笔者想感谢我们的同胞林的实验室同事阿马尔奇什蒂和Bijal帕特尔他们与一些组织学和细胞培养的实物援助。资金来自韦恩州立大学纳米医学奖学金（CBP）提供的启动资金和心血管病研究所种子格兰特（MTL）。

1.细胞培养制备<ol><li>利用商业购买的人主动脉平滑肌细胞。 </li><li>保持的88.6％由平滑肌细胞的生长培养基中的细胞231培养基，0.1％每种重组人胰岛素（RH-胰岛素），重组人成纤维细胞生长因子（RH-FGF），重组人表皮生长因子（RH-FGF）的，和抗坏血酸;和5％每胎牛血清（FBS）和L-谷氨酰胺;和1％抗生素/抗真菌剂。 注意:每个生长因子，FBS和L-谷氨酰胺购买作为血管培养基上生长套件。 </li><li>改变媒体每48小时直至细胞为约90％汇合，并准备用于组织播种。 </li><li>存储单元在扩展媒体之间变化的孵化器。 </li></ol> 2. 3D打印机插入和定制的硅胶的制备模压板<ol><li>使用商业三维打印机（ 例如 ，复制迷你）用于3D打印板插入。 <ol><li>使用运EN来源的三维设计软件，如搅拌机创建印刷外壳和职位的3D模型。 </li><li>通过.STL格式允许便携的3D打印机的软件出口模型的驱动程序文件。 </li><li>产生使用聚装入三维打印机打印讯息壳和外壳（乳酸）灯丝（PLA）。 </li><li>继印刷，执行70％-100％的乙醇溶液消毒每次插入一个30分钟的浸泡。 </li></ol></li><li>制备1:10固化剂至基部（PDMS）的有机硅聚合物的聚（二甲基硅氧烷）的聚合物混合物中并允许在室温下将混合物脱气10分钟。 <ol><li>定义用作很小（35 MM）的培养皿规格，中间体（60 MM），和大（100毫米）。 </li><li>加入2毫升，4毫升和6毫升未固化的硅的每个小，中和大板，分别和创建跨陪替氏培养皿的整个底部的薄层。 </li><li>浇PDMS到创建中小板的帖子100毫米板至7mm的高度，并允许在60℃的热板上固化大约2-3小时。然后用一个5毫米的活检穿孔，以冲压出圆柱形立柱。使用未固化的PDMS少量固定每个PDMS气缸和每个小板的中心。 </li><li>用于中间和大板，以在板的底部之前完成的PDMS的固化，将3D印刷讯息分别为10毫米，直径为20mm的中心到每个中间和大板，。对于大板，另外放置一个3D印刷外壳中从柱直径等距66.7毫米。 <ol><li>允许每个菜在敞开的空气在60℃下固化的热板上约2-3小时，使18小时对于聚合物的脱气。固定的印刷组件，以在适当的区域和方向的板， 如图1中所见。 </li></ol></li><li> 30分钟添加70％乙醇，用30％的蒸馏水中的溶液，以所有PLA的内部消毒工商业污水附加费，然后覆盖每个板。 </li><li>小心吸从每个板的乙醇，并允许风干。 </li><li>安排在生物安全柜（BSC），每个板旁边的盖子，面朝上。暴露板和盖子对UV光在BSC下进行30分钟，以完成灭菌。无菌技术用紫外线照射后的每一步进行。 </li></ol></li></ol> 3.纤维蛋白水凝胶的制备与平滑肌细胞和板的维护播种<ol><li>混合含有0.5毫升，1.1毫升和1.81毫升的量生长培养基+ 0.01％，TGF-β1为小，中和大板的大小，分别纤维蛋白凝胶的溶液。 <ol><li>添加40微升，88.4微升和凝血酶的145微升，从100U / mL的库存，以每小，中和大板的媒体，分别。用手轻轻旋转每块板，以保证凝血酶媒体内混合均匀。 </li><li>接下来，添加1601，L，354微升和580微升血纤维蛋白原，从20毫克的库存/毫升，逐滴圆到凝血酶媒体混合物到每个小，中和大板，分别用手轻轻旋转，以确保混合和水凝胶的分配连成一片层。 </li><li>允许的水凝胶以固化在室温下10-15分钟。 </li></ol></li><li> Trypsinize平滑肌细胞根据标准方法扩大150 MM细胞培养板和离心机。所得粒料应重新悬浮于3毫升分化培养基的组成的98％ - 231媒体，1％FBS和1％抗生素/抗真菌剂。 <ol><li>大力滴定上下2毫升吸管混合细胞，打破了所有的细胞团块。计数与血球细胞和/毫升为小，中和大板，分别创建2×10 6个细胞/ mL，1.0×10 7个细胞/ mL和1.4×10 7个细胞的细胞悬浮液。 </li><li>加入1毫升每个细胞悬液的intOA对应的50 mL锥形标有小，中和大。设置额外的50 mL锥形以这种方式为需要的每个额外的组织环。 </li><li>分化培养基添加到每个锥形分别获得2毫升，4毫升和5毫升为每个小，中和大血管的最后播种量。然后，小心吸管细胞溶液滴在每个相应板准备的水凝胶的顶部。 </li><li>代替板到37℃的培养箱中5％CO 2。 </li></ol></li><li>更改分化培养基每隔48-72小时每块板。在较大的板的情况下，改变介质最初在24小时后，然后改变它每48-24小时，以补偿该大的细胞接种密度。 <ol><li>经过2-4天的环会朝后已经完全在收缩，加入10微升，20微升和TGF-β1的35微升至每个小，中和大环，分别为。暴露于TGF-β后1为至少24小时，环准备被处理。 </li></ol></li></ol> 4.血管构造与维修大会<ol><li>最终的血管构造的制造之前，有专门的容器创建以保存完成的容器。 <ol><li>对于小容器，通过切割一个2英寸节断的50mL聚碳酸酯锥形管的顶创建环堆叠一个高大板，然后PDMS胶切割边缘成35毫米板。使用锥形盖作为板盖。 </li><li>对于中等和大型船舶高大环堆叠板，切割1.75英寸直径聚碳酸酯管插入2.5寸段长度作为高大的壁板。对于高大的板裤，削减0.125英寸厚的聚碳酸酯板成2英寸直径的圆块。使用丙烯酸溶剂水泥，结合聚碳酸酯管段的圆形切割片。从60毫米培养皿的盖子高高盘用盖子。 </li><li>三维pRINT讯息5，10和20毫米直径和50毫米的长度。 </li><li>加入10 mL未固化的硅的每个容器。在此之前的PDMS的完全固化，集中放置在步骤4.1.3创建的每个岗位到每个小，中和大容器。 </li><li>允许在热板上集合固化至60℃2-3小时。 </li><li>用70％乙醇与30％的蒸馏水中的溶液30分钟消毒。 </li><li>小心地从每个容器吸出乙醇，然后允许在BSC风干。接下来，安排在每个板放在旁边的盖子，正面朝上罩容器。使容器到BSC在UV光下为另外的30分钟进一步灭菌。使用无菌技术与紫外线照射后的每一步。 </li></ol></li><li>使用非常细镊子，小心翼翼地从它的后删除的每个卷紧平滑肌水凝胶环和转移到其相应的更大的容器与高大的帖子。 <ol><li>用的一对在每一个手钳和解除该环的一侧从柱，然后其他。要小心保护和维护流明。 </li><li>执行这一双手方法转移，首先一面，那么环的另一端滑动到高大的职位。使用温和的，渐进的动作，和周的工作，慢慢地荡推到高大的职位。接着堆叠组织环，直到所需的容器长度已经获得，每个环将大约1-2毫米的长度，以完成构建物。 </li></ol></li><li>随着定位在高大的3D打印的职位环形堆叠，转板，使岗位与工作表面平行。 <ol><li>用微量，在100U / mL的浓度轻轻添加凝血酶40,80和160微升在每个小，中和大容器的外表面，分别。同时加入了凝血酶，缓慢旋转板，以确保即使是结构的所有表面覆盖。这将是用于固定环形堆叠构建体在施工后的最初几天的纤维蛋白胶基。 </li><li>接着，以20毫克/毫升的浓度添加纤维蛋白原的40，80和160μL到每个小，中和大构建体，分别使用一个微量吸移管而迅速转动构建体。凝血酶和纤维蛋白原快速设置成一个坚固的凝胶混合后。由于固化时间短，迅速和均匀地涂在纤维蛋白原越好。 </li></ol></li><li>添加20mL的分化培养基到每个容器保持的构建体。直到需要的地方船只到37℃培养箱。更换介质每3-5天。 </li></ol>

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作者什么都没有透露。

午夜凶铃堆叠方法提出过电流血管组织工程技术构建多重优势。该RSM可以适应通过简单地定制职位和外壳尺寸创建任意大小的人类的船只。我们的方法允许对无聚合物完全人类细胞并迅速降解在身体的自然伤口愈合过程中发现的载体材料组成的工程化血管的发展。聚合物移植物已知会引起再狭窄在诊所和如果包含在工程化的移植物可能成为问题。细胞接种数目需要修改每个不同尺寸的组织环。细胞数目到播种表面积的曲线图在图5中示出从该种子数目可以近似和/或外推。应当指出的是，这里使用的细胞类型是人类主动脉平滑肌细胞。适应RSM中不同类型的细胞，细胞的大小和增殖率需要被考虑考虑和最佳播种密度。例如，我们还创建使用RSM人成纤维细胞环，并已发现至少2倍相比平滑肌细胞需要的细胞数量。容器的任何期望的长度可通过增加环的建立。环堆栈已培养了长达2个月，并保持稳定。中间和大环都保持在即使它们各自在60毫米和100毫米的钢板构成，分别由在100毫米的钢板外壳放置适当1500微米的壁厚。这示出了用于控制和获得适当的壁厚为给定的容器外壳的效用。在步骤3.3.1 TGF-β1中加入，因为它是已知刺激胶原蛋白产生19和具有紧固环的观察到的效果。一旦环已经完全卷起，TGF-β1的一个剂量在最后步骤中加入，并且这些环是准备使用1天后。<html> TGF-β1的确增强胶原产生环中，如在所述三色图像（ 图2）。 在小环细胞更圆而紧凑，而在2大尺寸，沿外边缘的细胞显示一定程度的对准的与组织的边缘，并与其他对准细胞一起。后者可指示细胞的成熟的后期阶段，从更高的细胞内容在较大的环的演变，并因此间信号的更大程度鼓励成熟。在较大的环纤维蛋白胶interspersion可能意味着更大的电池片作为倾向于他们推出小幅倍。呈现这种现象的病理图像获得的1日之后完成环滚上调因而是可以理解的纤维蛋白胶，这需要2周文化降解，仍然会存在。培养这些环为至少2周应降解纤维蛋白胶，留下一个完全的蜂窝结构的后面。 核苷酸"&gt;α平滑肌肌动蛋白（SMA）构成的薄丝促进收缩和原肌球蛋白是一个收缩蛋白。20，21两者SMA和原肌球蛋白在中间存在于所有尺寸的环，具有最强，最均匀地分布信号环。这种现象可能是由于更高程度的细胞密度和组织，刺激在收缩手法发展的增加。 弹性模量表示的环的弹性，从小到大环上的增加ê表明在胶原蛋白和弹性蛋白的生产的增加。极限抗拉强度是没有打破由环忍受的最高强度。破坏强度是组织故障点。对于环，UTS等于FS。为血管，UTS比FS更大，这表明在容器的极限强度是从所有环归因于机械贡献的组合在容器中，故障点是由于最弱环。 我们的工程船舶的实力奠定了在千帕范围内，而天然人的船舶有兆帕范围内的优势。为了加强我们实现这一天然血管的血管，我们正在调查技术来增加细胞外基质的生产，即胶原蛋白和弹性。促进胶原蛋白和弹性蛋白的生产生长因子目前被应用到我们的戒指，调查拉伸性能是否会增加。 除了机械性能，肌肉收缩的功能的措施相关的容器的性能。肌肉刺激和收缩通过因素如乙酰胆碱和肾上腺素能用来测试肌肉收缩力。这样的实验正在考虑为我们今后的研究。 总的来说，我们的结果表明，环堆积方法可以很容易地扩展实现了工程范围内血管组织的大小。扩展到人体最大的血管，如40毫米管腔直径主动脉，可能会需要营养血管，微血管大型血管内自然存在的，这对我们的实验室目前正在开发的发展。另外，内皮细胞层（ 即内膜），通常线介质层的内腔是用于在容器中建立适当的血流动力学是重要的。我们的实验室目前正在创作内膜在我们的SMC环形堆叠使用人血管内皮细胞。有了这些技术相结合，工程船将有更大的适用性诊所。

在治疗冠状动脉疾病（CAD）的，患者自身的血管被收获作为用于旁路手术移植物材料。然而，通常情况下，危重病人没有可行的船只捐赠给自己，在他们这样做的情况下，供体部位造成相当大的额外伤害，并有感染的严重风险。 1个工程血管移植物可以填补这方面的需求。可扩展性是用于工程船舶最重要的，以满足广泛的患者容器的尺寸要求。然而，对于工程船本发明的方法不容易可扩展的，并且通常需要复杂的模具或聚合物支架的再制造。大多数工程化移植物或者使用被接种的成纤维细胞的血管平滑肌或血管内皮细胞的聚合物管状支架;或滚动围绕心轴的细胞片以形成组织管。在临床试验中的两个工程血管移植物是基于脱细胞化ð聚合物ECM平台。 2，3，4可用于血管修复使用聚合物移植物已经公知的有通畅的问题，这可能会出现与具有持续的聚合物存在下，接枝的长期应用的一个主要问题。管状模具已被用于制造完全细胞容器，5，6，7，8，9，10，11，12，13，该程序将需要定制模具额外的设计和刀具制造，以生产各种尺寸的船只。 本文所描述的方法包括一种新颖的技术，用于创建易于扩展工程化血管移植物使用定制的3D印刷插片和传统的培养板中。 14个细胞接种到具有中心柱和外壳的插入板。后控制管腔直径，并允许细胞单层自组装到组织的环。该环的外壳厚度控制，最终容器的因而壁厚。然后完成组织环被堆叠以形成管状，血管移植物。这种方法的优点，被称为"环堆积的方法，"是任何粘附细胞类型可以接种到板设置和组织环或需要的所期望的应用的任何尺寸的管子可以通过简单地修改引导插入物来产生。在组织的组织工程创建环比较技术仍然难以规模，15，16，要求模具的再制造为每个所需的尺寸。此外，血管移植物使用这种方法，可以制造由在2-3周D，几周更快相比其他工程血管。 6对于诊所，这个时间差可以使在恶化患者的治疗中的显著差异。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Human Aortic Smooth Muscle Cells&#160;</td>
			<td>ATCC</td>
			<td>PCS-100-012</td>
			<td>vascular smooth muscle cells</td>
		</tr>
		<tr>
			<td>Medium 231</td>
			<td>Gibco (Life Technologies&#160;</td>
			<td>M-231-500</td>
			<td>media specific to vascular smooth muscle cells</td>
		</tr>
		<tr>
			<td>Human Aortic Smooth Muscle Cell Growth Kit&#160;</td>
			<td>ATCC</td>
			<td>PSC-100-042</td>
			<td>growth factors for maintaining vascular smooth muscle cell viability</td>
		</tr>
		<tr>
			<td>Replicator Mini 3D printer&#160;</td>
			<td>MakerBot&#160;</td>
			<td>N/A</td>
			<td>3D printer</td>
		</tr>
		<tr>
			<td>Poly(lactic acid) 3D ink (PLA)</td>
			<td>MakerBot&#160;</td>
			<td>N/A</td>
			<td>3D printer filament</td>
		</tr>
		<tr>
			<td>Poly(dimethlysiloxane) (PDMS)</td>
			<td>Ellworth Adhesives&#160;</td>
			<td>3097358-1004</td>
			<td>polymer for gluing plate parts</td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Hyclone Labratories, Inc.</td>
			<td>SH30256.01</td>
			<td>fibrin gel component</td>
		</tr>
		<tr>
			<td>Thrombin&#160;</td>
			<td>Sigma Life Sciences</td>
			<td>F3879-5G</td>
			<td>fibrin gel component</td>
		</tr>
		<tr>
			<td>Tranforming Growth Factor-Beta 1&#160;</td>
			<td>PeproTech</td>
			<td>100-21</td>
			<td>growth factor for stimulating collagen production</td>
		</tr>
		<tr>
			<td>Hemocytometer&#160;</td>
			<td>Hausser Scientific Co.</td>
			<td>3200</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>Polycarbonate tubing&#160;</td>
			<td>US Plastics&#160;</td>
			<td>PCTUB1.750X1.625</td>
			<td>material for making tall, ring stacking plates</td>
		</tr>
		<tr>
			<td>Polycarbonate sheet&#160;</td>
			<td>Home Depot</td>
			<td>409497</td>
			<td>material for making tall, ring stacking plates</td>
		</tr>
		<tr>
			<td>Adhesive polymer solvent&#160;</td>
			<td>SCIGRIP</td>
			<td>10799</td>
			<td>material for making tall, ring stacking plates</td>
		</tr>
		<tr>
			<td>Instron&#160; 5940</td>
			<td>Instron</td>
			<td>N/A</td>
			<td>tensile testing machine</td>
		</tr>
		<tr>
			<td>U-Stretch</td>
			<td>Cell Scale</td>
			<td>N/A</td>
			<td>tensile testing machine</td>
		</tr>
		<tr>
			<td>Smooth Muscle Actin&#160;</td>
			<td>MA5-11547</td>
			<td>Thermo Fisher</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>Tropomyosin</td>
			<td>MA5-11783</td>
			<td>Thermo Fisher</td>
			<td>antibody</td>
		</tr>
	</tbody>
</table>

scaling of engineered vascular grafts using 3d printed guides and the ring stacking method

冠状动脉疾病仍然死亡的主要原因，影响数百万美国人。随着缺乏可用的自体血管移植，移植工程提供住院治疗的巨大潜力。然而，工程化血管移植物通常是不容易扩展，因此需要定制模具或聚合物管的制造，以定制到不同的尺寸，构成一个费时和昂贵的做法。人体动脉的范围中管腔直径约2.0-38毫米，在约0.5-2.5毫米的壁厚。我们创建了一个方法，称为"环堆积法"，其中所需要的细胞类型的组织的大小可变环，这里演示血管平滑肌细胞（SMC），可以通过中心的职位导游来控制管腔直径创建壳和外壳口授血管壁的厚度。然后，这些组织环堆叠以创建管状结构，模仿血管的自然形式。该船长度be。通过简单的叠加构成所需的长度需要环的数量量身定做。与我们的技术中，管状形式，类似于血管，组织可以容易地在各种尺寸和长度的制造符合临床和患者的需要。

这里演示是3点不同的工程化血管移植物的尺寸（ 图1）的制造，显示出环堆积方法（RSM）是可伸缩的。为了证明适用性，所述3种不同的容器尺寸选择关联于实际的人类血管尺寸的左前降支（小;内腔直径= 4 MM）17，降主动脉（中间体;内腔直径= 10毫米），升主动脉（大;管腔直径= 20mm）的18。壁厚为小环约500微米，两者的中间和大环约1,500微米。证实每个容器是通过层叠6个环，等同于约6mm的小血管和9毫米的中间和大血管的长度建造。长度是基于每个单独的环的壁厚。 揭示了组织学分析在所有环尺寸高度细胞（ 图2）。红色物质标定纤维蛋白胶。在小环，残余的血纤维蛋白凝胶的少量看到的环的外边缘。在较大的环，一些纤维蛋白凝胶用蜂窝内容穿插。在马松染色，胶原蛋白的产生（由蓝色标记）的指示可以在中间和大环中可以看出。 以确定下列环的形成细胞的表型，组织环用免疫荧光分析抗体对α-平滑肌肌动蛋白（SMA）和肌球蛋白（ 图3）。所有的戒指尺寸为阳性两种抗体，验证平滑肌表型的维持。 于不同尺寸的环进行拉伸试验，以确定它们的机械性能（ 图4）。 U型舒展，一个mechani校准测试装置中，使用对拉伸测试小型和中间环和容器，而在Instron用于拉伸试验的大环和血管。弹性模量（E），极限拉伸强度（UTS）和破坏强度（FS）数据的收集。与实力的增强相关联增加环和船只的大小观察到的趋势一致。 细胞接种所需创建的各种尺寸的环数与播种表面积（ 图5）增加近似线性。为了创造更大的环，还需要至少14百万细胞以创建腹主动脉尺寸环。 六环栈，或容器，测试它们承受流能力进行测试。构建物装入定制灌注系统（ 图6），并进行在流速为100至417毫升/分钟流动为至多5分钟。船只能够承受流动。在容器端部中观察到轻微的泄漏，在连接器连接到灌注系统。 <img alt="图1" src="/files/ftp_upload/55322/55322fig1.jpg" /> 图1: 经缩放的工程化血管的构建。 A）的比例的工程血管，与板制备，细胞接种和容器建筑物开始的处理的图。展示三种不同尺寸的B）中的环和C）的船只。 D）代表大血管完全生物，类似天然组织。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55322/55322fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55322/55322fig2.jpg" /> 图2:组织学分析。 </st<html>荣&gt; H＆E和Masson三色染色显示整个所有的戒指尺寸环厚度可行的细胞结构。三色污渍揭示由蓝（蓝色箭头）表示胶原蛋白的生产领域。大环显示的血纤维蛋白凝胶穿插，可能是因为在细胞片的相对较大的表面积的折叠。比例尺:小圈= 200微米;中间环= 200微米;和大环= 0.5毫米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55322/55322fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55322/55322fig3.jpg" /> 图3:平滑肌标志物免疫荧光分析。所有的戒指尺寸为阳性平滑肌收缩蛋白α平滑肌肌动蛋白（SMA）和原肌球蛋白（Tm）的。比例尺= 200微米。负载/ 55322 / 55322fig3large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/55322/55322fig4.jpg" /> 图4:拉力试验分析。戒指和船只的各种规模的应力 - 应变曲线显示，在环/容器尺寸增加关联强度增加的总趋势。环和船只沿圆周捉襟见肘。从图评估参数为弹性模量，极限抗拉强度和破坏强度（列于表1）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55322/55322fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55322/55322fig5.jpg" /> 图5:细胞种植数量相关性播种冲浪王牌区。基于人类主动脉平滑肌细胞。表面面积被定义为在中心柱和板壁或外壳之间的环的形成板的面积。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55322/55322fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/55322/55322fig6.jpg" /> 图6:受到灌注分析六环船只。 A）定制的灌注系统流量试验。 B）的工程化容器装入灌注系统。三个容器灌注测试，泄漏的流动条件下长达5分钟。容器保持下流动稳定，在连接到系统管容器端连接器轻微泄漏。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55322/55322fig6large.jpg" target="_blank">请点击这里查看</a>一个更大的版本这个数字。 <img alt="动画图1" src="/files/ftp_upload/55322/55322vid1.jpg" /> 动画图1:通过一个精心设计的容器灌注流量的示范。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55322/55322RSM_Engineered_Vessel_Perfusion.mp4" target="_blank">请点击此处观看该视频。 （右键点击下载）。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td></td><td colspan="2">小</td><td colspan="2">中间</td><td colspan="2">大</td></tr><tr><td rowspan="3">戒指</td><td>弹性模量（kPa）的</td><td> 13.6±2.25 </td><td rowspan="3"> （N = 6） </td><td> 14.5±1.2 </td><td rowspan="3"> （N = 3） </td><td> 17.2±2.2 </td><td rowspan="3"> （N = 4） </td></tr><tr><td>极限抗拉强度（K霸） </td><td> 34.5±10.2 </td><td> 39.6±2.98 </td><td> 50.9±10.6 </td></tr><tr><td>破坏强度（千帕） </td><td> 34.5±10.2 </td><td> 39.6±2.98 </td><td> 50.9±10.6 </td></tr><tr><td rowspan="3">容器</td><td>弹性模量（kPa）的</td><td> 49.7±2.80 </td><td rowspan="3"> （N = 3） </td><td> 59.8±3.90 </td><td rowspan="3"> （N = 2） </td><td> 79.8±10.1 </td><td rowspan="3"> （N = 2） </td></tr><tr><td>极限拉伸强度（千帕） </td><td> 115±6.90 </td><td> 137±12.0 </td><td> 192±86.9 </td></tr><tr><td>破坏强度（千帕） </td><td> 96.2±12.2 </td><td> 60.7±12.1 </td><td> 173±92.2 </td></tr></tbody></table> 表1:经缩放的环和容器的拉伸性能。

Watch this Scientific Journal Video about Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method at JoVE.com

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

可扩展的工程血管会提高临床适用性。使用容易可观3D印刷指南，创建并堆叠成管状形式，形成血管移植血管平滑肌的环。移植物的大小可通过简单地改变三维印刷导向大小，以满足人冠状动脉的尺寸范围。

工程血管移植缩放使用3D打印指南和环堆叠方法

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

scaling-engineered-vascular-grafts-using-3d-printed-guides-ring

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.3K 次观看.  Wayne State University. 可扩展的工程血管会提高临床适用性。使用容易可观3D印刷指南，创建并堆叠成管状形式，形成血管移植血管平滑肌的环。移植物的大小可通过简单地改变三维印刷导向大小，以满足人冠状动脉的尺寸范围。 

视频: Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

基于生物工程，人造血管使用脉动生物反应器

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

科研

生物工程

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

医疗器械的使用3D印刷模具的液体注射成型快速，低成本原型

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large animal model. A step by-step protocol for inter-positional placement of the TEV and real-time digital assessment of the TEV and native carotid arteries is described here. In vivo monitoring is made possible by the implantation of flow probes, catheters and ultrasonic crystals (capable of recording dynamic diameter changes of implanted TEVs and native carotid arteries) at the time of surgery. Once implanted, researchers can calculate arterial blood flow patterns, invasive blood pressure and artery diameter yielding parameters such as pulse wave velocity, augmentation index, pulse pressures and compliance. Data acquisition is accomplished using a single computer program for analysis throughout the duration of the experiment. Such invaluable data provides insight into TEV matrix remodeling, its resemblance to native/sham controls and overall TEV performance in vivo.

Surgical Technique for the Implantation of Tissue Engineered Vascular Grafts and Subsequent In Vivo Monitoring

A step-by-step protocol for the inter-positional placement of Tissue Engineered Vessels (TEVs) into the carotid artery of a sheep using end-to-end anastomosis and real-time digital assessment in vivo until animal sacrifice.

手术技术组织工程血管移植和后续的植入<em&gt;在体内</em&gt;监控

The development of Tissue Engineered Vessels (TEVs) is advanced by the ability to routinely and effectively implant TEVs (4-5 mm in diameter) into a large ...

Athymic Rat Model for Evaluation of Engineered Anterior Cruciate Ligament Grafts

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without intervention. Current treatment strategies include ligament reconstruction with either autograft or allograft, which each have their associated limitations. Thus, there is interest in designing a tissue-engineered graft for use in ACL reconstruction. We describe the fabrication of an electrospun polymer graft for use in ACL tissue engineering. This polycaprolactone graft is biocompatible, biodegradable, porous, and is comprised of aligned fibers. Because an animal model is necessary to evaluate such a graft, this paper describes an intra-articular athymic rat model of ACL reconstruction that can be used to evaluate engineered grafts, including those seeded with xenogeneic cells. Representative histology and biomechanical testing results at 16 weeks postoperatively are presented, with grafts tested immediately post-implantation and contralateral native ACLs serving as controls. The present study provides a reproducible animal model with which to evaluate tissue engineered ACL grafts, and demonstrates the potential of a regenerative medicine approach to treatment of ACL rupture.

Animal models are important tools for the evaluation of tissue-engineered grafts. This paper presents the protocol for preparing an electrospun biodegradable polymer graft for use in anterior cruciate ligament tissue engineering, as well as a surgical protocol for implantation in a rat model.

无胸​​腺大鼠模型工程前交叉韧带移植评估

Anterior cruciate ligament (ACL) rupture is a common ligamentous injury that often requires surgery because the ACL does not heal well without ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

工程三维细胞化胶原凝胶的血管组织再生

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

极化神经组织工程三维蚕丝胶原蛋白为基础的模式

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

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 ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

三维印刷多孔纤维素纳米复合水凝胶脚手架

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Evaluation of Left Ventricular Structure and Function using 3D Echocardiography

Three-dimensional (3D) quantification of the left ventricle (LV) provides significant added value in terms of diagnostic accuracy and precise risk stratification in various cardiac disorders. Recently, 3D echocardiography became available in routine cardiology practice; however, high-quality image acquisition and subsequent analysis have a steep learning curve. The present article aims to guide the reader through a detailed 3D protocol by presenting tips and tricks and also by highlighting the potential pitfalls to facilitate the widespread but technically sound use of this important technique concerning the LV. First and foremost, we show the acquisition of a high-quality 3D dataset with optimal spatial and temporal resolution. Then, we present the analytical steps toward a detailed quantification of the LV by using one of the most widely applied built-in software. We will quantify LV volumes, sphericity, mass and also systolic function by measuring ejection fraction and myocardial deformation (longitudinal and circumferential strain). We will discuss and provide clinical examples about the essential scenarios where the transition from a conventional echocardiographic approach to a 3D-based quantification is highly recommended.

In this article, we provide a step-by-step acquisition and analysis protocol for the volumetric assessment and speckle-tracking analysis of the left ventricle by 3D echocardiography, particularly focusing on practical aspects that maximize the feasibility of this technique.