Unraveling the Mechanism of Mechanotransduction in Hierarchical Collagen Fiber Formation - PROJECT SUMMARY Collagen fibers are the primary source of strength and function in tissues throughout the body, particular tendons, ligaments, and menisci. Cells organize these fibers hierarchically, assemble them from nm-wide fibrils, into larger fibers and fascicles, growing in size and strength with increasing mechanical demand. Injuries disrupt this organization, resulting in loss of function, pain, and decreased mobility. Unfortunately, collagen fibers do not regenerate after injury, nor in engineered replacements, creating a lack of repair options for torn tendons, ligaments, and menisci. Our long-term goal is to understand how cells regulate collagen fiber formation so to engineer functional replacements and drive repair in vivo for musculoskeletal tissues throughout the body. As a step towards this goal, the objective of this proposal is to explore how mechanical cues transmitted via cellular contraction and stretch-activated ion channels regulate ligament fibroblast’s development of hierarchical fibers. While cellular contraction forces, highly regulated by integrins, focal adhesion kinase (FAK), and the actomyosin network, are well established to play a major role in collagen fibril alignment, recent work has suggested stretch- activated ion channels, TRPV4 and Piezo1, also play independent and transient roles in regulating collagen organization. However, this work is largely confined to 2D surfaces, unorganized collagen gels, or mouse models, which all lack the larger fibers and fascicles that dominate human musculoskeletal tissues. Recently, we developed a novel culture device that guides cells to produce native-size hierarchically organized fibrils, fibers, and fascicles over 6 weeks of culture. This novel system provides the unique ability to finally dissect the mechanical cues of development and investigate how these forces guide cells to produce strong hierarchical fibers. We hypothesize mechanical signals via integrin-based contraction and stretch-activated ion channels are critical to cell-driven hierarchical fiber formation, with each regulating different aspects of fiber maturation, both with and without dynamic load. Specifically, we hypothesize that while cellular contraction via FAK is critical to progressive hierarchical development, TRPV4 will regulate alignment and remodeling at the fibril level, and Piezo1 will regulate matrix maturation via collagen crosslinking at the fiber and fascicle level. In Aim 1 we will evaluate the contribution of FAK, TRPV4, and Piezo1 in passive static culture when cell-generated contraction forces drive fiber formation and in Aim 2 we will evaluate how their contribution changes with dynamic mechanical stimulation. In both aims, we will investigate how each signaling mechanism, when inhibited or activated, alters collagen organization at the fibril, fiber, and fascicle length-scale, proteoglycan accumulation, and collagen crosslinking, all important to overall tissue function and mechanics. A better understanding of how mechanical cues drive cells to produce hierarchical fibers is integral not only to creating functional replacements, but also identifying therapeutic targets for regenerating collagen fibers after injury, reducing scar formation, and developing optimal rehabilitation protocols for regenerating musculoskeletal tissues throughout the body.
