この作業は、NIH(グランツ1R01AR072027-01、1R03AR069383-01)、NSFキャリアアワード(1653702)、コネチカット大学によって財政的にサポートされています。

1. JBNTの合成
注:JBNTモノマーは、以前に公開された11として準備されました。
<ol><li>化合物A1の合成<ol>
<li>2-シアノー酢酸8.50g、エチルカルバミン酸9.80gを25mLのトルエンと2.5mLのN、N-ジメチルホルムアミドで調製する。4.90 mLの塩化ホスホリルを滴下する。その後、混合物を70°Cに加熱し、1.5時間攪拌し続けます。</li>
		<li>反応混合物を室温まで冷却し、100gの氷水を注ぎます。酢酸エチル(3 x 250 mL)で水層を抽出し、100 mLのブラインで洗浄します。無水硫酸ナトリウムの上に有機層を乾燥させ、真空下で揮発性物質を濾過し、蒸発させて淡黄色の固体を得る。</li>
		<li>黄色の固体をシリカゲルフラッシュカラムクロマトグラフィー(3%メタノール/ジクロロメタン)に供し、白色粉末として化合物A1の15.61gを得た。</li>
	</ol></li>
	<li>化合物A2の合成<ol>
<li>化合物A1の3.12gと2.75gの炭酸カリウムを50mLのN、N-ジメチルホルムアミドに混ぜます。室温で2時間かき混ぜます。</li>
		<li>2.6 mLの炭素二硫化物を反応懸濁液に加えます。4時間かき混ぜ続けます。</li>
		<li>0°Cの反応混合物に絶対エタノールを加える。 黄色の沈殿物をろ過し、ジエチルエーテルで洗います。真空下で一晩乾燥し、淡黄色の固体として化合物A2の6.18gを得る。</li>
	</ol></li>
	<li>化合物A3の合成<ol>
<li>15 mLのアセトニトリルにヨウ化メチル2.7 mLを加えます。また、6.18gのA2を80mLの水/アセトニトリル(7:3比)を加えて化合物A2の溶液を別途調製する。両方の溶液を混合し、室温で30分間攪拌します。</li>
		<li>反応混合物を95°Cに加熱し、3時間撹拌し続けます。</li>
		<li>反応混合物を室温まで冷却し、次に真空下で揮発液を蒸発させます。</li>
		<li>残渣3xを酢酸エチル100mLで抽出し、ブラインで洗浄します。無水硫酸ナトリウムの上に有機層を乾燥させ、真空下で揮発物を濾過し、蒸発させて黄色の固体を得る。</li>
		<li>黄色の固体をシリカゲルフラッシュカラムクロマトグラフィー(30%酢酸エチル/ヘキサン)に供し、化合物A3の4.52gをライトイエロー固体として得る。</li>
	</ol></li>
	<li>化合物A4の合成<ol>
<li>20 mLのエタノールに925 μLのアライラミンを加えて、アライラミン溶液を調製します。この溶液を化合物A3の溶液に滴下して添加し、30min以上の無水エタノールの75mLに3.14gのA3を加えて調製した。その後、反応混合物を16時間加熱還流する。</li>
		<li>反応混合物を室温まで冷却し、揮発性物質を蒸発させて黄色の固体を与えます。</li>
		<li>黄色の固体をシリカゲルフラッシュカラムクロマトグラフィー(50%酢酸エチル/ヘキサン〜2%メタノール/ジクロロメタン)に供し、白色結晶として化合物A4の1.80gを得た。</li>
	</ol></li>
	<li>化合物A5の合成<ol>
<li>14 mLの絶対エタノールに2.05gの塩酸グアニジニウムと7.8mLのナトリウムエトキシド(エタノール中21重量%)を加える。混合物を45°Cに加熱し、15分間加熱します。</li>
		<li>不溶性物質を濾取し、40 mL絶対エタノールに2.70gのA4を加えて調製した化合物A4の溶液に直接濾液を加える。反応混合物を16時間加熱還流する。</li>
		<li>沈殿をろ過し、オフホワイト固体として化合物A5の2.65gを得る。</li>
	</ol></li>
	<li>化合物A6の合成<ol>
<li>24.9 mLのトリエチルアミンを、1分以上のテトラヒドロフランの120 mLに化合物A5の2.11gと4-ジメチルアミノピリジン1.10 gを加えたスラリー混合物にゆっくりと加える。室温で2分間かき混ぜます。</li>
		<li>20.7 mLのジテルブチルジカーボネートを反応混合物に滴下し、室温で40時間撹拌し続けます。</li>
		<li>20mLの水を加える。真空下で揮発性を蒸発させる。</li>
		<li>500mLの酢酸エチルで赤粘性残基を抽出する。その後、250mLの水、10%のクエン酸75mL、200mLの水で2倍、飽和炭酸ナトリウム200mL、ブライン200mLで洗浄します。無水硫酸ナトリウムの上に有機層を乾燥させ、フィルターをかけ、真空下で揮発を蒸発させて、赤/オレンジの固体を与えます。</li>
		<li>固体をシリカゲルフラッシュカラムクロマトグラフィー(0-20%酢酸エチル/ヘキサン)に供し、白い泡として化合物A6の3.87gを得た。</li>
	</ol></li>
	<li>化合物A7の合成<ol>
<li>化合物A6(2.93g)の溶液にN-メチルモルフォリンN酸化物(水中50重量%、1.64mL)を加え、室温で5分間攪拌します。</li>
		<li>3分間にわたって混合物に4%水溶液のオスミウム(4%水溶液)を加え、室温で24時間撹拌し続けます。</li>
		<li>溶液が無色になるまで1.0M水性亜硫酸ナトリウムで反応を焼き付けます。真空下で揮発性物質を取り除き、水中に白いスラリーを生じさせます。350mLのジクロロメタンで残留物を抽出し、150mLの水で洗浄し、150mLのブラインで洗浄します。</li>
		<li>有機層を無水硫酸ナトリウムで乾燥し、真空下で揮発性物質を濾過し、蒸発させて、白色固体として化合物A7の2.45gを得る。</li>
	</ol></li>
	<li>化合物A8の合成<ol>
<li>ニクロロメタン/水(6:1、35mL)の化合物A7の1.36gの混合物に760gの歯ごめきナトリウムを加え、室温で42時間攪拌します。</li>
		<li>不溶性物質をろ過し、真空下で濾液を濃縮します。得られた残渣を250mLのジクロロメタンで抽出し、100mLの水と100mLのブラインで洗浄します。無水硫酸ナトリウムの上に有機層を乾燥させ、真空下で揮発物をろ過し、蒸発させて粗生成物を与えます。</li>
		<li>粗生成物をシリカゲルフラッシュカラムクロマトグラフィー(5-40%酢酸エチル/ヘキサン、5%メタノール/ジクロロメタン)に供し、化合物A8の1.08gを得る。</li>
	</ol></li>
	<li>化合物A9の合成<ol>
<li>1,2-ジクロロエタンの15 mLに830mgの化合物A8の溶液を調製する。6-ベンジルオキシカルボニルアミノミノ-2-L-アミノ-ヘキサノ酸トリメチルシリルエチルエステル(649.5 mg)とN,N-ジイソプロピレチルアミン(591 μL)の混合物に滴下の溶液を1,2-ジクロロエタンの24 mLで加え、15分間室温で攪拌します。</li>
		<li>360.3 mgのトリアセトキシ水素化ナトリウムを溶液に加え、室温で24時間撹拌し続けます。</li>
		<li>反応混合物を6mLの水で焼き付けます。200mLのジクロロメタンで反応混合物3倍を抽出し、水に200mLのクエン酸を200mL、200mLの水で2倍、飽和炭酸ナトリウム200mL、そして200mLのブラインをその順に洗浄します。無水硫酸ナトリウムの上に有機層を乾燥させ、真空下で揮発物をろ過し、蒸発させて粗生成物を得る。</li>
		<li>粗生成物をシリカゲルフラッシュカラムクロマトグラフィー(5-30%酢酸エチル/ヘキサン)に供し、885mg化合物A9を生成する。</li>
	</ol></li>
	<li>JBNTモノマーの合成<ol>
<li>化合物A9(0.54g)の0.54gを94%トリフルオロ酢酸/チオアニソール溶液の10 mLに溶解し、室温で72時間攪拌します。</li>
		<li>反応混合物にジエチルエーテル(80 mL)を加え、遠心分離機を沈殿に加えます。上清を含む透明なトリフルオロ酢酸を注ぎ出し、ジエチルエーテルとメタノールで白色沈殿物を洗浄し、168.6 mgの粗JBNTモノマーを得る(補足ファイル1)。</li>
		<li>逆相カラムを用いて、高速液体クロマトグラフィー(HPLC)により粗JBNTモノマーを精製します。分離プログラムは以下にリストされています: 溶媒A:100%水;溶媒B:100%アセトニトリル;溶媒C:pH=1 HCl水溶液;流量:3 mL/分( 表1を参照)。</li>
		<li>約7.2分で最大のピークを収集します。</li>
	</ol></li>
</ol>2. JBNT/FNの製造
<ol><li>80 μLの1 mg/mL JBNT水溶液を40 μLの1 mg/mL FN水溶液とピペットに数回加えます。</li>
	<li>約10sのピペット処理後の白色固体懸濁液の有無を確認してください。 メモ:NM(補足ファイル2)の自己組み立てプロセスをキャプチャするためのビデオが撮影されました。</li>
</ol>3. 凍結乾燥した標本の観察
注: このステップは JBNT および FN から作られた NM の足場構造を示すために実行されます。
<ol><li>凍結乾燥材料観察のためにFN、JBNTおよびFN/JBNT NMの解決を準備しなさい。100 μg/mL の FN を 40 μL の蒸留水で希釈し、FN の 20 μg/mL 溶液を作ります。</li>
	<li>1 mg/mL の JBNT の溶液を 1 mg/mL の 5 μL の DI 水 45 μL に希釈して調製します。</li>
	<li>100 μg/mL FN の 10 μL と 1 mg/mL の JBNT の 5 μL を 35 μL の DI 水に混ぜて NM 溶液を形成します。</li>
	<li>FN、JBNT溶液、NM溶液をそれぞれ備えた96ウェルクリアラウンドボトムマイクロプレートの3つのウェルをコーティングします。各ウェルは、50 μLの溶液を受け取ります。</li>
	<li>凍結乾燥を行い、後述するように、NMの足場構造を可視化する。<ol>
<li>プレートを-80°Cで一晩凍らせます。</li>
		<li>凍結乾燥した器具と真空ポンプをオンにします。</li>
		<li>クールダウンボタンを押して、システムの温度を-50°Cに下げます。</li>
		<li>凍結したプレートを凍結した器具に入れ、器具の開口部をすべて閉じます。</li>
		<li>開始ボタンをクリックして、システムの圧力を 0.9 mbar に減らします。</li>
		<li>4時間後、 気管内 ボタンを押してバルブを開き、大気圧に対するシステム圧力を高めます。</li>
		<li>凍結乾燥した器具からプレートを取り出します。</li>
		<li>顕微鏡を使用して、FN、JBNTおよび得られた自己組み立てNMを観察する。材料のためのカメラでいくつかの写真を撮ります。</li>
	</ol>
</li>
</ol>4. 吸収スペクトル測定
メモ: FN および JBNT のスペクトルの変更を使用して、自己組み立て型 JBNT/FN NM12を提示します。
<ol><li>30 μL の 100 μg/mL FN と 15 μL の蒸留水と 5 μL の 1 mg/mL JBNT を混合して、JBNT/FN NM 溶液を調製します。白色固体懸濁液は、溶液を数回ピペットした後に見ることができる。 注:溶液中のJBNTとFNの最終濃度はそれぞれ100 μg/mLおよび60 μg/mLです。NM溶液に使用されたものと同じ濃度のJBNTおよびFN溶液も、制御溶液として調製した。</li>
	<li>JBNTコントロール溶液を調製するために、45μLの蒸留水でJBNTの1mg/mLの5μLを希釈します。</li>
	<li>30 μLの100 μg/mL FNを20 μLの蒸留水で希釈し、FN制御溶液を調製します。</li>
	<li>分光光度計( 材料表を参照)を用いて、FN、JBNT、JBNT/FN NM溶液のUV-Vis吸収スペクトルを測定し、NMの組立を特徴付けます。<ol>
<li>[UV-Vis]ボタンをクリックします。分光光度計の試験面を洗浄し、2μLの蒸留水を滴下します。190-850 nm からブランクとして測定します。</li>
		<li>分光光度計の試験面を洗浄し、その上にFN溶液の2μLを落とします。190 nm から 850 nm までの FN 溶液の UV-Vis 吸収スペクトルを測定します。</li>
		<li>分光光度計の試験面を洗浄し、JBNT溶液の2μLを落とします。JBNT溶液の吸収スペクトルを190 nmから850 nmまで測定します。</li>
		<li>分光光度計の試験面を洗浄し、JBNT/FN NM溶液の2μLを落とします。JBNT/FN NM溶液の吸収スペクトルを190 nmから850 nmまで測定します。</li>
	</ol>
</li>
</ol>5. 透過電子顕微鏡(TEM)用JBNT/FN NMの調製
<ol><li>基本的なプラズマクリーナーで負の染色の前にいくつかのグリッドを洗浄します( 材料表を参照)。</li>
	<li>2つの異なるプロセスに従って標本の陰性染色を行う。<ol>
<li>JBNTs水溶液の3mg/mLの3μLを、JBNT/FN NM溶液を3μLの分離されたグリッドに落とし、2分間放置します。その後、0.5%のウラニル酢酸(UA)溶液の100 μLで各グリッドをすすいでください。余分な溶液を濾紙で排出し、空気乾燥グリッド。</li>
		<li>JBNT/FN NM溶液の9 μLを3 μLの2.0%UA溶液と混合し、ピペットを数回混合します。グリッド上の混合溶液をピペットし、2分間グリッド上に保管します。フィルターペーパーを使用して、混合溶液をグリッドから取り除き、常温でグリッドを乾燥させます。</li>
	</ol>
</li>
	<li>最後に、ステップ5.2.1で染色されたJBNT/FN NM、およびTEMでステップ5.2.2で染色されたJBNT/FN NMの試料を特徴付ける。</li>
</ol>6. インビトロ生物学的機能アッセイ
<ol><li>200 μL の陰性制御(NC、PBS)、JBNT、FN、および JBNT/FN NM ソリューションを、それぞれ 8 ウェルチャンバー スライドの 1 つのウェルに追加します。</li>
	<li>凍結乾燥した8ウェルチャンバーカバーグラスを凍結乾燥させ、ウェルの底に材料をコーティングします。凍結乾燥のプロトコルは、ステップ 3.5 と同じです。</li>
	<li>その後、ウェル10,000個(パッセージ3)をウェルに加え、5%CO2で37°Cで24時間インキュベートします。</li>
	<li>インキュベーション後、培養液をウェルからピペットアウトし、1xPBS溶液で培養液をリンスする。</li>
	<li>細胞を持つウェルに100 μLの固定液を加え、5分間放置します。細胞を1倍PBSで穏やかにリンスします。</li>
	<li>1%トリトン-X 100〜0.1%を希釈。0.1%トリトンX 100の100 μLを細胞と共に各ウェルに加え、10分間放置します。細胞を1倍PBSで穏やかにリンスします。</li>
	<li>ローダミンファロイジンを1.65μMに希釈し、ローダミンファロイジン100μLを細胞と共に各ウェルに加え、室温で光から30分間保つチャンバーカバーグラスをインキュベートします。細胞を1倍PBSで穏やかにリンスします。</li>
	<li>蛍光顕微鏡で細胞画像をキャプチャします。</li>
	<li>各グループの蛍光画像のセル数をカウントします。各ウェルの3つのランダムな領域を平均化することによって、細胞の接着密度を計算します。</li>
	<li>すべてのデータを平均±標準偏差(SD)として表示します。片裾t検定を使用して統計分析を実行し、続いてp<0.05を伴う分散分析(ANOVA)を実行します。</li>
</ol>

<ol>
	<li>Yao, 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=Improved+mobilization+of+exogenous+mesenchymal+stem+cells+to+bone+for+fracture+healing+and+sex+difference.">Improved mobilization of exogenous mesenchymal stem cells to bone for fracture healing and sex difference.</a> Stem Cells. 34 (10), 2587-2600 (2016).</li><li>Salasznyk, R. M., Williams, W. A., Boskey, A., Batorsky, A., Plopper, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adhesion+to+vitronectin+and+collagen+I+promotes+osteogenic+differentiation+of+human+mesenchymal+stem+cells.">Adhesion to vitronectin and collagen I promotes osteogenic differentiation of human mesenchymal stem cells.</a> Journal of Biomedicine and Biotechnology. 1 (2004), 24-34 (2004).</li><li>Hadjiargyrou, M., O'Keefe, 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=The+convergence+of+fracture+repair+and+stem+cells:+interplay+of+genes,+aging,+environmental+factors+and+disease.">The convergence of fracture repair and stem cells: interplay of genes, aging, environmental factors and disease.</a> Journal of Bone and Mineral Research. 29 (11), 2307-2322 (2014).</li><li>De Becker, A., Riet, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homing+and+migration+of+mesenchymal+stromal+cells:+How+to+improve+the+efficacy+of+cell+therapy.">Homing and migration of mesenchymal stromal cells: How to improve the efficacy of cell therapy.</a> World Journal of Stem Cells. 8 (3), 73-87 (2016).</li><li>Chen, Y., Song, S., Yan, Z., Fenniri, H., Webster, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembled+rosette+nanotubes+encapsulate+and+slowly+release+dexamethasone.">Self-assembled rosette nanotubes encapsulate and slowly release dexamethasone.</a> International Journal of Nanomedicine. 6, 1035-1044 (2011).</li><li>Song, S., Chen, Y., Yan, Z., Fenniri, H., Webster, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembled+rosette+nanotubes+for+incorporating+hydrophobic+drugs+in+physiological+environments.">Self-assembled rosette nanotubes for incorporating hydrophobic drugs in physiological environments.</a> International Journal of Nanomedicine. 6, 101-107 (2011).</li><li>Fenniri, H., 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=Helical+Rosette+Nanotubes:+Design,+Self-Assembly,+and+Characterization.">Helical Rosette Nanotubes: Design, Self-Assembly, and Characterization.</a> Journal of the American Chemical Society. 123 (16), 3854-3855 (2001).</li><li>Chen, 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=Self-assembled+rosette+nanotube/hydrogel+composites+for+cartilage+tissue+engineering.">Self-assembled rosette nanotube/hydrogel composites for cartilage tissue engineering.</a> Tissue Engineering Part C Methods. 16 (6), 1233-1243 (2010).</li><li>Van den Bogaerdt, A. J., 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=Collagen+cross-linking+by+adipose-derived+mesenchymal+stromal+cells+and+scar-derived+mesenchymal+cells:+Are+mesenchymal+stromal+cells+involved+in+scar+formation.">Collagen cross-linking by adipose-derived mesenchymal stromal cells and scar-derived mesenchymal cells: Are mesenchymal stromal cells involved in scar formation.</a> Wound Repair and Regeneration. 17 (4), 548-558 (2009).</li><li>Erickson, H. P., Carrell, N., McDonagh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+molecule+visualized+in+electron+microscopy:+a+long,+thin,+flexible+strand.">Fibronectin molecule visualized in electron microscopy: a long, thin, flexible strand.</a> Journal of Cell Biology. 91 (3), 673-678 (1981).</li><li>Chen, Q., Yu, H. C., Chen, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> U. S. Patent. , (2017).</li><li>Zhou, L., 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=Self-assembled+biomimetic+Nano-Matrix+for+stem+cell+Anchorage.">Self-assembled biomimetic Nano-Matrix for stem cell Anchorage.</a> Journal of Biomedical Materials and Research. 108, 984-991 (2020).</li><li>Jones, M., Leroux, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymeric+micelles+-+a+new+generation+of+colloidal+drug+carriers.">Polymeric micelles - a new generation of colloidal drug carriers.</a> European Journal of Pharmaceutics and Biopharmaceutics. 48 (2), 101-111 (1999).</li><li>Singh, P., Schwarzbauer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+and+stem+cell+differentiation+-+lessons+from+chondrogenesis.">Fibronectin and stem cell differentiation - lessons from chondrogenesis.</a> Journal of Cell Science. 125, 3703-3712 (2012).</li><li>Martino, M. 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=Controlling+integrin+specificity+and+stem+cell+differentiation+in+2D+and+3D+environments+through+regulation+of+fibronectin+domain+stability.">Controlling integrin specificity and stem cell differentiation in 2D and 3D environments through regulation of fibronectin domain stability.</a> Biomaterials. 30 (6), 1089-1097 (2009).</li><li>Somaiah, C., 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=Collagen+promotes+higher+adhesion,+survival+and+proliferation+of+mesenchymal+stem+cells.">Collagen promotes higher adhesion, survival and proliferation of mesenchymal stem cells.</a> PLoS One. 10 (12), 0145068 (2015).</li><li>Ogura, 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=Differentiation+of+the+human+mesenchymal+stem+cells+derived+from+bone+marrow+and+enhancement+of+cell+attachment+by+fibronectin.">Differentiation of the human mesenchymal stem cells derived from bone marrow and enhancement of cell attachment by fibronectin.</a> Journal of Oral Science. 46 (4), 207-213 (2004).</li><li>Do, A. V., Khorsand, B., Geary, S. M., Salem, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Printing+of+scaffolds+for+tissue+regeneration+applications.">3D Printing of scaffolds for tissue regeneration applications.</a> Advanced Healthcare Materials. 4 (12), 1742-1762 (2015).</li><li>Shi, 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=Structurally+and+functionally+optimized+silk-fibroin-gelatin+scaffold+using+3D+printing+to+repair+cartilage+injury+in+vitro+and+in+vivo.">Structurally and functionally optimized silk-fibroin-gelatin scaffold using 3D printing to repair cartilage injury in vitro and in vivo.</a> Advanced Material. 29, 1701089 (2017).</li><li>Journeay, W. S., Suri, S. S., Moralez, J. G., Fenniri, H., Singh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+inflammatory+activation+by+self-assembling+Rosette+nanotubes+in+human+Calu-3+pulmonary+epithelial+cells.">Low inflammatory activation by self-assembling Rosette nanotubes in human Calu-3 pulmonary epithelial cells.</a> Small. 4 (6), 817-823 (2008).</li><li>Journeay, W. S., Suri, S. S., Moralez, J. G., Fenniri, H., Singh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rosette+nanotubes+show+low+acute+pulmonary+toxicity+in+vivo.">Rosette nanotubes show low acute pulmonary toxicity in vivo.</a> International Journal of Nanomedicine. 3 (3), 373-383 (2008).</li></ol>

ユペン・チェン博士は、NanoDeセラピューティクス社の共同創設者です。

本研究では、DNAに触発されたJBNTおよびFNで形成された自己組み立てバイオミメティックNMを開発した。JBNT溶液を調製する際には、JBNT凍結乾燥粉末は、PBSの代わりに水に溶解する必要があります。また、PBSの塩はNM繊維に束ね、画像の解像度を大幅に低下させることができるので、NMのナノフィブリル構造を観察したい場合は、NMも水中に組み立てる必要があります。
NMは?...

ヒト間葉系幹細胞(hMCs)は、異なる間葉系に沿った自己再生および自己分化の可能性を示しており、組織の再生および維持に役立つ1。分化ポテンシャルに基づいて、hMCCは間葉組織損傷および造血障害療法2の候補と考えられている。hMSCは、組織修復、血管新生、および炎症を軽減することによって創傷治癒を促進する能力を示した3。しかし、生化学的またはバイオマテリアルの援助がなければ、hMSCが目的の場所で標的組織に到達し、機能する効率は低い4である。様々な設計された足場が病変に付着するhMSCを引き付けるために利用されてきたが、成長板骨折のようないくつかの部位は、長い骨の真ん中に、不規則な形をした傷ついた部位に完全に適合しない可能性がある従来のプレハブ足場によって容易にアクセスできない。
ここでは、その場で自己集合し、標的領域に到達しにくい場所に注入できるバイオミメティックナノ材料を開発しました。注射用バイオ足場NMは、ヤヌスベースナノチューブ(JBNT)およびフィブロネクチン(FN)で構成されています。JBNTは、ロゼットナノチューブ(RNT)とも呼ばれ、DNA塩基対、具体的にはチミンとアデニン、ここでは5、6、7に由来する。図1に示すように、ナノチューブは、6分子の由来DNA塩基が水素結合を介して自己集合して平面6を形成する際に形成される。その後、6つの分子が、最大200〜300μmの強いパイスタッキング相互作用7を介して平面に積み重ねられます。JBNTsは、FNがコラーゲン繊維と反応するように形態学的にコラーゲン繊維を模倣するように設計されています。
FNは、高分子量粘着糖タンパク質であり、これは細胞外マトリックス(ECM)9に見られる。これらは、ECMの他の成分、特にコラーゲン10への幹細胞の付着を媒介することができる。我々は、FNが非共有結合を介して数秒でNMを形成するためにそれらに反応できるように、形態学的にコラーゲン繊維を模倣するようにJBNTを設計しました。したがって、NMは、従来製造された足場ではアクセスできなかった骨折部位に注入される有望なバイオ足場である。ここで、注射可能なNMは、生体外でhMSCのアンカレッジを増強する優れた能力を提示し、組織再生のための足場として役立つ可能性を発揮する。

<table>
	<tbody>
		<tr>
			<td>1,2-dichloroethane</td>
			<td>Alfa Aesar</td>
			<td>39121</td>
			<td></td>
		</tr>
		<tr>
			<td>2-cyanoacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>C88505</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Dimethylaminopyridine</td>
			<td>TCI America</td>
			<td>D1450</td>
			<td></td>
		</tr>
		<tr>
			<td>8 wells Chambered Coverglass</td>
			<td>Thermo Fisher</td>
			<td>155409</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>absolute ethanol</td>
			<td>Thermo Fisher</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34851</td>
			<td></td>
		</tr>
		<tr>
			<td>allylamine</td>
			<td>Sigma-Aldrich</td>
			<td>145831</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC32G</td>
			<td></td>
		</tr>
		<tr>
			<td>citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>251275</td>
			<td></td>
		</tr>
		<tr>
			<td>concentrated hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>Thermo Fisher</td>
			<td>15230147</td>
			<td></td>
		</tr>
		<tr>
			<td>dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td></td>
		</tr>
		<tr>
			<td>diethyl ether</td>
			<td>Sigma-Aldrich</td>
			<td>296082</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-tert-butyl dicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>361941</td>
			<td></td>
		</tr>
		<tr>
			<td>ethyl acetate</td>
			<td>Sigma-Aldrich</td>
			<td>319902</td>
			<td></td>
		</tr>
		<tr>
			<td>ethylcarbamate</td>
			<td>Sigma-Aldrich</td>
			<td>U2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Thermo Fisher</td>
			<td>PHE0023</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixative Solution (4 % formaldehyde prepared in PBS)</td>
			<td>Thermo Fisher</td>
			<td>R37814</td>
			<td></td>
		</tr>
		<tr>
			<td>guanidinium hydrochloride</td>
			<td>Alfa Aesar</td>
			<td>A13543</td>
			<td></td>
		</tr>
		<tr>
			<td>hexanes</td>
			<td>Sigma-Aldrich</td>
			<td>227064</td>
			<td></td>
		</tr>
		<tr>
			<td>Human mesenchymal stem cells</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>methyl iodide</td>
			<td>Sigma-Aldrich</td>
			<td>289566</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Diisopropylethylamine</td>
			<td>Alfa Aesar</td>
			<td>A17114</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide</td>
			<td>Sigma-Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Methylmorpholine N-oxide</td>
			<td>Alfa Aesar</td>
			<td>A19802</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetraoxide</td>
			<td>Alfa Aesar</td>
			<td>45385</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140163</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution</td>
			<td>Thermo Fisher</td>
			<td>20012050</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphoryl chloride</td>
			<td>Sigma-Aldrich</td>
			<td>201170</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>347825</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse phase column</td>
			<td>Thermo Fisher</td>
			<td>25305-154630</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine Phalloidin</td>
			<td>Thermo Fisher</td>
			<td>R415</td>
			<td></td>
		</tr>
		<tr>
			<td>silica gel</td>
			<td>TCI America</td>
			<td>S0821</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium ethoxide</td>
			<td>Alfa Aesar</td>
			<td>L13083</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium periodide</td>
			<td>Sigma-Aldrich</td>
			<td>71859</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>239313</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium sulfite</td>
			<td>Sigma-Aldrich</td>
			<td>S0505</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium triacetoxyborohydride</td>
			<td>Alfa Aesar</td>
			<td>B22060</td>
			<td></td>
		</tr>
		<tr>
			<td>spectrophotometer(NanoDrop One/One&#7580; UV-Vis)</td>
			<td>Thermo Fisher</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem Cell Growth Medium BulletKit</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td></td>
		</tr>
		<tr>
			<td>tetrahydrofuran</td>
			<td>Sigma-Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr>
			<td>thioanisole</td>
			<td>Sigma-Aldrich</td>
			<td>T28002</td>
			<td></td>
		</tr>
		<tr>
			<td>toluene</td>
			<td>Sigma-Aldrich</td>
			<td>179418</td>
			<td></td>
		</tr>
		<tr>
			<td>triethylamine</td>
			<td>Alfa Aesar</td>
			<td>A12646</td>
			<td></td>
		</tr>
		<tr>
			<td>trifluoroacetic acid</td>
			<td>Alfa Aesar</td>
			<td>A12198</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Thermo Fisher</td>
			<td>HFH10</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Thermo Fisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of a biomimetic nano-matrix with janus base nanotubes and fibronectin for stem cell adhesion

バイオミメティックNMは、幹細胞の停泊を高めることができる組織工学的な生物学的足場として機能するように開発されました。バイオミメティックNMは、水溶液中の自己集合体を介してJBNTおよびFNから形成される。JBNTは、内部疎水性中空チャネルおよび外親水性表面で200〜300 μmの長さを測定します。JBNT は正に充電され、FN は負の電荷を受けます。したがって、中性水溶液に注入すると、非共価結合を介して結合してNMバンドルを形成する。自己組立プロセスは、化学イニシエーター、熱源、またはUV光なしで数秒以内に完了します。NM溶液のpHがFNの等電点(pI 5.5-6.0)より低い場合、正に帯電したFNが存在するため、NMバンドルは自己放出します。
NMは、形態学的に細胞外マトリックス(ECM)を模倣することが知られており、したがって、注入スキャフォールドとして使用することができ、これはhMSC接着を強化するための優れたプラットフォームを提供する。細胞密度解析および蛍光イメージング実験は、NMsが陰性対照と比較してhMSCのアンカレッジを有意に増加させたことを示した。

我々の研究は、JBNTsとFNのNMの形成が速く、10秒で起こったことを発見した。 図2に示すように、JBNT溶液をFN溶液と混合し、数回配管した際に白い凝集が得られた。NMの形成プロセスは完全にバイオミメティックである。外部刺激は必要ありません。製造のプロセスは、13を架橋するための紫外線または化学開始剤に基づいているいくつかの新興バイオマ?...

Watch this Scientific Journal Video about Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion at JoVE.com

Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

このプロトコルの目的は、ヤヌスベースナノチューブ(JBNT)とフィブロネクチン(FN)を用いたバイオミメティックナノマトリックス(NM)の組み立てを示すものです。ヒト間葉系幹細胞(hMCs)と共培養すると、hMSCの接着を促す優れた生理活性を示します。

ヤヌスベースナノチューブと幹細胞接着のためのフィブロネクチンによるバイオミメティックナノマトリックスの製造

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed ...

fabrication-biomimetic-nano-matrix-with-janus-base-nanotubes

Research

JoVE Journal

Bioengineering

4.0K ビュー.  University of Connecticut. このプロトコルの目的は、ヤヌスベースナノチューブ(JBNT)とフィブロネクチン(FN)を用いたバイオミメティックナノマトリックス(NM)の組み立てを示すものです。ヒト間葉系幹細胞(hMCs)と共培養すると、hMSCの接着を促す優れた生理活性を示します。

ビデオ: Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

あらかじめ設計されたサイズと形状を用いるポリマー繊維およびバイオハイブリッド繊維のマイクロ流体製造

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

生物工学

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment2.
Coagulation Factor VIII (FVIII)3 is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A&#160;-&#160;a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting4. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete5. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa6,7.
In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.&#160;The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory&#160;to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.

脂質ナノチューブ上血液凝固第VIII因子の螺旋組織

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

マトリックス修飾により誘導される細胞接着及び脱接着におけるリアルタイムの変化を測定するために、細胞 - 基質インピーダンスと生細胞イメージングを使用して、

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

溶剤アシスト脂質二重（SALB）法による生体膜の作製

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

微細と3D微小血管の組立

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

セミフレキシブル高分子を研究する多目的ツールとして DNA ナノチューブ

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

作製と内皮下電気抵抗を直接定量化する統合型電極を用いたオンチップ器官システムの検証

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

デュアルのバイオミメティック基底膜として極薄 Porated 弾性ゲルの細胞培養

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

細胞播種用カスタム Agarose 井戸とティッシュ リング自己組織型の 3 D プリントを使用しての作製

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver MWCNTs were prepared by the reductive deposition of silver from an aqueous solution of AgNO3 on the oxidized MWCNTs. Given the unique color of the CNTs, it was not possible to apply them to the minimum inhibitory concentration or mitochondrial toxicity assays to evaluate the toxicity and antibacterial properties, since they would interfere with the assays. The inhibition zone and minimum bactericidal concentration for the Ag-MWCNTs were measured and Live/Dead and Trypan Blue assays were used to measure the toxicity and antibacterial properties without interfering with the color of the CNTs.

In this study, antimicrobial nanomaterials were synthesized by acidic oxidation of multiwalled carbon nanotubes and subsequent reductive deposition of silver nanoparticles. Antimicrobial activity and cytotoxicity tests were performed with the as-prepared nanomaterials.

銀ナノ粒子とその抗菌活性と細胞毒性の評価変更多層カーボンナノ チューブの合成

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...