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

描述（由申请人提供）：大量文献支持体外和体内成骨和血管生成信号之间的联系，但对其空间和时间协调如何影响成体基质/干细胞（ASC）分化和骨再生知之甚少。本研究利用我们的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）可以堆叠以产生准三维（3-D）结构的ASC片层的生产和ii）通过严格调控的miRNA模拟物递送的ASC的光控分化。这些技术的组合将允许在3-D模型中诱导血管生成和成骨因子的时空梯度，以研究分化线索的时间和幅度如何影响复杂骨折部位环境中的ASC细胞命运。因此，本项目的总体目标是开发一种体外准3-D ASC成骨模型，以在裸鼠颅骨缺损模型中建立时空调制的成骨和血管生成线索的效果以及这些结果的相关性。如上所述，我们已经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，该系统能够开发准3-D结构以用作模型组织。我们还展示了光激活等离子体基因递送系统（PGDs），其用作方便的诱导型基因递送载体。当与miRNA模拟物（miR-148 b和miR-132）组合时，PGDs已被证明分别在人脂肪来源的ASC中诱导从头成骨分化和血管生成分化。结合这两种方法将允许在准3-D ASC片内诱导时空分化梯度，并提高我们对基于ASC的骨修复中成骨和血管生成因子相互作用的理解。将在以下三个目标中检验该假设：目标1将评估细胞片层堆积对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。目的2将评估在时间和空间上改变具有成骨和血管生成miRNA模拟物的PGDs的光活化对准3-D干细胞片内的ASC的骨生成和血管生成的影响。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结果相关联。将在CD-1裸鼠颅骨缺损修复中测试目标1&2中确定的最佳3-D堆叠尺寸和时空诱导条件。在术后1、6和12周对动物实施安乐死，并对缺损部位的骨形成和血管化进行组织学、X线和CT评估。该项目的成功实施将使人们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分化成骨，此外，这项研究将改善基于基质/干细胞的治疗方法，用于修复临界尺寸的骨缺损。

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.