Osteocytes as Dynamic Cells

描述(申请人提供)：骨细胞占骨骼细胞的90%以上。然而，由于在体内获取这些细胞或分离它们进行体外研究存在困难，人们对它们的功能知之甚少。尽管骨细胞传统上被认为是一种不活跃的细胞，但越来越多的证据表明，这些细胞对机械负荷有反应，并能够改变其局部环境，这表明它们可能比之前认为的更活跃。在牙本质基质蛋白-1(DMP1)启动子的控制下，利用绿色荧光蛋白(GFP)靶向骨细胞的转基因小鼠，对颅骨器官培养中的活骨细胞进行了时间推移动态成像研究。令人惊讶的是，这些研究表明，骨细胞远不是一个静态细胞，而是高度动态的。嵌在骨陷窝中的骨细胞扩张和收缩它们的胞体，伸展和收缩它们的树突。骨细胞之间以及与骨表面活动细胞之间的树突连接似乎是暂时的，有连接建立也有断开。这些观察结果提出了这样一种可能性，即树突可能不是永久性的细胞间连接，而是可以随着影响骨细胞功能的刺激而改变的动态结构。这项研究的中心假设是，骨中的骨细胞是高度动态的细胞，它们的胞体和树突运动对它们的功能和植入过程至关重要。为了验证这一假设，将使用多学科方法，包括使用转基因小鼠模型对活的骨细胞和成骨细胞进行动态成像，以及对细胞体和树突运动的计算分析。在目标1中，我们将使用最先进的成像技术来确定活体骨移植中骨细胞及其树突的动态特性。我们将确定这些变化是否随着年龄和/或对已知的影响骨细胞功能的外部刺激的反应而变化，如低氧和前列腺素E2。细胞运动和肌动蛋白聚合/解聚的抑制剂对作为骨细胞功能终点的缝隙连接信号的影响将被确定。在目标2中，我们将使用骨形成器官培养模型来动态成像成骨细胞嵌入和分化为骨细胞。这将使用分别由Col1a1和DMP1启动子驱动的在成骨细胞中表达DsRed和在骨细胞中表达GFP的小鼠的头盖骨来完成。矿物质沉积也将被成像，以确定成骨细胞嵌入/分化的动力学如何与矿化相结合。细胞运动和肌动蛋白聚合/解聚的抑制剂对包埋和矿化的影响将被确定。这些探索性研究的成功完成将为促进骨细胞功能的研究提供新的创新模型，为成骨细胞向骨细胞的转化过程提供基本的见解，并为将骨细胞树突视为动态交互结构提供新的范式。这将对我们理解骨细胞在正常骨骼中的作用以及在骨质疏松症和骨软化等疾病中的作用具有重要意义。这些研究将为在1-2年内应用RO1奠定基础。这项研究与公共卫生有关，因为它将为骨细胞的功能提供非常新的见解，骨细胞是骨中的一种主要细胞类型，目前仍不清楚。骨细胞凋亡与骨质疏松和颌骨骨坏死等疾病有关，最近的证据表明，这种细胞类型在调节骨形成/矿化和调节磷酸盐平衡方面发挥着重要作用。因此，了解这种细胞类型的功能将有助于开发治疗骨质疏松症、骨软化和其他代谢性骨骼疾病的方法。

DESCRIPTION (provided by applicant): Osteocytes make up over 90% of the cells in bone. However, little is known about their function, due to difficulties in accessing these cells in vivo or isolating them for in vitro studies. Although the osteocyte has classically been viewed as an inactive cell, evidence is accumulating that these cells are responsive to mechanical loading and are able to modify their local environment, suggesting that they may be more active than previously thought. Using a transgenic mouse in which green fluorescent protein (GFP) is targeted to osteocytes under control of the dentin- matrix protein-1 (DMP1) promoter, we have performed time lapse dynamic imaging studies on living osteocytes in calvarial organ cultures. Surprisingly, these studies have revealed that, far from being a static cell, the osteocyte is highly dynamic. Osteocytes that were embedded within their lacunae expanded and contracted their cell bodies and extended and retracted their dendrites. Dendritic connections between osteocytes and with motile cells on the bone surface appeared to be transient, with connections being made and broken. These observations raise the possibility that dendrites, rather than being permanent intercellular connections, may be dynamic structures that can be altered in response to stimuli affecting osteocyte function. The central hypothesis for the proposed studies is that osteocytes within bone are highly dynamic cells and that their cell body and dendrite motions are critical to their function and to the embedding process. To test this hypothesis, multidisciplinary approaches will be used, including the use of transgenic mouse models for dynamic imaging of living osteocytes and osteoblasts, together with computational analysis of cell body and dendrite motions. In aim 1 we will use state-of-the-art imaging techniques to determine the dynamic properties of osteocytes and their dendrites in living bone explants. We will determine whether these change with age and/or in response to external stimuli known to affect osteocyte function, such as hypoxia and prostaglandin E2. The effects of inhibitors of cell motility and actin polymerization/ depolymerization on gap junctional signaling as an endpoint of osteocyte function will be determined. In aim 2 we will use a bone forming organ culture model to dynamically image osteoblasts embedding and differentiating into osteocytes. This will be done using calvaria from mice expressing DsRed in osteoblasts and GFP in osteocytes, driven by the Col1a1 and DMP1 promoters, respectively. Mineral deposition will also be imaged to determine how the dynamics of osteoblast embedding/differentiation are integrated with mineralization. The effects of inhibitors of cell motility and actin polymerization/depolymerization on embedding and mineralization will be determined. Successful completion of these exploratory studies will develop new and innovative models to facilitate research into osteocyte function, provide fundamental insights into the process of osteoblast to osteocyte transition and provide a new paradigm for viewing osteocyte dendrites as dynamic interactive structures. This will have major implications for our understanding the role of osteocytes in normal bone and in diseases, such as osteoporosis and osteomalacia. These studies will lay the foundation for an RO1 application in 1-2 years. This research is relevant to public health as it will provide highly novel insights into the function of the osteocyte, a major cell type in bone, which is still not well understood. Osteocyte apoptosis has been implicated in diseases such as osteoporosis and osteonecrosis of the jaw and recent evidence suggests that this cell type plays a major role in regulation of bone formation/mineralization and in regulation of phosphate homeostasis. Understanding the function of this cell type will therefore aid the development of treatments for osteoporosis, osteomalacia and other metabolic bone diseases.