4-D Imaging Cell/Scaffold Interplays During In Vivo Bone Repair Process

描述(申请人提供)：骨骼组织工程正在迅速接近人类应用阶段，尽管还没有开发出严格的临床前测试来了解细胞和环境决定成败的因素。由于传统的组织形成的组织学和分子标记不能指示骨形成的程度，因此对投资于支架的祖细胞的增殖特性以及宿主/供体对修复的相对贡献还没有得到了解。为了克服这个问题，我们开发或获得了一系列GFP报告转基因小鼠，以标记骨修复中主要细胞成分的来源和分化水平。小鼠模型已经被开发出来，利用2D冷冻组织学来评估这些事件，这种组织学保存了矿化组织中记者的活性。然而，在动态骨修复过程中的时间和空间细胞/支架相互作用的细节仍然不清楚。获得这些知识对于改进现有的或开发新的研究策略和疗法至关重要。因此，这项应用的主要目标是向组织工程界证明，有可能开发一种体内4D延时成像平台，以使用双光子显微镜显示小鼠颅骨缺损中细胞/支架/新骨的相互作用。这将是首次通过在动态骨再生过程中发射细胞特异性GFP信号来实时可视化3D细胞-支架修复系统。然后，我们将使用这个新建立的平台来查看我们研究小组最近创造的两个新型支架中骨骼发育的不同阶段。这一结果将被用来试图解释之前的观察结果，即无论种植的祖细胞类型如何，板层支架的成骨能力都优于细胞结构。我们推测，骨祖细胞最初在体内的分布、增殖和向成骨分化的进展方式决定了支架中骨形成的最终结果。4D成像平台的成功实施将对该领域产生变革，因为它将提供关于某一特定战略成败的基本信息。该平台将成为了解空间关系的主要成像基础，用于评价骨形成过程中细胞元素、细胞元素和细胞/支架相互作用之间的时间事件。它将使研究人员开始了解并最终优化支架设计和细胞参与者，以及生长因子选择和骨再生早期阶段的释放谱设计。这一知识基础对骨组织工程界将是至关重要的。 公共卫生相关性：美国每年进行超过130万次骨修复手术，这产生了对骨再生材料的巨大需求。然而，到目前为止，关于骨修复动态过程中支架与细胞之间的时空相互作用知之甚少。在这项研究中，我们建议建立一个4D活体成像平台，实时可视化支架内细胞活动和骨发育，从而指导我们设计更好的支架/细胞复合体用于骨修复。

DESCRIPTION (provided by applicant): Skeletal tissue engineering is rapidly approaching the stage for human application despite the fact that rigorous preclinical testing to understand the cellular and environmental determinants of success or failure has not been developed. As traditional histological and molecular markers of tissue formation do not indicate the degree of bone formation, the knowledge of the proliferative properties of the progenitor cells that invest in the scaffold and the relative host/donor contributions to the repair has not been achieved. To overcome this problem, we have developed or acquired a series of GFP reporter transgenic mice that mark the source and level of differentiation of the major cellular components in bone repair. Murine models have been developed to assess these events utilizing 2D cryo-histology that preserves the activity of the reporters in mineralized tissues. However, details of temporal and spatial cell/scaffold interactions during the dynamic bone repair process are still not understood. Gaining this knowledge is vital for improving existing, or developing new research strategies and therapies. Thus, the primary objective of this application is to demonstrate to the tissue engineering community that it is possible to develop an in vivo 4D time-lapse imaging platform to visualize cell/scaffold/new bone interplay in a mouse calvarial defect using 2-photon microscopy. This will be the first time that a 3D cell- scaffold repair system is visualized in real-time with the emission of cell-specific GFP signals during the dynamic bone regeneration. We will then use this newly established platform to view different stages of bone development in two novel scaffolds created recently within our research group. The results will be used in an attempt to interpret prior observations, that lamellar scaffolds have superior bone forming ability to that of the cellular structure, irrespective of the type of progenitor cells seeded. We hypothesize that the way the osteoprogenitor cells initially distribute, proliferate and progress toward osteogenic differentiation in vivo determines the ultimate outcome of bone formation in a scaffold. Successful implementation of the 4D imaging platform will be transformative to the field because it will provide the essential information as to why a particular strategy succeeds or fails. The platform will become the primary imaging base of understanding the spatial relationships, for appreciating temporal events between the cellular elements the cellular elements and cell/scaffold interactions during bone formation. It will allow investigators to begin to understand and ultimately optimize scaffold design and cellular participants as well as growth factor selection and release profile design for early stages of skeletal regeneration. This knowledge base will be crucial for the bone tissue engineering community. PUBLIC HEALTH RELEVANCE: More than 1.3 million bone-repair procedures are conducted per year in the USA, which created a huge demand in bone re-generating materials. However, very little is known to date about the temporal and spatial interactions between scaffold and cells during the dynamic process of bone repair. In this study, we propose to establish a 4D in vivo imaging platform to visualize in real-time cellular activities and bone development in scaffolds, and thereby guide us to design better scaffold/cell complex for bone repair.