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&lt;sup&gt;c&lt;/sup&gt; 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

Universidad de Cartagena UDEC

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

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

制备纤维蛋白三维支架干细胞培养应用

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

科研

生物工程

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

细胞外基质蛋白纳米纤维和纳米结构工程采用表面引发大会

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

优化人骨髓间充质干细胞附着在聚（ε己内酯）静电纺纱线

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

静电纳米纤维支架与渐变的纤维组织

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

扩大奈米基材使用缝合技术的细胞行为Nanotopographical调制

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

Dendrimer-based Uneven Nanopatterns to Locally Control Surface Adhesiveness: A Method to Direct Chondrogenic Differentiation

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

A method to obtain dendrimer-based uneven nanopatterns that permit the nanoscale control of local arginine-glycine-aspartic acid (RGD) surface density is described and applied for the study of cell adhesion and chondrogenic differentiation.

基于状的不均匀 Nanopatterns 对局部控制表面粘附的研究--一种直接软骨分化的方法

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations ...

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 ...

Fabrication of Gradient Nanopattern by Thermal Nanoimprinting Technique and Screening of the Response of Human Endothelial Colony-forming Cells

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular reactions. However, it is difficult to determine the relation between the size of a nanostructure and the responses of cells owing to the lack of proper screening tools. Here, we show the development of reproducible and cost-effective gradient nanopattern plates for the manipulation of cellular responses. Using anodic aluminum oxide (AAO) as a master mold, gradient nanopattern plates with nanopillars of increasing diameter ranges [120-200&#8239;nm (GP 120/200), 200-280&#8239;nm (GP 200/280), and 280-360&#8239;nm (GP 280/360)] were fabricated by a thermal imprinting technique. These gradient nanopattern plates were designed to mimic the various sizes of nanotopography in the ECM and were used to screen the responses of human endothelial colony-forming cells (hECFCs). In this protocol, we describe the step-by-step procedure of fabricating gradient nanopattern plates for cell engineering, techniques of cultivating hECFCs from human peripheral blood, and culturing hECFCs on nanopattern plates.

Here, we present a protocol for the fabrication of gradient nanopattern plates via thermal nanoimprinting and the method of screening responses of human endothelial progenitor cells to the nanostructures. By using the described technology, it is possible to produce a scaffold that can manipulate cell behavior by physical stimuli.

热 Nanoimprinting 技术制备梯度 Nanopattern 及其对人血管内皮细胞菌落形成的响应筛选

Nanotopography can be found in various extracellular matrices (ECMs) around the body and is known to have important regulatory actions upon cellular ...

Fabrication and Characterization of Layer-By-Layer Janus Base Nano-Matrix to Promote Cartilage Regeneration

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo uses. The addition of growth factors into these biomaterial scaffolds is generally done to provide an optimal cell culture environment, mediating cell differentiation and its subsequent functions. However, the growth factors in a conventional biomaterial scaffold are typically designed to be released upon implantation, which could result in unintended side effects on surrounding tissue or cells. Here, the DNA-inspired Janus base nano-matrix (JBNm) has successfully achieved a highly localized microenvironment with a layer-by-layer structure for self-sustainable cartilage tissue constructs. JBNms are self-assembled from Janus base nanotubes (JBNts), matrilin-3, and transforming growth factor beta-1 (TGF-&#946;1) via bioaffinity. The JBNm was assembled at a TGF-&#946;1:matrilin-3:JBNt ratio of 1:4:10, as this has been the determined ratio at which proper assembly into the layer-by-layer structure could occur. First, the TGF-&#946;1 solution was added to the matrilin-3 solution. Then, this mixture was pipetted several times to ensure sufficient homogeneity before the addition of the JBNt solution. This formed the layer-by-layer JBNm, after pipetting several times again. A variety of experiments were performed to characterize the layer-by-layer JBNm structure, JBNts alone, matrilin-3 alone, and TGF-&#946;1 alone. The formation of JBNm was studied with UV-Vis absorption spectra, and the structure of the JBNm was observed with transmission electron microscopy (TEM). As the innovative layer-by-layer JBNm scaffold is formed on a molecular scale, the fluorescent dye-labeled JBNm could be observed. The TGF-&#946;1 is confined within the inner layer of the injectable JBNm, which can prevent the release of growth factors to surrounding areas, promote localized chondrogenesis, and promote an anti-hypertrophic microenvironment.

This protocol describes the assembly of a layer-by-layer Janus base nano-matrix (JBNm) scaffold by adding Janus base nanotubes (JBNts), matrilin-3, and Transforming Growth Factor Beta-1 (TGF-&#946;1) sequentially. The JBNm was fabricated and characterized; additionally, it displayed excellent bioactivity, encouraging cell functions such as adhesion, proliferation, and differentiation.

促进软骨再生的逐层Janus碱基纳米基质的制备和表征

Various biomaterial scaffolds have been developed to guide cell adhesion and proliferation in hopes to promote specific functions for in vitro and in vivo ...

Fabricating Engineered Nanofibrillar Collagen with Directed Patterning

Production of Nanofibrillar Patterned Collagen for Tissue Engineering

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are designed to emulate the biophysical properties of native tissue, providing cellular phenotypic and morphological guidance that contributes to the restoration of the regenerative tissue niche. Collagen, a prevalent extracellular matrix protein, is a common component of these regenerative biomaterials due to its biocompatibility and other favorable properties. The current study describes a novel and straightforward method for the fabrication of engineered nanofibrillar collagen with directed fibril patterning. Through the manipulation of shear stress, temperature, and pH, collagen fibrillogenesis and alignment are precisely controlled without requiring specialized equipment. This approach allows for the creation of nanofibrillar collagen biomaterials that mimic the native structure of tissues exhibiting either anisotropic or isotropic characteristics. The flexibility in collagen nanofibril patterning not only facilitates the study of nanoscale patterning on cell behavior but also offers diverse possibilities for patterned tissue engineering applications.

The following protocol presents a shear-based extrusion method for the fabrication of collagen hydrogels with nanoscale patterned fibrils, which can be applied to a broad range of tissue engineering applications.

Regenerative biomaterials are designed to facilitate cell-material interactions to guide the repair of damaged tissues and organs. These materials are ...