IDR: Primary cilia as sensors of electric field during electrical stimulation induced hASC osteogenesis

IDR：初级纤毛作为电刺激诱导 hASC 成骨过程中电场的传感器

• 批准号: 1133427
• 负责人: Matthew Fisher
• 金额: $ 59.42万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1133427&HistoricalAwards=false
• 关键词: IDR; Primary; cilia; sensors; electric

This interdisciplinary research project, funded by the IDR program at NSF, explores the extracellular physical stimuli greatly affect stem cell fate and are necessary for successful tissue engineering of load bearing tissues. For patient specific tissue engineering to become a reality, an interdisciplinary approach encompassing multiple disciplines is required. The success of this project relies on the specific knowledge base of each of the four investigators in a broad range of interdisciplinary areas including stem cell biology and mechanobiology, biomechanics, electric fields at micron size scales, nano- and microfabrication, and advanced imaging techniques. Intellectual Merit: The investigators have unique preliminary data indicating that electrical stimulation of magnitude 1 V/cm accelerates human adipose derived stem cell (hASC) osteogenic differentiation and significantly increases their calcium accretion. Moreover, they have determined that hASC possess primary cilia. They hypothesize that the primary cilia bend in response to an applied electric field, and thus play a key mechanotransduction role during electrical stimulation; further, that this mechanotransduction preferentially induces hASC osteogenesis over alternate differentiation pathways. Thus, the overall objective of this project is to investigate the hypothesis that electrical stimulation of magnitude 1 V/cm applied at a frequency of 1 Hz will promote hASC osteogenesis that is dependent on intact primary cilia. To test this hypothesis, the investigators will complete the following 3 aims:SA 1: Determine effect of AC electric field of 1 V/cm applied at f = 1 Hz on hASC fate. Custom interdigitated electrodes (IDEs) will be fabricated in our laboratory. Human ASC response to electrical stimulation will be characterized for hASC viability, proliferation, cytokine production, and differentiation. SA 2: Determine role of primary cilia in the transduction of electrical stimulation to hASC and on hASC differentiation. Primary cilia will be inhibited both biochemically and with siRNA knockdown strategy. Human ASC without primary cilia will be seeded on custom IDEs and exposed to an AC electric field of 1 V/cm at a frequency of 1 Hz. Human ASC response will be characterized as described in SA 1 and compared with intact cilia cells.SA 3: Determine the physical response of the primary cilia to electrical stimulation by imaging the cells' primary cilia in the absence and presence of an electric field. Primary cilia will be imaged with Hoffman modulation in side-view by seeding the cells onto flexible substrates, which will then be folded and immobilized across the IDEs.Broader Impacts of the Proposed Work: If an AC electric field of 1 V/cm at f = 1 Hz preferentially induces hASC osteogenesis, then autologous hASC could be directly implanted in a critical defect and currently available, FDA-approved, clinical devices could be utilized for electrical stimulation of hASC to induce functional bone formation in vivo in a patient specific manner. The role of primary cilia as sensors of electrical stimulation has not been previously investigated. If primary cilia play a vital role in mechanotransduction and functional bone formation from hASC, future work can focus on biochemical or genetic means to modulate their expression, e.g., number and/or length of primary cilia, in a population of hASC. Longer primary cilia may be more mechanosensitive (e.g., less electrical stimulation required to bend the primary cilia), potentially allowing for formation of structurally and mechanically robust bone by hASC (this is not presently attained by chemical osteogenic supplements alone), even at a reduced electrical-stimulation field strength.Further broader impacts include: 1) advanced integration of research and education; and 2) promotion of participation of underrepresented groups. The investigators will use ongoing connections with universities, high schools, and industry to create a rigorous and compelling methodology for educational outreach. The PI will also host and mentor underrepresented minorities in her laboratory using the NCSU Reaching Incoming Students Enrichment (RISE) program for outreach to high school student groups, develop a workshop for Expanding Your Horizons (program to introduce junior high school girls to science and engineering), and provide extensive undergraduate research opportunities to underrepresented students in science and engineering. The involvement of women and underrepresented minority students will be particularly emphasized in the project, consistent with the PI?s long-term research policies.

这个跨学科的研究项目，由美国国家科学基金会的IDR计划资助，探索细胞外的物理刺激极大地影响干细胞的命运，是必要的成功的承重组织的组织工程。为了使患者特异性组织工程成为现实，需要一种涵盖多个学科的跨学科方法。该项目的成功依赖于四名研究人员在广泛的跨学科领域的具体知识基础，包括干细胞生物学和机械生物学，生物力学，微米尺度的电场，纳米和微细加工以及先进的成像技术。智力优势：研究人员有独特的初步数据表明，幅度为1 V/cm的电刺激加速了人脂肪源性干细胞（hASC）的成骨分化，并显着增加了它们的钙积累。此外，他们已经确定hASC具有初级纤毛。他们假设初级纤毛响应于施加的电场而弯曲，并且因此在电刺激期间发挥关键的机械转导作用;此外，这种机械转导优先诱导hASC骨生成而不是替代分化途径。因此，本项目的总体目标是研究以下假设：以1 Hz的频率施加1 V/cm量级的电刺激将促进依赖于完整的初级纤毛的hASC成骨。为了检验这一假设，研究者将完成以下3个目标：SA 1：确定以f = 1 Hz施加的1 V/cm AC电场对hASC命运的影响。将在我们的实验室制造定制的叉指电极（IDE）。将针对hASC活力、增殖、细胞因子产生和分化来表征人ASC对电刺激的响应。SA 2：确定初级纤毛在电刺激hASC的传导和hASC分化中的作用。初级纤毛将被生物化学地和用siRNA敲低策略抑制。将无初级纤毛的人ASC接种在定制IDE上，并暴露于1 Hz频率下1 V/cm的AC电场。将如SA 1中所述表征人ASC反应并与完整纤毛细胞进行比较SA 3：通过在不存在和存在电场的情况下对细胞的初级纤毛进行成像来确定初级纤毛对电刺激的物理反应。初级纤毛将通过将细胞接种到柔性基底上，在侧视图中使用霍夫曼调制成像，然后将其折叠并固定在IDE上。拟议工作的更广泛影响：如果在f = 1Hz下的1V/cm的AC电场优先诱导hASC成骨，则自体hASC可以直接植入关键缺陷中，并且目前可获得FDA批准，临床装置可用于hASC的电刺激，从而以患者特异性方式在体内诱导功能性骨形成。初级纤毛作为电刺激传感器的作用以前没有研究过。如果初级纤毛在hASC的机械转导和功能性骨形成中起重要作用，未来的工作可以集中在生物化学或遗传手段来调节它们的表达，例如，初级纤毛的数量和/或长度。较长的初级纤毛可能对机械更敏感（例如，弯曲初级纤毛所需的电刺激较少），甚至在降低的电刺激场强下，潜在地允许通过hASC形成结构和机械上坚固的骨（这目前还不能通过单独的化学成骨补充剂实现）。研究人员将利用与大学，高中和行业的持续联系，为教育推广创造一个严格和令人信服的方法。PI还将在她的实验室中使用NCSU接触新生丰富（RISE）计划来接待和指导代表性不足的少数民族，以推广到高中学生团体，开发一个扩大视野的研讨会（计划将初中女生介绍给科学和工程），并为科学和工程领域代表性不足的学生提供广泛的本科研究机会。该项目将特别强调妇女和代表性不足的少数民族学生的参与，与PI？的长期研究政策。