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

研究缺氧信号对脂肪干细胞成骨的作用

• 批准号: 9910771
• 负责人: Ashley Farris
• 金额: $ 4.65万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9910771
• 关键词: 3-Dimensional; 3D Print; Adipose tissue; Alkaline Phosphatase; Autologous Transplantation; Biochemical; Biocompatible Materials; Biological; Biological Assay; Bispecific Antibody 2B1; Blood Vessels; Bone Density; Bone Regeneration; Bone Transplantation; Calcium; Caliber; Calvaria; Cell Culture Techniques; Cell Death; Cell Differentiation process; Cell Survival; Cells; Cellular Metabolic Process; Characteristics; Collaborations; Conflict (Psychology); Data; Defect; Deposition; Direct Costs; Down-Regulation; Encapsulated; Environment; Expression Profiling; Extracellular Matrix; Fibrinogen; Future; Gene Expression; Genes; Geometry; Gold; Histology; Hypoxia; Hypoxia Inducible Factor; In Situ; In Vitro; Infiltration; Lead; Literature; Mature Bone; Measures; Mediating; Mediator of activation protein; Mesenchymal Stem Cells; Metabolic; Metabolism; Minerals; Modeling; Morbidity - disease rate; Mus; Osteogenesis; Outcome; Oxygen; Oxygen Consumption; Patients; Physiological; Polymers; Population; Procedures; Production; Proteins; Role; Signal Transduction; Site; Small Interfering RNA; Source; Stromal Cells; Structure; Techniques; Testing; Thick; Tissue Engineering; Tissues; Transfection; Transplantation; Treatment Efficacy; United States; Western Blotting; bone; craniofacial bone; design; experimental study; gain of function; genetic manipulation; hypoxia inducible factor 1; implantation; improved; in vitro Model; in vivo; insight; loss of function; microCT; mineralization; nanoparticle; novel; novel strategies; novel therapeutics; osteogenic; overexpression; overtreatment; polycaprolactone; response; scaffold; standard care; stem; stem cell therapy; stem cells; subcutaneous

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.

项目摘要/摘要：颅面骨移植在美国用于治疗超过 200,000 名患者 每年各州。自体移植是目前的治疗标准，具有多种缺点，包括供体部位 发病率和缺乏可用组织。一种有前途的替代方案是脂肪干/基质的组合 带有骨诱导生物材料支架的细胞（ASC），可以通过 3D 打印来模仿细胞的天然几何形状 缺陷。 ASC 可以很容易地通过非侵入性操作以高产率获得，并且已被证明 在体外强烈矿化，使其可用作干细胞来源。尽管有这些希望 由于ASCs的特性，其在体内再生骨的能力有限。主要假设 植入后的缺氧环境可能导致大量细胞死亡；然而，在初步 在体外实验中，我观察到在严重缺氧的培养物中 ASC 的存活率极好（>70%），但在 体外成骨分化、Runx2 表达和碱性磷酸酶 (ALP) 活性，两个常见的 成骨的标志物受到缺氧的抑制。此外，通过使用一种新颖的策略 通过向 3D 支架中接种的细胞输送氧气，我已经证明，原位提供氧气 在小鼠异位骨形成模型中，移植的 ASC 使体内形成的骨量增加了一倍。 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。 本研究的目的是调查缺氧与 ASC 之间的相互作用 成骨，克服了该领域的几个主要限制。首先，ASC在缺氧条件下成骨 使用 3D 体外骨形成模型进行定量研究 之前的文献研究了骨形成的影响 氧气对 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 3D 体外骨形成模型来量化骨形成的变化 缺氧导致的矿化、组织微结构、代谢和基因表达。广泛的基因 表达数据可能使我们能够识别氧依赖性 ASC 成骨的新介质。接下来，在 具体目标 2，我将使用增益研究缺氧信号传导与 ASC 成骨之间的相互作用 使用新型非病毒聚合物纳米粒子和释放氧气进行功能和功能丧失研究 脚手架。首先，我将确定是否可以通过以下方式模拟缺氧对 ASC 成骨的影响： 上调缺氧诱导因子-1α (HIF-1α) 下调 HIF-1α。第三，我会确定效果 ASC 成骨过程中的氧气释放。最后，在具体目标 3 中，我将研究 HIF-1α 增益的影响 - 颅骨缺损模型中 ASC 成骨的功能和功能丧失以及氧输送。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： 改善ASC体内成骨作用。