Cell Response to 3D Engineered Gradients of FGF-2

描述（由申请人提供）： 内源性可溶性生长因子的空间浓度梯度在发育、稳态和伤口愈合过程中起着指导细胞迁移、增殖和分化的关键作用。该授权申请解决了以下问题：在工程仿生细胞外基质（bECM）内以指定方向梯度固定的外源性固相生长因子是否是招募和引导周围宿主细胞进入bECM并促进所需生物学结果的有效方法？我们的目标是确定这种生长因子的传递模式是否与组织工程应用中未来的治疗发展相关。体外模型范例将包括人微血管内皮细胞、MG-63人前成骨细胞和人成人间充质干细胞对纤维蛋白bECM中固相人成纤维细胞生长因子-2（FGF-2）的反应。体内模型将是鸡胚绒毛尿囊膜（CAM）试验，以评估置于CAM上的纤维蛋白bECM/FGF-2结构内的定向血管生成反应。该研究将从纤维蛋白膜上FGF-2的二维模式系统地进展到纤维蛋白bECM内FGF-2的三维（3D）模式，并从体外分离的细胞反应到体内血管生成反应。总体假设是纤维蛋白bECM中固相FGF-2的空间梯度将在体外引导细胞迁移，与梯度方向性配准，并且细胞迁移通量响应于梯度模式将大于均匀分布的模式。在体内，与均匀分布相比，梯度设计的定向血管生成反应更大。bECM/FGF-2结构将采用固体自由成型制造打印工艺制造，该工艺在3D纤维蛋白bECM内产生生长因子的特定3D空间模式。基于荧光的量子点成像技术和荧光光学切片显微镜将用于验证打印结构并评估细胞和CAM响应。该研究将由多学科生物工程与内分泌学，先进制造和生物物理成像的专业知识进行。

DESCRIPTION (provided by applicant): Spatial concentration gradients of endogenous soluble growth factors play critical roles directing cell migration, proliferation, and differentiation during development, homeostasis, and wound healing. This grant application addresses the question: Could an exogenous solid-phase growth factor immobilized in specified directional gradients within an engineered biomimetic extracellular matrix (bECM) be an effective approach to recruit and guide surrounding host cells into a bECM and promote a desired biological outcome? The goal is to determine if this growth factor delivery paradigm would be relevant for future therapy development in tissue engineering applications. The paradigm in vitro models will include human microvascular endothelial cell, MG-63 human preosteoblastic cell, and human adult mesenchymal stem cell responses to solid-phase human fibroblast growth factor-2 (FGF-2) in fibrin bECMs. The in vivo model will be a chicken embryo chorioallantoic membrane (CAM) assay to assess directed angiogenic response within fibrin bECM/FGF-2 structures placed on the CAM. The research will systematically progress from two-dimensional patterns of FGF-2 on fibrin films to three-dimensional (3D) patterns of FGF-2 within fibrin bECMs, and from isolated cell responses in vitro to angiogenic responses in vivo. The overall hypotheses are spatial gradients of solid phase FGF-2 in a fibrin bECM will direct cell migration in vitro in register with gradient directionality, and cell migrational flux will be greater in response to gradient patterns than patterns with uniform distributions. And in vivo, a directed angiogenic response will be greater in designs with gradients in comparison with uniform distributions. The bECM/FGF-2 structures will be fabricated with a solid freeform fabrication printing process that produces specified 3D spatial patterns of growth factors within 3D fibrin bECMs. Fluorescent based quantum dot imaging technology and fluorescence optical sectioning microscopy will be used to validate printed structures and assess cell and CAM responses. The research will be carried out by multidisciplinary bioengineering with expertise in endocrinology, advanced manufacturing, and biophysical imaging.