这项研究得到了ZonMw的资助，作为LSH 2Treat计划（436001003）和荷兰肾脏基金会（14a2d507）的一部分。N.A.K.感谢欧洲研究理事会（851960）的支持。我们感谢荷兰科学研究组织（024.003.013）资助的引力计划"材料驱动再生"。

作者没有什么可透露的。

此处描述的生物反应器允许系统地评估剪切应力和循环拉伸对管状可重新吸附脚手架的炎症和组织再生的个体贡献和综合影响。这种方法还使血管构造能够进行各种各样的分析，在具有代表性的结果部分就说明了这一点。这些结果表明，不同的血液机载系统（即剪切和拉伸的不同组合）对 TEVG 结构的生长和改造都有独特的影响。这些见解通过这个体外平台收集，有助于优化新开发的原地 TEVG 的脚?...

心血管组织工程（TE）正在作为替代治疗选择，目前使用的永久心血管假肢（如血管移植，心脏瓣膜置换），这是不理想的大组患者1，2，3，4。许多抢手的应用包括组织工程血管移植 （TEVG）5，6和心脏瓣膜 （TEHV）7，8.最常见的是，心血管TE方法利用可重新吸收的生物材料（无论是天然的还是合成的），作为新组织形成的有启发性的支架。新组织的形成可以完全在体外设计，通过用细胞播种脚手架，并在植入前在生物反应器中培养（体外TE）9、10、11或直接就地植入，在原位植入合成脚手架时无需预先培养，以诱导体内（原位TE）12、13、14等新组织的形成。对于体外和原位心血管TE方法，成功的功能再生主要取决于宿主对植入结构的免疫反应和适当的生物力学负荷。
生物力学负荷对心血管TE的重要性是众所周知的。在心血管植入物中，填充脚手架的细胞暴露于循环拉伸和剪切压力，这些压力是血友病环境引起的。许多研究已经报告了（循环）拉伸对基质成分形成的刺激作用，如胶原蛋白16，17，18，19，糖氨酸（GAGs）20，和弹性蛋白21，22，由各种细胞类型。例如，黄等人证明，双轴拉伸利用血管生物反应器23，提高了胶原蛋白和弹性蛋白在体外特威格斯中的沉积和组织。虽然重点通常在于拉伸作为主要负载，但这些研究通常使用流驱动的生物反应器，其中样本也暴露在剪切流中。虽然对剪切应力对3D组织形成和炎症的孤立影响知之甚少，但提供了一些数据。例如，Hinderer等人和Eoh等人证明，剪切流除了3D脚手架微结构外，对于人类血管平滑肌肉细胞在体外模型系统24、25中形成成熟的弹性蛋白非常重要。总之，这些发现说明了环伸和剪切应激对心血管TE的相关性。
TE植入物成败的另一个重要决定因素是宿主对植入移植物26的免疫反应。这对于物质驱动的原位TE策略尤其重要，它实际上依赖于对脚手架的急性炎症反应来启动细胞流入和内源性组织形成和重塑的后续过程。巨噬菌体是功能组织再生的关键启动器，28、29、30等多项研究表明。类似于伤口愈合，组织的再生是由巨噬细胞和组织产生细胞之间的寄生虫信号，如成纤维细胞和肌纤维细胞31，32，33。除了协调新的组织沉积外，巨噬细胞还参与外来脚手架材料34、35的主动吸收。因此，体外对生物材料的宏年反应已被确定为植入物36、37、38体内成功的预测参数。
植入式脚手架的宏观反应取决于脚手架的设计特征，如材料组成和微观结构35，39，40。除了脚手架属性外，对脚手架及其与肌纤维细胞的相声的宏法响应也受到血液动力载荷的影响。例如，环状拉伸被证明是巨噬细胞表型41，42，43，44和细胞因子43，44，45，46在3D电子喷支架分泌物的重要调节剂。Battiston等人利用巨噬细胞和血管平滑肌肉细胞的共培养系统证明，巨噬细胞的存在导致弹性蛋白和GAG水平增加，中度循环拉伸水平（1.07-1.10）刺激胶原蛋白I和弹性蛋白47的沉积。在以往的著作中，我们已经证明剪切应力是单核细胞招募到3D电喷支架48，49，剪切应力和循环拉伸影响人类单核细胞和间质频闪细胞50之间的寄生虫信号的重要决定因素。Fahy等人证明，剪切流增加了人类单核细胞51的亲炎细胞因子的分泌。
综合起来，上述证据表明，对血液机载荷的充分理解和控制对心血管TE至关重要，因此必须考虑炎症反应来实现这一点。许多生物反应器以前曾被描述为体外52，53，54，55，56，57，58或前体内59，60，61心血管组织培养。然而，所有这些系统都旨在尽可能模仿生理血液机载量条件。虽然这在体外创建心血管组织或维持前体内培养物方面具有很高的价值，但此类系统不允许系统地研究个体线索的个体影响。这是因为在这些生物反应器中应用循环拉伸和剪切应力是由相同的加压流驱动的，而加压流与它们有着内在的联系。虽然用于精确多提示机械操作的微系统已用于 2D 基材62或 3D 水凝胶设置63，64，但此类设置不允许纳入弹性 3D 生物材料脚手架。
在这里，我们介绍了管状生物反应器系统的应用，该系统独特地实现了剪切应力和循环拉伸的脱钩，并有助于机械地研究其个体和组合效应。该系统允许测试各种组织工程血管移植物（例如，合成或自然起源，不同的微结构，各种孔隙）。为了有效地将剪切应力和拉伸的应用脱钩，生物反应器使用的关键概念是（1） 使用独特的泵系统分离剪切应力和拉伸控制，（2） 以计算驱动尺寸的"从内到外"方式刺激脚手架。通过使用流泵将流量应用于管状脚手架的外表面，而脚手架的周长则通过使用单独的应变泵来扩展安装脚手架的硅胶管。硅胶管和包含构造的玻璃管的尺寸是使用计算流体动力学模拟仔细选择和验证的，以确保脚手架上的剪切应力（由于流量）和环状拉伸（由于管膨胀）不会显著影响彼此。这种从内到外的设计有几个实用的理由。如果拉伸由发光流体压力（类似于生理负荷）施加，则固有要求样品设计无泄漏。此外，拉伸样品所需的压力将完全由样品刚度决定，样本刚度可能因样品而异，并且随着时间的推移，很难控制样品的拉伸。这种生物反应器将组织工程移植物安装在硅胶管周围，并允许在移植物的外壁上应用壁切变应力 （WSS），并从内部给移植物加压。这样，样品之间和样品内部的同等装载条件可以保证，而且，样品允许泄漏，就像多孔血管支架19一样。这种从内到外的生物反应器是专门用来系统研究剪切和/或拉伸的影响，而不是在体外进行原生血管的工程，而传统的血管生物反应器设置更适合于此。有关生物反应器设计图纸 的图 1A-B 及其相应的 表 1， 了解生物反应器主要组件背后的功能描述和原理。
生物反应器的使用是根据我们小组最近的一系列研究证明的，其中我们调查了剪切应力和循环拉伸对可再吸附电喷支架中炎症和组织形成的个体和组合影响，用于原位心血管组织19、43、44。为此，我们使用单体或共培养中的人类巨噬细胞和肌纤维细胞来模拟原位再生级联的各个阶段。我们已经证明，人类巨噬细胞的细胞因子分泌明显受到循环拉伸和剪切应力的影响，通过parcrine信号和直接接触19、43、44，通过这些脚手架中的人类肌纤维细胞影响基质沉积和组织。值得注意的是，这些研究表明，在剪切应力和拉伸的结合应用的情况下，对组织形成和炎症的影响要么由两个负载之一主导，要么有两个负载的协同效应。这些发现说明了将两个负载脱钩以更好地了解机械环境对 TE 过程的贡献的相关性。这一理解可用于系统地优化相关血液动力负荷系统中的脚手架设计参数。此外，来自这些控制良好的环境的机械数据可以作为正在开发的数字模型的输入，以预测原位组织重塑的过程，正如最近为TEVG65或TEHV66报告的那样，以进一步提高预测能力。

<table><tbody><tr><td>advanced Dulbecco&amp;rsquo;s modified EagleMedium (aDMEM)</td><td>Gibco</td><td>12491-015</td><td>cell culture medium for fibroblasts</td></tr><tr><td>Aqua Stabil</td><td>Julabo</td><td>8940012</td><td>prevent microorganism growth in bioreactor-hydraulic reservoir</td></tr><tr><td>Bovine fibrinogen</td><td>Sigma</td><td>F8630</td><td>to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td></tr><tr><td>Bovine thrombin</td><td>Sigma</td><td>T4648</td><td>to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804</td><td>to spin down cells and conditioned medium</td></tr><tr><td>Clamp scissor - &quot;kelly forceps&quot;</td><td>Almedic</td><td>P-422</td><td>clamp the silicone tubing and apply pre-stretch to the scaffold so the scaffold can be sutured into the engraved groove (autoclave at step 1, step 7)</td></tr><tr><td>CO2 cell culture incubators</td><td>Sanyo</td><td>MCO-170AIC-PE</td><td>for cell culturing</td></tr><tr><td>Compressed air reservoir</td><td>Festo</td><td>CRVZS-5</td><td>smoothing air pressure fluctuations and create time delays for pressure build-up</td></tr><tr><td>Custom Matlab script to calculate the maximum stretches</td><td>Matlab</td><td>R2017. The Mathworks, Natick, MA</td><td>calculate the minimum and maximum outer diameter of the electrospun scaffold</td></tr><tr><td>Data acquisition board</td><td>National Instruments</td><td>BNC-2090</td><td>data processing in between amplifier system and computer</td></tr><tr><td>Ethanol</td><td>VWR</td><td>VWRK4096-9005</td><td>to keep sterile working conditions</td></tr><tr><td>Fetal bovine calf serum (FBS)</td><td>Greiner</td><td>758087</td><td>cell culture medium supplement; serum-supplement</td></tr><tr><td>Flow culture chamber compartments, consisting of a pressure conduit with engraved grooves and small holes to apply pressure on silicone tubing, a screw thread, nose cone, top compartment with flow inlet and bottom compartment flow outlet, adapter bushing</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>flow culture chamber compartments (autoclave at step 1, step 7)</td></tr><tr><td>Glass Pasteur pipet</td><td>Assistant</td><td>HE40567002</td><td>apply vacuum on electrospun scaffold (autoclave at step 1)</td></tr><tr><td>Glass tubes of the flow culture chamber</td><td>Custon made, Equipment &amp;amp; Prototype Center, Eindhoven University of Technology</td><td>n.a.</td><td>part of the flow culture chamber (clean and store in 70% ethanol, at step 1 and 7)</td></tr><tr><td>GlutaMax</td><td>Gibco</td><td>35050061</td><td>cell culture medium amino acid supplement, minimizes ammonia build-up</td></tr><tr><td>High speed camera</td><td>MotionScope</td><td>M-5</td><td>to monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)</td></tr><tr><td>High speed camera lens - Micro-NIKKOR 55mm f/2.8 - lens</td><td>Nikon</td><td>JAA616AB</td><td>to monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)</td></tr><tr><td>Hose clip</td><td>ibidi GmbH</td><td>10821</td><td>block medium flow (autoclave at step 1, step 7)</td></tr><tr><td>Hydraulic reservoir with 8 screw threads for 8 flow culture chambers</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>to apply pressure to the silicone mounted constructs (clean outside with a paper tissue with 70% ethanol, rinse reservoir with 70% ethanol followed by demi water, at step 1 and 7)</td></tr><tr><td>Ibidi pump system (8x) including ibidi pump, PumpControl software, fluidic unit, perfusion set (medium tubing), air pressure tubing, drying bottles with orange silica beads</td><td>ibidi GmbH</td><td>10902</td><td>set up used to control the flow in the flow culture chambers. Note 1: the ibidi pumps were modified by the manufacturer to enable 200 mbar capacity. Note 2: can be replaced by pump system of other manufacturer, as long as same flow regimes can be applied.</td></tr><tr><td>Knives (no.10 sterile blades, individual foil pack) and scalpel handle (stainless steel, individually wrapped)</td><td>Swann Morton</td><td>0301; 0933</td><td>to cut the silicone tubing in the correct size for the scaffold and to cut the suture material</td></tr><tr><td>LabVIEW Software</td><td>National Instruments</td><td>version 2018</td><td>to control the stretch applied to the scaffolds</td></tr><tr><td>Laminar flow biosafety cabinet with UV light</td><td>Labconco</td><td>302310001</td><td>to ensure sterile working conditions. The UV is used to decontaminate everything that cannot be autoclaved, or touched after autoclaving</td></tr><tr><td>Large and small petri dishes</td><td>Greiner</td><td>664-160</td><td>for sterile working conditions</td></tr><tr><td>L-ascorbic acid 2-phosphate (vitamin C)</td><td>Sigma</td><td>A8960</td><td>cell culture medium supplement, important for collagen production</td></tr><tr><td>LED light cold source KL2500</td><td>Zeiss</td><td>Schott AG</td><td>to aid in visualization for the time lapse of the scaffolds during monitoring of the stretch</td></tr><tr><td>Luer (female and male) locks and connectors, white luer caps</td><td>ibidi GmbH</td><td>various, see (https://ibidi.com/26-flow-accessories)</td><td>to close or connect parts of the bioreactor and the ibidi pump (autoclave at step 1, step 7)</td></tr><tr><td>Measuring amplifier (PICAS)</td><td>PEEKEL instruments B.V.</td><td>n.a.</td><td>to amplify the signal from the pressure sensor and feedback to LabView</td></tr><tr><td>Medium reservoir (large syringes 60 mL) and reservoir holders</td><td>ibidi GmbH</td><td>10974</td><td>medium reservoir (autoclave at step 1, step 7)</td></tr><tr><td>Medium tubing with 4.25 mm outer diameter and 1 mm inner diameter</td><td>Rubber BV</td><td>1805</td><td>to allow for a larger flow rate, the ibidi medium tubing with larger diameter is used. Note: the part of medium tubing guided through the fluidic unit valves are the same as the default ibidi medium tubing</td></tr><tr><td>Motion Studio Software</td><td>Idtvision</td><td>2.15.00</td><td>to make the high speed time lapse images for stretch monitoring</td></tr><tr><td>Needle (19G)</td><td>BD Microlance</td><td>301700</td><td>together with thin flexible tubing used to fill the hydraulic reservoir with ultrapure water without adding air bubbles</td></tr><tr><td>Needle driver</td><td>Adson</td><td>2429218</td><td>to handle the needle of the nylon suture through the silicone tube (autoclave at step 1, step 7)</td></tr><tr><td>Paper tissues</td><td>Kleenex</td><td>38044001</td><td>for cleaning of the equipment with 70% ethanol</td></tr><tr><td>Parafilm</td><td>Sigma</td><td>P7793-1EA</td><td>quick fix if leakage occurs</td></tr><tr><td>Penicillin/streptomycin (P/S)</td><td>Lonza</td><td>DE17-602E</td><td>cell culture medium supplement; prevent bacterial contamination</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Sigma</td><td>P4417-100TAB</td><td>for storage and washing steps (autoclave at step 1)</td></tr><tr><td>Plastic containers (60 mL) with red screw caps</td><td>Greiner</td><td>206202</td><td>to prepare the fibrinogen solution</td></tr><tr><td>Pneumatic cylinder</td><td>Festo</td><td>AEVC-20-10-I-P</td><td>to actuate the Teflon bellow (clean with a paper tissue with 70% ethanol at step 1 and 7)</td></tr><tr><td>Polycaprolactone bisurea (PCL-BU) tubular scaffolds (3 mm inner diameter, 200 &amp;micro;m wall thickness, 20 mm length)</td><td>SyMO-Chem, Eindhoven, The Netherlands</td><td>n.a.</td><td>produced using electrospinning from 15% (w/w) chloroform (Sigma; 372978) polymer solutions. See Van Haaften et al Tissue Engineering Part C (2018) for more details</td></tr><tr><td>Pressure conduit without holes (for static control)</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>to mount electrospun tubes on silicon tubing (autoclave at step 1, step 7)</td></tr><tr><td>Pressure sensor and transducer</td><td>BD</td><td>TC-XX and P 10 EZ</td><td>the air pressure going to the pneumatic actuated pump is raised until it reaches the set pressure</td></tr><tr><td>Proportional air pressure control valve and pressure sensor</td><td>Festo</td><td>MPPES-3-1/8-2-010, 159596</td><td>provides compressed air to the pneumatic actuated pump</td></tr><tr><td>Roswell Park Memorial Institute 1640 (RPMI-1640)</td><td>Gibco</td><td>A1049101</td><td>cell culture medium for monocyte/macrophage</td></tr><tr><td>Safe lock Eppendorf tubes (1.5 mL)</td><td>Eppendorf</td><td>30120086</td><td>multiple applications (autoclave at step 1)</td></tr><tr><td>Sodium dodecyl sulfate solution 20%</td><td>Sigma</td><td>5030</td><td>Used to clean materials, at a concentration of 0.1%.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Silicone O-rings</td><td>Technirub</td><td>1250S</td><td>to prevent leakage (autoclave at step 1, step 7)</td></tr><tr><td>Silicone tubing (2.8 mm outer diameter, 400 um wall thickness)</td><td>Rubber BV</td><td>1805</td><td>to mount the electrospun tubes on the pressure conduits (autoclave at step 1)</td></tr><tr><td>Sterile tube (15 mL)</td><td>Falcon</td><td>352095</td><td>multiple applications</td></tr><tr><td>Suture, 5-0 prolene with pre-attached taper point needle</td><td>Ethicon, Johnson&amp;amp;Johnson</td><td>EH7404H</td><td>Prolene suture wire 5-0 (75cm length, TF taper point needle, 1/2 circle, 13 mm needle length)</td></tr><tr><td>Syringe (24 mL)</td><td>B. Braun Melsungen AG</td><td>2057932</td><td>to add the ultrapure water or medium to the hydraulic reservoir or flow culture chamber</td></tr><tr><td>Syringe filter (0.2 &amp;micro;m)</td><td>Satorius</td><td>17597-K</td><td>to filter the fibrinogen solution</td></tr><tr><td>T150 cell culture flask with filter cap</td><td>Nunc</td><td>178983</td><td>to degas culture medium</td></tr><tr><td>T75 Cell culture flask with filter cap</td><td>Nunc</td><td>156499</td><td>to culture static control samples</td></tr><tr><td>Teflon bellow</td><td>Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td><td>n.a.</td><td>to load the hydraulic reservoir (clean outside with a paper tissue with 70% ethanol at step 1 and 7)</td></tr><tr><td>Tray (stainless steel)</td><td>PolarWare</td><td>15-248</td><td>for easy transport of the fluidic culture chambers and the bioreactor from incubator to laminar flow cabinet and back (clean with a paper tissue with 70% ethanol before and after use)</td></tr><tr><td>Tweezers</td><td>Wironit</td><td>4910</td><td>sterile handling of individual parts (autoclave at step 1 and 7)</td></tr><tr><td>Ultrapure water</td><td>Stakpure</td><td>Omniapure UV 18200002</td><td>to correct for medium evaporation, mixed with aqua stabil mixed and used as hydraulic fluid. (autoclave ultrapure water at step 1)</td></tr><tr><td>UV light</td><td>Philips</td><td>TUV 30W/G30 T8</td><td>for decontamination of grafts and bioreactor parts before seeding</td></tr></tbody></table>

a multi-cue bioreactor to evaluate the inflammatory and regenerative capacity of biomaterials under flow and stretch

从转化的角度来看，使用可再吸附生物材料直接在体内诱导再生是一种有吸引力的策略。这种材料在植入时会引起炎症反应，这是随后材料吸收和新组织再生的驱动因素。这一战略，也被称为原位组织工程，旨在获得心血管置换，如组织工程血管移植。炎症和再生过程都由脚手架上的局部生物力学线索（即拉伸和剪切应力）决定。在这里，我们详细描述了定制开发的生物反应器的使用，这种反应器能够使管状脚手架上的拉伸和剪切应力脱钩。这允许系统和标准化地评估管状脚手架在控制良好的机械负载的影响下的炎症和再生能力，我们利用人类巨噬细胞和肌纤维细胞进行动态共培养实验来证明这一点。详细讨论了该方法的关键实际步骤:生物反应器的建造和建立、脚手架和细胞播种的制备、拉伸和剪切流的应用和维护以及样品采集分析。

该生物反应器的开发是为了研究剪切应激和循环拉伸对血管组织生长和3D生物材料脚手架改造的个体和组合效应。生物反应器的设计允许在各种装载条件下培养多达8个血管结构（图1A）。血管结构位于流动培养室（图1B），其中周长伸展和WSS都可以独立控制。流文化室的顶部隔间持有一个流直发器，以相对较短的沉降长度（图1C）?...

Watch this Scientific Journal Video about A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch at JoVE.com

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

本协议的目标是在管状电喷脚手架中执行人类巨噬细胞和肌纤维细胞的动态共生，以研究物质驱动的组织再生，使用生物反应器，使剪切应力和循环拉伸脱钩。

评估流和拉伸下生物材料的炎症和再生能力的多奎生物反应器

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

a-multi-cue-bioreactor-to-evaluate-inflammatory-regenerative-capacity

Research

JoVE Journal

Engineering

5.5K 次观看.  Eindhoven University of Technology. 本协议的目标是在管状电喷脚手架中执行人类巨噬细胞和肌纤维细胞的动态共生，以研究物质驱动的组织再生，使用生物反应器，使剪切应力和循环拉伸脱钩。

视频: A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

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

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

静电纤维组织工程的后期处理

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

组织工程生物反应器的双轴机械装载设计

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

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. ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

平行板灌注系统的体外模型研究了细菌对移植组织的粘附性

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

发育生物学

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

在生理体外模型和病理血流应用的血管细胞重塑调查

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has developed a low-cost perfusion bioreactor system that allows culture medium to be continuously perfused into a cell culture module and fractionated in a downstream module to measure dynamics on this scale. The system is constructed almost entirely from commercially available parts and can be parallelized to conduct independent experiments in conventional multi-well cell culture plates simultaneously. This video article demonstrates how to assemble the base setup, which requires only a single multichannel syringe pump and a modified fraction collector to perfuse up to six cultures in parallel. Useful variants on the modular design are also presented that allow for controlled stimulation dynamics, such as solute pulses or pharmacokinetic-like profiles. Importantly, as solute signals travel through the system, they are distorted due to solute dispersion. Furthermore, a method for measuring the residence time distributions (RTDs) of the components of the perfusion setup with a tracer using MATLAB is described. RTDs are useful to calculate how solute signals are distorted by the flow in the multi-compartment system. This system is highly robust and reproducible, so basic researchers can easily adopt it without the need for specialized fabrication facilities.

This paper presents a method to construct and operate a low-cost, multichannel perfusion cell culture system for measuring the dynamics of secretion and absorption rates of solutes in cellular processes. The system can also expose cells to dynamic stimulus profiles.

多流灌注生物反应器集成了出口分馏功能，用于动态细胞培养

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This ...

无泵多通道流式培养，用于维护和评估组织和细胞

Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell conditions required for the maintenance of function and viability. Toward this end, we have developed a multi-channel flow culture system to maintain tissue and cells in culture and continuously assess function and viability by either in-line sensors and/or collection of outflow fractions. The system combines 8-channel, continuous optical sensing of oxygen consumption rate with a built-in fraction collector to simultaneously measure production rates of metabolites and hormone secretion. Although it is able to maintain and assess a wide range of tissue and cell models, including islets, muscle, and hypothalamus, here we describe its operating principles and the experimental preparations/protocols that we have used to investigate bioenergetic regulation of isolated mouse retina, mouse retinal pigment epithelium (RPE)-choroid-sclera, and cultured human RPE cells. Innovations in the design of the system, such as pumpless fluid flow, have produced a greatly simplified operation of a multi-channel flow system. Videos and images are shown that illustrate how to assemble, prepare the instrument for an experiment, and load the different tissue/cell models into the perifusion chambers. In addition, guidelines for selecting conditions for protocol- and tissue-specific experiments are delineated and discussed, including setting the correct flow rate to tissue ratio to obtain consistent and stable culture conditions and accurate determinations of consumption and production rates. The combination of optimal tissue maintenance and real-time assessment of multiple parameters yields highly informative data sets that will have great utility for research in the physiology of the eye and drug discovery for the treatment of impaired vision.

作者聚焦:通过多通道流式培养推进眼生理学研究，实现最佳组织维护和实时评估 

Real-time analysis of live tissue yields important functional and mechanistic data. This paper describes the protocols and critical variables to ensure accurate and reproducible generation of data by a novel and pump-free multi-channel fluidics system that maintains and assesses a wide range of tissue and cell models.