JP2004535245A - Porous extracellular matrix scaffold materials and methods - Google Patents

Abstract

A method for making an implantable scaffold for repairing damaged or diseased tissue includes suspending a piece of extracellular matrix material in a liquid. These extracellular matrix materials and liquids are formed into certain materials. Thereafter, the liquid is removed to form a plurality of gaps in the material. Further, a porous implantable scaffold made by such a method is also disclosed.

Description

【００３５】
相対的な気孔寸法を可視化するために各サンプルの表面の一定の走査電子顕微鏡画像を撮影した。これらの気孔寸法は約６００ミクロン乃至約７００ミクロンである。上記実施例１に従って調製した各サンプルを示す一定の画像が図１において示されている。
【００３６】
種々の気孔寸法が図１乃至図３および図８におけるような一定の発泡体の外表面部および図４乃至図７におけるような一定の発泡体の断面のそれぞれの走査電子顕微鏡画像により決定できる。これらの画像はそれぞれの気孔寸法の範囲を決定するために標準的な市場において入手可能な画像分析ソフトウェアと共に使用可能である。図４乃至図６は一定の発泡体の中における気孔寸法を測定および推定するための適当な市場において入手可能なソフトウェアによる結果をそれぞれ示している。この技法を用いて上記実施例１の発泡体が６００ミクロン乃至７００ミクロンの範囲内の気孔を有していることが決定されている。なお、このサンプルはさらに小さな気孔を含むことも可能である。
【００３７】
実施例２
この実施例２は一定の比較的に中程度の気孔寸法および一定の比較的に中程度の材料密度を有する一定の多孔質なＳＩＳ支持骨格材料の製造方法を示している。このような支持骨格材料は上記の粉砕したＳＩＳ材料を遠心分離処理により固めて、一定の比較的に低い温度まで（すなわち、−８０℃まで）一定の比較的に速い速度で（上記実施例１に比較して）凍結した後に、結果として得られた素材を凍結乾燥処理することにより得られる。この手順は以下のようである。すなわち、最初に、粉砕したＳＩＳを上記実施例１に関連して説明されているように製造した。具体的に言えば、はさみで切断したＳＩＳのランナー片（約６インチの長さ）を一定の適当な粉砕装置の中に２回通すことにより水中において比較的に均一に懸濁しているＳＩＳ材料（約２００ミクロンの太さ×１ｍｍ乃至５ｍｍの長さの細く長いＳＩＳ線維）の一定の「スラリー（slurry）」を製造する。
【００３８】
次に、４個の空の２４個ウェル型プレートのそれぞれの質量を記録する。その後、無菌条件による一定の無菌フード下において、一定の約３ｍｌ分量の上記の粉砕したＳＩＳ材料を上記組織培養プレートの各ウェル中に入れる。さらに、この材料の完充状態のプレートのそれぞれの質量を記録する。
【００３９】
その後、以下の技法の使用により遠心分離処理するために各プレートの重量を釣り合わせる。すなわち、２個のプレートを天秤に置き、これらの２個のプレートが釣り合うまで、比較的に軽いプレートの各ウェルの間の領域にＲＯ水を加える。その後、これら２個のプレートを遠心分離装置の中において互いに反対側に配置して、７分間にわたり３０００ｒｐｍにおいて遠心分離処理する。この処理の終了後に、各プレートを遠心分離装置から除去して、これらから水を除く。次に、各プレートのそれぞれの質量を記録する。さらに、残りのプレートについてこの遠心分離および質量測定の処理を繰り返す。
【００４０】
その後、上記のプレートを各試料が完全に凍結するまで一定の−８０℃のフリーザーの中に入れる。それぞれの材料の量に応じて、完全に凍結する時間が、例えば、約１分乃至約３０分等のように変わり得る。その後、凍結した各プレートを上記フリーザーから除去して、一定の適当な凍結乾燥装置の中に入れて上記実施例１に関連して説明されているものと同様のパラメーター（すなわち、８時間にわたる１３℃の一次乾燥温度における第１の期間に続き、４時間にわたる３５℃の二次乾燥温度における第２の期間）において凍結乾燥処理する。
【００４１】
上記凍結乾燥工程の完了後に、各プレートを凍結乾燥装置から除去して、各プレートの質量を決定して記録した。この方法による結果が以下の表においてまとめられている。
【表２】

【００４２】
上記実施例１のサンプルの場合と同様に、相対的な気孔寸法を可視化するために上記実施例２に従って調製した各サンプルの表面の一定の走査電子顕微鏡画像を撮影した。さらに、この実施例２に従って調製した各サンプルを示す一定の画像が図２において示されている。また、気孔寸法を決定するための上述した技法により、このサンプルが約１００ミクロン乃至１５０ミクロンの範囲内の気孔を有していることが分かった。
【００４３】
実施例３
この実施例３は一定の比較的に小さな気孔寸法および一定の比較的に高い材料密度を有する一定の多孔質なＳＩＳ支持骨格材料の製造方法を示している。このような支持骨格材料は上記実施例２におけるよりもさらに高い一定の密度に上記の粉砕したＳＩＳ材料を固めて、液体窒素によりこれらのサンプルをフラッシュ凍結処理した後に、凍結乾燥処理を行なうことにより得られる。この手順は以下のようである。すなわち、最初に、粉砕したＳＩＳを上記実施例１および２に関連して説明されているように製造した。具体的に言えば、はさみで切断したＳＩＳのランナー片（約６インチの長さ）を一定の適当な粉砕装置の中に２回通すことにより水中において比較的に均一に懸濁しているＳＩＳ材料（約２００ミクロンの太さ×１ｍｍ乃至５ｍｍの長さの細く長いＳＩＳ線維）の一定の「スラリー（slurry）」を製造する。この処理の後に、結果として得られた素材を一定の積載重量下に遠心分離処理する。この積載重量は以下のように調製する。コバン（Coban）の４８個の切片を５０ｍｍの長さで５ｍｍの幅（無延伸状態）の部材片に切り出す。その後、これらの部材片を引き伸ばして１ｃｍの直径で２ｍｍの厚さの一定のポリエチレン・ディスクの外縁部の周りに巻き付ける。必要であれば、各切片を整形して、上記コバンの各切片を上記ディスクの周囲に２回巻き付けることもできる。
【００４４】
次に、４個の空の２４個ウェル型プレートのそれぞれの質量を記録する。その後、無菌条件による一定の無菌フード下において、一定の約３ｍｌ分量の上記の粉砕したＳＩＳ材料を上記組織培養プレートの各ウェル中に入れる。さらに、この材料の完充状態のプレートのそれぞれの質量を記録する。その後、上記実施例２に関連して説明されているように各プレートの重量を釣り合わせて遠心分離処理する。その後、水を各プレートから排出して、遠心分離処理したプレートのそれぞれの質量を記録する。
【００４５】
上記の処理の完了後に、各プレートの各ウェルを２：１の一定の比率で組み合わせることによりプレートの数を４個から２個に減らす。さらに、少量の材料のウェルを多量の材料の各ウェルと混合して各ウェルにおいてある程度に一貫している量のＳＩＳ材料を得る試みを行なう。その後、このようにして完充したプレートの質量を記録した。次に、上記コバンを巻き付けた各ポリエチレン・ディスクを各ウェルの中に入れた。その後、上記２個のプレートを上記実施例２に関連して説明されている技法によりその重量を釣り合わせた。次に、これらのプレートを５分間にわたり３０００ｒｐｍで遠心分離処理した。その後、各プレートを遠心分離装置から除去して、水をこれらから排除し、ポリエチレン・ディスクも除去する。次に、各プレートの質量を再び記録する。さらに、これら２個のプレートの重量を（上記実施例２に関連して説明されているのと同様の一定の様式で）再び釣り合わせて、各プレートを７分間にわたり３０００ｒｐｍで再び遠心処理した。この処理の終了後に、水を再び各プレートから除去して、各プレートの質量を再び記録する。
【００４６】
上記の遠心分離処理と同時に、一定の液体窒素槽を一定の広口型の液体窒素容器の中に液体窒素を注ぎ入れることにより調製する。上記の各プレートはこの槽が準備されるまで遠心分離装置の中に保持されている。その後、各プレートがこの槽の中に浸漬されて、約１５秒間にわたりこの液体の中に保持される。この窒素槽から取り出す際に、各プレートは解凍を避けるために一定の−８０℃のフリーザーの中に速やかに入れられる。その後、これらの凍結したプレートはフリーザーから取り出されて、一定の適当な凍結乾燥装置の中に配置されて上記実施例１および２に関連して説明されているのと同様のパラメーター（すなわち、８時間にわたる１３℃の一次乾燥温度における第１の期間に続き、４時間にわたる３５℃の二次乾燥温度における第２の期間）に従って凍結乾燥処理される。
【００４７】
上記凍結乾燥工程の完了後に、各プレートを凍結乾燥装置から除去して、各プレートの質量を決定して記録した。この方法による結果が以下の表においてまとめられている。
【表３】

【００４８】
上記実施例１および２の各サンプルの場合と同様に、相対的な気孔寸法を可視化するために上記実施例３に従って調製した各サンプルの表面の一定の走査電子顕微鏡画像を撮影した。また、この実施例３に従って調製した各サンプルを示す一定の画像が図３において示されている。さらに、気孔寸法を決定するための上記の技法を用いて、上記サンプルが約４０ミクロン乃至６０ミクロンの範囲内の気孔を有していることが分かった。このような気孔寸法は図４乃至図６においてそれぞれ矢印により示されている。
【００４９】
図１乃至図８の操作電子顕微鏡の各画像において示されているように、図示の各ＥＣＭ発泡体は複数の相互接続している気孔を定めている網状の天然に発生するＥＣＭによる一定の三次元的な網状構造を含んでいる。この発泡体はその高さ、幅、および厚さの全体を通してこれらの気孔を有している。これらの気孔は連続状であり相互接続して上記発泡体の外表面部（図１乃至図３および図８を参照されたい）から当該発泡体の内部（図４乃至図７の各断面を参照されたい）まで延びている複数の不規則に形付けられた相互接続している通路を定めている。また、これらの相互接続している通路は三次元的である。なお、上述したように、各気孔の寸法、すなわち、上記の相互接続している通路に対応する最大の寸法は上記のような製造方法を制御することにより制御可能である。
【００５０】
上記の相互接続している各通路はその移植片の内部における細胞の移動を容易にして、生体内における効率的な栄養素の交換を可能にしている。これらの相互接続している通路はまた種々の生体活性物質、生物学的に誘導されている物質（例えば、種々の刺激因子）、細胞および／または生物学的な潤滑剤、生体相容性の無機材料、合成ポリマーおよび生体ポリマー（例えば、コラーゲン）を移植の前に上記ＥＣＭの長さ、幅および厚さの全体を通して伝達する一定の手段も提供している。また、このような気孔により定められている相互接続している通路は、上記発泡体を化学的に架橋するために用いられる種々の化合物等のような、上記の製造方法の途中において用いられる種々の材料のための通路としても作用する。さらに、これらの相互接続している通路ならびに上記発泡体の外表面部は上記種々の材料が担持される種々の部位としても作用できる。
【００５１】
上記の各実施例において示されているように、上記処理の各パラメーターは特定の用途に対応する所望の多孔度を有する一定のＥＣＭ発泡体を製造するために変更可能である。例えば、種々の骨細胞に関連する種々の用途に対応する比較的に低い密度（および比較的に高い多孔度）を有する一定の発泡体を製造すること、および種々の軟骨細胞に関連する種々の用途に対応する比較的に高い密度（および比較的に低い多孔度）を有する一定の発泡体を製造することが望ましいと考えられる。
【００５２】
さらに、本明細書において記載されている種々のＥＣＭ発泡体は架橋することもできる。具体的に言えば、本明細書において記載されているＥＣＭ発泡体は化学的にまたは物理的に架橋できる。
【００５３】
上記の図１乃至図８の走査電子顕微鏡の各画像において見られるように、図示の各ＥＣＭ発泡体は天然に発生する細胞外基質の相互接続している部材片を含んでいる。さらに、図４乃至図７の各ＥＣＭ発泡体の断面の走査電子顕微鏡画像において示されているように、天然に発生する細胞外基質の相互接続している部材片は上記発泡体に不規則な形状の一定の三次元的な形態を有する一定の内表面部を備えている。また、上記ＥＣＭ発泡体の各面部の走査電子顕微鏡画像において示されているように、上記の相互接続している天然に発生する細胞外基質の部材片は上記発泡体に不規則な形状の三次元的な形態を有する一定の外表面部を備えている。これらの不規則な三次元的な形態および相互接続している各通路により、本発明の開示の種々のＥＣＭ発泡体は天然に発生するＥＣＭの一定の比較的に大きな表面積を形成している。さらに、このような天然に発生するＥＣＭの大きな表面積は種々の生物学的な物質、生物学的に誘導されている物質、細胞、生体相容性のポリマーおよび生体相容性の無機材料が移植前に固定することのできる一定の比較的に大きな表面積を提供していることにおいて有利であると言える。加えて、例示されている各ＥＣＭ発泡体は種々の細胞が生体内において結合可能である天然に発生するＥＣＭの一定の比較的に大きな表面積を形成している。
【００５４】
上記のＥＣＭ発泡体の各製品は上記以外のＥＣＭ製品の密度よりも実質的に低い密度を有して作成することができる。比較のために、市場において入手可能なレストア（RESTORE）（登録商標）製品である一定のＥＣＭ積層体の密度は０．４６６±０．０７４ｇ／ｃｃである。また、「デバイスイズ・フロム・ナチュラリー・オカーリング・バイオロジカリー・デライブド・マテリアルズ（Devices from Naturally Occurring Biologically Derived Materials）」を発明の名称とする米国特許出願第ＸＸ／ＸＸＸ，ＸＸＸ号（代理人明細書番号第２６５２８０−７１１４２号，ＤＥＰ−７４８）において記載されているような粉砕および硬質化したＳＩＳにより構成されている一定のＥＣＭ製品は０．７４７±０．０５９ｇ／ｃｃの一定の密度を有して作成されている。さらに、「メニスカス・リジェネレーション・デバイス・アンド・メソッド（Meniscus Regeneration Device and Method）」を発明の名称とする米国特許出願第ＸＸ／ＸＸＸ，ＸＸＸ号（代理人明細書番号第２６５２８０−７１１４１号，ＤＥＰ−７４５）において記載されているような増強したＳＩＳ積層体により構成されている一定のＥＣＭ製品は０．９３３±０．０６１ｇ／ｃｃの一定の密度を有して作成されている。
【００５５】
上述したように、本発明の開示のＥＣＭ発泡体は種々の生体活性物質、生物学的に誘導されている物質、細胞および／または刺激因子、生体相容性の無機材料および／または生体相容性のポリマー（例えば、種々の生体相容性の合成ポリマーおよび生体ポリマー）およびこれらの材料の２種類以上の組み合わせ物を製造時において組み合わせることができる。例示的に、種々の細胞が上記ＥＣＭ発泡体の三次元的な空間全体に接種可能であり、種々の生物学的な材料が製造時に上記ＥＣＭ発泡体において乾燥可能であり、種々の生物学的材料および上記ＥＣＭ発泡体が同時に凍結乾燥可能であり、さらに、種々の生物学的材料が上記ＥＣＭ発泡体に対して共役的に連結できる。なお、上記の各材料の１種類以上を上記ＥＣＭ発泡体の形成前にその原料のＥＣＭ材料に対して結合、架橋、またはその他の方法により組み込むことが考慮されている。あるいは、これらの材料は凍結乾燥処理後にその最終的なＥＣＭ発泡体に対して結合、架橋、またはその他の方法により組み込むことも可能であると考えられる。さらに、上記の方法の種々の組み合わせが使用可能である。例えば、共役的に連結しているＥＣＭ発泡体および生物学的な潤滑剤の一定の移植片を移植して、さらに同一のまたは異なる１種類以上の生物学的な潤滑剤の付加的な関節内注射をその手術中、手術後、または手術中および手術後の両方において行なうことができる。
【００５６】
上記の「生体活性物質（Bioactive agents）」は以下のような、すなわち、種々の化学走性物質、治療剤（例えば、種々の抗生物質、ステロイド系および非ステロイド系の鎮痛薬および抗炎症薬、免疫抑制剤および抗癌薬等のような抗拒絶物質）、種々のタンパク質（例えば、種々の短鎖ペプチド、骨形態発生性タンパク質、糖タンパク質およびリポタンパク質）、細胞結合媒介物質、生物学的に活性なリガンド、インテグリン結合性シーケンス、種々のリガンド、種々の増殖および／または分化物質（例えば、表皮増殖因子、ＩＧＦ−Ｉ、ＩＧＦ−ＩＩ、ＴＧＦ−βＩ−ＩＩＩ、増殖および分化の各因子、脈管内皮増殖因子、線維芽細胞増殖因子、血小板由来増殖因子、インスリン由来増殖因子および形質転換増殖因子、副甲状腺ホルモン、副甲状腺関連タンパク質、ｂＦＧＦ、ＴＧＦβ上科因子、ＢＭＰ−２，ＢＭＰ−４、ＢＭＰ−６、ＢＭＰ−１２、ソニック・ヘッジホッグ（sonic hedgehog）、ＧＤＦ５、ＧＤＦ６、ＧＤＦ８、ＰＤＧＦ）、特定の増殖因子のアップレギュレーションに影響を及ぼす小形分子、テネイシンＣ、ヒアルロン酸、コンドロイチン硫酸、フィブロネクチン、デコリン（decorin）、トロンボエラスチン、トロンビン由来ペプチド、ヘパリン結合性ドメイン、ヘパリン、ヘパラン硫酸、ＤＮＡフラグメントおよびＤＮＡプラスミドの１種類以上を含む。また、別の上記のような物質が整形外科の分野において治療的価値を有する場合には、これらの物質の少なくとも一部が本発明の開示の概念における有用性を有することが予想され、このような物質は特別に限定されない限りにおいて上記の「生体活性物質（（bioactive agent）および（bioactive agents））」の意味に当然に含まれると考えられる。
【００５７】
上記の「生物学的に誘導されている物質（Biologically derived agents）」は以下のような、すなわち、骨（自家移植片、同種移植片および異種移植片）および骨の種々の誘導体、例えば、半月板組織を含む軟骨（自家移植片、同種移植片および異種移植片）および種々の誘導体、靭帯（自家移植片、同種移植片および異種移植片）および種々の誘導体、例えば、粘膜下組織を含む腸管組織の種々の誘導体（自家移植片、同種移植片および異種移植片）、例えば、粘膜下組織を含む胃組織の種々の誘導体（自家移植片、同種移植片および異種移植片）、例えば、粘膜下組織を含む膀胱組織の種々の誘導体（自家移植片、同種移植片および異種移植片）、例えば、粘膜下組織を含む消化管組織の種々の誘導体（自家移植片、同種移植片および異種移植片）、例えば、粘膜下組織を含む呼吸器組織の種々の誘導体（自家移植片、同種移植片および異種移植片）、例えば、粘膜下組織を含む生殖器組織の種々の誘導体（自家移植片、同種移植片および異種移植片）、例えば、肝臓基底膜を含む肝臓組織の種々の誘導体（自家移植片、同種移植片および異種移植片）、皮膚組織の種々の誘導体、高濃度血小板血漿（ＰＲＰ）、低濃度血小板血漿、骨髄吸引液、鉱物質除去化骨基質、インスリン由来増殖因子、全血、フィブリンおよび血液クロットの１種類以上を含む。さらに、精製したＥＣＭおよびその他のコラーゲン供給源もまた上記の「生物学的に誘導されている物質」の中に含まれるべきであると考えられる。また、別の上記のような物質が整形外科の分野において治療的価値を有する場合には、これらの物質の少なくとも一部が本発明の開示の概念における有用性を有することが予想され、このような物質は特別に限定されない限りにおいて上記の「生物学的に誘導されている物質（（biologically derived agent）および（biologically derived agents））」の意味に当然に含まれると考えられる。
【００５８】
上記の「生物学的に誘導されている物質（Biologically derived agents）」はまた生体再造形可能な種々の膠原性（collageneous）の組織基質も含む。さらに、この表現の「生体再造形可能な膠原性組織基質（bioremodelable collagenous tissue matrix）」および「天然に発生する生体再造形可能な膠原性組織基質（naturally occurring bioremodelable collageneous tissue matrix）」は皮膚、動脈、静脈、心膜、心臓弁、硬膜、靭帯、骨、軟骨、膀胱、肝臓、胃、筋膜および腸管、腱、および同類の供給源から成る群から選択される天然組織から誘導されている種々の基質を含む。この「天然に発生する生体再造形可能な膠原性（collageneous）組織基質」は洗浄、加工、滅菌、および随意的に架橋されている基質材料を意味することを目的としているが、種々の天然の線維を精製して精製した天然の線維から一定の基質材料を再形成することは一定の天然に発生する生体再造形可能な膠原性（collageneous）組織基質の定義には含まれない。さらに、上記用語の「生体再造形可能な膠原性組織基質（bioremodelable collageneous tissue matrices）」は「種々の細胞外基質（extracellular matrices）」をその定義内に含む。
【００５９】
上記の「細胞（Cells）」は以下のような、すなわち、種々の軟骨細胞、線維軟骨細胞、骨細胞、骨芽細胞、破骨細胞、骨膜細胞、骨髄細胞、間葉細胞、間質細胞、幹細胞、胚芽幹細胞、脂肪組織から由来または誘導した前駆体細胞、末梢血液先祖細胞、成人組織から単離した幹細胞、遺伝的に形質転換した細胞、軟骨細胞および別の細胞の一定の組み合わせ物、骨細胞および別の細胞の一定の組み合わせ物、骨膜細胞および別の細胞の一定の組み合わせ物、骨髄細胞および別の細胞の一定の組み合わせ物、間葉細胞および別の細胞の一定の組み合わせ物、間質細胞および別の細胞の一定の組み合わせ物、幹細胞および別の細胞の一定の組み合わせ物、胚芽幹細胞および別の細胞の一定の組み合わせ物、成人組織から単離した前駆体細胞および別の細胞の一定の組み合わせ物、末梢血液先祖細胞および別の細胞の一定の組み合わせ物、成人組織から単離した幹細胞および別の細胞の一定の組み合わせ物、および遺伝的に形質転換した細胞および別の細胞の一定の組み合わせ物の１種類以上を含む。さらに、別の細胞が整形外科の分野において治療的価値を有していることが分かった場合には、これらの細胞の少なくとも一部が本発明の開示の概念における有用性を有することが予想され、このような細胞は特別に限定されない限りにおいて上記の「細胞（（cell）および（cells））」の意味に当然に含まれると考えられる。
【００６０】
上記の「生物学的な潤滑剤（Biological lubricants）」はヒアルロン酸ナトリウム等のようなヒアルロン酸およびその塩類、デルマタン硫酸等のようなグリコスアミノグリカン、ヘパラン硫酸、コンドロイチン硫酸およびケラタン硫酸、種々のムチン糖タンパク質（例えば、ルブリシン（lubricin））を含む骨膜液および骨膜液の種々の成分、ビトロネクチン、トリボネクチン（tribonectins）、関節軟骨表面領域タンパク質、表面活性リン脂質、潤滑性糖タンパク質Ｉ，ＩＩ、および雄鶏冠（rooster comb）ヒアルロネート等を含む。この「生物学的な潤滑剤」はまた英国リーズのデピュイ・インターナショナル社（DePuy International, Ltd.）から欧州において入手可能であり、イスラエル国レホボットのバイオ−テクノロジー・ジェネラル（イスラエル）社（Bio-Technology General （Israel） Ltd.）により製造されているアースリースＴＭ（ARTHREASETM）高分子量ヒアルロン酸ナトリウム、ニュージャージー州リッジフィールドのバイオマトリクス社（Biomatrix, Inc.）により製造されていて、ペンシルバニア州フィラデルフィアのワイエス−エイエルスト・ファーマシューティカルズ社（Wyeth-Ayerst Pharmaceuticals）により配給されているシンビスク（SYNVISC）（登録商標）ハイラン（Hylan）Ｇ−Ｆ２０、ニューヨーク州ニューヨークのサノフィ−シンセラボ社（Sanofi-Synthelabo, Inc.）から入手可能であり、イタリア国パジュアのフィデイアＳ．ｐ．Ａ社（FIDIA S.p.A）により製造されているハイラガン（HYLAGAN）（登録商標）ヒアルロン酸ナトリウム、および１％、１．４％および２．３％（眼科学的使用用）の濃度でニュージャージー州ピーパックのファーマシア・コーポレーション社（Pharmacia Corporation）から入手可能であるヘアロン（HEALON）（登録商標）ヒアルロン酸ナトリウム等のような種々の市販製品を含むことも目的としている。また、別の上記のような物質が整形外科の分野において治療的価値を有する場合には、これらの物質の少なくとも一部が本発明の開示の概念における有用性を有することが予想され、このような物質は特別に限定されない限りにおいて上記の「生物学的な潤滑剤（（biological luburicant）および（biological luburicants））」の意味に当然に含まれると考えられる。
【００６１】
上記の「生体相容性のポリマー（Biocompatible polymers）」は種々の合成のポリマーおよび種々の生体ポリマー（例えば、コラーゲン等）の両方を含むことを目的としている。このような生体相容性のポリマーの例はポリ（Ｌ−ラクチド）（ＰＬＬＡ）およびポリグリコリド（ＰＧＡ）等のような種々の［アルファ］−ヒドロキシカルボン酸のポリエステル、ポリ−ｐ−ジオキサノン（ＰＤＳ）、ポリカプロラクトン（ＰＣＬ）、ポリビニル・アルコール（polyvinyl alchohol）（ＰＶＡ）、ポリエチレン・オキシド（ＰＥＯ）、米国特許第６，３３３，０２９号および６，３５５，６９９号において開示されている種々のポリマー、および種々のプロテーゼ移植片の構成において利用されている種々のポリマーまたはコポリマーの任意の別の生体吸収性で生体相容性のポリマー、コポリマーまたは種々のポリマーまたはコポリマーの混合物を含む。加えて、新しい生体相容性で生体吸収性の材料が開発される場合に、これらの材料の少なくとも一部がその材料により種々の固定手段が作成可能になる有用な材料になることが予想される。従って、上記の材料は例示のみを目的として示されており、本発明の開示が特許請求の範囲において特別に記載されていない限り任意の特定の材料に限定されないことが当然に理解されるべきである。
【００６２】
上記の「生体相容性の無機材料（Biocompatible inorganic materials）」はヒドロキシアパタイト、全てのリン酸カルシウム、リン酸アルファ−トリカルシウム、リン酸ベータ−トリカルシウム、炭酸カルシウム、炭酸バリウム、硫酸カルシウム、硫酸バリウム、種々のリン酸カルシウムの多形体、セラミック粒子、およびこれらの材料の組み合わせ物等のような種々の材料を含む。また、別の上記のような物質が整形外科の分野において治療的価値を有する場合には、これらの物質の少なくとも一部が本発明の開示の概念における有用性を有することが予想され、このような物質は特別に限定されない限りにおいて上記の「生体相容性の無機物質（（biocompatible inorganic material）および（biocompatible inorganic materials））」の意味に当然に含まれると考えられる。
【００６３】
種々の生体活性物質、生物学的に誘導されている物質、細胞、生物学的な潤滑剤、生体相容性の無機材料、生体相容性のポリマーの種々の組み合わせ物が上記本発明の開示の種々の支持骨格材料と共に使用可能であることが予想される。
【００６４】
また、標準的な種々の滅菌技法が本発明の開示の種々の製品と共に使用可能であることが予想される。
【００６５】
例示的に、種々の軟骨細胞等のような生活細胞により接種する実施形態の一例において、一定の滅菌処理した移植片を種々の生活細胞により接種し、これに続いて、その使用した細胞型に対して適当な一定の培地内において包装することができる。例えば、ダルベッコ修飾イーグル培地（Dulbecco’s Modified Eagle Medium）（ＤＭＥＭ）を含む一定の細胞培養培地が、細胞型に対して適当と思われる濃度および輸送条件等において、種々の可欠アミノ酸、グルコース、アスコルビン酸、ピルビン酸ナトリウム、殺真菌薬、抗生物質等のような標準的な添加物と共に使用可能である。
【００６６】
本発明の開示のＥＣＭ発泡体が、２００２年６月１４日に出願されている「ハイブリッド・バイオロジック−シンセチック・バイオアブソーバブル・スキャフォルズ（Hybrid Biologic-Synthetic Bioabsorbable Scaffolds）」を発明の名称とする米国特許出願第１０／１７２，３４７号と共に、本特許出願と同時に出願されていて、それぞれの内容全体が本明細書に参考文献として含まれている、以下の特許出願、すなわち、「メニスカス・リジェネレーション・デバイス・アンド・メソッド（Meniscus Regeneration Device and Method）」を発明の名称とする米国特許出願第ＸＸ／ＸＸＸ，ＸＸＸ号（代理人明細書番号第２６５２８０−７１１４１号，ＤＥＰ−７４５）、「デバイスイズ・フロム・ナチュラリー・オカーリング・バイオロジカリー・デライブド・マテリアルズ（Devices from Naturally Occurring Biologically Derived Materials）」を発明の名称とする米国特許出願第ＸＸ／ＸＸＸ，ＸＸＸ号（代理人明細書番号第２６５２８０−７１１４２号，ＤＥＰ−７４８）、「カーテイレイジ・リペア・アパレイタス・アンド・メソッド（Cartilage Repair Apparatus and Method）」を発明の名称とする米国特許出願第ＸＸ／ＸＸＸ，ＸＸＸ号（代理人明細書番号第２６５２８０−７１１４３号，ＤＥＰ−７４９）、「ユニタリー・サージカル・デバイス・アンド・メソッド（Unitary Surgical Device and Method）」を発明の名称とする米国特許出願第ＸＸ／ＸＸＸ，ＸＸＸ号（代理人明細書番号ＤＥＰ−７５０）、「ハイブリッド・バイオロジック／シンセチック・ポーラス・エクストラセルラー・マトリクス・スキャフォルド（Hybrid Biologic/Synthetic Porous Extracellular Matrix Scaffolds）」を発明の名称とする米国特許出願第ＸＸ／ＸＸＸ，ＸＸＸ号（代理人明細書番号第２６５２８０−７１１４４号，ＤＥＰ−７５１）、「カーテイレイジ・リペア・アンド・リジェネレーション・デバイス・アンド・メソッド（Cartilage Repair and Regeneration Device and Method）」を発明の名称とする米国特許出願第ＸＸ／ＸＸＸ，ＸＸＸ号（代理人明細書番号第２６５２８０−７１１４５号，ＤＥＰ−７５２）、「カーテイレイジ・リペア・アンド・リジェネレーション・スキャフォルド・アンド・メソッド（Cartilage Repair and Regeneration Scaffolds and Method）」を発明の名称とする米国特許出願第ＸＸ／ＸＸＸ，ＸＸＸ号（代理人明細書番号第２６５２８０−７１１８０号，ＤＥＰ−７６３）、および「ポーラス・デリバリー・スキャフォルド・アンド・メソッド（Porous Delivery Scaffold and Method）」を発明の名称とする米国特許出願第ＸＸ／ＸＸＸ，ＸＸＸ号（代理人明細書番号第２６５２８０−７１２０７号，ＤＥＰ−７６２）において開示されているそれぞれの概念と共に組み合わせることが可能であると予想される。例えば、整形外科の種々の用途において、移植処理がその移植部位へのヒアルロン酸の投与を含む一定の治療プログラムを伴うかこれに従うことが望ましいと考えられる。
【００６７】
上記から分かるように、本発明の開示の概念は多数の利点を提供している。例えば、本発明の開示の概念は一定の支持骨格材料の設計の要求に対して適合するために変更可能な種々の機械的特性を有することのできる一定の多孔質の移植可能な支持骨格材料の製造方法を提供している。例えば、気孔寸法および材料密度を変更して一定の所望の機械的な構成を有する一定の支持骨格材料を提供することができる。とりわけ、このような支持骨格材料の気孔寸法および材料密度の多様性は内部を通過する一定の所望量の細胞移動を提供すると共に、一定の所望量の構造的な剛性を提供する一定の支持骨格材料を設計する場合に特に有用である。加えて、本発明の開示の概念によれば、移植における所望の細胞活性を可能にする適当な物理的な微小構造を有するだけでなく、種々のＥＣＭ等において天然に見られるような生物化学的組成（種々のコラーゲン、増殖因子、グリコスアミノグリカン等）を有している種々の移植可能な装置が製造可能になる。
【００６８】
天然に発生する細胞外基質は精製した細胞外基質に優る種々の利点を提供すると考えられるが、本発明の開示が精製した細胞外基質にも適用可能であることが考慮されている。従って、天然に発生する細胞外基質をその細胞外基質の物理的な粉砕の前に精製することが可能になると予想される。この精製は一定の細胞外基質を物理的に粉砕する前にコラーゲン以外の実質的に全ての物質を除去するためにその天然に発生する細胞外基質を処理することを含むと考えられる。また、この精製は種々の糖タンパク質、グリコスアミノグリカン、プロテオグリカン、脂質、非膠原性タンパク質および核酸（ＤＮＡおよびＲＮＡ）を実質的に除去するために実施できると考えられる。
【００６９】
上記の開示は種々の変更および代替的な形態が可能であるが、その特定の代表的な実施形態が各添付図面において例示のために示されていて詳細に説明されている。しかしながら、これらの開示されている特定の形態に本発明の開示を限定することを全く目的としていないこと、および、これとは逆に、この目的が当該開示の趣旨および範囲に該当する全ての変更例、等価物、および代替物を含むことであることが当然に理解されると考える。
【００７０】
本明細書において記載されている装置および方法の種々の特徴から生じる本発明の開示の複数の利点が存在している。さらに、本発明の開示における装置および方法の代替的な種々の実施形態が上記の記載されている特徴の全てを含まないがこれらの特徴の利点の少なくとも一部から依然として恩恵を受けている可能性があることに注目する必要がある。また、当該技術分野における通常の熟練者は本発明の開示における特徴の１個以上を含み、本発明の開示の趣旨および範囲内に該当する一定の装置および方法のそれぞれ固有の実施方法を容易に考え出すことができる。
【図面の簡単な説明】
【００７１】
上記の詳細な説明は特に以下の各図面に基づいている。
【図１】一定の比較的に大きな気孔寸法および一定の比較的に低い材料密度を有している一定の多孔質な網状のＳＩＳの連続気泡型支持骨格材料の表面を示している一定の走査電子顕微鏡写真による画像である。
【図２】一定の比較的に中程度の気孔寸法および一定の比較的に中程度の材料密度を有している一定の多孔質な網状のＳＩＳの連続気泡型支持骨格材料の表面を示している一定の走査電子顕微鏡写真による画像である。
【図３】一定の比較的に小さな気孔寸法および一定の比較的に高い材料密度を有している一定の多孔質な網状のＳＩＳの連続気泡型支持骨格材料の表面を示している一定の走査電子顕微鏡写真による画像である。
【図４】矢印により示されている気孔の一例を伴う一定の多孔質な網状のＳＩＳの連続気泡型支持骨格材料の断面を示している一定の走査電子顕微鏡写真による画像であり、この画像は図１乃至図３の各画像よりも大きな一定の倍率である。
【図５】矢印により示されている気孔の例を伴う一定の多孔質な網状のＳＩＳの連続気泡型支持骨格材料の断面を示している一定の走査電子顕微鏡写真による画像であり、この画像は図１乃至図３の各画像よりも大きな一定の倍率である。
【図６】矢印により示されている気孔の例を伴う一定の多孔質な網状のＳＩＳの連続気泡型支持骨格材料の断面を示している一定の走査電子顕微鏡写真による画像であり、この画像は図１乃至図３の各画像よりも大きな一定の倍率である。
【図７】一定の多孔質な網状のＳＩＳの連続気泡型支持骨格材料の断面を示している一定の走査電子顕微鏡写真による画像であり、この画像は図１乃至図３の各画像よりも大きな一定の倍率である。
【図８】一定の多孔質な網状のＳＩＳの連続気泡型支持骨格材料の表面を示している一定の走査電子顕微鏡写真による画像であり、この画像は図１乃至図３の各画像よりも大きな一定の倍率である。
【図９】凝集性のＳＩＳの部材片の一定の素材を示している一定の走査電子顕微鏡写真による画像である。
【図１０】凝集性のＳＩＳの部材片の一定の素材を示している一定の走査電子顕微鏡写真による画像である。Disclosure of the content of the invention
[0001]
Cross reference to related applications
U.S. Patent Application No. XX / XXX, XXX, co-pending "Meniscus Regeneration Device and Method" (Attorney Specification No. 265280-71141, US Patent Application No. XX / XXX, XXX, entitled "Devices from Naturally Occurring Biologically Derived Materials", DEP-745), "Devices from Naturally Occurring Biologically Derived Materials". (Attorney Specification No. 265280-71142, DEP-748), US Patent Application No. XX / XXX, entitled "Cartilage Repair Apparatus and Method". XXX (Attorney Statement No. 265280-7114 No. 3, DEP-749), U.S. Patent Application No. XX / XXX, XXX, entitled "Unitary Surgical Device and Method" (Attorney Specification No. DEP- 750), US Patent Application No. XX / XXX, XXX, entitled "Hybrid Biologic / Synthetic Porous Extracellular Matrix Scaffolds" US Patent Application No. 265280-71144, DEP-751), U.S. Patent Application No. entitled "Cartilage Repair and Regeneration Device and Method". XX / XXX, XXX (Attorney Statement No. 2652) 80-71145, DEP-752), US Patent Application No. XX / XXX, entitled "Cartilage Repair and Regeneration Scaffolds and Method". No. XXX (Attorney Specification No. 265280-71180, DEP-763), and US Patent Application No. XX entitled "Porous Delivery Scaffold and Method" / XXX, XXX (Attorney Specification No. 265280-71207, DEP-762), each of which is assigned to the same assignee as the present patent application. Each of which was filed at the same time as Re is incorporated herein by reference. Also, US Patent Application No. 10/172, filed June 14, 2002, entitled "Hybrid Biologic-Synthetic Bioabsorbable Scaffolds". No. 347 is also cross-referenced, and this patent application is assigned to the same assignee as the present patent application and is incorporated herein by reference.
[0002]
Field of disclosure
The present disclosure relates generally to an extracellular matrix scaffold, and more particularly to a porous extracellular matrix scaffold for repairing and regenerating bodily tissue and to create such scaffolds. Related to certain ways to.
[0003]
Background of the Invention
Various naturally occurring extracellular matrices (ECMs) have been used for tissue repair and regeneration. One such extracellular matrix is the small intestinal submucosa (SIS). SIS has been used to repair, support, and stabilize a variety of anatomical defects and traumatic injuries. Commercially available SIS material is derived from porcine small intestinal submucosa, which remodels the quality of its host when implanted into human soft tissue. It has further been taught that this SIS material provides a natural substrate with a three-dimensional microstructure and a biochemical composition that facilitates host cell growth and supports tissue remodeling. I have. In fact, SIS is known to contain various growth factors and various biological molecules such as glycosaminoglycans that assist in the repair of soft tissues in the human body. The SIS materials currently used in the orthopedic field are desiccated and layered in the form of patches to repair or regenerate soft tissues such as various tendons, ligaments and rotator cuffs, etc. Supplied in configuration.
[0004]
Although submucosal tissue of the small intestine is available, other sources of submucosal tissue are also known to be effective in tissue remodeling. These sources include, but are not limited to, the stomach, bladder, gastrointestinal tract, respiratory or genital mucosa, or the basement membrane of the liver. See, for example, U.S. Patent Nos. 6,379,710, 6,171,344, 6,099,567, and 5,554,389, each of which is incorporated herein by reference. It is.
[0005]
Further, although the SIS is most often derived from pigs, these various submucosal tissue materials can be derived or derived from sources other than pigs, including bovine and sheep sources. In addition, the ECM materials described above may also be affected by various factors, such as the source from which the ECM material is derived and the delamination procedure, and the like. Partial layers of tissue material can also be included.
[0006]
As used herein, washing, delamination and / or grinding of an extracellular matrix, or crosslinking collagen or various other components into the extracellular matrix, Included in the definition of naturally occurring extracellular matrix. The complete or partial removal of one or more components or auxiliary components of the above-described naturally occurring substrate is also included in the definition of the naturally occurring extracellular matrix. However, separating and purifying the natural component or ancillary component described above and reforming a substrate material from the purified natural component or ancillary component is a definition of the above-mentioned naturally occurring extracellular matrix. Is not included. References have also been made to SIS, but other naturally occurring extracellular matrices (eg, stomach, bladder, gastrointestinal tract, respiratory or genital mucosa, and liver basement membrane), or It will be understood that similar sources (eg, cows, pigs, sheep) and the like are within the scope of the present disclosure. Thus, in this patent application, the term "naturally occurring extracellular matrix" or "naturally occurring ECM" is used for washing, processing, sterilizing, and optionally It is intended to mean a cross-linked extracellular matrix material. The following U.S. Patents, which are incorporated herein by reference: U.S. Patent Nos. 6,379,710, 6,187,039, 6,176,880, 6,126,686, 6, Nos. 099,567, 6,096,347, 5,997,575, 5,993,844, 5,968,096, 5,955,110, 5,922,028, 5,885 Nos., 619, 5,788,625, 5,762,966, 5,755,791, 5,753,267, 5,733,337, 5,711,969, 5,645. Nos. 860, 5,641,518, 5,554,389, 5,516,533, 5,460,962, 5,445,833, 5,372,821, 5,352,463. No. 5, 281, 422, and Beauty No. 5,275,826 discloses the use of various ECM for regeneration and repair of various tissues.
[0007]
Manipulation of pore size, porosity, and interconnectivity of scaffolds is an important technology contributing to the field of tissue engineering (Ma and Zhang, 2001, Journal of the -Biomedical Material Research (J Biomed Mater Res), vol. 56 (4), pp. 469-474, Ma and Choi, 2001, Tissue Eng, 7 (1) ), Pp. 23-33), because consideration of the pore size and density / porosity of the scaffold may affect the behavior of various cells and the quality of the regenerated tissue. . In fact, several researchers have previously shown that pore size affects cell behavior in various porous, three-dimensional matrices. For example, it has been demonstrated in the art that the pore size of the scaffold needs to be at least 100 microns in order to produce proper bone regeneration (Klawitter et al., 1976, Journal of Theology). -Biomedical Material Research (J. Biomed. Mater. Res.), 10 (2), pp. 311-323). In the case of pore sizes and interconnectivity smaller than this pore size, poor quality bone will be regenerated, and if the pore size is between 10 and 40 microns, the bone cells will become soft fibrous vascular (White and Shors, 1991, Dent Clin North Am., Vol. 30, pp. 49-67). . Current consensus in bone regeneration studies indicates that the pore size required for bone regeneration is between 100 microns and 600 microns (Shors, 1999, Orthopedics. Clinical of North America, Orthop Clin North Am, Vol. 30 (4), 599-613, Wang, 1990, Nippon Seikeigeka Gakki Zasshi, 64 (Nippon Seikeigeka Gakki Zasshi). 9), pp. 847-859). Further, it is generally accepted that optimal bone regeneration occurs at pore sizes between 300 microns and 600 microns.
[0008]
Similarly, in the regeneration of soft orthopedic tissue such as ligaments, tendons, cartilage, fibrous cartilage, etc., the pore size of the scaffold is believed to have a certain substantial effect. For example, basic studies have shown that various chondrocytes show adequate protein expression (collagen type II) in various scaffolds with pore sizes on the order of 20 microns, with a nominal porosity of about 80 microns. Have been shown to produce type I collagen in various scaffolds having the following (Nehrer et al., 1997, Biomaterials, 18 (11), p. 769-776). . More recently, cells (fibroblasts and endothelial cells) from various ligaments, tendons and blood vessels have significantly different activities when cultured on various scaffolds with different pore sizes ranging from 5 to 90 microns. (Salem et al., 2002, Journal of Biomedical Material Research (J Biomed Mater Res), 61 (2), pp. 212-217).
[0009]
Summary of the Invention
According to one example embodiment, a method is provided for making an implantable scaffold for repairing damaged or diseased tissue. The method includes suspending, mixing, or otherwise placing a piece of a naturally occurring extracellular matrix material in a liquid. These naturally occurring extracellular matrix materials and liquids are formed into certain materials. Subsequently, the liquid is removed to form a plurality of gaps in the material. In one particular implementation of this exemplary embodiment, the liquid has been removed by lyophilizing the naturally occurring extracellular matrix material and the liquid in which the material is suspended. In such a manner, the liquid sublimes, thereby creating a plurality of gaps in the material.
[0010]
The density and pore size of the scaffold can be varied by controlling the freezing rate of the suspension. The amount of water that suspends the pieces of the naturally occurring extracellular matrix material described above can also be varied to control the density and pore size of the resulting scaffold.
[0011]
According to another exemplary embodiment, there is provided an implantable scaffold for repairing or regenerating tissue prepared by the above method.
[0012]
In another aspect, the present disclosure provides an implantable scaffold for repairing or regenerating body tissue. The scaffold comprises a porous body portion of naturally occurring extracellular matrix pieces that are interconnected to define the inner surface of the body portion. The inner surface has an irregular shape in a certain three-dimensional form.
[0013]
In another aspect, the present disclosure provides an implantable device for repairing or regenerating body tissue. The device comprises a three-dimensional reticulated foam containing a plurality of interconnected pores. These interconnected pores define a three-dimensional interconnected passage having various irregular shapes. Further, at least a portion of the reticulated foam contains a naturally occurring extracellular matrix.
[0014]
In another aspect, the present disclosure provides a method of making an implantable device for repairing or regenerating body tissue. The method comprises the steps of supplying a naturally occurring extracellular matrix material in the form of a raw material, grinding the naturally occurring extracellular matrix of the raw material in the presence of a liquid to produce a naturally occurring cell. Forming a slurry of the extracellular matrix, and lyophilizing the slurry of the naturally occurring extracellular matrix to form a reticulated foam of the naturally occurring extracellular matrix.
[0015]
In another aspect, the present disclosure provides a method of making an implantable scaffold for repairing or regenerating body tissue. The method comprises the steps of: supplying a naturally occurring extracellular matrix material in the form of a raw material; crushing the naturally occurring extracellular matrix of the raw material to form a coherent member of the naturally occurring extracellular matrix. Forming a piece, and freeze-drying the coherent member of the naturally occurring extracellular matrix to form a reticulated foam of the naturally occurring extracellular matrix.
[0016]
The implantable device disclosed herein is a three-dimensional porous scaffold of various ECMs such as SIS and the like. Therefore, certain implants based on the teachings of the present disclosure may not only have the appropriate biochemical composition (eg, various collagens, growth factors, glycosaminoglycans, etc. naturally found in various ECMs, etc.) but also in implants. Obviously, it has the dual advantage of also having the appropriate physical microstructure to allow the desired cellular activity. These implantable devices also correspond to various devices used in the treatment of diseased or damaged fibrocartilage, such as meniscus, diseased or damaged articular cartilage, and diseased or damaged bone. It is considered useful in therapeutic applications in the surgical field.
[0017]
The above and other features of the present disclosure will become apparent from the following description and the accompanying drawings.
[0018]
Detailed description of the drawings
The present disclosure relates to a porous scaffold for implantation in a patient to repair or regenerate damaged or diseased tissue. The porous scaffold is composed of certain naturally occurring extracellular materials. For example, the scaffold can be composed of SIS. As described in detail herein, both the material density and the pore size of the scaffold can be varied to meet the requirements of a given scaffold design.
[0019]
Such a porous scaffold can be produced by suspending a piece of extracellular matrix material in a liquid. As used herein, the term “piece” refers to any fiber, piece, ribbon, fragment, filament, small fragment, small piece, fragment, portion, flake, section, cut piece. , A cutting portion, or other portion of a solid or solid-like substance. Similarly, as used herein, the term “suspending” refers to a solid (eg, ECM debris) regardless of whether an actual suspension is formed. ) Into any liquid. Thus, the term "suspension" is intended to include any operation of mixing a solid in a liquid or any other operation of putting a solid in a liquid. As a result of this, the term "suspension" is not intended to be limited to various suspensions, but rather refers to a material having a solid present in a liquid. It is aimed at.
[0020]
In either case, the pieces of extracellular matrix material and the suspension of the liquid form a material, for example, in the form of a "slurry". Subsequently, the liquid is removed from the material to form a plurality of gaps in the material. This liquid can be removed in a number of different ways. For example, as described in more detail below, the liquid can be removed by sublimation in certain freeze-drying processes. Alternatively, the liquid may be removed by subjecting the suspension to either unheated vacuum processing or vacuum processing in a controlled heating process. Further, the liquid can be removed from the suspension by ultrasonic waves. Further, microwave energy, RF energy, UV energy, or any other type of energy (or a combination thereof) can also be utilized to remove liquid from the suspension. Further, the liquid may be removed from the suspension by forcing or suctioning air passing through the suspension. Further, it is also possible to remove the liquid by centrifuging the suspension. In addition, the liquid may include certain water-soluble fillers that are removed, for example, by using certain alcohols. In essence, the present disclosure includes the removal of liquid from the suspension by any liquid removal process.
[0021]
As suggested above, any method for removing liquids from the above suspensions can be utilized with any other method known by those skilled in the art, but the present invention Is disclosed herein by way of example with reference to a freeze-drying process (ie, freeze-drying). However, such descriptions are merely exemplary in nature, and the various methods for removing liquids from the suspensions described above meet the various requirements of certain scaffold designs or process designs. It will be understood that it is naturally available to do so.
[0022]
Also, as suggested above, one example of a useful method for producing the porous scaffold of the present disclosure is a freeze-drying method. In this case, a piece of extracellular matrix material is suspended in a liquid. Thereafter, the suspension is frozen and subsequently lyophilized. Due to the freezing of the suspension, the liquid becomes ice crystals. Thereafter, these ice crystals are sublimated under reduced pressure during the freeze-drying process, leaving gaps of the material in each of the space portions occupied by the ice crystals. The density and pore size of the resulting scaffold controls, inter alia, the rate of freezing of the suspension and / or the amount of water in which the extracellular matrix material is suspended at the beginning of the freezing process. It can be changed by the following.
[0023]
As a specific example of the above method, the production of a porous SIS scaffold by freeze-drying is described in detail. However, while this example described herein is described with reference to a SIS scaffold, a scaffold composed of a variety of different extracellular matrix materials may be used. It is to be appreciated that manufacturing can also be accomplished in a similar manner.
[0024]
The first step in producing a porous scaffold with a desired pore size and density is the procurement of milled SIS. Illustratively, scissor-cut SIS runner pieces (approximately 6 inches long) are commercially available from Urshel Laboratories (Valparaiso, Ind.) In certain 1700 series Comitrol. ) (Trademark) device. The SIS material is collected in a container at the output of the device after being processed in the presence of a liquid. Thereafter, the material is subjected to a second treatment under similar conditions by the device. In one example method, a liquid (eg, water) is introduced into the input of the device at the same time as the SIS material. The resulting material is a "slurry" of SIS material (approximately 200 microns thick x 1-5 mm long thin SIS fibers) suspended in a substantially uniform manner in water. slurry). Although the suspension is described herein as being formed as a by-product of the milling process, the SIS pieces may be replaced by other components known to those skilled in the art. It will be appreciated that it can be suspended in the liquid (ie, water) in a manner. In addition, other methods for grinding SIS are known, but for the purposes of the present disclosure, the milled SIS comprises ribbon-like or cord-like fibers, where such ECM and It will be appreciated that at least some of the individual pieces of SIS material have a length greater than their respective widths and thicknesses. Such fibers can be combined to form a felt-like material, if desired.
[0025]
Various parameters of the processing, including the choice of blades to use, whether or not to use water, the amount of water to use, the rotational speed of each blade, and the number of times the material passes through the device, are determined by the 1700 Series Commitrol ( Comitrol ™ equipment can be modified. As an example, with a constant flow rate of about 2 gallons of water per minute, and blade operation at a constant speed of about 9300 rpm, a cutting head 140084-10 and a constant VERICUT from Urschel Laboratories. ) A shield impeller can be used. The first pass through the device at these parameters produces fibrous SIS material of different sizes, and the second pass produces SIS fibers of constant, more uniform size. For example, the material thus pulverized is subjected to the following processing, that is, the above-mentioned pulverized SIS suspension or slurry is centrifuged, excess water is poured off, and the remaining slurry is removed from a certain dish. Into a test to determine if it is consistent with the desired material when used in connection with the various exemplary embodiments described herein. can do. That is, by hand, a small amount of the ground SIS material in the dish is picked between the thumb and index finger and gently lifted from the dish. Exemplarily, beyond the portion pinched between the thumb and index finger, at least a small amount of additional SIS is lifted with the pinched material ("pinch test"). That is, this further SIS material is lifted with the above described material between the thumb and index finger because the individual pieces of the ground SIS material are intermingled or entangled with each other.
[0026]
The terms "cohesive ECM (cohesive ECM)", "cohesive SIS (cohesive SIS)", "cohesive ECM pieces" and "cohesive SIS pieces" ) "Is a separate piece of ECM or SIS that remains cohesive under some conditions (ie, under gravity) regardless of the shape of one or more of the individual ECM or SIS pieces. Crushed or otherwise physically treated to produce ECM or SIS pieces that can be mixed or entangled (in the dry or wet state) to form a uniform material Used herein to refer to certain ECM or SIS materials, respectively. An example method for indicating ECM or SIS material containing such cohesive pieces is the "pinch test" described in the preceding paragraph. Testing of the final ECM or SIS product produced may also provide evidence that the base material is comprised of cohesive ECM or SIS pieces. Illustratively, the ECM or SIS pieces are relative to each other (or another in the mixture or slurry) to such an extent that they remain integral throughout the process used to produce the foam structure. Sufficiently cohesive (for the piece). Examples of such cohesive SIS member pieces are shown in the scanning electron micrograph images of FIGS. 9 and 10.
[0027]
Thereafter, the crushed SIS suspension is frozen and freeze-dried (ie, freeze-dried). In particular, the SIS suspension is frozen at a controlled rate of temperature drop to control the size of the ice crystals formed. After this freezing, the freeze-drying process allows the ice crystals to sublime directly to a constant vapor under reduced pressure and low temperature conditions, so that the material does not spontaneously melt. This leaves a plurality of pores or gaps in the space occupied by the ice crystals.
[0028]
Any commercially available freezer for freezing the suspension to a certain desired temperature can be used. Similarly, any commercially available lyophilization equipment can be used for the lyophilization process described above. One example of an apparatus for performing this lyophilization process is a Virtis Genesis (TM) series lyophilizer available commercially from SP Industries, Inc., Gardener, NY. is there.
[0029]
The various processing parameters of the above manufacturing method can be varied to produce various scaffolds of different pore sizes and material densities. For example, various scaffolds with different pore sizes and material densities by changing the rate at which the suspension freezes, the amount of water present in the suspension, or the compactness of the extracellular matrix material It is possible to produce the material.
[0030]
For example, to produce a variety of scaffolds having a relatively large pore size and a relatively low material density, the suspension of the extracellular matrix is maintained at a constant temperature of about -20 ° C. After freezing at a slow controlled rate (eg, -1 ° C / min or less), the resulting material may be lyophilized. Also, in order to produce various scaffolds having a constant relatively small pore size and a constant relatively high material density, the extracellular matrix material must be centrifuged prior to freezing. The liquid (eg, water) can then be compacted by removing a portion of it in a substantially uniform manner. Thereafter, the resulting extracellular matrix material is flash-frozen with liquid nitrogen and then freeze-dried. Also, in order to produce various scaffolds with a certain medium pore size and a certain medium material density, the extracellular matrix material must first be centrifuged before freezing. The liquid (eg, water) is then compacted by removing a portion thereof in a substantially uniform manner. Thereafter, the resulting extracellular matrix material is frozen at a constant relatively fast rate (eg, -1 ° C / min or more) to a constant temperature of about -80 ° C, after which the material is frozen. A drying process is performed.
[0031]
Example 1
This Example 1 illustrates a method of making a porous SIS scaffold having a relatively large pore size and a relatively low material density. Such a scaffold is obtained by freezing a ground suspension of SIS at a slow controlled rate (up to -1 ° C / min) up to -20 ° C. The procedure is as follows. That is, first, crushed SIS is manufactured as described above. Specifically, the SIS runner pieces (about 6 inches long) cut with scissors are placed into a suitable comminuting device such as the Urschel Comitrol device described above. The SIS thus pulverized is collected in a container at the output port of the above-mentioned apparatus. Thereafter, the collected material is subjected to a second treatment through the above apparatus under the same conditions as before. The resulting material is a constant "slurry" of SIS material (about 200 microns thick x 1-5 mm long thin SIS fibers) relatively uniformly suspended in water. It is.
[0032]
Next, a constant low-speed freezing ethanol tank was prepared as follows. That is, enough ethanol is poured into a flat-bottomed plastic container large enough to hold four 24-well culture plates to obtain a head of about 1 cm. The container is then placed in a constant -20 ° C freezer. In addition, record the mass of each of these four empty 24-well plates. Thereafter, under a sterile hood under sterile conditions, an approximately 3 ml aliquot of the above ground SIS material is placed into each well or well of the tissue culture plate. In addition, record the mass of each of the full plates of this material. Thereafter, these culture plates were naturally frozen overnight in the ethanol freezing tank.
[0033]
The frozen plate was then removed from the ethanol bath and placed in a suitable freeze-drying device, such as the Virtis Genesis series freeze-drying device described above. Next, without freezing the frozen SIS material spontaneously, the ice crystals were directly sublimated into steam under reduced pressure and low temperature conditions by freeze-drying. This leaves a plurality of pores or gaps in the space previously occupied by the ice crystals. In this case, the parameters used in the freeze-drying process include a first period at 13 ° C. primary drying temperature for 8 hours, followed by a second period at 35 ° C. secondary drying temperature for 4 hours.
[0034]
After the lyophilization step is completed, the plates are removed from the lyophilizer and the weight of each plate is determined and recorded. The results of this process are summarized in the following table.
[Table 1]

[0035]
Constant scanning electron microscope images of the surface of each sample were taken to visualize the relative pore size. These pore sizes are from about 600 microns to about 700 microns. An image showing each sample prepared according to Example 1 above is shown in FIG.
[0036]
Various pore sizes can be determined by respective scanning electron microscopy images of the outer surface of the foam as in FIGS. 1-3 and 8 and the cross-section of the foam as in FIGS. These images can be used with standard commercially available image analysis software to determine the range of the respective pore sizes. FIGS. 4-6 show the results from appropriate commercially available software for measuring and estimating pore size in a foam, respectively. Using this technique, it was determined that the foam of Example 1 above had pores in the range of 600 microns to 700 microns. Note that this sample can include even smaller pores.
[0037]
Example 2
This Example 2 illustrates a method of making a porous SIS scaffold having a relatively medium pore size and a relatively medium material density. Such a scaffold may be obtained by consolidating the above ground SIS material by centrifugation and at a relatively high rate to a relatively low temperature (ie, to -80 ° C) (see Example 1 above). After freezing (compared to), the resulting material is obtained by freeze-drying. The procedure is as follows. That is, first, ground SIS was produced as described in connection with Example 1 above. Specifically, SIS material that is relatively uniformly suspended in water by passing the SIS runner piece (about 6 inches long) cut with scissors twice through a suitable milling device. A constant "slurry" of (about 200 microns thick x 1 mm to 5 mm long narrow SIS fibers) is produced.
[0038]
Next, record the mass of each of the four empty 24-well plates. Thereafter, under a sterile hood under aseptic conditions, an approximately 3 ml aliquot of the above ground SIS material is placed into each well of the tissue culture plate. In addition, record the mass of each of the full plates of this material.
[0039]
Thereafter, the weight of each plate is balanced for centrifugation by using the following technique. That is, place the two plates on a balance and add RO water to the area between each well of the relatively light plate until the two plates are balanced. The two plates are then placed opposite each other in the centrifuge and centrifuged at 3000 rpm for 7 minutes. At the end of this treatment, each plate is removed from the centrifuge to remove water therefrom. Next, the respective mass of each plate is recorded. Further, the centrifugation and mass measurement are repeated for the remaining plates.
[0040]
The plate is then placed in a constant -80 ° C freezer until each sample is completely frozen. Depending on the amount of each material, the time for complete freezing can vary, for example, from about 1 minute to about 30 minutes. Thereafter, each frozen plate is removed from the freezer and placed in a suitable lyophilizer to obtain parameters similar to those described in connection with Example 1 above (ie, 13 hours over 8 hours). (First period at the primary drying temperature in ° C., second period at the secondary drying temperature of 35 ° C. over 4 hours).
[0041]
After completion of the lyophilization step, each plate was removed from the lyophilizer and the weight of each plate was determined and recorded. The results from this method are summarized in the table below.
[Table 2]

[0042]
As with the sample of Example 1 above, a fixed scanning electron microscope image of the surface of each sample prepared according to Example 2 above was taken to visualize the relative pore size. Further, certain images showing each sample prepared according to this Example 2 are shown in FIG. Also, the techniques described above for determining pore size have shown that the sample has pores in the range of about 100 microns to 150 microns.
[0043]
Example 3
Example 3 illustrates a method of making a porous SIS scaffold with a relatively small pore size and a relatively high material density. Such a support skeleton material is obtained by consolidating the above ground SIS material to a constant density higher than that in Example 2 above, flash-freezing these samples with liquid nitrogen, and then performing freeze-drying. can get. The procedure is as follows. That is, ground SIS was first manufactured as described in connection with Examples 1 and 2 above. Specifically, SIS material that is relatively uniformly suspended in water by passing the SIS runner piece (about 6 inches long) cut with scissors twice through a suitable milling device. Produce a "slurry" of (about 200 microns thick x 1 mm to 5 mm long thin SIS fibers). After this treatment, the resulting material is centrifuged under a constant loading weight. This loading weight is prepared as follows. Forty-eight pieces of Coban are cut into 50 mm long, 5 mm wide (unstretched) pieces. The pieces are then stretched and wrapped around the outer edge of a 1 cm diameter, 2 mm thick, constant thickness polyethylene disc. If desired, each section can be shaped and each section of the covan can be wrapped twice around the disc.
[0044]
Next, record the mass of each of the four empty 24-well plates. Thereafter, under a sterile hood under aseptic conditions, an approximately 3 ml aliquot of the above ground SIS material is placed into each well of the tissue culture plate. In addition, record the mass of each of the full plates of this material. Thereafter, the weight of each plate is balanced and centrifuged as described in connection with Example 2 above. The water is then drained from each plate and the mass of each centrifuged plate is recorded.
[0045]
After completion of the above process, the number of plates is reduced from four to two by combining the wells of each plate in a fixed 2: 1 ratio. In addition, an attempt is made to mix wells of a small amount of material with each well of a large amount of material to obtain a somewhat consistent amount of SIS material in each well. Thereafter, the mass of the plate thus completed was recorded. Next, each polyethylene disk wrapped with the covan was placed in each well. Thereafter, the two plates were balanced in weight by the technique described in connection with Example 2 above. The plates were then centrifuged at 3000 rpm for 5 minutes. Thereafter, each plate is removed from the centrifuge to remove water therefrom and the polyethylene disc. Next, the mass of each plate is recorded again. Further, the weights of the two plates were rebalanced (in a similar manner as described in connection with Example 2 above) and each plate was centrifuged again at 3000 rpm for 7 minutes. After the end of the treatment, the water is again removed from each plate and the weight of each plate is recorded again.
[0046]
Simultaneously with the centrifugation, the liquid nitrogen tank is prepared by pouring liquid nitrogen into a wide-mouthed liquid nitrogen container. Each of the above plates is held in the centrifuge until the bath is ready. Thereafter, each plate is immersed in the bath and held in the liquid for about 15 seconds. Upon removal from the nitrogen bath, each plate is quickly placed in a constant -80 ° C freezer to avoid thawing. These frozen plates are then removed from the freezer and placed in an appropriate lyophilizer and placed in the same parameters as described in connection with Examples 1 and 2 above (ie, 8 Lyophilization according to a first period at a primary drying temperature of 13 ° C. over time, followed by a second period at a secondary drying temperature of 35 ° C. over 4 hours).
[0047]
After completion of the lyophilization step, each plate was removed from the lyophilizer and the weight of each plate was determined and recorded. The results from this method are summarized in the table below.
[Table 3]

[0048]
As in the case of the samples of Examples 1 and 2 above, a fixed scanning electron microscope image of the surface of each sample prepared according to Example 3 above was taken to visualize the relative pore size. Also, certain images showing each sample prepared according to this Example 3 are shown in FIG. In addition, using the techniques described above for determining pore size, it was found that the samples had pores in the range of about 40 microns to 60 microns. Such pore sizes are indicated by arrows in FIGS. 4 to 6, respectively.
[0049]
As shown in the operating electron microscope images of FIGS. 1-8, each of the ECM foams shown is a constant tertiary by a network of naturally occurring ECM defining a plurality of interconnected pores. Contains the original meshwork. The foam has these pores throughout its height, width, and thickness. These pores are continuous and interconnected from the outer surface of the foam (see FIGS. 1 to 3 and 8) to the interior of the foam (see each section in FIGS. 4 to 7). A plurality of irregularly-shaped interconnecting passages extending to the right side. Also, these interconnecting passages are three-dimensional. As described above, the size of each pore, that is, the maximum size corresponding to the interconnecting passage, can be controlled by controlling the above-described manufacturing method.
[0050]
The interconnecting passages facilitate the movement of cells within the implant and allow for efficient nutrient exchange in vivo. These interconnecting passages may also include various bioactive substances, biologically derived substances (eg, various stimulators), cellular and / or biological lubricants, biocompatible substances. It also provides a means for delivering inorganic materials, synthetic polymers and biopolymers (eg, collagen) throughout the length, width and thickness of the ECM prior to implantation. Also, the interconnecting passages defined by such pores may be used in the course of the manufacturing process, such as various compounds used to chemically crosslink the foam. It also acts as a passage for the material. Further, these interconnecting passages as well as the outer surface of the foam can also serve as various sites on which the various materials are carried.
[0051]
As shown in the above examples, the parameters of the above process can be varied to produce an ECM foam having a desired porosity corresponding to a particular application. For example, producing certain foams with relatively low density (and relatively high porosity) corresponding to different applications associated with different bone cells, and different foams associated with different chondrocytes. It may be desirable to produce a foam having a relatively high density (and relatively low porosity) corresponding to the application.
[0052]
Further, the various ECM foams described herein can also be crosslinked. Specifically, the ECM foams described herein can be chemically or physically crosslinked.
[0053]
As seen in the scanning electron microscopy images of FIGS. 1-8 above, each ECM foam shown includes interconnecting pieces of naturally occurring extracellular matrix. Furthermore, as shown in the scanning electron microscopy images of the cross-section of each ECM foam in FIGS. 4-7, the interconnecting pieces of naturally occurring extracellular matrix are irregular in the foam. An inner surface portion having a three-dimensional shape is provided. Also, as shown in the scanning electron microscopy images of each side of the ECM foam, the interconnected naturally occurring extracellular matrix pieces are irregularly shaped tertiary territories of the foam. It has an outer surface having an original shape. Due to these irregular three-dimensional morphologies and interconnecting passages, the various ECM foams of the present disclosure form a relatively large surface area of naturally occurring ECM. In addition, the large surface area of such naturally occurring ECMs is implanted with a variety of biological materials, biologically derived materials, cells, biocompatible polymers and biocompatible inorganic materials. It can be advantageous to provide a relatively large surface area that can be fixed in front. In addition, each ECM foam illustrated forms a relatively large surface area of a naturally occurring ECM to which various cells can bind in vivo.
[0054]
Each of the above ECM foam products can be made having a substantially lower density than the density of other ECM products. For comparison, the density of certain ECM laminates, which are commercially available RESTORE® products, is 0.466 ± 0.074 g / cc. In addition, US Patent Application No. XX / XXX, XXX, entitled "Devices from Naturally Occurring Biologically Derived Materials," Certain ECM products composed of crushed and hardened SIS as described in US Pat. No. 265,280-71142, DEP-748) have a constant density of 0.747 ± 0.059 g / cc. It is created with. Further, U.S. Patent Application No. XX / XXX, XXX, entitled "Meniscus Regeneration Device and Method" (Attorney Patent No. 265280-71141, DEP) An ECM product composed of an enhanced SIS laminate as described in -745) has been made with a density of 0.933 ± 0.061 g / cc.
[0055]
As mentioned above, the ECM foams of the present disclosure can be used to produce various bioactive substances, biologically derived substances, cells and / or stimulators, biocompatible inorganic materials and / or biocompatible substances. Insoluble polymers (eg, various biocompatible synthetic polymers and biopolymers) and combinations of two or more of these materials can be combined during manufacture. Illustratively, various cells can be inoculated throughout the three-dimensional space of the ECM foam, various biological materials can be dried in the ECM foam during manufacture, and various biological materials can be inoculated. The material and the ECM foam can be lyophilized simultaneously, and various biological materials can be conjugated to the ECM foam. It is contemplated that one or more of the above materials may be combined, crosslinked, or otherwise incorporated into the raw ECM material prior to forming the ECM foam. Alternatively, it is contemplated that these materials could be bonded, cross-linked, or otherwise incorporated into the final ECM foam after the lyophilization process. In addition, various combinations of the above methods can be used. For example, implanting an implant of a conjugated ECM foam and a biological lubricant, and then adding additional intra-articular joints of one or more biological lubricants of the same or different type Injections can be performed during, after, or both during and after surgery.
[0056]
The above "bioactive agents" include the following: various chemotactic agents, therapeutic agents (eg, various antibiotics, steroidal and non-steroidal analgesics and anti-inflammatory drugs, Various proteins (eg, various short peptides, bone morphogenetic proteins, glycoproteins and lipoproteins), cell binding mediators, biologically Active ligands, integrin binding sequences, various ligands, various growth and / or differentiation substances (eg, epidermal growth factor, IGF-I, IGF-II, TGF-βI-III, growth and differentiation factors, pulse Duct endothelial growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-derived growth factor and transforming growth factor, parathyroid hormone, parathyroid Gland-related proteins, bFGF, TGFβ superfamily factors, BMP-2, BMP-4, BMP-6, BMP-12, sonic hedgehog, GDF5, GDF6, GDF8, PDGF), specific growth factors One type of small molecule that affects up-regulation, tenascin-C, hyaluronic acid, chondroitin sulfate, fibronectin, decorin, thromboelastin, thrombin-derived peptide, heparin binding domain, heparin, heparan sulfate, DNA fragment and DNA plasmid Including the above. Also, where another such material has therapeutic value in the field of orthopedics, it is anticipated that at least some of these materials will have utility in the concepts of the present disclosure. Such substances are considered to be included in the meaning of the above-mentioned “bioactive agents (bioactive agents) and (bioactive agents)” unless otherwise specified.
[0057]
The above "biologically derived agents" are as follows: bone (autografts, allografts and xenografts) and various derivatives of bone, e.g. Cartilage (autograft, allograft and xenograft) including plate tissue and various derivatives, ligament (autograft, allograft and xenograft) and various derivatives such as intestinal tract including submucosal tissue Various derivatives of tissue (autografts, allografts and xenografts), for example various derivatives of gastric tissue including submucosa (autografts, allografts and xenografts), for example submucosa Various derivatives of bladder tissue, including tissues (autografts, allografts and xenografts), for example, various derivatives of gastrointestinal tissues including submucosa (autografts, allografts and xenografts) For example, various derivatives of respiratory tissue, including submucosal tissue (autograft, allograft and xenograft), for example, various derivatives of genital tissue including submucosal tissue (autograft, allograft) And xenografts), for example, various derivatives of liver tissue, including liver basement membrane (autografts, allografts and xenografts), various derivatives of skin tissue, high platelet plasma (PRP), low concentrations Contains one or more of platelet plasma, bone marrow aspirate, mineral-depleted bone matrix, insulin-derived growth factor, whole blood, fibrin and blood clots. Further, it is believed that purified ECM and other collagen sources should also be included in the "biologically derived material" described above. Also, where another such material has therapeutic value in the field of orthopedics, it is anticipated that at least some of these materials will have utility in the concepts of the present disclosure. Such substances are considered to be naturally included in the meaning of the above-mentioned "biologically derived agents" and "biologically derived agents" unless otherwise specified.
[0058]
"Biologically derived agents" as described above also include various collagenous tissue matrices that are bioremodelable. Furthermore, the terms "bioremodelable collagenous tissue matrix" and "naturally occurring bioremodelable collageneous tissue matrix" in this expression refer to skin, arteries Derived from a natural tissue selected from the group consisting of veins, pericardium, heart valve, dura mater, ligament, bone, cartilage, bladder, liver, stomach, fascia and intestinal tract, tendons, and the like Includes various substrates. This "naturally occurring bioremodelable collagenous tissue matrix" is intended to mean a substrate material that has been washed, processed, sterilized, and optionally cross-linked, but may include various natural materials. Purifying fibers and reforming a substrate material from purified natural fibers is not included in the definition of a naturally occurring bioremodelable collagenous tissue matrix. In addition, the term "bioremodelable collageneous tissue matrices" includes within its definition "various extracellular matrices".
[0059]
The above-mentioned "cells" are as follows: various chondrocytes, fibrochondrocytes, osteocytes, osteoblasts, osteoclasts, periosteal cells, bone marrow cells, mesenchymal cells, stromal cells, Stem cells, embryonic stem cells, precursor cells derived or derived from adipose tissue, peripheral blood progenitor cells, stem cells isolated from adult tissue, genetically transformed cells, certain combinations of chondrocytes and other cells, bone A certain combination of cells and another cell, a certain combination of periosteal cells and another cell, a certain combination of bone marrow cells and another cell, a certain combination of mesenchymal cells and another cell, stroma A certain combination of cells and another cell, a certain combination of stem cells and another cell, a certain combination of embryonic stem cells and another cell, precursor cells isolated from adult tissue and Certain combinations of peripheral blood progenitor cells and other cells, certain combinations of stem cells and other cells isolated from adult tissue, and genetically transformed cells and other cells. Includes one or more of certain combinations of cells. Furthermore, if another cell is found to have therapeutic value in the field of orthopedics, it is expected that at least some of these cells will have utility in the concepts of the present disclosure. Such cells are considered to be included in the meaning of the above-mentioned "cells (cells)" unless specifically limited.
[0060]
The above-mentioned "biological lubricants" include hyaluronic acid and its salts such as sodium hyaluronate, glycosaminoglycan such as dermatan sulfate, heparan sulfate, chondroitin sulfate and keratan sulfate. Periosteal fluid and various components of periosteal fluid, including mucin glycoproteins (eg, lubricin), vitronectin, tribonectins, articular cartilage surface region proteins, surface active phospholipids, lubricious glycoproteins I, II, and Includes rooster comb hyaluronate and the like. This "biological lubricant" is also available in Europe from DePuy International, Ltd., Leeds, UK, and is available in Rehobot, Israel from Bio-Technology (Israel). ARTHREASETM), a high molecular weight sodium hyaluronate manufactured by General (Israel) Ltd., manufactured by Biomatrix, Inc. of Ridgefield, NJ, and manufactured by Wyeth of Philadelphia, PA. -SYNVISC (R) Hylan G-F20 distributed by Wyeth-Ayerst Pharmaceuticals, Sanofi-Synthelabo, Inc., New York, NY. Available from , Fideia S. of Pajua, Italy p. HYLAGAN® sodium hyaluronate, manufactured by Company A (FIDIA SpA), and at concentrations of 1%, 1.4% and 2.3% (for ophthalmic use) Peapack, NJ It is also intended to include various commercial products such as HEALON® sodium hyaluronate, available from Pharmacia Corporation of the United States. Also, where another such material has therapeutic value in the field of orthopedics, it is anticipated that at least some of these materials will have utility in the concepts of the present disclosure. Such substances are considered to be naturally included in the meaning of "biological lubricants (biological luburicant) and (biological luburicants)" unless otherwise specified.
[0061]
The above "biocompatible polymers" are intended to include both various synthetic polymers and various biopolymers (eg, collagen, etc.). Examples of such biocompatible polymers are polyesters of various [alpha] -hydroxycarboxylic acids, such as poly (L-lactide) (PLLA) and polyglycolide (PGA), poly-p-dioxanone (PDS) ), Polycaprolactone (PCL), polyvinyl alcohol (PVA), polyethylene oxide (PEO), various polymers disclosed in U.S. Patent Nos. 6,333,029 and 6,355,699. And any other bioabsorbable and biocompatible polymer, copolymer or mixture of various polymers or copolymers utilized in the construction of various prosthetic implants. In addition, as new biocompatible, bioabsorbable materials are developed, it is anticipated that at least some of these materials will be useful materials from which various fixation means can be made. You. Accordingly, it is to be understood that the above materials are shown by way of example only and that the disclosure of the present invention is not limited to any particular material unless specifically stated in the claims. is there.
[0062]
The above "biocompatible inorganic materials" are hydroxyapatite, all calcium phosphates, alpha-tricalcium phosphate, beta-tricalcium phosphate, calcium carbonate, barium carbonate, calcium sulfate, barium sulfate, It includes various materials such as various calcium phosphate polymorphs, ceramic particles, and combinations of these materials. Also, where another such material has therapeutic value in the field of orthopedics, it is anticipated that at least some of these materials will have utility in the concepts of the present disclosure. Natural substances are considered to be naturally included in the meaning of the above-mentioned "biocompatible inorganic materials (biocompatible inorganic materials) and (biocompatible inorganic materials)" unless otherwise limited.
[0063]
Various combinations of various bioactive substances, biologically derived substances, cells, biological lubricants, biocompatible inorganic materials, biocompatible polymers are disclosed in the present invention. It is expected that it can be used with a variety of scaffold materials.
[0064]
It is also envisioned that various standard sterilization techniques can be used with the various products of the present disclosure.
[0065]
Illustratively, in one embodiment of an inoculation with living cells, such as various chondrocytes, etc., a sterilized graft is inoculated with various living cells, followed by the cell type used. It can be packaged in an appropriate medium. For example, certain cell culture media, including Dulbecco's Modified Eagle Medium (DMEM), may contain various essential amino acids, glucose, ascorbic acid at concentrations and transport conditions deemed appropriate for the cell type. , Sodium pyruvate, fungicides, antibiotics and the like.
[0066]
The ECM foam of the present disclosure is entitled "Hybrid Biologic-Synthetic Bioabsorbable Scaffolds," filed June 14, 2002. The following patent application filed concurrently with this patent application with US patent application Ser. No. 10 / 172,347, the entire contents of which are incorporated herein by reference: U.S. Patent Application No. XX / XXX, XXX (Attorney Specification No. 265280-71141, DEP-745), entitled "Meniscus Regeneration Device and Method", "Device" Is From Naturally Ocaring Biologically Derived Materi U.S. Patent Application No. XX / XXX, XXX (Attorney Specification No. 265280-71142, DEP-748), entitled "Devices from Naturally Occurring Biologically Derived Materials", "Cartage Rage Repair Apparatus" US Patent Application No. XX / XXX, XXX (Attorney Specification No. 265280-71143, DEP-749), entitled "Cartilage Repair Apparatus and Method", "Unitary Surgical Co., Ltd." US Patent Application No. XX / XXX, XXX (Attorney Specification No. DEP-750), entitled "Unitary Surgical Device and Method", "Hybrid Biologic / Synthetic Porous"・ Extracellular matrix scaffold (Hybrid Biologic / S Synthetic Porous Extracellular Matrix Scaffolds), U.S. Patent Application No. XX / XXX, XXX (Attorney Patent No. 265280-71144, DEP-751), "Cartage Rage Repair and Regeneration." US Patent Application No. XX / XXX, XXX (Attorney Specification No. 265280-71145, DEP-752), entitled "Cartilage Repair and Regeneration Device and Method", "Carteage Rage" US Patent Application No. XX / XXX, XXX, entitled "Cartilage Repair and Regeneration Scaffolds and Method" (Attorney Patent Number 265280-71180) No., DEP-763) and "Porous Deliver" US Patent Application No. XX / XXX, XXX, entitled "Porous Delivery Scaffold and Method" (Attorney Specification No. 265280-71207, DEP-762), entitled "Porous Delivery Scaffold and Method". It is anticipated that they can be combined with each of the concepts being described. For example, in various orthopedic applications, it may be desirable for the implantation procedure to be accompanied or followed by a treatment program that involves the administration of hyaluronic acid to the site of the implantation.
[0067]
As can be seen from the above, the concepts of the present disclosure provide a number of advantages. For example, the concepts of the present disclosure may be applied to a porous implantable scaffold that can have various mechanical properties that can be modified to meet the requirements of a scaffold design. We provide a manufacturing method. For example, pore size and material density can be varied to provide a scaffold with a desired mechanical configuration. Among other things, the variety of pore sizes and material densities of such scaffold materials provides a desired amount of cell migration therethrough and a scaffold that provides a desired amount of structural rigidity. It is particularly useful when designing materials. In addition, according to the concepts of the present disclosure, not only have the appropriate physical microstructure to allow for the desired cellular activity in transplantation, but also biochemicals such as those found naturally in various ECMs and the like. Various implantable devices having different compositions (various collagens, growth factors, glycosaminoglycans, etc.) can be produced.
[0068]
While naturally occurring extracellular matrix is believed to provide various advantages over purified extracellular matrix, it is contemplated that the disclosure of the present invention is also applicable to purified extracellular matrix. It is therefore expected that the naturally occurring extracellular matrix will be able to be purified prior to physical grinding of the extracellular matrix. This purification may involve treating the naturally occurring extracellular matrix to remove substantially all material other than collagen before physically grinding the extracellular matrix. It is also believed that this purification can be performed to substantially remove various glycoproteins, glycosaminoglycans, proteoglycans, lipids, non-collagenous proteins and nucleic acids (DNA and RNA).
[0069]
While the above disclosure is susceptible to various modifications and alternative forms, specific exemplary embodiments thereof have been shown by way of example in each of the accompanying drawings and have been described in detail. However, it is not intended at all to limit the disclosure of the invention to the specific forms disclosed, and, conversely, all modifications that fall within the spirit and scope of the disclosure. It is to be understood that it is intended to include examples, equivalents, and alternatives.
[0070]
There are several advantages of the present disclosure resulting from various features of the devices and methods described herein. Furthermore, alternative embodiments of the apparatus and method in the present disclosure may not include all of the features described above but may still benefit from at least some of the advantages of these features. It should be noted that there is. In addition, one of ordinary skill in the art would include one or more of the features in the disclosure of the present invention and would readily be able to easily implement each specific implementation of certain apparatus and methods falling within the spirit and scope of the present disclosure. I can figure it out.
[Brief description of the drawings]
[0071]
The above detailed description is particularly based on the following drawings.
FIG. 1 is a scan showing a surface of a porous reticulated SIS open-cell scaffold having a relatively large pore size and a relatively low material density. It is an image by an electron micrograph.
FIG. 2 illustrates the surface of an open-celled, reticulated SIS open-cell scaffold material having a relatively medium pore size and a relatively medium material density. 5 is an image from a certain scanning electron micrograph.
FIG. 3 is a scan showing a surface of a porous reticulated SIS open cell scaffold having a relatively small pore size and a relatively high material density. It is an image by an electron micrograph.
FIG. 4 is an image from a scanning electron micrograph showing a cross-section of an open-celled, reticulated SIS open-cell scaffold with an example of pores indicated by arrows; The fixed magnification is larger than each image of FIGS. 1 to 3.
FIG. 5 is an image from a scanning electron micrograph showing a cross-section of an open-celled scaffold of a porous reticulated SIS with examples of pores indicated by arrows; The fixed magnification is larger than each image of FIGS. 1 to 3.
FIG. 6 is an image from a scanning electron micrograph showing a cross section of an open-celled scaffold of a porous reticulated SIS with examples of pores indicated by arrows; The fixed magnification is larger than each image of FIGS. 1 to 3.
FIG. 7 is an image from a scanning electron micrograph showing a cross section of an open-cell scaffold of a porous, reticulated SIS, which is larger than the images of FIGS. 1-3. Constant magnification.
FIG. 8 is an image from a scanning electron micrograph showing the surface of an open-celled support framework of a porous, reticulated SIS, which is larger than the images of FIGS. 1-3. Constant magnification.
FIG. 9 is an image from a scanning electron micrograph showing a material of a cohesive SIS piece.
FIG. 10 is an image from a scanning electron micrograph showing a material of a cohesive SIS piece.

Claims (99)

In a method of making an implantable scaffold for repairing or regenerating body tissue,
A method comprising suspending a piece of a naturally occurring extracellular matrix material in a liquid, and lyophilizing the naturally occurring extracellular matrix material and the liquid. 2. The method of claim 1, further comprising freezing the extracellular matrix material and the liquid to form ice crystals with the liquid, wherein the freezing step is performed prior to the lyophilization step. 3. The method of claim 2, wherein the freeze-drying step further comprises sublimating the ice crystals directly into steam in the presence of a vacuum. The method of claim 1, wherein the freeze-drying step comprises sublimating the liquid to form a porous body portion. 2. The method of claim 1, further comprising the step of grinding the extracellular matrix material into its pieces. The extracellular matrix material is a material selected from the group consisting of small intestinal submucosa, bladder submucosa, gastric submucosa, gastrointestinal submucosa, respiratory submucosa, genital submucosa, and liver basement membrane The method of claim 1, comprising: 2. The method of claim 1, further comprising flash freezing the extracellular matrix material and the liquid prior to the lyophilization step. The method of claim 1, further comprising the step of consolidating the piece of naturally occurring extracellular matrix material prior to the lyophilization step. 2. The method of claim 1, further comprising the step of centrifuging the naturally occurring extracellular matrix material piece prior to the lyophilization step. 2. The method of claim 1, further comprising freezing the naturally occurring extracellular matrix material and the liquid at a controlled rate of temperature drop. The method of claim 10, wherein the freezing step comprises a variable rate of temperature drop to change the pore size of the scaffold. In addition, the following: a bioactive substance, a biologically derived substance, various cells, a biological lubricant, a biocompatible inorganic material, and a 2. The method of claim 1, comprising adding at least one of the following biocompatible polymers. In a method of making an implantable scaffold for repairing or regenerating body tissue,
Suspending a piece of a naturally occurring extracellular matrix material in a liquid;
Forming a piece of the naturally occurring extracellular matrix member and the liquid into a material; and removing the liquid to form a plurality of gaps in the material. 14. The method of claim 13, wherein said removing comprises sublimating said liquid. 14. The method of claim 13, wherein said removing comprises evaporating said liquid. Further, supplying a naturally occurring extracellular matrix in the form of a raw material, and forming the naturally occurring extracellular matrix member piece by grinding the naturally occurring extracellular matrix material 14. The method of claim 13, comprising the step of: wherein the piece of naturally occurring extracellular matrix is smaller than the form of the naturally occurring extracellular matrix material. Wherein the naturally occurring extracellular matrix material is from the group consisting of small intestinal submucosa, gastric submucosa, respiratory submucosa, gastrointestinal submucosa, genital submucosa, bladder submucosa, and liver basement membrane 14. The method of claim 13, comprising a selected material. 14. The method of claim 13, wherein said forming step comprises the step of consolidating said piece of naturally occurring extracellular matrix material. In addition, the following: a bioactive substance, a biologically derived substance, various cells, a biological lubricant, a biocompatible inorganic material, and a 14. The method of claim 13, comprising adding at least one of the following biocompatible polymers. An implantable scaffold for repairing or regenerating body tissue;
Prepared by a method comprising suspending a piece of a naturally occurring extracellular matrix material in a liquid, and lyophilizing the naturally occurring extracellular matrix material and the liquid. An implantable scaffold comprising a porous body portion that is provided. The method for preparing the porous body further comprises freezing the naturally occurring extracellular matrix material and the liquid to form ice crystals with the liquid, wherein the freezing step comprises freeze-drying. 21. The implantable scaffold of claim 20, performed prior to the step. 22. The implantable scaffold of claim 21, wherein the freeze-drying step further comprises sublimating the ice crystals directly into steam in the presence of a vacuum. 21. The implantable scaffold of claim 20, wherein the freeze-drying step comprises sublimating the liquid. 21. The implantable scaffold of claim 20, wherein the method for preparing the porous body portion further comprises the step of milling the naturally occurring extracellular matrix material into pieces. Wherein the naturally occurring extracellular matrix material is from the group consisting of small intestinal submucosa, gastric submucosa, bladder submucosa, gastrointestinal submucosa, respiratory submucosa, genital submucosa, and liver basement membrane 21. The implantable scaffold of claim 20, comprising a selected material. 21. The implantable support of claim 20, wherein the method for preparing the porous body portion further comprises flash freezing the naturally occurring extracellular matrix material and the liquid prior to the lyophilization step. Skeletal material. 21. The implantable scaffold of claim 20, wherein the method for preparing the porous body portion further comprises the step of solidifying the piece of naturally occurring extracellular matrix material prior to the lyophilization step. . 21. The implantable support of claim 20, wherein the method for preparing the porous body portion further comprises centrifuging the piece of naturally occurring extracellular matrix material prior to the lyophilization step. Skeletal material. In addition, the following: a bioactive substance, a biologically derived substance, various cells, a biological lubricant, a biocompatible inorganic material, and a 21. The implantable scaffold of claim 20, comprising at least one of the following biocompatible polymers: An implantable scaffold for repairing or regenerating body tissue;
Suspending a piece of a naturally occurring extracellular matrix material in a liquid;
Forming a piece of the extracellular matrix member and the liquid into a material; and removing the liquid to form a plurality of gaps in the material. An implantable scaffold comprising a porous body portion of the invention. 31. The implantable scaffold of claim 30, wherein the removing comprises sublimating the liquid. 31. The implantable scaffold of claim 30, wherein the removing comprises evaporating the liquid. 31. The implantable scaffold of claim 30, wherein the method for preparing the porous scaffold further comprises the step of grinding the naturally occurring extracellular matrix material into pieces. Wherein the naturally occurring extracellular matrix material is from the group consisting of small intestinal submucosa, gastric submucosa, bladder submucosa, gastrointestinal submucosa, respiratory submucosa, genital submucosa, and liver basement membrane 31. The implantable scaffold of claim 30, comprising a selected material. 31. The implantable scaffold of claim 30, wherein the forming step comprises a step of solidifying the piece of the naturally occurring extracellular matrix material. In addition, the following: a bioactive substance, a biologically derived substance, various cells, a biological lubricant, a biocompatible inorganic material, and a 31. The implantable scaffold of claim 30, comprising at least one of the following biocompatible polymers: An implantable scaffold for repairing or regenerating body tissue;
It comprises a porous body of naturally occurring extracellular matrix pieces interconnected to define an inner surface having an irregularly shaped three-dimensional configuration. Implantable scaffold. The interconnected naturally occurring extracellular matrix pieces define a plurality of gaps in the body portion, and the gaps further mix the substrate pieces with water to provide a constant wetness. Sized by controlling the size of each gap by forming the raw material in a frozen state and freezing the water in a controlled manner to control the size of the frozen water crystals 38. The implantable scaffold of claim 37. 38. The implantable support of claim 37, wherein at least a portion of the interconnected naturally occurring extracellular matrix pieces define pores having a nominal pore size of 100 microns to 700 microns. Skeletal material. 40. The implantable support of claim 39, wherein at least a portion of the interconnecting naturally occurring extracellular matrix pieces define pores having a nominal pore size of 300 microns to 700 microns. Skeletal material. 38. The implantable scaffold of claim 37, wherein at least a portion of the interconnected naturally occurring extracellular matrix pieces define pores having a nominal pore size of less than 100 microns. material. In addition, the following: a bioactive substance, a biologically derived substance, various cells, a biological lubricant, a biocompatible inorganic material, and a 38. The implantable scaffold of claim 37, comprising at least one of the following biocompatible polymers: A tertiary implant including a plurality of interconnecting pores defining a three-dimensional interconnecting passage having an irregular shape in an implantable device for repairing or regenerating body tissue. An implantable device comprising an original open-celled foam, wherein at least a portion of the foam comprises a naturally-occurring bioremodelable collagenous tissue matrix. 44. The implantable device of claim 43, wherein the foam comprises an interconnecting piece of a naturally occurring bioremodelable collagenous tissue matrix. The foam has an inner surface, wherein the inner surface has an irregularly shaped three-dimensional morphology, a naturally occurring bioremodelable collagenous tissue matrix. 44. The implantable device of claim 43, comprising: In addition, the following: a bioactive substance, a biologically derived substance, various cells, a biological lubricant, a biocompatible inorganic material, and a 44. The implantable device of claim 43, comprising at least one of the following biocompatible polymers: The naturally occurring bioremodelable collagenous tissue matrix comprises: small intestine submucosa, gastric submucosa, bladder submucosa, gastrointestinal submucosa, respiratory submucosa, 44. The implantable device of claim 43, comprising at least a portion of at least one of genital submucosa and hepatic basement membrane. 44. The implantable device of claim 43, wherein at least a portion of the device has a nominal pore size of 100 microns to 700 microns. 44. The implantable device of claim 43, wherein a nominal pore size of at least a portion of the device is between 300 microns and 700 microns. 44. The implantable device of claim 43, wherein at least a portion of the device has a nominal pore size of less than 100 microns. 44. The implantable device of claim 43, wherein the foam has a constant density between about 0.005 g / cc and 0.5 g / cc. An implantable device for repairing or regenerating body tissue, comprising a porous reticulated body portion with a matrix of a naturally occurring bioremodelable collagenous tissue. . The naturally occurring bioremodelable collagenous tissue matrix is as follows: small intestine submucosa, gastric submucosa, bladder submucosa, gastrointestinal submucosa, respiratory submucosa, 53. The implantable device of claim 52, comprising at least a portion of at least one of genital submucosa and hepatic basement membrane. In addition, the following: a bioactive substance, a biologically derived substance, various cells, a biological lubricant, a biocompatible inorganic material, and a 53. The implantable device of claim 52, comprising at least one of the following biocompatible polymers: 53. The implantable device of claim 52, wherein at least a portion of the reticulated body portion has a nominal pore size of 100 microns to 700 microns. 53. The implantable device of claim 52, wherein at least a portion of the mesh body portion has a nominal pore size of 300 microns to 700 microns. 53. The implantable device of claim 52, wherein at least a portion of the reticulated body portion has a nominal pore size of less than 100 microns. 53. The method of claim 52, wherein at least a portion of the mesh body defines a plurality of interconnecting pores defining a three-dimensional interconnecting passage having an irregular shape. Implantable device. In a method of making an implantable device for repairing or regenerating body tissue,
Supplying a naturally occurring extracellular matrix material in the form of a raw material;
Grinding the naturally occurring extracellular matrix of the raw material in the presence of a liquid to form a slurry of the naturally occurring extracellular matrix, and freezing the slurry of the naturally occurring extracellular matrix Drying to form an open-cell foam of naturally occurring extracellular matrix. 60. The method of claim 59, wherein the open cell foam of the naturally occurring extracellular matrix comprises a molecule other than collagen, and wherein the molecule other than collagen is present in the form of a source of the naturally occurring extracellular matrix. The described method. In addition, the following: a bioactive substance, a biologically derived substance, various cells, a biological lubricant, a biocompatible inorganic material, and a 60. The method of claim 59, comprising adding at least one of the following biocompatible polymers. In a method of making an implantable scaffold for repairing or regenerating body tissue,
Supplying a naturally occurring bioremodelable collagenous tissue matrix in the form of a raw material;
Grinding the naturally occurring bioremodelable collagenous tissue matrix of the raw material to form a coherent piece of the naturally occurring bioremodelable collagenous tissue matrix. Lyophilizing the coherent pieces of the naturally occurring bioremodelable collagenous tissue matrix to form the naturally occurring bioremodelable collagenous tissue matrix. A method comprising forming a reticulated foam. The naturally occurring bioremodelable collagenous tissue matrix reticulated foam comprises molecules other than collagen, wherein the molecules other than collagen are in the form of the naturally occurring extracellular matrix material. 63. The method of claim 62, which is present. In addition, the following: a bioactive substance, a biologically derived substance, various cells, a biological lubricant, a biocompatible inorganic material, and a 63. The method of claim 62, comprising adding at least one of the following biocompatible polymers. In a method of making an implantable scaffold for repairing or regenerating body tissue,
A constant derived from a tissue selected from the group consisting of small intestinal submucosa, gastric submucosa, bladder submucosa, respiratory submucosa, gastrointestinal submucosa, genital submucosa, and hepatic basement membrane Supplying the extracellular matrix material of
Physically pulverizing the extracellular matrix to form a coherent member of the extracellular matrix, and freeze-drying the coherent member of the extracellular matrix to form a constant open cell form of the extracellular matrix; Forming a foam of the above. 66. The method of claim 65, wherein the extracellular matrix comprises a naturally occurring extracellular matrix, and wherein the step of physically grinding the extracellular matrix comprises physically grinding the naturally occurring extracellular matrix. Method. 66. The method of claim 65, further comprising purifying the extracellular matrix before physically grinding the extracellular matrix. 66. The method of claim 65, further comprising treating the extracellular matrix to substantially remove various glycoproteins, glycosaminoglycans, proteoglycans, lipids, non-collagenous proteins and nucleic acids. 66. The method of claim 65, further comprising treating the extracellular matrix prior to physically grinding the extracellular matrix to remove substantially all material other than collagen. 66. The method of claim 65, wherein physically grinding the extracellular matrix comprises physically grinding the extracellular matrix in the presence of a liquid. A product made by the method of claim 65. In addition, the following: a bioactive substance, a biologically derived substance, various cells, a biological lubricant, a biocompatible inorganic material, and a 72. The product of claim 71 comprising at least one of the following biocompatible polymers: In a method of making an implantable device for repairing or regenerating body tissue,
A certain extracellular matrix material selected from the group consisting of small intestinal submucosa, gastric submucosa, bladder submucosa, respiratory submucosa, gastrointestinal submucosa, genital submucosa, and liver basement membrane Supplying, and physically grinding said extracellular matrix in the presence of a liquid to form a slurry of extracellular matrix. 74. The method of claim 73, further comprising freeze-drying the slurry of extracellular matrix to form a reticulated foam of the extracellular matrix. The extracellular matrix material comprises a naturally occurring extracellular matrix material, and the step of physically grinding the extracellular matrix in the presence of a liquid comprises the step of physically crushing the naturally occurring extracellular matrix in the presence of a liquid. 74. The method of claim 73, comprising the step of physically pulverizing. 74. The method of claim 73, further comprising purifying the extracellular matrix before physically grinding the extracellular matrix. 74. The method of claim 73, further comprising treating the extracellular matrix to substantially remove various glycoproteins, glycosaminoglycans, proteoglycans, lipids, non-collagenous proteins and nucleic acids such as DNA and RNA. The described method. 67. A product made according to the method of claim 66. In addition, the following: a bioactive substance, a biologically derived substance, various cells, a biological lubricant, a biocompatible inorganic material, and a 79. The product of claim 78, comprising at least one of the following biocompatible polymers: In a method of making an implantable scaffold for repairing or regenerating body tissue,
A certain extracellular matrix material selected from the group consisting of small intestinal submucosa, gastric submucosa, bladder submucosa, respiratory submucosa, gastrointestinal submucosa, genital submucosa, and liver basement membrane Supplying step,
A method comprising the steps of suspending a piece of extracellular matrix material in a liquid, and freeze-drying the piece of extracellular matrix material and the liquid. In addition, the following: a bioactive substance, a biologically derived substance, various cells, a biological lubricant, a biocompatible inorganic material, and a 81. The method of claim 80, comprising adding at least one of the following biocompatible polymers. In a method of making an implantable scaffold for repairing or regenerating body tissue,
A certain extracellular matrix material selected from the group consisting of small intestinal submucosa, gastric submucosa, bladder submucosa, respiratory submucosa, gastrointestinal submucosa, genital submucosa, and liver basement membrane Supplying step,
Suspending the piece of extracellular matrix material in a liquid;
Forming a piece of the naturally occurring extracellular matrix member and the liquid into a material; and removing the liquid to form a plurality of gaps in the material. In addition, the following: a bioactive substance, a biologically derived substance, various cells, a biological lubricant, a biocompatible inorganic material, and a 83. The method of claim 82, comprising adding at least one of the following biocompatible polymers. A slurry containing a coherent piece of a naturally occurring extracellular matrix in a liquid. In a slurry of an extracellular matrix material containing a coherent piece of extracellular matrix in a certain liquid, the extracellular matrix material may be a small intestinal submucosa, a gastric submucosa, a bladder submucosa, a respiratory organ. A slurry selected from the group consisting of submucosa, gastrointestinal submucosa, genital submucosa, and liver basement membrane. In a method of treating defective cartilage in a joint of a patient,
Implanting a biocompatible device into the patient's body at the joint,
The device comprises a foam comprising a naturally occurring extracellular matrix having a plurality of interconnected pores, wherein at least a portion of the interconnected pored extracellular matrix is about 30 microns. A method having a constant nominal pore size of ~ 100 microns. In a method of treating defective cartilage in a joint of a patient,
A certain extracellular matrix material selected from the group consisting of small intestinal submucosa, gastric submucosa, bladder submucosa, respiratory submucosa, gastrointestinal submucosa, genital submucosa, and liver basement membrane Supplying step,
Forming the extracellular matrix material into a foam having a plurality of interconnected pores, wherein at least a portion of the interconnected pored extracellular matrix is between 30 microns and 100 microns. And having the nominal pore size of B. and further comprising implanting the foam at the patient's joint. In a method of treating diseased or damaged bone,
Implanting a device into the diseased or damaged bone,
The device comprises a foam comprising a naturally occurring extracellular matrix having a plurality of interconnected pores, wherein at least a portion of the extracellular matrix has a nominal pore size greater than about 200 microns. A method having interconnecting pores having dimensions. In a method of treating diseased or damaged bone,
A certain extracellular matrix material selected from the group consisting of small intestinal submucosa, gastric submucosa, bladder submucosa, respiratory submucosa, gastrointestinal submucosa, genital submucosa, and liver basement membrane Supplying step,
Forming the extracellular matrix material into a foam having a plurality of interconnected pores, wherein the interconnected pores have an irregular shape and are three-dimensional. At least a portion of the extracellular matrix extending and having the interconnecting pores has a nominal pore size of greater than 200 microns and further has the disease or injury. Implanting said foam into bone. An implantable device for repairing or regenerating body tissue, comprising a porous, naturally occurring extracellular matrix, wherein the porous matrix has a density of less than 0.1 g / cc. Having an implantable device. 91. The implantable device according to claim 90, wherein the porous substrate has a constant density of less than 0.04 g / cc. 92. The implantable device of claim 91, wherein said porous substrate has a constant density of less than 0.01 g / cc. 91. The implantable device of claim 90, wherein said porous, naturally occurring extracellular matrix comprises SIS. 90. The implantable device of claim 90, wherein the porous substrate comprises a reticulated foam. 91. The implantable device of claim 90, wherein the porous substrate comprises an open cell foam. 91. The implantable device of claim 90, wherein the porous substrate includes a plurality of pores defining a three-dimensional interconnecting passage having an irregular shape. A method of processing an extracellular matrix material, comprising: grinding the extracellular matrix material in the presence of a liquid. The extracellular matrix material comprises small intestinal submucosa,
100. The method of claim 97, wherein said milling step comprises milling said small intestinal submucosa in the presence of said liquid. The liquid comprises water,
100. The method of claim 97, wherein said milling step comprises milling said extracellular matrix material in the presence of water.