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

用于检查骨细胞-成骨细胞相互作用和反应的 3D 共培养系统

• 批准号: 8621841
• 负责人: RUSSELL P MAIN
• 金额: $ 21.01万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-11 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8621841
• 关键词: Address; Age-Related Bone Loss; Architecture; Autocrine Communication; Beds; Biochemical; Bone Tissue; Cell Culture Techniques; Cell physiology; Cells; Cellular biology; Coculture Techniques; Collagen; Computer Simulation; Data; Development; Environment; Fostering; Fracture; Future; Gap Junctions; Gene Expression; Genetic Models; Giant Cells; Goals; Health; Human; Hydroxyapatites; In Vitro; Knowledge; Liquid substance; Measures; Mechanics; Mediating; Mediator of activation protein; Methods; Minerals; Mission; Modeling; Monitor; Morbidity - disease rate; Osteoblasts; Osteocytes; Osteogenesis; Osteoporosis; Outcome; Paracrine Communication; Pathologic; Pathway interactions; Phosphoric Monoester Hydrolases; Physiology; Positioning Attribute; Process; Prostaglandins; Psyche structure; Public Health; Quality of life; Reaction Time; Regulation; Relative (related person); Research; Risk; Role; Signal Transduction; Skeleton; Source; Stimulus; Surface; System; Technology; Testing; Time; Transgenic Mice; Translational Research; Vent; Wasting Syndrome; autocrine; base; bone; bone cell; bone loss; cellular targeting; design; effective therapy; experience; extracellular; flexibility; flu; fluid flow; improved; in vivo; innovation; new technology; novel; osteosarcoma; paracrine; prevent; public health relevance; response; shear stress; skeletal; skeletal disorder; therapy development; transcription factor

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.

描述（由申请人提供）：当前的2D共培养模型，用于检查成骨细胞 - 骨细胞相互作用及其对环境刺激的反应无法概括这些细胞在骨架中形成的3D细胞合胞体，这代表了我们的基本差距，以阐明我们的方法，用于阐明vivo boneation In vivo Boneation的方法。由于骨细胞在2D中的行为与在3D条件下的行为不同，因此该间隙的持续存在代表了一个重要的障碍，它将继续限制利用合成代谢途径的治疗和预处理骨骼浪费疾病（如骨质疏松）的疗法的发展。促使该提案中研究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疗法来治疗骨骼疾病。该提案的目的是开发一种新型的3D共培养系统，该系统在体内成骨细胞骨细胞相互作用及其对骨细胞指导的物理刺激的反应。中心假设是，该3D共培养系统将在体内整骨细胞生理学和间隙连接介导的成骨细胞的调节，以响应于骨细胞的液体流动。这些研究的理由是，这种创新的3D共培养系统将改变骨细胞之间的物理和生化相互作用如何在现实的体外环境中进行检查。在研究团队在骨骼机械生物学，体外流体负荷模型和计算机械分析方面的经验的指导下，将通过追求三个特定目标来测试中心假设：（1）开发一种3D复合构建体，该构建体模型并在体内骨质骨体内复制的本地骨骼结构并重现； （2）表征了基质包裹的骨细胞对流经3D构建体的流体流动的自分泌反应； （3）表征成骨细胞对骨细胞定向的液流量的间隙连接介导的旁分泌反应。在AIM＃1中，将优化3D矿物 - 胶原蛋白构建体的组成，以促进骨形成和骨细胞分化。在AIM＃2中，将通过测量通过SOST和TCF/LEF活性来测量典型的Wnt-Catenin信号传导的实时变化来确定基质包裹的骨细胞对lacunar管流体剪切应力的自分泌反应。在AIM＃3中，将成骨细胞播种在富含骨细胞的3D构建体上，但仍免受直接流体流的屏蔽，以检查骨连接介导的成骨细胞对骨细胞定向流动流动刺激的反应。该应用中提出的研究具有创新性，因为它将验证一种新型的3D矿物 - 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骨组织共培养模型之间是独特的，它在其灵活性中以从各种来源和遗传模型中融合了骨细胞。拟议的研究具有重要意义，因为将原代人或转基因小鼠细胞纳入这种新技术的未来应用中，将加速转化研究，旨在发现细胞转录因子的机械作用，并评估其作为骨骼浪费疾病的药理疗法的疗效。