Name: expansion of two-dimension electrospun nanofiber mats into three-dimension scaffolds
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds at JoVE.com

这项工作得到了国家卫生研究院国家普通医学研究所 (2p20 gm103440-06 和1r01gm123081 至 j. x.)、otis glebe 医学研究基金会 ne lb606 和内布拉斯加州医科大学启动资金的资助中心。

下文概述的所有体内程序都得到了内布拉斯加州大学医学中心 iacuc 委员会的批准。
1. 准备标准电纺解决方案
<ol><li>在一个20毫升的玻璃管里在二氯甲烷 (dcm) 和 n, n-二甲基甲酰胺 (dmf) 的溶剂混合物中溶解2克聚 (-己内酯), 浓度为 10% (w/v), 浓度为 4: 1 (w/v)。 注意: 将 dcm 和 dmf 放在通风良好的引擎盖中, 以避免暴露在烟雾中。不要将 dcm 暴露在塑料材料中。</li>
	<li>将玻璃...

<ol>
	<li>Chen, S., 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=Recent+advances+in+electrospun+nanofibers+for+wound+healing.">Recent advances in electrospun nanofibers for wound healing.</a> Nanomedicine. 12 (11), 1335-1352 (2017).</li><li>Khandalavala, K., Jiang, J., Shuler, F. D., Xie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+Nanofiber+Scaffolds+with+Gradations+in+Fiber+Organization.">Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization.</a> Journal of Visualized Experiments. (98), e52626 (2015).</li><li>Xie, J., Li, X., Xia, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Put+electrospun+nanofibers+to+work+for+biomedical+research.">Put electrospun nanofibers to work for biomedical research.</a> Macromolecular Rapid Communication. 29 (22), 1775-1792 (2008).</li><li>Xie, 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=Nanofiber+membranes+with+controllable+microwells+and+structural+cues+and+their+use+in+forming+cell+microarrays+and+neuronal+networks.">Nanofiber membranes with controllable microwells and structural cues and their use in forming cell microarrays and neuronal networks.</a> Small. 7 (3), 293-297 (2011).</li><li>Xie, 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=Radially+aligned,+electrospun+nanofibers+as+dural+substitutes+for+wound+closure+and+tissue+regeneration+applications.">Radially aligned, electrospun nanofibers as dural substitutes for wound closure and tissue regeneration applications.</a> ACS. 4 (9), 5027-5036 (2010).</li><li>Xie, 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="Aligned-to-random"+nanofiber+scaffolds+for+mimicking+the+structure+of+the+tendon-to-bone+insertion+site.">"Aligned-to-random" nanofiber scaffolds for mimicking the structure of the tendon-to-bone insertion site.</a> Nanoscale. 2 (6), 923-926 (2010).</li><li>Jiang, 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=Expanded+3D+Nanofiber+Scaffolds:+Cell+Penetration.">Expanded 3D Nanofiber Scaffolds: Cell Penetration.</a> Neovascularization, and Host Response. Advanced Healthcare Materials. 5 (23), 2993-3003 (2016).</li><li>Jiang, 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=Expanding+Two-Dimensional+Electrospun+Nanofiber+Membranes+in+the+Third+Dimension+by+a+Modified+Gas-Foaming+Technique.">Expanding Two-Dimensional Electrospun Nanofiber Membranes in the Third Dimension by a Modified Gas-Foaming Technique.</a> ACS Biomaterials Science &amp; Engineering. 10 (1), 991-1001 (2015).</li><li>Jiang, 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=CO2-expanded+nanofiber+scaffolds+maintain+activity+of+encapsulated+bioactive+materials+and+promote+cellular+infiltration+and+positive+host+response.">CO2-expanded nanofiber scaffolds maintain activity of encapsulated bioactive materials and promote cellular infiltration and positive host response.</a> Acta Biomaterialia. 68, 237-248 (2018).</li><li>Chen, S., 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=Nanofiber-based+sutures+induce+endogenous+antimicrobial+peptide.">Nanofiber-based sutures induce endogenous antimicrobial peptide.</a> Nanomedicine. 12 (10), 2597-2609 (2017).</li><li>Dhand, 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=Bio-inspired+crosslinking+and+matrix-drug+interactions+for+advanced+wound+dressings+with+long-term+antimicrobial+activity.">Bio-inspired crosslinking and matrix-drug interactions for advanced wound dressings with long-term antimicrobial activity.</a> Biomaterials. 138, 153-168 (2017).</li><li>Jiang, 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=Local+sustained+delivery+of+25-hydroxyvitamin+D3+for+production+of+antimicrobial+peptides.">Local sustained delivery of 25-hydroxyvitamin D3 for production of antimicrobial peptides.</a> Pharmaceutical Research. 32 (9), 2851-2862 (2015).</li><li>Jiang, 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=1&#945;,+25-dihydroxyvitamin+D3-eluting+nanofibrous+dressings+induce+endogenous+antimicrobial+peptide+expression.">1&#945;, 25-dihydroxyvitamin D3-eluting nanofibrous dressings induce endogenous antimicrobial peptide expression.</a> Nanomedicine (Lond). 13 (12), 1417-1432 (2018).</li><li>Ma, B., Xie, J., Jiang, J., Shuler, F. D., Bartlett, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+nanofiber+scaffolds+for+orthopedic+tissue+repair+and+regeneration.">Rational design of nanofiber scaffolds for orthopedic tissue repair and regeneration.</a> Nanomedicine. 8 (9), 1459-1481 (2013).</li></ol>

研究了通过 co2 降压技术将传统的二维电纺纳米纤维垫转化为扩展的三维支架。传统的二维纳米纤维垫通过亚临界 co2 流体成功地展开。关键的步骤是在优化的条件下制造2d 纳米纤维垫, 并在不变形边缘的情况下切割垫子 (例如, 使用锋利的手术剪刀)。与传统的二维垫相比, 这种 co2 膨胀纳米纤维支架具有许多优点, 包括层状结构 (图 3a-b</st...

开发一种可植入患者体内的合成支架, 以帮助组织修复和再生的概念, 已经渗透了几十年的再生医学领域。理想的合成支架用于诱导细胞从周围的健康组织迁移, 提供了细胞播种, 粘附, 信号转导, 增殖和分化的架构, 支持血管化, 允许充分的氧合和营养传递, 并促进宿主免疫活动, 以确保植入后成功1。此外, 它还可作为嵌入抗菌分子的载体, 以帮助伤口愈合1,3,6,7, 8,9.控制合成支架中这些生物分子的时间释放的能力是工程支架1时考虑的另一个理想属性。
电纺已成为生产纳米纤维支架1、2、3、4、5、6的一种应用。以前试图创建一个纳米纤维脚手架, 如这里讨论的, 已经做了不同程度的成功。然而, 传统的纳米纤维支架实现这些目标的能力有限。传统的纳米纤维支架大多是二维垫1,3。这些非膨胀支架密集地与小毛孔大小;这限制了细胞的浸润、迁移和分化, 因为它没有促进与体内1、7、8、9相似的环境。为此, 建立了新的3d 电纺纳米纤维支架制备技术, 以修正二维纳米纤维垫固有的缺陷。这些技术导致3d 脚手架;然而, 由于生产方法需要水溶液和冷冻干燥程序, 它们的适用性有限。这种处理导致纳米纤维的随机分布, 没有受限制的组织, 适当的厚度, 或所需的孔隙率, 以提供足够的纳米粒子线索, 细胞迁移和增殖所需的。这些因素导致以前的3d 电纺纳米纤维支架缺乏足够的模仿活组织 1,7,8, 9.
最近, 利用硼氢化钠 (nabh4) 水溶液处理和预先设计的模具, 开发了一种具有更好的细胞外基质 (ecm) 仿生的扩展三维支架,以帮助更好地控制所产生的脚手架的形状7,8。然而, 这种方法并不理想, 因为它需要使用水溶液, 化学反应, 和冷冻干燥, 可能会干扰聚合物和任何封装的生物分子是水溶性的。使用的添加剂也可能在组织再生过程中引起副作用 8,9。本文概述的 co2 膨胀方法大大减少了加工时间, 消除了对水溶液的需求, 并在比以前更大程度上保留了生物活性分子的数量和功能已建立的方法9。
在以前的研究中, 抗生素、银、1α、25二羟基维生素 d3和抗菌肽 ll-37 分别加载到纳米纤维支架中, 并结合起来研究这些支架释放剂的潜力。进一步帮助伤口愈合9,10,12,13。为了演示这种纳米纤维支架膨胀的方法, 将将荧光染料 coumarine 6 加载到支架中, 以证明将支架嵌入各种所需化合物的潜力。这种膨胀纳米纤维支架与包封的生物活性分子结合在组织再生、伤口愈合、三维组织模型的创建和药物局部传递方面具有巨大的潜力。

<table>
	<tbody>
		<tr>
			<td>Polycaprolactone</td>
			<td>Sigma-Aldrich</td>
			<td>440744</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Dimethlyformamide</td>
			<td>Fisher Chemical</td>
			<td>D-199-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane</td>
			<td>Fisher Chemical</td>
			<td>AC61093-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Coumarin 6</td>
			<td>Sigma-Aldrich</td>
			<td>546283</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotating Steel Drum</td>
			<td>customized</td>
			<td></td>
			<td>This serves as a collector during electrospinning.</td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>Fisher Scientific</td>
			<td>14-831-200</td>
			<td>Coaxial spinning requires two single syringe pumps.</td>
		</tr>
		<tr>
			<td>Revolver</td>
			<td>Lab Net International</td>
			<td>H5600</td>
			<td>Adjustable lab rotator for mixing solutions</td>
		</tr>
		<tr>
			<td>Hypodermic Needle (27G x 1 1/2&#34;)</td>
			<td>EXCELINT International Co</td>
			<td>26426</td>
			<td>This is part of the example customized coaxial nozzel shown.</td>
		</tr>
		<tr>
			<td>Hypodermic Needle (21G x 1 1/2&#34;)</td>
			<td>EXCELINT International Co</td>
			<td>26416</td>
			<td>This is part of the example customized coaxial nozzel shown.</td>
		</tr>
		<tr>
			<td>High Voltage DC Power Supply</td>
			<td>Gamma High Voltage Research</td>
			<td>ES30</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope</td>
			<td>FEI</td>
			<td>Nova 2300</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Zeiss</td>
			<td>Axio Imager 2</td>
			<td></td>
		</tr>
		<tr>
			<td>LL 37 ELISA Kit</td>
			<td>Hycult Biotech</td>
			<td>HK321-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

expansion of two-dimension electrospun nanofiber mats into three-dimension scaffolds

电纺已成为生产合成的, 功能性支架的首选技术, 由于仿生细胞外基质和易于控制的组成, 结构和直径的纤维。然而, 尽管有这些优点, 传统的电纺纳米纤维支架还是存在着不组织的纳米纤维取向、低孔隙率、小孔径和主要是二维垫子等局限性。因此, 非常需要开发一种新的工艺来制造能够克服上述限制的电纺纳米纤维支架。在此, 概述了一种新颖而简单的方法。传统的二维纳米纤维垫被转化为具有所需厚度、间隙距离、孔隙率和纳米粒子的三维支架, 以便通过次临界co2流体的降压, 实现细胞播种和增殖。除了为组织再生提供支架外, 这种方法还提供了封装生物活性分子 (如用于局部药物输送的抗菌肽) 的机会。co2膨胀纳米纤维支架在组织再生、伤口愈合、三维组织建模和外用药物输送方面具有巨大的潜力。

通过对亚临界co2流体的降压将传统的二维电纺纳米纤维垫扩展到三维支架的效果表现在不同的容量上: 支架的厚度从未经处理时的1毫米增加到 2.5 mm。19.2 毫米, 分别进行1和 2 co2 处理 (图 3-a-c)。孔--对细胞幼苗至关重要的结构特征--也以与厚度增加相对应的方式增加 (图 3c)。未处理的垫子的孔隙率分?...

Watch this Scientific Journal Video about Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds at JoVE.com

Expansion of Two-dimension Electrospun Nanofiber Mats into Three-dimension Scaffolds

本文介绍了通过对亚临界co2流体的降压, 将传统的二维电纺纳米纤维垫扩展为三维 (3d) 支架的技术。这些增强型支架是3d 的, 紧密地模仿细胞纳米粒子的线索, 并保留包存在纳米纤维中的生物分子的功能。

将二维电纺纳米纤维垫扩展到三维支架中

Electrospinning has been the preferred technology in producing a synthetic, functional scaffold due to the biomimicry to extracellular matrix and the ease ...

该协议报告了传统的电喷纳米纤维膜从2D到3D的转化，通过减压亚临界CO2流体，这是以前没有实现的。此方法消除了与以前的方法相关的许多问题，包括水溶液和化学反应的使用、多步骤过程、封装生物分子活性的丧失以及疏水聚合物的局限性。演示这个程序的是陈世轩，一个博士后从我实验室。在20英里玻璃管中，将两克PCL溶解在二氯甲烷和DF的溶剂混合物中，浓度为10%，比例为4比1。将玻璃管放入实验室旋转器中，直到溶液变清。解决方案可能在夜间混合。要设置电击装置，首先，将 PCL 溶液添加到 20 毫升注射器中，并附上 21 量表钝针。确保注射器中没有空气，并分离管。放置一个旋转钢桶，从针尖放置距离地面收集器 12 厘米的钢鼓。使用鳄鱼夹，将直流高压电源连接到针，并确保收集器接地。对于 20 毫升 PCL 溶液，使用直径为 20.27 毫米、流量为 0.5 毫升/小时设置注射器泵的参数。检查液滴是否在针尖形成。在微调器和距离微调器 20 厘米的接地收集器之间施加 20 千伏的电位。将对齐的纳米纤维垫收集在滚筒中，以 2，000 RPM 转速旋转。一旦 PCL 纳米纤维垫达到大约 1 毫米的厚度，收集它们。将 PCL 纳米纤维垫浸入液氮中五分钟。将 PCL 纳米纤维垫留在液氮中，用 0.5 毫米直径的冲孔冲压 PCL 纳米纤维垫。将 PCL 纳米纤维垫放入液氮中五分钟。用锋利的手术剪刀将垫子切成一厘米一厘米的正方形，同时浸入液氮中，以避免边缘变形。将切割垫放在 30 毫升离心管中，并加大约 1 克干冰。紧盖盖子，让干冰变成液态二氧化碳。一旦液体在管中形成，通过打开盖子快速释放压力。从管子上拆下并观察膨胀的脚手架。将脚手架放在新的离心管中，用干冰进行处理，并重复使用欲望厚度。在与细胞孵育之前，对环氧乙烷中膨胀的纳米纤维支架进行灭菌。通过二次处理后，通过压抑亚临界CO2流体将传统的2D电喷纳米纤维垫扩展到3D支架的有效性显示在左侧。脚手架的厚度从未经处理时的 1 毫米增加到 2.5 毫米，一次二氧化碳处理，到 19.2 毫米，用两次二氧化碳处理。脚手架的孔隙度从未经处理的垫子的79.5%增加到第一次处理后92.1%，第二次处理后增加到99.0%。这一点很重要，因为细胞渗透到脚手架的程度，因此其诱导再生的功效在很大程度上取决于孔隙度。SEM 图像显示，未经处理的 2D 垫的密集密包装纤维结构在与 CO2 膨胀后，被转换为有条不一致的分层结构，并配有对齐的纳米纤维。在 Vevo 研究中，通过皮下植入 CO2 膨胀的纳米纤维支架，为大鼠植入方形孔。这允许在孔内进行细胞迁移和增殖，以及在膨胀过程中创建的纳米纤维层内进一步渗透。从第1周到植入后第4周，与传统的纳米纤维垫相比，膨胀的脚手架显示形成的血管数量显著增加，多核巨型细胞数量显著增加。按照这个程序，不同的分子，包括生长因子，氨基调节化合物，止血剂，和抗微管剂可以并入纳米纤维垫，并在亚临界CO2流体中扩展。这种功能化的扩展纳米纤维支架可用于探索其他科学领域的新问题，如血液、感染的预防和治疗、免疫学以及组织再生和修复。有机溶剂是有毒的，应在化学罩中处理。此外，还应使用能够承受亚临界CO2流体高压的容器进行膨胀。

Research

JoVE Journal

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Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

科研

遗传学

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Canalostomy 为成人和新生小鼠内耳局部药物传递的外科治疗方法

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

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Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

一种简便、快速、定量的测定 DNA 蛋白 Crosslinks 在转染哺乳动物细胞中的研究

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

从前列腺癌的遗传工程小鼠模型生成肿瘤有机体

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

使用CRISPR/Cas9系统将基因挖空直接引入锥虫瘤的肌瘤阶段

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target ...

Use of Drosophila S2 Cells for Live Imaging of Cell Division

Use of Drosophila S2 Cells for Live Imaging of Cell Division

使用果蝇S2细胞进行细胞分裂的活成像

Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a ...

Characterization of Functionally Associated miRNAs in Glioblastoma and their Engineering into Artificial Clusters for Gene Therapy

胶质母细胞瘤中功能相关miRNA的特性及其工程在基因治疗的人工簇中

The biological relevance of microRNAs (miRNAs) in health and disease significantly relies on specific combinations of many simultaneously deregulated ...

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

使用冷冻胚胎进行转基因小鼠高效生产

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...