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: 10159731
• 负责人: Ashley Farris
• 金额: $ 2.78万
• 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 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10159731
• 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; 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; normoxia; 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：在美国，颅面骨移植用于治疗20多万名患者 每年一次。自体移植，目前的治疗标准，有多种缺点，包括供体部位。 发病率和可用组织的缺乏。一种有希望的替代方案是脂肪来源的干细胞/基质的组合。 细胞(ASCs)带有骨诱导生物材料支架，可以3D打印以模拟天然的几何形状 这个缺陷。ASCs可以很容易地通过非侵入性程序获得高产率，并已被证明 在体外矿化强劲，导致它们作为干细胞来源的用途。尽管有这些有希望的事情 由于ASCs的特性，在体内再生骨的能力有限。占主导地位的假设 植入后的低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，我观察到在严重低氧培养中ASC存活良好(70%)，但在 体外成骨分化、Runx2表达和碱性磷酸酶(ALP)活性，两种常见的 成骨的标志物，被低氧抑制。此外，通过使用一种新的战略来 将氧气输送到种植在3D支架中的细胞，我已经证明了在原位提供氧气 在小鼠异位骨形成模型中，移植的ASCs使体内形成的骨量增加了一倍。 因此，这一建议的主要前提是，ASCs体内骨形成的减少是由于 缺氧直接抑制ASC成骨。 本研究的目的是探讨低氧与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在低氧条件下会成骨 使用3D体外骨形成模型进行定量研究之前的文献检查了 氧对ASC分化的影响产生了相互矛盾的结果，因为它们依赖于 成骨作用。在特定的目标1中，我将使用骨形成的3D体外模型来量化 低氧引起的矿化、组织微结构、新陈代谢和基因表达。广泛性基因 表达数据可能使我们识别氧依赖的ASC成骨的新介质。下一步，在 具体目标2，我将研究低氧信号和ASC成骨之间的相互作用。 新型非病毒聚合物纳米粒子的功能和功能丧失研究及放氧 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，我将确定 ASC成骨过程中的氧释放。最后，在具体目标3中，我将研究低氧诱导因子-1α增益的影响。 在颅骨缺损模型中ASC成骨的功能、功能丧失和氧气输送。结果是 这些研究将加深对缺氧和成骨之间的理解，并为努力 促进ASC体内成骨。

项目成果

3D-printed oxygen-releasing scaffolds improve bone regeneration in mice.

• DOI: 10.1016/j.biomaterials.2021.121318
• 发表时间: 2022-01
• 期刊: Biomaterials
• 影响因子: 14
• 作者: Farris AL;Lambrechts D;Zhou Y;Zhang NY;Sarkar N;Moorer MC;Rindone AN;Nyberg EL;Perdomo-Pantoja A;Burris SJ;Free K;Witham TF;Riddle RC;Grayson WL
• 通讯作者: Grayson WL