組織工程的最終目標(biāo)是制造人體器官。然而，在這之前必須重建和表征三維組織。目前無法制造可操作的脈管系統(tǒng)，這限制了組織工程的發(fā)展?；诖?，美國(guó)海軍研究實(shí)驗(yàn)室Andre A. Adams和北卡羅萊納州立大學(xué)Michael A. Daniele團(tuán)隊(duì)使用微流體技術(shù)制造了獨(dú)立的小直徑血管，其具有可重現(xiàn)正常生物功能的組織細(xì)胞層。隨著培養(yǎng)時(shí)間的推移，內(nèi)皮細(xì)胞在腔壁上形成單層結(jié)構(gòu)并分泌細(xì)胞外基質(zhì)。在整合到含有成纖維細(xì)胞的三維凝膠中后，微血管延伸并形成中空分支，其與相鄰的毛細(xì)血管吻合成網(wǎng)。人工制造的微血管以及延伸出來的血管都支持流體和顆粒的灌注。以上所述微血管可以被設(shè)計(jì)為不同的直徑，并且可完成血管新生和融合，這將成為組織工程血管形成極具價(jià)值的工具。

該研究的目的是開發(fā)一種有效的人體血管構(gòu)建方法，這些制造的血管將通過血管生成，管腺增生和融合的自然過程來完成血管原始網(wǎng)絡(luò)的形成（圖1）。通過紫外（UV）光交聯(lián)聚乙二醇（PEG）和甲基丙烯酸明膠（GelMA）制備人內(nèi)皮微血管（HEMV），其可經(jīng)歷新血管生成以及與正常人血管成熟相關(guān)的其他發(fā)育過程。

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.

人臍靜脈內(nèi)皮細(xì)胞在制造過程中被引入HEMV的腔中; 3天后觀察到細(xì)胞貼附于腔壁。細(xì)胞持續(xù)擴(kuò)增到第10天并出現(xiàn)匯合的單層結(jié)構(gòu)（圖2a）。小動(dòng)脈包含多種細(xì)胞類型，通過在制造過程中將血管平滑肌細(xì)胞和周細(xì)胞引入微血管壁來建模，稱之為多細(xì)胞微血管（MCMV）。 內(nèi)皮細(xì)胞排列成管腔，而平滑肌細(xì)胞在第7-10天增殖以填充腔壁，并持續(xù)生長(zhǎng)至15天（圖2a）及以上。研究人員通過激光掃描共聚焦免疫熒光顯微鏡（LSCM）表征HEMV在不同時(shí)間點(diǎn)生物標(biāo)志物蛋白的表達(dá)和定位。內(nèi)皮生物標(biāo)志物CD31和血管內(nèi)皮鈣粘蛋白（VE-cadherin）在HEMV腔壁細(xì)胞中均有表達(dá)（圖2b）。利用該微血管進(jìn)行構(gòu)建體外組織需要從頭合成人源蛋白質(zhì)。因此，研究人員評(píng)估了包括膠原蛋白IV和層粘連蛋白在內(nèi)的細(xì)胞外基質(zhì)蛋白的時(shí)間依賴性沉積。12天后在HEMV的壁中觀察到人類膠原蛋白IV和人層粘連蛋白的顯著表達(dá)（圖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.

對(duì)完整HEMV的進(jìn)一步檢查發(fā)現(xiàn)駐留的內(nèi)皮細(xì)胞可以通過形成微血管壁促進(jìn)血管新生。因此，研究人員將它們包埋在含有人成纖維細(xì)胞的水凝膠中來模擬組織環(huán)境以測(cè)試HEMV的血管新生潛力。在含有原代成纖維細(xì)胞GelMA和基質(zhì)膠（matrigel）下包埋完整的HEMV時(shí)，可觀察到大量的新生血管（圖3a-c）。接下來，研究人員證實(shí)了新形成的芽是空心小管，平均管腔直徑為3-5μm（圖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).

本研究由美國(guó)海軍研究實(shí)驗(yàn)室Andre A. Adams團(tuán)隊(duì)和北卡羅萊納州立大學(xué)Michael A. Daniele團(tuán)隊(duì)共同完成，并于2017年5月發(fā)表于biomaterials。

論文信息：

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

論文鏈接：

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