Intermittent Cyclic Stretch of Engineered Ligaments Drives Hierarchical Collagen Fiber Maturation in a Dose- and Organizational-Dependent Manner.

Hierarchical collagen fibers are the primary source of strength in tendons and ligaments, however these fibers do not regenerate after injury or with repair, resulting in limited treatment options. We previously developed a culture system that guides ACL fibroblasts to produce native-sized fibers and fascicles by 6 weeks. These constructs are promising ligament replacements, but further maturation is needed. Mechanical cues are critical for development and in engineered tissues; however, the effect on larger fiber and fascicle formation is largely unknown. Our objective was to investigate whether intermittent cyclic stretch, mimicking rapid muscle activity, drives further maturation in our system to create stronger engineered replacements and to explore whether cyclic loading has differential effects on cells at different degrees of collagen organization to better inform engineered tissue maturation protocols. Constructs were loaded with an established intermittent cyclic loading regime at 5 or 10% strain for up to 6 weeks and compared to static controls. Cyclic loading drove cells to increase hierarchical collagen organization, collagen crimp, and tissue mechanics, ultimately producing constructs that matched or exceeded immature ACL properties. Further, the effect of loading on cells varied depending on degree of organization. Specifically, 10% load drove early improvements in mechanics and composition, while 5% load was more beneficial later in culture, suggesting a cellular threshold response and a shift in mechanotransduction. This study provides new insight into how cyclic loading affects cell-driven hierarchical fiber formation and maturation, which will help to develop better rehabilitation protocols and engineer stronger replacements.