NANO-PATTERNING OF BIOMATERIALS FOR BLOOD-VESSEL FORMATION IN ARTIFICIAL TISSUES

描述(申请人提供)：重要器官衰竭的患者可以接受全器官移植；但由于捐赠器官的供应有限，单靠移植不能满足公众的医疗需求。人工组织提供了捐赠器官的替代方案，但我们无法在这种结构中设计具有功能的微血管，这广泛地阻碍了临床有效人工组织的发展。因为所有人 组织规模的再生治疗需要灌流，形成功能性微血管的能力是最重要的。在植入没有微血管的大量人工组织后，内部的细胞会因缺氧而死亡，留下一层约0.2毫米厚的活细胞壳。人工血管化术将通过创建容量跨度的灌流网络和实现植入后快速的血管整合来防止这种情况发生。为了设计微血管系统，我们提出了一种新的生物材料策略：细胞-基质黏附配体的纳米级聚集。先前在2D表面的工作表明，配体的聚集通过受体聚集增加了生长因子的敏感性和运动性。使用动物模型的研究表明，受体聚集的分子干扰物的表达与分枝和成熟的减少有关。虽然配体聚集和受体聚集在热力学上是相关的，但纳米尺度的配体聚集是否会在3D、块状生物材料中导致形态上合适的微血管形成尚不清楚。为了回答这个问题，我们开发了一种纳米纤维生物材料，可以在特定的粘连配体的体积浓度和纳米级聚集的情况下制备。通过模仿天然细胞外基质的纳米级秩序，我们希望在体外实现器官型血管结构的形成。我们特别假设，黏附配体的聚集将上调导致血管形成的三个基本细胞过程 活体：(1)生长因子敏感性，(2)细胞运动，(3)血管分支和成熟。生长因子敏感性将通过测量增殖、代谢活性和蛋白酶分泌来评估。运动性，如细胞速度和持续长度的参数，以及细胞骨架组织将通过定量图像分析进行评估。分支和成熟度将通过适当标记的免疫染色和形态数据的计算分析来评估。通过结合非重叠技术，如组织特异性干细胞的共培养、生长因子输送和生物反应器/机械刺激，这里提出的生物材料可以进一步发展为再生医学的植入物。我的导师莎拉·海尔肖恩是基于蛋白质的材料工程专家，我们的合作者约翰·库克是微血管信号生物学的高级教授，他们制定了一个适当的培训计划来完成这个项目。

DESCRIPTION (provided by applicant): Patients suffering from failure of a vital organ can be treated with whole organ transplantation; transplantation alone, however, cannot meet the public's medical needs due to the limited supply of donated organs. Artificial tissues present an alternative to donated organs, but our inability to engineer functional microvessels within such constructs broadly prevents the development of clinically effective artificial tissues. Because all tissue- scale regenerative therapies require perfusion, the ability to form functional microvasculature is paramount. Upon implantation of bulk artificial tissues without microvasculature, cells on the inside die from lack of oxygen, leaving a shell of live cells about 0.2 mm thick. Artificial vascularization will prevent this by creating a volume- spanning perfusion-competent network and by enabling swift vascular integration after implantation. To engineer microvasculature, we propose a novel biomaterials strategy: nanoscale clustering of cell- matrix adhesion ligands. Previous work on 2D surfaces has shown that clustering of ligands increases growth factor sensitivity and motility via receptor clustering. Research using animal models has shown that expression of molecular disruptors of receptor clustering is associated with a decrease in both branching and maturation. Though ligand clustering and receptor clustering are related thermodynamically, it is unknown whether nanoscale ligand clustering will lead to morphologically appropriate microvasculature in a 3D, bulk biomaterial. To answer this question, we have developed a nanofibrous biomaterial that can be fabricated at a specified bulk concentration and nanoscale clustering of adhesion ligands. By mimicking the nanoscale order of the native the extracellular matrix, we expect to achieve organotypic blood-vessel structure formation in vitro. We specifically hypothesize that clustering of adhesion ligands will upregulate three essential cellular process that lead to formation of blood-vessels in vivo: (1) growth factor sensitivity, (2) cell motility, and (3) vessel branching and maturation. Growth factor sensitivity will be assessed by measuring proliferation, metabolic activity, and protease secretion. Motility, as parameterized by cell speed and persistence length, and cytoskeletal organization will be assessed by quantitative image analysis. Branching and maturation will be assessed by immunostaining for appropriate markers and computational analysis of morphological data. The biomaterials proposed here can be further developed as an implant for regenerative medicine by incorporating non- overlapping technologies such as co-culture of tissue-specific stems cells, growth factor delivery, and bioreactor/ mechanical stimulation. My mentor Sarah Heilshorn, an expert in protein-based materials engineering, and our collaborator John Cooke, a senior professor of microvascular signaling biology, have developed an appropriate training plan to accomplish this project.