Le Andrew V, Hatachi Go, Beloiartsev Arkadi, Ghaedi Mahboobe, Engler Alexander J, Baevova Pavlina, Niklason Laura E, Calle Elizabeth A
Department of Anesthesiology, Yale University, New Haven, Connecticut 06519, United States.
Division of Surgical Oncology, Department of Surgery, Nagasaki University Graduate School of Biomedical Sciences, Sakamoto 1-7-1, Nagasaki 852-8501, Japan.
ACS Biomater Sci Eng. 2017 Sep 11;3(9):2000-2010. doi: 10.1021/acsbiomaterials.6b00784. Epub 2017 Jun 30.
To date, efforts to generate engineered lung tissue capable of long-term function have been limited by incomplete barrier formation between air and blood and by thrombosis of the microvasculature upon exposure of blood to the collagens within the decellularized scaffold. Improved barrier function and resistance to thrombosis both depend upon the recapitulation of a confluent monolayer of functional endothelium throughout the pulmonary vasculature. This manuscript describes novel strategies to increase cell coverage of the vascular surface area, compared to previous reports in our lab and others, and reports robust production of multiple anticoagulant substances that will be key to long-term function in vivo once additional strides are made in improving barrier function. Rat lung microvascular endothelial cells were seeded into decellularized rat lungs by both the pulmonary artery and veins with the use of low-concentration cell suspensions, pulsatile, gravity-driven flow, and supraphysiological vascular pressures. Together, these strategies yielded 72.44 ± 10.52% endothelial cell nuclear coverage of the acellular matrix after 3-4 d of biomimetic bioreactor culture compared to that of the native rat lung. Immunofluorescence, Western blot, and PCR analysis of these lungs indicated robust expression of phenotypic markers such as CD31 and VE-Cadherin after time in culture. Endothelial-seeded lungs had CD31 gene expression of 0.074 ± 0.015 vs 0.021 ± 0.0023 for native lungs, = 0.025, and VE-Cadherin gene expression of 0.93 ± 0.22 compared to that of the native lung at 0.13 ± 0.02, = 0.023. Precursors to antithrombotic substances such as tissue plasminogen activator, prostacyclin synthase, and endothelial nitric oxide synthase were expressed at levels equal to or greater than those of the native lung. Engineered lungs reseeded with endothelial cells were implanted orthotopically and contained patent microvascular networks that had gas exchange function during mechanical ventilation on 100% O greater than that of decelluarized lungs. Taken together, these data suggest that these engineered constructs could be compatible with long-term function in vivo when utilized in future studies in tandem with improved barrier function.
迄今为止，生成具有长期功能的工程化肺组织的努力受到空气与血液之间屏障形成不完全以及脱细胞支架内血液暴露于胶原蛋白后微血管血栓形成的限制。改善屏障功能和抗血栓形成能力均取决于在整个肺血管系统中重现功能正常的内皮细胞汇合单层。与我们实验室和其他实验室之前的报道相比，本手稿描述了增加血管表面积细胞覆盖率的新策略，并报告了多种抗凝血物质的大量产生，一旦在改善屏障功能方面取得进一步进展，这些物质将是体内长期功能的关键。使用低浓度细胞悬液、搏动性重力驱动流和超生理血管压力，通过肺动脉和静脉将大鼠肺微血管内皮细胞接种到脱细胞大鼠肺中。综合这些策略，在仿生生物反应器培养3 - 4天后，与天然大鼠肺相比，这些策略使无细胞基质的内皮细胞核覆盖率达到72.44±10.52%。对这些肺进行免疫荧光、蛋白质印迹和PCR分析表明，培养一段时间后，CD31和VE - 钙黏蛋白等表型标志物表达强烈。内皮细胞接种的肺中CD31基因表达为0.074±0.015，而天然肺为0.021±0.0023，P = 0.025；VE - 钙黏蛋白基因表达为0.93±0.22，而天然肺为（此处原文有误，推测为0.13±0.02），P = 0.023。抗血栓物质的前体如组织纤溶酶原激活物、前列环素合酶和内皮型一氧化氮合酶的表达水平等于或高于天然肺。重新接种内皮细胞的工程化肺原位植入后，包含有功能的微血管网络，在100%氧气机械通气期间具有气体交换功能，优于脱细胞肺。综上所述，这些数据表明，当与改善的屏障功能一起用于未来研究时，这些工程构建体可能与体内长期功能兼容。
