Tunable Oxygen Delivery to Cells using Novel Microtank Technology

产品说明： 限制大的组织工程化移植物的临床应用的主要挑战之一是不能提供足够的氧气供应以促进血管向内生长和组织整合期间的细胞的植入后存活。作为 因此，细胞仅依赖于来自周围组织的有限氧气扩散。工程化移植物的内部区域迅速变得缺氧，导致大量细胞死亡、坏死核心的形成和移植物的失败。为了克服这些局限性，必须开发新的方法来提供持续的、局部的氧气输送，以在移植物植入和充分侵入宿主脉管系统带来含氧血液之间的关键时期维持细胞的活力。我们提出了一种利用中空聚合物微球的创新方法，该微球具有适当的材料特性，可促进高压下的氧负载并沿着压力梯度受控释放到紧邻的微环境中。通过改变微球的大小、聚合物壳的固有透氧性和厚度以及这些微罐填充的压力，我们建议调整氧气输送到嵌入水凝胶或生物材料支架中的细胞的量、速率和持续时间。这种多功能的创新技术在其 在组织工程和再生医学中的治疗应用中的氧气输送的精确时空控制的潜力。在这项研究中，我们将获得由聚偏二氯乙烯（PVDC）和聚丙烯腈（PAN）制成的市售微型罐，并将其分为两种不同的尺寸（38 - 45 �m和75 - 90 �m）和壳体厚度（2 � 0.5 �m和8 � 2 �m）。我们假设，我们可以调整氧气输送，以保持细胞的活力和生物学特性，使用这些组的微型坦克。该应用程序的目的是通过在两个特定目标中测试该假设来生成概念验证数据。在sp.目的1中，我们将描述这八组微型罐的特征，以确定破裂强度和随时间的氧气释放曲线。在Sp. Aim 2中，我们将评估使用这些微罐为体外缺氧（0%O2）或缺氧（2%O2）环境中培养的脂肪干细胞（ASC）提供持续氧气输送的潜力。随后，我们将测试向软骨细胞、ASCs和脂肪细胞提供适当氧气输送的潜力：三种细胞表型，它们被专门选择为在比氧摄取率（OUR）方面具有数量级差异。我们将评估细胞活力，增殖，基因表达（细胞存活和凋亡基因）和活性氧（ROS）的产生，这表明氧毒性。这些概念验证研究将为未来开发可生物降解的微型坦克和将组织工程移植物移植到小动物体内缺损部位奠定基础。

DESCRIPTION: One of the major challenges limiting the clinical use of large, tissue engineered grafts has been the inability to provide adequate oxygen supply to facilitate the post-implantation survival of cells during the period of vascular in-growth and tissue integration. As a consequence, cells rely solely on limited oxygen diffusion from surrounding tissue. The inner regions of engineered graft rapidly become hypoxic leading to massive cell death, the formation of necrotic cores, and failure of the graft. To overcome these limitations, it is imperative to develop novel methods to provide sustained, localized oxygen delivery to maintain the viability of cells during the critical period between the implantation of the graft and adequate invasion of host vasculature bringing oxygenated blood. We propose an innovative approach utilizing hollow polymeric microspheres, which have the appropriate material properties to facilitate oxygen loading at elevated pressures and controlled release along the pressure gradient into the immediate microenvironment. By varying the size of the microspheres, the intrinsic oxygen permeability and thickness of the polymer shell, and pressure to which these micro tanks are filled, we propose to tune the amount, rate, and duration of oxygen delivery to cells embedded in a hydrogel or biomaterial scaffold. This versatile, innovative technique is unprecedented in its potential for exquisite spatiotemporal control of oxygen delivery for therapeutic application in tissue engineering and regenerative medicine. In this study, we will obtain commercially available micro tanks made of polyvinylidene chloride (PVDC) and poly acrylonitrile (PAN) and sort into two different sizes (38 - 45 �m & 75 - 90 �m) and shell thicknesses (2 � 0.5 �m & 8 � 2 �m). We hypothesize that we can tune the oxygen delivery to maintain cellular viability and biological properties using these groups of micro tanks. The objective of the application is to generate proof- of-concept data by testing this hypothesis in two Specific Aims. In Sp. Aim 1, we will characterize these eight groups of micro tanks to determine rupture strength and the oxygen release profiles over time. In Sp. Aim 2, we will assess the potential for using these micro tanks to provide sustained oxygen delivery to adipose derived stem cells (ASCs) cultured in anoxic (0% O2) or hypoxic (2% O2) environments for up to two weeks in vitro. Subsequently, we will test the potential to provide appropriate oxygen delivery to chondrocytes, ASCs, and adipocytes: three cell phenotypes, which were specifically selected to have orders of magnitude differences in specific oxygen uptake rates (OURs). We will assess cell viability, proliferation, gene expression (for cell survival and apoptotic genes) and the generation of reactive oxygen species (ROS), which indicate oxygen toxicity. These proof-of-concept studies will formulate the foundation for future development of biodegradable micro tanks and transplantation of tissue engineered grafts to in vivo defect sites in small animals.