This project integrates advanced microscopy with a novel bioreactor for elucidating molecular, cellular and extracellular matrix interactions that guide angiogenic morphogenesis within a 3D tissue model. Though biologically motivated, implicit in this project is further characterization of nonlinear optical phenomena induced by ultrashort optical pulses an order of magnitude shorter than what is typically used in multiphoton microscopy and demonstration of its distinct advantages in live cell NLOM for the quantitative characterization of complex biological systems. The developed biaxial bioreactor will enable precise control of the mechanical environment in which 3D models of angiogenesis are cultured. Its strengths include the ability to control matrix density, stiffness and orientation. Combined with intravital NLOM, it will provide a powerful means to decipher dynamic cell-matrix interactions in culture and to gain new insight into mechanisms that enhance or inhibit angiogenesis.
Tissue engineering and regenerative medicine arose out of the need for replacement organs which far exceeds the number of donations. An approach to address the demand for replacement organs is to grow them in the lab. However, the size of tissues and organs grown in vitro is limited by the supply of nutrients and removal of waste products. In vivo, vascular blood vessels comprise an interconnected network that supplies nutrients and removes waste products. This vascular network is adaptable to changing needs such as supporting growing tissue or repairing wounds through the sprouting of new blood vessels by angiogenesis. In angiogenesis, endothelial cells that line existing blood vessels must detach and navigate the extracellular matrix all the while using this same matrix for leverage to move. Thus, a promising approach to growing utilitarian tissues and organs is to infiltrate them with blood vessels in a controlled manner. We hypothesized that by controlling the physical properties of tissue extracellular matrix, we could enhance or inhibit the movement of endothelial cells through the tissue, thereby controlling angiogenic patterning. The aims of this project included the development of enabling tools to manipulate and characterize the mechanical properties of the extracellular matrix as well as to characterize the movements of endothelial cells and patterning of resultant angiogenic vessels. For this we developed biaxial bioreactors that may be used to culture living tissue constructs under in-plane loads, characterize tissue mechanical responses, and couple with novel laser-based microscopy capable of high resolution imaging of cells, its movements, and its interactions with extracellular matrix proteins all the while maintaining a sterile environment free from contamination. As validation of our bioreactors, we were able to culture, mechanically test, and microscopically characterize evolving mechanical and microscopic properties of living tissue constructs over the time course of one month. With these enabling tools, we were able to mechanically and microscopically characterize angiogenic responses to tissue properties. Thus, with these tools, we were able to measure angiogenic responses to matrix stiffness and porosity. However, by changing density of extracellular matrix proteins, both stiffness and porosity change. To measure angiogenic responses to each property independently, a natural crosslinking agent transglutaminase was introduced to stiffen matrices without changing density. Thus, we measured angiogenic responses to matrix stiffness at constant porosity. Furthermore, with a series of matrices of different densities, crosslinking was used to create matrices of constant stiffness. The outcomes of this project demonstrated that the physical properties of extracellular matrix proteins may be used to modulate angiogenic responses. In addition, two graduate students were awarded the Ph.D. in biomedical engineering while working on this project. Also, activities of this project were used to provide research experiences for undergraduate students (who later matriculated to graduate programs) and to expose secondary school teachers to engineering research so that they could introduce engineering concepts to their students, increase student awareness of engineering, and encourage students to consider an engineering career.
