The objective of the research is to characterize the mechanical properties of the fibrillar form of fibronectin, and extracellular matrix structure that is essential for development and that is upregulated in pathologies such as cancer and atherosclerosis, through the analysis of a model system of fibronectin fibers and the use of a computational model built upon the stochastic properties of fibronectin molecules. Fibronectin is assembled into a unique material in vivo with extreme extensibility, leading to speculation that its litany of binding sites for cells and cell signaling molecules may be actuated by mechanical force. This proposal is unique in that it will quantify physical properties of fibronectin fibers that define its function in vivo over a wide range of mechanical strains and attempt to connect these physical properties with the molecular architecture of the fiber. The approach will utilize a technique for quantifying the molecular structure of fibronectin molecules in model fibers that will be compared with a computational model of fibronectin fiber mechanical properties that considers both molecular unfolding and entropic spring-like behavior of fibronectin molecules.
By combining all of these efforts, we expect this interdisciplinary proposal not only to generate a fundamental understanding of the underlying mechanisms governing mechanotransduction but also to have broad ranging implications in regenerative medicine and tissue engineering due to the fundamental role of fibronectin in vivo. It is surprising that despite our vast understanding of the importance of the physical environment on the behavior of virtually every studied cell, relatively little is known about the properties of native extracellular matrix structures. This program will be transformative in its capacity to promote new approaches in mechanotransduction research, as well as to immerse undergraduate and graduate students in a broad range of technological innovation. Active participation of both women and minority students will be fostered via a collaborative relationship with the Society of Women Engineers and Minority Engineers Society. Furthermore, this project will serve as a vehicle for the development of lab modules for courses at Boston University and Cornell University that are targeted for 3rd year undergraduate students.
The stiffness of tissues and organs is critically important to their functions, and alterations in stiffness from normal levels can be both a consequence and a cause of disease. For example, self palpation exams can be used to identify breast cancer due to the increased stiffness of the stroma surrounding the tumor. The materials that comprise the tissues and organs of mammalian organisms are composed of complex assemblies of proteins and sugars that are arranged in a particular architecture that defines their functions in vivo. How the mechanics of single and isolated molecules affects the collective mechanical behavior of larger supermolecular structures, and how these large (and dynamic) macromolecules interact with cells must be elucidated in order to understand how many biological materials, such as the extracellular matrix of cells, function. This is increasingly important since we now know that many cell types sense and respond to the rigidity of their surroundings. This project utilized combined experimental and computational approaches in order to determine how the mechanical properties of individual molecules leads to the bulk mechanical behavior that is sensed by cells. This project specifically studied fibronectin, a protein that assembles into fibers that are required for development and upregulated in diseased tissues. This project resulted in three primary research findings. First, a mathematical model was developed that uses a description of the mechanics of single molecules to predict the bulk material properties of fibronectin matrix. This model is unique since it does not use any fitting parameters, and it may therefore be useful for understanding the properties of other biological materials based on molecular mechanics. The second finding is that the molecular density of fibronectin matrix was determined for the first time. The molecular density was determined by applying ultraviolet microscopy to image single, micrometer-scale fibers. This is the first use of ultraviolet microscopy for such an application. Lastly, this project developed a novel test device for measuring the viscoelasticity of single fibronectin fibers. This device was used to show that fibronectin fibers creep over very extended time periods of up to 10 hours when loaded with a constant force. This finding of viscoelastic creep may be particularly important for mechanobiology since cells will not sense a static rigidity of fibronectin matrix fibers. This research program also facilitated the education and formal training of scientists and future engineers. Educationally, this project contributed to providing one graduate student with a broad, interdisciplinary project that incorporates research at molecular and microscopic length scales with in silico computer modeling. Next, this project also served as a basis for research and senior capstone design projects for numerous undergraduate students. Three of these students are now graduate students in PhD programs in biomedical engineering. The material researched in this project was also utilized for a module in the Upward Bound Math and Science program at Boston University, and more than 30 high school students from socioeconomically disadvantaged backgrounds were exposed to biomedical engineering through the lens of biomaterials.
