充分可溶性塩、そして１５ｍｇ／Ｌの濃度で弱可溶性塩を有する部分硬化ヒドロゲルを形成するように、与えられており、かつ部分硬化ヒドロゲル少なくとも４時間粘度の変化を受けない、上記キット。
２１．１−１６のいずれかによる動物注入用組成物を調製するためのキットであって、（ａ）多価カチオンで架橋結合された時にヒドロゲルを形成することができる生相容性ポリマー、（ｂ）多価金属カチオンの高度溶解性塩、及び（ｃ）多価カチオンの弱溶解性塩、からなる、上記キット。
２２．６−１１のいずれかによる動物注入用組成物を調製するためのキットであって、（ａ）多価カチオンで架橋結合された時にヒドロゲルを形成することができる生相容性ポリマー、（ｂ）多価金属カチオンの高度溶解性塩、及び（ｃ）多価カチオンの弱溶解性塩、からなり、その生相容性ポリマー及び弱溶解性塩が粉末状であり、任意に単一の容器中にある、上記キット。
２３．カルシウムイオンの結合のためにアルギン酸塩と競合する金属イオン封鎖剤を更に含む２２のキット。 This application is a partial continuation of US 08 / 762,733; 60 / 051,084; and 60 / 059,558, each of which is hereby expressly incorporated by reference. .
Background of the Invention
Field of Invention
The present invention is directed to a method of implanting a suspension of cells and / or hydrogel particles for the creation of new tissues in vivo.
Examination of related technologies
Contour anomalies, whether malformed, congenital or aesthetic, have recently required invasive surgical techniques for correction. In addition, shape anomalies that require additives often require a foreign body forming prosthesis with infection and ejection problems. Collagen has been used to create tissue bulk in certain applications (eg, endogenous sphincter deletion self-control), as Teflon® paste is used (bladder urethral reflux, vocal cord paralysis). Impossibility: “Contigen” used to correct Intrinsic Spincter Defectivity Inconence), but collagen has been shown to degrade rapidly (6-12 December) in the body to retain bulk It requires multiple treatments and is therefore unsatisfactory for correction of, for example, cranium and facial abnormalities, as well as alginate in other formulations has been shown to degrade in a few months in the body. Teflon paste particles are preferentially selectively from the injection site to the brain , Has been shown to migrate.
Tissue processing techniques using biocompatible polymer frameworks are an alternative to prosthetic materials currently used in craniofacial surgery, as well as a means to create organ equivalents to replace disease, defect, or injured tissue As has been explored. Tissue processing involves neotissue morphogenesis from constructs made from isolated cells and biocompatible polymers. Cells can be grafted to form a cartilage structure that is joined to a polymeric matrix. This is done by shaping the matrix prior to implantation to the desired anatomical structure and surgically implanting the shaped matrix, as described in US Pat. No. 5,041,138 to Vacanti et al. Is achieved.
A simple method of adding additional autogenous cartilage or bone to the craniofacial skeleton at a specific site and controlled size will reduce surgical trauma and eliminate the need for allografts or foreign body forming prostheses. If transplanted to the desired site or transplanted by simple application and multiple isolated cells can be joined, the skeleton of craniofacial cartilage could be increased with autologous cells but without undue surgery. Furthermore, successful transplantation of isolated cells will create the potential for increased tissue culture of cells.
Clearly, injection transplantation is preferred, which allows many isolated cells to be joined and the craniofacial cartilage skeleton to increase in autogenous tissue without extensive surgery. However, it has been shown that dissociated cells have not been successfully injected subcutaneously or into certain areas of the body such as the peritoneum. Cells are removed relatively quickly, presumably by phagocytic activity and cell death.
To develop tissue processing applicability to fields such as plastic surgery, expand tissue processing techniques to systems that deliver cell-polymer constructs with low invasiveness and remain at the delivery site. Has been investigated. A mixture of dissociated cells and a biocompatible polymer in the form of a hydrogel is used to form a cellular tissue and cartilage structure containing non-cellular material, and such mixture is degraded and removed. It is expected to detach from tissue or cartilage that is histologically and chemically equivalent to naturally occurring tissue or cartilage. Slowly polymerizing biocompatible, biodegradable hydrogels have proven to be useful as a means to deliver large numbers of isolated cells to a patient and create an organ equivalent or tissue (eg cartilage) ing. Such gels are believed to promote engraftment and provide a three-dimensional template for the growth of new cells, and the resulting tissue is more histologically natural in terms of composition. May be similar to the organization that exists.
Unlike the use of solid polymer systems to create cell-polymer constructs, liquid compositions that polymerize to form a gel support matrix are easier to mold and mold upon rebuilding or increasing upon order. Can be processed. In addition, the liquid polymer system has the potential to be used for delivery by injection, which is expected to be much less invasive than open implants. Calcium alginate gel has been proposed as a component in the means of delivering a large number of isolated chondrocytes to promote conjugation and cartilage formation. These initial studies were expanded in International Patent Publication No. WO 94/25080 to formulate slowly polymerized calcium alginate gels and use these gels to deliver large numbers of chondrocytes and generate new cartilage by injection. Has been.
In 1981, endoscopic treatment of vesicoureteral reflux was first introduced when polytetrafluoroethylene (Teflon®) was injected into the area below the patient's ureter. Over a decade, the search for the ideal injectable material has continued. The transfer of particles to the distal organ has raised concerns about the use of polytetrafluoroethylene paste. Loss of implant volume requires a high probability of re-treatment, which limits the usefulness of collagen. The ideal implant material is expected to be non-migrating, non-antigenic, and transported endoscopically, and to maintain its volume. Towards this end, long-term studies elucidating the effects of chondrocytes in vitro and in vivo have shown that alginate and biodegradable polymers embedded in chondrocytes can be delivered injectable and in vivo. It has been elucidated that it may serve as a synthetic substrate for the maintenance of cartilage structure, and the system is being tested for vesicoureteral reflux treatment in a porcine model. However, the behavior of the system was not successful.
Appropriate cells are suspended in M199 medium and this cell suspension is mixed with the same amount of 2% sodium alginate solution, then solid calcium sulfate powder is added to initiate the alginate cross-linking and the hydrogel To produce an injectable composition comprising cells. Such a composition is typically expected to contain 1% sodium alginate in a hydrogel with insoluble calcium sulfate in an amount of about 200 mg per ml of suspension. A small amount of calcium chloride can be carried into the composition from the cell suspension, but is usually less than 0.1 mg / ml in the final suspension. Experience has shown that such suspensions have an incubation period of approximately 1 hour, followed by a rapid viscosity increase, producing a relatively hard but brittle gel within 30 minutes of the viscosity increase. It is.
However, the hydrogel-cell suspension consistency described above is not fully satisfactory for use in injecting patients in need. In some cases, such hydrogel-cell suspensions harden before being injected into a patient. Such gels are useful for manufacturing preformed structures for implantation into patients to repair structural defects of known shape, but such preformed structures Requires extensive surgery to open a gap large enough to accept. On the other hand, some hydrogel-cell suspensions maintain a low viscosity until they are injected into the desired location in the body, but such suspensions will, of course, form the void or space that exists in the patient. It seems to be. Such gels cannot be used if there are no defined structural defects. Thus, to minimize the size of surgery required for implantation, a hydrogel-cell suspension that has sufficient consistency to maintain its shape but remains injectable when implanted in a patient. Turbidity is still needed. In particular, the disadvantages that seem to make it impossible to use existing injectable cartilage formulations in clinical trials are: (a) insufficient viscosity to separate tissue planes and do not overflow during injection (B) extending the time required to reach a viscosity high enough to attempt an injection; and (c) inconsistent performance between lots due to inadequate distribution of ingredients in the formulation. It is done.
Summary of invention
One object of the present invention is to provide a composition that can be injected into a surgical site but that accepts and maintains the desired shape after injection.
In particular, it is an object of the present invention to have an implant that has a consistency similar to POLYTEF® at the time of infusion and that retains the consistency for a time sufficient to allow proper application. It is to provide a possible composition.
It is another object of the present invention to provide an implant that does not migrate from a desired site, maintains its shape against gravity, and can be applied to a specific area by several means including pumping through an injection cannula. Is the purpose. These and other objectives may be suitable for one or more of the following specific examples.
In one aspect, the present invention provides a composition for injection into an animal comprising a biocompatible polymer that forms a stable partially cured hydrogel. When used for injection into animals, the composition is preferably a paste that resists flow under a force equal to gravity when formed into a block in vitro. Typically, the injectable paste is a suspension made primarily from irregularly shaped hydrogel particles with a linear dimension in the range of 30 microns to 500 microns, the injectable paste having at least 0.1 kgf. / Cm²The hydrogel in the particles has a gel strength of at least 3 kgf / cm²It has a gel strength of Such compositions can be prepared by creating a brittle hydrogel and passing the hydrogel through an orifice to break the hydrogel and form irregularly shaped particles; the resulting composition (containing the particles) is It looks like a paste that retains sufficient consistency to separate the tissue planes in the animal.
In a preferred embodiment, the biocompatible polymer in the composition of the present invention is an alginate, and the composition further comprises a highly soluble salt of a polyvalent metal cation, a weakly soluble salt of a polyvalent cation. Both highly soluble and weakly soluble salts may be calcium salts. Alternatively, the highly soluble salt is a calcium salt and the weakly soluble salt is selected from the group consisting of copper, calcium, aluminum, magnesium, strontium, barium, tin, and di-, tri- or tetra-functional organic cations. It can consist of a cation and an anion selected from the group consisting of low molecular weight dicarbonate ions, sulfates and carbonates. In a particularly preferred embodiment, the weakly soluble salt is calcium chloride and the mixture contains calcium chloride / calcium sulfate in a ratio of 0.001 to 100, more preferably 0.0075 to 1.0 by weight. Preferably, the composition comprises at least 0.5% alginate by weight and at least about 0.001 g calcium chloride per gram alginate. In another preferred embodiment, the composition further comprises a sequestering agent that competes with alginate for calcium ion binding, and the sequestering agent may comprise a phosphate anion.
Optionally, the injectable composition of the present invention, including a biocompatible polymer that forms a hydrogel, is a chondrocyte and other cells that form cartilage tissue, osteoblasts and other cells that form bone, muscle cells It may also include live cells such as fibroblasts as well as organ cells. Typically the cells will be dissociated cells and / or cell aggregates.
In another aspect, the present invention provides a kit for use in a method, which itself is a biocompatible polymer capable of forming a hydrogel (or the polymer is provided in the form of a ready-made weakly cross-linked hydrogel. And an orifice (typically the orifice will be a syringe needle or a cannula) through which the hydrogel is forced through and thereby breaks the weakly cross-linked hydrogel. In one embodiment, the present invention is a kit for preparing a partially cured hydrogel that is suitable for injection into an animal and will fully cure in vivo (1 A) a biocompatible polymer capable of forming a hydrogel when cross-linked with a multivalent cation, (2) a highly soluble salt of the multivalent cation, and (3) a weakly soluble salt of the multivalent cation. In a preferred embodiment, the kit comprises (1) a polymer, preferably an alginate, that forms a hydrogel when cross-linked by a divalent cation, (2) a sufficiently soluble salt comprising a divalent metal cation, preferably , Calcium chloride, and (3) a cation selected from the group consisting of copper, calcium, aluminum, magnesium, strontium, barium, tin, and di-, tri- or tetra-functional organic cations, and a low molecular weight dicarboxylic acid, A weakly soluble salt having an anion selected from the group consisting of sulfate ions and carbonate ions, preferably calcium sulfate. The kit is suitable for injection when the components are combined, optionally including mammalian cells, within 30 minutes, with a polymer at a concentration of 1.5%, 1 Partial cure with fully soluble salt at a concentration of 5 mg / L and weakly soluble salt at a concentration of 15 mg / LHydroIs provided to form a gel gel, and even its partially cured hydrogelIsNo change in viscosity for at least 4 hours.
The components of the kit can be in separate containers, or two or more components can be combined in the kit. Optionally, the biopolymer and at least one salt may be in powder form, and in a preferred embodiment, the biocompatible polymer bran and salt powder are combined in one container. More preferably, both the weakly soluble salt and the biocompatible polymer are powders and are combined in one container. In another embodiment, the kit includes a sequestering agent that competes with alginate for binding calcium ions, which can be placed in a separate container or combined with one or more other components. It may be.
In yet another aspect, the present invention is a method for treating a structural defect in an animal as needed for its treatment, comprising implanting a composition according to the present invention into the animal at the site of the defect. In one aspect, a method of treating a structural defect in an animal as needed for its treatment is (1) a partially effected biodegradable, biocompatible hydrogel, optionally comprising living cells Preparing a partially cured hydrogel, (2) breaking the hydrogel through an orifice under pressure to create a suspension containing hydrogel particles, and (3) removing the suspension at the site of the defect There is provided a method of treatment as described above, comprising the step of implanting in, wherein the hydrogel induces a fibrogenic response by surrounding the tissue and vascularization of the implant after implantation. Alternatively, a method of treating a structural defect in an animal as needed for its treatment comprises (1) preparing a biocompatible hydrogel consisting of a biopolymer or a modified biopolymer, optionally containing living cells, (2 ) Passing the hydrogel through the orifice under pressure to create a flowable composition with sufficient consistency to separate the tissue plane; and (3) implanting the flowable composition into the animal at the site of the defect. It consists of a process.
In another aspect, the present invention provides a method of treating structural defects in an animal as needed for its treatment, wherein a particle suspension of a partially cured biocompatible hydrogel is prepared and the hydrogel suspension is treated. Transplant the turbid material into the animal at the site of the defect and the hydrogel is intact but as a weakly cross-linked gel (eg gel strength = 6.59 kgf / cm²), Which results in a decrease in gel strength when it is injected at a rate of 10 ml / min through a 22-gauge or neulet (eg, gel strength = 0.231 kgf / cm²) Provide the above method. The composition used in this method forms a partially cured hydrogel that is suitable for injection into animals and is fully cured in vivo after injection.
In a preferred embodiment, the present invention is generally in the form of calcium chloride, preferably in approximately 15 minutes to initiate and only partially crosslink the biocompatible anionic polymer (eg, alginate). Provides a readily available cross-linking cation source at a sufficient concentration. More preferably, the alginate is present in an amount sufficient to introduce all of the water in the formulation into the loosely crosslinked gel (typically greater than about 0.5% alginate. Are present in such injectable cell suspensions). Such a formulation hardens quickly for immediate use in the operating room, but maintains a good “injectable” consistency over an extended period of time to allow the procedure to be completed. Preferably, the partially crosslinked hydrogel maintains this consistency for more than 24 hours. Even more preferably, the consistency is relatively stable for a sufficient amount of time to allow the hydrogel to be prepared and marketed in a partially crosslinked state. In general, the suspension when injected has a thick consistency that more easily divides the tissue when injected and is less subject to overflow from the injection site.
In a preferred embodiment, the present invention is a method for treating an (anatomical) structural defect in an animal, preferably at the site of the defect, preferably a soluble salt of a multivalent ion and a multivalent ion. Provided is a method for treating a suspension comprising a biodegradable, biocompatible polymer that forms a hydrogel when cross-linked by a multivalent ion in the form of a weakly soluble salt by implantation into an animal. The components are combined together to form a partially cured, injectable hydrogel, where the partially cured hydrogel forms an implant of the desired size and shape in situ, and the size and shape after implantation. And the animal cells penetrate the hydrogel implant to form a fibrous cell mass having substantially the same shape as the cured hydrogel.
[Brief description of the drawings]
FIG. 1 shows the time course of gel formation at 25 ° C. for alginate cross-linked with calcium sulfate powder and calcium chloride / calcium sulfate, respectively.
FIG. 2 shows the time course of gel formation at 37 ° C. for alginate cross-linked with calcium sulfate powder and calcium chloride / calcium sulfate, respectively.
FIG. 3 shows the time course of gel formation at 5 ° C. for alginate cross-linked with calcium sulfate powder and calcium chloride / calcium sulfate, respectively.
Detailed Description of the Invention
The present invention provides improved compositions that can be implanted into animals in tissue processing applications, as well as methods for implanting such compositions into animals by injection. The composition of the present invention comprises a biocompatible polymer in the form of a hydrogel. The composition of the present invention can be used without adding live cells to the material to be transplanted, in which case the transplant will pre-invade cells of the surrounding tissue over time and the initial transplant. A cell mass having a body shape is made, or live cells are included in the composition, whereby another type of cell can be introduced into the area where the composition has been implanted by injection.
Individual embodiments of the present invention are directed to forming a long-lasting tissue-like bulk in an animal that can mimic the properties of certain soft tissues. The compositions described for the present invention can be injected to form a persistent bulking material that lasts for a period of time sufficient to make a practical correction of tissue voids due to injury, surgery, or inherited malformations. The composition can be applied by incision or injection and then shaped after application. The injected material messes with shape and subsequent fiber cell proliferation and loose connective tissue invasion creates an organized tissue to correct existing voids and to hold the hydrogel where it was placed in the injection . The applied gel material is invaded by loose connective tissue to maintain the desired volume and shape. This introduced material and the resulting fiber inflow persists in vivo for a long time.
The gel according to the invention has the property of stimulating the fibrogenic response. As described herein, injection of an alginate gel into the subcutaneous portion facilitates infiltration of loose connective tissue into or around the injected gel segment. Through this response, the connective tissue eventually occupies a significant percentage of the bulk created by the injection of gel material. A suitable composition according to the present invention is an injectable paste comprising irregularly shaped particles, typically of a biocompatible polymer in the form of a hydrogel. The suspension separates tissue when injected and retains sufficient consistency (viscosity) to avoid spillage from the injection site. The properties of the gel suspension that stimulates the fibrogenic response include particle size, and the hydrogel properties of the particles are important for both stimulating the fibrotic response and immobilizing the implant at the injection site.
Preferably, the composition of the present invention appears to be thixotropic. In other words, the hydrogel is partially cured (weakly cross-linked) and as a result the hydrogel resists deformation to a certain point; however, as the force increases, the composition exhibits fluid behavior. In a preferred embodiment, a brittle hydrogel is made and broken to form irregularly shaped particles of the desired size, for example by forcing the hydrogel through an appropriately sized orifice. The resulting hydrogel suspension will retain its shape against gravity. The suspensions can be stacked to form a mound that maintains the same shape without other support. The resulting paste block or mound resists flow like a mass of a flat toothpaste paste, but some rounding of the edges can occur.
Preferably, the hydrogel of the present invention is a brittle material that breaks when forced through an orifice such as a needle or cannula. The consistency of the cured hydrogel can vary with changing conditions. For example, a high calcium ion concentration increases the degree of cross-linking of alginate, and a high temperature increases PLURONICS® hydrogel, other cross-linking. Once it has gelled, the partially cured hydrogel according to the present invention is stable and is equal to or less than the effect of gravity for a period of at least 2 hours, more preferably at least 24 hours, and often weeks. Holds the shape against the force. A fractured hydrogel is a suspension of irregularly shaped particles with dimensions that are a function of orifice size and pressure. Typically, the dimensions are in the range of 30 μm to 500 μm, preferably 40 μm to 300 μm. Appropriate orifice size and injection pressure can be determined by one of ordinary skill in the art. Effective particle size can be ascertained by monitoring the fiber formation response as described in Example 6.
The hydrogel is cured in the syringe barrel and then the pressure action on the syringe plunger pushes the composition out of the syringe. In a typical formulation, the composition is extruded from a syringe. In a typical formulation, the composition is partially cured and then pushed through a 22 gauge cannula at a rate of 10 ml / min as a high viscosity fluid, but severe damage to cells that can be contained in the composition There is no. In a suitable formulation, after passing through the cannula, the composition is POLYTEF®.pasteIt has a consistency of For example, the composition is a paste with a measured viscosity of 15,000 cPs at 25 ° C., and when the composition contains cells, extrusion of the composition at a rate of 10 ml / min through the 22 gauge cannula Substantially no breakage (ie, reduced cell viability by 25% or more).
The cell persistence and viability of the implant after injection and fibrogenesis can be measured by scanning electron microscopy, histology, and quantitative analysis with radioisotopes. Cell function can be measured by a combination of the techniques and functional measurements described above. Studies using labeled glucose, as well as studies using protein assays, can be performed to quantify cellular mass on polymer scaffolds. These cell mass studies can be linked to cell function tests to determine which cell mass is appropriate. In the case of chondrocytes, function is defined as the synthesis of cartilage matrix components (eg, proteoglycans, collagens) that can provide a suitable support for surrounding bound cells.
The biopolymer-containing composition of the invention is prepared in the form of an aqueous suspension of hydrogel particles. In general, the hydrogel compositions of the present invention typically include an aqueous medium, which itself typically includes one or more pharmaceutically acceptable salts and optionally other ingredients. For example, when the biopolymer is an alginate, the hydrogel is enriched with a solution of alginate (eg, soluble sodium alginate) in an amount sufficient to initiate alginate cross-linking to form a partially cured hydrogel. Made by mixing with a solution containing valence ions. Although the amount and type of salt can vary, normally the solute and solute concentration are selected to avoid undue stress on the animal when the composition is injected into animal tissue. When the hydrogel also contains living cells, the other components of the composition are compatible with the living cells, and preferably the composition contains factors that are advantageous for cell growth and maintenance.
polymer
The polymer material used in the implant according to the invention must form a hydrogel. Hydrogels are defined as materials that can be created when an organic polymer (natural or synthetic) is cross-linked through covalent, ionic or hydrogen bonds to trap water molecules and create a three-dimensional open lattice structure. . Examples of materials that can be used to form hydrogels include polysaccharides such as alginate, carrageenan, polyphosphadine, and ionic cross-linked polyacrylates, or cross-linked by temperature or pH, respectively. Block copolymers such as Pluronics® or Tetronics® polyethylene oxide-polypropylene glycol block copolymers are included.
In general, these polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions, and the polymers contain charged side chains, or monovalent ionic salts thereof. May be included. Examples of polymers with acidic side chains that can react with cations are poly (phosphazene), poly (acrylic acid), poly (methacrylic acid), copolymers of acrylic acid and methacrylic acid, poly (vinyl acetate) and sulfonated polystyrene. Sulfonated polymers such as Copolymers having acidic side chains obtained by reaction of acrylic or methacrylic acid and vinyl ether monomers or polymers can also be used. Examples of acidic groups are carboxylic acid groups, sulfonic acid groups, halogenated (preferably fluorinated) alcohol groups, phenolic OH groups and acidic OH groups.
Examples of polymers with basic side chains that can react with anions are polyvinylamine, polyvinylpyridine, polyvinylimidazoline, and some imino substituted polyphosphazines. Polymer ammonium or quaternary salts may also be formed from backbone nitrogen or pendant imino groups.
Polyphosphazenes are polymers that have a backbone composed of nitrogen and phosphorus separated by alternating single and double bonds. Each phosphorus atom is covalently bonded to two side chains (“R”). The repeating unit in the polyphosphazene has the general formula (I):

Here, n is an integer.
Suitable polyphosphazenes for cross-linking are acidic and have multiple side chains that can form salt bridges with di- or trivalent cations. Examples of preferred acidic side chains are carboxylic acid groups and sulfonic acid groups. Hydrolytically stable polyphosphazenes are formed from monomers having carboxylic acid groups that are crosslinked by divalent or trivalent cations such as Ca 2+ or Al 3+. By introducing a monomer having an imidazole, amino acid ester or glycol side chain group, a polymer decomposable by hydrolysis can be synthesized. For example, polyanionic [bis (carboxylatophenoxy)] phosphazene (PCPP) can be synthesized, which is cross-linked with dissolved polyvalent cations in an aqueous medium at room temperature or below to create a hydrogel matrix. .
Biodegradable polyphosphazenes have two different types of side chains: acidic side chains that can form salt bridges with polyvalent cations, and side groups that hydrolyze under in vivo conditions, such as imidazole groups, amino acid esters, glycerol And glucosyl. The term biodegradable as used herein is acceptable when exposed to a pH 6-8 physiological solution having a temperature of about 25 ° C-38 ° C in the desired application (usually in vivo therapy). It means a polymer that dissolves or degrades over a certain period of time, meaning a polymer that dissolves or degrades in about 5 years or less, most preferably in about 1 year or less. Side chain hydrolysis results in polymer collapse. Examples of hydrolyzable side chains are unsubstituted or substituted imidizole and amino acid esters in which the group is bonded to the phosphorus atom via an amino bond (polyamides in which both R groups are bonded in this way). Phosphazene polymer). For polyimidazole phosphazenes, some of the R groups on the polyphosphazene backbone are imidazole rings that are bonded to phosphorus in the backbone through a ring nitrogen atom. Other R groups are organic residues that do not participate in hydrolysis, methylphenoxy groups, or Allcock, etc.Macromolecules10: 824-830 (1977) and other groups described in the scientific literature.
Methods for the synthesis and analysis of various types of polyphosphazenes are described in Allcock H. et al. R. Etc.Inorg. Chem .11, 2548 (1972); Allcock et al.Macromolecule s16, 715 (1983); Allcock et al.Macromolecules 19, 1508 (1986); Allcock et al.Biomaterials, 19, 500 (1988); Allcock et al.Macromolecules21, 1980 (1988); Allcock et al.Inorcr. Chem. 21 (2), 515521 (1982); Allcock et al.Macromolecule les22, 75 (1989); U.S. Pat. Nos. 4,440,921, 4,495174 and 4,880,622; Allgock et al., U.S. Pat. No. 4,946,938 to Magill et al.Controlled Re lease 3,143 (1986), the teachings of which are specifically incorporated herein by reference.
Methods for synthesizing the other polymers are known to those skilled in the art. For example,Con cise Encyclopedia of Polymer Scienceas well asPolymeric Amines and Ammonium Salt s, E.M. See Goethals edit (Pergamen Press, Elmsford, NY 1980). Many polymers are commercially available, such as poly (acrylic acid).
A water-soluble polymer having a charged side chain is crosslinked by reacting the polymer with an aqueous solution containing a multivalent ion of opposite charge, and if the polymer has an acidic side chain, a polyvalent cation or If the polymer has basic side chains, a multivalent anion is used. Preferred cations for cross-linking polymers with acidic side chains to form hydrogels are divalent and trivalent cations such as copper, calcium, aluminum, magnesium, strontium, barium, and tin, and alkylammonium Salt, for example

Di-, tri- or tetra-functional cations such as Aqueous solutions of these anions are added to the polymer to form soft, highly swollen hydrogels and membranes. The higher the cation concentration or the higher the valence, the higher the degree of cross-linking of the polymer. It has been found to crosslink the polymer at concentrations as low as 0.005M.
Preferred anions for crosslinking the polymer to form a hydrogel are low molecular weight dicarboxylic acids such as terephthalic acid, sulfate ions, carbonate ions. An aqueous solution of these anionic salts is added to the polymer to form a soft, highly swollen hydrogel and membrane as in the case of the cation. The biocompatible polymer and cross-linking agent can be dissolved in any physiologically compatible solvent. For example, here, the common medium M-119 is used as a representative solvent.
A variety of polycations can be used to complex and stabilize the polymer hydrogel into a semipermeable surface membrane. Polymers with basic reactive groups such as amines and imines having a preferred molecular weight of 3,000-100,000 are examples of materials that can be used, such as polyethyleneimine and polylysine. These are commercially available. One polycation is poly (L-lysine), and examples of synthetic polyamines are polyethyleneimine, poly (vinylamine) and poly (allylamine). There are also natural polycations such as polysaccharides and chitosan.
Polyanions that can be used to react with basic surface groups on the polymer hydrogel to form a semipermeable membrane include polymers and copolymers of acrylic acid, methacrylic acid and other derivatives of acrylic acid, pendants such as sulfonated polystyrene. There are polymers with SO3H groups and polystyrene with carboxylic acid groups.
Polymers intended for use in the implants of the present invention include other natural, synthetic or modified biopolymers that form hydrogels under suitable conditions that are similar to the hydrogels described herein. Include. Suitable biopolymers include, for example, modified alginate and other biopolymers (see, eg, Putnam AJ; Mooney DJ (1996) “Tissue engineering using synthetic extracellular matrices”, Nature Medjcine, 24 (7); Mooney, D.J. (1996), “Tissue engineering with biogradable polymer matrixes”, Bajpai, Prophulla K (Ed), Proc. South. Biomed. Eng. , 1996; and Wong, WH and DJ Mooney "Synthesi. See 0817639195); and properties of biodegradable polymers used as synthetic matrices for tissue engineering "Atala, such as editing" Synthetic Biopolymer Scaffolds (Tissue Engineering), Birkhauser, 1997, ISBN.
Alginic acid biopolymer composition
In a preferred embodiment, the method of the present invention comprises forming a hydrogel by cross-linking of an acidic biopolymer with a polyvalent cation, wherein the effective availability of the polyvalent cation is controlled by dissolving a weakly soluble salt. This is to replenish soluble cations as they are absorbed by the crosslinks. Some of the polyvalent cations are provided by sufficiently soluble salts and are freely available (fast cross-linking ions), while the polyvalent cations required for complete cross-linking are provided by weakly soluble salts (slow cross-linking ions). It is done.The preferred polymer for this embodiment is alginate; however, the method of the invention can also be applied to various acidic polymers such as the biopolymer κ-carrageenan.In the following description, alginate is representative of any suitable polymer.
preferableBioThe preferred multivalent ion for use with the polymer (alginate) is calcium.Here, using the multivalent cation source for supplying the crosslinked cation of the present invention (where the cation source is selected to have a different solubility), the gelation properties and final gel properties are controlled. The method to do this will be described using calcium and calcium salts as representative crosslinking agents. Other multivalent ions having similar properties are, of course, within the expected scope of the present invention. The composition provided by the present invention also preferably includes a soluble buffer system depending on the polyvalent cation, and is usually a phosphate when the cation is a divalent or trivalent metal ion. . Phosphate and alginate compete for metal, whereby when the components of the composition are mixed, the alginate anion and phosphate anion compete for a limited amount of available cations, Alginate crosslinking in the composition is preempted.
In a representative example, the alginate of a biocompatible acidic biopolymer is cross-linked by a small amount of fully soluble calcium chloride and a large amount of weakly soluble calcium sulfate. The composition from which the hydrogel is formed contains a relatively high concentration of phosphate ions, buffers soluble calcium ion concentrations, and prevents complete cross-linking of alginate. When the composition is injected at the desired site, phosphate ions are diluted by diffusion into the surrounding biological fluid, and the concentration of soluble calcium ions increases locally. As the concentration of soluble calcium ions increases, calcium provides additional cross-linking in the alginate and the hydrogel hardens.Similar effects can be obtained for similar metal ions by buffering with phosphate anions or other similar systems (eg, biocompatible sequestering agents that buffer soluble cation concentrations in a similar manner). There is sex.
Alginate from seaweedcan getPolymannuronate and polyguluronate stretch union. Suitable alginate is commercially available. Individual alginate preparations have a measurable binding calcium capacity that is a function of mannuronate: guluronate (M / G). The effects resulting from changing the ratio of M and G residues are described in US Pat. No. 4,352,883, which is hereby incorporated by reference. A suitable highly purified alginate has an M / G ratio of 35/65.
Alginate is divalent in water at room temperatureCationsIonically cross-linked byCan form a hydrogel matrix. Because of this mild condition,Alginate isIt is the most commonly used polymer for hybridoma cell encapsulation as described, for example, in Lim US Pat. No. 4,352,883.In Lim's process, an aqueous solution containing the biological material to be encapsulated is suspended in a water-soluble polymer solution, the suspension is made into droplets, and the droplets are contacted with multivalent cations. The surface of the microcapsule is then cross-linked with a polyamino acid to form a semi-permeable membrane around the encapsulated material.
Hydrogel-like cell / polymer systems are used for tissue processing, eg cell suspensions such as chondrocytes in an alginate solution to which calcium salts have been added to initiate hydrogel formation There is. Typical of these systems is a chondrocyte / calcium alginate solution, where the isolated cell suspension is washed with a sodium alginate solution (eg, 0.1 M K₂HPO_ThreeIn, 0.135 M NaCl, pH 7.4) and a cell density of 20 × 10 6 cells / mL in a 1.0% alginate solution.(Cell density about 50 percent of the cell density of wild bovine articular cartilage)Give rise to The chondrocyte-sodium alginate suspension is stored on ice at 4 ° C. until use and 0.2 g of sterile CaSO / mL of cold chondrocyte-alginate solution prior to injection._FourAdd powder.
According to studies on the gel-forming properties of the formulations using calcium sulfate as a cross-linking ion source (see eg Example 1), such formulations can gel in as little as 16 minutes at human body temperature. Although it has been shown that it does not gel easily at room temperature. If the temperature of the alginate / cell suspension rises to such a point that gel formation is initiated within a reasonable amount of time (ie, in the operating room, at the level of minutes rather than hours), the composition of the composition It only takes a few minutes for the tensy to be suitable for injection. To prevent such formulations from gelling at room temperature, it is necessary to use a heating device to incubate the formulation prior to injection. In addition, the mixing and injection problems are CaSO _Four Around the particles, Ca in solution ⁺² In which CaSO is formed, and alginate is encapsulated _Four This is caused by the formation of a lump of mass that prevents injection through the cannula of the syringe. Therefore, it is necessary to attach a filter to the syringe in order to screen the mass formed by the more rapid gelling region in the suspension. These features increase the complexity in clinical application of this formulation and make it difficult to achieve consistency and gelling properties in every lot-to-lot transport.
In order to avoid the need to heat the mixture and to add a filter to the syringe, the inventor has developed a new formulation for injectable hydrogels used in tissue processing. This formulation provides rapid gel formation and long injectability of the gel suspension, giving more consistent performance.
In order to quickly form a consistency sufficient to separate the tissue planes during injection and prevent overflow while avoiding the problem of clogging of the cannula of the syringe, the present invention provides 2 A hydrogel-forming composition comprising a source of species is provided: wherein the two sources are one of a small amount of cation source (providing fully available cations) and the other of A large amount of cation source (provides sustained release of cross-linked ions as the gel is formed). The first cation source is a salt with high water solubility, ie having a solubility of at least 1 g / 100 ml, preferably at least 30 g / 100 ml, more preferably at least 60 g / 100 ml. Particularly preferred is a calcium salt, such as calcium chloride. The second cation source is a weakly soluble salt, ie having a solubility of less than 1 g / 100 ml, preferably less than 0.5 g / 100 ml, more preferably less than 0.3 g / 100 ml. Particularly preferred is a calcium salt, such as calcium sulfate.Calcium sulfate dihydrate (CaSO) in cold and hot water (100 ° C)_Four・ 2H₂The solubility of O) is 2.41 and 2.22 mg / mL, respectively.Also, any two salts of polyvalent cations having water solubility similar to calcium chloride and calcium sulfate are each suitable for use in the present invention. Either salt alone may provide sufficient cross-linking cations to produce a fully cured alginate hydrogel, but the use of two salts extends the cross-linking action over time and thereby partially cures. Extends the period during which the hydrogel (weakly cross-linked hydrogel) has a consistency suitable for injection via cannula.
“Quick” Ca Calcium is preferred ⁺² One of the sources, and its high solubility can speed up the gelation of alginate. Other calcium salts with equivalent solubility work in a similar manner. CaCl ₂ Experiments were performed to determine whether it would work alone. A concentration range of 1.0 to 6.6 mg / mL was tested and CaCl ₂ ・ 2H ₂ If the concentration of O is equal to or higher than 3.3 mg / mL, alginate and CaCl ₂ When the alginate gelled immediately at the interface with the solution, but less than or equal to 2.7 mg / mL, the alginate was used at a final concentration of 1.5% (w / v) Because it did not try to gel at all, CaCl ₂ It has been found that alone is unsuitable for crosslinking hydrogels. Depending on the alginate concentration and the desired gel strength, CaCl ₂ Can be in the range of 0.5 mg / mL to 3.0 mg / mL.
CaSO required to react stoichiometrically with typical sodium alginate _Four ・ 2H ₂ The amount of O is 0.309 mg / mg alginate. A typical known calcium sulfate crosslinked formulation is 20 mg CaSO / mg sodium alginate. _Four ・ 2H ₂ O powder or about 65 times the required amount is used. However, the mixing and injection problem is CaSO _Four Around the particles, Ca in solution ⁺² In which CaSO is formed, and alginate is encapsulated _Four This is caused by the formation of a lump of mass that prevents injection through the cannula of the syringe.
A ratio that produces a uniform gel with acceptable consistency within the required time is preferred. CaSO _Four / CaCl ₂ The preferred ratio depends on the polymer (eg, M / G ratio for alginate), the time required for gelation, the required tissue strength, and the like. As an example, 0.309 g CaSO / g alginate _Four ・ 2H ₂ For sodium alginate that can bind O, CaCl ₂ ・ 2H ₂ O / CaSO _Four ・ 2H ₂ The O / alginate ratio can be 1:10:10 and is preferably consistent with the predetermined amount of phosphate present in the formulation.
Suitable compositions can have a ratio that results in an acceptable consistency gel in about 14 minutes at room temperature. CaSO showing about 3 mg calcium chloride and 30 mg calcium sulfate per 30 mg alginate _Four / CaCl ₂ The ratio is 0.309 g of CaSO / g of sodium alginate. _Four ・ 2H ₂ Suitable for use with alginate that binds O. One skilled in the art can readily determine the stoichiometry of alginate with various calcium binding capacities, and calculate the similar molar ratios of weakly soluble, large amounts of soluble salts to obtain these An appropriate amount of alternative salt can be determined. The M / G ratio of alginate determines the Ca binding capacity. Preferably, the relative amounts of alginate, sufficiently soluble and weakly soluble salts, and optionally a soluble sequestering agent are such that the mixture forms a gel within 20 minutes and the gel resists gravity. It is expected to retain its shape, but at least 4 hours (this is the time to incubate the mixture at room temperature) is expected to be an amount that will allow injection without occluding the syringe cannula. The
As noted above, formulations using only a delayed soluble cation source consist of an undissolved salt and a more liquid portion that can be recovered and injected into an alginate-encapsulated mass. Cause distribution of ingredients. Such products are poorly reproducible in terms of consistency and may not be well characterized, controlled well, or modified to give various properties to the final formulation. The use of a rapid soluble cation source can also form a partition if the cation cannot be distributed throughout the mixture as a fully cross-linked gel is partially formed and can be easily injected Can not. These problems may be avoided by adding both weak solubility and a multivalent cation source available in large quantities. However, the inventors have improved over existing gels by mixing alginate with two cation sources in the presence of anions that sequester cations that provide excess cation binding capacity: extremely uniform mixing, And it has been found that a final formulation is produced that shows the production of a completely injectable and consistent suspension with controlled and uniform properties throughout.
As a result, the hydrogel-forming composition according to the present invention optionally comprises components that are expected to buffer the amount of cross-linking cation available to the biopolymer. The buffer component is usually an anionic sequestering agent, for example a phosphate anion. The rapid and complete cross-linking of alginate is ⁺² With a composition that competes with alginate in binding _Four Blocked with anions, thereby greatly reducing the gelation rate. The phosphate anion is typically present at about 0.1M, but the phosphate concentration can vary from 0.03M to 0.3M so that the gel time can be controlled. Also, other biocompatible sequestering agents that are expected to compete with alginate for calcium in a similar manner and thus prevent the formation of alginate-encapsulated salt particles are also contemplated by the present invention. .
The excess cation binding capacity and competition between anions and alginate for the available cations allows for complete available cations partitioning throughout the suspension. The product is homogeneous throughout and consistently produced, can be characterized by conventional test protocols, is fully available for application without loss of product, and desirable It can be adjusted to impart properties (eg, viscosity, gel strength, gelation time, etc.). Uniformity in this mixing has been demonstrated in a variety of mixing modes, such as vortex mixing or hand mixing in a mixing syringe, in a test tube, with magnetic and mechanical stirrers.
The degradation rate of alginate is not well documented and depends, for example, on the crosslinking method, the type of alginate, and the site of administration. Certain alginate gel formulations disintegrate very slowly in the human body due to the lack of enzymes that recognize polysaccharides. Moreover, alginate can be modified and / or crosslinked with various substances that increase or decrease its degradation rate. Alginates can be injected or applied to tissue spaces or voids and using two calcium sources to produce a weak gel that can be molded or cast to conform to the desired shape. Can be crosslinked. The manufacture of suitable compositions (except where the addition of cells in the formulations of the present invention is omitted) is described in detail in US application Ser. No. 08 / 762,733, which is incorporated by reference in its entirety. Is included in the disclosure.
Combined with the characteristics of alginate degradation and fibrogenic response to the formulations described herein, the present invention maintains the dimensions when injected and does not move, and further allows current tissue filling. Produces a permanent tissue-like body that can last significantly longer than the agent. A particularly beneficial feature is that this material can be injected through needles of various sizes, thereby allowing cystoscope or endoscopic applications with only a few surgical interventions. Become.
Cell source
The composition of the present invention includes genetically modified cells within a three-dimensional framework for efficient migration of large numbers of cells and for the promotion of graft joints for the purpose of creating new tissue or tissue equivalents. Can be used to give multiple cell types to do. It can also be used for immune protection of cell transplants by eliminating the host immune system while new tissues or tissue equivalents are growing.
Examples of cells that can be transplanted as described herein include chondrocytes and other cells that form chondrocytes, osteoblasts and other cells that form osteoblasts, muscle cells, fibroblasts and organs There are also cells, and expansions of isolated stem cells are also included.As used herein,Organ cells include hepatocytes, islet cells, intestinal cells, cells derived from the kidneys, and other cells that mainly synthesize, secrete or metabolize substances.
The cells to be included in the injectable hydrogel can be obtained directly from the donor, from cell culture of cells from the donor, or from an established cell culture line. In preferred embodiments, the cells are obtained directly from the donor, washed and transplanted directly in combination with the polymeric material. The cellular material may be dissociated single cells or agglomerated tissue (ie, aggregates of aggregated cells) or cell aggregates generated in vitro from dissociated cells. Cells can be cultured by methods known to those skilled in the art of tissue culture.
Cell persistence and viability after injection can be measured by scanning electron microscopy, histology, and quantitative analysis with radioisotopes. The function of the transplanted cells can be measured by a combination of the techniques and functional measurements described above. In the case of hepatocytes, for example, in vivo liver function studies can be performed by placing a cannula in the patient's common bile duct. As a result, bile can be collected little by little. Bile pigments are converted to asodipyrols by reaction with diazotized azodipyrrols ethyl anthranilate by high pressure liquid chromatography to observe basic tetrapyrols or with or without treatment with P-glucuronidase And then analyzed by thin layer chromatography. Diconjugated or monoconjugated bilirubin can also be measured by thin layer chromatography after alkaline methanolysis of the bound bile dye. In general, as the number of functioning transplanted hepatocytes increases, the level of conjugated bilirubin increases. In addition, simple liver function tests can be performed on items such as albumin production in blood samples.
Similar organ function tests can be performed by techniques known to those skilled in the art to determine the degree of cellular function after transplantation. For example, pancreatic islet cells can be distributed in a manner similar to that specifically used to transplant hepatocytes to achieve glucose regulation by proper secretion of insulin to cure diabetes. Other endocrine tissues can also be transplanted. Studies with labeled glucose, as well as studies with protein analysis, can be used to quantify cell mass on polymer scaffolds. These cell mass studies can be correlated with cell function studies to determine which are the appropriate cell masses.In the case of chondrocytes, function is defined as the synthesis of cartilage matrix components (eg, proteoglycans, collagens) that can provide a suitable support for surrounding bound cells.
In addition, chondrocyte progenitor cells or cells having the ability to differentiate into chondrocytes derived from pluripotent stem cells can be used instead of chondrocytes. Examples include fibroblasts or mesenchymal stem cells that differentiate to form chondrocytes. The term “chondrocytes” described herein includes such progenitor cells of chondrocytes.
Mixing protocol
The order of addition for the components that are reacted to form a hydrogel having a partially cured consistency can vary greatly. Typically, alginate, optionally containing a sequestering anion, is mixed with a cation source in one or more stages. Generally, alginate designed to solidify at a controlled rate upon mixing with a biodegradable liquid polymer such as the next multivalent ion salt is used. Changes in individual component concentrations and mixing incubation conditions (a) control the consistency of the material at the time of injection, and (b) control the time required to achieve an injectable consistency. And (c) can be modified to control the properties of the final cells in vivo to meet the specific requirements of the recipient tissue for proper texture and dimension retention.
The concentrations of the individual components, alone or in combination, change the various properties of the formulation both before and after application, (a) for injection and application, (b) good bonding of the implant and Specific requirements may be met to create the required properties and functions of the final gel and replacement tissue, or (c) for formulation manufacture, distribution and patient application. For example, injection of a hydrogel formulation into clogged tissue (eg muscle, subcutaneous) requires high viscosity to prevent spillage of the material. Viscosity changes include (1) selection of raw material viscosity (eg, low, medium or high viscosity alginate), (2) gel concentration (eg, 0.3% to 3 to achieve a wide range of gel viscosities). A range of 0.0% alginate can be used), (3) the amount of multivalent killer source to control the degree of partial cross-linking, or (4) to compete with alginate for cation binding. Many mechanisms such as the anion concentration given to can be achieved alone or in combination. Good penetration of the implant by various cell types, or the nature of the desired exchange tissue to be created, requires modification of the formulation components. For example, the creation of hard packed tissue (eg liver, cartilage) requires larger polymer chain lengths or concentrations (eg> 1.5% alginate and / or high guluron content alginate).
Control of the injectable time and the nature of the formulation prior to injection aids in the manufacture, distribution and application of the treatment according to the present invention. The processing time of the final formulation, or the time required to set the platform to the injectable consistency, is due to many methods, such as the amount of highly soluble cations provided and / or cation binding competition. The concentration can be controlled by adjusting the concentration of the anion applied to the substrate. For example, the time of injectable consistency can be controlled to allow several minutes of processing time (34-38 ° C.) or more than a month (4-8 ° C.), depending on the temperature. Temperature plays a very important role in the rate of gel formation. Increasing the temperature, for example, when the temperature is increased, as occurs when injecting the material into tissue at a temperature of 37 ° C., causes the material to cure and become a fully crosslinked hydrogel. 0.1x CaSO as shown in Example 1_Four・ 2H₂At the O level (ie 20 mg / mL in a compounded batch), the partially cured gel will set in 32 minutes at body temperature and can be infused for at least 90 minutes. This CaSO at 37 ° C_FourThe best case is that it still requires an excessive amount of time to reach an injectable consistency, is more complicated than desired, and contradicts what is desired.
The cation source can be selected to control gelation and gel properties and can be combined to control degradation. Compositions according to the present invention comprising a rapid and delayed cross-linking source give a more rapid viscosity increase and longer time to complete crosslinking compared to calcium sulfate only formulations. In a particularly preferred embodiment, the mixture forming the partially cured hydrogel will also contain significant amounts of phosphate anions. Typically, the phosphate concentration will be about 0.1 mole.
Increasing the concentration of alginate helps the gel retain more water, resulting in a thicker paste and a stronger gel, and also accelerates gel formation. Preferred alginate levels are at least 0.75%, more preferably 1.5% to 2.5% and up to 3.0% in the final cell-containing suspension forming the hydrogel.
In a preferred embodiment of the invention, the acidic biopolymer is sodium alginate, the fully soluble salt is calcium chloride, and the weakly soluble salt is calcium sulfate. Alginate is at least in solution0.75It is present in an amount of% by weight, preferably about 1.5%. Freely soluble salts give 10-15 mM calcium ions, while weakly soluble salts are dispersed in an alginate solution in an amount that provides about 8 times more calcium ions if completely dissolved. Given in powder form. In other words, it is supplied by two salts as a divalent metal crosslinker, i.e. provided by two salts of calcium chloride and calcium sulfate in a weight ratio of 1 part to 10 parts by weight. In a preferred method of making a hydrogel suspension, 9 parts of a 2% alginate solution is mixed with 2 parts of a suitable cell suspension medium such as M-199 and then contains 1.8% anhydrous calcium chloride. Mixed with 1 part of 18% calcium sulfate suspended in the same cell suspension medium. The final mixture is well mixed and used for injection into the tissue. A rapid increase in viscosity is expected to occur within 15 minutes of mixing all ingredients together. The consistency of the mixture should be sufficient so that the formation of the mixture is maintained against gravity, but will remain injectable at room temperature for at least 24 hours.
The preferred viscosity for injectable applications will vary depending on the intended use, but (a) the formulation container (eg syringe size, piston diameter, resistance required for proper accessibility, or application speed), ( b) the application device (catheter or needle length, diameter, composition), (c) the separation resistance of the receiving tissue and (d) the required distribution type (eg local application to the bone or organ surface, without draining) The balance of various parameters such as injection into the tissue.
Within the scope of the intent of the invention are kits used for the preparation and infusion of the compositions of the invention. Such kits contain the components of the composition in one or more containers and include a formulation container (eg, a mixing container or syringe) and / or a gel breaking orifice (eg, a cannula or needle). .
Therapeutic use
Typically, the hydrogel solution is injected directly into the patient site, where the hydrogel forms an implant and induces a fibrogenic response from the surrounding tissue. The loose connective tissue formed by the fibrogenic reaction holds the hydrogel particles in place, the tissue formation is accompanied by angiogenesis, and the angiogenic soft tissue body is generated and occupies its volume. Shi is established at the time of transplantation. Such hydrogel implantation can be used in a wide variety of reconstruction procedures, such as in-situ casting of the implant material, and can reconstruct three-dimensional tissue defects.
Alternatively, the cells may be contained in a hydrogel composition of the present invention or injected directly into a patient site where the hydrogel hardens into a matrix that disperses the cells. In other alternatives, the cells may be suspended in a biopolymer solution, where the solution is poured or injected into a fixed or inflatable mold having the desired anatomical shape, followed by A matrix that hardens and disperses the cells is formed and can be implanted into the patient. After implantation, the hydrogel eventually degrades, leaving only the resulting tissue. Such hydrogel-cell mixtures can be used to remodel three-dimensional tissue defects, such as casting cell implants on demand, filling pre-inserted inflatable molds or scaffolds, and so on. In general, it can be used for a wide variety of reconstruction techniques such as tissue transplantation. The present invention envisions transplantation using such a composition according to the present invention.
Chondrocytes, preferably autologous chondrocytes, are mixed with an in vivo liquid biodegradable, biocompatible polymeric material (eg, a coagulable alginate or other carrier) and the cell suspension is The treatment of forming vesicoureteral reflux and incontinence is described. Injection of the hydrogel suspension of the present invention into an area where reflux has occurred or an area where a filler is needed, in an amount effective to induce the formation of the body of tissue that provides the necessary control for the urinary tract May be. Such suspensions include biodegradable liquid polymers (eg, alginate), copolymers of guluronic acid and mannuronic acid, designed to solidify at a controlled rate when contacted with calcium salts. Such a hydrogel composition is injected at the desired site where a tissue-like body is formed to repair the defect. In addition, the hydrogel composition according to the present invention may contain cells such as chondrocytes in order to compensate for cell infiltration from surrounding tissues. This cell suspension is harvested and grown to a high density, allowed to elapse as needed, and subsequently designed to coagulate at a controlled rate when contacted with calcium salts. Included are chondrocytes mixed with a degradable liquid polymer (eg, alginate, a copolymer of guluronic acid and mannuronic acid). Such cells are mixed with the hydrogel-forming composition and subsequently injected at the desired site where they grow and repair the defect. Preferred are autologous chondrocytes because this method of treatment does not require FDA approval when using autologous cells.
A hydrogel based on a weakly cross-linked polymeric material such as alginate is applied to the defect area in an amount effective to produce a bulge of the bladder mucosa to repair the defect, for example, the bladder mucosa providing the necessary control for the urinary tract. Can be injected. Similarly, biodegradable polymers serve as synthetic substrates for injectable implants to maintain tissue structure within the human body. Such alginate suspension can be easily injected using a cystoscope, and the formed tissue can correct vesicoureteral reflux without causing any obstruction.
The ideal injectable material for endoscopic treatment of reflux is expected to be a natural filler that is non-antigenic, non-migrating, and stable in volume. This procedure can be performed in less than 15 minutes, in a short period of time for mask anesthesia, and on an outpatient basis with no need for hospitalization. There is no need for a bladder drain or a peri-bladder drain. All procedures are performed endoscopically and the bladder does not undergo surgical procedures, so there is no postoperative discomfort. The patient can return to normal activity levels almost immediately.
The gel material described herein can be formulated and separated in units where it is manufactured from sterilized components (which can be mixed to form the final gel material), or the final Can be supplied to the physician as any of the pre-filled syringes containing the appropriate cross-linked product.
This material may be used as a replacement for existing tissue fillers (eg, collagen paste; Teflon paste) in applications such as vocal cord paralysis and soft tissue damage voids.
Example
In order to facilitate a more complete understanding of the present invention, several examples are given below. However, the scope of the invention is not limited to the specific embodiments disclosed in these examples, and these examples are for illustrative purposes only.
Example 1
Effect of calcium sulfate concentration on gelation time
This experiment shows the effect of changing the calcium sulfate concentration in the absence of calcium chloride. At 0 ° C., 37 ° C., and room temperature (23 ° C.), the amount of calcium sulfate was maintained between 0.02 g / mL (0.1 ×) and 0.2 g / mL (1. 0x). 2% solution of sodium alginate (UP MVG grade with a M / G ratio of 35/65, manufactured by Pronova) in 0.1 molar phosphate buffer (pH = 7.4) containing 0.79% sodium chloride As prepared. Sodium alginate solution was diluted 1: 1 with M199 cell medium and calcium sulfate powder was added in amounts ranging from 20 mg / mL to 200 mg / mL. After mixing, the sample solution was kept at either ice bath temperature (0 ° C.), room temperature (RT), or body temperature (37 ° C.). Periodic gel formation was tested by injecting an aliquot of each sample onto a Mylar sheet.
Table A shows the results. Also shown is the gel formation time (shown as X) and the injection blocking time (symbol B). As is apparent from Table A, gel formation was not observed on the ice bath in the absence of added calcium chloride. At room temperature, gel formation occurred between 70-100 minutes and hydrogel injection was blocked within 20 minutes of that time. At 37 ° C, depending on the concentration of calcium sulfate, initial gelation occurred between 32 and 16 minutes, and injection blocking generally occurred within 15 minutes of the initial gelation.
These experiments show that no gelation occurred (nc) at 120C until 120 minutes. At room temperature, gelation started in 90 minutes for 0.2x and gelation started in 70 minutes for 1.0x. At body temperature, gelation begins in 16 minutes for the 1.0x formulation, and the gelation time decreases almost linearly with decreasing calcium sulfate concentration. At human body temperature, this calcium sulfate cross-linked hydrogel formulation may gel within 16 minutes, but only allows a few minutes for injection, plus it eliminates lot-to-lot variation in delivery and gelling properties. Therefore, there is a need to add a heating device and a filter attached to the syringe. Both of these requirements can add complexity to the clinical use of the formulation. Temperature plays a very important role in the gel formation rate. From Table A, 0.1x, ie 60 mg CaSO in blended batch._Four・ 2H₂In the amount of O, gelation occurred within 32 minutes at body temperature and could be infused for at least 90 minutes. CaSO at 37 ° C_FourThis best case scenario is that more time is still needed before injection, which is more complex and more varied than the target.

Example 2
Mixture of calcium chloride and calcium sulfate
In a preferred formulation, sodium alginate is dissolved in pH 7.4 K2HPO4 buffer. Cells were fed into M199 medium and then suspended in alginate solution. Store calcium sulfate powder in a 1.5 ml vial. Calcium chloride is dissolved in M199 medium at a concentration of 18 mg / mL and stored in a separate vial. In this application, CaCl₂The solution is CaSO_FourMix with powder, then mix with alginate, place in syringe, wait about 15 minutes and the formulation is ready to be injected.
In addition to test tube mixing on the vortex, experiments were also conducted to show that syringe mixing of this formulation exhibited the same gelling properties. Experiments were also performed to add chondrocytes into the formulation, which gelled successfully at room temperature for 14 minutes, but maintained injectability for more than 2 hours. Later experiments show that this formulation can maintain its injectability for over 2 weeks. Table 2 lists the ingredients of this formulation.

Example 3
Effect of Temperature on Gelation Time The time required for gelation of alginate when mixed with a calcium chloride / calcium sulfate salt mixture varies with temperature. Sodium alginate (UP MVG grade, manufactured by Pronova) was prepared as a 2% solution in 0.1 molar phosphate buffer (pH = 7.4) containing 0.79% sodium chloride. 2.25 ml alginate solution was mixed with 0.5 ml M199 cell culture medium. To this mixture was added 0.25 ml of cell culture medium containing 18 mg calcium chloride / ml and 45 mg suspended calcium sulfate powder. After mixing, each sample was held at a predetermined temperature and periodically tested for gel formation. The time at which gel formation in the two samples was detected is shown in the accompanying table. At room temperature (20-30 ° C), the gel formed in 13-30 minutes. It took 7 to 8 minutes to form a gel at 37 ° C.

Example 4
Formation of injectable alginate gel The sensitivity of the time required for gel formation was tested as described below, with slight variations in the concentration of the various components in the mixture. Solutions of sodium alginate, calcium chloride and calcium sulfate were prepared as described in Examples 1 and 2. However, one or another component was changed positively or negatively by 16% from the reference value. After mixing, the samples were kept at room temperature and periodically tested for gel formation.
Table 3 shows the gel formation time and the period during which the consistency suitable for injection was maintained. In the table, with respect to the amount of each component, the reference value is indicated by M, the value 16% higher than the reference value is indicated by H, and the value 16% lower than the reference value is indicated by L. Within these parameter variations, a gel time of 18 minutes plus or minus about 15% indicates that there is only a minor effect on the variation of certain components within this range. All of these solutions gelled within about 20 minutes, but the alginate gel consistency was still acceptable for infusion beyond 24 hours. Samples of this formulation, prepared in a similar manner, showed adequate injectable properties beyond one month after formulation.

Example 5
CaSO_FourPowdered CaCl₂/ CaSO_FourComparison to Mixture This experiment is performed to quantitatively characterize the gel and gelation process of the two formulations and to qualitatively determine the strength of the final gel. During the above experiments, the time required for gelation and gelation was determined by visual observation. By using a viscometer, the change in viscosity when the alginate begins to gel can be measured directly, and the relative strength of the gel can also be determined qualitatively.
CaSO_FourAlginate gelled with powder was prepared as described for Example 1 and CaCl₂/ CaSO_FourAn alginate gelled with a mixture of was prepared as described for Example 2. CaCl₂/ CaSO_FourSamples are prepared by syringe mixing and CaSO_FourPowder samples were prepared using a vortex mixing method. Syringe mixing is a very simple method that can be carried out easily and can give a very consistent product. It can also be used with approved biomedical instruments, creating a closed system that separates the product from exposure to the environment. Viscosity changes associated with the gelation process of these two systems at temperatures of 5 ° C., 25 ° C. and 37 ° C. are measured, and the results are plotted in FIGS.
At room temperature, CaCl₂/ CaSO_FourWhile the sample gelled and became a set point that can be injected in 13 minutes (set point that can be injected: gel consistency that can be stacked without collapsing)_FourThe single sample did not cure to gel until 56 minutes. CaCl before gelation₂/ CaSO_FourThe viscosity of the sample is about 665 cP and CaSO_FourThe single sample is 202 cP, and the former is about 3.3 times thicker than the latter. As the alginate begins to gel, its viscosity increases rapidly and reaches a very high value (FIG. 1), ranging from 1100 cP to 30,000 cP within 2 minutes. After gelation, CaCl₂/ CaSO_FourThe viscosity of the sample is CaSO_FourAbout twice that of a single sample (about 40,000 cP vs. 20,000 cP).
Gelation of both formulations is accelerated at body temperature. CaCl₂/ CaSO_FourThe sample started to gel in 4 minutes and CaSO_FourThe single sample begins to gel in 13 minutes. The viscosity before gelation is CaCl at 37 ° C.₂/ CaSO_FourSample and CaSO_FourAbout 520 cP and 135 cP, respectively, for a single sample. After gelation, CaCl₂/ CaSO_FourThe viscosity of the sample can reach 16,600 cp and CaSO_FourAbout 10 times higher than the viscosity of the single formulation (Figure 2), CaCl₂And CaSO_FourCan produce a stronger gel and allow better tissue separation for transplantation applications.
Gelation is very delayed at lower temperatures. Similarly, CaCl₂/ CaSO_FourSamples were CaSO at all time points of 5 ° C._FourHigher viscosity than single(Figure 3).
CaSO_FourCompared to a single formulation, CaCl₂And CaSO_Four1) Initial viscosity higher than 3-4 times; 2) Shorter time to reach injectable consistency; 3) Give higher viscosity after gelation and therefore better Give tissue separation. Example 6 Experiments Performed to Show Composition of Alginate Gels with Tissue Adaptability and Injectability Properties Compositions containing the ingredients listed in Table 4 below were prepared. The viscosity of this composition was about 1000 Cp immediately after mixing. Gelation occurred approximately 15 minutes after mixing, producing an intact but weak cross-linked gel (modulus = 6.59 kgf / cm²). When injected through a 22 gauge needle, the gel is crushed into irregularly shaped pieces (“crumbles”) resulting in a decrease in gel strength (modulus = 0.231 kgf / cm).²).
This material was injected subcutaneously into normal mice and the transplanted tissue was monitored for 6 months. Upon histological evaluation of the transplanted tissue, crumbles with dimensions ranging from 50 microns to 500 microns were observed. The initial volume of transplanted tissue lasted for 6 months. Histological evaluation over time shows loose connective tissue from the host encasing the crumble, and injection of the alginate gel of the composition described herein under the skin is wet of loose connective tissue in or around the injected gel fragment. To promote. This gel exhibits the characteristics of a fiber formation response, which causes the connective tissue to ultimately account for a significant percentage of the amount formed by the injection of the gel material.

For clarity of understanding, the invention has been described in some detail by way of illustration and example in connection with specific embodiments, but other aspects, advantages, and modifications may be found in the technical fields related to the invention. It will be apparent to those skilled in the art. The foregoing description and examples are intended to be illustrative and not limiting of the invention. Modifications of the above-described aspects for practicing the present invention will be apparent to those skilled in the fields of medicine, plastic surgery, tissue culture and / or related fields, which are also within the scope of the present invention, Limited only to the claims.
All publications and patent applications mentioned in the specification are indicative of the level of industry proficiency associated with the present invention. All publications and patent applications are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually included as a reference. include.
[Aspect of the Invention]
1. A composition comprising a biocompatible polymer for injection into an animal, characterized in that the biocompatible polymer forms a stable, partially cured hydrogel.
2. The composition of claim 1, wherein the composition is a paste that resists flow under a force equal to gravity as soon as it is molded into a block, the paste being suitable for injection into an animal.
3. 2. The composition of 2, wherein the injectable paste consists mainly of a suspension of irregularly shaped hydrogel particles, the particles having a diameter of 30 microns to 500 microns.
4). Injectable paste is at least 0.1 kgf / cm ² The hydrogel in the particles has a gel strength of at least 3 kgf / cm ² 3. A composition having a gel strength of
5. A composition is made by creating a brittle hydrogel and passing the hydrogel through an orifice, thereby breaking the hydrogel to form irregularly shaped particles, which separate the tissue planes in the animal The composition of claim 1 comprising particles that maintain sufficient consistency.
6). The composition of any of 1-5, wherein the biocompatible polymer is an alginate.
7). 6. The composition according to 6, further comprising a highly soluble salt of a polyvalent metal cation and a weakly soluble salt of a polyvalent cation.
8). 7. The composition according to 7, wherein the highly soluble salt and the weakly soluble salt are both calcium salts.
9. A highly soluble salt is calcium chloride and a weakly soluble salt is selected from the group consisting of copper, calcium, aluminum, magnesium, strontium, barium, tin and di-, tri- or tetra-functional organic cations; And an anion selected from the group consisting of low molecular weight dicarboxylate ions, sulfates and carbonates.
10. 9. The composition of 9, wherein the weakly soluble salt is calcium sulfate and the mixture comprises calcium chloride / calcium sulfate in a ratio of 0.0075 to 1.0 on a weight basis.
11. 9. The composition of 9, wherein the mixture comprises at least 0.5% by weight alginate and at least about 0.001 g calcium chloride per gram alginate.
12 9. The composition of 9, further comprising a sequestering agent that competes with alginate for binding calcium ions.
13. 12. The 12 compositions, wherein the sequestering agent comprises a phosphate anion.
14 The composition of any one of 1-13, wherein the composition also comprises living cells.
15. 14. A composition comprising 14 cells selected from the group consisting of chondrocytes and other cells forming cartilage tissue, osteoblasts and other cells forming bone, muscle cells, fibroblasts and organ cells.
16. The composition of any of 14-15, wherein the cells are selected from the group consisting of dissociated cells and cell aggregates.
17. A method of treating a structural defect in an animal as needed for its treatment, comprising transplanting a composition according to any of 1-16 into an animal at the site of the defect.
18. A method for treating structural defects in animals as needed for the treatment, comprising preparing a partially cured biodegradable, biocompatible hydrogel, optionally containing living cells Breaking the hydrogel through an orifice under pressure to create a suspension containing the hydrogel particles, and implanting the suspension into the animal at the site of the defect, The method of treatment, wherein after implantation, the fibrogenic response is induced by surrounding the tissue and vascularization of the implant.
19. A method of treating a structural defect in an animal as needed for its treatment, comprising preparing a biocompatible hydrogel comprising a biopolymer or a modified biopolymer, optionally containing living cells, and subjecting the hydrogel to pressure A fluid composition having sufficient consistency to separate the tissue plane through the orifice and implanting the fluid composition into the animal at the site of the defect.
20. A kit for preparing a partially cured hydrogel that is suitable for injection into an animal and that will fully cure in vivo, preferably forming a hydrogel when cross-linked by a divalent cation, preferably Alginate, a polymer, a sufficiently soluble salt containing a divalent metal cation, preferably calcium chloride, and copper, calcium, aluminum, magnesium, strontium, barium, tin, and di-, tri- or tetra- Consisting of a weakly soluble salt, preferably calcium sulfate, having a cation selected from the group consisting of functional organic cations and an anion selected from the group consisting of low molecular weight dicarboxylic acids, sulfate ions and carbonate ions, 30 minutes or less when the ingredients are combined with a suspension of mammalian cells Within, to give a partially cured hydrogel suitable for injection, having a polymer at a concentration of 1.5%, a sufficiently soluble salt at a concentration of 1.5 mg / L, and a weakly soluble salt at a concentration of 15 mg / L. The kit as described above, wherein the kit is partially cured hydrogel and is not subject to change in viscosity for at least 4 hours.
A kit for preparing a composition for animal injection according to any of 21.1-16, wherein (a) a biocompatible polymer capable of forming a hydrogel when crosslinked with a multivalent cation, ( The kit, comprising: b) a highly soluble salt of a polyvalent metal cation; and (c) a weakly soluble salt of a polyvalent cation.
A kit for preparing an animal injectable composition according to any of 22.6-11, wherein: (a) a biocompatible polymer capable of forming a hydrogel when crosslinked with a multivalent cation; b) a highly soluble salt of a polyvalent metal cation, and (c) a weakly soluble salt of a polyvalent cation, the biocompatible polymer and the weakly soluble salt being in powder form, optionally a single The kit in a container.
23. 22 kits further comprising a sequestering agent that competes with alginate for binding of calcium ions.

Claims (14)

A hydrogel composition for infusion into an animal to treat structural defects in the animal as needed for its treatment,
The hydrogel composition comprises an alginate, a highly soluble salt of a polyvalent metal cation, and a weakly soluble salt of a polyvalent cation,
The highly soluble salt of the polyvalent metal cation is calcium chloride, and the weakly soluble salt of the polyvalent cation is calcium sulfate.
Hydrogel composition. The hydrogel composition of claim 1, further comprising a sequestering agent that competes with alginate for binding of calcium ions. The hydrogel composition of claim 2, wherein the sequestering agent comprises a phosphate anion. The hydrogel composition of any of claims 1 to 3, wherein the hydrogel composition also comprises living cells. To prepare a partially cured hydrogel solution suitable for injection into animals and to treat structural defects in the animal as needed for its treatment, which will be fully cured in vivo A kit,
Alginate,
Calcium chloride, and
Contains calcium sulfate,
Within 30 minutes when the ingredients are combined with a suspension of mammalian cells, suitable for infusion, 1.5% alginate, 1.5 mg / L calcium chloride, and 15 mg A kit as described above, wherein the kit is provided to form a partially cured hydrogel having a calcium sulfate concentration of / L, and the partially cured hydrogel does not undergo a change in viscosity for at least 4 hours. A kit for preparing the hydrogel composition of claim 1, comprising (a) alginate, (b) calcium chloride, and (c) calcium sulfate. 7. The kit of claim 6, further comprising a sequestering agent that competes with alginate for binding of calcium ions. The kit of claim 7, wherein the sequestering agent comprises a phosphate anion. The kit according to any one of claims 6 to 8, wherein the hydrogel composition also contains living cells. A kit for preparing the hydrogel composition of claims 6-9, wherein (a), (b), and (c) are in powder form, optionally in a single container. A method of preparing a hydrogel composition for injection into an animal for treating structural defects in the animal as needed for the treatment comprising:
(A) preparing a hydrogel comprising partially crosslinked alginate, and (b) breaking the hydrogel by forcing it through an orifice to form a paste comprising irregularly shaped particles of the hydrogel Including
The hydrogel is crosslinked by a mixture of calcium chloride and calcium sulfate;
The above method. 12. The method of claim 11, wherein the hydrogel composition further comprises a sequestering agent that competes with alginate for binding of calcium ions. 13. The method of claim 12, wherein the sequestering agent comprises phosphate ions. 14. The method of claim 12 or 13, wherein the hydrogel composition also comprises live cells.

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