BACKGROUND: Diabetes is a worldwide epidemic, and the number of those affected is only growing. Diabetes is characterized by hyperglycemia due to the body’s inability to produce or properly use insulin. Hyperglycemia contributes to diabetic complications in several ways, one of which is promoting glycation. Glycation is the non-enzymatic glucosylation of proteins, and because glycation is adventitious, the process most commonly occurs on proteins with long half-lives, such as collagen. Glycation greatly changes collagen’s mechanical and biochemical properties. Glycation leads to the production of advanced glycation end products (AGEs) that have been shown to contribute to the complications seen in diabetes in one of two ways: establishment of crosslinks between molecules in the basement membrane of the extracellular matrix, altering cellular function, or interactions between AGEs and AGE receptors on the cell surface. Diabetes greatly impairs the body’s ability to heal wounds, and it is thought that the AGEs produced by glycation greatly contribute this phenomenon. However, it is not fully understood, what direct role AGEs and glycated collagen plays in the wound healing process. Three-dimensional microtissue models have been developed for the purpose of studying wound healing, and the creation of a three-dimensional microtissue with glycated collagen allows for investigation into the specific role that glycated collagen plays on both the mechanical and biochemical properties of the wound closure and the healing process. METHODS: In order to study the effect of glycated collagen on wound healing, a protocol to make glycated collagen must first be developed. To make glycated collagen, soluble rat-tail type I collagen will be incubated with 250mM ribose at 4°C for a minimum of five days to allow the collagen to become glycated. The glycated collagen will be used to make a collagen gel, and then papain buffer will digest the gel. The extent of glycation will be determined through quantifying the digested glycated collagen gel’s autofluorescence, absorbance, and changes that can be perceived visually. Once it is confirmed that the collagen has been glycated, it will be incorporated into a microtissue model based on a previously published protocol. The microtissue will then be wounded with a micromanipulator and 16-gauge needle, and visualized via time-lapse microscopy. The rate at which the wound closes will be compared in microtissues made with glycated collagen to those made with non-glycated collagen. RESULTS: Glycation of collagen was unable to be confirmed consistently by measuring the autofluorescence of the collagen gel digests. However, the absorbance of the collagen gel digest was used to determine that the collagen was 43.16% glycated and visual changes in the collagen gels made with glycated collagen was also observed. Microtissues were able to successfully form with the glycated collagen, and were able to be used to compare wound healing in normal microtissues against those made with glycated collagen.

It has been well established that the fundamental behaviors of mammalian cells are influenced by the physical cues that they experience from their surrounding environment. With respect to cells in our bodies, mechanically-driven morphological and phenotypic changes to our cells have been linked to responses critical to both normal development and disease progression, including lung, heart, muscle and bone disorders, and cancer. Although significant advancements to our understanding of cell behavior have been made using 2D cell culture methods, questions regarding how physical stretch guides cell behavior in more complex 3D biological systems remain unanswered. To address these questions, we used microfabrication techniques to develop vacuum-actuated stretchers for high throughput stretching and dynamic mechanical screening of 3D microtissue cultures. This thesis contains five research chapters that have utilized these devices to advance our understanding of how cells feel stretch and how it influences their behavior in a 3D matrix. In the first research chapter (chapter 2), we characterized how stretch is transferred from the tissue-level to the single-cell level and we investigated the cytoskeletal reinforcement response to long-term mechanical conditioning. In the second research chapter (chapter 3), we examined the effects of an acute dynamic stretch and found that 3D cultures soften through actin depolymerization to homeostatically maintain a mean tension. This softening response to stretch may lengthen tissues in our body, and thus may be an important mechanism by which airway resistance and arterial blood pressure are controlled. In the third and forth research chapters (chapter 4-5), we investigated the time dependencies of microtissues cultures and we found that their behavior differed from our knowledge of the rheological behavior of cells in 2D culture. Microtissues instead followed a stretched exponential model that seemed to be set by a dynamic equilibrium between cytoskeletal assembly and disassembly rates. The difference in the behavior from cells in 2D may reflect the profound changes to the structure and distribution of the cytoskeleton that occur when cells are grown on flat surfaces vs. within a 3D environment. In the fifth and final research chapter (chapter 6), we examined how mechanical forces may contribute to the progression of tissue fibrosis through activating latent TGF-β1. Our results suggest that mechanical stretch contributes to a feed forward loop that preserves a myofibroblastic phenotype. Together these investigations further our understanding of how cells respond to mechanical stimuli within 3D environments, and thus, mark a significant contribution to the fields of mechanobiology and cell mechanics.