Investigating the Role of Hypoxic Signaling on Adipose-Derived Stem Cell Osteogenesis

项目概要/摘要：颅面骨移植用于治疗美国超过20万例患者。 国家每年。自体移植，目前的治疗标准，有多个缺点，包括供体部位 发病率和可用组织的缺乏。一种有前景的替代方案是脂肪源性干细胞/基质的组合， 细胞（ASCs）与骨诱导生物材料支架，可以3D打印，以模仿天然几何形状， 缺陷。ASC可以很容易地从非侵入性程序中以高产量获得，并且已经显示出 在体外强烈矿化，导致它们作为干细胞来源的效用。尽管有这些有希望 由于ASCs的特性，ASCs在体内再生骨的能力有限。一个主要的假设是 植入后的缺氧环境可能导致大量细胞死亡;然而，在初步研究中， 在体外实验中，我观察到ASC在严重缺氧培养中有极好的存活率（>70%），但在缺氧培养过程中， 体外成骨分化，Runx 2表达和碱性磷酸酶（ALP）活性，两种常见的 骨生成的标志物被缺氧抑制。此外，通过使用一种新的策略， 将氧气输送到种植在3D支架中的细胞，我已经证明，原位提供氧气， 在鼠异位骨形成模型中，移植的ASC使体内形成的骨的量加倍。 因此，该提议的主要前提是ASC体内骨形成的减少是由于 缺氧直接抑制ASC成骨。 这项研究的目的是探讨缺氧和ASC之间的相互作用 骨生成，克服了该领域的几个主要限制。首先，缺氧条件下的ASC成骨将是 使用骨形成的3D体外模型进行定量研究 氧对ASC分化的影响产生了相互矛盾的结果，因为他们依赖于 成骨在具体目标1中，我将使用骨形成的3D体外模型来量化 矿化，组织微结构，代谢和基因表达由于缺氧。广泛的基因 表达数据可能使我们能够鉴定氧依赖性ASC成骨的新介质。接着在 具体目标2，我将研究缺氧信号和ASC成骨之间的相互作用，使用增益的， 使用新型非病毒聚合物纳米颗粒和释氧剂的功能和功能丧失研究 脚手架首先，我将确定缺氧对ASC成骨的影响是否可以通过 上调缺氧诱导因子-1 α（HIF-1α）下调HIF-1α。第三，我将确定 氧释放对ASC成骨的影响。最后，在具体目标3中，我将研究HIF-1α的作用， 功能和功能丧失以及氧输送对颅骨缺损模型中ASC成骨的影响。结果 这些研究将加深对缺氧和成骨之间的理解，并为以下方面的努力提供信息： 促进体内ASC成骨。

Project Summary/Abstract: Craniofacial bone grafts are used to treat over 200,000 patients in the United States annually. Autograft, the current standard of treatment, has multiple drawbacks including donor-site morbidity and lack of available tissue. A promising alternative is the combination adipose-derived stem/stromal cells (ASCs) with osteoinductive biomaterial scaffolds that can be 3D-printed to mimic the native geometry of the defect. ASCs can be readily obtained in high yields from non-invasive procedures and have been shown to mineralize robustly in vitro, leading to their utility as a stem cell source. Despite these promising characteristics, ASCs have demonstrated limited ability to regenerate bone in vivo. A predominant hypothesis is that the hypoxic environment following implantation may lead to massive cell death; however, in preliminary in vitro experiments, I have observed excellent (>70%) ASC survival in severely hypoxic culture, but during in vitro osteogenic differentiation, Runx2 expression and alkaline phosphatase (ALP) activity, two common markers for osteogenesis, are inhibited by hypoxia. Additionally, through the use of a novel strategy for delivering oxygen to cells seeded in 3D scaffolds, I have demonstrated that providing oxygen in situ to transplanted ASCs doubled the amount of bone formed in vivo in a murine ectopic bone formation model. Thus, the major premise of this proposal is that the reduced in vivo bone formation by ASCs is due to the direct inhibition of ASC osteogenesis by hypoxia. The objective of the proposed study is to investigate the interplay between hypoxia and ASC osteogenesis, overcoming several major limitations in the field. First, ASC osteogenesis in hypoxia will be studied quantitatively using a 3D in vitro model of bone formation Previous literature examining the impact of oxygen on ASC differentiation produced conflicting results, because they relied on qualitative metrics of osteogenesis. In Specific Aim 1, I will use a 3D in vitro model of bone formation to quantify changes in mineralization, tissue microarchitecture, metabolism, and gene expression due to hypoxia. Extensive gene expression data may allow us to identify novel mediators of oxygen-dependent ASC osteogenesis. Next, in Specific Aim 2, I will investigate the interplay between hypoxic signaling and ASC osteogenesis using gain-of- function and loss-of-function studies using novel non-viral polymeric nanoparticles and oxygen-releasing scaffolds. First, I will determine whether the effect of hypoxia on ASC osteogenesis can be simulated by upregulating hypoxia-inducible factor-1α (HIF-1α) downregulating HIF-1α. Third, I will determine the effect of oxygen release on ASC osteogenesis. Finally, in Specific Aim 3, I will study the effects of HIF-1α gain-of- function and loss-of-function and oxygen delivery on ASC osteogenesis in a calvarial defect model. The results of these studies will deepen the understanding between hypoxia and osteogenesis and inform efforts to improve ASC osteogenesis in vivo.