This work was partially funded through the Richard Barber Foundation and a Thomas Rumble Fellowship (TJW).

1。水/オイル/水ダブルエマルションマイクロス制作<ol><li>最初の脱イオン水中のポリビニルアルコール（PVA）vの溶液/ wの2％及び0.5％を準備する。透明になるまで50℃での溶液を撹拌し、これには数時間かかる場合があります。脱イオン水に2％v / vのイソプロピルアルコールの溶液を調製する。 </li><li>希望親水性タンパク質の水溶液を調製します。下の表は、例示的製剤を提供する。 </li></ol><img alt="表1" src="/files/ftp_upload/51517/51517table1highres.jpg" /> 表1：実施例プロテインソリューション以下のタンパク質溶液は、正常にこのプロトコルを使用してカプセル化され、エレクトロスピニングされている。必要に応じて他の親水性タンパク質溶液を使用することができる。 <ol start="3"><li> 50ミリリットルの遠心分離管に0.5％PVA溶液40mlを置き、脇に置きます。 </li><li> 15mlの遠心管中で65:35、300mgの溶解ジクロロメタン3ml中のポリ（乳酸 - コ - グリコール酸）（PLGA）。ボルテックスミキサーは、PLGAの溶解を促進するために使用することができる。 </li><li>タンパク質溶液200μl、2％PVA溶液4μlのを結合します。 PLGA溶液（ステップ1.4）にタンパク質混合物を注ぐ。ソリューションは、主に独立したままになります。 </li><li>氷水のビーカーにチューブを置きます。均一な乳白色のエマルションが作成されるまで、〜10ワット（RMS）でワンド超音波処理器を使用して、数秒（5-10）の解を攪拌する。 </li><li> 0.5％PVA（ステップ1.3）を含む50mlチューブ中にエマルジョンを注ぐ。 〜20秒間ボルテックスミキサーで高速でソリューションを混ぜる。解決策は、曇った外観を開発します。 </li><li> 2分間350rpmで撹拌プレート上200ミリリットルのビーカーや場所にエマルジョンを転送します。撹拌プレート上のビーカーに2％イソプロピルアルコール50mlを加える。混合物が硬化するDCMが蒸発させる1時間の​​最小値とPLGA攪拌継続できるようにします。 </li><li>転送さt彼は遠心管に溶液をマイクロスフェア。 </li><li> 3分間425×gで遠心分離する。マイクロスフェアは、チューブの底に集め、白が表示されます。慎重にマイクロスフェアの上に、チューブから上清を除去し、500ミリリットルボトルに保管してください。 </li><li>四分の三の完全かつ液体中のマイクロスフェアを再配布するためにそれを振っチューブを充填して脱イオン水でマイクロスフェアを洗い流す。 </li><li>繰り返します1.10＆1.11 4回繰り返します。 </li><li>最後のすすぎに続いて、他のサンプルで再び500ミリリットルボトル内の場所上清を除去。その後少なくとも24時間、凍結乾燥、遠心管に集めミクロスフェアを凍結する。 </li><li>光学顕微鏡下で、または走査型電子顕微鏡で微小球を可視化する。 60ミクロンよりも大きくないマイクロスフェアは、効果的なエレクトロスピニングのためのものです。マイクロスフェアが大きすぎる場合、より長い超音波処理またはボルテックス時間は、ステップ1.6または1.7で必要とされ得る。 </li> &lt;李&gt;は、-20℃の冷凍庫内で乾燥微小球を保管してください。 </li><li>オプション：ステップ1.10 30から500ミリリットルボトルにタンパク質の量をテストするために、製造者の指示に従って、タンパク質アッセイを使用してください。これは、製造工程において使用したものと溶液中の量を減算することにより、マイクロスフェア中にカプセル化タンパク質のパーセントを計算するために使用される。 </li></ol>注：PLGA溶液31ミリリットルにローダミンを2μg/を追加し、FITC結合タンパク質をカプセル化マイクロスフェア中のタンパク質の位置を視覚化するために、図1に例を示します。 マイクロスフェアと2。エレクトロスピニング<ol><li>電界紡糸溶液を調製する前に、37℃で溶解することによって脱イオン水に、光開始剤のw / v溶液0.5％を作成する。このプロセスには数時間かかることがあります。 </li><li> 29（バーディックらを参照してください。合成のための）2％W / Vメタクリル化ヒアルロン酸（MeHAを）を作成し、3％/ 900 kDのポリ（エチレンオキシド）（PEO）v及び0.05％wの脱イオン水vの光開始剤溶液/ wである。 <ol><li>所望の体積のためMeHAをとPEOの正確な量を計算します。例えば、電界紡糸溶液10mlをMeHAをとPEO 300mgの200mgのを必要とする。 </li><li>所望の最終体積（この例では9 ml）を90％の脱イオン水にPEOを溶解する。これには数時間かかることがあり、37℃で加熱撹拌プレートまたは水浴プロセスを加速するために使用することができる。 </li><li>次MeHAを追加し、透明になるまでソリューションを撹拌するボルテックスミキサーを使用しています。これはほんの数分かかります。 </li><li>最後に、残りの10％の体積（この例では1 ml）を充填するために0.5％の光開始剤溶液を添加する。 </li></ol></li><li> 400 mg / mlの最大所望の濃度でマイクロスフェアを追加します。マイクロスフェアが均等に溶液中に分散されるまで、ボルテックスミキサー上に溶液を混ぜる。 </li><li>シリンジにソリューションを移し、6インチの18ゲージのBLを添付UNT先端の針。 </li><li>シリンジポンプにシリンジを配置し、1.2ミリリットル/時間で分配するように設定します。 </li><li>収集プレートまたはマンドレル上にアルミ箔の層をテープで固定します。これは、完成した足場を簡単にクリーンアップとストレージが可能になります。回転マンドレルは、整列した繊維を作成するために使用されている。平板または固定マンドレルは、ランダムに配置された繊維になります。 </li><li>収集装置に高電圧電源からアース線を接続する。針に正のリードを接続します。 </li><li>ニードルチップと収集面間に15センチあるように、シリンジポンプ及び収集面を調整する。 </li><li>ソリューションは、注射器の最後に表示されている場合、ポリマーポンプを起動し、電圧源をオンにし、24 kVの電圧を設定する注意 ：電圧がオンにされると、システムの金属部分に触れないでください。チャージはまた、皮膚に帯電部分から短い距離をジャンプすることがあります。 </li><li> Dまで溶液を実行します。esired足場の厚さが達成される。完了すると、電圧源とシリンジポンプをオフにしてください。 </li><li>添付足場とホイルを外します。タンパク質を含む完成した足場は、-20℃の冷凍庫に保存されている。 </li></ol> 3タンパク質生物活性試験<ol><li>細胞培養培地を準備します。追加の10％v / vのウシ胎児血清ダルベッコ改変イーグル培地に1％v / vのL-グルタミン、および1％v / vのペニシリン - ストレプトマイシン。 </li><li>ウェルプレートに完全にフィットするカバーガラスを選択します。 </li><li>製造業者によって記載されるようにカバースリップを治療するために3-（トリメトキシシリル）プロピルメタクリレートを使用する。メタクリル化は、カバースリップの足場付着性を向上させます。 </li><li>エレクトロスピニングの前に取り外し可能な両面テープでelectrospinnerの収集領域へのメタクリル化カバーガラスを取り付けます。カバースリップ上に紡績することは取り扱いや閲覧が容易になります。 </li><li>上記のように所望の厚さにエレクトロスピニング。 </li><li> AFTERは慎重にマンドレルからカバースリップを削除エレクトロスピニング。明確な窒素室に足場コーティングしたカバーガラスを置き、全ての酸素がパージされることを確認してください。 </li><li> 15分間は10mW / cm 2の 365nmの光の下でチャンバーと足場を置きます。適切なサイズのウェルプレートに場所を架橋した後。足場側が上を向いていることを確認します。 </li><li> 30分間殺菌灯の下に置いて足場を滅菌する。所望であれば、フィブロネクチンまたは他のタンパク質は、細胞接着を強化するためのコーティングとして使用される。コー​​ト足場に、メーカーの指示に従ってください。 </li><li>収穫後根神経節（DRG）、以前にホーレンベック32によって記述。一つのDRGは、試験した各足場に覆わカバースリップのために必要とされます。 </li><li>ウェルプレートの各足場上のメディア100〜200μLを置きます。慎重メディア滴の各足場に1 DRGを置く。厚手の足場のために複数のメディアが必要になる場合があります。 DRGは、完全に水没する必要があり、floaませんティン。 </li><li>細胞は足場に付着させ4時間、37℃で足場およびDRGをインキュベートします。 </li><li>ウェルについての適切なレベルにメディアを記入し、バックインキュベーターに配置します。 4-6日間培養を続けます。 </li><li>インキュベーション期間の後、慎重に各ウェルから培地を除去し、穏やかにPBSで一回洗浄します。 / vパラホルムアルデヒドwの4％を用いて30分間、細胞を固定します。 </li><li>ニューロフィラメントに対する抗体染色を用いて染色する細胞。これは定量化のための神経突起伸長を可視化することができます。 DAPIは、細胞の核を表示するために使用することができる。例えば、染色プロトコルはSundararaghavanおよび同僚14によって記載された。 </li><li>蛍光顕微鏡を用いて細胞を視覚化する。 <ol><li>顕微鏡のステージ上ウェルプレートを置きます。 </li><li> DAPI用のフィルタおよび励起の設定を使用して、細胞塊を見つけます。 </li><li>セルたら拡張神経突起を可視化するために、FITCにフィルタを切り替えることである。 U収集し、全体の構造を見るために必要な数の画像を組み合わせ、顕微鏡でのステッチ機能を歌う。 DAPI、FITC及び明視野に対して繰り返します。 </li></ol></li></ol>

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	<li>Wrobel, M. R., Sundararaghavan, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+migration+in+neural+tissue+engineering.">Directed migration in neural tissue engineering.</a> Tissue Eng Part B Rev. , (2013).</li><li>Schmidt, C. E., Leach, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+tissue+engineering:+strategies+for+repair+and+regeneration.">Neural tissue engineering: strategies for repair and regeneration.</a> Annual Review of Biomedical Engineering. 5, 293-347 (2003).</li><li>Madduri, S., di Summa, P., Papaloizos, M., Kalbermatten, D., Gander, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+controlled+co-delivery+of+synergistic+neurotrophic+factors+on+early+nerve+regeneration+in+rats.">Effect of controlled co-delivery of synergistic neurotrophic factors on early nerve regeneration in rats.</a> Biomaterials. 31, 8402-8409 (2010).</li><li>Madduri, S., Gander, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factor+delivery+systems+and+repair+strategies+for+damaged+peripheral+nerves.">Growth factor delivery systems and repair strategies for damaged peripheral nerves.</a> J Control Release. 161, 274-282 (2012).</li><li>Madigan, N. N., McMahon, S., O'Brien, T., Yaszemski, M. J., Windebank, 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=Current+tissue+engineering+and+novel+therapeutic+approaches+to+axonal+regeneration+following+spinal+cord+injury+using+polymer+scaffolds.">Current tissue engineering and novel therapeutic approaches to axonal regeneration following spinal cord injury using polymer scaffolds.</a> Respir Physiol Neurobiol. 169, 183-199 (2009).</li><li>Sundararaghavan, H. G., Monteiro, G. A., Firestein, B. L., Shreiber, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+growth+in+3D+collagen+gels+with+gradients+of+mechanical+properties.">Neurite growth in 3D collagen gels with gradients of mechanical properties.</a> Biotechnol. Bioeng. 102, 632-643 (2009).</li><li>Hudson, T. W., Evans, G. R., Schmidt, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+strategies+for+peripheral+nerve+repair.">Engineering strategies for peripheral nerve repair.</a> Clin Plast Surg. 26, 617-628 (1999).</li><li>Kokai, L. E., Bourbeau, D., Weber, D., McAtee, J., Marra, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+growth+factor+delivery+promotes+axonal+regeneration+in+long+gap+peripheral+nerve+repair.">Sustained growth factor delivery promotes axonal regeneration in long gap peripheral nerve repair.</a> Tissue Eng Part A. 17, 1263-1275 (2011).</li><li>Bronzino, J. D., Peterson, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biomedical Engineering Handbook, Third Edition - 3 Volume Set: Tissue Engineering and Artificial Organs. , (2006).</li><li>Bell, J. H. A., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+generation+nerve+guides:+materials,+fabrication,+growth+factors,+and+cell+delivery.">Next generation nerve guides: materials, fabrication, growth factors, and cell delivery.</a> Tissue Eng Part B Rev. 18, 116-128 (2012).</li><li>Ruiter, G. C. W., Malessy, M. J. A., Yaszemski, M. J., Windebank, A. J., Spinner, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+ideal+conduits+for+peripheral+nerve+repair.">Designing ideal conduits for peripheral nerve repair.</a> Neurosurgical focus. 26, (2009).</li><li>Olakowska, E., Woszczycka-Korczy&#324;ska, I., J&#281;drzejowska-Szypu&#322;ka, H., Lewin-Kowalik, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+nanotubes+and+nanofibres+in+nerve+repair.+A+review.">Application of nanotubes and nanofibres in nerve repair. A review.</a> Folia Neuropathol. 48, 231-237 (2010).</li><li>Gunn, J., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyblend+nanofibers+for+biomedical+applications:+perspectives+and+challenges.">Polyblend nanofibers for biomedical applications: perspectives and challenges.</a> Trends Biotechnol. 28, 189-197 (2010).</li><li>Sundararaghavan, H. G., Masand, S. N., Shreiber, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+generation+of+haptotactic+gradients+through+3D+collagen+gels+for+enhanced+neurite+growth.">Microfluidic generation of haptotactic gradients through 3D collagen gels for enhanced neurite growth.</a> Journal of Neurotrauma. 28, 2377-2387 (2011).</li><li>Sundararaghavan, H. G., Metter, R. B., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+fibrous+scaffolds+with+multiscale+and+photopatterned+porosity.">Electrospun fibrous scaffolds with multiscale and photopatterned porosity.</a> Macromol Biosci. 10, 265-270 (2010).</li><li>Edalat, F., Sheu, I., Manoucheri, S., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Material+strategies+for+creating+artificial+cell-instructive+niches.">Material strategies for creating artificial cell-instructive niches.</a> Current Opinion in Biotechnology. 23, 820-825 (2012).</li><li>Annabi, N., 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=Synthesis+of+highly+porous+crosslinked+elastin+hydrogels+and+their+interaction+with+fibroblasts+in+vitro.">Synthesis of highly porous crosslinked elastin hydrogels and their interaction with fibroblasts in vitro.</a> Biomaterials. 30, 4550-4557 (2009).</li><li>Casta&#241;o, O., Eltohamy, M., Kim, H. -. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+technology+in+tissue+regeneration.">Electrospinning technology in tissue regeneration.</a> Methods Mol. Biol. 811, 127-140 (2012).</li><li>Chew, S. Y., Wen, J., Yim, E. K. F., Leong, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+release+of+proteins+from+electrospun+biodegradable+fibers.">Sustained release of proteins from electrospun biodegradable fibers.</a> Biomacromolecules. 6, 2017-2024 (2005).</li><li>Han, D., Gouma, P. -. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+bioscaffolds+that+mimic+the+topology+of+extracellular+matrix.">Electrospun bioscaffolds that mimic the topology of extracellular matrix.</a> Nanomedicine. 2, 37-41 (2006).</li><li>Prabhakaran, M. P., 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=Electrospun+biocomposite+nanofibrous+scaffolds+for+neural+tissue+engineering.">Electrospun biocomposite nanofibrous scaffolds for neural tissue engineering.</a> Tissue Eng Part A. 14, 1787-1797 (2008).</li><li>Xie, J., MacEwan, M. R., Schwartz, A. G., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+nanofibers+for+neural+tissue+engineering.">Electrospun nanofibers for neural tissue engineering.</a> Nanoscale. 2, 35-44 (2010).</li><li>Yao, L., O'Brien, N., Windebank, A., Pandit, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orienting+neurite+growth+in+electrospun+fibrous+neural+conduits.">Orienting neurite growth in electrospun fibrous neural conduits.</a> J. Biomed. Mater. Res. Part B Appl. Biomater. 90, 483-491 (2009).</li><li>Sill, T. J., von Recum, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning:+applications+in+drug+delivery+and+tissue+engineering.">Electrospinning: applications in drug delivery and tissue engineering.</a> Biomaterials. 29, 1989-2006 (2008).</li><li>Ifkovits, J. L., Sundararaghavan, H. G., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning+fibrous+polymer+scaffolds+for+tissue+engineering+and+cell+culture.">Electrospinning fibrous polymer scaffolds for tissue engineering and cell culture.</a> Journal of Visualized Experiments: JoVE. , (2009).</li><li>Sundararaghavan, H. G., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradients+with+depth+in+electrospun+fibrous+scaffolds+for+directed+cell+behavior.">Gradients with depth in electrospun fibrous scaffolds for directed cell behavior.</a> Biomacromolecules. 12, 2344-2350 (2011).</li><li>Meinel, A. J., Germershaus, O., Luhmann, T., Merkle, H. P., Meinel, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+matrices+for+localized+drug+delivery:+current+technologies+and+selected+biomedical+applications.">Electrospun matrices for localized drug delivery: current technologies and selected biomedical applications.</a> Eur J Pharm Biopharm. 81, 1-13 (2012).</li><li>Ji, W., 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=Bioactive+electrospun+scaffolds+delivering+growth+factors+and+genes+for+tissue+engineering+applications.">Bioactive electrospun scaffolds delivering growth factors and genes for tissue engineering applications.</a> Pharm. Res. 28, 1259-1272 (2011).</li><li>Burdick, J. A., Chung, C., Jia, X., Randolph, M. A., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+degradation+and+mechanical+behavior+of+photopolymerized+hyaluronic+acid+networks.">Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks.</a> Biomacromolecules. 6, 386-391 (2005).</li><li>P&#233;an, J. M., 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=Optimization+of+HSA+and+NGF+encapsulation+yields+in+PLGA+microparticles.">Optimization of HSA and NGF encapsulation yields in PLGA microparticles.</a> International Journal of Pharmaceutics. 166, 105-115 (1998).</li><li>Cartiera, M. S., Johnson, K. M., Rajendran, V., Caplan, M. J., Saltzman, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Uptake+and+Intracellular+Fate+of+PLGA+Nanoparticles+in+Epithelial+Cells.">The Uptake and Intracellular Fate of PLGA Nanoparticles in Epithelial Cells.</a> Biomaterials. 30, 2790-2798 (2009).</li><li>Hollenbeck, P. J., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neurons: Methods and Applications for the Cell Biologist. , (2003).</li><li>Boer, R., 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=Rat+sciatic+nerve+repair+with+a+poly-lactic-co-glycolic+acid+scaffold+and+nerve+growth+factor+releasing+microspheres.">Rat sciatic nerve repair with a poly-lactic-co-glycolic acid scaffold and nerve growth factor releasing microspheres.</a> Microsurgery. 31, 293-302 (2011).</li><li>Pujic, Z., Goodhill, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+compartment+diffusion+chamber+for+studying+axonal+chemotaxis+in+3D+collagen.">A dual compartment diffusion chamber for studying axonal chemotaxis in 3D collagen.</a> Journal of Neuroscience Methods. 215, 53-59 (2013).</li><li>Madduri, S., di Summa, P., Papalo&#239;zos, M., Kalbermatten, D., Gander, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+controlled+co-delivery+of+synergistic+neurotrophic+factors+on+early+nerve+regeneration+in+rats.">Effect of controlled co-delivery of synergistic neurotrophic factors on early nerve regeneration in rats.</a> Biomaterials. 31, 8402-8409 (2010).</li><li>Xu, X., 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=Polyphosphoester+microspheres+for+sustained+release+of+biologically+active+nerve+growth+factor.">Polyphosphoester microspheres for sustained release of biologically active nerve growth factor.</a> Biomaterials. 23, 3765-3772 (2002).</li><li>Yan, Q., Yin, Y., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+new+PLGL-RGD-NGF+nerve+conduits+for+promoting+peripheral+nerve+regeneration.">Use new PLGL-RGD-NGF nerve conduits for promoting peripheral nerve regeneration.</a> Biomed Eng Online. 11, (2012).</li><li>Gungor-Ozkerim, P. S., Balkan, T., Kose, G. T., Sarac, A. S., Kok, F. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+growth+factor+loaded+microspheres+into+polymeric+electrospun+nanofibers+for+tissue+engineering+applications.">Incorporation of growth factor loaded microspheres into polymeric electrospun nanofibers for tissue engineering applications.</a> J Biomed Mater Res A. , (2013).</li><li>Li, X., 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=Encapsulation+of+proteins+in+poly(L-lactide-co-caprolactone)+fibers+by+emulsion+electrospinning.">Encapsulation of proteins in poly(L-lactide-co-caprolactone) fibers by emulsion electrospinning.</a> Colloids Surf B Biointerfaces. 75, 418-424 (2010).</li><li>Wang, C. -. Y., 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=The+effect+of+aligned+core-shell+nanofibres+delivering+NGF+on+the+promotion+of+sciatic+nerve+regeneration.">The effect of aligned core-shell nanofibres delivering NGF on the promotion of sciatic nerve regeneration.</a> J Biomater Sci Polym Ed. 23, 167-184 (2012).</li><li>Liu, J. -. J., Wang, C. -. Y., Wang, J. -. G., Ruan, H. -. J., Fan, C. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+nerve+regeneration+using+composite+poly(lactic+acid-caprolactone)/nerve+growth+factor+conduits+prepared+by+coaxial+electrospinning.">Peripheral nerve regeneration using composite poly(lactic acid-caprolactone)/nerve growth factor conduits prepared by coaxial electrospinning.</a> J Biomed Mater Res A. 96, 13-20 (2011).</li></ol>

The authors have nothing to disclose.

多くの研究は、神経細胞が地形手がかり（繊維配向）および化学的手がかり（成長因子）1,2,10,11,35によって指示することができることを示している。エレクトロスピニングは、整列した繊維を作成するための容易な方法です。成長因子は、神経成長を促すが、神経導管（NC）にそれらを含めるために、持続放出のための方法が必要とされる。両方の手がかりとより堅牢なシステムを作...

神経組織工学における継続的な課題の一つは、神経が自然に成長する細胞外マトリックスを模倣神経導管（NC）を作成します。研究は、細胞が機械的、地形、接着剤及び化学的シグナル1-3を含め、環境内のいくつかの要因に反応することが示されている。この分野における主要な課題の一つは、信号の適切な組み合わせを決定し、細胞増殖4を支持するために長時間のキューを維持することができるシステムを製作される。末梢ニューロンが軟質基板を好むことが知られており、整列した繊維により誘導され、神経成長因子（NGF）5-7に応答する。週間の化学的手がかりを提供することができnCSが近い同種移植、神経修復8,9の現在のゴールドスタンダードと改善された機能回復を提供することが示されている。 さまざまな材料および製造方法は機械的およびトポグラフィーを生成するために使用することができアルは10-13頭出し。機械的な合図は、アプリケーションのクリティカル1,13のための適切な材料の選択を行うこと、選択された材料に固有である。製造方法は地形キューが相分離、自己集合および1,13エレクトロスピニングが挙げられる制御する。マイクロスケールの用途、マイクロ流体工学、フォトパターニング、エッチング、塩浸出、またはガスフォームに対して14〜17をも使用することができる。エレクトロスピニングは、その柔軟性と生産13,18-23のしやすさに組織培養のための繊維状基材を設計するための最も一般的な方法として登場しました。電界紡糸ナノファイバーは、それ自体を撃退および24を放電するために短いギャップを横切って伸張させるポリマー溶液に高電圧を印加することにより製造される。整列足場は、接地回転マンドレル上に繊維を収集することによって作成することができ、非整列足場は、固定プレート25上に収集される。接着シグナリングは、繊維状足場ウィットをコーティングすることによって達成することができる時間フィブロネクチンまたは26をエレクトロスピニングする前に、HAへのそのようなRGDなどの接着ペプチドを、結合させる。 成長因子などの化学信号は、それらが制御された放出のためのソースを必要とするので、長期間にわたって維持することが最も困難である。多くのシステムは成功のさまざまなレベルで電界紡糸繊維質のネットワークに制御された放出を追加しようとしてきた。これらの方法は、ブレンドの電界紡糸、エマルジ ​​ョンエレクトロスピニング、コアシェル電気紡糸およびタンパク質共役27を含む。さらに、エレクトロスピニングは、伝統的に考慮されなければならない、したがって、タンパク質の生物活性を維持し、タンパク質28の生存率に影響を与えることができる揮発性溶媒中で行われる。 このアプローチは、特に末梢神経成長のための調整可能な足場を作成するために、機械的、地形的、化学的および接着剤信号を結合対処しています。足場の力学を正確に合成することによって制御されるメタクリル化ヒアルロン酸（HA）。メタクリル化部位は、光反応性架橋剤を取り付けるために使用される。架橋された材料は、もはや水溶性ではなく、排他的に酵素29により分解される。架橋の量は、分解速度、力学と材料の他の物理的特性を変更します。 〜500 Paで引張弾性率を有する30％のメタクリル化とHAを使用して、神経組織の本来の力学に近く、一般的に、ニューロン26,29に好まれているソフトの基板を作成します。回転マンドレル上でエレクトロスピニングすると、地形のキューのための整列した繊維を作成するために使用されている。マイクロスフェアと一緒にエレクトロスピニングを使用すると、長期間にわたって足場内の化学信号を提供する。 NGFを含む神経突起成長のミクロスフェアをサポートするために、化学信号を作成するために使用される。 NGFは製造中に過酷な溶剤に遭遇しないように、ほとんどのエレ​​クトロスピン材料とは異なり、HAは水溶性である。 、SCAを粘着信号を追加するには、ffoldは、フィブロネクチンで被覆されている。 NGFはフィブロネクチン（接着剤）でコーティングされたマイクロスフェア（化学）を放出したソフト（機械）整列（地形）繊維：完成したシステムは、上記の信号の4つのすべてのタイプが含まれています。このシステムの製造および試験は、このプロトコルに記載されている。 プロセスは、水中油中水のダブルエマルジョンとマイクロスフェアの製造から始まる。エマルジョンは、界面活性剤、ポリビニルアルコール（PVA）で安定化される。内側の水相は、タンパク質が含まれています。それはジクロロメタン（DCM）に溶解PLGAシェル材料を含有する、油相に添加されると、界面活性剤は、DCMからタンパク質を保護する相の間の障壁を作成する。このエマルジョンは、ミクロスフェアの外表面を作成するためにPVAを含む別の水相中に分散しなくなる。安定したエマルジョンがDCMを蒸発させて攪拌される。洗浄および凍結乾燥した後は、乾いたマイクロスフェアの続きが残されているタンパク質をaining。 微小球が完了した後、それらを足場に電界紡糸される準備ができている。まず、電界紡糸溶液を調製。溶液の粘度は、適切な繊維形成にとって重要である。純粋なHAの解決策はこの要件を満たしていない。 PEOは、電界紡糸を可能にするために、担体ポリマーとして添加される。マイクロスフェアは、全体に分散マイクロスフェアと繊維状足場に得られた溶液とエレクトロに追加されます。 生産が完了すると、タンパク質は、その実行可能性を検証するためにテストする必要があります。これを行うには、NGFに応答する一次電池を使用することができる。このプロトコルは、8〜10日齢のニワトリ胚から後根神経節（DRG）を使用しています。セルの束は、NGFまたは空のあるもので満たされたマイクロスフェアを含む足場上に播種する。 NGFはまだ実行可能である場合は、NGFを含む足場上の強化された神経突起成長が表示されるはずです。 NGFはもはや実行可能でない場合、それは意志拡張するとコントロールのように表示されます神経突起を促進しない。 本明細書で説明正確な手順は、システムがさまざまな細胞および組織型に対して最適化することができる単純な物質への変更、エレクトロスピニング法、およびタンパク質で、しかし、神経支持体上に集光される。

<table border="0" cellpadding="0" cellspacing="0" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DAPI</td>
			<td style="width:208px;">Invitrogen</td>
			<td style="width:139px;">D1306</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Irgacure 2959</td>
			<td style="width:208px;">BASF</td>
			<td style="width:139px;">24650-42-8</td>
			<td style="width:291px;">Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PEO 900 kDa</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">189456</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Methacryloxethyl thiocarbamoyl rhodamine B</td>
			<td style="width:208px;">Polysciences, Inc.</td>
			<td style="width:139px;">23591-100</td>
			<td style="width:291px;">Prepare stock solution in DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Syringe Pump</td>
			<td style="width:208px;">KD Scientific</td>
			<td style="width:139px;">KDS100</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Power Source</td>
			<td style="width:208px;">Gamma High Voltage</td>
			<td style="width:139px;">ES30P-5W</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Motor</td>
			<td style="width:208px;">Triem Electric Motors, Inc</td>
			<td style="width:139px;">0132022-15</td>
			<td style="width:291px;">Must attach to a custom built mandrel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Tachometer</td>
			<td style="width:208px;">Network Tool Warehouse</td>
			<td style="width:139px;">ESI-330</td>
			<td style="width:291px;">Use to monitor mandrel speed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:269px;">Omnicure UV Spot Cure System with collimating adapter</td>
			<td style="width:208px;">EXFO</td>
			<td style="width:139px;">S1000</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Needles</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">14-825-16H</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Coverslips</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">12-545-81</td>
			<td style="width:291px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Polyvinyl Alcohol</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">P8136-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Isoporopyl Alcohol</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">I9030-500mL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td style="width:139px;">BP9703-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">BSA-FITC</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">080M7400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">&#946;-Nerve Growth Factor (NGF)</td>
			<td style="width:208px;">R&#38;D Systems</td>
			<td style="width:139px;">1156-NG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">65:35 Poly-Lactic-Glycolic-Acid (PLGA)</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">1001554270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dichloromethane</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">34856-2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Coomassie (Bradford) Protein Assay</td>
			<td style="width:208px;">Thermo Scientific</td>
			<td align="right" style="width:139px;">1856209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">3-(Trimethoxysilyl)propyl methacrylate</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td align="right" style="width:139px;">1001558456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fibronectin</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">DMEM</td>
			<td style="width:208px;">Lonza</td>
			<td style="width:139px;">12-604F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">FBS</td>
			<td style="width:208px;">Atlanta Biologicals</td>
			<td style="width:139px;">S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PBS</td>
			<td style="width:208px;">Hyclone</td>
			<td style="width:139px;">SH30256.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Glutamine</td>
			<td style="width:208px;">Fisher Scientific</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Pen-Strep</td>
			<td style="width:208px;">Sigma-Aldrich</td>
			<td style="width:139px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Paraformaldehyde</td>
			<td style="width:208px;">Alfa Aesar</td>
			<td style="width:139px;">A11313&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrospinning growth factor releasing microspheres into fibrous scaffolds

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth. This system incorporates mechanical, topographical, adhesive and chemical signals. Mechanical properties are controlled by the type of material used to fabricate the electrospun fibers. In this protocol we use 30% methacrylated Hyaluronic Acid (HA), which has a tensile modulus of ~500 Pa, to produce a soft fibrous scaffold. Electrospinning on to a rotating mandrel produces aligned fibers to create a topographical cue. Adhesion is achieved by coating the scaffold with fibronectin. The primary challenge addressed herein is providing a chemical signal throughout the depth of the scaffold for extended periods. This procedure describes fabricating&#160;poly(lactic-co-glycolic acid) (PLGA) microspheres that contain Nerve Growth Factor (NGF) and directly impregnating the scaffold with these microspheres during the electrospinning process. Due to the harsh production environment, including high sheer forces and electrical charges, protein viability is measured after production. The system provides protein release for over 60 days and has been shown to promote primary nerve cell growth.

85％のタンパク質カプセル化微小球は直径50±14μmで、一貫して足場に生産され、エレクトロれています。サイズは、3つの別個の製造バッチから微小球のサンプルを撮像することによって決定した。商業ラボソフトウェアを使用して測定光学顕微鏡および長さに撮影された画像。 図1は、サイズ分布のヒストグラムを示す。封入率は、製造プロセス中に脱出したタンパク質を定量?...

Watch this Scientific Journal Video about Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds at JoVE.com

Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

繊維状足場に微小球を離すエレクトロ成長因子

This procedure describes a method to fabricate a multifaceted substrate to direct nerve cell growth.  This system incorporates mechanical, topographical, ...

electrospinning-growth-factor-releasing-microspheres-into-fibrous

Research

JoVE Journal

Bioengineering

12.2K ビュー.  Wayne State University. This protocol combines electrospinning and microspheres to develop tissue engineered scaffolds to direct neurons. Nerve growth factor was encapsulated within PLGA microspheres and electrospun into Hyaluronic Acid (HA) fibrous scaffolds. The protein bioactivity was tested by seeding the scaffolds with primary chick Dorsal Root Ganglia and culturing for 4-6 days.

ビデオ: Electrospinning Growth Factor Releasing Microspheres into Fibrous Scaffolds

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

エレクトロスピニングの基礎：最適化ソリューションと装置のパラメータ

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

生物工学

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 ...

Axon Stretch Growth: The Mechanotransduction of Neuronal Growth

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata are separated from their axon terminals due to skeletal growth of the enlarging organism (Weiss 1941; Gray, Hukkanen et al. 1992). This mechanotransduction induces a secondary mode of neuronal growth capable of accommodating continual elongation of the axon (Bray 1984; Heidemann and Buxbaum 1994; Heidemann, Lamoureux et al. 1995; Pfister, Iwata et al. 2004).
Axon Stretch Growth (ASG) is conceivably a central factor in the maturation of short embryonic processes into the long nerves and white matter tracts characteristic of the adult nervous system. To study ASG in vitro, we engineered bioreactors to apply tension to the short axonal processes of neuronal cultures (Loverde, Ozoka et al. 2011). Here, we detail the methods we use to prepare bioreactors and conduct ASG. First, within each stretching lane of the bioreactor, neurons are plated upon a micro-manipulated towing substrate. Next, neurons regenerate their axonal processes, via growth cone extension, onto a stationary substrate. Finally, stretch growth is performed by towing the plated cell bodies away from the axon terminals adhered to the stationary substrate; recapitulating skeletal growth after growth cone extension.
Previous work has shown that ASG of embryonic rat dorsal root ganglia neurons are capable of unprecedented growth rates up to 10mm/day, reaching lengths of up to 10cm; while concurrently resulting in increased axonal diameters (Smith, Wolf et al. 2001; Pfister, Iwata et al. 2004; Pfister, Bonislawski et al. 2006; Pfister, Iwata et al. 2006; Smith 2009). This is in dramatic contrast to regenerative growth cone extension (in absence of mechanical stimuli) where growth rates average 1mm/day with successful regeneration limited to lengths of less than 3cm (Fu and Gordon 1997; Pfister, Gordon et al. 2011). Accordingly, further study of ASG may help to reveal dysregulated growth mechanisms that limit regeneration in the absence of mechanical stimuli.

A unique tissue engineering method was developed to elongate numerous nerve fibers in culture by recapitulating axon stretch growth; a form of nervous system growth whereby nerves elongate in conjunction with growth of the enlarging body.

軸索のストレッチの成長：神経成長のメカノトランスダ

During pre-synaptic embryonic development, neuronal processes traverse short distances to reach their targets via growth cone. Over time, neuronal somata ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

上皮成長因子受容体をターゲットとしたゼラチンベースのエンジニアNanovectorsを使用してヒト膵臓癌細胞における遺伝子デリバリーとトランスフェクション

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

マウス胚性幹細胞からの神経組織エンジニアリングのためのポリエレクトロ繊維状足場（グリセロール - ドデカン）

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

相関顕微鏡を用いて、無傷の細胞における上皮成長因子受容体の化学量論を学びます

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Polyelectrolyte Complex for Heparin Binding Domain Osteogenic Growth Factor Delivery

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone fusion. Large doses of highly potent biological agents are required due to growth factor instability as a result of rapid enzymatic degradation as well as carrier inefficiencies in localizing sufficient amounts of growth factor at implant sites. Hence, strategies that prolong the stability of growth factors such as BMP-2/NELL-1, and control their release could actually lower their efficacious dose and thus reduce the need for larger doses during future bone regeneration surgeries. This in turn will reduce side effects and growth factor costs. Self-assembled PECs have been fabricated to provide better control of BMP-2/NELL-1 delivery via heparin binding and further potentiate growth factor bioactivity by enhancing in vivo stability. Here we illustrate the simplicity of PEC fabrication which aids in the delivery of a variety of growth factors during reconstructive bone surgeries.

Self-assembled polyelectrolyte complexes (PEC) fabricated from heparin and protamine were deposited on alginate beads to entrap and regulate the release of osteogenic growth factors. This delivery strategy enables a 20-fold reduction of BMP-2 dose in spinal fusion applications. This article illustrates the benefits and fabrication of PECs.

ヘパリン結合ドメイン骨形成成長因子の送達のための高分子電解質複合体

During reconstructive bone surgeries, supraphysiological amounts of growth factors are empirically loaded onto scaffolds to promote successful bone ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein (HDL)-mimicking nanoparticles (NPs). HDLs are endogenous NPs and have been explored as vehicles for the delivery of therapeutic agents. Various methods have been developed to prepare HDL-mimicking NPs. However, they are generally complicated, time consuming, and difficult for industrial scale-up. In this study, one-step homogenization was used to mix the excipients and form the prototype NPs. NGF is a water-soluble protein of 26 kDa. To facilitate the encapsulation of NGF into the lipid environment of HDL-mimicking NPs, protamine USP was used to form an ion-pair complex with NGF to neutralize the charges on the NGF surface. The NGF/protamine complex was then introduced into the prototype NPs. Apolipoprotein A-I was finally coated on the surface of the NPs. NGF HDL-mimicking NPs showed preferable properties in terms of particle size, size distribution, entrapment efficiency, in vitro release, bioactivity, and biodistribution. With the careful design and exploration of homogenization in HDL-mimicking NPs, the procedure was greatly simplified, and the NPs were made scalable. Moreover, various challenges, such as separating unloaded NGF from the NPs, conducting reliable in vitro release studies, and measuring the bioactivity of the NPs, were overcome.

Simple homogenization was used to prepare novel, high-density, lipoprotein-mimicking nanoparticles to encapsulate nerve growth factor. Challenges, detailed protocols for nanoparticle preparation, in vitro characterization, and in vivo studies are described in this article.

神経成長因子カプセル化のための新規HDL模倣ナノ粒子の調製とキャラクタリゼーション

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...

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 ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

脱軟骨由来のマトリックス足場の作製

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...