Gurjarpadhye Abhijit Achyut, DeWitt Matthew R, Xu Yong, Wang Ge, Rylander Marissa Nichole, Rylander Christopher G
1 School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University , Blacksburg, Virginia.
2 Department of Electrical and Computer Engineering, Virginia Polytechnic Institute and State University , Blacksburg, Virginia.
Tissue Eng Part C Methods. 2015 Jul;21(7):758-66. doi: 10.1089/ten.TEC.2014.0345. Epub 2015 Jan 30.
Lumen endothelialization of bioengineered vascular scaffolds is essential to maintain small-diameter graft patency and prevent thrombosis postimplantation. Unfortunately, nondestructive imaging methods to visualize this dynamic process are lacking, thus slowing development and clinical translation of these potential tissue-engineering approaches. To meet this need, a fluorescence imaging system utilizing a commercial optical coherence tomography (OCT) catheter was designed to visualize graft endothelialization.
C7 DragonFly™ intravascular OCT catheter was used as a channel for delivery and collection of excitation and emission spectra. Poly-dl-lactide (PDLLA) electrospun scaffolds were seeded with endothelial cells (ECs). Seeded cells were exposed to Calcein AM before imaging, causing the living cells to emit green fluorescence in response to blue laser. By positioning the catheter tip precisely over a specimen using high-fidelity electromechanical components, small regions of the specimen were excited selectively. The resulting fluorescence intensities were mapped on a two-dimensional digital grid to generate spatial distribution of fluorophores at single-cell-level resolution. Fluorescence imaging of endothelialization on glass and PDLLA scaffolds was performed using the OCT catheter-based imaging system as well as with a commercial fluorescence microscope. Cell coverage area was calculated for both image sets for quantitative comparison of imaging techniques. Tubular PDLLA scaffolds were maintained in a bioreactor on seeding with ECs, and endothelialization was monitored over 5 days using the OCT catheter-based imaging system.
No significant difference was observed in images obtained using our imaging system to those acquired with the fluorescence microscope. Cell area coverage calculated using the images yielded similar values. Nondestructive imaging of endothelialization on tubular scaffolds showed cell proliferation with cell coverage area increasing from 15 ± 4% to 89 ± 6% over 5 days.
In this study, we showed the capability of an OCT catheter-based imaging system to obtain single-cell resolution and to quantify endothelialization in tubular electrospun scaffolds. We also compared the resulting images with traditional microscopy, showing high fidelity in image capability. This imaging system, used in conjunction with OCT, could potentially be a powerful tool for in vitro optimization of scaffold cellularization, ensuring long-term graft patency postimplantation.
生物工程血管支架的管腔内内皮化对于维持小口径移植物的通畅并防止植入后血栓形成至关重要。遗憾的是，目前缺乏可视化这一动态过程的非破坏性成像方法，从而延缓了这些潜在组织工程方法的研发和临床转化。为满足这一需求，设计了一种利用商用光学相干断层扫描（OCT）导管的荧光成像系统来可视化移植物的内皮化。
采用C7 DragonFly™血管内OCT导管作为激发光谱和发射光谱的输送与采集通道。将聚-dl-乳酸（PDLLA）电纺支架接种内皮细胞（ECs）。在成像前，接种的细胞用钙黄绿素乙酰甲酯处理，使活细胞在蓝色激光激发下发出绿色荧光。通过使用高保真机电组件将导管尖端精确地定位在标本上，对标本的小区域进行选择性激发。将所得荧光强度映射到二维数字网格上，以生成单细胞水平分辨率的荧光团空间分布。使用基于OCT导管的成像系统以及商用荧光显微镜对玻璃和PDLLA支架上的内皮化进行荧光成像。计算两组图像的细胞覆盖面积，以对成像技术进行定量比较。将管状PDLLA支架接种ECs后置于生物反应器中，并使用基于OCT导管的成像系统监测5天内的内皮化情况。
使用我们的成像系统获得的图像与荧光显微镜获得的图像相比，未观察到显著差异。利用这些图像计算出的细胞面积覆盖率得出相似的值。管状支架内皮化的非破坏性成像显示细胞增殖，细胞覆盖面积在5天内从15±4%增加到89±6%。
在本研究中，我们展示了基于OCT导管的成像系统获得单细胞分辨率并量化管状电纺支架内皮化的能力。我们还将所得图像与传统显微镜进行了比较，显示出图像能力的高保真度。该成像系统与OCT结合使用，可能成为体外优化支架细胞化的有力工具，确保植入后移植物的长期通畅。
