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

描述（由申请人提供）：尽管尚未开发出用于了解成功或失败的细胞和环境决定因素的严格临床前测试，但Skeleton组织工程正在迅速接近人类应用阶段。由于传统的组织形成的组织学和分子标记物不能指示骨形成的程度，因此尚未获得对投资于支架的祖细胞的增殖特性以及对修复的相对宿主/供体贡献的知识。为了克服这个问题，我们已经开发或获得了一系列GFP报告基因转基因小鼠，这些小鼠标记了骨修复中主要细胞组分的来源和分化水平。已经开发了小鼠模型，以利用2D冷冻组织学评估这些事件，其保留了矿化组织中报告基因的活性。然而，在动态骨修复过程中，时间和空间的细胞/支架相互作用的细节仍然不清楚。获得这些知识对于改善现有或开发新的研究策略和疗法至关重要。 因此，本申请的主要目的是向组织工程界证明，可以开发体内4D延时成像平台，以使用双光子显微镜观察小鼠颅骨缺损中的细胞/支架/新骨相互作用。这将是第一次在动态骨再生过程中实时可视化3D细胞-支架修复系统，并发射细胞特异性GFP信号。然后，我们将使用这个新建立的平台，在我们研究小组最近创建的两种新型支架中观察骨发育的不同阶段。这些结果将用于解释先前的观察结果，即无论接种的祖细胞类型如何，层状支架都具有优于细胞结构的骨形成能力。我们假设骨祖细胞最初的分布、增殖和向体内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性影响，因为它将提供有关特定战略成功或失败原因的重要信息。该平台将成为理解空间关系的主要成像基础，用于理解骨形成过程中细胞元素之间的时间事件以及细胞/支架相互作用。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骨骼再生早期阶段的生长因子选择和释放曲线设计。这一知识基础将是至关重要的骨组织工程界。 公共卫生相关性：美国每年进行超过130万例骨修复手术，这对骨再生材料产生了巨大的需求。然而，目前对骨修复动态过程中支架和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在体内成像平台，实时显示支架中的细胞活动和骨发育，从而指导我们设计更好的骨修复支架/细胞复合物。

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.