Distributed vasculogenesis from modular agarose-hydroxyapatite-fibrinogen microbeads.
Distributed vasculogenesis from modular agarose-hydroxyapatite-fibrinogen microbeads.
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DOI：
10.1016/j.actbio.2017.03.050
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发表时间：
2017-06
影响因子：
9.7
通讯作者：
Stegemann JP
中科院分区：
文献类型：
--
作者：
Rioja AY;Daley ELH;Habif JC;Putnam AJ;Stegemann JP
严重肢体缺血会损害四肢的血液循环，引起疼痛、伤口愈合中断和潜在的组织坏死。治疗性血管生成旨在直接修复受损的微血管以恢复血液流动。在这项研究中，我们开发了模块化的微型结构，旨在具有强大的处理质量，允许体外预培养，并促进微血管形成。微珠基质由琼脂糖（AG）基底组成，以防止聚集，结合纤维蛋白原（FGN）和/或羟基磷灰石（HA）的细胞粘附成分。制备并表征了人脐静脉内皮细胞（HUVEC）和成纤维细胞共培养微珠。微珠的直径一般在80 ~ 100微米之间，随着FGN和HA的加入，微珠的尺寸逐渐增大。HA的加入提高了微珠的产率，也提高了FGN在基质内分布的均匀性。各微珠类型细胞活力均较高。当细胞种子微球包埋在纤维蛋白水凝胶中时，在7天内观察到HUVEC的发芽和相邻微球之间的融合。与AG、AG+HA或AG+FGN微珠相比，在纤维蛋白中嵌入微珠前再进行7天的预培养，AG+HA+FGN微珠中的HUVEC网络长度显着增加。重要的是，相对于由纯纤维蛋白组成的控制微珠，复合微珠导致更均匀和广泛的内皮网络形成。这些结果表明AG+HA+FGN微球支持HUVEC在相邻微球内部和之间发芽，并能促进外部基质的分布血管化。这种模块化微组织可以通过快速重建微血管网络来治疗缺血组织。
Critical limb ischemia impairs circulation to the extremities, causing pain, disrupted wound healing, and potential tissue necrosis. Therapeutic angiogenesis seeks to repair the damaged microvasculature directly to restore blood flow. In this study, we developed modular, micro-scale constructs designed to possess robust handling qualities, allow in vitro pre-culture, and promote microvasculature formation. The microbead matrix consisted of an agarose (AG) base to prevent aggregation, combined with cell-adhesive components of fibrinogen (FGN) and/or hydroxyapatite (HA). Microbeads encapsulating a co-culture of human umbilical vein endothelial cells (HUVEC) and fibroblasts were prepared and characterized. Microbeads were generally 80–100 microns in diameter, and the size increased with the addition of FGN and HA. Addition of HA increased the yield of microbeads, as well as the homogeneity of distribution of FGN within the matrix. Cell viability was high in all microbead types. When cell-seeded microbeads were embedded in fibrin hydrogels, HUVEC sprouting and inosculation between neighboring microbeads were observed over seven days. Pre-culture of microbeads for an additional seven days prior to embedding in fibrin resulted in significantly greater HUVEC network length in AG+HA+FGN microbeads, as compared to AG, AG+HA or AG+FGN microbeads. Importantly, composite microbeads resulted in more even and widespread endothelial network formation, relative to control microbeads consisting of pure fibrin. These results demonstrate that AG+HA+FGN microbeads support HUVEC sprouting both within and between adjacent microbeads, and can promote distributed vascularization of an external matrix. Such modular microtissues may have utility in treating ischemic tissue by rapidly re-establishing a microvascular network.