Defense Date
2026
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Engineering
First Advisor
Jennifer Puetzer
Second Advisor
Seth Cheatham
Third Advisor
Peter Pidcoe
Fourth Advisor
Hank Donahue
Fifth Advisor
Christopher Lemmon
Abstract
Hierarchical collagen fibers are the primary source of strength in tendons and ligaments; however, these fibers largely do not regenerate after injury or with repair, resulting in limited treatment options. We previously developed a static culture system that guides anterior cruciate ligament (ACL) fibroblasts to produce native-sized fibers and early fascicles by 6 weeks. These constructs are promising ligament replacements, but further maturation is needed. Mechanical cues are critical for development in vivo and in engineered tissues;however, the effect on larger fiber and fascicle formation is largely unknown. The collective focus of this work was to drive further maturation in our system to create stronger engineered replacements, as well as to glean insight into cellular response to differing loading patterns based on strain, strain rate, and frequency at different degrees of collagen organization. We found that intermittent cyclic load at 5% or 10% strain drove cells to increase hierarchical collagen organization, collagen crimp, and tissue tensile properties. Further, the effect of loading on cells varied depending on degree of organization. Specifically, 10% load drove early improvements in tensile properties and composition, while 5% load was more beneficial later in culture, suggesting a shift in mechanotransduction (Chapter 1). From this outcome, we hypothesized that a progressively decreasing cyclic strain, as organization increases, would better drive cells to produce more mature hierarchical fibers, resulting in significantly stronger replacements. Contrary to this hypothesis, neither decreasing nor increasing stepped cyclic strain led to further tissue maturation.The stepped cyclic strain may have led to repeated disruption of cellular tensional homeostasis and thus repeated breakdown and remodeling of collagen. This led to a weaker, less organized construct than steady load (Chapter 2). In an effort to challenge, rather than disrupt tensional homeostasis, we evaluated whether combined slow-elongation and steady-intermittent cyclic stretch results in synergistic fiber maturation. We found that the combination of both loads led to synergistic improvements in fiber organization, composition, and tissue strength (Chapter 3). Lastly, we investigated the effect of intermittent cyclic load at different frequencies rather than differing strains and found that the effect of frequency had an inverse dose-response with a 0.5 Hz frequency resulting in stronger hierarchical fiber formation compared to a 2 Hz frequency (Chapter 4). Load at 0.5 Hz frequency drove the greatest maturation by 6 weeks of culture, with most of the improvements developing later in culture, while 2 Hz load drove early improvements in composition, but maturation was reduced later in culture once cells were anchored on aligned fibers, suggesting a shift in mechanotransduction or a cellular threshold in response to frequency of load. Collectively, physiologically relevant loading patterns were able to drive the development of some of the most mature tissue engineered ACLs to date. 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.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
8-4-2026