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：颅面骨移植用于美化200,000多名患者 每年的状态。当前治疗标准自体移植具有多个缺点，包括供体现场 发病率和缺乏可用的组织。有望的替代方法是脂肪衍生的茎/基质组合 细胞（ASC）具有骨诱导的生物材料支架，可以被3D打印以模仿天然的几何形状 缺陷。 ASC可以从非侵入性程序中很容易获得，并已证明 在体外矿化强度，导致它们作为干细胞来源的效用。尽管有这些承诺 特征，ASC表现出有限的体内骨骼再生能力。主要假设 是植入后的低氧环境可能导致大规模细胞死亡；但是，初步 在体外实验中，我观察到在严重低氧培养中的出色（> 70％）ASC生存期，但在IN期间 体外成骨分化，Runx2表达和醇磷酸酶（ALP）活性，两个常见 缺氧抑制成骨的标志物。另外，通过使用新型策略 将氧气输送到3D支架中的细胞中，我已经证明了原位提供氧气 移植的ASC在鼠生态骨形成模型中体内形成的骨骼量增加了一倍。 这是该提议的主要前提是，ASC的体内骨形成减少是由于 缺氧直接抑制ASC成骨的作用。 拟议的研究的目的是研究缺氧与ASC之间的相互作用 成骨，克服了该领域的几个主要局限性。首先，缺氧的ASC成骨发生将是 使用3D骨形成的3D体外模型进行定量研究，先前的文献检查了 ASC分化的氧气产生了冲突的结果，因为它们是根据定性指标营救的 成骨。在特定目标1中，我将使用3D体外骨形成模型来量化 由于缺氧引起的矿化，组织微体系结构，代谢和基因表达。广泛的基因 表达数据可能使我们能够鉴定出氧依赖性ASC成骨的新型介体。接下来，在 特定的目标2，我将利用 - 使用获得的增长来研究低氧信号传导和ASC成骨的相互作用 使用新型的非病毒聚合纳米颗粒和释放氧气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 上调低氧诱导因子-1α（HIF-1α）下调HIF-1α。第三，我将确定 ASC成骨的氧气释放。最后，在特定的目标3中，我将研究HIF-1α的效果 在颅不足模型中，功能和功能丧失和氧递送对ASC骨的发生。结果 这些研究将加深缺氧和成骨的理解，并为 改善体内的ASC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