| Summary: | Thesis (Ph.D.)--Boston University === Cells respond to a number of cues that affect how they interact with their surrounding environment, such as topology, the presentation of adhesive ligands, and stiffness. Recent advancements in the field ofmechanobiology have revealed that one of the main ways in which cells sense these cues is through contractile forces. Mechanobiology research seeks to understand how environmental cues affect the forces that cells exert on their surronnding environment and how these mechanical forces are communicated to the cell and transformed into biochemical signals. Therefore, quantitative methods have been developed to determine cell contractility on soft, optically transparent, deformable surfaces by quantifying substrate deformation in terms of cellular traction forces. However, the currently available tools that are used to study cell interactions are limited in their applicability due to the need for specialized technical expertise that is not amenable to the widespread adaptation of these techniques. Therefore, we have sought to develop a novel traction force microscopy technique known as micropattem traction microscopy. With this technique, we hope to greatly simplify the current traction force microscopy techniques and provide a method which will be able to be adopted by a wide range of laboratories.
This dissertation describes the process ofthe development and application of this novel traction force technique to probe questions in mechanobiology that have not been previously broached due to the lack of appropriate tools. The technique itself uses
indirect microcontact printing to create a regularized array of fluorescent protein onto a glass substrate, which is then transferred to an optically transparent, soft, elastic polyacrylamide hydrogel. Cells, limited by their ability to adhere only to patterned regions, will deform the pattern at these defined points. Thus, with knowledge of the bulk elastic properties ofthe substrate and a priori knowledge of the pattern, we are able to quantify the force a cell is exerting without its removal. We also developed and released a robust, automated MATLAB program that will aid users in the calculation of traction forces so that people with limited experience with programming can utilize the program without significant investments into training. This indirect approach allows for not only individual proteins, but also for multiple, spatially distinct, fluorescent proteins such as fibronectin and gelatin to be simultaneously patterned onto this surface as well. The ability to pattern multiple proteins in a spatially defmed region significantly aids in giving users control over as many parameters as possible. Finally, we will explore the current and future potential that this technique has to offer to researchers in the field of mechanobiology.
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