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

本跨学科研究项目由美国国家科学基金会（NSF） IDR项目资助，旨在探索细胞外物理刺激对干细胞命运的巨大影响，以及对承载组织成功的组织工程所必需的。为使患者特异性组织工程成为现实，需要一种涵盖多学科的跨学科方法。该项目的成功依赖于四位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，纳米和微制造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，1 V/cm量级的电刺激可加速人脂肪源性干细胞（hASC）的成骨分化，并显著增加其钙的积累。此外，他们还确定hASC具有初级纤毛。他们假设初级纤毛在电场作用下弯曲，因此在电刺激过程中发挥了关键的机械传导作用；此外，这种机械转导比其他分化途径优先诱导hASC成骨。因此，本项目的总体目标是研究以1hz频率施加1v /cm大小的电刺激将促进依赖于完整初级纤毛的hASC成骨的假设。为了验证这一假设，研究人员将完成以下3个目标：SA 1：确定以f = 1 Hz施加1 V/cm的交流电场对hASC命运的影响。定制的交叉指状电极（IDEs）将在我们的实验室制造。人类ASC对电刺激的反应将以hASC活力、增殖、细胞因子产生和分化为特征。SA 2：确定初级纤毛在电刺激传导到hASC和hASC分化中的作用。初级纤毛将受到生物化学和siRNA敲低策略的抑制。没有初级纤毛的人类ASC将被播种在定制的ide上，并暴露在频率为1hz的1v /cm的交流电场中。人类ASC反应的特征将在SA 1中描述，并与完整的纤毛细胞进行比较。SA 3：通过在无电场和有电场的情况下对细胞的初级纤毛成像，确定初级纤毛对电刺激的物理反应。初级纤毛将通过Hoffman调制在侧视图中成像，通过将细胞播种到柔性底物上，然后将其折叠并固定在ide上。拟议工作的更广泛影响：如果1 V/cm、f = 1 Hz的交流电场优先诱导hASC成骨，那么自体hASC可以直接植入关键缺陷，目前fda批准的临床设备可以用于电刺激hASC，以患者特定的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被研究过。如果初级纤毛在hASC的机械转导和功能性骨形成中起着至关重要的作用，那么未来的工作可以集中在生化或遗传手段来调节它们的表达，例如，在hASC群体中，初级纤毛的数量和/或长度。更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更少），即使在降低的电刺激场强下，也可能通过hASC形成结构和机械坚固的骨骼（目前仅通过化学成骨补充剂无法实现）。更广泛的影响包括：1)研究和教育的先进整合；2)促进弱势群体的参与。调查人员将利用与大学、高中和工业界的持续联系，为教育推广创造一种严谨而令人信服的方法。PI还将在她的实验室中利用NCSU接触新生（RISE）计划与高中学生群体进行接触，并在其实验室中接待和指导未被充分代表的少数民族，开发扩展视野（向初中女生介绍科学和工程的计划）研讨会，并为未被充分代表的科学和工程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，这与PI？美国的长期研究政策。

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.