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

描述(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，但对它们的时空协调如何影响成人基质/干细胞(ASC)分化和骨再生知之甚少。这项研究利用我们实验室最近描述的两种技术来探索血管生成和成骨信号的协调；i)生产可堆叠以产生准三维(3-D)结构的ASCs薄片；ii)通过严格调控的miRNA模拟递送来光控制ASCs的分化。这些技术的结合将允许在三维模型中诱导血管生成和成骨因子的时空梯度，以研究分化信号的时机和大小如何影响复杂骨折部位环境中ASC细胞的命运。因此，我们这个项目的总体目标是开发一种ASC成骨的体外准三维模型，以建立时空调控的成骨和血管生成信号的作用，并在裸鼠颅骨缺损模型中研究这些结果的相关性。如上所述，我们已经开发了一种利用热可逆的甲基纤维素水凝胶聚合物和自动化细胞操纵系统来生产ASC细胞片的方法，使得能够开发准三维结构作为模型组织。我们还展示了一种光激活的胞浆基因传递系统(PGDS)，它是一种方便的、可诱导的基因传递工具。当与miRNA模拟物、miR-148B和miR-132相结合时，PGDS已被证实分别诱导人脂肪来源的ASCs向新生成骨和血管生成分化。这两种方法的结合将允许在准三维ASC片内诱导时空分化梯度，并提高我们对成骨和血管生成因子在ASC骨修复中的相互作用的理解。这一假设将在以下三个目标中得到验证：目标1将评估细胞片堆积对ASC活性和分化的影响。将ASC细胞片堆叠成1、2、5、10和20层，培养长达28天，以确定对ASC活性、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PGDS与成骨和血管生成miRNA模拟物的光激活对准三维干细胞片内ASCs成骨和血管生成的影响。在目标3中，我们将在12周裸鼠颅骨缺损模型中将体外结果与体内结果相关联。在目标1和2中确定的最佳3-D堆叠大小和时空诱导条件将在修复CD-1裸鼠的颅骨缺损中进行测试。动物在术后1周、6周和12周被安乐死，并接受组织学、X射线和�CT评估缺损处的骨形成和血管形成。该项目的成功实施将使我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向骨的分化，此外，这项研究将导致改进基于基质/干细胞的治疗方法，用于关键大小的骨缺损修复。

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.