微流控技术制备微血管并实现血管新生

组织工程的最终目标是制造人体器官。然而，在这之前必须重建和表征三维组织。目前无法制造可操作的脉管系统，这限制了组织工程的发展。基于此，美国海军研究实验室Andre A. Adams和北卡罗莱纳州立大学Michael A. Daniele团队使用微流体技术制造了独立的小直径血管，其具有可重现正常生物功能的组织细胞层。随着培养时间的推移，内皮细胞在腔壁上形成单层结构并分泌细胞外基质。在整合到含有成纤维细胞的三维凝胶中后，微血管延伸并形成中空分支，其与相邻的毛细血管吻合成网。人工制造的微血管以及延伸出来的血管都支持流体和颗粒的灌注。以上所述微血管可以被设计为不同的直径，并且可完成血管新生和融合，这将成为组织工程血管形成极具价值的工具。

该研究的目的是开发一种有效的人体血管构建方法，这些制造的血管将通过血管生成，管腺增生和融合的自然过程来完成血管原始网络的形成（图1）。通过紫外（UV）光交联聚乙二醇（PEG）和甲基丙烯酸明胶（GelMA）制备人内皮微血管（HEMV），其可经历新血管生成以及与正常人血管成熟相关的其他发育过程。

Fig. 1. Neovascularization strategy and implementation. 1. Human endothelial cells (EC) are encapsulated in a bio-macromolecular tubule using a hydrodynamic shaping device. The resultant human endothelial microvessel (HEMV) matures with a coherent endothelial cell lumen. Temporal events a-e (left) correspond to experimental micrographs (right). (a) Representative composite image of ten adjacent fields (10X) taken along the length of a single HEMV immunostained with anti-CD31 (green) demonstrates the ability to produce long continuous HEMVs. Scale, 500 mm; inset 50 mm (b) A confluent monolayer of endothelial cells form along the luminal face of the microvessel. 2. Once embedded in a three-dimensional matrix, the HEMV develop a primitive microvasculature network through traditional vascularization processes, (c) HEMV angiogenesis, the sprouting of endothelial growths from the original HEMV into an extracellular matrix containing dermal fibroblasts (DAPI-stained nuclei, blue), (d) HEMV tubulogenesis (arrowhead), the hollowing of sprouts to support fluid transport, (e) HEMV anastomosis, adevelopmental process whereby neighboring sprouts form connections (arrowheads) establishing a closed-loop system for circulation. Scale, (b + d) 25 mm; (c + e) 50 mm.

人脐静脉内皮细胞在制造过程中被引入HEMV的腔中; 3天后观察到细胞贴附于腔壁。细胞持续扩增到第10天并出现汇合的单层结构（图2a）。小动脉包含多种细胞类型，通过在制造过程中将血管平滑肌细胞和周细胞引入微血管壁来建模，称之为多细胞微血管（MCMV）。 内皮细胞排列成管腔，而平滑肌细胞在第7-10天增殖以填充腔壁，并持续生长至15天（图2a）及以上。研究人员通过激光扫描共聚焦免疫荧光显微镜（LSCM）表征HEMV在不同时间点生物标志物蛋白的表达和定位。内皮生物标志物CD31和血管内皮钙粘蛋白（VE-cadherin）在HEMV腔壁细胞中均有表达（图2b）。利用该微血管进行构建体外组织需要从头合成人源蛋白质。因此，研究人员评估了包括胶原蛋白IV和层粘连蛋白在内的细胞外基质蛋白的时间依赖性沉积。12天后在HEMV的壁中观察到人类胶原蛋白IV和人层粘连蛋白的显著表达（图2c-d）。

Fig. 2. Characterization of cell-laden HEMV. (a) Top, depicts a 20-day time course showing endothelial cell attachment to the inner luminal face of the HEMV, forming vessel mimics similar in size and cellularity to capillaries and venules. Lower, multi-cell microvessel composed of endothelial cells (lumen) and smooth muscle cells/pericytes (outer-walls) creates an arteriole-like mimic. Scale, 50 mm. Arrowheads depicts smooth muscle cell/pericyte placement and outgrowth in walls. (b) Laser scanning confocal microscopy identifies cell surface protein expression in day 12 HEMV. Endothelial cells express both CD31 (green) and VE-cadherin (red), confirming the cell type and the presence of critical adherens junctions necessary for proper endothelial function. CD31 (green); VE-cadherin (red); DAPI (blue nuclear stain) overlay is also shown. Both orthogonal and 3D views confirm the hollow, tubule morphology of the created HEMV. Scale, 50 mm (c and d) Anti-collagen IV and anti-laminin show accumulation of newly secreted human matrix protein. Top panels show representative HEMV at time zero (T0); bottom panel shows microvessels analyzed at day 12. Day 12 HEVM were observed with a 6.5-fold increase in collagen IV (p < 0.001); similarly laminin exhibited a 13-fold increase in expression (p < 0.001) when compared to T0 microvessels. Scale, 100 mm.

对完整HEMV的进一步检查发现驻留的内皮细胞可以通过形成微血管壁促进血管新生。因此，研究人员将它们包埋在含有人成纤维细胞的水凝胶中来模拟组织环境以测试HEMV的血管新生潜力。在含有原代成纤维细胞GelMA和基质胶（matrigel）下包埋完整的HEMV时，可观察到大量的新生血管（图3a-c）。接下来，研究人员证实了新形成的芽是空心小管，平均管腔直径为3-5μm（图3d-e）。

Fig. 3. HEMV undergoing neovascularization. (aec) Day 17 panoramic images (10X) of representative embedded HEMV showing angiogenic sprouting throughout fibroblasts matrices (DAPI). Both GFR-Matrigel? and 4%-GelMA support angiogenesis. Lower right micrograph, 20X confocal orthogonal views showing HEMV remain hollow (arrowhead) during angiogenic growth. Scale, 500 mm; inset 50 mm. (b) No statistical difference (p > 0.59) between the matrices was observed when comparing the number of na?ve sprouts present/10X field. (c) Sprout lengths from four individual GFR-Matrigel? embedded HEMV were quantified. Lengths varied from 50 to 1290 mm, with a median of 547 mm after 21 days. n ? 38 (d) Depicts a panoramic image focusing on a single sprout (center) stained with anti-CD31 (green) and DAPI (blue). Four separate positions along the microvessel (60X) are also shown. Adjacent orthogonal views show the hollow nature of the sprouts. Inset iii, denoted with an asterisk, shows the displacement of a sprout nucleus to the outer edge of the sprout. Close inspection of anti-CD31 reveals extensive filopodial projections, indicating continual expansion through 21 days. Scale, 200 mm; inset 25 mm. (e) Upper (10X) and lower panels (20X) show representative HEMV undergoing neovascularization. Arrowheads depict multiple anastomoses, whereby a single sprout connects with a neighboring growth. Overlaid images anti-CD31 (green) and DAPI (blue) are shown right. Scale, 50 mm (10X); 25 mm (20X).

本研究由美国海军研究实验室Andre A. Adams团队和北卡罗莱纳州立大学Michael A. Daniele团队共同完成，并于2017年5月发表于biomaterials。

论文信息：

Kyle A. DiVito, Michael A. Daniele*, Steven A. Roberts, Frances S. Ligler, Andre A. Adams*. Microfabricated blood vessels undergo neoangiogenesis. Biomaterials, 2017, 138:142.

论文链接：

https://www.sciencedirect.com/science/article/pii/S014296121730323X