Name: scaling of engineered vascular grafts using 3d printed guides and the ring stacking method
Uploaded: 2017-03-27T11:30:00.000+00:00
Duration: 9 min 38 s
Description: Watch this Scientific Journal Video about Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method at JoVE.com

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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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?...

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><tbody><tr><td>Human Aortic Smooth Muscle Cells&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</td><td>MakerBot&amp;nbsp;</td><td>N/A</td><td>3D printer</td></tr><tr><td>Poly(lactic acid) 3D ink (PLA)</td><td>MakerBot&amp;nbsp;</td><td>N/A</td><td>3D printer filament</td></tr><tr><td>Poly(dimethlysiloxane) (PDMS)</td><td>Ellworth Adhesives&amp;nbsp;</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&amp;nbsp;</td><td>Sigma Life Sciences</td><td>F3879-5G</td><td>fibrin gel component</td></tr><tr><td>Tranforming Growth Factor-Beta 1&amp;nbsp;</td><td>PeproTech</td><td>100-21</td><td>growth factor for stimulating collagen production</td></tr><tr><td>Hemocytometer&amp;nbsp;</td><td>Hausser Scientific Co.</td><td>3200</td><td>for cell counting</td></tr><tr><td>Polycarbonate tubing&amp;nbsp;</td><td>US Plastics&amp;nbsp;</td><td>PCTUB1.750X1.625</td><td>material for making tall, ring stacking plates</td></tr><tr><td>Polycarbonate sheet&amp;nbsp;</td><td>Home Depot</td><td>409497</td><td>material for making tall, ring stacking plates</td></tr><tr><td>Adhesive polymer solvent&amp;nbsp;</td><td>SCIGRIP</td><td>10799</td><td>material for making tall, ring stacking plates</td></tr><tr><td>Instron 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&amp;nbsp;</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?...

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

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

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

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 ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

组织工程血管在小鼠模型的生成和嫁接

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

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. ...

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 ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

逐步在泰瑟尔脚手架上播种，研究发芽血管

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

Standardized Technique of Aortic Valve Re-implantation for Valve-sparing Aortic Root Replacement

Despite the obvious advantages of the preservation of a normal aortic valve during aortic root replacement, the complexity of valve sparing procedures prevents a number of cardiac surgeons from incorporating them into their practice. The aim of this protocol is to describe a simplified and user-friendly technique of an aortic valve-sparing root replacement (VSRR) procedure by re-implantation of the aortic valve. Proper selection of patients and limitations of the technique are discussed.
In 54 consecutive patients, normal appearing aortic valves were re-implanted in a commercially available polyester prosthesis with pre-shaped sinuses by a simplified and standardized technique. Placement of the first row of the proximal suture line, choice of the prosthesis size, and adjustment of the height of the commissures of the patient to the fixed height of the sinus portion of the prosthesis were slightly modified from the reference techniques with the aim of increasing its feasibility for use by other cardiac surgeons. Early mortality and morbidity as well as 5-year survival, freedom from aortic valve reoperation, and freedom from recurrent moderate regurgitation were collected in all patients.
Thirty-day mortality, re-sternotomy for bleeding, re-sternotomy for mediastinitis, and the incidence of stroke were very low, 1.8% for each (1 of 54). No patient required permanent pace-maker implantation. At 5 years, survival, freedom from aortic valve reoperation, and freedom from recurrent moderate regurgitation were 97.5%, 95.2%, and 91.6%, respectively.
Mid-term results of our standardized technique of re-implantation of the aortic valve for valve-sparing aortic root replacement are very good and compare with more complex techniques reported by experienced surgeons. By following the present protocol of the standardized re-implantation technique, a greater number of cardiac surgeons can perform this procedure with comparable good results.

Valve-sparing aortic root replacement has the advantage of preserving the patient&#39;s own aortic valve. The complexity of the reported techniques to date restricts their use to a limited number of cardiac surgeons. This protocol describes step-by-step a standardized technique reproducible by a greater number of cardiac surgeons.

主动脉瓣重置术治疗瓣膜备用主动脉根置换的规范化技术

Despite the obvious advantages of the preservation of a normal aortic valve during aortic root replacement, the complexity of valve sparing procedures ...

医学

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. 
Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. 
This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

Engineered flaps require an incorporated functional vascular network. In this protocol, we present a method of fabricating a 3D printed tissue flap containing a hierarchical vascular network and its direct microsurgical anastomoses to rat femoral artery.

使用3D打印技术制造工程血管瓣

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal symptoms. These anomalies may be associated with other congenital heart diseases. It is hard to identify the accurate anatomic vessel location from two-dimensional imaging data, such as computed tomography. As an additive manufacturing method, three-dimensional (3-D) printing can covert the acquired imaging data into 3-D physical models. This protocol describes the procedure for modeling the volumetric DICOM imaging into 3-D data and printing it as an anatomically realistic 3-D model. Using this model, surgeons can identify the vessel location of complex aortic anomalies, which is helpful for pre-operative planning and intra-operative guidance.

Here, we present a protocol to use three dimensional printed models for pre-operative planning and intra-operative reorganization of complicated vascular locations when handling a congenital aortic anomaly.

复杂主动脉异常的三维打印

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...