著者らは、NIHのディレクターの新イノベーター賞（1DP2 OD006499-01 AAと1DP2 AJEにOD006460-01〜）からの支援、そして科学技術（ナノメディシンの優秀なUCサンディエゴセンター）キング·アブドゥルアズィーズ市に感謝しています。我々は、原稿上の彼女の批判的なコメントのためにジェシカ·ムーア氏に感謝したいと思います。

1。蛍光標識アクリロイル-PEG-RGDSの合成<ol><li>室温でアルゴン下、ジメチルスルホキシド（DMSO）のモル比で1.2:1:2とNNジイソプロピルエチルアミン（DIPEA）アクリロイル-PEGスクシンイミジルカルボキシメチルエステル（3400グラム/モルaPEG-SCMは、PEG MW）とRGDSペプチドを反応一晩。 </li><li>飛行のマトリックス支援レーザー脱離/イオン化時間（MALDI-TOF）質量分析法によって共役を確認してください。 MALDIターゲットとドライでの試料スポットにaPEG-RGDS反応液の1μlを添加します。ユニバーサルMALDIマトリックスのテトラヒドロフラン（THF）と1分間ボルテックスの飽和溶液を調製します。同じサンプルのスポットにこのソリューションの約1μlを添加する。比較のためにaPEG-SCMの手順を繰り返します。ロードし、分析します。 aPEG-RGDSの分子量はaPEG-SCM（ 図2）より大きくなければなりません。 </li><li>蛍光色素を結合するために、溶解のAlexa Fluor 350カルボン酸（succinimydylエステル）の等モル量を追加DMSOの最小容量で、1.1からaPEG-RGDS反応溶液に、室温で一晩アルゴン下で反応する。 </li><li>日当たり少なくとも二回透析液を交換し、1,000:1体積比で48時間、4℃でのDI-H 2 Oに対して（MW 3500 Da）は透析によってaPEG-RGDS-350を精製する。 </li><li>精製LabconcoフリーゾーンPlusまたは同等のフリーズドライシステムでaPEG-RGDSを-350、-20℃で保存をフリーズドライ</li></ol> 2。 RGDS-350層を交互に使用した2DのPEGゲルの2Dモールドと製作の準備<ol><li>疎水性ガラススライドを準備します。真空オーブン内のガラス皿にきれいなガラススライドを配置します。 80熱が°Cで30分間完全に表面を乾燥させる。場所はドラフト内でスライドと皿と優しくコート表面全体に30秒間揺らし、各スライドに250μlのSigmacoteを追加します。徹底的にLEAがでで5分間ずつ2回浸漬し、蒸留水で洗浄した後、100％メタノールでコーティングされたスライドをすすぎトン10ミリリットル。 </li><li> 10ミリメートル生検パンチでシリコーンスペーサー（0.8mm厚）を切り取ります。 </li><li>シリコーンスペーサーとSigmacote処理したスライドガラスをオートクレーブ。 </li><li>最終濃度（ 例えば 40％、変化得イオジキサノールの異なる量（水中60％原液）と混合PEGアクリレート（PEGDA）前駆体（最終濃度15パーセントw / v）のことで、個々のマイクロチューブ内の各々の層のための解決策を策定する30％、20％と10％）、リン酸塩残量を補うことは傾斜密度の解を得るために、緩衝生理食塩水（PBS）。同様にして、交互層のために、イオジキサノールおよびPBSでミックスaPEG-RGDS-350（終濃度8 mm）が様々な濃度（ 例えば 35％、25％、15％）を得た。 </li><li>様々な層（各層の溶液1ml当たりの株式溶液10μl）の各溶液に（2 - ヒドロキシ-4&#39;-（2 - ヒドロキシエトキシ）-2 - メチルプロピオフェノン、N-ビニルピロリドンで333mg/ml株）光開始剤を追加します。光開始剤Isは光に敏感であるように、金型内のゲルの階層化する前に重合を防止するために最後に追加された。 </li><li>このプロトコルの後続ステップは、無菌性を確保するためにバイオセーフティキャビネット内で実行されます。 </li><li>滅菌した1mlシリンジおよび0.2μmフィルターを用いて、各溶液をろ過滅菌を。 2 Sigmacore処理されたガラススライド、 図1に示すように配置するクランプとの安全の間にスペーサーを挟むことにより、金型設定を組み立てます。 </li><li>密度の低い溶液（35％イオジキサノールと例えば aPEG-RGDS-350）に続く最初の最も密な溶液（40％イオジキサノールと例えば PEGDA）を追加することで、階層化ゲルをキャストします。 図1に示すように、所望の組成と密度のいくつかの層を達成するために代替の階層化を繰り返します。 </li><li>ポータブルUVR-9000ランプを使用して3分間365nmの光でモールドを照射します。重合したゲルは、5分間硬化させることを許可します。クランプを外し、そっと上部のガラスを持ち上げるスライドやカビ、成層DGMPゲルは、スライド上に残ります。無菌のスパチュラを使用して、慎重に滅菌PBSまたは洗浄用培地を含む50 mlチューブにゲルを置きます。 </li><li>密度調節剤、光開始剤、及び未反応のポリマーを除去するために一日に少なくとも二回バッファを交換し、1,000:1体積比でPBS中の重合ゲルを洗浄します。代わりに、PBSを細胞増殖培地と交換することができる。 PBSまたはステップ3で概説細胞培養実験のための増殖培地中DGMPゲルを保管してください。 </li><li>交互層を可視化するために、VersaDocゲルドキュメンテーションユニットのサンプルトレイ上の定規に沿ってDGMPゲル（これらのゲルは、細胞培養のため使用できません）を手配。 350 nmのゲルを公開。露光時間は、フルオロフォアの濃度に応じて異なります。 DGMPヒドロゲル中に濃い青とバンドを交互に異なる化学組成（ 図3）の離散的な層の形成を実証している。 </li></ol><ol><li> RGDSペプチドを組み込んだゲルは、そのようなC2C12筋芽細胞などの接着依存性細胞を使用しています。 </li><li>ゆっくりバイオセーフティキャビネット内で無菌のセルスクレイパーを用いて48ウェル細胞培養プレートのウェルにDGMPゲル（PBS中に保存）を挿入します。 </li><li>プリ暖かい増殖培地（10％v / vのウシ胎児血清および1％v / vの100xのペニシリン - ストレプトマイシン溶液を補充したダルベッコ改変イーグル培地またはDMEM）およびPBS 37℃に設定した水浴℃で</li><li> PBSでC2C12細胞3倍の60％コンフルエントのプレート（10 mm）を洗浄します。 0.25％トリプシン-EDTAの1mlを添加し、2分間37℃でインキュベートすることにより、PBSおよび収穫細胞を吸引。増殖培地とカウント細胞で細胞を​​再懸濁する。 C2C12筋芽細胞（20,000細胞/ cm 2）と同様にゲル含有細胞培養DGMPシード。 5％CO 2/95％相対湿度で37℃で細胞をインキュベートします。再しないように注意してください4時間後にそっと交換媒体、軽く接着した細胞を移動します。 </li><li> 24時間後、DGMPゲルのRGDS含有層上にC2C12筋芽細胞の付着は落射蛍光および位相差顕微鏡（ZEISS AXIOVERT 200）で確認することができる。 </li></ol>

<ol>
	<li>Place, E. S., Evans, N. D., Stevens, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexity+in+biomaterials+for+tissue+engineering.">Complexity in biomaterials for tissue engineering.</a> Nat. Mater. 8, 457-470 (2009).</li><li>Liu, V. A., Jastromb, W. E., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+protein+and+cell+adhesivity+using+PEO-terminated+triblock+polymers.">Engineering protein and cell adhesivity using PEO-terminated triblock polymers.</a> J. Biomed. Mater. Res. 60, 126-134 (2002).</li><li>Sharma, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+zonal+organization+into+tissue-engineered+cartilage.">Designing zonal organization into tissue-engineered cartilage.</a> Tissue Eng. 13, 405-414 (2007).</li><li>Hahn, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photolithographic+patterning+of+polyethylene+glycol+hydrogels.">Photolithographic patterning of polyethylene glycol hydrogels.</a> Biomaterials. 27, 2519-2524 (2006).</li><li>Kizilel, S., Sawardecker, E., Teymour, F., Perez-Luna, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+formation+of+covalently+bonded+hydrogel+multilayers+through+surface+initiated+photopolymerization.">Sequential formation of covalently bonded hydrogel multilayers through surface initiated photopolymerization.</a> Biomaterials. 27, 1209-1215 (2006).</li><li>Harley, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+multiphase+osteochondral+scaffold.+II.+Fabrication+of+a+mineralized+collagen-glycosaminoglycan+scaffold.">Design of a multiphase osteochondral scaffold. II. Fabrication of a mineralized collagen-glycosaminoglycan scaffold.</a> J. Biomed. Mater. Res. A. 92, 1066-1077 (2010).</li><li>Cuchiara, M. P., Allen, A. C., Chen, T. M., Miller, J. S., West, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilayer+microfluidic+PEGDA+hydrogels.">Multilayer microfluidic PEGDA hydrogels.</a> Biomaterials. 31, 5491-5497 (2010).</li><li>Roam, J. L., Xu, H., Nguyen, P. K., Elbert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+of+protein+concentration+gradients+mediated+by+density+differences+of+poly(ethylene+glycol)+microspheres.">The formation of protein concentration gradients mediated by density differences of poly(ethylene glycol) microspheres.</a> Biomaterials. 31, 8642-8650 (2010).</li><li>Gleghorn, J. P., Lee, C. S., Cabodi, M., Stroock, A. D., Bonassar, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesive+properties+of+laminated+alginate+gels+for+tissue+engineering+of+layered+structures.">Adhesive properties of laminated alginate gels for tissue engineering of layered structures.</a> J. Biomed. Mater. Res. A. 85, 611-618 (2008).</li><li>Karpiak, J. V., Ner, Y., Almutairi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density+gradient+multilayer+polymerization+for+creating+complex+tissue.">Density gradient multilayer polymerization for creating complex tissue.</a> Adv. Mater. 24, 1466-1470 (2012).</li><li>Pierschbacher, M. D., Ruoslahti, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+attachment+activity+of+fibronectin+can+be+duplicated+by+small+synthetic+fragments+of+the+molecule.">Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule.</a> Nature. 309, 30-33 (1984).</li><li>Kaushik, G., Fuhrmann, A., Cammarato, A., Engler, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=+In+Situ+Mechanical+Analysis+of+Myofibrillar+Perturbation+and+Aging+on+Soft,+Bilayered+Drosophila+Myocardium."> In Situ Mechanical Analysis of Myofibrillar Perturbation and Aging on Soft, Bilayered Drosophila Myocardium.</a> Biophysical Journal. 101, 2629-2637 (2011).</li></ol>

著者らは、開示することは、競合する利害関係はありません。

DGMPは高価な機器に依存しない多層ゲルを調製するためのシンプルな戦略です。このプロトコルは、コラーゲンやヒアルロン酸などの他の生体適合性材料を使用して、足場を作成するために適応させることができます。生理活性小分子は、例えば細胞接着促進RGDSペプチドのため、層の間の合図の混入を防ぐために、ポリマーマトリックスに係留することができます。マトリックスメッシュのサイズに応じて、彼らは、ハイドロゲル10を通って拡散しにくいようなタンパク質は、化学的結合を必要とせずに異なる層にカプセル化することができます。ここでは、イオジキサノール（Nycoprep）は、以前の生細胞アプリケーション用に使用されている不活性密度修飾子を使用していました。例えば、ショ糖、ブドウ糖などの他の密度調節剤を使用することもできます。セトリング時間（t s）を変化させることによって、人は、必要に応じて二つの層の間のインタフェースが（長いセトリング時間はスムーズな遷移を与える）またはスムーズな遷移を生成するために微調整することができます&lt;&gt; 10（商標）。たとえば、DGMPゲル層の間のスムーズな遷移が走化性などの細胞プロセスを研究する生物学的な手がかりの連続勾配を生成するために使用することができる。 ゲル剛性に密度調整剤の効果を15％aPEGdaゲルのため、図5に示されている。PEGDAとイオジキサノール濃度の関数としての剛性と気孔率のより完全な特性評価現在評価されている。この例ではPEGDA濃度が比較的高いですが、我々は、することなくゲルに比べて30％のイオジキサノールを含むゲルは60％以上の弾性率を観察した。ゲル剛性の変化を変調マクロマー濃度や架橋密度を調整することができる。 我々はまた、ポリアクリルアミドおよびPEG前駆体10を使用して 3D多層ゲルを作成するDGMP手法を適用している。濃度又はプレポリマーの架橋度を変化させることができにおける構造変化3Dで偏成長および移行などの細胞の挙動を調査するために使用することができる足場、。 要約すると、DGMPは、生物医学と基礎研究を幅広いアプリケーション向けに生体適合性材料の様々な2Dおよび3Dの足場を製造するために適用することができる、柔軟なテクニックです。

<table border="1"><tbody><tr><td> 試薬またはインストゥルメント </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>ポリエチレングリコールsuccinimydylカルボキシメチル（α-PEG-SCM） </td><td> Laysan </td><td> 120から64 </td></tr><tr><td> Polyethelyeneグリコールジアクリレート（PEGDA） </td><td> Dajacラボ</td><td> 9359 </td></tr><tr><td>アルギニン - グリシン - アスパラギン酸 - セリン（RGDS） </td><td>アメリカンペプチド</td><td> 49-01-4 </td></tr><tr><td> N、N -ジイソプロピルエチルアミン（DIPEA） </td><td>シグマ</td><td> D125806 </td></tr><tr><td>ジメチルスルホキシド（DMSO） </td><td>シグマ</td><td> D2438 </td></tr><tr><td> N、N-ジメチルホルムアミド（DMF） </td><td>フィッシャー</td><td> D119-4 </td></tr><tr><td>テトラヒドロフラン（THF） </td><td>フィッシャー</td><td> T397 </td></tr><tr><td>透析カセット（3500ダ） </td><td>サーモサイエンティフィック</td><td> 66330 </td></tr><tr><td>のAlexa Fluor 350カルボン酸エステルsuccinimydyl </td><td>ライフテクノロジーズ</td><td> - 10168 </td></tr><tr><td> Sigmacote </td><td>シグマ</td><td> SL2 </td></tr><tr><td>シリコーンスペーサー</td><td>グレンジャー</td><td> 1MWA4 </td></tr><tr><td>生検パンチ</td><td> Acuderm </td><td> P1025（10ミリメートル） P850（8 mm）の</td></tr><tr><td> Dulbeccoリン酸（DPBS）緩衝生理食塩水</td><td> Hyclone社</td><td> SH30028 </td></tr><tr><td>イオジキサノール（NycoPrep） </td><td>フィッシャー</td><td> NC9388846 </td></tr><tr><td> 2 - ヒドロキシ-4&#39;-（2 - ヒドロキシエトキシ）-2 - メチル</td><td>シグマ</td><td> 410896 </td></tr><tr><td>ダルベッコ改変イーグル培地（DMEM） </td><td>ライフテクノロジーズ</td><td> 11054 </td></tr><tr><td>ウシ胎仔血清</td><td>ライフテクノロジーズ</td><td> 10082 </td></tr><tr><td>ペニシリン - ストレプトマイシン</td><td>ライフテクノロジーズ</td><td> 15140 </td></tr><tr><td> C2C12筋芽細胞</td><td> ATCC </td><td> CRL-1772 </td></tr><tr><td> MALDI </td><td>ブルカー</td><td> N / A </td></tr><tr><td> UVR-9000 </td><td> Bayco </td><td> UVR-9000 </td></tr><tr><td> VersaDoc </td><td>バイオ·ラッド</td><td> N / A </td></tr></tbody></table>

density gradient multilayered polymerization (dgmp): a novel technique for creating multi-compartment, customizable scaffolds for tissue engineering

種類と生物学的刺激の濃度（ 例えば 、成長因子、阻害剤、または小分子）やマトリックス構造（ 例えば組成、濃度、またはマトリックスの剛性が）空間にわたって変化、調査の広い範囲を可能にする複雑な組織培養マトリックス、これらの変数は、細胞分化、移動、および他の現象にどのような影響を与えるかについて。階層化行列を作成する上での大きな課題は、各レイヤ1からの個々の成分の拡散せず、層界面の構造的完全性を維持している。これを達成するための現在の方法は、光パターニング2-3、リソグラフィ4、シーケンシャルfunctionalization5、6を凍結乾燥、マイクロフルイディクス7、またはそれらの多くは、洗練された計測機器や技術的なスキルを必要とする遠心分離8が含まれています。その他は9層の剥離につながる可能性があります個々の層の連続した添付ファイルに依存している</&gt;（商標）。 DGMPは密度10を変化させるレイヤーを作成するなどのような不活性イオジキサノール密度修飾子を使用することで、これらの問題を克服しています。密度修飾子は任意のプレポリマーまたは生物活性分子と混合することができるので、DGMPは各足場層をカスタマイズすることができます。彼らは水のままで単に密度調節剤の濃度を変化させることによって、隣接する層の混合を防ぐ。その後のシングルステップ重合は、各層が異なる化学的および機械的特性を有する構造的に連続多層足場を生じさせる。密度修飾子は、簡単に個々の層またはそのコンポーネントの摂動なしですすぎ十分に除去することができる。この技術は、それゆえ様々なサイズ、形状、材料のハイドロゲルを作成するための適している。 交互の層がRGDS-350を組み込むた2D-ポリエチレングリコール（PEG）でゲルを製造するためのプロトコルは、以下の通りです。我々は、PEG bを使用それは、生体適合性および不活性であるecause。 RGDS、細胞接着ペプチド11は 、生物学的な手がかりの空間的制約を示すために使用され、フルオロフォア（アレクサフルオル350）の接合は、私たちは視覚的に様々な層を区別することができますされています。この手順は、他の材料に適合させることができる（ 例えば、コラーゲン、ヒアルロン酸など）と10いくつかの修正を加えて3Dゲルを作製するために拡張することができます。

MALDI-TOF分析はアクリロイル-PEG（ 図2）にRGDSペプチドの結合を確認します。ゲルイメージングは、光重合開始剤（ 図3A）の後RGDS-350（青）の交互層を明らかにする。 図3Aに示すように、2D DGMPゲルサイズはシリコーン型（左10ミリメートル; 8ミリメートル、右）の直径に基づいて変化させることができ、したがって、複数のアッセイにおける使用のために容易にカスタマイズ可能です-このケースに収まるように48ウェル細胞培養プレート（ 図3B）。 DGMPゲル上で培養したC2C12筋芽細胞の落射蛍光および位相差顕微鏡は、細胞接着ペプチド（RGDS）の区画を示す、RGDS-350-含有するPEG層（ 図4）で選択的結合を示しています。 <img src="/files/ftp_upload/50018/50018fig1.jpg" alt="図1" fo:content-width="4.5in" fo:src="/files/ftp_upload/50018/50018fig1highres.jpg"/> 図1。分子weigMALDI-TOFはRGDSペプチドの結合の後に得られたaPEG-RGDSにaPEG-SCMを比較することにより、HT分析。 <img src="/files/ftp_upload/50018/50018fig2.jpg" alt="図2" fo:content-width="6in" fo:src="/files/ftp_upload/50018/50018fig2highres.jpg"/> 図2。 DGMPゲル作製の模式図。勾配が積層された後、彼らは光重合続いメスインターフェイスを作成するために時間の期間（T S） を変化させるために解決させることができる。成層DGMPゲルは簡単にさらに使用するために金型から取り出すことができます。 <a href="https://www.jove.com/files/ftp_upload/50018/50018fig2large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 <img src="/files/ftp_upload/50018/50018fig3.jpg" alt="図3" /> 図3光重合後に得られた）2次元多層ゲルは350nmとVersaの白色光チャネルを使用して画像化ドクゲルドキュメンテーションユニット。画像が白でRGDSを含む交互層を明らかにする。B）は DGMPゲルの挿入を48ウェル細胞培養皿にグレースケール。 <img src="/files/ftp_upload/50018/50018fig4.jpg" alt="図4" /> 図4 DGMPゲル（スケールバーは50μm）上に成長させたC2C12筋芽細胞の位相コントラストと落射蛍光画像を合併。 <img src="/files/ftp_upload/50018/50018fig5.jpg" alt="図5" /> 図5。ゲル表面の弾力性に関するイオジキサノールの効果。2 nNの力トリガ12を使用して、以前に確立された方法を用いて架橋したPEG基板の静的サンプルの原子間力顕微鏡の測定は。 * P &lt;0.05、** P &lt;0.01。

Watch this Scientific Journal Video about Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering at JoVE.com

Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

ここでは、組織工学のための異なった層の間の連続的なインタフェースを備えた生体適合性、階層化行列を作成するための独自の戦略を説明します。このような足場は、様々な生物学的、化学的または機械的な手がかりによって細胞の挙動を調節するのに理想的なカスタマイズ可能な環境を提供することができます

密度勾配多層重合（DGMP）：マルチコンパートメントを作成するための新規な技術、組織工学のためのカスタマイズ可能な足場

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix ...

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Bioengineering

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

12.4K ビュー.  University of California, San Diego. ここでは、組織工学のための異なった層の間の連続的なインタフェースを備えた生体適合性、階層化行列を作成するための独自の戦略を説明します。このような足場は、様々な生物学的、化学的または機械的な手がかりによって細胞の挙動を調節するのに理想的なカスタマイズ可能な環境を提供することができます

ビデオ: Density Gradient Multilayered Polymerization DGMP: A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

動脈組織工学におけるエラストマーのPGSの足場

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

生物工学

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

組織工学のためのエレクトロ繊維のポスト処理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

3次元細胞培養のための自己申告足場

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past studies have evaluated the performance of the reactor for the removal of COD1 and nitrogen species2-4 by heterotrophic and chemoautotrophic bacteria, respectively. The HDBR design eliminates the requirement for external flocculation/sedimentation processes while still yielding effluent containing low suspended solids. In this study, the HDBR is applied as a photobioreactor (PBR) in order to characterize the nitrogen removal characteristics of an algae-based photosynthetic microbial community. As previously reported for this HDBR design, a stable biomass zone was established with a clear delineation between the biologically active portion of the reactor and the recycling reactor fluid, which resulted in a low suspended solid effluent. The algal community in the HDBR was observed to remove 18.4% of total nitrogen species in the influent. Varying NH4+ and NO3- concentrations in the feed did not have an effect on NH4+ removal (n=44, p=0.993 and n=44, p=0.610 respectively) while NH4+ feed concentration was found to be negatively related with NO3- removal (n=44, p=0.000) and NO3- feed concentration was found to be positively correlated with NO3- removal (n=44, p=0.000). Consistent removal of NH4+, combined with the accumulation of oxidized nitrogen species at high NH4+ fluxes indicates the presence of ammonia- and nitrite-oxidizing bacteria within the microbial community.

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

多様な微生物群集の高密度栽培のための新しいバイオリアクター

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

繊細な組織操作のためのカスタマイズ可能なソフトロボット空気圧グリッパ装置のロッドベースの試作

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

新生児心臓足場：再生研究のための新しい行列

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

組織工学的応用の制御可能な形態の三次元 Poly(ε-caprolactone) 足場のエレクトロスピニング記述を溶かす

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

軟骨と皮膚組織類縁ミキシング ユニット Bioink Precellularization の小説のパッシブ バイオプリンティング

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

内耳組織工学用マウス蝸牛を Decellularizing するためのプロトコル

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

三次元ペプチド足場で哺乳動物の細胞を培養

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...