Modulating Cell Phenotype during Tubulogenesis through 3D Micropatterning

描述（由申请人提供）：血管生成是微血管形成的关键过程，将营养和氧气输送到目标细胞和组织。在这个过程中，内皮细胞对特定的细胞外信号作出反应，使它们从现有的血管迁移，形成小管，这一过程被称为管生成。然而，这些信号机制的改变或破坏可能导致不健康血管结构的形成，表明疾病状态（即癌症）。在这个提议中，我们的目标是使用微图案生物材料来控制内皮细胞微环境的时空元素，主要由粘合剂、机械和扩散/可溶性线索组成。通过观察和表征内皮细胞如何操纵和协调其局部微环境的反应，我们可以根据其与个体信号的特定相互作用对其相应的细胞表型和小管网络进行分类。为了实现这一目标，我们将首先创建一个基于双光子的模式策略，能够通过使用正交光化学在三维（3D）聚乙二醇（PEG）水凝胶中平行固定多个生物分子。此外，我们将加入新的功能，以允许瞬时修改散装水凝胶的性质。使用这项技术，我们将在水凝胶中创建具有大块和局部（图案）生长因子的粘附配体图案。通过首先控制生长因子的空间引入，我们将研究如何在3D中控制微管发生事件（即分支）的启动。

DESCRIPTION (provided by applicant): Angiogenesis is a critical process in the formation of microvasculature to deliver nutrients and oxygen to target cells and tissues. During this process, endothelial cells respond to specific extracellular signals that cause them to migrate from existing vessels and form tubules through a process called tubulogenesis. Alterations or disruptions in these signaling mechanisms, though, can lead to the formation of unhealthy vessel structures, indicative of disease states (i.e. cancers). In this proposal, we aim to use micropatterned biomaterials to control the spatiotemporal elements of endothelial cell microenvironments, composed primarily of adhesive, mechanical, and diffusible/soluble cues. By observing and characterizing how endothelial cells manipulate and coordinate responses from their local microenvironment, we can classify their corresponding cellular phenotypes and tubule networks based upon their specific interactions with individual cues. To accomplish this, we will first create a two-photon-based patterning strategy capable of immobilizing multiple biomolecules in parallel within three dimensional (3D) poly(ethylene glycol) (PEG) hydrogels through the use of orthogonal photochemistries. In addition, we will incorporate new functionalities to allow the bulk hydrogel properties to be transiently modified. Using this technology we will create patterns of adhesive ligands within the hydrogel with bulk and localized (patterned) growth factors. By first controlling the spatial introduction of growth factos, we will investigate how we can control the initiation of tubulogenic events (i.e. branching) in 3D. Furthermore, by employing the growth factors involved in wound healing: platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF), we will investigate how the order in which each growth factor is encountered as well as the display (i.e., bulk or locally immobilized) of the individual growth factors effects the relatie structure of the tubule network. Finally, we will explore the temporal introduction of these growth factors and how their incorporation during tubulogenesis can alter, disrupt, or reinforce endothelial cell responses. From these studies, we anticipate that we can control the branching, elongation, and overall structure of the tubules that are formed. To verify this we will create a comprehensive method to characterize and classify tubule networks as well as the phenotype of the endothelial cells, themselves. Finally, the variations in the spatial and temporal introduction should enable us to decouple these effects to create a tubulogenic model which will allow us to "pre-program" 3D cellular microenvironments to drive specific tubulogenic and phenotypic outcomes.