A 3D co-culture system for examining osteocyte-osteoblast interactions and respon

描述（由申请人提供）：目前用于检查骨细胞-成骨细胞相互作用及其对环境刺激的反应的2D共培养模型不能重现这些细胞在骨骼中形成的3D细胞合胞体，这代表了我们用于阐明体内骨形成调控机制的方法中的根本性空白。由于骨细胞在2D条件下的行为与3D条件下不同，因此这种差距的持续存在代表了一个重大障碍，该障碍将继续限制利用合成代谢途径治疗和预防骨骼消耗性疾病（如骨质疏松症）的疗法的发展。本提案中的长期研究目标是通过了解骨形成成骨细胞的骨细胞调节来开发新的体内物理和药理学疗法来治疗骨骼疾病。本提案的目的是开发一种新的三维共培养系统，再现体内骨细胞-成骨细胞相互作用及其对骨细胞定向物理刺激的反应。中心假设是该3D共培养系统将再现体内骨细胞生理学和响应于骨细胞定向的流体流动的成骨细胞的间隙连接介导的调节。这些研究的基本原理是，这种创新的3D共培养系统将改变如何在现实的体外环境中检查骨细胞之间的物理和生化相互作用。在研究团队在骨骼机械生物学、体外流体载荷模型和计算力学分析方面的经验指导下，中心假设将通过追求三个特定目标进行测试：（1）开发一种模拟天然骨结构并再现体内骨细胞生理学的3D复合材料结构;（2）表征基质包埋的骨细胞对通过3D结构的流体流动的自分泌反应;（3）在3D中表征成骨细胞对骨细胞导向的流体流动的差距连接介导的旁分泌反应。在目标#1中，将优化3D矿物质-胶原构建体的组成以促进骨形成和骨细胞分化。在目标#2中，将通过Sost和TCF/Lef活性测量经典Wnt-连环蛋白信号传导的实时变化来确定基质包埋的骨细胞对腔隙性小管流体剪切应力的自分泌反应。在目标#3中，将成骨细胞接种在骨细胞富集的3D构建体上，但屏蔽直接流体流动，以检查成骨细胞对骨细胞定向流动刺激的差距连接介导的反应。本申请中提出的研究是创新性的，因为它将验证新型3D矿物质-胶原共培养系统，以阐明骨细胞定向的物理刺激在调节骨形成中的作用，并且在3D骨组织共培养模型中是独特的，因为它灵活地掺入来自各种来源和遗传模型的骨细胞。拟议的研究是重要的，因为在这种新技术的未来应用中纳入原代人类或转基因小鼠细胞将加速旨在发现细胞转录因子的机制作用并评估其作为骨骼消耗性疾病药物治疗靶点的疗效的转化研究。

DESCRIPTION (provided by applicant): Current 2D co-culture models for examining osteocyte-osteoblast interactions and their response to environ- mental stimuli cannot recapitulate the 3D cellular syncytium formed by these cells in the skeleton, representing a fundamental gap in our approach for elucidating regulatory mechanisms of in vivo bone formation. Because bone cells behave differently in 2D than in 3D conditions, continued existence of this gap represents a significant barrier that will continue to limit development of therapies harnessing anabolic pathways to treat and pre- vent skeletal wasting diseases, like osteoporosis. The long-term research goal motivating the studies in this proposal is to develop new in vivo physical and pharmacological therapies to treat skeletal disease through an understanding of osteocyte regulation of bone-forming osteoblasts. The objective of this proposal is to develop a novel 3D co-culture system that reproduces in vivo osteocyte-osteoblast interactions and their responses to osteocyte-directed physical stimuli. The central hypothesis is that this 3D co-culture system will reproduce in vivo osteocyte physiology and gap junction-mediated regulation of osteoblasts in response to osteocyte- directed fluid flow. The rationale for these studies is that this innovative 3D co-culture system will transform how physical and biochemical interactions among bone cells can be examined in a realistic in vitro environment. Guided by the research team's experience in skeletal mechanobiology, in vitro fluid loading models, and computational mechanical analyses, the central hypothesis will be tested by pursuing three specific aims: (1) Develop a 3D composite construct that models native bone architecture and reproduces in vivo osteocyte physiology; (2) Characterize the autocrine response of matrix-embedded osteocytes to fluid flow through the 3D construct; and (3) Characterize the gap junction-mediated paracrine response of osteoblasts to osteocyte- directed fluid flow in 3D. In Aim #1, the composition of the 3D mineral-collagen construct will be optimized to promote bone formation and osteocyte differentiation. In Aim #2, the autocrine response of matrix-embedded osteocytes to lacunar canalicular fluid shear stress will be determined by measuring real-time changes in canonical Wnt-catenin signaling through Sost and TCF/Lef activity. In Aim #3, osteoblasts will be seeded on the osteocyte-enriched 3D construct, but shielded from direct fluid flow, to examine the gap junction-mediated response of osteoblasts to osteocyte-directed flow stimuli. The research proposed in this application is innovative because it will validate a novel 3D mineral-collagen co-culture system to elucidate the role of osteocyte- directed physical stimuli in regulating bone formation and is unique among 3D bone tissue co-culture models in its flexibility to incorporate bone cells from various sources and genetic models. The proposed research is significant because incorporating primary human or transgenic mouse cells in future applications of this novel technology will accelerate translational research aimed at discovering the mechanistic role of cellular transcription factors and evaluating their efficacy as targets for pharmacologic therapies for skeletal wasting diseases.