Name: nondestructive monitoring of degradable scaffold-based tissue-engineered blood vessel development using optical coherence tomography
Uploaded: 2018-10-03T14:00:00
Description: Watch this Scientific Journal Video about Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography at JoVE.com

我们感谢广东省科技规划项目 (2016B070701007) 支持这项工作。

1. 可降解 PGA 支架基组织工程容器培养
<ol><li>PGA 脚手架制作<ol>
<li>将 PGA 网 (19 毫米直径和1毫米厚), 用0.3 缝合环氧乙烷 (17 厘米长, 5.0 毫米直径, 5-0 毫米厚) 消毒。</li>
		<li>将聚四氟乙烯 (聚四氟乙烯, 1cm 长度) 缝合到 PGA 网格的每一端, 重叠2毫米。</li>
		<li>在1摩尔/升氢氧化钠中用手蘸 PGA 支架1分钟, 调整网格的空间结构, 用组织培养级水浸泡三次, 每组2分钟。每次用纸巾轻轻拍干脚手架。然后用?...

<ol>
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生成具有类似于原生血管的结构和机械特性的工程容器, 可缩短临床使用时间, 是血管工程的终极目标。光学成像技术允许可视化组织工程化的血管特异成分, 它不能监测整个文化和暴露移植到一个文化环境中的个体结构, 而不损害不孕7。本文将培养室与灌注系统分离。相对独立的灌流系统, 保证了养殖过程中污染风险的降低和导丝的安置。同时, 该腔内成像方式采用了 TEBVs 的简?...

组织工程血管 (TEBVs) 是最有希望的材料作为理想的血管移植1。为了使移植物在临床上有用, 以类似的结构和功能特性作为本机血管, 多项技术的设计, 以维持血管功能2,3。虽然在植入和 III 期临床研究4中, 已有工程血管可接受的通畅率, 但长期培养和高成本也显示了监测 TEBVs 发展的必要性。对仿生化学-机械环境中 TEBVs 细胞外基质 (ECM) 的生长、重塑和适应过程的理解可以为血管组织工程的发展提供重要的信息。
跟踪小直径工程船舶发展的理想策略5应为无损、无菌、纵向、三维和定量。TEBVs 在不同的培养条件下可以用这种成像方式评估, 甚至包括血管移植前后的变化。描述生活工程船的特征的策略是必要的。光学成像技术允许组织沉积和生物材料的可视化和量化。其他优点是可以使深组织和无标签成像与高分辨率6,7。然而, 图像特异分子和不易接近的光学设备用于实时监测是一个重要的实际障碍, 限制了非线性光学显微学的广泛应用。光学相干层析成像 (OCT) 是一种用于指导心脏介入治疗8的广泛应用的临床工具, 具有血管内成像方式。文献报道了 OCT 的方法, 以评估 TEBVs9,10的壁厚, 加上肯定成像方式的血管组织工程研究。然而, 没有观察到工程血管生长和重塑的动力学。
本论文详细介绍了四周培养的生物降解高分子支架 TEBVs 的制备方法。人脐动脉血管平滑肌细胞 (HUASMCs) 在生物反应器中扩展并播种成多孔可降解羟基酸 (PGA) 支架。生物降解聚合物在组织工程的临时基质中发挥作用, 并具有一定的降解率11。为了保证支架降解与新组织形成之间的适当匹配, ECM 和 PGA 支架是有效血管重塑的关键因素。该灌注系统模拟了本机的生物力学微环境, 在压力刺激下保持了一致的变形。
提出的协议的目的是描述一个相对简单和无损的战略, TEBVs 成像和长期监测的文化。该协议可用于不同培养条件下工程船舶形态变化和厚度测量的可视化。此外, 还可以对组织工程支架中聚合物基材料的降解进行分析, 以进行鉴定。采用扫描电子显微镜 (SEM) 和偏光显微镜相结合的方法, 对细胞外基质分布和 PGA 降解的相关和定量进行了研究, 有助于评估脚手架退化与 OCT 成像相结合。

<table border="0" cellpadding="0" cellspacing="0" style="width:797px;" width="797">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PGA mesh</td>
			<td style="width:265px;">Synthecon</td>
			<td style="width:149px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">silicone tube</td>
			<td style="width:265px;">Cole Parmer</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">connector</td>
			<td style="width:265px;">Cole Parmer</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">intravascular OCT system</td>
			<td style="width:265px;">St. Jude Medical, Inc</td>
			<td style="width:149px;">ILUMIEN&#8482; OPTIS&#8482; SYSTEM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">scanning electron microscopic</td>
			<td style="width:265px;">Philips&#160;</td>
			<td style="width:149px;">FEI Philips XL-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polarized microscope</td>
			<td style="width:265px;">Olympus</td>
			<td style="width:149px;">Olympus BX51</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:216px;">sutures</td>
			<td style="width:265px;">Johnson &#38; Johnson</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:216px;">pulsatile pump</td>
			<td style="width:265px;">Guangdong Cardiovascular Institute</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LightLab Imaging software</td>
			<td style="width:265px;">St. Jude Medical, Inc</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

nondestructive monitoring of degradable scaffold-based tissue-engineered blood vessel development using optical coherence tomography

具有类似天然血管的结构和机械性质的工程化血管移植有望满足日益增长的动脉旁路需求。用脉冲刺激对可降解高分子支架基组织工程血管 (TEBVs) 的生长动力学和重塑过程进行表征, 对血管组织工程具有重要意义。光学成像技术是监测工程组织血管化的有力工具, 能够实现实时文化中的高分辨率成像。本文通过光学相干层析成像 (OCT) 对 TEBVs 在长期培养中的生长和重塑进行了无损、快速的实时成像策略。评价几何形态学, 包括血管重塑过程、壁厚、不同培养时间点的 TEBV 厚度比较和脉动刺激的存在。最后, OCT 提供了实际的可能性, 实时观察在重建组织中的聚合物在脉动刺激或不和在每一个容器段, 通过与评估聚合物降解使用扫描电子显微镜 (SEM) 和偏光显微镜。

三维培养系统由生物反应器中的一个培养室和一个封闭流体循环10、13 (图 1) 的灌注系统组成。OCT 成像导管插入到 Y 形接头的远端, 并将其拉回硅胶管中进行成像。在生物反应器培养过程中, OCT 成像首次被用于描述生物降解高分子支架基 TEBVs 的结构表征。
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Watch this Scientific Journal Video about Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography at JoVE.com

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

基于光学相干断层扫描的可降解支架组织工程血管发育的无损监测

可生物降解高分子支架组织工程血管脉冲刺激实时培养过程中的非破坏性和长周期监测的步进协议本文介绍了光学相干层析成像方法。

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

该方法有助于回答血管组织工程领域关键问题，如监测工程师血管生长动态和长期培养过程中重塑。使用光学相干断层扫描（OCT）的主要优点是，它是一种现成的实时和无损成像策略，用于描述工程容器的结构特征和改造过程。演示程序也将是刘尚民，和马文涛，我的实验室技术人员。要开始此过程，请根据文本协议中所述，将 PGA 脚手架在每升氢氧化钠中浸渍 PGA 脚手架一分钟，以调整网格的空间结构。然后，将脚手架浸泡在组织培养级水中三次，每次两分钟。每次用纸巾轻轻拍干脚手架。然后，用鼓风机将发动机罩中的脚手架晾干一小时。为了组装用于 OCT 成像的生物反应器和 y-结，首先，将自行开发的玻璃圆柱形生物反应器、PGA 支架、硅胶管、生物兼容管、连接器、搅拌棒和设备浸泡在 95% 乙醇罐中组装两个小时。将 PGA 支架通过生物反应器的侧臂，用连接器连接到一侧，以及使用用于提供 OCT 导丝的 y 结到另一侧。同样地在生物反应器中组装另一个 PGA 脚手架。用 4-0 缝合线拧紧聚四氟乙烯，将聚四氟乙烯安装到生物反应器唇上。将生物反应器再次放入乙醇罐中一小时，然后随着鼓风机在发动机罩中干燥过夜。现在，通过标准的外植技术将人类脐动脉平滑肌细胞从人类脐动脉中分离出，在将细胞保存到PGA支架中的原因中，避免生物反应器底部任何细胞悬浮液滴滴。此外，在细胞座后尽快用硅胶塞盖盖住生物反应器，以防止污染。扩大并保持细胞在平滑的肌肉生长介质。将人体脐动脉平滑肌细胞在上述培养基中每毫升500万细胞的浓度播种到PGA脚手架上。在生物反应器中放置一个 sir 棒后，盖住硅胶塞盖，该盖有一个进料管和三个短管段，用于气体交换。将 PTFE 0.22 微米过滤器连接到每个换气管，并在进给管上安装一个肝素盖。以每分钟 13 轮的搅拌速度调整搅拌杆。然后，将玻璃生物反应器、硅胶塞盖和PGA脚手架组装到培养系统中。让细胞坚持45分钟，每15分钟倾斜一次生物反应器，并站在左侧和右侧。现在，将LOO-OH-YEE泵、磷酸盐缓冲盐水袋和驱动器与生物相容管连接，以创建大量系统。打开驱动器以用 PBS 填充管子。将整体生物反应器放在加湿培养箱中，在 37 摄氏度下 CO2 为 5%。用450毫升的培养剂填充培养室。在静态培养下种植种子脚手架一周，每三到四天更换培养基，通过喂食管吸气一半的旧培养基，用同等数量的新鲜培养基重新填充反应器。为了准备用于 OCT 成像的大量系统，请泵送 PBS 袋中的液体，通过生物相容管循环并回袋。打开驾驶员的功率，以每分钟 60 次的频率和 120 毫米汞的输出收缩压调节泵设置。根据组织工程血管培养的需要调整机械参数。单击运行按钮，使大量系统正常工作。在静态培养一周后，通过迭代加压生物相容管，为容器提供三周的固定脉动模拟。使用光源确保轴向分辨率为 10 到 20 微米，图像深度为 1 到 2 毫米，以基于频域 OCT 血管内成像系统识别组织工程血管（TEVBs）的结构。打开电源开关并打开图像采集软件 将光纤成像导管连接到驱动电机和光控制器，并实现导管自动退机功能。将图像采集速率的参数设置为每秒 10 帧，自动回退速度为每秒 10 毫米。现在，用18规格的针头通过肝素盖将成像导管连接到y-结。将导管放入硅胶管中，在将 PGA 支架加载到生物反应器上之前，确定 PGA 网的结构紧固性。现在，将导管尖放在感兴趣的区域。调整回拉设备并检查图像质量。以一天、四天、七天、十天、14天、17天、21天和 28 天的文化时间为每位 TEBV 获取图像。通过实时观察TEBV微观结构（包括表面形态、内部结构和组成）按顺序保存这些图像。重复测量三次，每次对工程容器进行可靠的测量。使用图像捕获软件在整个测试过程中捕获一系列图像。要使用图像分析软件测量组织工程血管壁厚，首先选择要分析的图像。然后，单击跟踪工具，使用软件自动识别组织工程血管的内侧，并手动绘制外侧草图。屏幕上将显示厚度图。重复测量五次，以可靠地测量结构。考虑使用两个独立调查员有义务获取信息。打开培养物完成后放置在生物反应器上的硅胶塞盖，丢弃培养基。松开生物反应器唇中的聚四氟乙烯，用剪刀从聚四氟乙烯的外侧切割硅胶管。从生物反应器中采集TEBV，并把它们切成部分进行扫描电子显微镜检查。拉出支撑硅胶管，用 4% 的功率甲醛固定部分。执行常规组织学染色与质量的三色和西兰红检查胶原蛋白和PGA的形态。要评估PGA含量和胶原蛋白成分，请通过偏振显微镜观察具有红色染色的组织学样本。OCT图像与经过四周培养后对特博夫组织病理学的发现进行比较。与组织学评估相比，OCT 图像显示了紧凑的微结构和特定组件。培养基、硅胶管、TEBV 和 PGA 片段可见。马森的三色染色表明胶原纤维分布在一定方向，以及工程容器介质层的PGA残留物。天狼星红色染色通过偏振显微镜显示PGA残留物和胶原蛋白成分。工程容器的扫描电子显微图展示了紧凑的显微结构。同时，这种宫内成像模式采用无损、易监测的TEBV，包括长期培养中的改造过程和视觉形态原位。如这里所示，通过TEBV厚度的比较，重塑发生得更早，形态变化在动态组中表现得更为明显。这项技术为血管组织工程领域的研究人员探索结构特征和工程容器的长期改造过程铺平了道路。通过极化显微镜与 OCT 成像清晰显示聚合物残留物可能有助于评估脚手架的降解。

Research

JoVE Journal

Bioengineering

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

科研

生物工程

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

在一个容器的支架部署在监测的墙力学

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

Integrated Photoacoustic Ophthalmoscopy and Spectral-domain Optical Coherence Tomography

集成光声眼底镜检查和谱域光学相干断层扫描

Both the clinical diagnosis and fundamental investigation of major ocular diseases greatly benefit from various non-invasive ophthalmic imaging ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

协议相对液力评估的三单张聚合物阀

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

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

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

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

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. ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

斜扫描激光检眼镜 (奥斯陆) 与光学相干层析成像 (OCT) 的多模态视网膜成像

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

心脏内侧燃光导管,用于监测细胞代谢,使用渗透哺乳动物心脏的透射吸收光谱

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

用于大鼠圆周食管重建的组织工程移植

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

使用光学漫反射相关光谱，基于脑血流的休息状态功能连接

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...