Spatiotemporal Modulation of Osteogenesis in a 3-D Stromal/Stem Cell Model

3-D 基质/干细胞模型中成骨的时空调节

• 批准号: 8979685
• 负责人: Daniel J. Hayes
• 金额: $ 35.75万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2016-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8979685
• 关键词: 3-Dimensional; Adipose tissue; Adult; Affect; AlamarBlue; Alkaline Phosphatase; Angiogenic Factor; Animals; Blood Vessels; Bone Regeneration; CD-1 Nude Mouse; Calcium; Calvaria; Cell Survival; Cell Therapy; Cell model; Cells; Complex; Cues; Culture Media; DNA; Defect; Development; Environment; Evaluation; Fracture; Gene Delivery; Gene Expression; Goals; Health; Human; Hydrogels; In Vitro; Lead; Life; Light; Literature; Location; Mechanics; Mesenchymal Stem Cells; Metabolic; Methods; Methylcellulose; MicroRNAs; Modeling; Mus; Musculoskeletal; Nude Mice; Operative Surgical Procedures; Osteocalcin; Osteogenesis; PECAM1 gene; Polymers; Positioning Attribute; Process; Production; Qualifying; Research; Role; Signal Transduction; Site; Spatial Distribution; Staining method; Stains; Stem cells; Structure; System; Techniques; Testing; Thick; Time; Tissue Engineering; Tissue Model; Tissues; Untranslated RNA; Variant; Vascular Endothelial Growth Factors; Vascularization; X-Ray Computed Tomography; adult stem cell; angiogenesis; base; bone; clinically relevant; comparative; gene delivery system; improved; in vivo; innovation; laser capture microdissection; mineralization; nanoplasmonic; osteogenic; osteopontin; photoactivation; plasmonics; repaired; scaffold; spatiotemporal; stem cell differentiation; von Willebrand Factor

DESCRIPTION (provided by applicant): A substantial body of literature supports linkages between osteogenic and angiogenic signals both in vitro and in vivo, but little is known about how their spatial and temporal coordination impacts the differentiation of adult stromal/stem cells (ASC) and bone regeneration. This study explores the coordination of angiogenic and osteogenic signals utilizing two techniques, recently described by our labs; i) the production ASCs sheets that can be stacked to generate quasi three- dimensional (3-D) structures and ii) the photo-controlled differentiation of ASCs through tightly regulated miRNA mimic delivery. The combination of these techniques will allow the induction of spatiotemporal gradients of angiogenic and osteogenic factors in a 3-D model to study how the timing and magnitude of differentiation cues impact ASC cell fate in the complex fracture site environment. Thus, our overall goal for this project is the development of an in vitro, quasi 3-D model of ASC osteogenesis to establish the effect of spatiotemporally modulated osteogenic and angiogenic cues and correlation of these results in a nude mouse calvarial defect model. As noted above we have developed a method of ASC cell sheet production utilizing a thermally reversible methylcellulose hydrogel polymer and automated cell manipulation system enabling the development of quasi 3-D structures to serve as model tissues. We have also demonstrated a light activated Plasmonic Gene Delivery system (PGDs), which serves as a convenient inducible gene delivery vehicle. When combined with miRNA mimics, miR-148b and miR-132, PGDs have been demonstrated to induce de novo osteogenic and angiogenic differentiation in human adipose derived ASCs, respectively. Combining both these methods will allow the induction of spatiotemporal differentiation gradients within the quasi 3-D ASC sheets and improve our understanding of the interplay of osteogenic and angiogenic factors in ASC based bone repair. The hypothesis will be tested in the following three Aims: Aim 1 will assess the impact on cell sheet stacking on ASC viability and differentiation. ASC cell sheets will be stacked into 1, 2, 5, 10 and 20 layers and cultured for up to 28 days to determine the impact on ASC viability, proliferation and differentiation potential. Aim 2 will assess the impact of temporally and spatialy varying the light activation of PGDs with osteogenic and angiogenic miRNA mimics on the osteogenesis and angiogenesis of ASCs within the quasi 3-D stem cells sheets. In aim 3 we will correlate in vitro results with in vivo in a 12-week nude mouse calvarial defect model. Optimal 3-D stack sizes and spatiotemporal induction conditions determined in Aim 1&2 will be tested in the repair of a calvarial defect in a CD-1 nude mouse. Animals will be euthanized at 1,6 and 12 weeks post-surgery and undergo histological, x-ray and �CT evaluation for bone formation and vascularization of the defect site. The successful conduct of the project will provide a greater understanding of how spatiotemporally coordinated expression of osteo- and angiogenic factors impact ASC differentiation into bone and additionally, this research will lead to improved stromal/stem cell based therapies for critical sized bone defect repair.

描述（由申请人提供）：大量文献支持体外和体内成骨信号和血管生成信号之间的联系，但对于它们的空间和时间协调如何影响成体基质/干细胞（ASC）和成体基质/干细胞（ASC）的分化知之甚少。这项研究利用我们实验室最近描述的两种技术探索血管生成和成骨信号的协调；i) 可以堆叠以生成准三维 (3-D) 的生产 ASC 片。结构和 ii) 通过严格调控 miRNA 模拟传递来控制 ASC 的分化，这些技术的结合将允许在 3D 模型中诱导血管生成和成骨因子的时空梯度，以研究分化的时间和程度。因此，我们该项目的总体目标是开发 ASC 成骨的体外准 3D 模型，以建立时空调节的效果。裸鼠颅骨缺损模型中的成骨和血管生成线索以及这些结果的相关性如上所述，我们开发了一种利用热可逆甲基纤维素水凝胶聚合物和自动化细胞操作系统生产ASC细胞片的方法，从而能够开发准3-D。我们还展示了光激活等离子体基因传递系统（PGD），当与 miRNA 模拟物结合时，它可以作为一种方便的诱导基因传递载体。 miR-148b 和 miR-132、PGD 已被证明可分别诱导人脂肪来源的 ASC 中的从头成骨和血管生成分化，结合这两种方法将允许在准 3-D ASC 表内诱导时空分化梯度并改善。我们对基于 ASC 的骨修复中成骨和血管生成因子相互作用的理解 该假设将在以下三个目标中得到检验： 目标 1 将评估细胞片堆叠对 ASC 活力和分化的影响 将 ASC 细胞片堆叠成 1、2、5、10 和 20 层并培养长达 28 天，以确定对 ASC 活力、增殖和分化潜力的影响。将评估使用成骨和血管生成 miRNA 模拟物在时间和空间上改变 PGD 的光激活对准 3-D 干细胞内 ASC 的成骨和血管生成的影响在目标 3 中，我们将在 12 周的裸鼠颅骨缺损模型中将体外结果与体内相关联。目标 1 和 2 中确定的最佳 3-D 堆栈大小和时空诱导条件将在颅骨缺损的修复中进行测试。 CD-1裸鼠将在手术后1.6周和12周处死，并接受组织学、X射线和CT评估骨形成和血管化。该项目的成功进行将有助于更好地了解骨和血管生成因子的时空协调表达如何影响 ASC 分化为骨，此外，这项研究还将改进基于基质/干细胞的关键尺寸疗法。骨缺损修复。