Doctor of Philosophy
Physiology and Pharmacology
Musculoskeletal Health Research
Dixon, S. Jeffrey
Holdsworth, David W.
Our body’s functioning depends on the ability of cells to sense and react to their local mechanical environment; this process is known as mechanotransduction. Despite the importance of understanding how cells interact with mechanical stimuli, the specific mechanisms governing such processes have yet to be elucidated. Using microscopy to detect the early responses of living cells to mechanical loads and forces would be a critical step towards further understanding cellular mechanotransduction. Dynamic and high-frequency cyclical loads are relevant to human physiology and disease. Yet, modern microscopy systems are not capable of delivering the appropriate mechanical stimuli to live cell cultures. To address this deficiency, we developed a suite of mechanostimulation platforms that provide precise and relevant loads and forces to cell cultures during simultaneous microscopic analysis. We developed a motion-control system capable of precisely delivering vibrations to live cells during real-time microscopy. Using this system, we found that vibration of osteoblastic cells does not elicit acute elevation of cytosolic free calcium, but did desensitize responses to later stimulation with extracellular ATP. We next developed and validated a technique for the practical fabrication of microfluidic channels. In contrast to the effect of vibration, osteoblastic cells were found to respond to changes in fluid shear stress with transient elevation in the concentration of cytosolic free calcium. Lastly, we developed a system to apply disturbed fluid flow to live cells during real-time imaging. This system was used to demonstrate changes in the concentration of cytosolic free calcium in human endothelial cells exposed to laminar and disturbed flow. Our findings indicate that different forms of mechanical stimuli activate distinct signaling pathways in cells. Moreover, these new technologies will facilitate investigations of the signaling pathways activated by dynamic mechanical stimulation of a variety of cell types, in particular those of the skeletal and vascular systems.
Summary for Lay Audience
Our bodies have the capability to respond and adapt to mechanical loads and forces. Cells, the functional units of our body, have the machinery responsible for sensing and responding to these forces. For example, during physical activity such as running, the cells in our bones sense the repeated force applied to the body, and can signal to strengthen the bone tissue if necessary. Indeed, over time, increases or decreases in physical activity can strengthen or weaken our bones, respectively. In general, physical forces play an important role in the regular healthy function of the human body, and also in the development of many serious diseases, including bone disorders like osteoporosis and vascular diseases like atherosclerosis. Therefore, it is important to understand the processes by which cells carry out these functions. However, these processes have been difficult to study for many reasons. For one, there are several different kinds of forces that can interact with cells, including shear stress due to fluid flow and acceleration due to vibration. Another reason is that it is technically challenging to observe the immediate changes in cell activity when a specific force is applied.
In this thesis, we describe the development of new technologies that will allow scientists to study the early responses of living cells to the application of controlled physical forces. The first study describes a tool that allows scientists to vibrate cells back-and-forth hundreds of times per second, while only moving them less than the width of a human hair. The second study describes a tool that allows scientists to easily and affordably flow fluid over cells in a controlled way. The final study describes a tool that allows scientists to apply fluid flow to cells in a way similar to that seen in diseased blood vessels. These tools are all integrated with existing microscopes and cellular-imaging techniques. Our findings indicate that vibration and fluid flow activate different biological control systems within the interior of the cell. Further studies using these devices may aid in the development of new medical treatments for diseases like osteoporosis and atherosclerosis.
Lorusso, Daniel, "Dynamic mechanostimulation of live cells during real-time microscopy" (2019). Electronic Thesis and Dissertation Repository. 6480.
Available for download on Sunday, August 22, 2021