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

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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.