JP7434293B2 - ナノ粒子被覆コラーゲン移植物 - Google Patents
ナノ粒子被覆コラーゲン移植物 Download PDFInfo
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- JP7434293B2 JP7434293B2 JP2021513933A JP2021513933A JP7434293B2 JP 7434293 B2 JP7434293 B2 JP 7434293B2 JP 2021513933 A JP2021513933 A JP 2021513933A JP 2021513933 A JP2021513933 A JP 2021513933A JP 7434293 B2 JP7434293 B2 JP 7434293B2
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Description
a) 組織との一体化及び血管新生を促進するような方法で相互に接続する細孔;
b) 最終的にコラーゲン含有膜が正常組織に置き換わるような生分解性及び/又は生体吸収性;
c) 細胞の付着、増殖及び分化を促進する表面の化学的性質;
d) 強度と柔軟性、並びに
e) 低い免疫原性。
(i) コラーゲン含有組織を分離し、エタノール溶液中で組織をインキュベートする;
(ii) 含まれている非コラーゲン性タンパク質を変性させるために、無機塩及び陰イオン性界面活性剤を含む第1の溶液中で、工程(i)のコラーゲン含有組織をインキュベートする;
(iii) 無機酸を含む第2の溶液中で、工程(ii)で得たコラーゲン含有組織を前記材料中のコラーゲンが変性するまでインキュベートする;並びに
(iv) 無機酸を含む第3の溶液中で、工程(iii)で得たコラーゲン含有組織を、前記組織内のコラーゲン束が整列することを可能にするのに十分な時間、同時機械的刺激とともにインキュベートする、ここで、前記機械的刺激は、コラーゲン含有組織に周期的に張力を加えることを含む
によって製造してもよい。
(i) コラーゲン含有組織の所定の期間の引き延ばし;
(ii) コラーゲン含有組織の所定の期間の弛緩;並びに
(iii) 工程(i)及び(ii)のn回の繰り返し(nは1以上の整数)
を含んでいてもよい。
歯科用骨再生誘導法でCEマークが承認されているCelGro(登録商標)コラーゲン膜をOrthocell Ltd, Australiaから入手した。銀の70nmナノ粒子濃縮溶液はSuzhou ColdStones Technology Co., Ltd.(Jiangsu, China)から購入した。
70nm銀ナノ粒子を含有するAgNP濃縮溶液(濃度20mg/mL)を、0.6、0.8、1.0及び1.2mg/mLに希釈した。以下の試験に応じて、コラーゲン膜を1.0、1.5又は2.0cmの正方形に切り取った。化学用のすべての試薬はSigma-Aldrich(Steinheim, Germany)から購入し、精製することなく使用した。
スパッタリングによるAgNP被覆コラーゲン膜は、高周波マグネトロンスパッタリング(Hummer BC-20 DC/RF Sputter System, AnatechUSA)による直接堆積によって製造した。高純度の銀(99.99%、Ezzi Vision Pty Ltd, Australia)を銀のソースとして使用した。コラーゲン膜は、スパッタリング槽内の試料台に両面テープで固定して、スパッタリング中の安定性を確保した(Jiang et al., Surface and Coatings Technology, 2010. 204(21-22): p. 3662-3667;Song et al., Thin Solid Films, 2011. 519(20): p. 7079-7085)。スパッタリングの前に、スパッタリング槽を一晩(約10時間)真空シールし3.0×10-7Torrとした。スパッタリング中に、流量20sccmのArガス(純度99.99%)をスパッタリング槽にパージした。スパッタリングは、100WのDC電力を10分間印加して、17℃、1×10-2Torrで行った。コラーゲン膜と銀の間の距離は12cmであった。
抗菌活性試験
試験生物と適切な培養液の混合物を用いて、0.5~10.0のマクファーランド濁度標準を調製した。目で比較した後、抗菌試験のために0.5マクファーランド濁度標準を選択した。寒天プレートを調製するために、各ペトリ皿に15mlの溶原培地(LB)寒天を注ぎ、固化させた。100μlの黄色ブドウ球菌(株:ATCC 6538P)又は緑膿菌(株:ATCC 9027)の細菌懸濁液を固体LB寒天培地の表面に均一に分散し、沈降させた。異なる銀濃度を有する超音波AgNP被覆コラーゲン膜とスパッタリングAgNP被覆コラーゲン膜を、直径5mmの円形に切り取り、細菌懸濁液で覆われたLB寒天培地の表面に置いた。被覆されていないコラーゲン膜を対照とした。LB寒天-細菌-AgNP被覆コラーゲン膜のプレートを37℃で96時間インキュベートし、24時間ごとに、阻害ゾーンを各膜の周囲に細菌の増殖がない面積(mm2)として測定した。
C3H101/2細胞を細胞毒性と生存率を試験するために使用し、RAW264.7細胞をサイトカイン放出を測定するために使用した。両方の細胞株を、5%CO2を含む加湿雰囲気中、37℃でインキュベートした。C3H101/2細胞は、10%ウシ胎児血清(FBS、Gibco(登録商標))並びに1%ストレプトマイシン及びペニシリン混合物を添加した最小必須培地(MEMα、Gibco(登録商標))で培養した。RAW264.7細胞は、10%ウシ胎児血清(FBS、Gibco(登録商標))並びに1%ストレプトマイシン及びペニシリン混合物を添加したダルベッコ改変イーグル培地(DMEM+GlutaMAX(登録商標)-I)で培養した。
この実験では、C3H10細胞を用い細胞増殖及び細胞生存率を試験した(Vangsness et al., Clinical orthopaedics and related research, 1997, 337: p. 267-271)。AgNP被覆コラーゲン膜から放出されたAgNPの細胞毒性を評価するために、AgNP被覆コラーゲン膜(直径1cm)にC3H10細胞を3×103細胞の密度で播種し、24時間インキュベートして付着させた。超音波で被覆した膜から放出されたAgNPの細胞毒性を評価するために、AgNPを被覆していないコラーゲン膜(直径1cm)にC3H10細胞を3×103細胞の密度で播種し、最終濃度1.86×10-6mg/mLのAgNPが補充された培地で培養した。
PureLinkTM RNA Mini Kit(Invitrogen, ThermoFisher Scientific, USA)を製造元の指示に従って使用し、培養したC3H101/2細胞から全RNAを単離した。QuantiTec Reverse Transcription kit(Qiagen)を使用して相補的DNA(cDNA)を合成した。iQTM SYBR(登録商標)Green Supermixを製造元の指示に従って使用して、リアルタイムPCRを行った。骨形成遺伝子(RUNX2、ALP、OPN)の相対的な発現量を、ハウスキーピング遺伝子(36B4)に対して正規化することによって得た。炎症性サイトカイン遺伝子発現試験では、1、2及び4時間前に、AgNP被覆膜に播種したRAW264.7細胞に100ng/mlのLPSで刺激した。RNA抽出、cDNA合成及びq-PCRを上述したように行った。TNFα及びIL-6の発現量を得、ハウスキーピング遺伝子(36B4)に対して正規化した。選択した遺伝子のプライマーを表1に示す。
AgNP被覆コラーゲン膜上の付着細胞の成長及び増殖は、共焦点レーザー走査顕微鏡画像で可視化した。C3H101/2細胞を、96ウェルプレート中のAgNP被覆コラーゲン膜に、3.0×104生細胞/cm2の細胞密度で播種した。24時間のインキュベーション後、膜をPBSで3回穏やかに洗浄した。細胞の固定には4%パラホルムアルデヒドを使用し(室温で20分)、続いてPBSで3回洗浄した。ローダミンファロイジン(5単位/mL;Biotium, USA)を用い、細胞骨格を暗所で30分間染色した。PBSで3回洗浄した後、Hoechst(分子プローブ、Eugene, USA)を用い、核を暗所で15分間染色し、続いてPBSで3回洗浄した。すべての標本は、共焦点レーザー走査型顕微鏡(CLSM;Nikon A1, Nikon, Japan)を使用して可視化した。
すべてのデータを平均±標準偏差として表示する。一元配置分散分析(ANOVA)を行い、群間の有意差を求め、p<0.05である場合に有意であると見なした。
骨との一体化と感染の予防は、歯槽骨の再建において最も重要である。この実験では、抗菌活性及び抗炎症活性を組み合わせた2種のバリア膜を開発し、AgNP被覆コラーゲン膜を生成する2種の被覆方法の有効性を評価した。AgNP溶液によるコラーゲン膜の超音波処理は、均一な分布と制御可能な沈着を備えた膜を効果的に生成することが見出された。抗菌効果と細胞毒性を検討することで被覆濃度を確定した。この実験で開発されたAgNP被覆コラーゲン膜は、骨再生を誘導する可能性、黄色ブドウ球菌及び緑膿菌に対する優れた抗菌効果を示し、効果的な抗炎症及び骨形成誘導能を実証した。
以下に、本願の出願当初の請求項を実施の態様として付記する。
[1]移植可能なコラーゲン含有医療機器を製造する方法であって、前記方法はコラーゲン含有医療機器を金属マイクロ粒子及び/又は金属ナノ粒子で被覆する工程を含み、コラーゲン含有医療機器を被覆する前記工程は超音波処理工程であり、前記コラーゲン含有医療機器は、金属マイクロ粒子及び/又は金属ナノ粒子で被覆されていない医療機器と比較して、移植時に抗菌及び抗炎症活性を示す、方法。
[2]前記金属マイクロ粒子及び/又は金属ナノ粒子は、銀及び銅又はその組合せからなる群から選択される金属を含む、[1]に記載の方法。
[3]前記コラーゲン含有医療機器はコラーゲン含有膜である、[1]に記載の方法。
[4]前記コラーゲン含有医療機器は宿主生物の内部に送達されるか、in vitroで使用される、[1]に記載の方法。
[5]前記宿主生物はヒト又は動物である、[1]に記載の方法。
[6]前記被覆は前記機器の少なくとも一部を覆う、[1]に記載の方法。
[7]前記被覆は、天然ポリマー、合成ポリマー、金属、金属酸化物、酸化物、金属窒化物、ホウ酸塩、セラミック、ジルコニア、同種移植用硬組織、同種移植用軟組織、異種移植用硬組織、異種移植用軟組織、カーボンナノ構造、炭素、ガラス、天然材料、生体適合材料をさらに含む、[1]に記載の方法。
[8]前記金属マイクロ粒子及び/又は金属ナノ粒子のサイズは、約0.5nm~約500nmである、[1]に記載の方法。
[9]前記被覆は、感染の処置、感染の防止、炎症の処置、炎症の防止、細胞接着の促進、バイオフィルム形成の防止、バイオフィルム形成の阻害、細胞増殖の促進、生体系若しくは非生体系との結合の促進、細胞機能の増加若しくは減少、薬物及び/若しくは生物活性剤の送達、又は、材料の宿主組織とのより良い一体化の確保、の少なくとも1つを実現することができる、[1]に記載の方法。
[10]前記被覆は金属マイクロ粒子及び金属ナノ粒子を含む、[1]に記載の方法。
[11]前記被覆は金属ナノ粒子を含む、[1]に記載の方法。
[12]前記被覆は金属ナノ粒子及び/又は金属マイクロ粒子の1つ以上の層を含む、[1]に記載の方法。
[13]前記1つ以上の層は銀のナノ粒子を含む、[12]に記載の方法。
[14]前記医療機器は、整形外科用インプラント、歯科用インプラント、獣医学用補綴材料、足場又は組織工学のマトリックスである、[1]に記載の方法。
[15]前記整形外科用インプラントは、股関節インプラント、膝関節インプラント又は肩関節インプラントである、[14]に記載の方法。
[16]前記歯科用インプラントは、アバットメントである[14]に記載の方法。
[17]インプラントにおけるバイオフィルム形成を阻害する方法であって、前記方法は、バイオフィルムの形成及び/又は細菌の増殖を防ぐために、銀のナノ粒子で被覆されたコラーゲン含有膜で前記インプラントを覆う工程を含む、方法。
[18]前記バイオフィルムは、細菌、真菌又は原生動物のバイオフィルムである、[17]に記載の方法。
[19]前記インプラントは、整形外科用インプラント、歯科用インプラント、足場又は組織工学のマトリックスである、[17]に記載の方法。
[20]医療機器又はインプラントにおける微生物のコロニー形成を阻害する方法であって、前記方法は、微生物のコロニー形成を防ぐために、銀のナノ粒子で被覆されたコラーゲン含有膜で前記機器又はインプラントを覆うことを含む、方法。
[21]銀のナノ粒子で被覆されたコラーゲン含有医療機器を含む包装物であって、前記銀のナノ粒子は超音波処理によって適用されており、前記機器は気密容器又は真空パックされた容器に密封されている、包装物。
Claims (15)
- 移植可能なコラーゲン含有膜を製造する方法であって、前記方法はコラーゲン含有膜を銀ナノ粒子で被覆する工程を含み、コラーゲン含有膜を被覆する前記工程は超音波浴中での超音波処理工程であり、前記コラーゲン含有膜は、銀ナノ粒子で被覆されていないコラーゲン含有膜と比較して、移植時に抗菌及び抗炎症活性を示し、前記コラーゲン含有膜は80%(w/w)以上の編目構造のI型コラーゲンの繊維又は束を含む、方法。
- 前記コラーゲン含有膜を、濃度0.6mg/mL、0.8mg/mL又は1.0mg/mLの銀ナノ粒子で被覆することを含む、請求項1に記載の方法。
- 前記コラーゲン含有膜は宿主生物の内部に送達されるか、in vitroで使用される、請求項1に記載の方法。
- 前記宿主生物はヒト又は動物である、請求項3に記載の方法。
- 前記被覆は前記コラーゲン含有膜の少なくとも一部を覆う、請求項1に記載の方法。
- 前記被覆は、天然ポリマー、合成ポリマー、金属、金属酸化物、酸化物、金属窒化物、ホウ酸塩、セラミック、ジルコニア、同種移植用硬組織、同種移植用軟組織、異種移植用硬組織、異種移植用軟組織、カーボンナノ構造、炭素、ガラス、天然材料、生体適合材料をさらに含む、請求項1に記載の方法。
- 前記銀ナノ粒子のサイズは、約0.5nm~約500nmである、請求項1に記載の方法。
- 前記被覆は、感染の処置、感染の防止、炎症の処置、炎症の防止、細胞接着の促進、バイオフィルム形成の防止、バイオフィルム形成の阻害、細胞増殖の促進、生体系若しくは非生体系との結合の促進、細胞機能の増加若しくは減少、薬物及び/若しくは生物活性剤の送達、又は、材料の宿主組織とのより良い一体化の確保、の少なくとも1つを実現することができる、請求項1に記載の方法。
- 前記被覆は銀ナノ粒子の1つ以上の層を含む、請求項1に記載の方法。
- 前記コラーゲン含有膜は、整形外科用インプラント、歯科用インプラント、獣医学用補綴材料、足場又は組織工学のマトリックスから選択される医療機器を被覆する、請求項1に記載の方法。
- 前記整形外科用インプラントは、股関節インプラント、膝関節インプラント又は肩関節インプラントである、請求項10に記載の方法。
- 前記歯科用インプラントは、アバットメントである請求項10に記載の方法。
- 請求項1~9のいずれか一項に記載の方法によって製造された移植可能なコラーゲン含有膜。
- 請求項13に記載の移植可能なコラーゲン含有膜で被覆された、整形外科用インプラント、歯科用インプラント、獣医学用補綴材料、足場又は組織工学のマトリックスから選択される医療機器。
- 銀のナノ粒子で被覆されたコラーゲン含有膜を含む包装物であって、前記銀のナノ粒子は超音波浴中での超音波処理によって適用されており、前記コラーゲン含有膜は気密容器又は真空パックされた容器に密封されており、前記コラーゲン含有膜は80%(w/w)以上の編目構造のI型コラーゲンの繊維又は束を含む、包装物。
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