这项工作由NIH（赠款1R01AR072027-01，1R03AR069383-01）、NSF职业奖（1653702）和康涅狄格大学提供财政支持。

1. JBNT的合成
注：JBNT单体是如前11年出版的。
<ol><li>化合物A1的合成<ol>
<li>在 25 mL 甲苯和 2.5 mL N、二甲基甲酰胺中准备含有 8.50 克 2 氰乙酸和 9.80 克乙基碳酸的溶液。向下添加 4.90 mL 氯化磷。然后将混合物加热至70°C，并保持搅拌1.5小时。</li>
		<li>冷却反应混合物到室温，倒入100克冰水。用乙酸乙酸乙酯（3 x 250 mL）提取水层，用100mL盐水清洗。将有机层干燥在硫酸氢钠上，过滤并蒸发真空下的挥发物，以获得淡黄色固体。</li>
		<li>将黄色固体受制于二氧化硅凝胶闪光柱色谱（3% 甲醇/二氯甲烷），以产生 15.61 克复合 A1 作为白色粉末。</li>
	</ol></li>
	<li>化合物A2的合成<ol>
<li>将3.12克化合物A1和2.75克碳酸钾混合在50mL的N-二甲基酰胺中。在室温下搅拌2小时。</li>
		<li>在反应悬浮物中添加2.6 mL的二氧化碳。继续搅拌4小时。</li>
		<li>在 0 °C 下将绝对乙醇添加到反应混合物中。 过滤黄色沉淀物，用二乙醚清洗。在真空下干燥过夜，产生6.18克的复合A2作为淡黄色固体。</li>
	</ol></li>
	<li>化合物A3的合成<ol>
<li>在15mL的丙酮三烯中加入2.7 mL的甲基碘化物。此外，通过添加 6.18 克 A2 至 80 mL 的水/丙酮三醇（7：3 比），单独准备复合 A2 溶液。混合溶液，在室温下搅拌30分钟。</li>
		<li>将反应混合物加热至95°C，并保持搅拌3小时。</li>
		<li>将反应混合物冷却到室温，然后在真空下蒸发挥发物。</li>
		<li>用 100 mL 乙酸乙酯提取残留物 3 倍，用盐水清洗。将有机层干燥在硫酸钠上，过滤并蒸发真空下的挥发物，获得黄色固体。</li>
		<li>将黄色固体受制于二氧化硅凝胶闪光柱色谱（30% 乙酸乙酯/六烷），以产生 4.52 克的复合 A3 作为浅黄色固体。</li>
	</ol></li>
	<li>化合物A4的合成<ol>
<li>在20升乙醇中加入925μL的阿利胺，准备阿利胺溶液。将此溶液向下添加到复合 A3 溶液中，通过在 75 mL 的绝对乙醇中添加 3.14 克 A3，超过 30 分钟。然后加热反应混合物倒流16小时。</li>
		<li>冷却反应混合物到室温，蒸发挥发物，给一个黄色的固体。</li>
		<li>将黄色固体受制于二氧化硅凝胶闪光柱色谱（50% 乙酸乙酯/六烷至 2% 甲醇/二氯甲烷），以产生 1.80 克复合 A4 作为白色晶体。</li>
	</ol></li>
	<li>化合物A5的合成<ol>
<li>在14 mL的绝对乙醇中加入2.05克盐酸钾和7.8毫克乙酰胺钠（乙醇21wt%）。将混合物加热至45°C 15分钟。</li>
		<li>过滤掉不溶性材料，将过滤液直接加入化合物A4的溶液中，通过在40 mL绝对乙醇中加入2.70克A4来制备。将反应混合物加热至倒流 16 小时。</li>
		<li>过滤沉淀物，获得2.65克复合A5作为白色固体。</li>
	</ol></li>
	<li>化合物A6的合成<ol>
<li>在1分钟内加入2.11克化合物A5和1.10克4-迪米西拉米诺皮里丁，在120mL的四氢喃中缓慢地加入24.9 mL的三乙胺。在室温下搅拌2分钟。</li>
		<li>将20.7 mL的二丁基二碳酸酯滴入反应混合物中，并在室温下保持搅拌40小时。</li>
		<li>加入20mL的水来抑制反应。在真空中蒸发挥发物。</li>
		<li>用500mL乙酸酯提取红色粘性残留物。然后，用250mL的水、75 mL的10%柠檬酸、2倍的200mL水、200mL的饱和碳酸氢钠和200mL的盐水洗净。将有机层干燥在硫酸钠上，过滤并蒸发真空下的挥发物，以产生红色/橙色固体。</li>
		<li>将固体受制于二氧化硅凝胶闪光柱色谱（0-20%乙酸乙酯/六烷），以产生3.87克复合A6作为白色泡沫。</li>
	</ol></li>
	<li>化合物A7的合成<ol>
<li>加入N-甲基甲基肌酰胺N-氧化物（水中50 wt.%，1.64 mL）滴入丙酮/水中复合A6（2.93克）溶液（8：1，58.5 mL），在室温下搅拌5分钟。</li>
		<li>在 3 分钟内将四氧化硅（4% 水溶液）滴入混合物中，并在室温下保持搅拌 24 小时。</li>
		<li>用1.0M水性硫酸钠淬灭反应，直到溶液变色。清除真空下的挥发物，在水中产生白色泥浆。用350mL二氯甲烷提取残留物，然后用150 mL的水清洗，然后用150mL的盐水清洗。</li>
		<li>将有机层干燥在硫酸氢钠上，过滤并蒸发真空下的挥发物，产生2.45克复合A7作为白色固体。</li>
	</ol></li>
	<li>化合物A8的合成<ol>
<li>在35 mL的二氯甲烷/水（6：1，35 mL）中加入760克硫化钠，在室温下搅拌42小时。</li>
		<li>过滤掉不溶性材料，将过滤剂浓缩在真空下。用250mL二氯甲烷提取产生的残留物，然后用100mL的水和100mL的盐水清洗。将有机层干燥在硫酸氢钠上，过滤并蒸发真空下的挥发物，以提供粗制滥干的产品。</li>
		<li>将粗产品受制于二氧化硅凝胶闪光柱色谱（5-40%乙酯/六烷，然后5%甲醇/二氯甲烷），产生1.08克化合物A8。</li>
	</ol></li>
	<li>化合物A9的合成<ol>
<li>在15 mL的1，2-二氯甲烷中准备830毫克复合A8的溶液。将滴水溶液添加到 6 苯氧碳化物-2-L-氨基-六角酸三酯乙酯 （649.5 毫克） 的混合物中 和N，N-二甲基胺（591μL）在24 mL的1，2二氯乙烷超过3分钟，并在室温下搅拌15分钟。</li>
		<li>在溶液中加入360.3毫克三乙氧氢钠，在室温下保持搅拌24小时。</li>
		<li>用6mL的水淬灭反应混合物。提取反应混合物 3 倍与 200 mL 二氯甲烷和洗涤 200 mL 的 10% 柠檬酸在水中， 2x 与 200 mL 的水， 200 mL 饱和碳酸氢钠， 和 200 mL 盐水按该顺序。将有机层干燥在硫酸氢钠上，过滤并蒸发真空下的挥发物，以获得粗制滥干的产品。</li>
		<li>将粗产品受制于二氧化硅凝胶闪光柱色谱（5-30% 乙酸乙酯/六烷），产生 885 毫克化合物 A9。</li>
	</ol></li>
	<li>JBNT单体合成<ol>
<li>溶解 0.54 克化合物 A9 （0.54 g） 在 10 mL 的 94% 三氟乙酸/硫化物溶液中，在室温下搅拌 72 小时。</li>
		<li>将二乙醚（80 mL）添加到反应混合物中，将沉淀下来的离心机。倒出含有超常剂的透明三氟乙酸，用二乙醚和甲醇清洗白色沉淀物，产生168.6毫克粗JBNT单体（补充文件1）。</li>
		<li>使用反相柱用高性能液体色谱 （HPLC） 净化粗 JBNT 单体。隔离方案如下：溶剂A：100%水：溶剂B：100%丙酮三甲酰：溶剂C：pH=1 HCl水溶液;流速：3 mL/分钟（见表1）。</li>
		<li>收集最大峰值约7.2分钟。</li>
	</ol></li>
</ol>2. JBNT/FN的制造
<ol><li>将 80μL 的 1 毫克/mL JBNT 水溶液添加到 40 μL 的 1 毫克/mL FN 水溶液和移液器中多次。</li>
	<li>检查在管道输送约10s后是否有白色固体悬架。 注：拍摄了一段视频来捕捉NM的自组装过程（补充文件2）。</li>
</ol>3. 对冻虫标本的观察
注：执行此步骤是为了显示由 JBNT 和 FN 制成的 NM 的脚手架结构。
<ol><li>为亲血材料观察准备 FN、JBNT 和 FN/JBNT NM 解决方案。用 40 μL 蒸馏水稀释 10 μL 的 10 μl FN，使 FN 的溶液达到 20μg/mL。</li>
	<li>通过稀释 45 μL DI 水中 1 毫克/mL JBNT 的 5 μL，准备 100 毫克/mL 的 JBNT 溶液。</li>
	<li>将 100μg/mL FN 的 10 μL 和 5 μL 的 1 毫克/mL 的 JBNT 混合在 35 μL 的 DI 水中，形成 NM 解决方案。</li>
	<li>分别用 FN、JBNT 解决方案和 NM 解决方案涂上三口 96 井透明圆底微板的井。每口井可获得 50μL 的解决方案。</li>
	<li>执行低血友病（如下所述）以可视化 NM 的脚手架结构。<ol>
<li>在 -80 °C 下将盘子冷冻过夜。</li>
		<li>打开冻素化仪器和真空泵。</li>
		<li>按 下冷却 按钮，将系统温度降低到 -50 °C。</li>
		<li>将冷冻板放入冻毒仪器中，并关闭仪器的所有开口。</li>
		<li>单击 "开始 "按钮，将系统压力降低到 0.9 mbar。</li>
		<li>4小时后，按下 气压 按钮并打开阀门，以增加系统对大气的压力。</li>
		<li>把盘子从嗜酸的仪器里拿出来。</li>
		<li>使用显微镜观察 FN、JBNT 和由此产生的自组装 NM。用相机拍几张照片作为材料。</li>
	</ol>
</li>
</ol>4. 吸收光谱测量
注：使用FN和JBNT光谱的变化来呈现自组装的JBNT/FN NM12。
<ol><li>将 30 μL 的 100μg/mL FN 与 15 μL 蒸馏水和 5 μL 的 1 毫克/mL JBNT 混合，以准备 JBNT/FN NM 解决方案。多次管道输送溶液后，可以看到白色固体悬架。 注：解决方案中 JBNT 和 FN 的最终浓度分别为 100μg/mL 和 60 μg/mL。JBNT 和 FN 解决方案与 NM 解决方案中使用的解决方案浓度相同，也作为控制解决方案编写。</li>
	<li>用 45 μL 蒸馏水稀释 1 毫克/mL 的 JBNT 5 μL，以准备 JBNT 控制解决方案。</li>
	<li>用 20 μL 蒸馏水稀释 30 μL 的 100μg/mL FN，以准备 FN 控制解决方案。</li>
	<li>用光谱仪（见 材料表）测量 FN、JBNT 和 JBNT/FN NM 解决方案的紫外线吸收光谱，以描述 NM 的组装。<ol>
<li>单击 紫外线-维斯 按钮。清洁光谱仪的测试表面，并在上面滴2微升蒸馏水。从190-850nm测量为空白。</li>
		<li>清洁光谱仪的测试表面，并在上面投下 2 μL FN 溶液。测量 FN 溶液的紫外线-维斯吸收光谱从 190 nm 到 850 nm。</li>
		<li>清洁光谱仪的测试表面，并在上面投下 2 μL 的 JBNT 溶液。测量 JBNT 解决方案的吸收光谱从 190 nm 到 850 nm。</li>
		<li>清洁光谱仪的测试表面，并在上面投下 2 μL 的 JBNT/FN NM 溶液。测量 JBNT/FN NM 解决方案的吸收光谱从 190 nm 到 850 nm。</li>
	</ol>
</li>
</ol>5. 为传输电子显微镜（TEM）准备JBNT/FN NM
<ol><li>用基本等离子清洁剂在负染色前清洁几个网格（见 材料表）。</li>
	<li>在两个不同的过程后对标本进行负染色。<ol>
<li>在分离的网格上滴下 3 μL 的 1 毫克/mL 的 JBNT 水溶液和 3 μL 的 JBNT/FN NM 溶液，然后离开 2 分钟。然后用 100 μL 的 0.5% 醋酸尿酸酯 （UA） 溶液冲洗每个网格。用滤纸排干多余的溶液，空气使网格干燥。</li>
		<li>将 JBNT/FN NM 解决方案的 9 μL 与 3 μL 的 2.0% UA 溶液混合，并多次移液器。将混合溶液放在网格上，并将其保留在网格上 2 分钟。使用滤纸从网格中去除混合溶液，并在室温下干燥网格。</li>
	</ol>
</li>
	<li>最后，将 JBNT、JBNT/FN NM 的标本与步骤 5.2.1 染色，JBNT/FN NM 的标本与 TEM 染色为步骤 5.2.2。</li>
</ol>6. 体外生物功能检测
<ol><li>将 200μL 的负控制 （NC、PBS）、JBNT、FN 和 JBNT/FN NM 解决方案分别添加到 8 井室滑梯的一口井中。</li>
	<li>用冻干仪器将8口井室的遮盖玻璃冷冻干燥，将材料涂在井底。冷冻干燥协议与步骤 3.5 相同。</li>
	<li>然后在井中加入10，000个细胞/井hMSC（通道3），在37°C下用5%的CO2孵育24小时。</li>
	<li>孵化后，从井中移出细胞培养介质，用1x PBS溶液冲洗培养物。</li>
	<li>将 100μL 的固定溶液添加到每个带细胞的油井中，然后离开 5 分钟。用 1x PBS 轻轻冲洗细胞两次。</li>
	<li>稀释 1% 特里顿-X 100 至 0.1%。将 100 μL 的 0.1% Triton-X 100 添加到每个井中，并离开 10 分钟。用 1x PBS 轻轻冲洗细胞两次。</li>
	<li>将罗达明法洛丁稀释至1.65微米。用细胞将100μL的罗达明·法洛丁加入每口井，并在室温下孵育室内的盖玻璃，使光线保持30分钟。用 1x PBS 轻轻冲洗细胞两次。</li>
	<li>使用荧光显微镜捕捉细胞图像。</li>
	<li>计算每组荧光图像上的细胞数。通过每个油井中平均三个随机区域来计算细胞粘附密度。</li>
	<li>将所有数据显示为平均±标准偏差 （SD）。使用单尾 t 测试进行统计分析，然后分析 p<0.05 的方差 （ANOVA），这被认为具有统计学意义。</li>
</ol>

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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>

陈玉鹏博士是纳米德治疗公司的联合创始人。

在这项研究中，我们开发了一种自组装的仿生NM，它是由DNA启发的JBNT和FN形成的。在准备 JBNT 解决方案时，JBNT 亲血粉应该溶解到水中，而不是 PBS 中，因为 PBS 会导致 JBNT 的聚集，从而抑制其组装。此外，如果我们想要观察NM的纳米纤维结构，NM也应该在水中组装，因为PBS中的盐会与NM纤维捆绑在一起，从而大大降低图像的分辨率。
NM已显示出巨大的潜力，作为一个新的组织工?...

人类间质干细胞（hMSCs）已经显示出沿着不同的间质血统自我更新和自我分化的潜力，这有助于组织1的再生和维持。基于分化潜力，hMSC被认为是间质组织损伤和造血障碍治疗2的候选者。hMSC已经显示出通过增加组织修复，血管生成和减少炎症3促进伤口愈合的能力。然而，如果没有生化或生物材料的帮助，hMSC在预期位置达到目标组织和功能的效率很低。虽然各种工程脚手架已被利用来吸引 hMSC 粘附在病变上，但一些部位（如长骨中间的生长板断裂）不容易被传统的预制脚手架使用，这些脚手架可能无法完全适应不规则形状的受伤部位。
在这里，我们开发了一种生物仿生纳米材料，可以就地自组装，并注射到难以到达的目标区域。注射生物脚手架NM由Janus基纳米管（JBNTs）和纤维素（FN）组成。JBNTs，也被称为罗塞特纳米管（RNTs），来自DNA碱基对，特别是胸腺素和腺苷，这里5，6，7。如图1所见，纳米管形成时，六个分子的衍生DNA碱基对自组装通过氢键形成平面6。然后，六个分子通过强大的 pi 堆叠相互作用7在平面上相互堆叠，长度可达 200-300μm。JBNT 旨在从形态上模仿胶原纤维，以便 FN 能够对它们做出反应。
FN是一种高分子量的胶粘剂糖蛋白，可以在细胞外基质（ECM）9中找到。这些可以调解干细胞附着在ECM的其他成分，特别是胶原蛋白10。我们设计了 JBNT 来形态模仿胶原纤维，以便 FN 能够在几秒钟内通过非价粘接与它们做出反应，形成 NM。因此，NM 是一个有希望的生物脚手架，可注射到传统制造的脚手架无法到达的骨折部位。在这里，注射NM提供了一个极好的能力，以提高hMSC锚定体外，显示其潜力，作为组织再生的脚手架。

<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

一种仿生NM被开发为组织工程生物脚手架，可以增强干细胞锚定。生物仿生NM由JBNT和FN通过水溶液中的自组装而形成。JBNT 的长度为 200-300μm，内有疏水空心通道和外部亲水表面。JBNT 带正电荷，FN 收取负费用。因此，当注入中性液态溶液时，它们通过非价粘合粘合在一起，形成 NM 束。自组装过程在几秒钟内完成，没有任何化学启动器、热源或紫外线。当 NM 解决方案的 pH 度低于 FN 的等电点（pI 5.5-6.0）时，NM 捆绑包将由于带正电荷 FN 的存在而自行释放。
众所周知，NM 在形态上模仿细胞外矩阵 （ECM），因此可以用作注射脚手架，这为增强 hMSC 粘附性提供了绝佳的平台。细胞密度分析和荧光成像实验表明，与负控制相比，NMS显著增加了 hMSC 的锚定。

我们的研究发现，JBNT和FN的NM形成速度很快，在10秒内发生。如图2所示，当JBNT溶液与FN溶液混合并多次吹笛时，获得白色絮凝。NM的形成过程完全是生物仿生。无需外部刺激。制造过程比一些新兴生物材料容易得多，这些生物材料基于紫外线或化学启动器，用于交叉链接13。
我们捕获并分析了 FN、JBNT 和 NM（图 3）?...

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

该协议的目的是展示与Janus碱纳米管（JBNTs）和纤维素（FN）的生物仿生纳米三角星（NM）的组装。当与人类间质干细胞 （hMSC） 共同培养时，NMs 在鼓励 hMSC 粘附方面表现出出色的生物活性。

用Janus碱纳米管和纤维素制造用于干细胞粘附的仿生纳米矩阵

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 次观看.  University of Connecticut. 该协议的目的是展示与Janus碱纳米管（JBNTs）和纤维素（FN）的生物仿生纳米三角星（NM）的组装。当与人类间质干细胞 （hMSC） 共同培养时，NMs 在鼓励 hMSC 粘附方面表现出出色的生物活性。

视频: 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.

预设计尺寸和形状的聚合物纤维和生物贿赂纤维的微流体制造

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

科研

生物工程

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.

凝血因子VIII的脂质纳米管螺旋组织

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.

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

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.

生物膜制作的溶剂辅助脂质双分子层（SALB）方法

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.

缩微和3D微血管大会

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.

DNA 纳米管作为研究半聚合物的万能工具

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.

用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

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.

超薄 Porated 弹性水凝胶作为双细胞培养的仿生基底膜

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.

用 3 d-印刷模具制作细胞播种和组织环自组装用的自定义琼脂糖井

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.

银纳米粒子改性多壁碳纳米管的合成及其抗菌活性和细胞毒性的评价

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