Name: fabrication of a biomimetic nano-matrix with janus base nanotubes and fibronectin for stem cell adhesion
Uploaded: 2020-05-10T14:45:00
Description: 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

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...

<ol>
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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）?...

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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 ...

Research

JoVE Journal

Bioengineering

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